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The author(s) shown below used Federal funds provided by the U.S. Department of Justice and prepared the following final report:
Document Title: Synthesis and Analytical Profiles for Regioisomeric and Isobaric Amines Related to MDMA, MDEA and MBDB: Differentiation of Drug and non-Drug Substances of Mass Spectral Equivalence
Author: C. Randall Clark, Ph.D. Document No.: 236243
Date Received: October 2011 Award Number: 2006-DN-BX-K016
This report has not been published by the U.S. Department of Justice. To provide better customer service, NCJRS has made this Federally-funded grant final report available electronically in addition to traditional paper copies.
Opinions or points of view expressed are those
of the author(s) and do not necessarily reflect the official position or policies of the U.S.
Department of Justice.
1
Final Technical Report
Synthesis and Analytical Profiles for Regioisomeric and Isobaric Amines Related to MDMA, MDEA and MBDB: Differentiation of Drug and non-Drug Substances of
Mass Spectral Equivalence
Award Number: 2006-DN-BX-K016
Submitted By: C. Randall Clark, Ph.D. Auburn University
Abstract: This project has focused on issues of resolution and discriminatory capabilities in
controlled substance analysis providing additional reliability for forensic evidence and
analytical data on regioisomeric and isobaric phenethylamines of forensic interest. The broad
objective of this research is improved specificity in the analytical methods used to identify
MDMA, MBDB, MDEA, and related phenethylamine controlled substances. This improved
specificity comes from methods which allow the forensic analyst to eliminate non-drug
imposter substances as the source of analytical data matching that of the controlled drug
substance. This project has developed methods to discriminate between the
methylenedioxyphen-ethylamine drugs and those regioisomeric and isobaric molecules having
the same molecular weight and major fragments of equivalent mass (i.e. identical mass
spectra). The work has emphasized those analytical methods commonly employed in forensic
laboratories for confirmation of drug identity: gas chromatography, mass spectrometry,
infrared spectrometry. When compounds exist which produce the same mass spectrum (same
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
2
MW and fragments of equivalent mass) as the drug of interest, the identification by GC-MS
must be based entirely upon the ability of the chromatographic system to resolve these
substances.
The initial phase of this work involved the organic synthesis of the direct
regioisomeric substances related to MDMA, MBDB, and MDEA. We also prepared
representative samples of the indirect regioisomeric molecules (such as the 2, 3, and 4-
methoxymethcathinones) and the isobaric substances, compounds of the same nominal mass
but different elemental composition (for example 2, 3, and 4-ethoxyphenethylamines and the
10 different ring substitution patterns for the methoxy-methyl-phenethylamines). Additional
compounds were prepared in related categories to further refine the developed analytical
methods and confirm/challenge experimental observations. A number of deuterium labeled
analogs has been prepared to establish mass spectral fragmentation patterns. Other isomeric
series were prepared such as the methoxymethylene-substitution phenyl ring pattern for the
MDMA and the MBDB side chain series and the 2-, 3-, and 4-ethoxyphenyl compounds for
the MBDB series. Overall, in this project more than 90 phenethylamines having a
regioisomeric or isobaric relationship to MDMA or MBDB have been synthesized and
evaluated.
The chromatographic retention properties for each series of isomers have been
evaluated by gas chromatographic techniques on a variety of stationary phases. These studies
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
3
established structure-retention relationships (elution orders) for the side chain regioisomers and
the isobaric amines on a number of stationary phases.
Mass spectrometry studies have focused on the underivatized free amines and have
clearly established the significant similarity in the mass spectrum for the side chain and ring
regioisomers. Derivatization studies have established methods for differentiation of side chain
substitution patterns yielding unique fragment ions of significant abundance in the EI mass
spectrum for these compounds. The perfluoroacyl derivatives provide maximum mass spectral
information for compound individualization and in a number of regioisomeric subsets the HFBA
derivatives provide more data for molecular individualization than PFPA or TFA derivatives. A
detailed background study established the advantages of perfluoroacyl derivatives over other
alkyl or aryl amide derivatives. A second detailed background study provided the scientific
evidence for the mechanism of fragmentation producing the unique m/z 110, 160 and 210 ions
for the methamphetamine side chain in the TFA, PFPA and HFBA derivatives respectively. The
mass spectral studies clearly show that the individual side chain isomers of m/z58 and m/z72 can
be identified by specific fragment ions. Additionally unique ions help to point the direction of
investigation for ring substitution pattern. For example the unique fragment ions at m/z107 in the
spectrum of ethoxy-substituted phenethylamines allows its’ differentiation from
methylenedioxyphenethylamines. Vapor phase infrared spectrometry following separation by gas
chromatography (GC-IRD) provided confirmation of the nature and position of aromatic ring
substituent.
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
4
TABLE OF CONTENTS
Abstract……………………………………………………………………………………1
Excutive Summary……………………………………………………………………….9
I.Introduction…………………………………………………………………………….16
I.1 Statement of the Problem ……………………………………………………….....16
I.2 Literature Citations and Review……………………………………………………20
I.3 Statement of Hypothesis or Rationale for the Research……………………………...47
II. Methods (Narrative Descriptions of the Experimental Design) ……………………...48
II.1 Synthesis Design ……………………………………………………………………48
II.2 Methods (Synthetic materials, and procedures) …………………………………..57
II.2.1 Materials……………………………………………………………………….57
II.3 Analytical Methods……………………………………………………………...…58
II.3.1 Instruments……………………………………………….………………….…58
II.3.2 GC- Columns………………………………………………………………..…59
III. Results………………………………………………………………………………61
Chapter 1…………………………………………………………………………….62
Mass Spectrometry and Gas Chromatographic Studies on Direct Regioisomers to 3,4-MDMA…………………………………………….…………..62 Mass spectral studies of direct regioisomers of 3,4-MDMA (methylenedioxyphenyl substituted side-chain regioisomers) …… ….……………62
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
5
Mass spectral studies of perfluroacyl derivatives of 3,4- MDMA and their direct regioisomers………………………………………67
Gas chromatographic separation of perfluroacyl derivatives of 3,4- MDMA and their direct regioisomers………………………………………83
Conclusions of Chapter 1…………………………………………………………88
Chapter 2……………………………………………………………………………89
Mass Spectrometry and Gas Chromatographic Studies of Indirect Regioisomers Related to 3,4-MDMA, the Methoxymethcathinones. …………89
Mass spectral studies of the undreivatized and the perfluroacyl derivatives of the methoxymethcathinones…………………………………………………90
Gas chromatographic Separation of 2,3-MDMA , 3,4-MDMA from the Methoxymethcathinnes………………………………………………………...…101
Conclusion of Chapter 2……………………………………………………….…106
Chapter 3……………………………………….…………………………………107
Mass Spectrometry and Gas Chromatographic Studies on Acylated Derivatives of a Series of Side Chain Regioisomers of 2-Methoxy-4-Methyl Phenethylamines…………………………………………………………………107
Mass Spectral Studies on Chain Regioisomers of 2-Methoxy-4-Methyl Phenethylamines…………………………………………………………………107
Gas Chromatographyic separation of the perflouroacyl derivatives of 2- methoxy-4-methylphenethylamines from 2,3- and 3,4-MDMA……………123
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
6
Conclusion of Chapter 3………………………………………..……………………127
Chapter 4…….……………………………………………………………………128
GC-MS Analysis of Acylated Derivatives of the Side Chain Regioisomers of 4-Methoxy-3-Methyl Phenethylamines Related to Methylenedioxymethamphetamine………..128
Mass Spectrometral studies of 4-methoxy-3-methyl phenethylamines along with 2,3- and 3,4- MDMA……………………………………….…………………….128 Gas Chromatography of the regioiosmeric4-methoxy-3-methyl Phenethylamines and 2,3- and 3,4-MDMA………………………………………146 Conclusions of Chapter 4………………………………………………….……...149
Chapter 5……………………………………………………….…………………....150
Gas Chromatographic and Mass Spectral Studies on Acylated Side Chain Regioisomers of 3-Methoxy-4-methyl-phenethylamine and 4-Methoxy-3-methylphenethylamine…150
Mass Spectral Studies on Side Chain Regioisomers of 3-Methoxy-4-methyl-phenethylamine and 4-Methoxy-3-methylphenethylamine……………………...150
Gas Chromatography of 3-Methoxy-4-methyl-phenethylamine, 4-Methoxy-3-methyl-phenethylamine, 2,3- and 3,4-MDMA………………………………….173
Conclusions of Chapter 5…………………………………………………………..176
Chapter 6……………………………….…………………………………………177
Mass Spectral and Gas Chromatographic Studies of Methoxy Methyl Methamphetamines Isobaric to 3, 4-MDMA. ……………………………………………………………177 Mass Spectral Studies of Methoxy Methyl methamphetamines Isobaric to 3,4-MDMA………………………………………………….……….177
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
7
Mass Spectral Studies of Perfluroacyl Derivatives Methoxymethyl Methamphetamines compared to 2,3- and 3,4-MDMA………………………………………………183 Gas chromatographic separation of the perfluroacyl derivatives of ring substituted methoxy methyl methamphetamines, 2,3- and 3,4- MDMA. ………200
Conclusions of Chapter 6………………………………………………………..211
Chapter 7…………………………………………………………………………….213
GC–MS Analysis of Ring and Side Chain Regioisomers of Ethoxyphenethylamines………………………………………………………….213 Mass spectrometry of Ring and Side Chain Regioisomers of Ethoxyphenethylamines………………………………………………………….213 Gas chromatography of Ring substituted Ethoxy Phenethylamines, 2,3- and 3,4-MDMA. …………………………………………………………….234
Conclusions of Chapter 7…………………………………………………………238
Chapter 8……………………………………………………………………...239
GC-MS Studies on Direct Side Chain Regioisomers Related to Substituted Methylenedioxyphenethylamines; MDEA, MDMMA and MBDB……………...239 Mass Spectral Studies……………………………………………………………239 Gas chromatography of Side Chain Regioisomeric Phenethylamines Related to the Controlled Substances MDEA, MDMMA, and MBDB. ………………….257
Conclusion of Chapter 8………………………………………………………...260
Chapter 9…………………………………………………………………...260
GC-MS Evaluation of a series of Acylated Derivatives of 3,4-Methylenedioxymethamphetamine .......................................................................260
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
8
Mass Spectrometry of Acylated Derivatives of 3,4-Methylenedioxymethamphetamine………………………………………………261
Gas Chromatography………………………………….………………………….275
Conclusions of Chapter 9…………………………………………………………..277
IV. Conclusions……………………………………………………………………278
IV.1 Discussion of findings. …………………………………………………………278
IV.2 Implications for policy and practice. …………………………………………...278
IV.3 Implications for further research………………………………………………..279
V. References………………………………………………………………………….281
VI. Dissemination of Research Findings………………………………………………293
VI.1 Scientific Publications Related to this Project…………………………………….293
VI.2 Scientific Presentations Related to this Project…………………………..……….298
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
9
Executive Summary: In this project, we have synthesized over 90 regioisomeric and isobaric
phenethylamines of mass spectral equivalence and evaluated the analytical profiles of these
amines. The work has emphasized those analytical methods commonly employed in forensic
laboratories for confirmation of drug identity: gas chromatography, mass spectrometry,
infrared spectrometry. The synthetic efforts focused on those regioisomeric and isobaric
substances most likely to appear in future forensic samples. In some cases compounds were
prepared to complete a set of all the possibilities in a series when some of the regioisomers
have already appeared in clandestine samples. In other cases the compounds prepared were
available directly or indirectly from commercially available precursor substances. We have
collected, cataloged and categorized the mass spectra for the free amines and several
derivatives of each of the 90 amines; studied the gas chromatographic retention properties of
various subsets of the amines and their derivatives; collected and evaluated vapor phase
infrared spectra for these compounds. As of the date of this writing (January, 2011) twenty
scientific publications resulting from this area of research are in print or accepted for
publication in Forensic Science International and Journal of Chromatographic Science. A few
other manuscripts related to this project are yet to be written. Additionally 12 presentations
have been made at national meetings during this project period.
The overall goal of this project was a comprehensive study of those regioisomeric
and isobaric amines capable of producing mass spectra equivalent to those of MDMA,
MBDB and MDEA. The availability of all the necessary compounds to establish and
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
10
prove the structure-retention, structure-fragmentation and other structure-property
analytical experiments was the starting point for this research. The mass spectra of
phenethylamines are characterized by a base peak formed from an amine initiated alpha-
cleavage reaction of the carbon-carbon bond of the ethyl linkage between the aromatic
ring and the amine group. In methamphetamine and ring substituted methamphetamines
(for example 3,4-methylenedioxymethamphetamine), the alpha-cleavage reaction yields
the substituted imine fragment at m/z 58 and the benzyl fragment or substituted benzyl
fragment at the appropriate mass.
C N
C N C N
C N C N
H
CH3
H
H
CH3
CH3
H
HH
CH2CH3
HH
H
H3C
H3C
H3C
H
HCH3CH2
H
m/z=58, C4H8N+
Regioisomeric Forms of m/z 58
In this project the substituted benzyl fragment for 3,4-MDMA and its
regioisomeric and isobaric equivalents occurs at m/z 135. There are four side chain
regioisomers of methamphetamine with the potential to yield mass spectra essentially
equivalent to methamphetamine yielding regioisomeric imine fragment ions in their
electron ionization mass spectra at m/z 58. Thus, for every individual ring substitution
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
11
pattern there are five side chains yielding equivalent m/z 58 fragment ions (the
methamphetamine side chain and four regioisomeric equivalents). The ring substitution
pattern in methylenedioxymethamphetamine (MDMA) doubles the number of isomers to ten,
the five side chain isomers in which the methylenedioxy-ring is substituted in a 3,4-manner
and the five in which the methylenedioxy-ring is substituted in a 2,3-pattern. Thus, there are
nine other amines with the potential to produce a mass spectrum essentially the same as that of
3,4-MDMA. Most of these potential designer analogues have the strong possibility to be
misidentified as 3,4-MDMA based on mass spectrometry. In addition to the ten amines
containing the methylenedioxy-group, other substitution patterns have the potential to produce
mass spectra with fragments of equivalent mass to those of 3,4-MDMA.
O
O
58
H3C
H3CO
58
EtO
58 58
H3CO
H3CO
58O
H3CO
58H3C
Example of Regioisomeric and Isobaric Compounds in this Study
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
12
For example, the reported stimulant methoxy-methcathinones are uniquely isomeric
with the MDMAs having the same MW, elemental composition and expected major fragment
ions of equal mass to those of the MDMAs. Additionally isobaric substitution patterns
especially those likely to undergo few if any unique/characteristic fragmentations such as
methoxy-methyl disubstitution of the methamphetamine aromatic ring, ethoxy-
monosubstitution as well as methoxymethylene- monosubstitution are compounds with the
potential to produce El mass spectra essentially equivalent to that of 3,4-MDMA. Each
methoxy-methyl, methoxymethylene- or ethoxy-substitution pattern would produce 5-side
chain isomers analogous to methamphetamine with the potential to yield mass spectra
equivalent to 3,4-MDMA. The o, m, and p-ethoxy substituents would yield a total of 15-
isomers, 15 for the methoxymethylenes and the methoxy-methyl disubstituted aromatic ring
system (10 unique disubstitution patterns) can result in over 50 isomers.
Representative samples have been prepared in this study for each aromatic ring substitution
pattern and each side chain regioisomer. A complete set of all side chain regioisomers was
prepared in enough categories to confirm mass spectral methods for specific side chain
identification. The number of side chain regioisomers for the MBDB series doubles to 20 for
each ring substitution pattern and the base peak for these compounds is m/z72. In this project
efforts were concentrated on preparing compounds necessary to confirm methods developed
for the MDMA isomers (m/z58). When the number of mass spectral equivalent isomeric
substances is relatively small, chromatographic separation and reference standard
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
13
availability is not a major concern. However, the continued designer exploration of some
drug categories will likely produce even greater numbers of regioisomeric and isobaric
substances especially among the phenethylamines. As the number of compounds having
mass spectral equivalence increases so does the challenge of specific identification via
chromatographic resolution and other analytical methods. In this project we have
systematically examined the analytical profiles of compounds from each structural
category. The results of the work will allow the forensic analyst to differentiate among
most of the regioisomeric and isobaric equivalents of MDMA without the need for
reference standards of all these compounds.
The mass spectra in almost all the compounds prepared in this study showed the
expected m/z 58 and 135 ions with no features for specific differentiation. Upon
derivatization with perfluoroacyl reagents such as trifluoroacetyl, pentafluoropropionyl
and heptafluorobutryl characteristic and distinguishing ions were obtained in many cases.
These are specific ions which occur for differentiation and not just changes in the relative
abundance of common ions. In every case specific ions to identify the side chains were
obtained. The individualization is the result of fragmentation of the alkyl carbon-nitrogen
bond yielding hydrocarbon fragments at m/z 148, 162 and 176 as well as other unique
fragments from these regioisomeric amides. The N-ethylamides show a specific ion to
identify the ethyl group by loss of mass 28 and the methamphetamine side chain yields a
unique ion at m/z110, 160 and 210 for the TFA, PFPA and HFBA derivatives
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
14
respectively. Thus, following chemical derivatization the variations in regioisomeric
m/z58 side chains can be specifically identified. This is possible due to specific ions
identifying the number of carbons attached directly to the aromatic ring in an
uninterrupted manner and the nature of the substituent attached to nitrogen. During the
course of this work the mass spectral fragmentation mechanisms and proof of structure
for these diagnostic ions have been confirmed by homologation experiments and
deuterium labeling studies. These additional compounds were synthesized in order to
confirm the details of the observed mass spectral fragmentation pathways.
In an additional fundamental study, a series of acylation reagents were evaluated to
find the reagent yielding maximum specific fragmentation information as well as appropriate
chromatographic properties. The perfluoroacyl derivatives of MDMA show excellent
chromatographic properties and unique mass spectral fragment ions. The perfluoroalkyl
amides of TFAA, PFPA and HFBA yield a unique series of mass spectral fragments at
m/z 110, 160 and 210 respectively. Additionally, the base peaks in these mass spectra
occur at relatively higher masses at m/z 154, 204 and 254 respectively. These ions are the
perfluoroacylimines resulting from loss of 135 mass units (3,4-methylenedioxybenzyl)
from the molecular ion. This (M-135)+ species also occurs for the acetyl, propionyl and
butyryl amides however these ions rearrange through loss of the acyl group to yield a
common ion at m/z 58. Thus, the base peak for the non-fluorinated hydrocarbon
derivatives is the same as that observed for the underivatized MDMA, m/z 58. The
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
15
formyl, d3-acetyl, d5-benzoyl and pentafluorobenzoyl derivatives provided
chromatographic and confirmatory mass spectral evidence for fragmentation pathways.
In many cases the ring substituent can be identified following perfluoroacylation
as well. In some cases the position of ring substitution is not available from the mass
spectral data. In all cases compounds having identical mass spectra in the derivatized and
underivatized form, can be differentiated by vapor phase infrared spectrometry. GC-IRD
studies yield individual spectra to differentiate primarily between ring substituents. Thus
these techniques in concert provide molecular individualization for most of these
compounds. The differentiation by IRD of the methcathinones is straight forward due to
the carbonyl absorption in this set of compounds.
Similar studies for the precursor substances synthesized in this study have helped
to provide analytical details for the substituted phenylacetones and 2-butanones and
related ketones. Additionally the tertiary amines cannot yield stable amide derivatives for
side chain or ring substituent identification. These compounds can only be differentiated
by underivatized mass spectra, vapor phase infrared spectrometry and other techniques.
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
16
Main Body of the Final Technical Report
I. Introduction
I.1 Statement of the Problem
3,4-Methylenedioxymethamphetamine, MDMA, is a Schedule I drug under the
Controlled Substances Act (CSA). Primarily illicitly manufactured in and trafficked from
Europe, MDMA is the most popular of the club drugs. DEA reporting indicates widespread
abuse of this drug within virtually every city in the United States. The mass spectrum is often
the confirmatory piece of evidence in the identification of drugs in the forensic laboratory.
While the mass spectrum is often considered a specific "fingerprint" for an individual
compound, there are other substances which produce very similar or almost identical mass
spectra. Many of these compounds which yield the same mass spectrum are positional isomers
of side-chain or aromatic ring substituents. Such compounds having MS-equivalency and
similar elution properties, perhaps coelution, represent a serious analytical challenge for the
forensic chemist. The ability to distinguish between these isomers directly enhances the
specificity of the analysis for the controlled substance. If other compounds exist which have
the potential to produce the same mass spectrum as the drug of interest then the identification
by GC-MS must be based entirely upon the ability of the chromatographic system to separate
the isomeric substance from the actual drug. Those substances which coelute with the drug of
abuse will be misidentified. The ultimate concern then is "if the laboratory has never analyzed
the counterfeit substances, how can the analyst be sure that these compounds would not coelute
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17
with the controlled drug?" The courts expect forensic drug chemistry to identify a substance
as an individual compound, not report it as an unknown member of a large group of
regioisomeric/isobaric substances. Methamphetamine is an excellent example which illustrates
the possibility of counterfeit amines based on side chain isomerism. The El mass spectrum for
methamphetamine is dominated by the m/z 58 fragment and the m/z 91 fragment with very
little molecular ion at m/z 149. There are four other side chain isomers of methamphetamine
having a molecular weight of 149 which yield an imine fragment of m/z58 and an
unsubstituted benzyl fragment of m/z 91.
NH2
H3C CH3
PhentermineC10H15N, MW=149
MethamphetamineC10H15N, MW=149
NH
H CH3
CH3
NH2
1-Phenyl-2-aminobutaneC10H15N, MW=149
N
N,N-DimethylphenethylamineC10H15N, MW=149
NH
N-EthylphenethylamineC10H15N, MW=149
C2H5
CH3
CH3
C2H5
Methamphetamine and Regioisomeric Side Chains Yielding m/z 58.
However, the high resolution capabilities of capillary GC coupled with the limited number of
compounds (a total of five for the methamphetamines having an unsubstituted aromatic ring)
can solve the issue in this case. Aromatic ring substitution makes this issue much more
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
18
challenging in forensic drug analysis. When the number of mass spectral equivalent
isomeric substances is relatively small, chromatographic separation and reference
standard availability is not a major concern. However, the continued designer exploration
of some drug categories will likely produce even greater numbers of regioisomeric and
isobaric substances especially among the phenethylamines. As the number of compounds
having mass spectral equivalence increases so does the challenge of specific
identification via chromatographic resolution and other analytical methods.
Major Fragment Ions for Substituted Methamphetamines
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19
The results of this project allow the forensic analyst to differentiate among most
of the regioisomeric and isobaric equivalents of MDMA and minimizes the need for a
large number of reference standards of all these compounds.
I.2 Literature Citations and Review
I.2.1 Introduction
Designer drug exploration of the methylenedioxyphenethylamines has produced
several drugs of abuse in recent years. These derivatives include 3,4-
methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA)
and 3,4-methylenedioxyethyl-amphetamine (MDEA), 3,4-methylenedioxyphenyl-2-
butanamine (BDB) and 2-methylamino-1-(3,4-methylenedioxyphenyl)butane (MBDB).
The methylenedioxy-derivatives of amphetamine and methamphetamine represent the
largest group of designer drugs (Figure I.2-1).
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20
O
O
R1
N
R3R2
MDA: R1 = CH3, R2 = R3 = H
MDMA: R1 = CH3, R2 = CH3, R3 = H
MDEA: R1 = CH3, R2 = C2H5, R3 = H
BDB: R1 = C2H5, R2 =R3 = H
MBDB: R1 = C2H5, R2 = CH3, R3 = H
Figure I.2-1: Chemical structures of methylenedioxyphenalkyl amine derivatives.
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21
MDMA, also known as eccstcy, with its stimulant and hallucinogenic effects in humans
is the most commonly used derivative of this series and has become a major drug of
abuse in recent years. In 2003, the estimated annual production of ecstacy worldwide was
100-125 tons and the estimated retail price for that drug alone was 63.74 billion US
dollars for 1.4 billion tablets. In the same year eight million people were reported to
abuse Ecstacy alone [UNODCCP, 2003]. In 2005, the reported consumption of ecstacy
among US students indicated that 1.7 % of the eight grade students in the US had
consumed the drug and this increased to 2.6% among tenth grade students and as high as
3.0 % among students in grade twelve [MTF, 2005]. About 0.1 % of the global
population (age 15 and above) consume ecstasy and significantly higher ratios, 0.5 –
2.4%, have been reported from countries in the Oceania region, Western Europe and
North America. West Europe and North America together account for almost 85% of
global consumption. Use of ecstasy, however, is increasingly spreading to developing
countries as well.
Tablets or capsules are the most common way to administer MDMA, however the
powder also can be snorted, smoked or—in rare cases—injected (TCADA 2002). Drug
effects may last up to six hours and are known to include the production of profoundly
positive feelings, empathy for others, elimination of anxiety, and extreme relaxation.
MDMA is also said to suppress the need to eat, drink, or sleep, enabling users to endure
two- to three-day parties. For a while, it has been clear that many tablets sold as ecstasy
do not always contain MDMA as an active substance and in many cases these tablets are
notoriously impure [Ecstasy Data, 1996-2006]. Ecstasy tablets are prepared in clandestine
laboratories and, during most of the last decade; Western Europe has been the world’s
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22
major manufacturing region. The most frequently mentioned country of origin was
Belgium followed by Germany, the UK, Spain and the USA. The most frequently
mentioned source countries located in Eastern Europe were the Baltic countries, Poland
and Belarus. China, Indonesia and Thailand were the most frequently reported source
countries located in Asia. In Africa, the Republic of South Africa, and in South America,
Colombia, were identified as source countries for ecstasy [UN ODCCP, 2003].
The goal of clandestine manufacturers is often to prepare substances with
pharmacological profiles that are sought after by the user population. Clandestine
manufacturers are also driven by the desire to create substances that circumvent existing
laws. In Europe, as a result of the substance-by-substance scheduling approach, the
appearance of new substances cannot be immediatley considered as illicit drugs. This
offers room for clandestine experimentation into individual substances within a class of
drugs with similar pharmacological profiles, perhaps yielding substances of increased
potency. In the USA, continued designer exploration has resulted in legislation
(Controlled Substances Analog Act) to upgrade the penalties associated with clandestine
use of all compounds of a series. Thus, identification of new MDA derivatives and other
designer drugs is essential and a significant task for forensic laboratories.
I.2.2 Analytical Methods Used to Identify and Separate 3,4-MDMA
I.2.2.1 Spectroscopy
I.2.2.1.1 Mass spectrometry
Gas chromatography-mass spectrometry (GC-MS) is the main tool used for the
detection and identification of unknown drugs in forensic and other drug screening
laboratories. The mass spectra of 3,4-methylenedioxyamphetamines are characteristic and
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23
can be used for differentiation from other phenethylamines [Noggle et al., 1988]. The
electron impact (EI) mass spectra for 3,4-methylenedioxyamphetamines exhibit weak to
extremely weak molecular ions which, in some cases, may not be evident without
computer enhancement. The base peaks result from the alpha cleavage involving the
carbon-carbon bond of the ethyl linkage between the aromatic ring and the amine
nitrogen producing the products shown in Scheme I.2.2-1. Furthermore a peak at m/z
135, which corresponds to methylenedioxybenzyl cation, is characterstic for these
compounds.
Unfortunately, the EI technique is often insufficient, alone, to discriminate
between structurally similar phenethylamines. The amine dominated fragmentation
reactions in these compounds often yields low mass fragments of similar mass and little
(if any) molecular ion species. The differentiation of these compounds is only achieved
by means of derivatization and chromatographic methods. Borth et al., reported
regioisomeric differentiation of 2,3- and 3,4-methylenedioxyphenalkylamines by using
collision-induced dissociation (CID) mass spectrometry under EI and chemical ionization
(CI) [Borth et al., 2000].
In a recent study, [Sachs and Woo, 2007] described the use of the low mass
region between mass 39 and 58 for differentiation between methamphetamine and its four
possible regioisomers. All five regioisomeric phenethylamines yield m/z 58 as the imine
base peak therefore the additional two carbons of the side chain are bonded to the
fragment imine species. The reported method [Sachs and Woo, 2007] for differentiation
between the compounds uses m/z 39 as a reference ion and compares its relative
abundance to the ions at m/z 41, m/z 42 and m/z 56. The relative abundance of each of
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24
the three ions (m/z 41, 42 and 56) was characteristic for each of the five side chain
regioisomers. The use of the m/z 39 as a pseudo-internal standard was platform
independent and reported to be consistent among numerous published reports on similar
compounds [Sachs and Woo, 2007].
O
OHN
R
O
OHN
R
EI
70ev NH
R
O
O
m/z = 163
O
O
CH2
m/z = 135
R m/z H 44CH3 58C2H5 72C3H7 86
Scheme I.2.2-1: Mass fragments (EI) of 3,4-methylenedioxyamphetamines.
Methamphetmaine was the only one of the five regioisomeric amines which
showed both m/z 41 and m/z 56 in greater abundance than m/z 39. The mechanistic
fragmentation process for the formation of each of these diagnostic low mass ions was
described in detail. However, the disadvantage of the method is the relatively low
intensity of these low mass ions and the lack of a unique and characteristic ion for each of
the regioisomeric side chains (i.e. all five compounds show fragment ions at m/z 39, 41,
42 and 56 and differentiation is based on the relative abundance of these ions).
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25
I.2.2.1.2 Nuclear magnetic resonance (NMR)
NMR is a nondestructive flexible technique that can be used for the simultaneous
identification of pure compounds and even mixtures of compounds in one sample. Its
advantages, compared to GC-MS techniques, include stereochemical differentiation and
the capability to analyze nonvolatile compounds. However, the lack of use in forensic
laboratories can be attributed to the high cost of instrumentation and the poor sensitivity
of NMR. The chemical shift data with proton assignments, appropriate proton-proton J-
coupling as well as 13C-NMR shift assignments for MDMA as the free base and
hydrochloride salt in deuterochloroform has been reported [Dal Cason et al., 1997].
Solid state NMR also can be used for analytical purposes in much the same way as
solution NMR. The observed chemical shifts however differ in the solution and solid
states because of conformational freezing and packing effects. Lee et al. reported the
differences in chemical shifts of the solid state NMR and solution NMR for MDMA [Lee
et al., 2000]. This study described the differences between chemical shifts of
MDMAHydrochloric acid in ecstasy tablets and pure crystals.
I.2.2.1.3 Infrared (IR) spectroscopy
The absorption of IR radiation is also considered one of the non-destructive
techniques that can be used for the identification of organic molecules. The region from
1250 to 600 cm-1 is generally classified as the “fingerprint region” and is usually a result
of bending and rotational energy changes of the molecule as a whole. However since the
clandestine samples are usually impure, overlapping absorptions of different molecules
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26
present in the sample becomes a possibility. Hence, this region is not useful for
identifying functional groups, but can be useful for determining whether or not samples
are chemically identical. Common absorptions for 3,4-methylenedioxyamphetamines
were reported [Young, 2000]. Because of the polymorphic crystalline structure of the
hydrochloride salt compared to the base form, different infrared spectra were determined
for MDMA hydrochloride alone [Shulgin, 1986]. Near infrared (NIR) spectroscopy
(1100-2500 nm) offers a possibility for fast and nondestructive screening of ecstasy
tablets. Modern NIR equipment is portable and easy to handle so that confiscated samples
can be measured on the spot and in real time. It has been shown that differentiation of
placebo, amphetamine and ecstasy samples is possible [Sonderman and Kovar, 1999].
Infra red detector coupled with GC also had significant application in the
identification of controlled substances in presence of their isomers. Generally isomers
other than optical isomers yield different IR spectra. Diastereomers resulting from two or
more chiral centers also exhibit different infrared spectra [Lanig et al, 2003].
Polymorphism in the solid phase can yield variations in the infrared spectrum of an
individual compound. Analytes in the solid phase can crystallize in different ways,
allowing for different crystal forms and different intermolecular interactions resulting in
significant variability in the IR spectrum of an individual compound [Lanig et al, 2003].
GC-FTIR spectroscopy is characterized by scanning quickly enough to obtain IR spectra
of peaks eluting from the capillary columns. Thus, this technique combines the separation
power of GC with the identification power of IR and is not affected by polymorphism
since the IR spectra are obtained in the vapor phase. GC-IR and MS have been
successfully used for the identification of some amphetamine isomers [Duncan and
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27
Soine, 1988]. The GC-IR Spectra of methamphetamine and phentermine have been
previously reported [Kempfert, 1988] along with other non-phenethylamine forensic
compounds.
I.2.2.1.4 Raman spectroscopy
Raman spectroscopy also has been used to study ecstasy tablets [Bell et al., 2000a].
The use of far-red (785 nm) excitation reduces interfering background luminescence and
therefore the level of fluorescence background, even in untreated samples, is sufficiently
low which makes it possible to obtain good quality data in reasonable times. It was
shown that Raman methods can be used to distinguish between ecstasy analogs and the
compounds can be identified even in a mixture with bulking agents in ecstasy tablets. The
spectra can also be used to identify bulking agents, the relative concentration of drug to
bulking agent and the degree of hydration of the active compounds. Bell et al. found that
composition profiling by Raman methods is a fast and effective method of discriminating
between ecstasy tablets manufactured in different ways and with different drug feed
stocks [Bell et al., 2000b]. Recently a surface-enhanced Raman scattering (SERS)
spectroscopy method was developed for a rapid determination of MDMA in illicit
samples [Sagmuller B. et al., 2001].
I.2.2.1.5 Ultra violet (UV) spectroscopy
The 3,4-methylenedioxyphenyl group is a strong chromophore in the UV range
with two major absorption bands at 285 nm and 235 nm range with the absorptivity
slightly higher at 285 nm [Noggle et al., 1987]. Because of their common chromophore, a
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28
variety of 3,4-methylenedioxyamphetamines and 3,4-methylenedioxyphenyl-2-
butanamines show similar UV absorption properties and therefore are not discriminated
by UV-spectroscopy [Clark et al., 1995].
I.2.2.2 Chromatography
Chromatography has played a key role in forensic drug separation and identification
over the past 35 years. Gas chromatography (GC) coupled with several detection methods
such as flame ionization (FID), nitrogen phosphorus (NPD), electron capture (EC), and
mass spectrometry (MS) has been used for the analysis of different N-substituted
analogues of 3,4-methylenedioxyamphetamines. The determination of MDMA, MDA
and MDEA in biological samples has mainly been carried out using GS-MS [Clauwaert
et al., 2000; Peters FT et al., 2005]. Actually, GC-MS is considered the method of choice
in forensic laboratories. High-performance liquid chromatography (HPLC) with different
detectors is another often applied technique in the determination of N-substituted analogs
of 3,4-methylenedioxyamphetamines. Most of the literatures describe the identification
and quantitative determination of MDA, MDMA, and MDEA, and their metabolites in
biological samples. Derivatization is mainly required in the analysis of biological
samples to improved sensitivity and selectivity, while such a technique is not usually
applied in the analysis of non-biological samples such as drug forms.
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29
I.2.2.2.1 High-performance liquid chromatography (HPLC)
I.2.2.2.1.1 Analysis of biological samples.
The determination of MDA, MDMA and MDEA in biological samples has been
done by HPLC with various detectors, such as ultraviolet (UV), diode array detection
(DAD), fluorescence (FL), electrochemical (ED), mass spectrometry (MS), and mass
spectrometry –mass spectrometry (MS-MS) and these studies are reviewed in Table I.2-1.
Table I.2-1: HPLC methods to separate 3,4-methylenedioxyamphetamines from
biological matrices.
Separated compounds Matrix Column Detector Reference
MDA, MDEA, MDMA, MBDB
Oral fluids Kromasil 100 C8
FL Concheiro et al. 2005
MDA, MDEA, MDMA urine octadecyl C18 FL De Costa JL et al., 2004
R-MDA, S-MDA, R-HME, S-HME, R-MDE, S-MDE
Plasma urine
chiral-CBH FL Buechler J et al. 2003
MDA, MDMA, MDEA, amphetamine, methamphetamine, ephedrine
plasma oral fluids
Hypersil BDS C18
MS-MS Wood M. et al. 2003
MDMA, MDA, amphetamine
Blood, urine, postmortem tissue
Hypersil BDS phenyl
MS Mortier KA et al., 2002
R-MDA, S-MDA, R-MDMA, S-MDMA, R-MDEA, S-MDEA
plasma ChiralDex, LiCrospher 60 RP-select B
FL Brunnenberg and Kovar, 2001
MDA, MDMA, MDEA
blood, vitreous
Hypersil BDS C18
FL Clauwaert et al., 2000
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30
humor, urine
MDA, MDEA, MDMA, MBDB, 2-CB and some phenethylamines
serum Superspher 100 RP 18
APCI-MS Bogusz et al., 2000
MDA, MDMA, MDEA
hair PLRP-S FL Tagliaro et al., 1999
amphetamine, methamphetamine, MDA, MDMA, MDEA
urine Silica column (APEX)
UV-visible
Talwar et al., 1999
MDA, MDMA, MDEA, MBDB
urine, serum, saliva
LiChrocart-Li-Crospher 100 RP-18
FL Mancinelli et al., 1999
amphetamine, methamphetamine, MDA, MDMA, MDEA
serum Superspher Select B a ECOcart
APCI-MS, DAD/UV
Bogusz et al., 1997
MDA, MDMA
plasma, urine
Spherisorb ODS-1
DAD Helmlin et al., 1996
MDA, MDMA, MDEA
whole blood Whatman silica Partisphere
ED Michel et al., 1993
I.2.2.2.1.2 Analysis of non-biological samples.
Reversed phase LC is the main technique used for separation and quantitation of the
active substances, usually 3,4-methylenedioxy-amphetamines, in ecstasy tablets. Usually
the column of the choice is conventional C18 or a base-deactivated C18 stationary phase
(Table I.2-2). Mancinelli et al. have developed a HPLC-fluorimetric procedure suitable
for different matrices, dosage forms and biological samples, to determine MDA, MDMA,
MDEA, and MBDB [Mancinelli et al., 1999]. This procedure was carried out in basic
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31
isocratic conditions and because of the high pH (11.4) the analytes are non-ionized. The
analysis time is less than 10 min and the first eluting compound is MDA (4.94 min)
followed by MDEA (6.77 min), MBDB (7.53 min) and last eluting one is MDMA (9.50
min).
Sadeghipour and Veuthey also reported the use of fluorimetric detection in
determination of MDA, MDMA, MDEA, and MBDB [Sadeghipour and Veuthey, 1997].
The selectivity of the method was verified not only with common substances, which can
appear in seized tablets, but also with some drugs of abuse such as cocaine, morphine,
amphetamine, methamphetamine, etc. The selectivity of the method is based on that only
methylenedioxylated amphetamines are natively fluorescent and therefore detectable. The
optimal mobile phase condition was determined as 20 mM NaH2PO4 solution (adjusted to
pH 3.8)-acetonitrile (85:15, v/v). Under these conditions, the analytes were ionized and
analysis time was less than 6 min. The elution order was MDA, MDMA, MDEA and the
last eluting compound was MBDB.
In addition, Sadeghipour et al. have developed a reversed-phase LC method with
UV detection for separation and quantitation of five amphetamines (amphetamine,
methamphetamine, MDA, MDMA, and MDEA) and ephedrine in the presence of
adulterants in illicit drugs [Sadeghipour et al., 1997]. The comparison of a regular
reversed phase column (RP18 Nucleosil 100) and a base-deactivated column (RP18-AB
Nucleosil 100) was demonstrated and as a result, the base-deactivated phase gave higher
efficiency and lower peak asymmetry and capacity factors which allowed a rapid
separation of amphetamines with good resolution. The mobile phase composition was
optimized by studying the influence of pH, buffer composition and the organic solvent
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32
type. The best results were obtained with acetonitrile concentrations between 7 and 10%
and with phosphate buffer (pH adjusted to between 3.4 and 3.8).
The reversed phase separation of N-alkyl MDAs has been achieved on a C18 stationary
phase and a ternary mobile phase [Noggle et al., 1987 and 1988]. The ternary mobile
phase consisted of pH 3 phosphate buffer, acetonitrile, and methanol containing
triethylamine. The use of triethylamine as a competing base (silanophile) was necessary
on Bondapak C18 stationary phase to prevent peak tailing.
Table I.2-2: HPLC methods to separate 3,4-methylenedioxyamphetamines from
non-biological matrices.
Separated compounds Column Detector Reference
MDA, MDMA, MDEA, MBDB
LiChrocart-LiCrospher 100 RP-18
FL Mancinelli et al., 1999
MDA, MDMA; MDEA; MBDB
FL Sadeghipour and Veuthey, 1997
MDA, MDMA, MDEA, and other phenethylamines
RP18 Nucleosil 100, RP18-AB Nucleosil 100
FL Sadeghipour et al., 1997
MDEA, MDMMA, MBDB, MDP-3-MB
Bondclone C18 UV Clark et al., 1996
MDA, MDMA, MDEA, MDMMA
Bondclone C18 UV Clark et al., 1995a
BDB, MBDB, MDP-2-EB, MDP-2-MMB, MDP-2-OHB
Bondclone C18 UV Clark et al., 1995a
MDP-3-B, MDP-3-MB MDP-3-EB, MDP-3-MMB, MDP-3-OHB
Bondclone C18 UV Clark et al., 1995a
MDMA, BDB, MDP-3-B
Bondclone C18 UV Clark et al., 1995a
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33
MDA, MDMA, NOHMDA
Nucleosil C18 UV Valaer et al., 1990
MDA, MDMA, NOHMDA
Deltabond C8 UV Valaer et al., 1990
MDA, MDMA, MDMMA, MDEA, N-isopropyl MDA, N-n-propyl MDA, NOHMDA
Bondapak C18 UV Noggle et al., 1988
MDA, MDMA, MDEA, N-isopropyl MDA, N-n-propyl MDA, N-isobutyl MDA, N-n-butyl MDA
Bondapak C18 UV Noggle et al., 1987
The elution order of the N-alkyl MDAs is based on the relative lipophilicity of the
derivatives and thus retention increases with the size of the alkyl chain on the nitrogen.
The N,N-dimethyl MDA (MDMMA) elutes before N-ethyl MDA (MDEA), and the
branched isopropyl derivative elutes before N-n-propyl MDA [Noggle et al., 1988].
Under these conditions the N-hydroxy MDA (NOHMDA) has the highest retention (25
min) which has been explained by higher polarity and lower basicity of the compound
compared to the other N-alkyl MDAs. The pKa value for NOHMDA has been determined
by titration to be 6.22, which is much lower than the pKa values of other N-alkyl MDAs
(cirka 10) [Valaer et al., 1990]. The separation of NOHMDA from MDA and MDMA
was improved by using a Deltabond C8 stationary phase which enabled the separation
without the need for competing base and in a shorter analysis time (10 min) [Valaer et al.,
1990].
Clark et al. reported the separation studies of N-substituted MDAs, N-substituted-2-
butanamines and N-substituted-3-butanamines [Clark et al., 1995a]. The separation of the
compounds was accomplished using a C18 stationary phase (Bondclone C18) with an
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34
acidic mobile phase. The elution order under these conditions was according to the size
of the N-substituent. The N-substituted MDAs eluted in the same order as previously;
MDA (5.08 min), MDMA (5.87 min) and MDEA (6.68 min). The elution order of N-
substituted-2-butanamines and N-substituted-3-butanamines was similar. The three-
carbon side chain propanamines (MDAs) had lower capacity factors than the 2-and 3-
butanamines when comparing compounds with identical N-substituents. In addition, 3-
butanamines display higher capacity factors than the 2-butanamines of the same N-
substituent in every case. When compounds with the same molecular weight as MDMA
were compared, it was found that MDMA eluted first, followed by BDB and the last
compound to elute was MDP-3-B [Clark et al., 1995a]. The separation of compounds
with the same molecular weight as MDEA was also studied under the same conditions
and the results are shown in Table I.2-3 [Clark et al., 1996].
Table I.2-3: Reversed phased LC separation of MDEA and its regioisomers.
Compound Rt (min)
MDMMA 7.93
MDEA 8.77
MBDB 10.91
MDP-3-MB 12.60
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35
I.2.2.2.2 Gas chromatography (GC)
I.2.2.2.2.1 Analysis of biological samples.
Publications on GC procedures with different detectors used for the determination
of 3,4-methylenedioxyamphetamines from biological samples are reviewed in Table I.2-
4. A mass spectrometer is the most specific detector for drug testing. Several manuscripts
describe drug testing using GC with less specific detectors. For example Drummer et al.
identified amphetamine, methamphetamine, MDMA, MDA, and other drugs of forensic
interest in blood using GC-NPD [Drummer et al., 1994]. In addition, the analysis has
been performed by a dual channel GC combined with a NP and an EC detector [Lillsunde
et al., 1996]. Ortuño et al. stated that when analyzing plasma samples with GC-NPD,
good chromatographic separation and adequate sensitivity of underivatized compounds
can be achieved but the same approach is not applicable for urine [Ortuño et al., 1999].
When GC-MS is used for screening blood samples, the selected ion monitoring (SIM)
mode is often applied [Marquet et al., 1997].
Table I.2-4: GC procedures for the identification and/or quantification of 3,4-
methylene-dioxy amphetamines from biological samples.
Separated compounds Matrix Column Detector Reference
Enantiomers of MDEA, MDMA, MDA
blood HP5-MS MS(EI) Peters FT et al., 2005
Enantiomers ofMDA, MDMA, MDEA, amphetamine, methamphetamine
urine MS(EI) Paul BD et al., 2004
MDMA, MDEA hair DB5-MS MS (SIM) Girord and Staub, 2000
MDA, MDMA, MDEA, MBDB
serum DB5-MS MS (SIM) Weinman et al., 2000
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36
MDMA and its metabolites (MDA, HMMA, HMA)
plasma, urine
Ultra-2 NPD Ortuño et al., 1999
R and S-MDMA and its chiral metabolites (MDA, HMMA, HMA)
urine DB5-MS MS (EI, PCI) deBoer et al., 1997
MDA, MDMA, MDEA, amphetamine, methamphetamine
blood HP5-MS MS (EI, SIM)
Marquet et al., 1997
MDA, MDMA, MDEA, amphetamine, methamphetamine
urine SPB-5 MS (EI, CI, SIM)
Dallakian et al., 1996
MDA, MDMA, MDEA, MBDB
urine FSC HP-5 MS (EI, SIM)
Kronstrand, 1996
MDA, MDMA, amphetamine, methamphetamine, etc.
blood FSC HP-5 NPD, ECD Lillsunde et al., 1996
MDA, MDMA, MDEA, BDB, MBDB
urine HP1 MS (SIM) Maurer, 1996
MDA, MDMA, amphetamine, methamphetamine
blood FSC BP-5 NPD Drummer et al., 1994
MDA, MDMA, MDEA urine FSC DP-5 MS (CI, SIM)
Lim et al., 1992
I.2.2.2.2.2 Analysis of non-biological samples.
O’Connell and Heffron established a GC-MS procedure to determine the principal
amphetamines, MDMA, MDEA, MDA, cocaine and pharmacologically active impurities
in ecstasy tablets [O’Connell and Heffron, 2000]. The tablets were ground into powders
and dissolved in ethanol. The column used was a HP-1 fused-silica capillary column
column (60 m x 0.25 mm id) with a 1 m film thickness of methylsilicone.
Lillsunde and Korte have reported a method for analyzing 12 ring and N-substituted
amphetamine-derivatives in body fluids or seized materials by GC combined either with
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37
MS, EC or NP detector [Lillsunde and Korte, 1991]. GC-MS was used for identification
with a packed column, 2% SP-2110 / 1% SP-2510. GC-ECD and GC-NPD was used for
quantitation with a fused silica capillary column, SE-54. Derivatization was done with
heptafluorobutyric anhydride and then most of the 12 amphetamines examined were
separated and their retention times are listed in Table I.2-5. Only MDEA and 2,5-
dimethoxy-4-ethylamphetamine as well as 3,4,5-trimethoxyamphetamine and 3-methoxy-
4,5-methylenedioxyamphetamine co-eluted. On the other hand, the mass spectra are
characteristic and can be used to distinguish these compounds from each other.
Table I.2-5: Retention times (min) of HFBA derivatives of amphetamines on packed
and capillary columns [Lillsunde and Korte, 1991].
Compound (HFBA-derivative) Rt
(packed column) Rt
(capillary column)
amphetamine 3.3 2.74
methamphetamine 3.8 3.61
N-ethylamphetamine 4.1 3.95
4-methoxyamphetamine 5.8 4.77
MDA 7.5 5.77
2,5-dimethoxyamphetamine 6.9 6.24
MDMA 7.5 6.87
2,5-dimethoxy-4-ethylamphetamine 7.8 7.23
MDEA 7.8 7.25
3,4,5-trimethoxyamphetamine 8.8 7.76
3-methoxy-4,5-methylenedioxyamphetamine 8.8 7.77
4-bromo-2,5-dimethoxyamphetamine 10.2 8.70
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38
Gas chromatographic separation of MBDB, MDEA, N,N-dimethyl-3,4-
methylenedioxy-amphetamine (MDMMA) and N-methyl-1-(3,4-methylenedioxyphenyl)-
3-butanamine (HMDMA) has been achieved on a 12 m x 0.20 mm id methylsilicone
column (HP-1) [Noggle et al., 1995]. These compounds have the same molecular weight
and similar retention properties (Table I.2-6). HMDMA has the highest retention time
and the other three compounds elute over approximately 0.25 minutes with MDEA and
MDMMA eluting before MBDB. In this system compounds having the C3 carbon side
chain attached to the aromatic ring elute before the two aryl-C4 butanamines.
The use of mass spectrometry as a detector does not provide significant data for
differentiation among MDEA, MBDB and MDMMA since these regioisomeric
compounds also yield regioisomeric fragment ions of equal mass. HMDMA can be easily
distinguished from the other three compounds because the difference in mass spectrum
produced by substitution of the methylamino group at the 3-position of the butanamine
side chain. Pentafluoropropionylamide (PFPA) derivativatization of MDEA, HMDMA,
and MBDB, was used to improve mass spectrometric differentiation [Clark et al., 1996].
The GC analysis of the three PFPA derivatives showed slightly higher retention for
derivatized compounds and they eluted in the same order as underivatized compounds.
Tertiary amines such as MDMMA do not form stable PFPA-derivatives and therefore it
can be easily identified since the mass spectrum remains unchanged after acylation with
PFPA.
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39
Table I.2-6: GC separation of MDEA, MDMMA, MBDB and HMDMA.
Compound Structure Rt
MDEA HN
O
O
7.23
MDMMA N
O
O
7.35
MBDB O
OHN
7.50
HMDMA O
O
N
H
7.68
I.2.3 General Problems for Identification and Separation.
Regioisomerism at the aromatic ring and the alkyl side-chain of the
methylenedioxyalkylamines produces a variety of compounds that have very similar
analytical properties. The methylenedioxy ring can be fused to the aromatic in a 2,3- or
3,4-pattern, yielding regiosiomerism of the aromatic portion of the molecule.
Identification of 2,3-methylenedioxy-phenalkylamines is of importance to forensic
chemists, although those compounds are not as likely to appear on the clandestine
market. Alkyl side-chain regioisomerism is most significant when imine fragments of
equivalent mass and similar abundance appear in the mass spectra of these compounds.
Some of the alkyl side-chain regioisomers can be differentiated by derivatization [Clark
et al., 1995b], but for example 2,3- and 3,4-methylenedioxyphenalkyl-amines cannot be
differentiated by derivatization [DeRuiter et al., 1998].
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40
Further more there are other compounds that do not necessarily contain the
methylenedioxy ring, however they constitute indirect regioisomeric and isobaric
substances related to the drug of abuse 3,4-MDMA. These substances have the same
molecular weight and are capable of producing mass spectra similar to 3,4-MDMA. The
co-elution of one or more of these substances with MDMA remains a possibility.
Additionally the lack of reference materials for these substances complicates the
identification procedure.
I.2.3.1 Differentiation of regioisomeric 2,3- and 3,4-methylenedioxy phenalkyl
amines and isobaric substances related to MDMA and MDEA by
chromatographic methods.
The analytical properties of the 2,3-MDAs, such as 2,3-MDA, 2,3-MDMA, 2,3-
MDEA, and 2,3-MDMMA, has been compared to the corresponding 3,4-MDAs [Casale
et al., 1995]. The EI mass spectra of 2,3- and 3,4-MDAs showed fragments of the same
mass with only slight differences in relative intensity. The distinguishable difference is
the relative abundance of ions at m/z 135 and m/z 136. Such differences cannot be
considered significant from the analytical point of view, in particular when the analytes
must be detected in a chromatographic run and the mass spectra of the compounds of
interest show interferences from co-chromatographing substances. Gas chromatographic
studies of the regioisomers on the methylsilicone stationary phase showed that all four
2,3-MDAs were easily resolved from its respective 3,4-regiosiomers and their retention
times were significantly less than the corresponding 3,4-substituted MDA. Naturally, the
regioisomeric MDAs could each be differentiated by proton NMR via variances in both
chemical shifts and overall peak patterns. However, NMR is not a technique, which is
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41
commonly used in the forensic laboratories and therefore the differentiation has usually
depended on chromatographic and mass spectrometric methods.
DeRuiter et al. studied liquid chromatographic and mass spectral methods to
differentiate 2,3- and 3,4-methylenedioxyphenyl ring substitution regioisomers of
MDMA, BDB, MDEA, and MBDB [DeRuiter et al., 1998]. The derivatization of the
side-chain regioisomers to pentafluoropropionylamide (PFPA) derivatives enabled
differentiation by mass spectroscopy. The fragmentation of the bond between the alkyl
side-chain and the nitrogen in the PFPA-derivatized amines yielded prominent ions that
identified the nature of the hydrocarbon chain attached directly to the aromatic ring. The
reversed-phase LC system consisting of a C18 stationary phase (Hypersil Elite C18) and a
mobile phase of 30% methanol in pH 3 phosphate buffer gave an excellent separation of
regioisomeric MDMA and BDBs. The first eluting compound was 3,4-MDMA followed
by 2,3-MDMA and then 3,4-BDB and last eluting compound was 2,3-BDB. A similar
elution order was obtained when regioisomeric MDEA and MBDBs were separated on
the same stationary phase but by using a different mobile phase composition. The
optimum isocratic separation was achieved when the mobile phase was 10% acetonitrile
in pH 3 phosphate buffer. The two compounds with the C3 alkyl chain attached to the
aromatic ring eluted first; the 3,4-MDEA eluted before the 2,3-MDEA, and the two
compounds with C4 alkyl group showed greater retention. The observed elution order was
the same; first are eluting compounds with shorter alkyl chains attached to the aromatic
ring and secondly, the 3,4-regioisomers elute before the 2,3-regioisomers, for several C18
stationary phases in both methanol- and acetonitrile-modified acidic aqueous systems. In
addition, four regioisomers of 3,4-MDMA and 3,4-MDEA were analyzed by GC on a
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42
methylsilicone stationary phase under temperature-programmed conditions. 3,4-MDMA
and its regioisomers eluted over a 0.6 min time window from 6.7 to 7.3 min and 3,4-
MDEA and its regioisomers produced an elution range of 0.5min (7.0 to 7.5 min). The
elution order of the compounds was not mentioned.
Aalberg et al. reported the use of Dry Lab software for the separation of direct
and indirect regioisomers related to the drug of abuse 3,4-MDMA and 3,4 MDEA using
LC. The Dry lab software uses initial four runs under identical conditions using two
different gradient times and two different temperatures. By entering the collected
retention data along with column dimensions, mobile phase composition, flow rate and
column efficiency, the software will generate three dimensional resolution map and
suggest the best conditions for separating the desired compounds in a physical mixture.
Aalberg reported the 10 direct side chain and ring regioisimers of MDMA
including N-ethyl-3,4-methylenedioyphenylethanamine, N,N-dimethyl-3,4-methylene-
dioxyphenethanamine, 3,4-methylenedioxyphetramine, 3,4-methylenedioxyBDB and
their 2,3- ring regioisomers. The HPLC study showed the rentention time increase with
side chain length of the uninterrupted hydrocarbon attached to the aromatic ring. Base
line separation of these compounds was carried out on an XTerra column at 40C with tG
of 90 min (initial 3% methanol, final 30% methanol in pH 3 phosphate buffer). On
Supelcosil ABZ+Plus column a better sepration was obtained at 41C with a tG of 230
min (initial 3% methanol, final 40% methanol in pH 3 phosphate buffer). Dry lab helped
in determining the optimum isocratic separation of these compounds on a Supelcosil
ABZ+Plus with 2% methanol and pH 3 phosphate buffer at 45C [Aalberg et al. 2003].
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43
The indirect regioisomers of 3,4-MDMA included in the Aalberg study were p-
ethoxymethamphetamine, 1-p-ethoxyphenyl)-2-butanamine, 4-methoxy-3-methylmeth-
amphetamine, 1-(3-methyl-4-methoxyphenyl)-2-butanamine, ,-dimethyl-1-(3-methyl-
4-methoxyphenyl)-2-ethanamine, N-methyl-1-(2-methoxyphenyl)-1-methyl-2-propan-
amine, N-methyl-1-(3-methoxyphenyl)-1-methyl-2-propanamine, N-methyl-1-(4-
methoxyphenyl)-1-methyl-2-propanamine and p-methoxymethcathinone. The sepration
of these substances was only achieved on Supelcosil ABZ+Plus and XTerra columns,
when acetonitrile was used as an organic modifier in the mobile phase. Separations
conditions were gradient mobile phase with a gradient time of 20.90 min at 40C, starting
from 14% and ending at 26% acetonitrile in pH 3 phosphate buffer. It was very hard to
get a base line separation for both N-methyl-1-(3-methoxyphenyl)-1-methyl-2-
propanamine and N-methyl-1-(4-methoxyphenyl)-1-methyl-2-propanamine because of
the similarity of their retention properties. The Supelcosil ABZ+Plus offered the best
resolution of all the 19 compounds with the gradient mobile phase (initial mobile phase
consisted of 3% acetonitrile and pH 3 phosphate buffer, and the final composition, 9.6%
acetonitrile, was reached in 35 min) at room temperature [Aalberg et al. 2003].
Aalberg et al. also reported gas chromatographic separations for the above
described substances. For the 2,3- and 3,4- regioisomers directly related to the drug of
abuse MDMA, optimum separation was obtained using a 35% phenylmethylsilicone
phase (DB35MS) and temperature programming of 1800C as intial temperature and an
increase in rate of 0.3 0C/ minute. These conditions were determined by the retention
modeling software, Dry Lab [Aalberg et al., 2004].
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44
Other indirect ring and side chain regioisomers and isobaric substances related to
MDMA were separated on non polar Ultra 2 column (25 m, 0.2 mm, 0.33m) with a
temperature program rate of 7.3C/min. The analysis time was decreased on a narrow
pore column (Hp-5) by optimizing the temperature program through segmented
temperature ramp using Dry Lab software. A base line separation of all the 19 direct,
indirect and isobaric substances related to MDMA was not obtained in the study [Aalberg
et al., 2004].
I.2.3.2 Differentiation of regioisomeric 2,3- and 3,4-methylenedioxyphen-
alkylamines and isobaric substances by mass spectroscopic methods.
The underivatized methylenedioxyphenalkylamines give virtually identical EI mass
spectra containing mainly intense immonium ions as mentioned earlier. The
differentiation of some methylenedioxyphenalkylamines can be achieved by means of
derivatization using various chromatographic methods. The use of tandem mass
spectrometry (MS-MS) is reported to give additional information contained in the
collision induced dissociation (CID) mass spectra of molecular ions using EI and
especially methane CI [Borth et al., 2000a]. CID mass spectra are obtained by parent ions
colliding with an inert gas during the passage through the reaction chamber of a tandem
mass spectrometer.
In their study Borth et al. were able to differentiate 18 regioisomeric methylene-
dioxyphenyl-2-propanamines and methylenedioxyphenyl-2-butanamines (Figure I.2-2).
The mixture of compounds was analyzed by GC on a methylsilicone stationary phase and
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45
an insufficient separation was obtained. Several compounds (3a and 1c or 4a, 2c and 3b
or 4b, 3c and 3d or 4c and 4d) co-eluted.
In general, the molecular ion CID mass spectra using EI were distinct, except in the
case of compounds 4e and 4f the molecular ions did not have sufficient intensity for
recording daughter ion mass spectra. The EI-CID mass spectra of all 2,3-
methylenedioxyphenethylamines derivatives (1a-d and 3a-d) are dominated by
immonium base peak ions with the general formula [CnH2n+nN]+ (m/z 44, 58, 72, etc.)
resulting from an -cleavage reaction. In contrast the EI-CID mass spectra of 3,4-
methylenedioxy isomeric compounds show significant or base peaks ions at m/z 136.
This signal was explained to be due to a rearrangement of nitrogen H atoms to the
aromatic ortho position eliminating an imine or by a specific six-center H-rearrangement
of a -H-atom of the alkyl side chain to the aromatic ring eliminating a neutral enamine
[Borth et al., 2000a].
The CI-CID spectra of all ring substituted regioisomers generate a very intense or
base peak ion at m/z 135 via cleavage of the benzyl bond by the charge of ammonium
cation [Borth et al., 2000b]. The 2,3-methylenedioxy isomeric compounds formed a
significant [C7H7O2]+ ion at m/z 123 with the mass of a protonated
methylenedioxybenzene. The 3,4-methylenedioxy ring-substituted compounds do not
show this ion to a significant extent, but they show a significant homologous [C8H9O2]+
ion at m/z 137 by a formally benzylic bond cleavage. Therefore, the study of Borth et al
using CI-CID mass spectrometry suggests that, the ion at m/z 123 indicates the 2,3-ring-
substituted phenethylamine isomers and the ion at m/z 137 the 3,4-ring-substituted
isomers.
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46
NR2R1
OO
NR2R1
O
O
a: R1 = H; R2 = H a: R1 = H; R2 = H
b: R1 = H; R2 = CH3 b: R1 = H; R2 = CH3
c: R1 = H; R2 = C2H5 c: R1 = H; R2 = C2H5
d: R1 = CH3; R2 = CH3 d: R1 = CH3; R2 = CH3
1 2
NR2R1
OO
NR2R1
O
O
a: R1 = H; R2 = H a: R1 = H; R2 = H
b: R1 = H; R2 = CH3 b: R1 = H; R2 = CH3
c: R1 = H; R2 = C2H5 c: R1 = H; R2 = C2H5
d: R1 = CH3; R2 = CH3 d: R1 = CH3; R2 = CH3
e: R1 = H; R2 = n-C3H7
f: R1 = H; R2 = i-C3H7
3 4
Figure I.2-2: Chemical structures of 18 regioisomeric methylenedioxyphenyl-2-
propanamines and methylenedioxyphenyl-2-butanamines [Borth et
al., 2000a].
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47
I.3 Statement of Hypothesis or Rationale for the Research
Analytical specificity in forensic drug analysis requires knowledge of all possible
isomers in order to identify an individual compound and eliminate all other possibilities.
Knowledge of the properties of all the possible regioisomeric/isobaric substances is
essential in order to specifically identify any one of these compounds. The precursor
chemicals are available to synthesize any of these compounds and several have already
appeared in clandestine drug samples. It is perhaps a matter of time until one appears
which has GC retention properties and MS equivalence to MDMA or MBDB. The goal
of this work is to have the data and methods for differentiation available to prevent
misidentification at that future time.
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48
II. Methods (Narrative Descriptions of the Experimental Design)
II.1 Synthesis Design
Synthetic Methods Not Available
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49
Synthetic Methods Not Available
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50
Synthetic Methods Not Available
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51
Synthetic Methods Not Available
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52
Synthetic Methods Not Available
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53
Synthetic Methods Not Available
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54
Synthetic Methods Not Available
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55
Synthetic Methods Not Available
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56
Synthetic Methods Not Available
Synthetic Methods Not Available
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57
Synthetic Methods Not Available
II.2 Methods (Synthetic materials):
Synthetic Methods Not Available
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58
II.3 Analytical Methods II.3.1 Instruments
GC-MS analysis was performed on two diggerent GC-MS. the first GC-MS is an
HP-5890 GC coupled with a HP-5970 mass selective detector (Hewlett Packard, Palo
Alto , CA) using Helium (grade 5.0) as carrier gas. The mass spectrometer was operated
on the electron impact (EI) mode using ionization voltage of 70 ev and a source
temperature of 230 oC [Instrument 1]. Samples were dissolved in HPLC grade
acetonitrile (Fisher Scientific NJ, USA) and manually introduced (1 µL), individually and
in a physical mixture using a 10µL Hamilton syringe (Hamilton Co., Reno Nevada,
USA).
The Second GC-MS is GC–MS analysis was performed using an Agilent
Technologies (Santa Clara, CA) 7890A gas chromatograph and an Agilent 7683B auto
injector coupled with a 5975C VL Agilent mass selective detector. The mass spectral
scan rate was 2.86 scans/s. The GC was operated in splitless mode with a helium (grade
5) flow rate of 0.7 mL/min and the column head pressure was 10 psi (68.946 kPa). The
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59
MS was operated in the electron impact (EI) mode using an ionization voltage of 70 eV
and a source temperature of 230 8C. The GC injector was maintained at 250 8C and the
transfer line at 280 0C [ Instrument 2]
GC–IRD studies were carried out on a Hewlett-Packard 5890 Series II gas
chromatograph and a Hewlett-Packard 7673 auto injector coupled with a Hewlett-
Packard 5965B infrared detector obtained from Analytical Solutions and Providers,
Covington, Kentucky. The vapor phase infrared detector (IRD) spectra were recorded in
the range of 4000–550 cm_1 with a resolution of 8 cm_1 and a scan rate 1.5 scans/s. The
IRD flow cell temperature as well as the transfer line was 280 8C and the GC was
operated in the splitless mode with a carrier gas (helium grade 5) flow rate of0.7 mL/min
and a column head pressure of 10 psi[ Instrument 3].
H1NMR for the identification of raction intermediates were collected for samples
dissolved in deuterated chloroform using a Brucker Avance 250 (250MHz) instrument
with the chemical shifts being reported as ppm downfield from tetramethylsilane
[Instrument 4]
II.3.2 GC- Columns
Different capillary GC columns were evaluated throughout the course of this
work, however only columns showed best compromises between resolution and
analysis time are illustrated in Table II.1. All columns used were purchased from
Restek Corporation (Bellefonte PA, USA) and have the same dimensions, 30m x
0.25mm-I.d. column coated (fd) with o.25 µm. Inlet pressure was converted
according to the constant flow mode and the total flow was 60 ml/min. The
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60
injection was in the split mode with an injector temperature at 250C except for
Rt-βDEXcst-TM, the injector temperature was adjusted to 200C as
recommended by the manufacturer.
Table II.1: List of columns used and their compostion.
Column Name Column Composition
Rtx-1 100% Dimethyl polysiloxane
Rtx-5 95% dimethyl-5%diphenyl polysiloxane
Rtx-35 65% dimethyl-35%diphenyl polysiloxane
Rxi-50 50% phenyl to 50% methyl polysiloxane
Rtx-200 trifluoropropyl methyl polysiloxane
Rt-βDEXcst-TM 14% cyanopropyl phenyl – 86 %
dimethylpolysiloxane
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61
III. Results During the course of this project we have prepared a large number of ring and side-chain
substituted phenethylamines and compared and evaluated the analytical properties of
these compounds. This work has included the 5 side chain isomers of 3,4-
methylenedioxy ring system yielding the m/z 58 base peak in the EI-mass spectrum (3,4-
MDMA and the 4 side chain isomers as well as the equivalent 2,3-methylenedioxy series
of compounds. Additional studies have characterized the 2-, 3-, and 4-methoxymeth-
cathinones which can be considered indirect isomers since these compounds have the
same elemental composition as the MDMA series.
A number of individual synthetic and analytical studies have evaluated the 2-, 3-, and 4-
ethoxyphenethylamines as well as the 10 different ring substitution patterns for the
methoxy-methyl-phenethylamines. Furthermore the 2-, 3-, and 4-methoxy-alpha-methyl-
methamphetamines were prepared and evaluated as well.
The methamphetamine side-chain isomer of every ring substitution pattern was prepared
and evaluated in this study.
A series of all ten of the MBDB (m/z 72) side chain isomers was prepared and evaluated
as well as of the more common side chains for several other ring substitution patterns.
Well over 90 compounds were evaluated in this project which allowed us to confirm
specific structure property relationships for differentiation among most of these isomers.
The next chapters provide the details of specific studies as an example of each category
of compounds evaluated in this project.
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62
Chapter 1 Mass Spectrometry and Gas Chromatographic Studies on Direct
Regioisomers to 3,4-MDMA.
There are nine other methylenedioxy-substituted phenethylamines with the
potential to produce a mass spectrum essentially the same as 3,4-MDMA, five of them
being 2,3-methylenedioxy ring substituted regioisomers.
Mass spectral studies of direct regioisomers of 3,4-MDMA (methylenedioxyphenyl substituted side-chain regioisomers).
The mass spectra of the phenethylamine drugs of abuse including 3,4-MDMA are
characterized by a base peak formed by an -cleavage reaction involving the carbon-
carbon bond of the ethyl linkage between the aromatic ring and the amine. In 3,4-MDMA
(MW=193) the -cleavage reaction yields the 3,4-methylenedioxybenzyl fragment at
mass 135/136 (for the cation and the radical cation, respectively) and the substituted
imine fragment at m/z 58. Thus, the mass spectrum for 3,4-MDMA contains major ions
at m/z 58 and 135/136 as well as other ions of low relative abundance.
There are nine other methylenedioxy substituted regioisomers (a total of ten
compounds) of the 3,4-MDMA molecule (MW=193) which yield -cleavage fragments
at m/z 58 and 135/136 during analysis by mass spectrometry (Scheme 1.1). The mass
spectra in Figure 1.1 are for the ten possible direct regioisomers of the MDMA molecule.
The first five side chain regioisomers show the methylenedioxy-group fused to the
aromatic ring in a 3,4-manner while in compounds 6 through10 the substitution is in the
2,3-manner. All compounds show the expected fragments (m/z 58 and 135), and in
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63
addition, most of the compounds show the molecular ion at m/z 193. In a direct
comparison of the 3,4-regioisomers versus the 2,3-regioisomers with the identical side
chain, the major difference is the greater relative abundance of the radical cation at m/z
136 for the 3,4-substitution pattern in most cases.
Clark et al [1996, 1998] and Aalberg et al. [2000, 2003] in previous work have
described the analytical properties of these unique direct regioisomeric equivalences to
the drug of abuse 3,4-methylenedioxymethamphetamine. These results illustrated that the
mass spectrum alone cannot be used to identify an individual compound within this group
to the exclusion of all others. Their observations [Aalberg et al., 2000 and 2004]
illustrated that 3,4-MDMA (3) and N-ethyl-2,3-methylenedioxyphenyl-2-ethanamine (7)
co-eluted using some common gas chromatographic stationary phases and conditions.
However, additional studies [Aalberg et al., 2004] have identified capillary gas
chromatographic phases and conditions for the complete resolution of compounds 1-10.
Optimum separation was obtained using a 35% phenylmethylsilicone phase (DB35MS)
and temperature programming conditions determined by retention modeling software
(Dry Lab).
While these studies have shown that all ten compounds can be resolved, the lack
of mass spectral specificity makes the specific identification of MDMA (with the
exclusion of all other regioisomers) a significant challenge. The lack of available
reference samples for all ten of these regioisomeric molecules further complicates the
individual identification of any one of these substances. When other compounds exist
with the potential to produce the same or nearly identical mass spectrum as the drug of
interest, the identification by gas chromatography-mass spectrometry (GC-MS) must be
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64
based primarily upon the ability of the chromatographic system to separate the
“counterfeit substance” from the actual drug of interest. If not, those substances co-
eluting with the target drug in chromatographic systems could be misidentified as the
target drug. Without the appropriate standards a thorough method validation is not
possible, and thus co-elution of drug and non-drug combinations would remain a
possibility. Furthermore, the ability to distinguish between these regioisomers directly
enhances the specificity of the analysis for the target drugs of interest
O
O
H2C C N
C2H8
O
O
CH2
C N
C2H8
m/z = 135
m/z = 58
70ev
MW = 193
Scheme 1.1: General mass spectral fragmentation for the methylenedioxy regioisomers
of MDMA (compounds 1-10).
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65
Figure 1.1: Structures and mass spectra of methylenedioxy substituted side-chain
regioisomers.
Structure of regioisomers Mass spectrum
N
O
O
1
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
m/z-->
Abundance
Average of 10.543 to 10.576 min.: 00050920.D (-)58
135775142 88 17810565 193117 163148
NH
O
O
2
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
5000
10000
15000
20000
25000
30000
35000
m/z-->
Abundance
Average of 12.065 to 12.124 min.: 00050920.D (-)58
136
7751 9165 19339 105 149119 163 176
O
O NH
CH3 3
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
140000
m/z-->
Abundance
Average of 10.673 to 10.721 min.: 00050920.D (-)58
7751 13591 19365 14939 105 119 163 176
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66
O
O NH2
4
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
20000
40000
60000
80000
100000
120000
140000
160000
m/z-->
Abundance
Average of 9.734 to 9.766 min.: 00050920.D (-)58
42 7751 1359165 193147119105 177162
O
O NH2
5
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
5000
10000
15000
20000
25000
30000
m/z-->
Abundance
Average of 13.441 to 13.516 min.: 00050920.D (-)58
136
7741 51164106 19365 91 121 147 176
N
OO
6
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000
m/z-->
Abundance
Average of 8.898 to 8.930 min.: 00050920.D (-)58
77 1355142 1788810565 117 147 161
NH
OO
7
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
10000
20000
30000
40000
50000
60000
70000
80000
90000
m/z-->
Abundance
Average of 10.840 to 10.867 min.: 00050920.D (-)58
1357751
8842 10565 178148 162 19111895
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67
NH
CH3
OO
8
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
m/z-->
Abundance
Average of 9.405 to 9.443 min.: 00050920.D (-)58
77 1355142 89 105 17865 192162118 14895
NH2
OO
9
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
5000
10000
15000
20000
25000
m/z-->
Abundance
Average of 8.380 to 8.428 min.: 00050920.D (-)58
42 77 9151 65 193148135105 119 160
NH2
OO
10 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
0
2000
4000
6000
8000
10000
12000
14000
16000
m/z-->
Abundance
Average of 11.622 to 11.660 min.: 00050920.D (-)58
7741 13551 16419310565 91 116 147
Mass spectral studies of perfluroacyl derivatives of 3,4- MDMA and its direct regioisomers
The perfluoroacylated derivatives of the eight primary and secondary amines
(Compounds 2-5 and 7-10) were prepared and evaluated for their ability to individualize
the GC-MS properties of the compounds in this uniquely regioisomeric series and to
maintain or improve chromatographic resolution. Of course, the two tertiary amines,
compounds 1 and 6, would not form stable amide derivative.
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68
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
m/z-->
AbundanceScan 530 (9.772 min): 110903-1.D
42
51
77
91
119
135
148
160
176
204
220 244252 272 294 309 324
339
Acylation of the amines significantly lowers the basisty of nitrogen and can allow
other fragmentation pathways to play a more prominent role in the mass spectrum [F. W.
McLafferty et al, 1993]. The mass spectra for the eight pentaflouropropionyl and
heptflourobutryl amides are shown in Figures 1.2 and 1.3, respectively
From these spectra a common peak occurs at m/z 204 and 254 which corresponds
to the loss of 135 mass units from the molecular ions at 339 and 389 for PFPA and HFBA
amides, respectively.
Figure 1.2: Mass Spectra of the PFPA derivatives of compounds (2-5) and (7-10).
O
O
N C2F5
O
a
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69
O
O
N C2F5
O
b
O
O
HN C2F5
O
c
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
500000
1000000
1500000
2000000
m/z-->
AbundanceScan 494 (9.469 min): 110903-2.D
42
51
77
91
119
135
147
162
176
204
220 241 259269282 300 324
339
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
20000
40000
60000
80000
100000
120000
140000
m/z-->
AbundanceScan 393 (8.305 min): 110903-3.D
41
59
77
91
119
135
145
164
176
204
324
339
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70
O
O
NH
C2F5O
d
O
O
N C2F5
O
e
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
m/z-->
AbundanceScan 448 (8.935 min): 110903-4.D
41
51
77
89
119
135
147161
176
204
220 292310
339
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
20000
40000
60000
80000
100000
120000
140000
160000
180000
m/z-->
AbundanceScan 460 (9.077 min): 110903-5.D
4151
77
91119
135
148
160
176
204
220
339
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71
O
O
N C2F5
O
f
O
O
HN C2F5
O
g
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
m/z-->
AbundanceScan 431 (8.749 min): 110903-6.D
42
51 7791
119 135
147
162
177
204
220
339
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
m/z-->
AbundanceScan 357 (7.881 min): 110903-7.D
41
59
77
91
119
135
146
164
176
204
324
339
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72
O
O
HN C2F5
O
h
Figure 1.3: Mass Spectra for the HFBA derivatives of compounds (2-5) and (7-
10).
O
O
N c3F7
O
a
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
2000
4000
6000
8000
10000
12000
14000
16000
18000
m/z-->
AbundanceScan 400 (8.385 min): 110903-8.D
41
5179
105
135
161
176
204
339
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
m/z-->
AbundanceScan 810 (13.125 min): 20304-4.D
42
6977
105119
148
169
178197
226
254
279 302 322 360374
389
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73
O
O
N C3F7
O
b
O
O
HN C3F7
O
c
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
m/z-->
AbundanceScan 887 (14.016 min): 30204-2.D
42
6977
105
131
135
162
192
210
220
254
263 291 310 332 350 374
389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
65000
70000
75000
80000
85000
m/z-->
AbundanceScan 722 (12.083 min): 20404-4.D
41
59
77
105119
135
169
176
214
254
374
389
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74
O
O
NH
C3F7O
d
O
O
N C3F7
O
e
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
m/z-->
AbundanceScan 825 (13.292 min): 30204-4.D
41
69
77
105 131
135
161
176
214
226
254
296302 330360361
389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
m/z-->
AbundanceScan 810 (13.125 min): 20304-4.D
42
6977
105119
148
169
178197
226
254
279 302 322 360374
389
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75
O
O
N C3F7
O f
O
O
HN C3F7
O
g
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
m/z-->
AbundanceScan 776 (12.730 min): 12804-6.D
42
6977
105
119
135
162
192
210
220
254
263 291 313 339350 374
389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
m/z-->
AbundanceScan 665 (11.431 min): 12804-7.D
42
59
77
105119
135
169
176
214
226
254
277 295305320 350374
389
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76
O
O
HN C3F7
O h
These ions at m/z 204 and 254 is the PFPA and HFBA imine species likely
formed from the alpha cleavage of the amide nitrogen to eliminate the 2, 3- and 3, 4-
methylenedioxybenzyl radical, thus the m/z 204 and 254 ions in PFPA and HFBA amides
are analogous to m/z 58 in the underivatized species because all these ions represent the
(M-135)+ species. The general fragmentation pattern and structures for the m/z 204 and
254 ions are shown in Scheme 1.2. The methylenedioxybenzyl cation at m/z 135 is a
fragment common to all the spectra in Figures 1.2 and 1.3. The decreased role for alpha
cleavage reaction in the fragmentation of these amides allows the formation of ions more
diagnostic of each individual side chain isomer. Acylation, and in particular the
perflouroacylation, weakens the bond between nitrogen and the alpha-carbon of the
substituted methylenedioxyphenethyl group, allowing the formation of charged
hydrocarbon species of increased relative abundance. These hydrocarbons of varying
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
m/z-->
AbundanceScan 734 (12.244 min): 12804-8.D
41
6977
105 131
135
161
176
214
226
254
265280 311 330
360
370
389
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77
mass significantly individualize the mass spectra and provide specific structure
information.
Scheme 1.2: Formation of the (M-135)+ ions in the perfluoroacyl-derivatives of the
regioisomeric amines.
The mass spectra in Figures 1.2 and 1.3 illustrate the role of hydrocarbon
fragments at m/z 148, 162 and 176 in the electron impact mass spectral differentiation
among these side chain regioisomers.
The spectra for the N-ethyl derivatives in Figures 1.2a, 1.2e and 1.3a, 1.3e) show
a base peak at m/z 148 corresponding to the alkane radical cation which occurs from
hydrogen rearrangement and subsequent fragmentation of the alkyl carbon to nitrogen
bond of the phenethylamine side chain (Scheme 1.3).
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78
O
O
CH C
R1
R2
N R3
R4
H
CO
O
O
HC C
R1
R2
N R3
R4
CHO
O
O
HC C
R1
R2
R1=R2=H, m/z=148R1=H, R2=CH3, m/z = 162R1=R2=CH3, m/z = 176R1=H, R2=C2H5, m/z = 176
Scheme 1.3: Mechanism for the formation of the alkane radical cation in the
perfluoroacyl-derivatives of the regioisomeric amines.
This ion at m/z 148 would only occur for the N-ethyl regioisomer. The spectra in
Figures 1.2b, 1.2f and 1.3b, 1.3f show the 2,3- and 3,4-methylenedioxyphenylpropane
hydrocarbon ion at m/z 162, identifying this molecules as the PFPA and HFBA
derivatives of 2,3- and 3,4-methyelenedioxymethamphetamines, respectively. The
proposed mechanism for the formation of the hydrocarbon fragment is illustrated in
Scheme 2.3. The spectra for the PFPA and HFBA derivatives of the primary amines 4, 5,
9 and 10 show ions at m/z 176 from the corresponding 2,3- or 3,4-methylenedioxyphenyl
alkyl radical cation. This m/z 176 results from hydrogen rearrangement and subsequent
fragmentation of alkyl carbon to nitrogen bond. The lower abundance of m/z 176 for the
2, 3- and 3, 4-methylenedioxyphentramines (compounds 4 and 9) may be attributed to
steric inhibition of hydrogen transfer in the alpha, alpha-dimethyl substitution pattern.
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79
While the alkene ions at 148, 162, and 176 help to identify the side chain
regioisomers, one complicating factor in the PFPA derivatives for the N-
ethylphenethylamines (Figures 1.2a and 1.2e) is the appearance of an ion at m/z 176 in
addition to the base peak at m/z 148. The 176 ion suggests a four carbon chain directly
attached to the aromatic ring as occurs for the alpha-ethyl- and alpha, alpha-dimethyl-
phenethylamines (Figures 1.2 c, d, g, h and 1.3 c, d, g, h). The m/z 176 ion in the spectra
for the PFPA derivatives of the N-ethyl regioisomers (Figures 1.2a and 1.2e) is a
rearrangement of the m/z 204 ion resulting in the loss of mass 28 (the N-ethyl group) via
hydrogen transfer (Scheme 1.4). This coincidental common mass from two different
fragmentation pathways is confirmed by examining the mass spectra for the HFBA
derivatives of the N-ethyl-phenethylamines shown in Figure 1.3a and 1.3e. The loss of 28
mass units from the acylimine fragment at m/z 254 yields the equivalent fragment ion at
m/z 226. Thus, the HFBA derivatives may offer more unique characteristic ions for
individualization of these regioisomeric substances.
Scheme 1.4: Mechanism for the loss of mass 28 from the perfluoroacyl-N-ethyl-
imine cation.
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80
A comparison of the PFPA derivatives for 3,4- and 2,3-
methylenedioxymethamphetamine (Figures 2.2b and 2.2f) with the HFBA derivatives
(Figures 1.3b and 1.3f) indicated unique ions at m/z 160 and m/z 210. This mass
difference of 50 (CF2) suggests these ions contain the perfluoroalkyl group for each
derivative, C2F5 and C3F7, respectively the suggested mechanism of forming these masses
can be illustrated in Scheme 1.5. Additional information about these ions can be obtained
by a comparison of the mass spectra for the PFPA and HFBA derivatives of 3,4-MDMA
and NCD3-3,4-MDMA (MDMA-d3) in Figure 1.4. The corresponding ions in these
spectra occur at m/z 163 and 213 and the equivalent ions for the derivatives of d5-MDMA
also occur at m/z 163 and 213. Thus, an analysis of the masses of the components which
make up the fragment at m/z 160 for example include C2F5 (119 mass units) and CH3 (15
mass units) leaving only a mass of 26 available for the total of 160. The mass 26 would
correspond to CN and the proposed mechanism for the formation of (C2F5CNCH3)+ is
shown in Scheme 1.5. An equivalent fragmentation pathway has been reported [C.R.
Clark et al, 1995] for methamphetamine.
R2CNH3C
m/z = 160 (R2 = C2F5)m/z = 210 (R2 = C3F7)
H3C N
CHO
R2
CH3
H3C N
CH
C
O
R2
CH3
m/z = 204 (R2 = C2F5)m/z =254 (R2 = C3F7)
Scheme 1.5: Formation of m/z 160 and 210 from the PHPA and HFBA derivatives of 2,3- and 3,4-MDMA
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81
Figure 1.4: Mass spectra of the PFPA and HFBA derivatives for the d3 and d5- MDMA.
O
O
N
CD3
C2F5
O
a
O
O
N
CD3
C2F5
OD
H D
b
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
m/z-->
AbundanceScan 402 (8.407 min): 51104-4.D
45
5177
89
119
135
147
162
179
207
223 269 303
342
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
5000
10000
15000
20000
25000
30000
35000
40000
45000
m/z-->
AbundanceScan 405 (8.411 min): 51104-2.D
41
5178
119
136
163
208
344
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82
O
O
N
CD3
C3F7
O
C
O
O
N
CD3
C3F7
OD
H D
d
50 100 150 200 250 300 3500
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
m/z-->
AbundanceScan 401 (8.386 min): 51104-3.D
45
6977
105
131
135
162
194
213
223
257
263 313 332 353 373
392
50 100 150 200 250 300 3500
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
m/z-->
AbundanceScan 399 (8.372 min): 51104-1.D
46
6978
107 133
136
164
196
213
258
315
394
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83
The spectra for the derivatives of d3- and d5-MDMA in Figure 2.4 also lend
support to the proposed mechanism for the formation of the alkene fragment at m/z 162
illustrated in Scheme 1.3. The exact structure for the d5-MDMA is shown in Scheme 1.6,
the benzylic position contains one hydrogen and one deuterium and the transfer
fragmentation can occur to remove either species to form the alkene radical cation. Thus,
the resulting alkene can yield ions at m/z 163 or 164 depending on the probability of
transfer.
Scheme 1.6: Formation of m/z 163 and 164 in the perfluoroacyl-derivatives of d5-
MDMA.
Gas chromatographic separation of perfluroacyl derivatives of 3,4- MDMA and their direct regioisomers
The PFPA and HFBA derivatives of the eight primary and secondary amines were
compared on two stationary phases using capillary columns of the same dimensions, 30m
x 0.25mm and 0.25um depth of film using instrument 1. Previous studies on the
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84
chromatographic properties of the underivatized compounds [Aalberg et al., 2000 and
2004 ] have shown that other compounds in this series co-eluted with MDMA using some
common gas chromatographic stationary phases and conditions.
The stationary phases compared were the relatively nonpolar phases, 100%
dimethyl polysiloxane (Rtx-1) and 95% dimethyl-5% diphenyl polysiloxane (Rtx-5).
Several temperature programs were evaluated and the best compromises between
resolution and analysis time were used to generate the data in Table 8 and the
chromatograms in Figures 1.5 and 1.6
The two chromatograms for the PFPA derivatives in Figure 19 were generated
using two temperature programs. The first program used was to hold the column
temperature at 100 oC for 1 minute, ramped to 180 oC at 9 oC/minute, hold at 180 oC for 2
minutes ramp to 200 oC at 10 oC/minute [TP-1]. This program was used to separate
compounds 2-5 and 7-10 in their HFBA and PFPA forms on Rtx-5. The same program
was also used to separate the PFPA derivatives of the same compounds on Rtx-1. The
second temperature program used to resolve the HFBA derivatives of compounds 2-5 and
7-10 on Rtx-1. The program was set up to hold the column temperature at 70 oC for 1
minute, ramped to 150 oC at 7.5 oC/minute, hold at 150 oC for 2 minutes ramp to 250 oC
at 10 o /minute[ TP-2].
The resulting elution order and resolution are quite similar. In fact the elution
order is the same for all the chromatograms in Figures 1.5 and 1.6. The chromatograms
show that the 2,3-isomer elutes before the corresponding 3,4-isomer for all the side chain
regioisomers. For example, 2,3-MDMA elutes before 3,4-MDMA, and this pattern holds
for all side chain regioisomers. When the ring substitution pattern is held constant (ie 2,3-
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85
or 3,4-) and the side chain elution order is evaluated the two secondary amides elute
before the two tertiary amides. Additionally, in every case in this limited set of
compounds the branched side chain elutes before the straight chain isomer when the ring
substitution pattern and the degree of amide substitution are constant. Therefore, the 2,3-
phentermine-PFPA elutes first followed by 2,3-BDB-PFPA (both secondary amides),
then 2,3- MDMA-PFPA, and N-ethyl 2,3-methylenedioxyphenethylamine-PFPA the two
tertiary amides. Perhaps the most useful information in these chromatograms is the
relative elution of the derivatized controlled substance MDMA and its closest eluting
regioisomeric equivalents. Both the PFPA and HFBA derivatives of MDMA elute
between the N-ethyl-2,3- and 3,4-methylenedioxyphenethylamine PFPAs and HFBAs,
both the N-ethyl regioisomers show very distinct mass spectra with several characteristic
ions to differentiate these compounds from the drug of abuse MDMA. Thus,
derivatization methods coupled with both chromatographic and mass spectral procedures
can allow for the complete characterization of the side chain substitution pattern for these
ten uniquely isomeric substances, however, the HFBA derivatives offer more unique
fragment ions for additional discrimination among these regioisomeric substances.
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86
Abundance
Time (minutes)
Figure 1.5: Capillary gas chromatographic separation of the PFPA derivatives of
compounds (2-5) and (7-10). Columns used: A Rtx-1; B Rtx-5.
10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.500
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
140000
150000
Time-->
AbundanceTIC: 111103-4.D
9.00 10.00 11.00 12.00 13.00 14.00 15.000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
Time-->
AbundanceTIC: 30304-14.D
9
4
10
8
5
2
9
4
10
8
7
3
A
2
5
B
7 3
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87
Abundance
Time (minutes) Figure 1.6: Capillary gas chromatographic Separation of HFBA derivatives of
compounds (2-5) and (7-10). Columns used: A Rtx-1; B Rtx-5
20.00 21.00 22.00 23.00 24.00 25.00 26.000
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
Time-->
AbundanceTIC: 20404-2.D
8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
Time-->
AbundanceTIC: 30204-13.D
9
9
4
4
10
10
8
8
5
5
7
7
3
3
2
2
A
B
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88
Conclusions of Chapter 1
3,4-MDMA and nine other methylenedioxyphenethylamines are a unique subset
of regioisomeric molecules; each compound has a molecular weight of 193 and yields a
base peak at m/ z 58 in the MS from the loss of the corresponding methylenedioxybenzyl
group. Thus, the traditional EI MS provides little stru ctural information for diff e
rentiating among these 10 compounds. Because of the unique similarity of these
compounds by MS, the specific identification of a compound such as 3,4-MDMA
requires methods to eliminate any of the other nine isomers.
This elimination process may be accomplished on the basis of chromatography
alone but would ultimately require the analyst to use reference samples of each of the 10
amines. The reference samples would be necessary to determine if any of the isomeric
methylenedioxyphenethylamines coeluted with 3,4-MDMA. Derivatization of the eight
primary and secondary amines with various acylating agents yields amides with similar
resolution to the underivatized amines by capillary GC on RTX-1 and RTX- 5 stationary
phases. However the perfluoroacyl derivatives significantly individualize the mass
spectra for these amides and allow for specific identification. The individualization is the
result of fragmentation of the alkyl carbon–nitrogen bond yielding hydrocarbon
fragments at m/ z 148, 162, and 176, as well as other unique fragments from these
regioisomeric amides. The PFPA and HFBA derivatives are essentially equivalent for
chromatographic purposes. However, the HFBA derivatives offer more unique fragment
ions for additional discrimination among these regioisomeric substances.
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89
Chapter 2
Mass Spectrometry and Gas Chromatographic Studies of Indirect Regioisomers Related to 3,4-MDMA, the
Methoxymethcathinones.
The methoxymethcathinones constitutes a set of indirect regioisomers of the
controlled drug substance 3,4-methylenedioxymethamphetamine. The various isomeric
forms of the methoxymethcathinones have mass spectra essentially equivalent to 3, 4-
MDMA, all have molecular weight of 193 and major fragment ions in their electron
ionization mass spectra at m/z 58 and 135/136. For these individual regioisomers the m/z
135/136 ion is the methoxybenzoyl carbocation (C8H7O2) + not the methylenedioxybenzyl
carbocation (C8H7O2)+. The specific identification and differentiation between these
compounds and the drug of abuse 3,4-MDMA must be based on a combination of mass
spectral data as well as chromatographic resolution of these regioisomeric substances.
The mass spectra of the underivatized methoxymethcathinones as well as their
perfluroacetyl derivatives will be compared with 3,4- and 2,3- MDMA. The
chromatographic separation of the underivatized and derivatized methoxymethcathinones
from 2,3- and 3,4-methylenedioxymethamphetamines will be discussed.
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90
Mass spectral studies of the underivatized and the perfluoroacyl derivatives of the methoxymethcathinones
Methoxymethcathinones represent a potentially significant challenge for
analytical drug chemistry. All the regioisomeric methoxymethcathinones have the same
molecular weight (193) and the same side chain as the drug of abuse 3,4-MDMA.
The mass spectra for the three methoxymethcathinone regioisomers (Figure 2.1)
show a base peak at m/z 58 as seen for 2,3- and 3,4-MDMA (Figure 2.1). The major
fragmentation pattern for the methoxymethcathinones is shown in Scheme 2.1. The
methoxybenzoyl (C8H7O2)+ fragment has the same mass and empirical formula as the
methylenedioxybenzyl (C8H7O2)+ cation occurring at m/z 135. Furthermore the m/z 58
ion in the methoxymethcathinones is the same imine structure as that obtained in the
mass spectra of both 3,4- and 2,3-MDMA.
O
O
HN
OCH3
HN
O
EI70ev
EI70 ev
HC NH
MW = 193MW=193
m/z = 58EI70 ev
eI70 ev
O
O
CH2
OCH3
O
m/z = 135 m/z = 135
C8H7O2+ C8H7O2
+
Scheme 2.1: EI fragmentation pattern of the underivatized 2,3- ,3,4-MDMAs and
the methoxymethcathinones
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91
Figure 2.1: Mass spectra of the underivatized methoxymethcathinones. Structure of regioisomers Mass spectrum
1
HN
O
OCH3
2
OCH3
HN
O
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
500000
1000000
1500000
2000000
2500000
3000000
3500000
m/z-->
AbundanceScan 570 (10.338 min): 30104-9.D
42
58
63
77
80
92
105108 121
135
146147 160 175178 193
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
m/z-->
AbundanceScan 597 (10.670 min): 30104-10.D
42
58
6477
86
92107108 121
135136 148 162164 178 191
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92
HN
O
H3CO
3
Since the mass spectra of the methoxymethcathinones is almost identical to the
drug of abuse 3,4-MDMA, perfluoroacylated derivatives of 3,4- and 2,3-
methylenedioxymethamphetamines and their regioisomeric secondary amines, ortho,
meta and para-methoxymethcathinones, were prepared and evaluated in an effort to
individualize their mass spectra and to improve chromatographic resolution. Acylation
of the amines significantly lowered the basicity of nitrogen and allowed other
fragmentation pathways to play a more prominent role in the mass spectrum. The mass
spectra for the five pentaflouropropionyl and heptflourobutryl amides are shown in
Figures 2.2 and 2.3, respectively.
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
m/z-->
AbundanceScan 683 (11.658 min): 30104-11.D
42
58
64
77
79
92
107108 121
135
137 150 162165 178 191
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93
Figure 2.2: Mass spectra of the PFPA derivatives of 3,4-MDMA (a); 2,3-MDMA
(b) ;ortho (c); meta (d) and para (e)-methoxymethcathinones.
O
O
N C2F5
O
a
O
O
N C2F5
O
b
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
500000
1000000
1500000
2000000
m/z-->
AbundanceScan 494 (9.469 min): 110903-2.D
42
51
77
91
119
135
147
162
176
204
220 241 259269282 300 324
339
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
m/z-->
AbundanceScan 431 (8.749 min): 110903-6.D
42
51 7791
119 135
147
162
177
204
220
339
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94
OCH3
N
O
C2F5O
c
N
O
C2F5O
OCH3 d
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
5000
10000
15000
20000
25000
30000
m/z-->
AbundanceScan 401 (8.399 min): 11903-10.D
41
56
77
92107
135
160
204
339
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
400000
m/z-->
AbundanceScan 388 (8.212 min): 110903-9.D
4256
77
92 119
135
137
160
176
204
220 300 339
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95
N
O
C2F5O
H3CO
e
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
10000
20000
30000
40000
50000
60000
m/z-->
AbundanceScan 460 (9.072 min): 11903-11.D
41
6477
92 107
135
160176
204
339
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96
Figure 2.3: Mass Spectra for the HFBA derivatives of 3,4-MDMA (a); 2,3-MDMA
(b); ortho (c); meta (d) and para (e)-methoxymethcathinones.
O
O
N C3F7
O
a
O
O
N C3F7
O
b
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
m/z-->
AbundanceScan 887 (14.016 min): 30204-2.D
42
6977
105
131
135
162
192
210
220
254
263 291 310 332 350 374
389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
m/z-->
AbundanceScan 776 (12.730 min): 12804-6.D
42
6977
105
119
135
162
192
210
220
254
263 291 313 339350 374
389
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97
OCH3
N
O
C3F7O
c
N
O
C3F7O
OCH3
d
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
500000
1000000
1500000
2000000
2500000
3000000
3500000
m/z-->
AbundanceScan 739 (12.309 min): 30204-9.D
42
69
77
92
120
135
169176
210
220
254
289 313322 350 370 389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
m/z-->
AbundanceScan 762 (12.559 min): 30204-10.D
42
69
77107
119
135
169
176
210
220
254
285 346 370
389
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98
N
O
C3F7O
H3CO
e
From these spectra a common peak occurs at m/z 204 and 254 which
corresponds to the loss of 135 mass units from the molecular ions at 339 and 389 for
PFPA and HFPA amides. This ion at m/z 204 and 254 is the PFPA and HFPA imine
species likely formed from the alpha cleavage of the amide nitrogen to eliminate the 2, 3-
and 3, 4-methylenedioxybenzyl and methoxybenzoyl radicals. Thus the m/z 204 and 254
in PFPA and HFPA amides are analogous to m/z 58 in the underivatized species because
all these ions represent the (M-135) + species. The general fragmentation pattern and
structures for the m/z 204 and 254 ions are shown in Scheme 2.2
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
m/z-->
AbundanceScan 855 (13.647 min): 30204-11.D
42
69
77
92
119
135
169176
210
220
254
285 313 358374389
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99
The relative abundances for the m/z 204 and 254 ions are always higher in the 3,4- and
2,3-methylenedioxymethamphetamines than in the methoxymethcathinones The
methylenedioxybenzyl and the methoxybenzoyl cations at m/z 135 are fragments
common to all the spectra (Figures 3.2 and 3.3). The relative abundance of m/z 135 in
perfluoroacyl derivatives of methoxymethcathinones is higher than that observed for 3,4-
and 2,3-methylenedioxymethamphetamines. The m/z 135 ion is the base peak in the mass
spectra for the derivatives of the methoxymethcathinones likely due to the additional
carbonyl site for initial radical cation formation in these compounds.
HC N
CH3
CH3
O
R
m/z = 204 (R = C2F5)m/z =254 (R = C3F7)
O
O
N
CH3
CH3
R
O
NR
CH3
CH3O
O
H3CO
EI/ 70evEI/ 70ev
Scheme 2.2: Formation of m/z 204 and m/z 254 for the PFPA and HFBA derivatives
of 2,3-, 3,4-MDMAs and methoxymethcathinones
The decreased role for alpha cleavage reaction in the fragmentation of these
amides as a result of perflouroacylation which weakens the bond between nitrogen and
the alpha-carbon of the substituted methylenedioxyphenethyl group, allowing the
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100
formation of charged hydrocarbon species of increased relative abundance. These
hydrocarbons of varying mass significantly individualize the mass spectra and provide
specific structural information. The mass spectra in Figures 2.2 and 2.3 illustrate the role
of the hydrocarbon fragment at m/z 162 in the electron impact mass spectral
differentiation among these regioisomeric compounds. The spectra in Figures 2.2a, 2.2b,
2.3a and 2.3b show the 2,3- and 3,4-methyelenedioxyphenylpropene radical cation at m/z
162, identifying these molecules as the PFPA and HFBA derivatives of 2,3- and 3,4-
methyelenedioxy methamphetamines, respectively. The formation of the m/z 162 ion has
been described chapter 1 and requires the transfer of hydrogen from the benzylic carbon.
This fragmentation mechanism does not take place in the PFPA and HFBA derivatives of
the methoxymethcathinones due to the absence of benzylic hydrogen (Scheme 2.3). One
can conclude that the presence of alkene ions at m/z 162, can be used to identify the side
chain of 3,4- and 2,3-methyelenedioxymethamphetamines and exclude the regioisomeric
methoxymethcathinones. Conversely, the base peak at m/z 135 as well as the absence of
the m/z 162 ion would identify one of these substances as a methoxymethcathinone
regioisomer.
A comparison of the PFPA derivatives between the 3,4- and 2,3-
methylenedioxymethamphetamine (Figures 2.2a and 2.2b) with their HFBA derivatives
(Figures 3.3a and 3.3b) indicates unique ions at m/z 160 and m/z 210. This mass
difference of 50 (CF2) suggests these ions contain the perfluoroalkyl group for each
derivative, C2F5 and C3F7, respectively. These unique ions have been fully characterized
in chapter 1 using deuterated analogs of 3,4-MDMA and methamphetamine. These ions
at m/z 160 and 210 are the result of a rearrangement decomposition of ions 204 and 254
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101
respectively (Scheme 1.2). The m/z 204 and 254 ions have the same structure whether
generated from derivatives of the MDMAs or the methoxymethcathinones. Therefore
these unique ions do not provide any information to differentiate between the two groups
of substances.
O
O
C N R
O
HHO
O
N R
OH
H
O
O
CH
H
m/z 162
N
O
RO
H3CO R = C2F5, PFPA derivativeR= C3F7, HFBA derivative
Scheme 2.3: Benzylic hydrogen transefer to form m/z 162 occur only in MDMA
perfluroaceyl derivatives but not for methoxymethcathinones.
Gas chromatographic separation of 2,3-MDMA and 3,4 MDMA from
the methoxymethcathinones
Gas chromatographic properties of the PFPA and HFBA derivatives of the 2,3-
and 3,4-methylenedioxymethamphetamines and 2-, 3-, and 4-methoxymethcathinones
were compared on two stationary phases using capillary columns of the same dimensions,
30m x 0.25mm and 0.25um depth of film. The stationary phases compared in this study
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102
were the relatively nonpolar phases, 100% dimethyl polysiloxane (Rtx-1) and 95%
dimethyl-5% diphenyl polysiloxane (Rtx-5). The underivatized compounds were not
completely resolved with 2-methoxymethcathinone co-eluting with 3-
methoxymethcathinone using these common gas chromatographic stationary phases and
some common temperature programming conditions (Figure 2.4).
The PFPA and HFBA derivatives showed improved resolution when compared to
the underivatized amines. Several temperature programs were evaluated and the best
compromise between resolution and analysis time were used to generate the
chromatograms in Figures 2.5 and 2.6.
The two chromatograms for the PFPA derivatives (Figure 2.5) were generated
using two different temperature programs; the resulting elution order and resolution are
quite similar. In fact the elution order is the same for all the chromatograms shown in
Figures 2.5 and 2.6. In each case the derivatized 2-methoxymethcathinone (compound 1)
eluted first followed closely by the 3-methoxymethcathinone derivative (compound 2).
The third compound to elute is the 2,3-MDMA derivative (compound 4) and the
derivatized 4-methoxymethcathinone (compound 3) is the forth peak in each
chromatogram. The derivatized form of 3,4-MDMA (compound 5) showed the greatest
retention in each chromatogram.
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103
Time (minutes)
Figure 2.4: Capillary gas chromatogram for a physical mixture of underivatized
3,4 MDMA, 2,3-MDMA and methoxymethcathinones.
11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.000
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
Time-->
AbundanceTIC: 111703-6.D
1+2
43
5
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104
Abundance
Time (minutes)
Figure 2.5: Capillary gas chromatographic separation of PFPA derivatives of
compounds 1-5. Columns used: A Rtx-1; B Rtx-5.
1 2
3
5
8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
Time-->
AbundanceTIC: 30304-16.D
1
2
4 3 5
A
B
7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.500
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
Time-->
AbundanceTIC: 11903-14.D
4
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105
Abundance
Time (minutes) Figure 2.6: Capillary gas chromatographic separation of HFBA derivatives of
compounds 1-5. Columns used; A: Rtx-1; B: Rtx-5.
4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time-->
AbundanceTIC: 12904-4.D
9.00 10.00 11.00 12.00 13.00 14.00 15.000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
6500000
Time-->
AbundanceTIC: 30204-14.D
A
B
1
5
3
4
2
1
2
4
3
5
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106
Conclusion of Chapter 2
3,4- and 2,3-MDMA and 2-, 3-, and 4-methoxymethcathinone are a unique subset of
regioisomeric molecules; each compound has a Mw of 193 and yields a base peak for the
N-methyl imine of acetaldehyde at m/z 58 in the mass spectrum from the loss of the
corresponding C8H7O2 methylenedioxybenzyl and methoxybenzoyl groups, respectively.
Thus, the traditional EI-MS provides little structural information for differentiating
among these five compounds. Because of the unique similarity of these compounds by
MS, the specific identification of a compound such as 3,4-MDMA requires methods to
eliminate the other four isomers. This elimination process may be accomplished on the
basis of chromatography alone but would ultimately require the analyst to use reference
samples of each of the five amines. Derivatization of these amines with various acylating
agents yields amides with improved resolution compared with the underivatized amines
by capillary GC on Rtx-1 and Rtx-5 stationary phases. These perfluoroacyl derivatives
significantly individualize the mass spectra for these amides. The individualization is the
result of fragmentation of the alkyl carbon–nitrogen bond, yielding hydrocarbon
fragments at m/z 162 as well as other unique fragments from the MDMA amides that
were not formed for the methoxymethcathinones. The PFPA and HFBA derivatives are
essentially equivalent for chromatographic purposes; however, the HFBA derivatives
offer more unique fragment ions for additional mass spectral discrimination among these
regioisomeric substances.
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107
Chapter 3
Mass Spectrometry and Gas Chromatographic Studies on Acylated
Derivatives of a Series of Side Chain Regioisomers of 2-Methoxy-4-
Methyl Phenethylamines
In this chapter GC-MS studies are described for a series of side chain isomers of 2-
methoxy-4-methylphenethylamines. These isomers have an isobaric relationship (equal
mass, different elemental composition) to the controlled substance 3,4-MDMA.
Mass Spectral Studies on Chain Regioisomers of 2-Methoxy-4-Methyl
Phenethylamines
Mass spectrometry is the primary method for confirming the identity of drugs and
other substances of abuse in forensic samples. The five side chain regioisomers of 2-
methoxy-4-methylphenethylamine (Figure 3.1, Compounds 1-5) have the potential to
yield mass spectra essentially equivalent to 3,4-MDMA (and 2,3-MDMA). All have
molecular weight of 193 and major fragment ions in their electron ionization mass
spectra at m/z 58 and 135/136 (Figure 3.2). The m/z 58 ion in the methoxy methyl
phenethylamine is regioisomeric with that obtained in the mass spectra of both 2,3 and
3,4-MDMA. Additionally, the isobaric methoxy methyl benzyl (C9H11O)+ fragments
have the same nominal mass as the methylenedioxybenzyl (C8H7O2)+ cation occurring at
m/z 135 (Scheme 3.1). The individual mass spectra for 2,3- and 3,4-MDMA are also
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108
presented in Figure 3.2 (Compounds 6 and 7). The data in Figure 3.2 show that the mass
spectra do not provide any major fragment ions to differentiate among these compounds.
Figure 3.1: Structures of the side chain regioisomers of 2-methoxy-4-methylphenethylamines, and 2,3- and 3,4-MDMA.
H3C
N
OCH3
1
H3C
HN
OCH3
2
H3C
HN
OCH3 3
H3C
NH2
OCH3
4
H3C
NH2
OCH3
5
HN
O
O
6
O
O
HN
7 (3,4-MDMA)
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109
HC
N
CH3
CH3
m/z = 135
m/z = 58
MW = 193
H2C
CH2
CH
CH3
HN
CH3
O
O
H2C
O
O
CH2
EI 70ev
H
H3C
H3C
C N
C2H8
OCH3
OCH3
CN
C2H8
m/z = 135
Scheme 3.1: EI fragmentation pattern for compounds 1-7. Figure 3.2: Mass Spectra of the underivatized amines 1-7.
H3C
N
OCH3 1
40 60 80 100 120 140 160 180 2000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
m/z-->
AbundanceAverage of 10.725 to 10.848 min.: 71707--6.D
41 51
58
65 77
91
92 103 121122
136
149162163 176 191193
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110
H3C
HN
OCH3 2
H3C
HN
OCH3
3
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
m/z-->
AbundanceAverage of 9.175 to 9.253 min.: 110606-1.D
42
58
597778
91
103 115120 133135 149 160162
176188
191
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
m/z-->
AbundanceAverage of 9.961 to 10.261 min.: 71707--7.D
42
58
657779
91 105
115121 131136
146 160164 178 189192
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111
H3C
NH2
OCH3
4
H3C
NH2
OCH3
5
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
m/z-->
AbundanceAverage of 10.234 to 10.415 min.: 71707--8.D
41
58
6577
81
91 105
115121136
146148 161 176178
193
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
140000
150000
160000
170000
180000
190000
200000
m/z-->
AbundanceAverage of 11.092 to 11.635 min.: 71707--9.D
41
58
65
7779
91 105
117
121
136
137
149
164
176 193
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112
HN
O
O
6
O
O
HN
7
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
m/z-->
AbundanceAverage of 9.215 to 9.325 min.: 110706-1.D
42
58
63
77
7991 105
117120
135
146148 163 174178 192
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
m/z-->
AbundanceAverage of 9.657 to 9.790 min.: 110606-6.D
42
58
63
77
8991 105
117120
135
137 148 162 174178 191
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113
OCH3H3C
N
R1 R2 X
R3
O
R1+R2+R3 = C2H7 C2F5= X,, MW = 339 C3F7= X, MW= 389
OCH3H3C
m/z = 135
N
R1
R2
R3
X
O
m/z = 204, X= C2F5m/z = 254, X= C3F7
OCH3H3C
R1
R2
m/z = 148, R1=R2=Hm/z = 162, R1= CH3; R2=Hm/z = 176, R1= CH3; R2= CH3m/z = 176,R1= C2H5; R2= H
H3C
m/z = 105
Scheme 3.2: Mass spectral fragmentation products for the acylated 2-methoxy-4-methyl phenethylamines.
In the next phase of this study various perfluoroacylated derivatives of the
regioisomeric primary and secondary amines were prepared and evaluated in an effort to
individualize their mass spectra and provide unique marker ions for specific
identification. Generally, acylation of the amines significantly lowers the basicity of
nitrogen and can allow other fragmentation pathways to play a more prominent role in the
resulting mass spectrum.
The mass spectra for the pentafluoropropionyl and heptfluorobutyryl amides are
shown in Figures 3.3 and 3.4, respectively. From these spectra a common peak occurs at
m/z 204 and 254 which corresponds to the loss of 135 mass units from the molecular ions
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114
at 339 and 389 for PFPA and HFBA amides. The m/z 204 and 254 ions in the PFPA and
HFBA amides are analogous to m/z 58 in the underivatized species because all these ions
represent the (M-135)+ species. The general fragmentation pattern and structures for the
m/z 204 and 254 ions are shown in Scheme 3.2. The 2-methoxy-4-methylbenzyl cation
(m/z 135) and the methylenedioxybenzyl cation (m/z 135) are fragments common to all
spectra in Figures 3.3 and 3.4. However, the 2-methoxy-4-methylbenzyl cation at m/z
135 in the perfluoroacyl derivatives shows a very high relative abundance. Indeed the m/z
135 ion is the base peak in all the PFPA derivatives of compounds 2,4,5 and in the HFBA
derivatives of compounds 4 and 5. The remaining two HFBA derivatives of compounds 2
and 3 and the PFPA derivative of compound 3 show the m/z 135 ions as a major
fragment of at least 90% relative abundance. This would suggest that the perfluoroacyl
derivatives of compounds 2, 3, 4 and 5 offer a distinct discrimination between the
methylenedioxy and the 2-methoxy-4-methyl substitution patterns based on the
difference in relative abundances of the substituted benzyl cation at m/z 135.
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115
Figure 3.3: Mass Spectra of the PFPA derivatives of compounds 2-7.
H3C
N C2F5
O
OCH3
2
H3C
N C2F5
O
OCH3 3
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
750000
m/z-->
AbundanceAverage of 17.255 to 17.432 min.: 72407-8.D
42 65 77
91
119
135148
162
176
204
220 237 272 294 309 324
339
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
m/z-->
AbundanceAverage of 15.319 to 15.441 min.: 72407-9.D
42
56 79 91
105
135
147
162
190
204
220 237 269282 308 324339
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116
H3C
NH C2F5
O
OCH3
4
H3C
NH C2F5
O
OCH3
5
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
m/z-->
AbundanceAverage of 14.531 to 14.675 min.: 72407-10.D
42
59
79 91
105
135
146164
176
204
215 257 282 306 324
339
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
m/z-->
AbundanceAverage of 14.995 to 15.129 min.: 72407-11.D
41
65
79 91
105
135
145
161
176
204
220 237246 278292 310
339
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117
N
O
O C2F5
O
6
O
O
N C2F5
O
7
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
m/z-->
AbundanceAverage of 16.557 to 16.723 min.: 72407-12.D
42
56 77
91
119 135
147
162
177
204
220233 259263 300 324
339
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
m/z-->
AbundanceAverage of 18.845 to 19.066 min.: 72407-13.D
42
51
77
91
119
135
147
162
176
204
220 237 259269282 300 324
339
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118
Figure 3.4: Mass spectra of the HFBA derivatives of compounds 2-7.
H3C
N C3F7
O
OCH3 2
H3C
N C3F7
O
OCH3 3
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
340000
360000
380000
m/z-->
AbundanceAverage of 17.802 to 17.982 min.: 72407-1.D
42
69
91
92 119
148
169
178197
226
254
322 344 370
389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
m/z-->
AbundanceAverage of 15.736 to 15.857 min.: 72407-2.D
42
6991
105
115
135
162
192
210
220
254
309 358374389
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119
H3C
NH C3F7
O
OCH3 4
H3C
NH C3F7
O
OCH3
5
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
m/z-->
AbundanceAverage of 14.994 to 15.094 min.: 72407-3.D
41
59
91
105
121
135
169
176
214
226
254
273 313 350 374
389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
m/z-->
AbundanceAverage of 15.458 to 15.613 min.: 72407-4.D
41
6979
105
121
135
161
176
214
226
254
266 296313328360361
389
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120
N
O
O C3F7
O
6
O
O
N C3F7
O
7
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
m/z-->
AbundanceAverage of 17.093 to 17.314 min.: 72407-5.D
42
6977
105131
135
162
192
210
220
254
263 291 309 339350 370
389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
750000
m/z-->
AbundanceAverage of 19.525 to 19.788 min.: 72407-7.D
42
6977
105
131
135
162
191
210
220
254
263 293309 350 370
389
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121
The decreased role for the alpha cleavage reaction in the fragmentation of these
amides allows the formation of more diagnostic ions of each individual isomer. Acylation
weakens the bond between nitrogen and the alkyl carbon of the phenethyl side chain,
allowing the formation of charged side chain specific hydrocarbon species of increased
relative abundance. These hydrocarbons of varying mass significantly individualize the
mass spectra and provide specific structure information. The spectra for the N-ethyl
isomer (compound 2) in Figures 3.3-2 and 3.4-2 show a base peak at m/z 148
corresponding to the alkene radical cation which occurs from hydrogen rearrangement
and subsequent fragmentation of the alkyl carbon to nitrogen bond of the phenethylamine
side chain (see Scheme 3.2). This ion at m/z 148 would only occur for the N-ethyl
regioisomer. The spectra in Figures 3.3-3, 3.3-6, 3.3-7, 3.4-3, 3.4-6 and 3.4-7 show the
substituted phenylpropene hydrocarbon radical cation at m/z 162, identifying these
molecules as the PFPA and HFBA derivatives of 2-methoxy-4-methylmethamphetamine
and the 2,3-, 3,4-methylenedioxymethamphetamines respectively. The spectra for the
PFPA and HFBA derivatives of the primary amines (compounds 4 and 5) show ions at
m/z 176 from the corresponding substituted phenylbutene radical cation. The lower
abundance of m/z 176 for the 4-methoxy-3-methylphentramine (compound 4) may be the
result of steric inhibition of hydrogen transfer in the alpha, alpha-dimethyl substitution
pattern.
While the alkene ions at 148, 162, and 176 help to identify the side chain
regioisomers, one complicating factor in the PFPA derivatives for the N-
ethylphenethylamines (Figure 3.3-2) is the appearance of an ion at m/z 176 in addition to
the base peak at m/z 148. The 176 ion suggests a four carbon chain directly attached to
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122
the aromatic ring as occurs for the alpha-ethyl- (compound 5) and alpha, alpha-dimethyl-
(compound 4) phenethylamines (Figures 3.3-4, 3.3-5 and 3.4-4, 3.4-5). The m/z 176 ion
in the spectra for the PFPA derivatives of the N-ethyl regioisomers (Figure 3.3-2) is a
rearrangement of the m/z 204 ion resulting in the loss of mass 28 (the N-ethyl group) via
hydrogen transfer This coincidental common mass from two different fragmentation
pathways is confirmed by examining the mass spectra for the HFBA derivatives of the N-
ethyl-phenethylamines shown in Figure 3.4-2. The loss of 28 mass units from the
acylimine fragment at m/z 254 yields the equivalent fragment ion at m/z 226. Thus, the
HFBA derivatives may offer more characteristic ions for individualization of these
regioisomeric substances.
A comparison of the mass spectra for the PFPA and HFBA derivatives of all three
ring substituted methamphetamines (Compounds 3, 6 and 7) indicates unique ions at m/z
160 and m/z 210 (see Figures 3.3-3, 3.3-6, 3.3-7; 3.4-3, 3.4-6, and 3.4-7). This mass
difference of 50 (CF2) suggests these ions contain the perfluoroalkyl group for each
derivative, C2F5 and C3F7 respectively. This ion has been described in previous chapters
as the cationic methylated nitrile C2F5CNCH3 or C3F7CNCH3. The equivalent
fragmentation further supported by the analysis of the mass spectra of the PFPA and
HFBA derivatives of d3- and d5-MDMA in previous studies [Chapter 1]. This
fragmentation pathway appears unique to the acylated N-methyl C-methyl pattern
(methamphetamine side chain).
The presence of relatively high abundances of the m/z 105 ion in the spectra of
the PFPA and HFBA derivatives of compound 3 allows for significant discrimination of
this 2-methoxy-4-methylmethamphetamine from the two isobaric methylenedioxy-
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123
methamphetamines isomers (compounds 6 and 7). This ion at m/z 105 represents the loss
of 30 mass units (formaldehyde, CH2O) from the methoxymethylbenzyl cation at m/z
135. Previous reports suggested that the m/z 105 ion is characteristic of all o-methoxy
substitution patterns regardless the position of the methyl group on the aromatic ring
[Chapter 6]. However, the absence of this fragment from the spectra of the derivatives of
compound 2 suggests the necessity of further structure fragmentation studies for these
compounds.
Gas Chromatographyic separation of the perflouroacyl derivatives of 2- methoxy-4-
methylphenethylamines from 2,3- and 3,4-MDMA
The PFPA and HFBA derivatives of the six primary and secondary amines were
compared on two stationary phases using two GC-MS systems, The relatively nonpolar
100% dimethyl polysiloxane (Rtx-1) and the more polar trifluoropropyl methyl
polysiloxane (Rtx-200). Several temperature programs were evaluated and one program
showing the best compromise between resolution and analysis time was used to generate
the chromatograms in Figures 3.5 and 3.66. The elution order was the same for both
stationary phases and all temperature programs evaluated in this study. The
chromatograms of the perfluoroacyl compounds show that the 2,3-MDMA elutes before
the corresponding 3,4-isomer, which elutes last. When the ring substitution pattern is held
constant (2-methoxy-4-methyl), the side chain elution order is secondary amides before
the tertiary amides and in this limited set of examples branched isomers elute before the
more linear ones. Therefore, the amides of compound 4 elute first followed by the amide
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124
of compound 5 (both secondary amides), then the amides of compound 3 (the
methamphetamine side chain) and finally the amides of compound 2 (the more linear of
these two tertiary amides). The amides of 2,3-MDMA elute between the secondary and
tertiary amides in this group of compounds.
Perhaps the most useful information in these chromatograms is the relative elution of the
derivatized controlled substance 3,4-MDMA and its closest eluting regioisomeric and
isobaric equivalents. Both the PFPA and HFBA derivatives of 3,4-MDMA elute last and
the N-ethyl-2-methoxy-4-methylphenethylamine PFPAs and HFBAs are the closest
eluted compounds in the 2-methoxy-4-methyl phenethylamine series. The isobaric
acylated N-ethyl amides show very distinct mass spectra with several characteristic ions
to differentiate it from the corresponding amides of the drug of abuse 3,4-MDMA. Thus,
derivatization methods coupled with both chromatographic and mass spectral procedures
can allow for the complete differentiation of the side chain substitution pattern of the 2-
methoxy-4-methylphenethylamines from 3,4-MDMA and its regioisomer, 2,3-MDMA.
Additionally in this limited study, the Rtx-200 stationary phase provided the greatest
resolution of the amides of compound 7 (3,4-MDMA) from all the other isomers.
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125
Abundance
Time (minutes) Abundance
Time (Minutes) Figure 3.5: Chromatograms of the PFPA (A) and HFBA (B) derivatives of
compounds 2-7. GC-MS system 1, Rtx-200 column.
4
4
5
3
3
2
2
7
7
65
6
12.00 14.00 16.00 18.00 20.00 22.00 24.000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
Time-->
AbundanceTIC: 72507-6.D
10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
Time-->
AbundanceTIC: 72507-4.D
A
B
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126
Abundance
10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
Time-->
Abundance
TIC: 0801207-14.D\data.ms
Time (minutes)
Abundance
10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
Time-->
Abundance
TIC: 0801207-07.D\data.ms
Time (minutes)
Figure 3.6: Chromatograms of the PFPA (A) and HFBA (B) derivatives of
compounds 2-7. GC-MS system2, Rtx-1 column.
4
5
3
6 2
7
72
6
3
5
4
A
B
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127
Conclusion of Chapter 3
Differentiation of the side chain substitution pattern of the 2-methoxy-4-
methylphenethylamines from 3,4-MDMA and its regioisomer, 2,3-MDMA was
accomplished using a combination of gas chromatography and mass spectrometry.
Derivatization of the primary and secondary amines with various acylating agents yields
amides with improved resolution compared to the underivatized amines by capillary gas
chromatography on Rtx-1 and Rtx-200 stationary phases.
Additionally, the perfluoroacyl derivatives significantly individualize the mass spectra for
these amides and allow for unambiguous identification. The individualization results
from fragmentation of the alkyl carbon-nitrogen bond yielding characteristic hydrocarbon
fragments at m/z 105, 148, 162, and 176 as well as other unique fragments.
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128
Chapter 4
GC-MS Analysis of Acylated Derivatives of the Side Chain
Regioisomers of 4-Methoxy-3-Methyl Phenethylamines Related to
Methylenedioxymethamphetamine
In this chapter all the side chain regioisomeric possibilities of 4-methoxy-3-
methyl phenethylamines, as a representative of the m/z58 side chain regioisomers of the
methoxy group in the 4 position, will be compared from the mass spectral and
chromatographic aspects to 2,3- and 3,4-MDMA.
Mass Spectral studies of 4-methoxy-3-methyl phenethylamines along with 2,3- and
3,4- MDMA
Mass spectrometry is the primary method for confirming the identity of drugs and
other substances of abuse in forensic samples. The mass spectra of phenethylamines are
characterized by a base peak formed from an amine initiated alpha-cleavage reaction
involving the carbon-carbon bond of the ethyl linkage between the aromatic ring and the
amine. In 3,4-methylenedioxymethamphetamine (MW=193) the alpha-cleavage reaction
yields the substituted imine fragment at m/z 58 and the 3,4-methylenedioxybenzyl
fragment at mass 135/136 (for the cation and the radical cation, respectively). Thus, the
mass spectrum for 3,4-methylenedioxymethamphetamine contains major ions at m/z 58
and 135/136 as well as other ions of low relative abundance. There are a total of five
regioisomeric forms of the m/z 58 imine species.
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129
The five side chain regioisomers of 4-methoxy-3-methylphenethylamine
(Compounds 1-5) have the potential to yield mass spectra essentially equivalent to 3,4-
MDMA and 2,3-MDMA ( structures shown in Figure 4.1). All have molecular weight of
193 and major fragment ions in their electron ionization mass spectra at m/z 58 and
135/136 (Figure 4.2). The isobaric methoxy methyl benzyl (C9H11O)+ fragments have the
same mass as the methylenedioxybenzyl (C8H7O2)+ cation occurring at m/z 135.
Furthermore the m/z 58 ion in the methoxy methyl phenethylamine is regioisomeric with
that obtained in the mass spectra of both 2,3 and 3,4-MDMA (Scheme 4.1). The
individual mass spectra for 2,3- and 3,4-MDMA are also presented in Figure 4.2
(Compounds 6 and 7).
Figure 4.1: Structures of side chain regioisomers of methamphetamine
N
1
N,N-Dimethylphetheylamine
HN
2
N-ethylphenethylamine
HN
3
Methamphetamine
NH2
4
α, α-Dimethylphenethylamine
NH2
5
1-Phenyl-2-butanamine
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130
Figure 4.2: Mass Spectra of the undreivatized amines:
H3CO
CH3
N
1
H3CO
CH3
HN
2
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
200000
400000
600000
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1600000
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2000000
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2400000
m/z-->
AbundanceAverage of 9.175 to 9.253 min.: 110606-1.D
42
58
597778
91
103 115120 133135 149 160162
176188
191
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
m/z-->
AbundanceAverage of 9.792 to 9.928 min.: 110606-2.D
41
58
65 77
80
91
103 117121
136
137 149163 174176 191
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131
H3CO
CH3
HN
3
H3CO
CH3
NH2
4
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
m/z-->
AbundanceAverage of 9.352 to 9.497 min.: 110606-3.D
42
58
65 7782
91
103 115121
136
137 147 162 174178 191
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
m/z-->
AbundanceAverage of 9.053 to 9.210 min.: 110606-4.D
41
58
65 7782
91
103 115121
135
137 148 161 175178
193
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132
H3CO
CH3
NH2
5
HN
O
O
6
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
200000
400000
600000
800000
m/z-->
AbundanceAverage of 10.110 to 10.279 min.: 110606-5.D
41
58
59 747891
103105121
132
136
148 159164 176 193
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
m/z-->
AbundanceAverage of 9.215 to 9.325 min.: 110706-1.D
42
58
63
77
7991 105
117120
135
146148 163 174178 192
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133
O
O
HN
7
HC
N
CH3
CH3
m/z = 135
m/z = 58
MW = 193
H2C
C
N
CH2
CH
CH3
HN
CH3
O
O
H2C
m/z = 135
O
O
CH2
EI 70ev
H
H3CO
CH3
CH3
H3CO
C2H8
C N
C2H8
Scheme 4.1: EI fragmentation pattern of the underivatized compounds 1-7
This lack of mass spectral specificity in addition to the possibility of
chromatographic co-elution, with 3,4-MDMA, could result in misidentification of the
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
m/z-->
AbundanceAverage of 9.657 to 9.790 min.: 110606-6.D
42
58
63
77
8991 105
117120
135
137 148 162 174178 191
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134
target drug. Furthermore, the lack of available reference samples could complicate the
individual identification of any one of these substances. This constitutes a significant
analytical challenge where the specific identification by gas chromatography-mass
spectrometry (GC-MS) must be based primarily upon the ability of the chromatographic
system to separate the regioisomeric/isobaric non-drug substance from the actual drug of
interest. Additionally, the ability to distinguish between these regioisomers directly
enhances the specificity of the analysis for the target drugs of interest.
In the next phase of this study various perfluoroacylated derivatives of the
regioisomeric primary and secondary amines were prepared and evaluated in an effort to
individualize their mass spectra and provide unique marker ions for specific
identification. Acylation of the amines significantly lowers the basicity of nitrogen and
can allow other fragmentation pathways to play a more prominent role in the resulting
mass spectrum.
The mass spectra for the twelve pentafluoropropionyl and heptfluorobutryl
amides are shown in Figures 4.3 and 4.4, respectively. From these spectra a common
peak occurs at m/z 204 and 254 which corresponds to the loss of 135 mass units from the
molecular ions at 339 and 389 for PFPA and HFPA amides. This ion at m/z 204 and 254
is the PFPA and HFPA imine species likely formed from the alpha cleavage of the amide
nitrogen to eliminate the 4-methoxy-3-methylbenzyl radical as well as the 2, 3- and 3, 4-
methylenedioxybenzyl radical. Thus the m/z 204 and 254 ions in the PFPA and HFPA
amides are analogous to m/z 58 in the underivatized species because all these ions
represent the (M-135)+ species. The general fragmentation pattern and structures for the
m/z 204 and 254 ions are shown in Scheme 4.2. The 4-methoxy-3-methylbenzyl cation
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135
and the methylenedioxybenzyl cation (m/z 135) are fragments common to all spectra in
Figures 4.3 and 4.4. However, the 4-methoxy-3-methylbenzyl cation at m/z 135 in the
perfluoroacyl derivatives shows a very high relative abundance. Indeed the m/z 135 ion is
the base peak in all the PFPA derivatives of compounds 2-5 and in the HFBA derivatives
of compounds 4 and 5. The remaining two HFBA derivatives of compounds 2 and 3
show the m/z 135 ions as a major fragment of at least 90% relative abundance. This
would suggest that the pentafluoroacyl derivatives offer a distinct discrimination between
the methylenedioxy and the 4-methoxy-3-methyl substitution patterns based on the
difference in relative abundances of the substituted benzyl cation at m/z 135.
The decreased role for the alpha cleavage reaction in the fragmentation of these
amides allows the formation of ions more diagnostic of each individual isomer. Acylation
weakens the bond between nitrogen and the alkyl carbon of the phenethyl side chain,
allowing the formation of charged hydrocarbon species of increased relative abundance.
These hydrocarbons of varying mass significantly individualize the mass spectra and
provide specific structure information. The mass spectra in Figures 4.3 and 4.4 illustrate
the role of hydrocarbon fragments at m/z 148, 162 and 176 in the electron impact mass
spectral differentiation among these regioisomers.
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136
Figure 4.3: Mass Spectra of the PFPA derivatives of compounds 2-7.
H3CO
CH3
N C2F5
O
2 3
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 3600
100000
200000
300000
400000
500000
600000
700000
m/z-->
AbundanceAverage of 12.672 to 12.805 min.: 111306-3.D
42 65 77
91
119
135148
176
191
204
220 272281294 324
339
355
H3CO
CH3
N C2F5
O
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
m/z-->
AbundanceAverage of 12.109 to 12.332 min.: 111306-4.D
42
5677
91
119
135
147
162
176
204
220228 259269282 300 324
339
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137
H3CO
CH3
NH C2F5
O
4
H3CO
CH3
NH C2F5
O
5
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
m/z-->
AbundanceAverage of 10.712 to 11.001 min.: 111306-5.D
42
59
77
91
119
135
146164
176
204
220 237 257263 282 306 324
339
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
1000000
1200000
m/z-->
AbundanceAverage of 11.537 to 11.727 min.: 111306-6.D
41
65 77
91
119
135
147
161
176
204220 237246 263 294 310320
339
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138
N
O
O C2F5
O
6
O
O
N C2F5
O
7
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
m/z-->
AbundanceAverage of 11.758 to 11.926 min.: 111306-2.D
42
56 77
91
119135
147
162
177
204
220 242 259263 282 300 324
339
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
m/z-->
AbundanceAverage of 12.777 to 13.078 min.: 111306-1.D
42
5177
91
119
135
147
162
191
204
220 243 259269282 300 324
339
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139
Figure 4.4: Mass spectra of the HFBA derivatives of compounds 2-7
H3CO
CH3
N C3F7
O
2
H3CO
CH3
N C3F7
O
3
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
m/z-->
AbundanceAverage of 13.152 to 13.339 min.: 111306-9.D
42
69
77
91
119
148
169
178197
226
254
271281 313322 341 370
389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
750000
800000
m/z-->
AbundanceAverage of 12.605 to 12.795 min.: 11130610.D
42
69
77
91
120
135
162
191
210
220
254
256 281 313319 341 374
389
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140
H3CO
CH3
NH C3F7
O
4
H3CO
CH3
NH C3F7
O
5
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
m/z-->
AbundanceAverage of 11.193 to 11.472 min.: 11130611.D
42
59
77
91
121
135
169
176
214214
254
256 281 313330 356374
389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
m/z-->
AbundanceAverage of 12.087 to 12.298 min.: 11130612.D
41
69
77
91
120
135
161
176
207226
254
255 281 313328344360
389
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141
N
O
O C3F7
O
6
O
O
N C3F7
O
7
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
m/z-->
AbundanceAverage of 12.311 to 12.479 min.: 111306-8.D
42
6977
105131
135
162
192
210
220
254
256 281 313327 350 374
389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
m/z-->
AbundanceAverage of 13.268 to 13.512 min.: 111306-7.D
42
6977
105131
135
162
191
210
220
254
263 293310 332 350 370
389
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142
H3CO
CH3
N
H3CO
CH3
N
R
O
O
N
N
O
O
R
R
O
O
O
R
O
H2C
N R
O
N R
O
HN R
O
N R
O
N R
O
R=C2F5 , m/z =339R=C3F7 , m/z =389
R=C2F5 , m/z =204R=C3F7 , m/z =254
H3CO
CH3
NH R
O
H3CO
CH3
NH R
O
HN R
O
Scheme 4.2: Formation of the (M-135)+ ions in the perfluoroacyl derivatives of the
regioisomeric 4-methoxy-3-methylphenethyl amines, 2,3- and 3,4-MDMA.
The spectra for the N-ethyl isomer (compound 2) in Figures 4.3-2 and 4.4-2 show
a base peak at m/z 148 corresponding to the alkene radical cation which occurs from
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143
hydrogen rearrangement and subsequent fragmentation of the alkyl carbon to nitrogen
bond of the phenethylamine side chain (see Scheme 4.3). This ion at m/z 148 would only
occur for the N-ethyl regioisomer. The spectra in Figures 4.3-3, 4.3-6, 4.3-7, 4.4-3, 4.4-6
and 4.4-7 show the substituted phenylpropane hydrocarbon ion at m/z 162, identifying
these molecules as the PFPA and HFBA derivatives of 4-methoxy-3-
methylmethamphetamine and the 2,3-, 3,4-methylenedioxymethamphetamines
respectively. The proposed mechanism for the formation of the hydrocarbon fragment is
illustrated in Scheme 4.3.
CH
R5
H
R1
R2
N R3
O
R4
HC
R5
R1
R2
N R3
O
R4H
HC
R5
C
R1
R2
R3 = C2H5 (Compound 2) CH3 (Compounds 3,6,7) H2 (Compound 4,5)R4 = C2F5, PFPA Derivatives C3F7, HFBA DerivativesR5 = 4-Methoxy-3-methyphenyl (Compounds 2-5) = 2,3-Methylenedioxyphenyl (Compound 6) =3,4-Methylenedioxyphenyl (compound 7)
R1=R2 = H, m/z= 148 (Compound 2)R1= H, R2= CH3, m/z =162 (Compounds 3,6,7)R1=R2 = CH3, m/z =176 (Compound 4)R1=H, R2= C2H5, m/z=176 (Compound 5)
Scheme 4.3: Mechanism for the formation of the alkene radical cation in the
perfluroacyl derivatives ofthe regioisomeric 4-methoxy-3-methylphenethyl amines,
2,3- and 3,4-MDMA.
However the base peak of m/z 135 in the spectra of the PFPA derivative of
compound 3 offer a significant discrimination of 4-methoxy-3-methylmethamphetamines
over its two isobaric methylenedioxymethamphetamines isomers (compounds 6 and 7) .
The spectra for the PFPA and HFBA derivatives of the primary amines (compounds 4
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144
and 5) show ions at m/z 176 from the corresponding substituted phenylbutene radical
cation. This m/z 176 results from hydrogen rearrangement and subsequent fragmentation
of alkyl carbon to nitrogen bond. The lower abundance of m/z 176 for the 4-methoxy-3-
methylphentramine (compound 4) may be attributed to steric inhibition of hydrogen
transfer in the alpha, alpha-dimethyl substitution pattern.
While the alkene ions at 148, 162, and 176 help to identify the side chain
regioisomers, one complicating factor in the PFPA derivatives for the N-
ethylphenethylamines (Figure 4.3-2) is the appearance of an ion at m/z 176 in addition to
the base peak at m/z 148. The 176 ion suggests a four carbon chain directly attached to
the aromatic ring as occurs for the alpha-ethyl- (compound 5) and alpha, alpha-dimethyl-
(compound 4) phenethylamines (Figures 4.3-4, 4.3-5 and 4.4-4, 4.4-5). The m/z 176 ion
in the spectra for the PFPA derivatives of the N-ethyl regioisomers (Figure 4.3-2) is a
rearrangement of the m/z 204 ion resulting in the loss of mass 28 (the N-ethyl group) via
hydrogen transfer (see Scheme 4.4). This coincidental common mass from two different
fragmentation pathways is confirmed by examining the mass spectra for the HFBA
derivatives of the N-ethyl-phenethylamines shown in Figure 4.4-2. The loss of 28 mass
units from the acylimine fragment at m/z 254 yields the equivalent fragment ion at m/z
226. Thus, the HFBA derivatives may offer more characteristic ions for individualization
of these regioisomeric substances.
A comparison of the mass spectra for the PFPA and HFBA derivatives of all three
ring substituted methamphetamines (Compounds 3, 6 and 7) indicates unique ions at m/z
160 and m/z 210 (see Figures 4.3-3, 4.3-6, 4.3-7; 4.4-3, 4.4-6, and 4.4-7). This mass
difference of 50 (CF2) suggests these ions contain the perfluoroalkyl group for each
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145
derivative, C2F5 and C3F7 respectively. An analysis of the masses of the components
which make up the fragment at m/z 160 for example include C2F5 (119 mass units) and
CH3 (15 mass units) leaving only a mass of 26 available for the total of 160. The mass 26
would correspond to CN and the proposed mechanism for the formation of
(C2F5CNCH3)+ is shown in Scheme 4.5. An equivalent fragmentation pathway has been
further supported by the analysis of the mass spectra of the PFPA and HFBA derivatives
of d3- and d5-MDMA in chapter 1.
H2C N
CH2
CH2H
RC
O
H2C N
H
R
O
R=C2F5, m/z = 204R=C3F7, m/z = 254
R=C2F5, m/z = 176R=C3F7, m/z = 226
Scheme 4.4: Mechanism for the loss of mass 28 from the perfluoroacyl-N-ethyl-
imine cation.
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146
N
H3C
CHH3C O
C
R
N C
OCH
RH3C
H3C
N C RH3C
R= C2F5, m/z= 204R= C3F7, m/z= 254
R= C2F5, m/z= 160R= C3F7, m/z= 210
Scheme 4.5: Mechanism for the formation of the m/z 160 and m/z 210 ions in the
mass spectra of the perfluoroacyl derivatives of the ring substituted
methamphetamines (Compounds 3, 6 and 7).
Gas Chromatography of the regioisomeric 4-methoxy-3-methylphenethyl amines,
2,3- and 3,4-MDMA.
The PFPA and HFBA derivatives of the six primary and secondary amines were
compared on two stationary phases of the same dimensions, 30 m x 0.25 mm and 0.25
µm depth of film. The stationary phases compared in this study were the relatively
nonpolar phase 100% dimethyl polysiloxane (RTX-1) and the more polar trifluoropropyl
methyl polysiloxane (Rtx-200). Several temperature programs were evaluated and one
program showing the best compromise between resolution and analysis time was used to
generate the chromatograms in Figure 4.5. The chromatograms of the perfluoroacyl
compounds show that the 2,3-MDMA elutes before the corresponding 3,4-isomer, which
elutes last. When the ring substitution pattern is held constant (4-methoxy-3-methyl), the
side chain elution order is secondary amides before the tertiary amides and in this limited
set of examples branched isomers elute before the more linear ones. Therefore, the
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147
amides of compound 4 elute first followed by the amide of compound 5 (both secondary
amides), then the amides of compound 3 (the methamphetamine side chain) and finally
the amides of compound 2 (the more linear of the tertiary amides). The amides of 2,3-
MDMA elute between the secondary and tertiary amides in this group of compounds.
Perhaps the most useful information in these chromatograms is the relative elution of the
derivatized controlled substance 3,4-MDMA and its closest eluting regioisomeric and
isobaric equivalents. Both the PFPA and HFBA derivatives of 3,4- MDMA elute last and
the N-ethyl-4-methoxy-3-methylphenethylamine PFPAs and HFBAs are the closest
eluted compounds in the 4-methoxy-3-methyl phenethylamine series. The isobaric N-
ethyl amides show very distinct mass spectra with several characteristic ions to
differentiate it from the corresponding amides of the drug of abuse 3,4-MDMA. Thus,
derivatization methods coupled with both chromatographic and mass spectral procedures
can allow for the complete differentiation of the side chain substitution pattern of the 4-
methoxy-3-methylphenethylamines from 3,4-MDMA and its regioisomer, 2,3-MDMA.
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148
Abundance
Time (minutes) Abundance
Time (Minutes) Figure 4.5: GC Chromatograms of the PFPA (A) and HFBA (B) derivatives of
compounds 2-7. Rtx-200 column.
12.00 14.00 16.00 18.00 20.00 22.00 24.000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
3200000
Time-->
AbundanceTIC: 121206-1.D
12.00 14.00 16.00 18.00 20.00 22.000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
Time-->
AbundanceTIC: 121206-4.D
4
4
5
5
6
6
3
3
2
2
7
7
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149
Conclusions of Chapter 4
3,4-MDMA , 2,3-MDMA and the five side chain regioisomers of 4-methoxy-3-
methyl phenethylamines are a unique subset of regioisomeric and isobarc molecules;
each compound has a molecular weight of 193 and yields a base peak at m/z 58 in the
mass spectrum from the loss of the corresponding methylenedioxybenzyl and 4-
methoxy-3-methylbenzyl groups respectively. Thus the traditional electron impact mass
spectrum provides little structural information for differentiating among these seven
compounds. Because of the unique similarity of these compounds by mass spectrometry,
the specific identification of a compound such as 3,4-MDMA requires methods to
eliminate other regioisomeric and isobaric substances.
This elimination process could be accomplished on the basis of chromatography
alone but ultimately would require the analyst to use reference samples of the other
substances. Derivatization of the primary and secondary amines with various acylating
agents yields amides with improved resolution compared to the underivatized amines by
capillary gas chromatography on TRx-1 and TRX-200 stationary phases. Additionally,
the perfluoroacyl derivatives significantly individualize the mass spectra for these amides
and allow for specific identification. The individualization is the result of fragmentation
of the alkyl carbon-nitrogen bond yielding characteristic hydrocarbon fragments at m/z
148, 162 and 176 as well as other unique fragments.
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150
Chapter 5
Gas Chromatographic and Mass Spectral Studies on Acylated Side
Chain Regioisomers of 3-Methoxy-4-methyl-phenethylamine and 4-
Methoxy-3-methyl-phenethylamine
This chapter describes the MS and GC behavior of a set of selected compounds
having nonequivalent ring substituents at the 3- and 4-position of the aromatic ring. Thus,
these ten compounds represent all the possible regioisomeric methoxy methyl
phenethylamines having the same 3,4 substitution pattern as 3,4-MDMA.
Mass Spectral Studies on Side Chain Regioisomers of 3-Methoxy-4-methyl-
phenethylamine and 4-Methoxy-3-methyl-phenethylamine
Mass spectrometry is the primary method for confirming the identity of drugs and
related substances in forensic samples. The mass spectra of phenethylamines are
characterized by a base peak formed from an amine initiated alpha-cleavage reaction
involving the carbon-carbon bond of the ethyl linkage between the aromatic ring and the
amine. In 3,4-methylenedioxymethamphetamine (MW=193) the alpha-cleavage reaction
yields the substituted imine fragment at m/z 58 and the 3,4-methylenedioxybenzyl
fragment at mass 135/136 (for the cation and the radical cation, respectively). Thus, the
mass spectrum for 3,4-methylenedioxymethamphetamine contains major ions at m/z 58
and 135/136 as well as other ions of low relative abundance [ Chapter 1].
The side chain regioisomers of 3-methoxy-4-methyl phenethylamine and 4-
methoxy-3-methyl phenethylamine (Compounds 1-10, Figure 5.1) have the potential to
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151
yield a mass spectrum essentially equivalent to 3,4-MDMA. All have molecular weight
of 193 and major fragment ions in their electron ionization mass spectra at m/z 58 and
135/136 (Figure 5.2). The individual mass spectra for 2,3- and 3,4-MDMA are also
presented in Figure 5.2 (Compounds 11 and 12). The isobaric methoxy-methyl-benzyl
(C9H11O)+ fragments have the same mass as the methylenedioxybenzyl (C8H7O2)+ cation
occurring at m/z 135. Furthermore the m/z 58 ion in the ring substituted methoxy-methyl
phenethylamine is regioisomeric with that obtained in the mass spectra of both 2,3 and
3,4-MDMA (Scheme5.1). This lack of mass spectral specificity for the isomers shown in
Figure 5.2, in addition to the possibility of chromatographic co-elution with 3,4-MDMA,
could result in misidentification in this series of drugs and drug-like substances
Figure 5.1: Structures of Side Chain Regioisomers of 3-Methoxy-4-methyl-
phenethylamine, 4-Methoxy-3-methyl-phenethylamine, 2,3- and 3,4-MDMA
H3C
OCH3
N
N,N-Dimethyl-3-methoxy-4-methyl
phenethylamine 1
H3CO
CH3
N
N,N-Dimethyl-4-methoxy-3-methyl
phenethylamine 6
H3C
OCH3
HN
N-Ethyl-3-methoxy-4-methyl
phenethylamine 2
H3CO
CH3
HN
N-Ethyl-4-methoxy-3-methyl
phenethylamine 7
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152
H3C
OCH3
HN
3-Methoxy-4-methyl methamphetamine
3
H3CO
CH3
HN
4-Methoxy-3-methyl methamphetamine
8
H3C
OCH3
NH2
3-Methoxy-4-methyl phentramine
4
H3CO
CH3
NH2
3-Methoxy-4-methyl phentramine
9
H3C
OCH3
NH2
3-Methoxy-4-Methyl BDB
5
H3CO
CH3
NH2
4-Methoxy-3-Methyl BDB
10
HN
O
O
11
(2,3-MDMA)
O
O
HN
12
(3,4-MDMA)
.
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153
HC
N
CH3
CH3
m/z = 135 m/z = 58
MW = 193
H2C
CH2
CH
CH3
HN
CH3
O
O
H2C
O
O
CH2
EI 70ev
H
C N
C2H8
CN
C2H8
m/z = 135
CH3
CH3
H3CO
H3CO
Scheme 5.1: Structures of the major EI fragments for 3-Methoxy-4-methyl-
phenethylamine, 4-Methoxy-3-methyl-phenethylamine, 2,3- and 3,4-MDMA
Figure 5.2: Mass Spectra of 3-Methoxy-4-methyl-phenethylamine, 4-Methoxy-3-
methyl-phenethylamine, 2,3- and 3,4-MDMA.
H3C
OCH3
N
1
30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
m/z-->
Abundance
Average of 12.281 to 12.316 min.: 071022-01.D\data.ms58.1
91.142.1 77.0103.1 135.1 149.1115.0 193.1176.0161.9
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154
H3C
OCH3
HN
2
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
65000
70000
m/z-->
Abundance
Average of 13.073 to 13.143 min.: 071022-02.D\data.ms58.1
136.1
91.177.044.0 103.1 149.1121.1 191.1176.1161.1
H3C
OCH3
HN
3
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
1700000
1800000
1900000
2000000
2100000
2200000
m/z-->
Abundance
Average of 12.391 to 12.520 min.: 071022-03.D\data.ms58.1
91.1135.1
77.1103.142.1 120.1 178.1162.1147.1 192.1
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155
H3C
OCH3
NH2
4
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
750000
800000
850000
900000
950000
1000000
m/z-->
Abundance
Average of 11.995 to 12.024 min.: 071022-04.D\data.ms58.1
91.1135.1
77.1 178.142.1 103.1 121.1 161.1148.1 191.1
H3C
OCH3
NH2
5
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
340000
m/z-->
Abundance
Average of 13.359 to 13.435 min.: 071022-05.D\data.ms58.1
136.1
91.1
41.1 77.1121.1 164.1103.1
149.1 193.1176.1
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156
H3CO
CH3
N
6
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
750000
800000
850000
900000
950000
1000000
1050000
1100000
1150000
1200000
1250000
m/z-->
Abundance
Average of 12.391 to 12.461 min.: 071022-06.D\data.ms58.1
91.142.1 135.177.1 149.1 193.2103.1 120.1 176.1162.1
H3CO
CH3
HN
7
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
4000
4200
4400
4600
m/z-->
Abundance
Average of 13.528 to 13.767 min.: 071022-07.D\data.ms58.1
136.144.0
91.0
77.0121.0 191.1149.0
103.0 176.0
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157
H3CO
CH3
HN
8
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
750000
800000
850000
900000
950000
1000000
1050000
1100000
1150000
m/z-->
Abundance
Average of 12.619 to 12.683 min.: 071022-08.D\data.ms58.1
136.191.1
77.142.1 121.1103.1 162.1 178.1150.1 192.2
H3CO
CH3
NH2
9
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
m/z-->
Abundance
Average of 12.240 to 12.386 min.: 071022-09.D\data.ms58.1
135.191.1
42.1 178.177.0 121.1103.1 161.1148.1 193.1
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158
H3CO
CH3
NH2
10
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000
32000
34000
36000
38000
40000
42000
44000
46000
48000
50000
m/z-->
Abundance
Average of 13.662 to 14.321 min.: 071022-10.D\data.ms58.1
136.1
91.1
41.1 121.074.5
164.1103.1 149.1
176.1 191.1
HN
O
O
11
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
m/z-->
Abundance
Average of 12.444 to 12.555 min.: 080907-06.D\data.ms58.1
77.1 135.042.1 88.6 105.1 178.1 192.1118.1 163.1147.1
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159
O
O
HN
12
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
m/z-->
Abundance
Average of 13.027 to 13.167 min.: 080907-07.D\data.ms58.1
135.077.042.1 88.6 105.0 178.1148.1 193.1163.1120.1
The second phase of this study involved the preparation and evaluation of various
perfluoroacylated derivatives of the regioisomeric primary and secondary amines, in an
effort to individualize their mass spectra via formation of unique marker ions and
improved chromatographic resolution. Acylation of the amines generally lowers the
basicity of nitrogen and can allow other fragmentation pathways to play a more
prominent role in the resulting mass spectrum. Of course the tertiary amine (compounds 1
and 6) do not form a stable amide derivative.
The mass spectra for the twenty pentafluoropropionyl and heptfluorobutryl
amides are shown in Figures 5.3 and 5.4, respectively. From these spectra a common
peak occurs at m/z 204 and 254 which corresponds to the loss of 135 mass units from the
molecular ions at 339 and 389 for PFPA and HFPA amides. This ion at m/z 204 and 254
is the PFPA and HFPA imine species likely formed from the alpha cleavage of the amide
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160
nitrogen to eliminate the methoxy-methyl-benzyl or methylenedioxybenzyl radical. Thus
the m/z 204 and 254 in PFPA and HFPA amides are analogous to m/z 58 in the
underivatized species because all these ions represent the (M-135)+ species (Scheme 5.2).
The 3-methoxy-4-methylbenzyl cation, 4-methoxy-3-methylbenzyl cation and the
methylenedioxybenzyl cation (m/z 135) are fragments common to all spectra in Figures
5.3 and 5.4.
Figure 5.3: Mass spectra of the PFPA derivatives of compounds 2-5 and 7-12
H3C
OCH3
N C2F5
O
2
40 60 80 100 120 140 160 180 200 220 240 260 280 300 3200
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
m/z-->
Abundance
Average of 15.976 to 16.209 min.: 071023-11.D\data.ms148.1
176.0
204.1
339.191.1 119.0
56.1272.1 310.1229.1 251.0
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161
H3C
OCH3
N C2F5
O
3
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
m/z-->
Abundance
Average of 15.358 to 15.737 min.: 071023-12.D\data.ms204.1
162.1
135.1
42.1 91.1339.1
65.1 300.1228.1 269.0113.0
H3C
OCH3
NHC2F5
O
4
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
m/z-->
Abundance
Average of 13.895 to 14.117 min.: 071023-13.D\data.ms204.1
136.1
176.1
59.1 339.1
91.1
306.1237.1 282.1260.1114.0
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162
H3C
OCH3
NH C2F5
O
5
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
m/z-->
Abundance
Average of 14.822 to 14.979 min.: 071023-14.D\data.ms176.1
135.1
204.1
339.1
91.141.1
310.165.1 237.1 282.1113.0 260.1
H3CO
CH3
N C2F5
O
7
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
m/z-->
Abundance
Average of 16.495 to 16.839 min.: 071023-15.D\data.ms135.1
91.1176.0
339.1204.165.142.1 272.1 294.0229.1113.0
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163
H3CO
CH3
N C2F5
O
8
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
m/z-->
Abundance
Average of 15.807 to 16.250 min.: 071023-16.D\data.ms135.1
162.1
204.1
91.142.1
339.165.0
269.0228.0 308.0
H3CO
CH3
NHC2F5
O
9
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
m/z-->
Abundance
Average of 14.111 to 14.665 min.: 071023-17.D\data.ms135.1
204.0
176.1
91.059.0 339.1
263.1 306.1237.1113.1
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164
H3CO
CH3
NH C2F5
O
10
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
m/z-->
Abundance
Average of 15.148 to 15.358 min.: 071023-18.D\data.ms135.1
176.1
91.1 339.1
41.1 204.165.1 310.1263.1237.0113.0 288.1
N
O
O C2F5
O
11
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
m/z-->
Abundance
Average of 15.597 to 15.941 min.: 071023-19.D\data.ms204.1
162.1
339.1119.042.1 77.1
300.1263.1241.1
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
165
O
O
N C2F5
O
12
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1800000
2000000
m/z-->
Abundance
Average of 16.769 to 17.048 min.: 071023-20.D\data.ms204.1
162.1
135.1
339.177.142.1105.1
269.0 300.1243.1
Figure 5.4: Mass Spectra of the HFBA derivatives of compounds 2-5 and 7-12
H3C
OCH3
N C3F7
O
2
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
200000
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800000
1000000
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3000000
m/z-->
Abundance
Average of 16.611 to 16.786 min.: 071023-01.D\data.ms148.1
226.0
254.1
389.191.1
117.156.1 178.0
322.1 360.1282.0
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166
H3C
OCH3
N C3F7
O
3
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
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3200000
m/z-->
Abundance
Average of 15.953 to 16.221 min.: 071023-02.D\data.ms254.1
162.1
210.0
135.1
91.142.1
389.1
350.1293.1 319.0
H3C
OCH3
NHC3F7
O
4
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
3200000
m/z-->
Abundance
Average of 14.431 to 14.723 min.: 071023-03.D\data.ms254.1
136.1176.1
59.1
389.191.1 214.0
307.1 356.1280.0
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167
H3C
OCH3
NH C3F7
O
5
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
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600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
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1800000
1900000
2000000
m/z-->
Abundance
Average of 15.358 to 15.661 min.: 071023-04.D\data.ms176.1
135.1
254.1
389.1
91.1226.041.1
360.1307.1 334.1280.0
H3CO
CH3
N C3F7
O
7
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
m/z-->
Abundance
Average of 17.089 to 17.386 min.: 071023-05.D\data.ms135.1
91.1
226.0
169.0 254.0 389.1
65.1196.9 322.140.1 359.1279.1
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168
H3CO
CH3
N C3F7
O
8
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
1700000
1800000
1900000
2000000
2100000
2200000
2300000
m/z-->
Abundance
Average of 16.425 to 16.740 min.: 071023-06.D\data.ms135.1
162.1
254.1
210.0
91.1
42.1
389.1
319.0293.1 350.1
H3CO
CH3
NHC3F7
O
9
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
3200000
3400000
3600000
m/z-->
Abundance
Average of 14.764 to 15.218 min.: 071023-07.D\data.ms135.1
254.1
176.1
91.159.1 389.1
214.0313.1 356.1288.0
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169
H3CO
CH3
NH C3F7
O
10
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
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1200000
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3000000
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3800000
m/z-->
Abundance
Average of 15.731 to 16.267 min.: 071023-08.D\data.ms135.1
176.1
91.1389.1
254.141.1226.0 360.166.0 313.1288.0201.0
N
O
O C3F7
O
11
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
3200000
3400000
m/z-->
Abundance
Scan 2013 (16.320 min): 071023-09.D\data.ms254.1
162.1210.1
135.1389.142.1 77.1
105.1350.1291.0 320.1
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170
O
O
N C3F7
O
12
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
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400000
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700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
1700000
1800000
1900000
2000000
2100000
2200000
2300000
m/z-->
Abundance
Average of 17.357 to 17.841 min.: 071023-10.D\data.ms254.1
162.1
135.1210.0
77.142.1 389.1
105.1
310.0 350.1282.9
N
R1 R2 X
R3
O
R1+R2+R3 = C2H7 C2F5= X,, MW = 339 C3F7= X, MW= 389
N
R1
R2
R3
X
O
m/z = 204, X= C2F5m/z = 254, X= C3F7
R1
R2
CH3
XCNH3C
m/z = 160 (X = C2F5)m/z = 210 (X = C3F7)Cpds ,3,8,11 and 12m/z = 148, R1=R2=H,cpd 2 and 7
m/z = 162, R1= CH3; R2=H,cpds ,3,8, 11 and 12m/z = 176, R1= CH3; R2= CH3, cpds 4 and 9m/z = 176,R1= C2H5; R2= H, cpds 5 and 10
H3CO
H3CO
CH3
Scheme 5.2: Structures of the major EI fragments for the perfluoroacylated 3-
Methoxy-4-methyl-phenethylamines and 4-Methoxy-3-methyl-phenethylamines
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171
Indeed the m/z 135 ion is the base peak in all the PFPA and HFBA derivatives of
compounds 7-10 and this increased relative intensity may serve as an indicator ion for
discrimination of the 4-methoxy-3-methyl ring substitution pattern from other ring
substitution patterns in this study. The decreased role for alpha cleavage reaction in the
fragmentation of these amides allows the formation of ions more diagnostic of each
individual isomer. Acylation weakens the bond between nitrogen and the alpha-carbon
allowing the formation of charged hydrocarbon species of increased relative abundance.
These hydrocarbons of varying mass identify the number of carbons attached directly to
the aromatic ring. The mass spectra in Figures 5.3 and 5.4 show hydrocarbon fragments
at m/z 148, 162 and 176 for a two-carbon, three-carbon and four-carbon chain attached
directly to the aromatic ring.
The spectra for the N-ethyl derivatives in Figures 5.3-2, 5.-7, 5.4-2 and 5.4-7
show a base peak at m/z 148 corresponding to the two-carbon alkene radical cation which
occurs from hydrogen rearrangement and subsequent fragmentation of the alkyl carbon to
nitrogen bond of the phenethylamine side chain. This ion at m/z 148 would only occur for
the N-ethyl regioisomer. The relative abundance of both m/z 148 and 135 offer a clear
discrimination of compound 2 from its direct regioisomer (compound 7) as well as from
the other isomers. The spectra in Figures 5.3-3, 5.3-8, 5.3-11, 5.3-12, 5.4-3, 5.4-8, 5.4-11
and 5.4-12 show the ring substituted methoxy-methyl or the
methylenedioxyphenylpropene hydrocarbon ion at m/z 162 (the three-carbon alkene
radical cation), identifying this molecules as the PFPA and HFBA derivatives containing
the methamphetamine side chain, compounds 3, 8, 11 and 12 respectively. The spectra
for the PFPA and HFBA derivatives of the primary amines 4, 5, 9 and 10 show ions at
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172
m/z 176 from the four-carbon alkene radical cation of the 3-methoxy-4-methyl- and 4-
methoxy-3-methyl-phenethylamines. This m/z 176 results from hydrogen rearrangement
and subsequent fragmentation of the alkyl carbon to nitrogen bond yielding the
methoxymethyl-phenylbutene radical cation. The lower abundance of m/z 176 for
compounds 4 and 9 may be attributed to steric inhibition of hydrogen transfer in the
alpha, alpha-dimethyl substitution pattern.
While the alkene ions at 148, 162, and 176 help to identify the side chain
regioisomers, one complicating factor in the PFPA derivatives for the N-
ethylphenethylamines (Figures 5.3-2 and 5.3-7) is the appearance of an ion at m/z 176 in
addition to the base peak at m/z 148. Based on the previous discussion, the m/z 176 ion
suggests a four carbon chain directly attached to the aromatic ring as occurs for the alpha-
ethyl- and alpha, alpha-dimethyl-phenethylamines (Figures 5.3-4, 53-5, 5.3-9, 5.3-10 and
5.4-4, 5.4-5, 5.4-9, 5.4-10). The m/z 176 ion in the spectra for the PFPA derivatives of
the N-ethyl regioisomers (Figures 5.3-2 and 5.3-7) is a rearrangement of the m/z 204 ion
resulting in the loss of mass 28 (the N-ethyl group) via hydrogen transfer. This
coincidental common mass from two different fragmentation pathways is confirmed by
examining the mass spectra for the HFBA derivatives of the N-ethyl-phenethylamines
shown in Figures 5.4-2 and 5.4-7. The loss of 28 mass units from the acylimine fragment
at m/z 254 yields the equivalent fragment ion at m/z 226. Thus, the HFBA derivatives
may offer more characteristic ions for individualization of these regioisomeric substances
compared to the PFPA derivatives.
A comparison of the PFPA derivatives for compounds 3, 8, 11 and 12 (Figures
5.3-3, 5.3-8, 5.3-11 and 5.3-12) with their HFBA derivatives (Figures 5.4-3, 5.4-8, 5.4-
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173
11 and 5.4-12) indicates unique ions at m/z 160 and m/z 210. This mass difference of 50
(CF2) suggests these ions contain the perfluoroalkyl group for each derivative, C2F5 and
C3F7 respectively. Additional information about these ions were obtained in previous
deuterium labeling studies which confirmed that methyl group on nitrogen is a part of this
resulting fragment. The remaining mass 26 would correspond to CN and the proposed
structures of m/z 160 and 210 are shown in Scheme 5.2.
Gas Chromatography of 3-Methoxy-4-methyl-phenethylamine, 4-Methoxy-3-
methyl-phenethylamine, 2,3- and 3,4-MDMA
The PFPA and HFBA derivatives of the primary and secondary amine side chain
regioisomers of the ring substituted methoxy methyl phenethyl amines, 2,3-MDMA and
3,4-MDMA were compared on a 100% dimethyl polysiloxane (Rtx-1) stationary phase.
Several temperature programs were evaluated and one program showing the best
compromise between resolution and analysis time was used to generate the
chromatograms in Figure 5.5. The chromatograms show that when the ring substitution
pattern is held constant (i.e. 3-methoxy-4-methyl- or 4-methoxy-3-methyl-) the two
secondary amides elute before the two tertiary amides. Additionally, in every case in this
limited set of compounds the branched side chain elutes before the straight chain isomer
when the ring substitution pattern and the degree of amide substitution are constant,
regardless of the derivatizing agent. Therefore, the alpha, alpha-dimethyl isomer elutes
first followed by the alpha-ethyl isomer (both secondary amides), then the
methamphetamine, and N-ethyl phenethylamines (the two tertiary amides).
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174
When the side chain is held constant, the 3-methoxy-4-methyl ring substitution
pattern elutes before the 4-methoxy-3-methyl ring substitution pattern and this elution
order is the same for both the PFPA and the HFBA derivatives. Perhaps the most useful
information in these chromatograms is the relative elution of the derivatized controlled
substance 3,4-MDMA and its closest eluting regioisomeric equivalents. Both the PFPA
and HFBA derivatives of 3,4-MDMA elute after the N-ethyl-3-methoxy-4-methyl
phenethylamine and 4-methoxy-3-methylphenethylamine PFPAs and HFBAs, both the
N-ethyl regioisomers show very distinct mass spectra with several characteristic ions to
differentiate these compounds from the drug of abuse 3,4-MDMA. Thus, derivatization
methods coupled with chromatographic and mass spectral procedures can allow for the
characterization and differentiation of these ten uniquely isomeric substances. The
individualization is possible without the need for reference samples of all these uniquely
similar substances.
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175
11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00
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Time-->
Abundance
TIC: 071031-02.D\data.ms
11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
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1500000
1600000
Time-->
Abundance
TIC: 071101-01.D\data.ms
Figure 5.5: GC Separation of the PFPA (A) and HFBA (B) derivatives of
compounds 2-5 and 7-12; Rtx-1 column.
4
9
5
10 3
11 8
2 12
4
9
5 10 3
11
8
2
7 12
A
B
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176
Conclusions of Chapter 5
3,4-MDMA , 2,3-MDMA and ten side chain regioisomers of 3-methoxy-4-
methyl-phenethylamine and 4-methoxy-3-methyl-phenethylamine are a unique subset of
regioisomeric and isobaric molecules; each compound has a molecular weight of 193 and
yields a base peak at m/z 58 in the mass spectrum from the loss of the corresponding
methylenedioxybenzyl or the mass equivalent isobaric ring substituted methoxy methyl
benzyl groups. Thus the traditional electron impact mass spectrum provides little
structural information for differentiating among these ten compounds.
Derivatization of the eight primary and secondary amines with various acylating
agents yields amides which significantly individualize the mass spectra for these amides
and allow for specific identification. The individualization is the result of fragmentation
of the alkyl carbon-nitrogen bond yielding hydrocarbon fragments at m/z 148, 162 and
176 as well as other unique fragments from these regioisomeric amides. The PFPA and
HFBA derivatives are essentially equivalent for chromatographic purposes however; the
HFBA derivatives offer more unique fragment ions for additional discrimination among
these regioisomeric substances. Chromatographic resolution of the acylated amines was
achieved on relatively non polar stationary phase Rtx-1 (100% dimethyl polysiloxane).
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177
Chapter 6
Mass Spectral and Gas Chromatographic Studies of Methoxy Methyl
Methamphetamines Isobaric to 3, 4-MDMA.
Isobaric substances are compounds of the same nominal mass but with different
elemental composition. There are isobaric substances, which do not contain the
methylenedioxy substitution pattern in the aromatic ring, yet they are able to yield the
same major fragments in their mass spectra as the controlled drug substance MDMA.
Among these MDMA isobaric substances are the ring substituted methoxy methyl
methamphetamines.
Mass Spectral Studies of Methoxy Methyl methamphetamines Isobaric to 3,4-MDMA
The mass spectrum of 3,4-MDMA is characterized by a base peak formed by an
-cleavage reaction involving the carbon-carbon bond of the ethyl linkage between the
aromatic ring and the amine. In 3,4-MDMA (MW=193) and its direct regioisomer, 2,3-
MDMA, the -cleavage reaction yields the 3,4-methylenedioxybenzyl and 2,3-
methylenedioxybenzyl fragments at mass 135/136 (for the cation and the radical cation,
respectively) and the substituted imine fragment at m/z 58. Thus, the mass spectrum for
3,4-MDMA contains major ions at m/z 58 and 135/136 as well as other ions of low
relative abundance.
The various methoxy methyl ring substitution patterns of methamphetamine have
the potential to yield mass spectra essentially equivalent to 3,4-MDMA and 2,3-MDMA,
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178
all have molecular weight of 193 and major fragment ions in their electron ionization
mass spectra at m/z 58 and 135/136 (Figure 6.1). The isobaric methoxy methyl benzyl
(C9H11O)+ fragments have the same mass as the methylenedioxybenzyl (C8H7O2)+ cation
occurring at m/z 135. Furthermore the m/z 58 ion in the methoxymethamphetamine is the
same imine structure as that obtained in the mass spectra of both 3,4 and 2,3-MDMA
(Scheme 6.1).
HC
NH
CH3
CH3
m/z = 135
m/z = 58
70ev
MW = 193
HN
H3CO
H3C
CH2H3CO
H3C
Scheme 6.1: General mass spectral fragmentation for the ring substituted methoxy
methyl methamphetamines.
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179
Figure 6.1: Structures and mass spectra of ring substituted methoxy methyl
methamphetamines
Structures
Mass spectra
HN
CH3OCH3
CH3 1
HN
CH3OCH3H3C
2
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
m/z-->
AbundanceScan 208 (6.163 min): 110605-3.D
42
58
6577
82
91105
115121 135 146147 163 174178 192
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
m/z-->
AbundanceScan 895 (14.093 min): 100605-2.D
42
58
657779
91 105
115121136
146147 160 174178 192194
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180
HN
CH3OCH3
H3C
3
HN
CH3OCH3
CH3
4
HN
CH3CH3
OCH3
5
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
m/z-->
AbundanceScan 869 (13.789 min): 100605-3.D
42
58
657779
91105
115121136
146147 160 178 192
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
m/z-->
AbundanceScan 226 (6.372 min): 110605-2.D
42
58
6577
82
91105
115121135
146147 160 174178 193
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
m/z-->
AbundanceScan 971 (14.956 min): 11206-2.D
42
58
657779
91
105115121 135
136 147 162 174178 192
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181
HN
CH3H3C
OCH3
6
HN
CH3
H3C
OCH3
7
HN
CH3
cH3
OCH3
8
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
3200000
m/z-->
AbundanceScan 935 (14.406 min): 100605-4.D
42
58
657779
91
103 115120135
137 147 163 174178 191
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
500000
1000000
1500000
2000000
2500000
3000000
m/z-->
AbundanceScan 241 (6.496 min): 122005-1.D
41
58
59 777991
103 115121 131135
147 161162 178 192
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
m/z-->
AbundanceScan 964 (14.898 min): 11206-1.D
42
58
65 7782
91
103 115121135
146147 162 174178 193
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182
HN
CH3H3CO
CH3
9
HN
CH3CH3H3CO
10
The mass spectra for the ten ring substituted methoxy methyl methamphetamines
in Figure 6.1 show only the major fragment ion at equivalent masses. This lack of mass
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
m/z-->
AbundanceScan 958 (14.711 min): 100605-5.D
42
58
65 7782
91
103 115121
136
137 147 162 174178 192
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
500000
1000000
1500000
2000000
2500000
3000000
3500000
m/z-->
AbundanceScan 916 (14.336 min): 111005-3.D
42
58
63
77
79 89 105117120
135146148 163 178 192
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183
spectral specificity in addition to the possibility of chromatographic co-elution, with 3,4-
MDMA, could result in misidentification of the target drug. Furthermore, the lack of
available reference samples for all ten of these isobaric molecules complicates the
individual identification of any one of these substances. This constitutes a significant
analytical challenge, where the use of gas chromatography-mass spectrometry (GC-MS)
must be based primarily upon the ability of the chromatographic system to separate the
“counterfeit substance” from the actual drug of interest. Additionally, the ability to
distinguish between these regioisomers directly enhances the specificity of the analysis
for the target drugs of interest.
Mass Spectral Studies of Perfluroacyl Derivatives Methoxymethyl Methamphetamines compared to 2,3- and 3,4-MDMA
The perfluoroacylated derivatives of the ten methoxy methyl methamphetamines
were prepared and evaluated for their ability to individualize the mass spectral properties
of these compounds and to maintain or improve chromatographic resolution. Acylation of
the amines significantly lowers the basicisty of nitrogen and can allow other
fragmentation pathways to play a more prominent role in the mass spectrum. These
perfluoroacyl derivatives allowed the unique fragmentation reactions which characterized
and served to individualize the side chain regioisomers. However, in these substances the
perfluoroacyl derivatives were less successful at differentiating the 2,3- from the 3,4- ring
substitution pattern of the identical side chain. Since all ten of the methoxy methyl
methamphetamines have the same side chain and have ten different substitution patterens,
perfluroacylation may not allow for complete compound individualization based only on
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184
the observed mass spectrum. The mass spectra for the ten pentafluoropropionyl (PFPA)
and heptflourobutryl (HFBA) amides are shown in Figures 6.2 and 6.3. For comparative
purposes in this chapter 2,3- and 3,4-MDMA will be given the compounds number 11
and 12.
Figure 6.2: Mass spectra of the PFPA derivatives of methoxy methyl
methamphetamines, 2,3- and 3,4-MDMA
N
CH3
C2F5
O
OCH3
CH3
a
N
CH3
C2F5
O
H3COCH3
b
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
750000
m/z-->
AbundanceScan 602 (10.711 min): 12806-1.D
42
5677
91
105
135
147
162
190
204
220 292 308 324339
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
m/z-->
AbundanceScan 651 (11.286 min): 12806-2.D
42
56 7991
105
135
147
162
190
204
220 269282 308 324
339
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185
N
CH3
C2F5
O
H3C
OCH3
c
N
CH3
C2F5
O
CH3
OCH3 d
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
m/z-->
AbundanceScan 631 (11.053 min): 12806-3.D
42
56 7791
105135
147
162
190
204
220 263 282 308320
339
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
m/z-->
AbundanceScan 676 (11.576 min): 12806-4.D
42
56 7991
105
135
147
162
190
204
220 282 308 324
339
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186
N
CH3
C2F5
O
CH3
OCH3 e
N
CH3
C2F5
O
OCH3
H3C
f
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
m/z-->
AbundanceScan 753 (12.383 min): 12806-5.D
42
5677
91119
135147
162
176
204
220 300 324
339
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
m/z-->
AbundanceScan 685 (11.683 min): 12806-7.D
42
5677
91 119 135
147
162
176
204
220 300 324
339
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187
N
CH3
C2F5
O
OCH3
H3C
g
N
CH3
C2F5
O
OCH3
CH3
h
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
m/z-->
AbundanceScan 705 (11.909 min): 12806-8.D
42
5677
91119
135147
162
176
204
339
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
400000
m/z-->
AbundanceScan 729 (12.185 min): 12806-11.D
42
5677
91 119135
147
162
177
204
324
339
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188
N
CH3
CH3
C2F5
O
CH3
H3CO
i
j
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
m/z-->
AbundanceScan 728 (12.166 min): 12806-9.D
42
56
77
91
119
135
147
162
176
204
220 263 324
339
N
CH3
CH3
C2F5
O
CH3H3CO
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
m/z-->
AbundanceScan 770 (12.668 min): 12806-10.D
42
56 77
91 119
135
147
162
176
204
220
339
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189
O
O
N C2F5
O
k
O
O
N C2F5
O
l
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
500000
1000000
1500000
2000000
m/z-->
AbundanceScan 494 (9.469 min): 110903-2.D
42
51
77
91
119
135
147
162
176
204
220 241 259269282 300 324
339
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
m/z-->
AbundanceScan 431 (8.749 min): 110903-6.D
42
51 7791
119 135
147
162
177
204
220
339
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190
Figure 6.3: Mass spectra of the HFBA derivatives of methoxy methyl
methamphetamines, 2,3- and 3,4-MDMA
N
CH3
C3F7
O
OCH3
CH3 a
N
CH3
C3F7
O
H3COCH3
b
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
m/z-->
AbundanceScan 641 (11.177 min): 12806-14.D
42
69 91
105
115
135
162
192
210
220
254
256 342 389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
m/z-->
AbundanceScan 695 (11.785 min): 12806-15.D
42
69 91
105
115
135
162
192
210
220
254
269 313 332 358374
389
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191
N
CH3
C3F7
O
H3C
OCH3 c
N
CH3
C3F7
O
CH3
OCH3 d
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
m/z-->
AbundanceScan 671 (11.518 min): 12806-16.D
42
6991
105
115
135
162
192
210
220
254
268 313 332 358372
389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
m/z-->
AbundanceScan 720 (12.079 min): 12806-17.D
42
6991
105
115
135
162
192
210
220
254
269 332 358374
389
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192
N
CH3
C3F7
O
CH3
OCH3 e
N
CH3
C3F7
O
OCH3
H3C
f
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
m/z-->
AbundanceScan 791 (12.893 min): 12806-18.D
42
69 91105
131135
162
192
210
220
254
293310 350 374
389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
m/z-->
AbundanceScan 730 (12.204 min): 12806-6.D
42
69 91
105 131
135
162
191
210
220
254
278 310 332 350 374
389
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193
N
CH3
C3F7
O
OCH3
H3C
g
N
CH3
C3F7
O
OCH3
CH3
h
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
m/z-->
AbundanceScan 749 (12.415 min): 12806-19.D
42
69 91105 131
135
162
191
210
220
254
269 296 330 358374
389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
m/z-->
AbundanceScan 778 (12.750 min): 12806-20.D
42
6991
105115
135
162
192
210
220
254
273 293 313330 358374
389
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194
N
CH3
CH3
C3F7
O
CH3
H3CO
i
N
CH3
CH3
C3F7
O
CH3H3CO j
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
m/z-->
AbundanceScan 768 (12.637 min): 12806-21.D
42
69
91
92 120
135162
192
210
220
254
278 319
389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
m/z-->
AbundanceScan 819 (13.194 min): 12806-22.D
42
6991
105119
135
162
192
210
220
254
313330 372
389
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195
O
O
N C3F7
O
k
O
O
N C3F7
O
l
All these spectra show major high mass fragment ion at m/z 204 or 254
corresponding to the loss of 135 mass units from the molecular ion 339 and 389 from the
PFPA and HFBA amides, respectively. The ions at m/z 204 and 254 are the PFPA and
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
m/z-->
AbundanceScan 887 (14.016 min): 30204-2.D
42
6977
105
131
135
162
192
210
220
254
263 291 310 332 350 374
389
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
m/z-->
AbundanceScan 776 (12.730 min): 12804-6.D
42
6977
105
119
135
162
192
210
220
254
263 291 313 339350 374
389
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196
HFBA imine species likely formed from the alpha cleavage of the amide nitrogen to
eliminate the methoxymethylbenzyl radical. Thus the m/z 204 and 254 ions in PFPA and
HFBA amides are analogous to m/z 58 in the underivatized species because all these ions
represent the (M-135)+ species. The general fragmentation pattern and structures for the
m/z 204 and 254 ions are shown in Scheme 6.2. The methoxymethylbenzyl cation and
radical cation at m/z 135/136 is also a common fragment in most of the spectra in Figures
6.2 and 6.3. The identical fragmentation pathways for the PFPA and HFBA derivatives of
2,3- and 3,4-MDMA produced ions of the same structure at m/z 204 and 254 and isobaric
ions at equivalent masses for the benzylic species at m/z135/136.
N R
O
H3C
H3CO
EI 70 ev
N R
O
H3CO
H3C Scheme 6.2: Formation of m/z 204 (R=C2F5) and m/z 254 (R= C3F7) from
perfluoroaceyl derivatives of methoxy methyl methamphetamines.
Acylation, and in particular the perflouroacylation, weakens the bond between
nitrogen and the alpha-carbon of the substituted methoxy methyl phenethyl group,
allowing the formation of charged hydrocarbon species of increased relative abundance.
The mass spectra in Figures 6.2 and 6.3 illustrate the role of hydrocarbon fragments at
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197
m/z 162, 105 and 210 in the electron impact mass spectral differentiation among these
isobaric compounds.
The mass spectra for all derivatives (Figures 6.2 and 6.3) show a common peak at
m/z 162 corresponding to the alkene radical cation which occurs from hydrogen
rearrangement and subsequent fragmentation of the alkyl carbon to nitrogen bond of the
phenethylamine side chain (Scheme 6.3). The isobaric methylenedioxyphenylpropene
radical cation is observed in the mass spectrum of the PFPA and HFBA derivatives of
2,3- and 3,4-MDMA.The presence of the m/z 162 ion (as described previously) indicates
that a 3-carbon chain is attached directly to the aromatic ring in an uninterrupted manner.
The companion ion identifying the substituent on nitrogen as the N-methyl group occurs
at m/z160 in the PFPA derivatives (Figure 6.2) and at m/z 210 in the HFBA derivatives
(Figure 6.3)
Additionally the PFPA and HFBA derivatives of d3- and d5-MDMA, in Figure 1.4
and discussed earlier in chapter 1, also lend support to the proposed structure for the
formation of the alkene fragment at m/z 162 illustrated in Schemes 1.5 and 1.6 in chapter
1. A comparison of the PFPA derivatives (Figure 6.2) with the HFBA derivatives (Figure
6.3) indicated unique ions at m/z 160 and m/z 210. This mass difference of 50 (CF2)
suggests these ions contain the perfluoroalkyl group for each derivative, C2F5 and C3F7
respectively. The PFPA and HFBA derivatives of d3- and d5-MDMA, (Figure 1.4;
chapter 1) showed that the m/z 160 and m/z 210 ions contain the N-methyl group and
supports the proposed structure of the characteristic nitrile fragment.The suggested
mechanism of forming these masses was illustrated previously in Scheme 1.5; chapter 1.
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198
H3CO
H3C
CH
CH3
N
R
H
O
CH3
H3CO
H3C
HC
CH3
N
RHO
CH3
H3CO
H3C
HC
CH3
R = C2F5 PFPA derivatives R= C3F7 HFBA Derivatives
m/z= 162
Scheme 6.3: Formation of m/z 162 from perfluoacyl derivatives of methoxy methyl
methamphetamines.
The mass spectra of the 2-methoxy-substituted methyl methamphetamines
derivatives shown in Figure 6.2 a, b, c, and d for the PFPA derivatives and Figure 6.3 a,
b, c and d for the HFBA derivatives show a more prominent m/z 105 ion than the other
substitution patterns. This ion at m/z 105 represents the loss of 30 mass units
(formaldehyde, CH2O) from the methoxymethylbenzyl cation at m/z 135. The further
loss of formaldehyde (CH2O) from those benzylic cations having an ortho-methoxy
group can be attributed to a 1,6-hydride shift from the carbon of the methoxy group to the
methylene of the methoxymethyl benzyl cation followed by another hydride
rearrangement and loss of formaldehyde to give the methyl benzyl cation (Scheme 6.4).
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199
CH2
OCH2
H3CH
CH2
OCH2
H3C
CH2
H3C
H
H2C
H3C
H
m/z = 105 Scheme 6.4: Formation of m/z 105 form methoxymethylbenzyl radical.
As expected in this study, acylation of the side chain nitrogen in these isobaric
methamphetamines did not individualize the resulting mass spectra. Thus, differentiation
among these compounds and differentiation from 3,4-MDMA remains a significant
challenge for chromatographic studies. However, the mass spectra obtained for the PFPA
and HFBA derivatives do provide information which could allow these ten
methoxymethylmethamphetamines to be divided into three subsets based on the position
of the methoxy-group ring substitution.
The mass spectra of the PFPA and HFBA derivatives of the ortho-methoxy subset
(compounds 1-4) all show m/z 105 ion formed through the mechanism described in
Scheme 6.4. This ion does not occur to any significant extent in the derivatives of the
meta-methoxy of para-methoxy subsets. The m/z 105 ion is not observed in the mass
spectrum of the PFPA and HFBA derivatives of 2,3- and 3,4-MDMA. Thus the m/z 105
ion may distinguish this ortho-methoxy subset from MDMA but no specific ions were
observed to distinguish among the members of this subset, compounds 1-4.
The PFPA and HFBA derivatives of compounds 5-8 (methoxy group in the meta
postion of the aromatic ring) can be differentiated from the PFPA and HFBA derivatives
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200
of compounds 1-4 by the absence of m/z 105. Perfluroacylation did not offer an
advantage in distinguishing these compounds from each other and from the MDMAs.
The para-methoxy subset, the PFPA and HFBA derivatives of compounds 9 and
10, shows a very different distribution of ions than that observed for either of the other
two subsets. The low mass ions at m/z 135 and m/z 162 show a very high relative
abundance and actually appear as the base peak in several spectra. These are the only
derivatives not showing the perfluoroacylimine at m/z 204 or m/z 254 as the base peak.
In summary, perfluoroacylation did not allow mass spectrometry to individualize
these compounds. The presence of the m/z 105 ion suggests the methoxy group is in the
ortho-position of the aromatic ring while the significant abundance of the low mass ions
at m/z 135 and m/z 162 indicates the methoxy group is substituted at the para-position of
the aromatic ring. The meta-substituted aromatic ring methoxy group isomers did not
show any unique fragments to distinguish them from 2,3- and 3,4-MDMA.
Gas chromatographic separation of the perfluroacyl derivatives of ring substituted
methoxy methyl methamphetamines, 2,3- and 3,4- MDMA.
The underivatized and PFPA and HFBA derivatives of 2,3- and 3,4-MDMA and
their isobaric substituted methoxy methyl methamphetamines were compared on four
stationary phases using capillary columns of the same dimensions, 30m x 0.25mm and
0.25um depth of film.
Several temperature programs were evaluated, however only one program
yielding the best compromises between resolution and analysis time was used to generate
the twelve chromatograms for the PFPA and HFBA derivatives in Figures 6.5-6.10,
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201
respectively. The program (TP-2) was set up to hold the column temperature at 70 oC for
1 minute, ramped to 150 oC at 7.5 oC/minute, hold at 150 oC for 2 minutes and finally
ramped to 250 oC at 10 oC/minute. Only chromatograms generated on Rtx-1 and Rtx-35
are shown in Figures 6.5-6.10.
The co-elution of some underivatized, PFPA and HFBA derivatives of 2,3-, 3,4-
MDMA and their isobaric substances varied from one stationary phase to another.
Underivatized compound 2 co-elutes with compound 4 while underivatized compound 6
co-elutes with underivatized compounds 7 and 9 on Rtx-1 stationary phase. Each of the
PFPA and HFBA derivatives of compounds 8 and 9 co-elute, also the HFBA derivatives
of compounds 4 and 6 co-elute on the Rtx-1 stationary phase.
Four sets of underivatized compounds were found to co-elute on Rtx-5, 2,3-
MDMA co-elutes with compound 6, compound 2 co-elutes with compound 4, compound
5 co-elutes with compound 8 and finally compound 7 co-elutes with compound 9. In case
of the PFPA derivatives of the 2,3- , 3,4-MDMA and their isobaric substituted methoxy
methyl methamphetamine, there are two sets of compounds co-elute namely compound
11 co-elutes with compound 7 and compound 8 co-elutes with compound 9. Three sets of
the HFBA derivatives were found to co-elute, compound 11 co-elutes with compound 7,
compound 1 co-elutes with compound 3 and finally compound 5 co-elutes with
compound 8on the same column.
Three sets of underivatized compounds were found to co-elute on Rtx-35,
compound 4 co-elutes with compound 6, compound 5 co-elutes with compound 8 and
finally compound 7 co-elutes with compound 9. In case of the PFPA derivatives of the
2,3-, 3,4-MDMA and their isobaric substituted methoxy methyl methamphetamine, there
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202
are two sets of compounds which co-elute where compound 5 co-elutes with compound 9
and compound 4 co-elutes with compound 6. The HFBA derivatives of compounds 2, 8
and 9 co-elute on the same column.
The best resolution, among the evaluated columns, was achieved on Rtx-200 where in a
physical mixture of the 12 PFPA derivatized compounds yielded 11 peaks with
compounds 7 and 9 co-elute (Figure 6.4). The PFPA derivatives allow more distinctive
differentiation between compounds 7 and 9 based on mass spectrometry. Compound 9
has a base peak at m/z 135 compared to m/z 204 base peak for compound 7. Thus a
sample containing compound 7 or 9 could be identified based on mass spectrometry. The
similarity in chromatographic properties among these regioisomeric and isobaric
molecules in the derivatized and underivatized form provides for a significant
chromatographic challenge. However, all four of the stationary liquid phases evaluated in
this study successfully resolved 3,4-MDMA from the other isomers. The variation in
chromatographic selectivity among the phases resulted in various coelutions within the
isobaric methoxy methyl methamphetamines. Since mass spectrometry of the
perfluoroacyl derivatives of the isobaric methoxymethyl methamphetamines (compounds
1-10) successfully divided these compounds into subsets based on the ring position of the
methoxy group, the chromatographic properties were evaluated using the same subsets.
The chromatographic properties of each subset of compounds was compared to 2,3- and
3,4-MDMA.
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203
Abundance
Time (minutes)
Figure 6.4: Capillary gas chromatograph of a physical mixture of the PFPA
derivatives of methoxy methyl methamphetamines (Compounds 1-10), 2,3- (11) and
3,4-MDMA (12) . Column used: RTX-200.
In all the chromatographic studies 3,4-MDMA elutes last in every subset and in
every form (derivatized and underivatized). In the first subset (the ortho-methoxy
substituted aromatic ring), compounds 1-4, along with 2,3-MDMA and 3,4-MDMA, the
elution order of the perfluroacyl derivatives of these compounds was compound 1
followed by 3, 2, 4, 2,3-MDMA- and finally 3,4-MDMA (compound 12). The elution
order, on Rtx-1 and Rtx-35, was the same for this subset of compounds. ( Figures 6.5 and
6.6). This subset of isobaric amines was identified as having a significant m/z 105 ion in
their mass spectra.
In the second subset (the meta-methoxy substituted aromatic ring), compounds 5-
8 along with 2,3-MDMA and 3,4-MDMA, the PFPA derivative of compound 6 elutes
12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.000
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
Time-->
AbundanceTIC: 32006-12.D
1
3 2
4
6
11
7 + 9
8
5
10
12
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204
first followed by 2,3-MDMA-PFPA, 7-PFPA, 8-PFPA, 5-PFPA and finally 3,4-MDMA-
PFPA(compound 12); (Figure 6.7A). The elution order is the same for the HFBA
derivatives of the same compounds on Rtx-1 (Figures 6.8A). This elution order has
changed on Rtx-35, where compound 7 elutes before 2,3-MDMA, and most significantly
2,3-MDMA (compound 11) and 8 co-elute. This change in elution order takes place for
the PFPA and HFBA of these compounds (Figures 6.7b and 6.8b) which suggests that
Rtx-35 is not the best column for resolving this subset of compounds. It is this subset of
isobaric amines that showed no distinguishing characteristics (neither unique fragment
ions nor unique relative abundance of fragment ions) in their mass spectra.
In the third subset (the para-methoxy substituted aromatic ring), compounds 9, 10,
2,3-MDMA and 3,4-MDMA, the elution order of the perfluroacyl derivatives was 2,3-
MDMA followed by 9, 10 and finally 3,4-MDMA. The elution order was the same on
both Rtx-1 and Rtx-35 (Figures 6.9 and 6.10). It is this subset of compounds which
showed mass spectra most easily distinguished from the other subsets and from 2,3- and
3,4-MDMA.
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205
Abundance
Time (minutes)
Figure 6.5: Capillary gas chromatographic separation of PFPA derivatives of
compounds 1-4, 2,3-MDMA and 3,4-MDMA. Columns used: A RTX-1;
B RTX-35.
15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.500
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
Time-->
AbundanceTIC: 13106-6.D
1
3
2 4
11
12
15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
Time-->
AbundanceTIC: 21306-2.D
1
3
2 4
11
12
A
B
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
206
Abundance
Time (minutes)
Figure 6.6: Capillary gas chromatographic separation of HFBA derivatives of
compounds 1-4, 2,3-MDMA and 3,4-MDMA. Columns used: A RTX-
1; B RTX-35.
14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.000
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
1700000
Time-->
AbundanceTIC: 20106-9.D
1
3
2 11
12
16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.500
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
Time-->
AbundanceTIC: 21306-7.D
1
3 2 4
11
12
A
B
4
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
207
Abundance
Time (minutes)
Figure 6.7: Capillary gas chromatographic separation of PFPA derivatives of
compounds 5-8, 2,3-MDMA and 3,4-MDMA. Columns used: A
RTX-1; B RTX-35.
14.00 15.00 16.00 17.00 18.00 19.00 20.000
200000
400000
600000
800000
1000000
Time-->
AbundanceTIC: 13106-9.D
6
8
5 1
12
6
7
8 + 11
5
A
B
15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
Time-->
AbundanceTIC: 21306-3.D
7
11
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
208
Abundance
Time (minutes) Figure 6.8: Capillary gas chromatographic separation of HFBA derivatives of
compounds 5-8, 2,3-MDMA and 3,4-MDMA. Columns used: A
RTX-1; B RTX-35.
6
11 7
5
12
17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.500
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
Time-->
AbundanceTIC: 21306-6.D
6
7
8+11
5
12
16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.000
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
750000
Time-->
AbundanceTIC: 20106-12.D
A
B
8
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209
Abundance
.
Time (minutes)
Figure 6.9: Capillary gas chromatographic separation of PFPA derivatives of
compounds 9-10, 2,3-MDMA (11) and 3,4-MDMA(12) . Columns
used: A RTX-1; B RTX-35.
15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.500
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
3200000
3400000
Time-->
AbundanceTIC: 13106-10.D
9
10
12
17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00 22.500
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
Time-->
AbundanceTIC: 21306-5.D
11
9
10
12
A
B
11
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
210
Abundance
Time (minutes)
Figure 6.10: Capillary gas chromatographic separation of HFBA derivatives of
compounds 9-10, 2,3-MDMA(11) and 3,4-MDMA(12). Columns
used: A RTX-1; B RTX-35.
16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.500
100000
200000
300000
400000
500000
600000
700000
800000
900000
Time-->
AbundanceTIC: 20106-16.D
11
9
10
12
16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.500
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
Time-->
AbundanceTIC: 21306-8.D
11
9
10
12
A
B
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211
Conclusions of Chapter 6
The methoxy methyl methamphetamines have an isobaric relationship to the 2,3-
and 3,4-methylenedioxymethamphetamines and represent a unique analytical challenge
in forensic drug chemistry. Each of these compounds has a molecular weight of 193 and
yields a base peak for the N-methyl imine of acetaldehyde at m/z 58 in the mass
spectrum. This ion represents the loss of 135 mass units from the molecular ion
corresponding to the substituted benzyl species, C8H7O2 (methylenedioxybenzyl) and
C9H11O (methoxy methyl benzyl). Thus, the traditional electron impact mass spectrum
provides little structural information for differentiating among these 12 compounds.
Because of the unique similarity of these compounds by MS, the specific identification of
a compound such as 3,4-MDMA requires methods to eliminate the other isomers. The
ring-substituted methoxy methyl methamphetamines were evaluated for their
chromatographic and mass spectral properties compared to 2,3- and 3,4-MDMA. The
mass spectra studies showed almost identical mass spectra for all 10 methoxy methyl
methamphetamines and 2,3- and 3,4-MDMA.
The perfluoroacylated derivatives of the 10 methoxy methyl methamphetamines
were prepared and evaluated for their ability to individualize the mass spectra of these
compounds and to maintain or improve chromatographic resolution. The
perfluoroacylation process did not produce unique mass spectra characteristic of each
compound. However, derivatization did subdivide the methoxy methyl
methamphetamines into subgroups based on the position of the methoxy group on the
aromatic ring relative to the alkylamine side-chain. The presence of the m/z 105 ion
suggests the methoxy group is in the 2-position relative to the alkylamine side-chain, and
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212
the significant abundance of the low mass ions at m/z 135 and m/z 162 indicates the
methoxy group is substituted at the 4-position of the aromatic ring. The 3-substituted
aromatic ring methoxy group isomers did not show any unique fragments to distinguish
them from 2,3- and 3,4-MDMA as the perfluoroacylated derivatives.
Different stationary phases and temperature programs were used in an effort to
separate the methoxy methyl methamphetamines from 2,3- and 3,4-MDMA. The best
resolution among the evaluated columns was achieved on Rtx-200, where a mixture of
the 12 compounds gave 11 peaks and only the PFPA derivatives of methoxy methyl
methamphetamines 7 and 9 coelute. The perfluoroacyl derivatives of the methoxy methyl
methamphetamines were divided into three subsets based on the ring substitution pattern
of the methoxy group. The derivatives of the individual subsets were each resolved on
Rtx-1.
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213
Chapter 7
GC–MS Analysis of Ring and Side Chain Regioisomers of
Ethoxyphenethylamines
The target compounds of this chapter are a series of ring substituted ethoxy
phenethylamines with molecular weight 193 and the potential to produce mass spectra
with major fragment ions at m/z 58 for the imine and m/z 135/136 for the substituted
benzyl fragment. The major fragment ions observed in the EI mass spectrum for 3,4-
MDMA (MW = 193) occur at equivalent masses.
Mass spectrometry of Ring and Side Chain Regioisomers of Ethoxyphenethylamines
MS is the primary method for confirming the identity of drugs and related
substances in forensic samples. The mass spectra of phenethylamines are characterized
by a base peak formed from an amine initiated alpha-cleavage reaction involving the
carbon–carbon bond of the ethyl linkage between the aromatic ring and the amine. In 3,4-
methylenedioxymethamphetamine (MW = 193) the alpha-cleavage reaction yields the
substituted imine fragment at m/z 58 and the 3,4-methylenedioxybenzyl fragment at mass
135/136 (for the cation and the radical cation, respectively). Thus, the mass spectrum for
3,4-methylenedioxymethamphetamine contains major ions at m/z 58 and 135/136 as well
as other ions of low relative abundance. The ring substituted ethoxy methamphetamines
(Compounds 1–3, Figure 7.1) have the potential to yield a mass spectrum essentially
equivalent to 2,3- and 3,4-MDMA. There are five possible side chain regioisomers for
each one of these ring substitution patterns.
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214
The five side chain regioisomers of 2-ethoxyphenethylamine included in this study are
shown in Figure 7.1 (Compounds 1, 4–7). All five side chain regioisomers have
molecular weight of 193 and major fragment ions in their electron ionization mass
spectra at m/z 58 and 135/136 (Scheme 7.1). The individual mass spectra for 2,3- and
3,4-MDMA are also presented in Scheme 7.1 (Compounds 8 and 9). The isobaric ethoxy
benzyl (C9H11O)+ fragments have the same mass as the methylenedioxybenzyl (C8H7O2)+
cation occurring at m/z 135. Furthermore the m/z 58 ion in the 2-ethoxyphenethylamines
is regioisomeric with that obtained in the mass spectra of both 2,3 and 3,4-MDMA
(Figure 7.2).
HC
N
CH3
CH3
m/z = 135
m/z = 58
MW = 193
H2C
CH2
CH
CH3
HN
CH3
O
O
H2C
O
O
CH2
EI 70ev
H
C N
C2H8
CN
C2H8
m/z = 135
OC2H5
OC2H5
Scheme 7.1: EI fragmentation pattern for ring substituted ethoxy phenethylamines. Figure 7.1: Structures of the ring substituted ethoxymethamphetamines, side chain regioisomerics of 2-ethoxyphenethylmethamphetamines, and 2,3- and 3,4-MDMA.
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215
HN
OC2H5 1
HN
OC2H5 2
HN
C2H5O
3
N
OC2H5
4
HN
OC2H5 5
NH2
OC2H5
6
NH2
OC2H5 7
HN
O
O
8
O
O
HN
7 (3,4-MDMA)
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216
This lack of mass spectral specificity for the isomers shown in Figure 7.2, in addition to
the possibility of chromatographic co-elution with 3,4-MDMA, could result in
misidentification in this series of drugs and drug-like substances.
Figure 7.2: Mass Spectra of compounds ring substituted ethoxy Phenethylamines,
2,3- and 3,4-MDMA. HN
OC2H5 1
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
m/z-->
Abundance
Average of 11.377 to 11.593 min.: 092607-01.D\data.ms58.1
77.1 91.1 107.142.1 135.1 178.1119.1 149.1 192.1163.1
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217
HN
OC2H5 2
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
750000
800000
850000
m/z-->
Abundance
Average of 12.170 to 12.298 min.: 092607-02.D\data.ms58.1
77.0107.142.1 91.0 135.1
178.1120.1 148.1 192.2162.1
HN
C2H5O 3
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
5000
10000
15000
20000
25000
30000
35000
m/z-->
Abundance
Average of 12.555 to 12.706 min.: 092607-03.D\data.ms58.1
107.044.077.0 135.091.0 121.0 148.0 178.1162.1 191.1
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
218
N
OC2H5 4
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
m/z-->
Abundance
Average of 11.097 to 11.231 min.: 080907-01.D\data.ms58.1
91.142.1 77.1121.1107.1 193.1133.1 146.1 162.1 178.1
HN
OC2H5 5
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
m/z-->
Abundance
Average of 11.972 to 12.182 min.: 080907-02.D\data.ms58.1
136.191.177.0107.1 121.142.1 193.2149.1 178.1163.1
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
219
NH2
OC2H5 6
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
750000
800000
850000
900000
950000
1000000
1050000
1100000
1150000
1200000
1250000
1300000
1350000
1400000
1450000
1500000
1550000
m/z-->
Abundance
Average of 11.098 to 11.232 min.: 080907-04.D\data.ms58.1
77.091.042.1 107.1
178.1135.1149.1119.0 161.1 193.1
NH2
OC2H5 7
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
50000
100000
150000
200000
250000
300000
350000
400000
450000
m/z-->
Abundance
Average of 12.257 to 12.403 min.: 080907-05.D\data.ms58.1
91.077.0 136.141.1 107.1164.1121.0 150.1 193.1178.1
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
220
HN
O
O
8
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
m/z-->
Abundance
Average of 12.444 to 12.555 min.: 080907-06.D\data.ms58.1
77.1 135.042.1 88.6 105.1 178.1 192.1118.1 163.1147.1
O
O
HN
9
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
m/z-->
Abundance
Average of 13.027 to 13.167 min.: 080907-07.D\data.ms58.1
135.077.042.1 88.6 105.0 178.1148.1 193.1163.1120.1
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
221
In the next phase of this study, various Acylated derivatives of the ring substituted
ethoxy methamphetamines and the primary and secondary amines of the side chain
regioisomers of 2-ethoxyphenethylamine were prepared and evaluated in an effort to
individualize their mass spectra and provide unique marker ions for specific
identification. Acylation of the amines significantly lowers the basicity of nitrogen and
can allow other fragmentation pathways to play a more prominent role in the resulting
mass spectrum. Of course the tertiary amine (compound 4) does not form a stable amide
derivative. The mass spectra for the pentafluoropropionyl and heptafluorobutyryl amides
are shown in Figures 7.3 and 7.4, respectively. Perfluroacyl derivatives of the ring
substituted ethoxy methamphetamines (Compounds 1–3) have almost identical mass
spectra with the derivatized 2,3- and 3,4-MDMA (Compounds 8 and 9) except for a
unique ion at m/z 107. This ion at m/z 107 represents the loss of 28 mass units (ethylene,
C2H4) fromthe ethoxybenzyl cation at m/z 135 (Scheme 7.2). Although the relative
abundance varies significantly, them/z 107 ion is present in all mass spectra of the PFPA
and HFPA of compounds (1–3 and 5–7) and offers a unique fragment ion to discriminate
these compounds from 3,4- and 2,3-MDMA. The m/z 107 ion is also present in relatively
low abundance in the mass spectra of the underivatized ethoxy amines shown in Figure
7.2.
Figure 7.3: Mass Spectra of the HFBA derivatives of ring substituted ethoxy
Phenethylamines, 2,3- and 3,4-MDMA.
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222
N
OC2H5
C3F7
O
1
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
1700000
1800000
1900000
2000000
2100000
2200000
2300000
2400000
m/z-->
Abundance
Average of 14.787 to 14.892 min.: 092607-22.D\data.ms254.1
162.1
210.0
135.1
91.1
42.1
389.1342.1309.0283.1
N C3F7
O
OC2H5 2
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
m/z-->
Abundance
Average of 15.819 to 15.900 min.: 092607-23.D\data.ms254.0
162.1
210.0
42.1135.1107.177.1
389.1350.1309.0282.0
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223
N C3F7
O
C2H5O 3
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
m/z-->
Abundance
Average of 16.518 to 16.629 min.: 092607-24.D\data.ms162.1
254.0
135.1
107.1
210.0
42.1 77.0
389.1319.0 360.1291.1
N C3F7
OC2H5
O
5
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
750000
800000
850000
900000
950000
m/z-->
Abundance
Average of 15.591 to 15.702 min.: 080707-6.D\data.ms148.1
226.091.1
254.1
120.1389.1
56.1 178.0342.1295.1
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224
NH
OC2H5
C3F7
O
6
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
m/z-->
Abundance
Average of 13.878 to 13.965 min.: 080707-6.D\data.ms254.1
136.1
59.1 107.1176.1
214.0
389.1
350.1302.0
NH
OC2H5
C3F7
O
7
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
m/z-->
Abundance
Average of 14.221 to 14.315 min.: 080707-6.D\data.ms135.1
176.1
107.1
254.1
77.1
41.1
389.1226.0
360.1324.1285.0
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225
N
O
O C3F7
O
8
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
m/z-->
Abundance
Average of 16.058 to 16.168 min.: 092607-25.D\data.ms254.0
162.1
210.0
135.042.1 389.177.0
105.0
350.0308.9282.9
O
O
N C3F7
O
9
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
m/z-->
Abundance
Average of 17.264 to 17.410 min.: 092607-26.D\data.ms254.0
162.1
135.0
210.0
77.042.1
389.1105.0
318.9 350.1292.9
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226
Figure 7.4: Mass Spectra of the PFPA derivatives of the ring substituted ethoxy
Phenethylamines, 2,3- and 3,4-MDMA.
N
OC2H5
C2F5
O
1
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
3200000
m/z-->
Abundance
Average of 14.286 to 14.519 min.: 092607-27.D\data.ms204.1
162.1
135.1
91.142.1
339.165.0 294.1233.0113.1 258.9
N C2F5
O
OC2H5 2
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
m/z-->
Abundance
Average of 15.259 to 15.446 min.: 092607-28.D\data.ms204.0
162.1
119.042.1 77.0339.1
300.1254.0 278.0232.0
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227
N C2F5
O
C2H5O 3
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
m/z-->
Abundance
Average of 15.912 to 15.993 min.: 092607-29.D\data.ms162.1 204.0
135.1107.0
42.1 77.0
339.1268.9 309.9240.9
N C2F5
OC2H5
O
5
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
140000
150000
m/z-->
Abundance
Average of 15.032 to 15.125 min.: 080708-14.D\data.ms148.1
91.1
176.0
204.0
119.0
339.156.0
292.1244.9
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228
NH
OC2H5
C2F5
O
6
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
50000
100000
150000
200000
250000
300000
350000
400000
450000
m/z-->
Abundance
Average of 13.394 to 13.476 min.: 080708-14.D\data.ms204.0
136.1
59.1 107.1176.1
339.184.0 282.1245.0 311.0
NH
OC2H5
C2F5
O
7
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
m/z-->
Abundance
Average of 13.726 to 13.802 min.: 080708-14.D\data.ms135.1
176.1
107.1
204.077.041.1
339.1310.1274.0250.1
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229
N
O
O C2F5
O
8
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
100000
200000
300000
400000
500000
600000
700000
800000
900000
m/z-->
Abundance
Average of 15.463 to 15.580 min.: 092607-30.D\data.ms204.0
162.1
119.042.1 339.177.0
262.9 300.1240.9140.7
O
O
N C2F5
O
9
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
m/z-->
Abundance
Average of 16.646 to 16.804 min.: 092607-31.D\data.ms204.0
162.1
135.0
77.042.1 339.1
105.0268.9 300.1242.9
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230
OH2C
H3C
O HO
CH2
CH2
H2C
H
CH2CH2
OH2C
H3C
CH2R
HO
CH2
R
HO
CH2
and / or
Scheme 7.2: mass spectral fragmentation of ethoxyphenethylamines yielding cation
at m/z 107
These spectra in Figures 7.3 and 7.4 show a common peak at m/z 204 and 254,
which corresponds to the loss of 135 mass units from the molecular ions at 339 and 389
for the PFPA and HFBA amides. This ion at m/z 204 and 254 is the PFPA and HFBA
imine species formed from the alpha cleavage of the amide nitrogen to eliminate the
ethoxy benzyl radical or themethylenedioxybenzyl radical. Thus the m/z 204 and 254
ions in the PFPA and HFBA amides are analogous to m/z 58 in the underivatized species
because all these ions represent the (M-135)+ species. The general fragmentation pattern
and structures for the m/z 204 and 254 ions are shown in Scheme7.3. The ethoxy benzyl
cation (m/z 135) and the methylenedioxybenzyl cation (m/z 135) are fragments common
to all spectra in Figures 7.3 and 7.4. However, the ethoxy benzyl cation in the
perfluoroacyl derivatives shows a very high relative abundance in some of these isobaric
compounds.
Indeed them/z 135 ion is the base peak in the PFPA andHFBA derivatives of Compound
7. This would suggest that the perfluoroacyl derivatives offer some level of
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231
discrimination between the methylenedioxy and ethoxy ring substitution patterns based
on the difference in relative abundances of the substituted benzyl cation at m/z 135.
N
R1 R2 X
R3
O
R1+R2+R3 = C2H7 C2F5= X,, MW = 339 C3F7= X, MW= 389
N
R1
R2
R3
X
O
m/z = 204, X= C2F5m/z = 254, X= C3F7
OC2H5
R1
R2
OC2H5
XCNH3C
m/z = 160 (X = C2F5)m/z = 210 (X = C3F7)Cpds 1,2,3,8 and 9m/z = 148, R1=R2=H,cpd 5
m/z = 162, R1= CH3; R2=H,cpds 1,2,3,8 and 9m/z = 176, R1= CH3; R2= CH3, cpd 6m/z = 176,R1= C2H5; R2= H, cpd 7 Scheme7.3: Mass spectral fragmentation products for the acylated ethoxy
phenethylamines.
The decreased role for the alpha cleavage reaction in the fragmentation of these
amides allows the formation of ions more diagnostic of individual side chain isomers.
Acylation weakens the bond between nitrogen and the alkyl carbon of the phenethyl side
chain, allowing the formation of charged hydrocarbon species of increased relative
abundance. These hydrocarbons of varying mass significantly individualize the mass
spectra and provide specific structural information. The mass spectra in Figures 7.3 and
7.4 illustrate the role of hydrocarbon fragments at m/z 148, 162, and 176 in the electron
impact mass spectral discrimination among the side chain regioisomers. The spectra for
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232
the N-ethyl isomer (compound 5) in Figures 7.3-5 and 7.4-5 show a base peak at m/z 148
corresponding to the alkene radical cation which occurs from hydrogen rearrangement
and subsequent fragmentation of the alkyl carbon to nitrogen bond of the phenethylamine
side chain (see Scheme 7.3). This ion at m/z 148 would only occur for the N-ethyl
regioisomer. The spectra in Figures 7.3-1, 7.3-2, 7.3-3, 7.3-4,7.3-9, 7.4-1, 7.4-2, 7.4-3,
7.4-4 and 7.4-9 show the substituted phenylpropane hydrocarbon ion at m/z 162,
identifying these molecules as the
PFPA and HFBA derivatives of 2-, 3-, 4-ethoxymethamphetamines and the 2,3-, 3,4-
methylenedioxymethamphetamines, respectively. However, the base peak of m/z 162 and
relatively high abundance m/z 107 in the spectra of the perfluroacyl derivatives of
compound 3 offer a significant discrimination of 4-ethoxymethamphetamines over the 2-,
3-ethoxymethamphetamines and the two isobaric methylenedioxymethamphetamines
isomers (Compounds 1, 2, 8, and 9 in Figures 7.3 and 7.4). The spectra for the PFPA and
HFBA derivatives of the primary amines (compounds 6 and 7 in Figure 7.3 and 7.4)
show ions at m/z 176 from the corresponding substituted phenylbutene radical cation.
The lower abundance of m/z 176 for the 2-ethoxy phentermine (Compound 6) may be
attributed to steric inhibition of hydrogen transfer in the alpha, alpha-dimethyl
substitution pattern.
While the alkene ions at 148, 162, and 176 help to identify the side chain
regioisomers, one complicating factor in the PFPA derivatives for the N-
ethylphenethylamines [Figure 7.4-5] is the appearance of an ion at m/z 176 in addition to
the base peak at m/z 148. The 176 ion might suggest a four carbon chain directly attached
to the aromatic ring as occurs for the alpha-ethyl- (Compound 7) and alpha, alpha-
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233
dimethyl- (Compound 6) phenethylamines [Figures 7.3-6, 7.3-7, and 7.4-6, 7.4-7]. The
m/z 176 ion in the spectra for the PFPA derivatives of the N-ethyl regioisomers [Figure
7.4-5] is a rearrangement of the m/z 204 ion resulting in the loss of mass 28 (the N-ethyl
group) via hydrogen transfer. This coincidental common mass from two different
fragmentation pathways is confirmed by examining the mass spectra for the HFBA
derivatives of the N-ethyl-phenethylamines shown in Figure 7.3-5. The loss of 28 mass
units from the acylimine fragment at m/z 254 yields the equivalent fragment ion atm/z
226. Thus, the HFBA derivatives may offer more characteristic ions for individualization
of these regioisomeric substances.
A comparison of the mass spectra for the PFPA and HFBA derivatives of all ring
substituted methamphetamines (Compounds 1, 2, 3, 8, and 9) indicates unique ions at m/z
160 and m/z 210 [see Figures 7.3-1, 7.3-2, 7.3-3, 7.3-8, 7.3-9, 7.4-1, 7.4-2, 7.4-3, 7.4-8
and 7.4-9]. This mass difference of 50 (CF2) suggests these ions contain the
perfluoroalkyl group for each derivative, C2F5 and C3F7, respectively. An evaluation of
the masses of the components which make up the fragment at m/z 160 for example
include C2F5 (119 mass units) and CH3 (15 mass units).
Previous deuterium labeling studies have confirmed that methyl group on nitrogen is a
part of this resulting fragment (Chapter 1). The remaining mass 26 would correspond to
CN and the proposed structures of m/z 160 and 210 are shown in Scheme 7.3. An
equivalent fragmentation pathway has been reported for methamphetamine and further
supported by the analysis of the mass spectra of the PFPA and HFBA derivatives of d3-
and d5-MDMA in a previous study (Chapter1).
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234
Gas Chromatography of Ring substituted Ethoxy Phenethylamines, 2,3- and 3,4-
MDMA.
The PFPA and HFBA derivatives of the ring substituted ethoxy
methamphetamines and the primary and secondary side chain regioisomers of the 2-
ethoxyphenethylamine were compared on the relatively non polar 100% dimethyl
polysiloxane (Rtx-1).
Four physical mixtures were prepared, two containing the HFBA and the PFPA
derivatives of 2-, 3-, and 4-ethoxymethamphetamine along with 2,3- and 3,4 MDMA,
while the other two contained the side chain regioisomers of 2-ethoxyphenethylamine
(Compounds 1, 5, 6 and 7) with the isobaric 2,3- and 3,4-MDMAs. Several temperature
programs were evaluated and one program showing the best compromise between
resolution and analysis time was used to generate the retention data in Table I and the
chromatograms in Figures 7.5 and 7.6. Chromatograms shows that 3,4- MDMA has the
highest affinity for the stationary phase among this set of compounds both in the free
amine form and the two amide forms investigated in this study.
The chromatograms in Figure 7.5 show the separation of the PFPA (Figure 7.5A)
and HFBA (Figure 7.5B) derivatives of the five compounds having the methamphetamine
side chain. This common side chain allows for a comparison of relative stationary phase
affinity as a function of ring substituents in this set of compounds. The 2-ethoxy
substitution pattern shows the least retention and compound 1 has the lowest retention
time. The 3-ethoxymethamphetamine (Compound 2) elutes second followed by 2,3-
MDMA (Compound 8) then the 4-ethoxy substituent compound 3. The compound
showing the highest retention time and eluting last is 3,4-MDMA (Compound 9). Among
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235
the ethoxy substituents the 2-isomer shows the least retention followed by the 3-isomer,
and the 4-ethoxy isomer has the greatest retention on the Rtx-1 phase. The elution order
is the same for both the PFPA and HFBA derivatives with the HFBA derivatives having
slightly greater retention under identical chromatographic conditions.
The chromatograms in Figure 7.6 show the separation of the side chain regioisomers as
the ring substitution pattern is held constant (2-ethoxy). The side chain elution order is
secondary amides (Compounds 6 and 7) before the tertiary amides (Compounds 1 and 5)
and in this limited set of examples branched isomers elute before the more linear ones.
Therefore, the amides of compound 6 elute first followed by the amide of compound 7
(both secondary amides), then the amides of compound 1 (the methamphetamine side
chain) and finally the amides of compound 5 (the more linear of the tertiary amides).
The methylenedioxy isomers (Compounds 8 and 9) elute later than any of the side chain
isomers of the 2-ethoxy ring substitution pattern. Thus, these chromatographic results
coupled with the mass spectral data allow for the individualization of each member of
this series. The derivatized side chain isomers separated in Figure 7.6 can be
differentiated by unique fragment ions in their mass spectra in addition to the well
resolved chromatographic results. The compounds having a common side chain are well
resolved in Figure 7.5 and them/z 107 fragment ion allows for the identification of those
compounds having the ethoxy ring substituent.
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236
1
2
8
3
9
1
3
8
9
2
A
B
11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
Time-->
Abundance
TIC: 070927-08.D\data.ms
11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
Time-->
Abundance
TIC: 070927-07.D\data.ms
Figure 7.5: GC Separation of the PFPA (A) and HFBA (B) derivatives of
compounds 1-3,8 and 9; Rtx-1 column
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237
6
7 1
5
8
9
6
7
1 5
8 9
10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
Time-->
Abundance
TIC: 080708-14.D\data.ms
10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
6500000
7000000
7500000
8000000
8500000
Time-->
Abundance
TIC: 080707-6.D\data.ms
Figure 7.6: GC Separation of the PFPA (A) and HFBA (B) derivatives of
compounds 1, 5,6,7,8 and 9; Rtx-1 column
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238
Conclusions of Chapter 7
3,4-MDMA, 2,3-MDMA, and the three ring substituted ethoxy
methamphetamines are a unique subset of regioisomeric and isobaric molecules; each
compound has a molecular weight of 193 and yields a base peak at m/z 58 in the mass
spectrum from the loss of the corresponding methylenedioxybenzyl and ring substituted
ethoxybenzyl groups, respectively. The traditional electron impact mass spectrum
provides little structural information for differentiating among these seven compounds.
Derivatization with acylating agents yields amides with improved resolution compared to
the underivatized amines by capillary gas chromatography on the Rtx-1 stationary phase.
Additionally, the perfluoroacyl derivatives significantly individualize the mass
spectra for these amides and allow for specific identification. The ring substituted ethoxy
methamphetamines are characterized from 2,3- and 3,4-MDMA by the presence of m/z
107. Side chain regioisomers yield unique hydrocarbon fragment ions at m/z 148, 162
and 176.
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239
Chapter 8
GC-MS Studies on Direct Side Chain Regioisomers Related to
Substituted Methylenedioxyphenethylamines; MDEA, MDMMA and
MBDB
There are an additional seven direct side chain regioisomeric phenethylamines
related to the controlled substances MDEA, MDMMA, and MBDB. The structures of this
set of compounds are shown in Figure 8.1.
A previous report from our research group included the synthesis and mass spectral
evaluation of these ten regioisomeric compounds. The mass spectra of the underivatized
compounds provided very little structural information for the specific differentiation
among these regioisomers even though these regioisomers were separated by capillary
gas chromatography.
In this chapter, derivatization of the primary and seconday amines was carried out in
an effort to obtain more specific ions that would help discriminating among the members
of this set of compounds.
Mass Spectral Studies
Mass spectrometry is the primary method for confirming the identity of drugs in
forensic samples. Figure 8.2 shows representative electron impact mass spectra of the
three drugs of abuse; MDEA, MBDB and MDMMA, involved in the study (Compounds
6, 7, 9). The MS spectra of all ten underivatized regioisomers were previously reported.
The mass spectra of the ten regioisomeric compounds (MW=207) are characterized by a
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240
base peak at m/z 72 formed by α-cleavage reaction involving the carbon–carbon bond of
the ethyl linkage between the aromatic ring and amine nitrogen.
Figure 8.1: Structures of the side chain regioisomeric phenethylamines related to
MDEA, MDMMA, and MBDB.
O
O
NH2
1
1-(3,4-Methylenedioxyphenyl)-2-
pentanamine
O
O
NH2
2
1-(3,4-Methylenedioxyphenyl)-3-
methyl-2-butanamine
O
O
NH2
3
1-(3,4-Methylenedioxyphenyl)-2-methyl-2-butanamine
O
O
NH
5 N-propyl -1-(3,4-
Methylenedioxyphenyl)-2-ethanamine
O
O
NH
5 N-isopropyl-1-(3,4-
Methylenedioxyphenyl) -2-ehanamine
O
O
NH
6
MDEA 3,4-Methylenedioxy-N-
ethylamphetamine
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241
O
O
NH
7
MBDB
N-methyl-1-(3,4-Methylenedioxyphenyl)-2-butanamine
O
O
HN
8
N-methyl-3,4-methylenedioxyphentermine
O
O
N
9
MDMMA 3,4-Methylenedioxy-N,N-
dimethylamphetamine
O
O
N
10
N-ethyl-N-methyl-3,4-
methylenedioxyphenyl)-2-ethanamine
Other less abundant peaks were observed at m/z 135/136 from the 3,4-
methylenedioxybenzyl cation and radical cation fragments, respectively as well as other
ions of low relative abundance. The EI mass spectra of these regioisomers show some
variation in the relative intensity of the major ions with only one or two minor ions that
might be considered side-chain specific fragments. Thus, the ultimate identification of
any one of these amines with the elimination of the other nine regioisomeric substances
can not depend on their mass spectra alone.
This lack of mass spectral specificity in addition to the possibility of
chromatographic co-elution with any of the drugs of abuse; MDEA, MBDB and
MDMMA could result in misidentification of the target drug. Furthermore, the lack of
available reference samples for the seven regioisomeric 3,4-methylenedioxy-
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242
phenethylamines compounds complicates the individual identification of any one of these
substances. This constitutes a significant analytical challenge, where the specific
identification by GC–MS must be based primarily upon the ability of the
chromatographic system to separate the regioisomeric substance from the actual drug of
interest. Additionally, the ability to distinguish between these regioisomers directly
enhances the specificity of the analysis for the target drugs of interest.
Figure 8.2: EI Mass Spectra of MDEA (compound 6) MBDB (compound 7) and
MDMMA (compound 9).
O
O
NH
6
30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 2100
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
m/z-->
Abundance
Scan 1404 (12.270 min): 080506-27.D\data.ms72.1
44.1
135.1
105.1163.1 192.191.1 148.1 206.2121.157.1 176.1
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243
O
O
NH
7
30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
1700000
1800000
m/z-->
Abundance
Average of 12.888 to 12.964 min.: 080506-28.D\data.ms72.1
135.1
57.1 88.742.1 178.1105.1 120.1 148.1 163.1 206.2191.2
O
O
N
9
30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
3200000
3400000
3600000
3800000
4000000
4200000
4400000
m/z-->
Abundance
Scan 659 (7.678 min): 090223-01.D\data.ms72.1
135.042.1 56.1
105.091.1 147.1 163.1 192.1121.0 177.1 204.1
Various perfluoroacyl derivatives of the regioisomeric primary and secondary
amines (Compounds 1-8) were prepared and evaluated in an effort to individualize their
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244
mass spectra and maintain or improve chromatographic resolution. Acylation of the
amines significantly lowers the basicity of nitrogen and can allow other fragmentation
pathways to play a more prominent role in the mass spectrum. The trifluoroacetyl,
pentafluoropropionyl and heptafluorobutryl derivatives of compounds 1-8 were evaluated
for their ability to individualize the mass spectra through the formation of specific
fragments. Compounds 9 and 10 are tertiary amines and do not form a stable amide
derivative.
The mass spectra for the eight pentaflouropropionyl (PFPA) amides are shown in
Figure 8.3. The spectra for the PFPA derivatives are representative of all the
perfluoroacyl amides in this study. From these spectra, a common peak occurs at m/z 218
which corresponds to the loss of 135 mass units from the molecular ion at 353. This ion is
the PFPA imine species, likely formed from the α-cleavage of the amide nitrogen to
eliminate the 3,4-methylenedioxybenzyl fragment at m/z 135. Thus this ion is analogous
to m/z 72 in the underivatized species because it represents the (M–135)+ species.
The decreased role for the α-cleavage fragmentation of these amides allows the
formation of more diagnostic ions for each individual isomer. Acylation, and in particular
the perflouroacylation, weakens the bond between nitrogen and the α-carbon of the
substituted phenethyl group, allowing the formation of charged hydrocarbon species of
increased relative abundance. These alkenes (radical cations) of varying mass are formed
due to the transfer of a benzylic hydrogen to the ionized carbonyl oxygen followed by the
loss of a neutral amide species. The resulting alkene radical cations (3,4-
methylenedioxyphenylalkenes) significantly individualize the mass spectra and provide
specific structural information. The mass spectra in Scheme 8.1 illustrate the role of the
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245
alkene fragments at m/z 148, 162, 176 and 190 in identification of these regioisomers.
These ions identify the number of carbons in the hydrocarbon chain attached directly to
the aromatic ring in an uninterrupted manner.
Figure 8.3: Mass spectra of the PFPA derivatives of compounds 1-8.
O
O
HN C2F5
O
1
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
m/z-->
Abundance
Average of 8.255 to 8.302 min.: 090219-09.D\data.ms135.1
190.1
353.177.1 161.1
55.1105.0 218.1
310.1280.1252.1
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246
O
O
HN C2F5
O2
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
m/z-->
Abundance
Average of 8.185 to 8.214 min.: 090219-10.D\data.ms135.1
190.1
353.1
77.155.1218.1
164.0105.1310.1280.1252.1
O
O
NH C2F5
O3
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
m/z-->
Abundance
Average of 8.179 to 8.214 min.: 090219-11.D\data.ms218.1
135.0
55.1
164.0 190.1 353.177.1
105.0 324.1296.1241.1 269.0
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247
O
O
N
O C2F5
4
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
6500000
m/z-->
Abundance
Average of 8.593 to 8.634 min.: 090219-12.D\data.ms148.1
176.0353.1
77.1 119.043.1218.1
244.1 324.1294.1266.1
O
O
N
O C2F5
5
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340
500000
1000000
1500000
2000000
2500000
3000000
3500000
m/z-->
Abundance
Average of 8.511 to 8.535 min.: 090219-13.D\data.ms148.1
176.0
353.143.1 77.1 119.0
218.1 244.1 309.1280.1
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248
O
O
N
O C2F5
6
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
6500000
m/z-->
Abundance
Average of 8.360 to 8.406 min.: 090219-14.D\data.ms218.1
162.1
190.0
135.1
77.1353.1
105.151.0
309.1273.1243.1
O
O
N
C2F5O
7
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
m/z-->
Abundance
Average of 8.436 to 8.476 min.: 090219-15.D\data.ms218.1
176.1
135.0
42.177.1 353.1
105.1324.1269.1 293.0244.1
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249
O
O
N
C2F5O
8
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
m/z-->
Abundance
Average of 8.389 to 8.407 min.: 090219-16.D\data.ms218.1
160.0
135.0
56.1
353.1103.179.1 190.1 282.1 310.1251.9
Further examination of the mass spectra of the PFPA derivatives for the eight
regioisomers (Figure 8.3) indicates unique ions at m/z 160 for both compounds 7 and 8.
An analysis of the masses of the components, which make up the fragment at m/ z 160
include for example C2F5 (119 mass units) and CH3 (15 mass units), leaving only a mass
of 26 available for the total of 160. The mass 26 would correspond to CN and the
proposed mechanism for the formation of (C2F5CNCH3)+ is shown in Scheme 8.2. The
analogous ion occurs at m/z 110 and m/z 210 for the TFA and HFBA derivatives,
respectively. An equivalent fragmentation pathway has been previously reported and this
fragment is characteristic of N-methyl substituted phenethylamine such as compound 7
and 8.
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250
O
O
CH
H
C
R1
N
R2
R3
X
CO
O
O
HC
C
R1
N
R2
R3
X
CHO
O
O
HC
C
R1
R2
R1=H, R2= C3H7, m/z=190, cpd 1R1=H, R2= CH(CH3)2, m/z=190, cpd 2R1=CH3, R2= C2H5, m/z= 190, cpd 3R1=H, R2=H, m/z=148, cpds 4 and 5R1=H, R2= CH3, m/z=162, cpd 6R1=H, R2= C2H5, m/z= 176, cpd 7R1=CH3, R2= CH3, m/z= 176, cpd 8
X= CF3, TFA C2F5, PFPA C3F7, HFBA
Scheme 8.1: Mechanism of the formation of the alkene radical cation in the perfluoroacyl derivatives of the primary and secondary regioisomeric amines
N
R1
CH3
m/z = 168, X = CF3
= 218, X = C2F5
=268, X = C3F7
X
O
N
O
X
CH3
R1N C XH3C
m/z = 110, X = CF3
= 160, X = C2F5
= 210, X = C3F7
N X
CH3
OR1
O
OR2
R1 = H, R2 = C2H5, cpd 7R1 = CH3, R2 = CH3, cpd 8X = CF3, TFA C2F5, PFPA
C3F7, HFBA
R2
R2
Scheme 8.2: Formation of m/z 110, 160, 210 for compounds 7 and 8
An important fragmentation pathway characteristic to the PFPA derivative of
compound 6 (the only N-ethyl compound in this limited series) and compounds 4, 5,
which are N-propyl and isopropyl amines is illustrated in Scheme 8.3. This results in the
formation of fragment ions at m/z 190 (Compound 6) or m/z 176 (Compounds 4, 5) for
their PFPA amides. These ions came from the imine fragment at m/z 218 through
hydrogen rearrangement and subsequent cleavage of the N-alkyl group on the nitrogen.
This occurs only with the N-alkyl group of ethyl or larger.
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251
N
H3C
CH2
m/z = 168, X = CF3 = 218, X = C2F5 =268, X = C3F7
X
O
Compound 6 m/z = 140, X = CF3 = 190, X = C2F5 = 240, X = C3F7Compound 4, 5 m/z = 126, X = CF3 = 176, X = C2F5 = 226, X = C3F7
N X
CH2
OCH3
O
O
R1=H, R2=H Compound 6R1=H, R2=CH3 Compound 4R1=CH3, R2=CH3 Compound 5
H
CHR2
H
R1
NH
H3C X
O
H
R2
R1
Scheme 8.3 Formation of m/z 140, 190 and 240 for compound 6 and m/z 126, 176 and 226 for compounds 4 and 5.
Derivatization categorized this limited set of compounds into three subset groups
depending on the mass recorded for base peak. The first group includes compounds 1 and
2 having m/z 135 as the base peak in their PFPA derivatives. The second group comprises
compounds 4 and 5 having a base peak at m/z 148. Finally, the third group includes
compounds 3, 6, 7 and 8 which are characterized by having the fragment ions at m/z 218
as the base peak.
Moreover, derivatization also enables the discrimination among some members
within the same group. For the second group, compounds 4 and 5 could be differentiated
by the difference in the relative abundance of the fragment ions at m/z 176. Among the
members of the third group, compounds 7 and 8, which are the only two N-methyl
compounds in this series, could be characterized by the fragment ions at m/z 160. In
addition, they can also be differentiated by inverse intensities of the relative abundances
of fragment ions at m/z 160 and 176.
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252
The fragment ion m/z 190 in the mass spectra of the PFPA derivative of compound 6
(Scheme 8.3) can be confused with the same mass fragment ion found in the mass spectra
of the PFPA derivatives of compounds 1-3 formed by another fragmentation pathway
(Scheme 8.1). The TFA and HFBA derivatives eliminated such confusion through the
observance of analogous ions at m/z 140 and 240 for both derivatives respectively formed
through the same fragmentation pathway shown in Scheme 11.3. Figure 11.4 shows
representative mass spectra of the TFA derivatives of compounds 1-3 and 6.
Figure 8.4: Mass spectra of the TFA derivatives of compounds 1-3 and 6.
O
O
HN CF3
O
1
40 60 80 100 120 140 160 180 200 220 240 260 280 3000
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
m/z-->
Abundance
Average of 8.307 to 8.336 min.: 090219-01.D\data.ms135.0
190.1
303.177.0161.055.1
105.1242.1222.0 264.1 284.1
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253
O
O
NH CF3
O
2
40 60 80 100 120 140 160 180 200 220 240 260 280 3000
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
m/z-->
Abundance
Average of 8.197 to 8.226 min.: 090219-02.D\data.ms135.1
190.1
303.1
77.055.0
168.1105.0
259.0228.1 281.0
O
O
NH CF3
O
3
40 60 80 100 120 140 160 180 200 220 240 260 280 3000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
m/z-->
Abundance
Scan 766 (8.302 min): 090219-03.D\data.ms168.1
135.1
55.1
303.1
190.1114.077.1
274.1232.0211.0 254.0
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254
O
O
N
O CF3
6
40 60 80 100 120 140 160 180 200 220 240 260 280 3000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
m/z-->
Abundance
Average of 8.476 to 8.506 min.: 090219-06.D\data.ms168.1
140.1
77.1303.1
51.1 105.1
205.1 264.1236.1 284.1
The ion at m/z 176 can be formed through two different fragmentation pathways
(Scheme 8.1 and 8.3) for the PFPA derivatives of compounds 4, 5, 7 and 8. The use of
TFA and HFBA as derivatizing agents resolved this problem by providing characteristic
peaks at m/z 126 and 226 for the TFA and HFBA derivatives of compounds 4 and 5,
respectively. Figure 8.5 shows representative mass spectra of the HFBA of these four
compounds. Hence, TFA and HFBA derivatives provide more structure specific fragment
ions than the PFPA analog.
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255
Figure 8.5: Mass spectra of the HFBA derivatives of the compounds 4, 5, 7 and 8.
O
O
N
O C3F7
4
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 4000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
m/z-->
Abundance
Average of 8.680 to 8.756 min.: 090219-20.D\data.ms148.1
226.0
403.177.143.1
268.1119.0177.1 374.1344.1294.1
O
O
N
O C3F7
5
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 4000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
m/z-->
Abundance
Average of 8.622 to 8.651 min.: 090219-21.D\data.ms148.1
226.0
43.1403.177.1
105.1268.1194.1 294.1 359.1330.0
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256
O
O
N
C3F7O
7
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 4000
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000
32000
34000
36000
38000
40000
42000
44000
46000
48000
50000
52000
m/z-->
Abundance
Average of 8.535 to 8.564 min.: 090219-23.D\data.ms268.1
176.1
135.0
210.0
42.0
69.0
403.1
240.0100.0
373.9
O
O
N
C3F7O
8
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 4000
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
1700000
m/z-->
Abundance
Average of 8.512 to 8.529 min.: 090219-24.D\data.ms268.1
210.0
176.1
135.056.1
103.1 403.1239.9 331.9 360.1306.0
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257
Gas Chromatography of Side Chain Regioisomeric Phenethylamines Related to the
Controlled Substances MDEA, MDMMA, and MBDB.
GC-MS is a powerful tool that combines the identification power of mass
spectrometry with the separation power of gas chromatography. The separation of a
physical mixture of the compounds 1-10 in the underivatized form was the subject of a
previous report from our laboratory.
Several stationary phases and different temperature programs were evaluated in
an effort to resolve the TFA, PFPA and HFBA derivatives in this study. The Rtx-200
phase provided adequate resolution for these compounds of closely related retention
properties. However, different temperature programs were necessary for resolving each
set of perfluoroacyl derivatives.
The separation was performed using three temperature programs for each
derivative studied. Program one was used to separate the trifluoroacetyl (TFA)
derivatives and consisted of an initial hold at 100°C for 1.0 min, ramped up to 180°C at a
rate of 12°C min-1, held at 180°C for 2.0 min then ramped to 200°C at a rate of 10°C min-
1 and held at 200°C for rest of the run duration. The second program was used to resolve
the pentafluoropropionic anhydride (PFPA) derivatives. It started at 100°C for 1.0 min,
ramped up to 180°C at a rate of 7.5°C min-1, held at 180°C for 2.0 min then ramped to
200°C at a rate of 10°C min-1 and held at 200°C. The third program utilized to separate
the heptafluorobutyric anhydride (HFBA) derivatives had an initial oven temperature at
100°C for 1.0 min, followed by temperature increase at a rate of 9°C min-1 to reach
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258
180°C, held at 180°C for 2.0 min then ramped to 200°C at a rate of 10°C min-1 and held
at 200°C for the remaining of the run.
Figure 8.6 shows the chromatographic separation of all derivatized forms (TFA,
PFPA and HFBA) of compounds 1-8. The chromatogram shows that the derivatives of
the primary amines (compounds 1-3), the most branched side chain (compound 3) elutes
first and the least branched (compound 1) elutes last. For the secondary amines
(compounds 4-8), the perfluroamides elute in a similar pattern. Thus the compound of
greatest side chain branching (compound 8) elutes first followed by compound 6, 7, 5 and
the most linear tertiary amide (compound 4) elutes last. The resolution of the PFPA and
HFBA derivatives was better than that of the TFA derivatives. However both TFA and
HFBA derivatives provide more structurally specific ions in the mass spectrum to help in
discriminating among these compounds. Hence, the HFBA derivatives are considered the
best choice to discriminate by mass spectrometry and resolve by gas chromatography this
set of regioisomeric phenethylamine derivatives.
18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
Time-->
Abundance
TIC: 090406-3.D\data.ms
2 8 3
1
6
7 5
4
A
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259
18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
1700000
1800000
1900000
2000000
2100000
2200000
2300000
2400000
2500000
Time-->
Abundance
TIC: 090406-9.D\data.ms
17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
Time-->
Abundance
TIC: 090407-2.D\data.ms
Figure 8.6 Gas chromatographic separation of A: TFA; B: PFPA and C: HFBA derivatives of compounds (1-8), Column used; Rtx-200.
3 8 2 6 1
7 5
4 B
3
6
8
21
7
5
4
C
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260
Conclusion of Chapter 8
Three regioisomeric methylenedioxyphenethylamines (MDEA, MDMMA, and
MBDB), having the same molecular weight and major mass spectral fragments of
equivalent mass, have been reported as drugs of abuse. Seven additional
phenethylamines have a side chain regioisomeric relationship with the controlled
substances. Derivatization of the eight primary and secondary amines with various
perfluroacylating agents yields amides which significantly individualized their mass
spectra and allowed for specific side chain identification. The individualization is the
result of formation of unique marker ions or as a result of differences in the relative
abundances of some common ions. The trifluoroacetyl and heptafluorobutryl derivatives
offer more unique fragment ions over pentafluoropropionyl derivatives. Chromatographic
resolution of the perfluroacyl amides was achieved on a relatively polar stationary phase
Rtx-200.
Chapter 9
GC-MS Evaluation of a Series of Acylated Derivatives of 3,4-
Methylenedioxymethamphetamine
The aim of this chapter is to study and compare the mass spectral properties of
MDMA after derivatization with different reagents commonly used for amine acylation.
The second task is to evaluate the retention properties of each acyl derivative under
chromatographic conditions regularly used by forensic chemists. The derivatizing agents
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261
used in this study included trifluoroacetic anhydride (TFAA), pentafluoropropionic
anhydride (PFPA), heptafluorobutric anhydride (HFBA), pentafluorobenzoyl chloride,
acetic anhydride, propionic anhydride , butyric anhydride and benzoyl chloride.
Additionally, the formyl, d3-acetyl and d5-benzoyl derivatives were prepared to confirm
mass spectral fragmentation pathways.
Mass Spectrometry of Acylated Derivatives of 3,4-
Methylenedioxymethamphetamine
Mass spectrometry is the principal method used for confirming the identity of
drugs and related substances in forensic samples. Figure 9.1 shows the structure of 3,4-
MDMA and the mass spectrum for the underivatized amine. The mass spectrum for 3,4-
MDMA is presented here primarily for comparison with the various acylated derivatives
reported in this study. The fragmentation of phenethylamines is generally characterized
by a base peak from an amine initiated alpha-cleavage reaction breaking the carbon-
carbon bond of the ethyl linkage between the aromatic ring and the nitrogen atom of the
amine group. With regards to 3,4-MDMA, initial ionization of nitrogen followed by
cleavage of the alpha bond yields the m/z 58 imine cation, the base peak in the MDMA
mass spectrum.
Figure 9.1: Mass Spectrum of the underivatized MDMA
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262
O
O
HN
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 1900
100000
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1000000
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m/z-->
Abundance
Average of 13.027 to 13.167 min.: 080907-07.D\data.ms58.1
135.077.042.1 88.6 105.0 178.1148.1 193.1163.1120.1
Formation of the molecular ion via ionization of the aromatic pi-electrons
followed directly by alpha-cleavage yields the benzylic cation at m/z 135 and hydrogen
rearrangement from the equivalent molecular ion followed by alpha-cleavage initiated at
the distant radical site yields the radical cation at m/z 136 in Figure 9.1. The structures for
these fragment ions are shown in Scheme 9.1
The initial group of derivatives evaluated in this study were the simple alkyl
amides of 3,4-MDMA obtained by derivatization with acetic, propionic and butyric
anhydrides. Acylation of the amine results in significantly lower nitrogen basicity and
this often results in the formation of unique and characteristic fragment ions.
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263
.
HC
N
CH3
CH3
m/z = 58
O
O
O
O
CH2
H
m/z = 135
HN
EI 70eV
O
O
CH2
m/z = 136
H
H
Scheme 9.1. EI fragmentation pattern for the underivatized MDMA.
Figure 9.2 (A, B and C) shows the mass spectra for the homologous acetyl,
propionyl and butyryl amides. These three spectra show molecular ions of low relative
abundance at m/z 235, 249 and 263 respectively. Additionally, a second homologous
series of fragment ions (M-135)+ occurs at m/z 100, 114 and 128 suggestive of the
acylated imine fragment for each of the amides. The mass spectrum in Figure 9.2D
provides evidence to support these structural assignments and shows the equivalent ion at
m/z 103 for the d3-acetamide. The structures for the acylated imine fragments included in
this study are shown in scheme 9.2
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264
.
N
X
O
X = CF3, 289 = C2F5, 339 = C3F7, 389 = C6F5, 387 = H, 221 = CH3, 235 = C2H5, 249 = C3H7, 263 = C6H5, 297 = CD3, 238 = C6D5, 302
N
X
O
m/z = 154, X = CF3 = 204, X = C2F5 = 254, X = C3F7 = 252, X = C6F5 = 86, X = H = 100, X = CH3 = 114, X = C2H5
= 128, X = C3H7 = 162, X = C6H5
= 103, X = CD3 = 167, X = C6D5
O
O
Scheme 9.2: Imine fragmentation pattern of acylated derivatives of MDMA.
The spectra in Figure 9.2A, B and C show two prominent ions occurring at the
same mass for all three derivatives. The base peak for these derivatives is the m/z 58 ion
and this ion is equivalent to the base peak in the underivatized amine whose mass
spectrum is shown in Figure 9.1.
The m/z 58 ion in the spectrum of these derivatives is likely the result of
hydrogen rearrangement from the alkyl group of the acyl imine fragments at m/z 100,
114 and 128 to yield the common imine fragment at m/z 58. This fragmentation pathway
is illustrated in Scheme 9.3. Furthermore, the mass spectrum in Figure 9.2D for the d3-
acetamide shows the deutrated imine species at m/z 59 providing support for the
structural assignment and fragmentation pathway
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265
Figure 9.2. Mass spectra of acetyl, propanyl, butryl and d3-Acetyl amides of MDMA
O
O
N CH3
O
A
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 2400
100000
200000
300000
400000
500000
600000
700000
800000
m/z-->
Abundance
Average of 22.947 to 23.186 min.: 071105-10.D\data.ms58.1
162.1
100.1
135.177.143.1
235.1192.1118.1 177.1 220.1
O
O
N C2H5
O
B
40 60 80 100 120 140 160 180 200 220 2400
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
m/z-->
Abundance
Average of 25.809 to 26.118 min.: 071108-18.D\data.ms58.1
162.1
114.1
135.1
77.1
42.1249.1192.198.1 220.1
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266
O
O
N C3H7
O
C
40 60 80 100 120 140 160 180 200 220 240 2600
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
m/z-->
Abundance
Scan 4487 (30.740 min): 080204-10.D\data.ms58.1
162.1
128.1
77.1
41.1 105.1263.1192.1 220.1146.1 246.1
O
O
N CD3
O
D
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 2400
50000
100000
150000
200000
250000
300000
350000
400000
450000
m/z-->
Abundance
Average of 22.597 to 23.046 min.: 071113-20.D\data.ms59.1
162.1
103.1
135.177.1
42.1 238.1193.1119.1 178.1 220.1
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267
Figure 9.3. Mass Spectra of the formyl, benzoyl and d5-benzoyl amides of MDMA.
O
O
N H
O
A
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 2300
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
m/z-->
Abundance
Average of 21.735 to 21.997 min.: 071121-04.D\data.ms162.186.1
58.1
135.0
105.142.1 221.1121.0 191.1177.0 206.972.0
O
O
N
O
B
40 60 80 100 120 140 160 180 200 220 240 260 280 3000
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
m/z-->
Abundance
Average of 65.304 to 66.236 min.: 071121-04.D\data.ms105.0
162.1
77.1
135.051.1297.1191.1 251.9
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268
167
O
O
N
O
D
D
D
D
DC
40 60 80 100 120 140 160 180 200 220 240 260 280 3000
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
m/z-->
Abundance
Scan 10447 (65.479 min): 071126-20.D\data.ms110.1
162.1
82.1
135.154.1
191.1 302.1
The formamide (N-formyl) and benzamide (N-benzoyl) derivatives were prepared
and evaluated to determine the scope of the hydrogen rearrangement in the further
decomposition of the acyl imine species. The mass spectra of these compounds are shown
in Figure 9.3A-B. As shown in Scheme 9.3, the hydrogen of the formyl group does
transfer in significant abundance; however the resulting m/z 58 fragment is not the base
peak for this compound. The decreased likelihood of hydrogen transfer through the 3-
membered ring transition leaves the formyl imine species (m/z 86) as the more prominent
ion in its mass spectrum. The benzamide in Figure 9.3B does not show any indication of
a m/z 58 ion demonstrating that the aromatic hydrogens do not migrate in the same
manner as the hydrogen of the alkyl groups in the previously described spectra.
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269
N
C
O
R
H
H
R=H, m/z = 100R= CH3, m/z =114R= C2H5, m/z = 128
N
H
m/z = 58
N
H
O
m/z = 86
Scheme 9.3 Formation of m/z 58 from the N-formyl, acetyl propionyl and butyryl
imine fragments
NO
O
XO
HH
NO
O
XHO
H
O
O
H
m/z= 162 Scheme 9.4 Formation of m/z 162 in all amide derivatives of MDMA
The other common fragment seen in all the mass spectra in Figure 9.2 and those
in Figure 9.3A and B is the m/z 162 ion. This fragment ion is the 3,4-
methylenedioxyphenylpropene radical cation occurring through hydrogen transfer from
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270
the propyl group to the carbonyl oxygen followed by loss of the neutral amide species.
This mechanism for formation of the m/z 162 radical cation is shown in Scheme 9.4. An
interesting uniqueness was observed in the mass spectrum of the benzamide derivative in
Figure 9.3B. Two separate fragmentation pathways can yield ions at m/z 162, the 3,4-
methylenedioxyphenylpropene radical cation and the benzoylimine cation. Figure 9.3C
shows confirmation of this point with the mass spectrum for the d5-benzamide. The d5-
benzoylimine is shifted to m/z 167 as expected while the 3,4-
methylenedioxyphenylpropene fragment remains at m/z 162. Additionally, the benzoyl
fragment (C6H5CO+) at m/z 105 in Figure 9.3B is shifted to m/z 110 for the d5-benzoyl
derivative.
The mass spectra for all the derivatives described to this point have some
disadvantages for the identification of amines such as 3,4-
methylenedioxymethamphetamine. The derivatives formed from alkanoic carboxylic acid
anhydrides (acetic, propionic and butyric) fragment to yield base peaks at m/z 58
equivalent to the underivatized amines. Thus without the aid of chromatographic
separation, the completeness of the derivatization process itself could be questioned. The
loss of significant ion current as m/z 58 is a drawback to the use of these derivatives for
forensic analysis and the only unique major ion for identification of 3,4-MDMA is the
m/z 162 ion and this ion appears regardless of the acylating species. The benzamide
derivative yields significant ion current at unique masses such as m/z 105 and 162.
However the benzamide derivative of 3,4-MDMA was observed to have quite high GC
retention properties as will be described later in this study. These relatively high retention
properties may limit this derivative utility in some applications.
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271
The mass spectra for a series of perfluoroacyl derivatives of 3,4-MDMA are
shown in Figure 9.4. One of the major features of these spectra is the absence of m/z 58
ion. This ion does not occur since no hydrogen is available for migration from the acyl
portion of these derivatives. The spectra in Figure 9.4A, B, C and D further show
common ions at m/z 135 and m/z 162. These ions are the 3,4-methylenedioxybenzyl
cation and the 3,4-methylenedioxyphenylpropene radical cation respectively. The
formation of these ions occurs in an analogous manner to that described earlier in this
report. The three perfluoroalkyl derivatives, trifluoroacetyl, pentafluoropropionyl and
heptafluorobutyryl amides show several ions in a homologous series separated by 50
mass units (CF2). The m/z 154, m/z 204 and m/z 254 ions in these three spectra represent
the perfluoroacylimine species (see Scheme 9.2). These ions are the base peaks in the
three spectra and without hydrogen atom to migrate from the acyl group, no m/z 58
decomposition ion is formed. Additionally, these spectra do not show any evidence of
analogous fluorine transfer decomposition products.
The second homologous series of ions in Figure 9.4A, B and C occur at m/z 110,
160 and 210 respectively. This series of ions differing by 50 mass ions (CF2) indicates
that the perfluoro fragment is a component of these ions. Previous studies in chapter 1
using d3-3,4-MDMA in which the three deuterium atoms are bonded to the N-methyl
group (N-CD3) have shown that all three deuterium labels are contained in the m/z 160
ion for the PFPA derivative. This leaves only 26 mass units (CN) to account for all the
mass of the m/z 160 ion. The mechanism and account for these ions is shown in Scheme
17.5.
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272
Figure 9.4. Mass spectra of the fluorinated acyl derivatives of MDMA.
O
O
N CF3
O
A
40 60 80 100 120 140 160 180 200 220 240 260 2800
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40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
m/z-->
Abundance
Average of 16.920 to 17.025 min.: 071121-04.D\data.ms154.0
135.0
110.0
77.1289.142.1
204.0 250.1176.0 222.1 269.9
O
O
N C2F5
O
B
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
340000
m/z-->
Abundance
Average of 16.670 to 16.722 min.: 071121-04.D\data.ms204.0
162.1
135.0
77.142.1 339.1
105.1
269.9 300.0242.9
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273
O
O
N C3F7
O
C
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
1700000
1800000
1900000
2000000
2100000
2200000
2300000
m/z-->
Abundance
Average of 17.357 to 17.841 min.: 071023-10.D\data.ms254.1
162.1
135.1210.0
77.142.1 389.1
105.1
310.0 350.1282.9
O
O
N
O
F
F
F
F
FD
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
m/z-->
Abundance
Scan 6747 (43.913 min): 080204-11.D\data.ms195.0
162.1
252.1
135.144.077.1
105.0 225.1 387.1281.0
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274
N
m/z = 154, X = CF3
= 204, X = C2F5 = 254, X = C3F7
X
O
N
O
X N C X
m/z = 110, X = CF3 = 160, X = C2F5 = 210, X = C3F7
Scheme 9.5: Formation of m/z 110, 160 and 210 in the spectra of the TFAA, PFPA
and HFBA derivatives of MDMA.
Based on our studies of numerous phenethylamines, this mechanism appears to be
specific for the perfluoroacyl derivatives of N-methyl substituted compounds. Next
chapter will address the scope of this mechanism.
While this project did not examine any perfluoroacyl derivatives in this
homologous series beyond the heptafluorobutyramide, the perfluorooctanoyl (PFO)
derivatives of 3,4-MDMA and methamphetamine have been reported. These derivatives
show a major fragment ion at m/z 410 which extends this series of rearrangement ions
(see Scheme 9.5) to higher chain homologues. The additional four CF2 groups in the PFO
derivative beyond the HFBA derivative account for the 200 additional mass units. The
work of Westphal et al further described the PFO derivative of d5-MDMA and clearly
showed that only three deuterium labels were incorporated into the analogous fragment
ion at m/z 413. The base in the mass spectrum of the PFO derivative of MDMA occurs at
m/z 454 which is the perfluoroacylimine species and analogous to the base peaks in
Figure 9.4A, B and C. Thus these data are consistent with the experimental observations
reported in this study.
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275
The remaining spectrum in Figure 9.4D is for the pentafluorobenzamide and the
base peak at m/z 195 is the pentafluorobenzoyl cation. This ion is analogous to the m/z
105 and m/z 110 ions for the benzoyl and d5-benzoyl cations respectively (Scheme 9.6).
These ions would be major fragments in the benzamide of most amines and thus do not
provide ions characteristic of 3,4-MDMA.
N
X
O
X = C6F5, 387 = C6H5, 297 = C6D5, 302
X
O
m/z = 195, X = C6F5 = 105, X = C6H5
= 110, X = C6D5
O
O
Scheme 9.6. Formation of m/z 195, 105 and 110 from the pentafluorobenzoyl,
benzoyl and d5-benzoyl derivatives of MDMA.
Gas Chromatography The chromatogram in Figure 9.5 provides a comparison of the acyl group
structure and relative GC retention properties on a nonpolar stationary phase, 100%
dimethylpolysiloxane, Rtx-1. Several temperature programs were evaluated in this
project and one program showing the best compromise between resolution and analysis
time was used to generate the chromatogram in Figure 9.5.
The perfluoroalkylamides are the first group of derivatives to elute on the
dimethylpolysiloxane stationary phase. These amides elute much earlier than their
corresponding non-fluorinated hydrocarbon counterparts. Both the fluorinated and non-
fluorinated alkyl derivatives elute before the aromatic amides. In every case of direct
comparison, the perfluoro species eluted much earlier than its non-fluorinated
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276
hydrocarbon counterpart. The most obvious example of this effect can be seen for the
benzamides in the last two peaks in the chromatogram (Figure 9.5). The high relative
retention for the benzamides coupled with the lack of major fragment ions could limit
their value in MDMA analysis and identification. The alkyl hydrocarbon derivatives
provide significantly increased chromatographic retention yet show the m/z 58 ion as the
base peak as does the derivatized amine, MDMA. Thus, the perfluoroalkyl derivatives
offer excellent chromatographic properties as well as a significant number of
characteristic fragment ions for MDMA identification.
10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
340000
Time-->
Abundance
TIC: 080204-13.D\data.ms
Figure 9.5. Gas chromatographic separation of MDMA derivatives. Peaks: 1, PFPA; 2, TFAA; 3, HFBA; 4, N-formyl; 5, acetyl; 6, propoinyl; 7, butyrl; 8, pentaflurobenzoyl and 9, benzoyl amides.
1
2
3 4
5
7
8
9
6
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277
Conclusions of Chapter 9
The perfluoroacyl derivatives of MDMA show excellent chromatographic
properties and unique mass spectral fragment ions. The perfluoroalkyl amides of TFAA,
PFPA and HFBA yield a unique series of mass spectral fragments at m/z 110, 160 and
210 respectively. Additionally, the base peaks in these mass spectra occur at relatively
higher masses at m/z 154, 204 and 254 respectively. These ions are the
perfluoroacylimines resulting from loss of 135 mass units (3,4-methylenedioxybenzyl)
from the molecular ion. This (M-135)+ species also occurs for the acetyl, propionyl and
butyryl amides however these ions rearrange through loss of the acyl group to yield a
common ion at m/z 58. Thus, the base peak for the non-fluorinated hydrocarbon
derivatives is the same as that observed for the underivatized MDMA, m/z 58. The
formyl, d3-acetyl, d5-benzoyl and pentafluorobenzoyl derivatives provided
chromatographic and confirmatory mass spectral evidence for fragmentation pathways.
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278
IV. Conclusions
IV.1 Discussion of Findings.
The compounds investigated in this project are a series of closely related
regioisomeric and isobaric amines having mass spectra essentially equivalent to that of
MDMA, MDEA and MBDB. Mass spectral equivalence in the underivatized amines
shows that MS methods do not provide confirmation of drug identity and molecular
individualization. Additional scientific data for molecular individualization can be
obtained following simple chemical derivatization with perfluoroacylating reagents.
Perfluoroacylamines provide specific and unique mass spectral fragments
for side chain and some ring substituent identification. Vapor phase infrared spectrometry
(GC-IRD) provides additional confirmation for other ring substituents and for those
compounds not forming stable acylation products. Thus the results of this research have
provided improved specificity in the analytical methods used to identify these
phenethylamine controlled substances. This improved specificity comes from methods
which allow the forensic analyst to eliminate non-drug imposter substances as the source
of analytical data matching that of the controlled drug substance.
IV.2 Implications for policy and practice:
The results of this project allow the forensic analyst to differentiate among most
of the regioisomeric and isobaric equivalents of MDMA. This can be accomplished
without having access to a large number of reference standards of these compounds.
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279
IV.3 Implications for further research:
Perhaps the first implication from the results of this work is an educational
program to efficiently and effectively transfer the findings to practicing forensic analysts.
While we have published extensively on this subject in the scientific literature, a one or
two day short course would allow us to put these data in the hands of forensic drug
chemists in an organized and effective manner. The course would include background
educational material on mass spectra and infrared interpretation as well as
chromatographic theory. The course could be offered in conjunction with regional
meetings of forensic scientists over a period of a year to 18 months and reach numerous
individual forensic scientists.
Retention index studies for all the ring substituted amines of the same side-chain,
especially the methamphetamine side-chain compounds. Since the methamphetamine
side chain is perhaps the most likely to appear in new designer drugs, retention indexing
studies on standardized GC columns and stationary phases could further decrease the
need for reference standards in identification of these regioisomeric and isobaric amines.
Such studies should be done in cooperation with collaborators in forensic science
laboratories.
Liquid Chromatographic studies on unique stationary phases to provide
retention/separation/resolution data on as large a subset of these compounds as possible.
Again concentrating on those compounds having the same side chain structure. These are
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280
the compounds for which mass spectrometry provides the least information for molecular
individualization.
In concert with LC studies MS-MS techniques can be explored for evidence of
specific fragment ions identifying aromatic ring substitution patterns. The initial phase of
such a study would be to compare the fragment patterns for all the possible regioisomeric
substitution patterns in each series. Since we have all the compounds available already,
our group is well positioned to begin this study immediately.
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
281
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This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
294
VI. Dissemination of Research Findings VI.1 Scientific Publications Related to this Project:
Ashley L Thigpen, Jack DeRuiter and C Randall Clark, “GC-MS Studies on the
Regioisomeric 2,3- and 3,4-Methylenedioxyphenethylamines Related to MDEA,
MDMMA and MBDB,” J. Chromatogr. Sci., 45, 229 (2007).
Tamer Awad, Jack DeRuiter and C. Randall Clark, “Chromatographic and Mass Spectral
Studies on Methoxy Methyl Methamphetamines Related to 3,4-Methylenedioxy-
methamphetamine,” J. Chromatogr. Sci., 45, 466 (2007).
Tamer Awad, Jack DeRuiter and C. Randall Clark, “GC-MS Analysis of Regioisomeric
Ring Substituted Methoxy Methyl Phenylacetones,” J. Chromatogr. Sci., 45, 458 (2007).
Tamer Awad, C. Randall Clark and Jack DeRuiter, GC-MS Analysis of Acylated
Derivatives of the Side Chain Regioisomers of 4-Methoxy-3-methyl-phenethylamines
Related to Methylenedioxymethamphetamine,” J. Chromatogr. Sci., 45, 477 (2007).
Tamer Awad, Jack DeRuiter and C. Randall Clark, “GC-MS Analysis of Acylated
Derivatives of a Series of Side Chain Regioisomers of 2-Methoxy-4-Methyl-
Phenethylamines,” J. Chromatogr. Sci., 46, 375-380 (2008).
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
295
Tarek Belal, Tamer Awad, Jack DeRuiter and C. Randall Clark, “GC-MS Studies on
Acylated Derivatives of 3-Methoxy-4-methyl- and 4-Methoxy-3-methyl-
phenethylamines: Regioisomers Related to 3,4-MDMA,” Forensic Science International,
178, 61-82 (2008).
Tamer Awad, Jack DeRuiter and C. Randall Clark, “GC- MS Analysis of Ring and Side
Chain Regioismoers of Ethoxyphenethylamines,” J. Chromatogr. Sci., 46, 671-679
(2008).
Ashley L. Thigpen Tamer Awad, Jack DeRuiter and C. Randall Clark
“GC-MS Studies on the Regioisomeric Methoxy-Methyl-Phenethylamines Related to
MDEA, MDMMA and MBDB,” J. Chromatogr. Sci., 46, 900-906 (2008).
C. Randall Clark, Tamer Awad and Jack DeRuiter, “GC-IRD Analysis of Regioisomeric
Substituted Phenethylamines of Mass Spectral Equivalence,” Chromatography Today, 1
#4, 27-30 (2008).
Tamer Awad, Jack DeRuiter, Tarek Belal and C. Randall Clark, “Gas Chromatographic
and Mass Spectral Studies on the Acylated Side Chain Regioisomers of 3-Methoxy-4-
Methyl Phenethylamine and 4-Methoxy-3-Methyl Phenethylamine,” J. Chromatogr. Sci.,
47, 279-286 (2009).
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
296
Tarek Belal, Tamer Awad, C. Randall Clark and Jack DeRuiter, “GC-MS Evaluation of a
series of Acylated Derivatives of 3,4-Methylenedioxy-methamphetamine,” J.
Chromatogr. Sci., 47, 359-364 (2009).
Tamer Awad, Tarek Belal, Jack DeRuiter, Kevin Kramer, and C. Randall Clark,
“Comparison of GC-MS and GC-IRD methods for the differentiation of
methamphetamine and regioisomeric substances,” Forensic Science International, 185,
67–77 (2009).
Tarek Belal, Tamer Awad, Jack DeRuiter and C. Randall Clark, “GC-IRD Methods for
the Identification of Isomeric Ethoxyphenethylamines and Methoxymethcathinones,”
Forensic Science International, 184, 54–63 (2009).
Hadir M. Maher, Tamer Awad and C. Randall Clark, “Differentiation of the
Regioisomeric 2, 3, and 4-Trifluoromethylphenylpiperazines (TFMPP) by GC-IRD and
GC-MS,” Forensic Science International, 188, 31-39 (2009).
Hadir M. Maher, Tamer Awad, Jack DeRuiter and C. Randall Clark, “GC–MS Studies on
Acylated Derivatives of Dimethoxyamphetamines (DMA): Regioisomers Related to 2,5-
DMA,” Forensic Science International, 192, 115-125 (2009).
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
297
Tamer Awad, Tarek Belal, C. Randall Clark, Jack DeRuiter, “GC-IRD Studies on
Regioisomeric Ring Substituted Methoxy Methyl Phenylacetones Related to 3,4-
Methylenedioxyphenylacetone,” Forensic Science International, 194, 39-48 (2010).
Hadir M. Maher, Tamer Awad, Jack DeRuiter and C. Randall Clark
“GC–IRD Methods for the Identification of Some Tertiary Amine Regioisomers of
MDMA,” Forensic Science International, 199, 18–28 (2010).
Abdullah M. Al-Hossaini, Tamer Awad, Jack DeRuiter and C. Randall Clark, “GC-MS
and GC-IRD Analysis of Ring and Side Chain Regioisomers of Ethoxyphenethylamines
Related to the Controlled Substances MDEA, MDMMA and MBDB,” Forensic Science
International, 200, 73-86 (2010).
Tamer Awad, Tarek Belal, Hadir M. Maher, C. Randall Clark and Jack DeRuiter. “GC-
MS Studies on Side Chain Regioisomers Related to Substituted Methylenedioxy-
phenethylamines; MDEA, MDMMA and MBDB” J. Chromatogr. Sci., 48, 726-732
(2010).
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
298
VI.2 Scientific Presentations Related to this Project:
Synthesis and Analytical Profiles for Regioisomeric and Isobaric Amines Related to
MDMA, MDEA and MBDB: Differentiation of Drug and non-Drug Substances of Mass
Spectral Equivalence—Progress Presentation
C. Randall Clark, 2007 General Forensics R&D Grantees Meeting, , San Antonio, Texas,
February 2007.
Synthesis and Analytical Profiles for Regioisomeric and Isobaric Amines Related to
MDMA, MDEA and MBDB: Differentiation of Drug and non-Drug Substances of Mass
Spectral Equivalence—Progress Presentation
C. Randall Clark, 2007 General Forensics R&D Advisory Group, Scottsdale, Arizona,
October 2007.
GC-MS Analysis of the Ring and Side Chain Regioisomers of Ethoxyphenethylamines
C. Randall Clark, Tamer Awad and Jack DeRuiter. American Chemical Society National
Meeting, New Orleans, Louisiana, USA; April 10, 2008.
Synthesis and Analytical Profiles for Regioisomeric and Isobaric Amines Related to
MDMA, MDEA and MBDB: Differentiation of Drug and non-Drug Substances of Mass
Spectral Equivalence—Progress
C. Randall Clark, 2009 General Forensics R&D Grantees Meeting, Denver, Colorado.
February 2009.
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
299
GC-IRD Identification of Isomeric Phenethylamines of Mass Spectral Equivalence
C. Randall Clark, Tamer Awad, Tarek Belal and Jack DeRuiter, Pittsburgh Conference,
Chicago, Illinois, March 8, 2009.
GC-MS Studies on Side Chain Regioisomers Related to the Substituted
Methylenedioxyphenethylamines MDEA, MDMMA AND MBDB, Tamer Awad, Jack
DeRuiter and C. Randall Clark, Pittsburgh Conference, Chicago, Illinois, March 8, 2009.
GC-MS and GC-IRD Studies on the Ring Isomers of N-Methyl-1-Methoxyphenyl-1-
Methyl-2-Propanamines Related to 3,4-MDMA, Tamer Awad, Hadir M. Maher, Jack
DeRuiter and C. Randall Clark, American Chemical Society Meeting, Salt Lake City,
Utah, April, 2009.
Abdullah M. Al-Hossaini, Tamer Awad, Jack DeRuiter and C. Randall Clark,
“GC-MS and GC-IRD Studies on Ethoxy- and Methoxymethyl-phenethylamines:
Isobaric Substances Related to the Methylenedioxyphenethylamine Drugs,”
Pittsburg Conference on Analytical Chemistry (Pittcon), March 2, 2010, Orlando, FL.
Tamer Awad, Karim M.Abdel-Hay, Jack DeRuiter and C. Randall Clark
“Comparison of GC-IRD and GC-MS Methods for the Identification of Isomeric Drug
Substances: Piperazines and Phenethylamines,” Pittsburg Conference on Analytical
Chemistry (Pittcon), March 4, 2010, Orlando, FL.
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.
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C. Randall Clark, “Comparison of GC-MS and GC-IRD Methods for the Identification of
Isomeric Substituted Phenethylamine and Related Drug Substances,” Southwestern
Association of Forensic Scientists (SWAFS), Grapevine TX, Sept 23, 2010.
This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.