Evaluation of Designer Amphetamine Interference in GC–MS Amine ConfirmationProcedures

Justin M. Holler*, Shawn P. Vorce, Jessica L. Knittel, Brittany Malik-Wolf, Barry Levine and Thomas Z. Bosy

Division of Forensic Toxicology, Armed Forces Medical Examiner System, Dover AFB, Dover, DE 19902, USA

*Author to whom correspondence should be addressed. Email: justin.m.holler.ctr@mail.mil

In recent years, a class of new designer drugs commonly referred toas ‘bath salts’ have made their way to the illicit drug market. The mostcommon drugs encountered are designer amphetamines and cathi-nones. Many analytical methods for analysis and identification of bathsalts have been published, but there has been little reported on theirimpact on existing gas chromatography–mass spectrometry (GC–MS) amine confirmation methods. Due to structural similarities, the po-tential exists that designer amphetamines may interfere with methodsused for analysis of sympathomimetic amines. Methiopropamine,4-fluoroamphetamine, 4-fluoromethamphetamine (4-FMA) and4-methylamphetamine were examined for potential interference withimmunoassays and GC – MS confirmation analysis utilizing threederivatization procedures: R(-)-a-methoxy-a-trifluoromethylphenyla-cetyl chloride (R-MTPAC), heptafluorobutyric anhydride (HFBA) andchlorodifluoroacetic anhydride (ClF2AA). Significant cross-reactivitywas observed with all the four compounds on the Syva Emitw II PlusAmphetamines and Roche KIMS Amphetamines II immunoassays.Laboratories utilizing GC–MS selected-ion-monitoring confirmationmethods with R-MTPAC, HFBA or ClF2AA derivatives could experiencepotential chromatographic and mass spectral interferences from 4-flur-oamphetamine, 4-FMA and methiopropamine in the form of ion ratioand quantitative failures. Careful ion selection, proper selectivity andspecificity studies during method validation and rigid chromatographicand spectral acceptance criteria are required to assure the robustnessand accuracy of GC–MS methods.

Introduction

Complications in the gas chromatography–mass spectrometry

(GC–MS) analysis of amphetamines and sympathomimetic

amines have arisen over time. In the early years of the federal

workplace drug testing program, artifactual production of

methamphetamine was observed in the presence of high con-

centrations of ephedrine or pseudoephedrine (PE). This

phenomenon was eventually traced to an injection port tempera-

ture above 1808C (1). To address this phenomenon, the

Substance Abuse and Mental Health Administration imposed an

additional requirement before a positive result for methampheta-

mine could be reported. Urine samples containing methampheta-

minewere reported positive only if they had concentrations above

the 500 ng/mL cutoff and contained at least 200 ng/mL of am-

phetamine (2). This amphetamine requirement remains in place

today, although the cutoffs were changed in 2010 (3).

Another complication in the analysis of sympathomimetic

amines is the potential for chromatographic interference from

other structurally related amine compounds. These amines may

be endogenous, such as phenethylamine or tyramine, or they

may be structurally similar drugs that are taken therapeutically

or are abused. One group of abused drugs that have the potential

to interfere with the analysis of amphetamine and methampheta-

mine are the designer drugs commonly referred to as ‘bath salts.’

These drugs have gained in popularity over the past 5–10 years

and include designer amphetamines as well as designer cathi-

nones. As with many designer drugs, simple and subtle modifica-

tions are made to the base structure of a controlled compound in

an attempt to circumvent legal restrictions imposed by various

government agencies (4–7) and to elude detection. For example,

4-fluoroamphetamine (4-FA) and 4-methylamphetamine (4-MA)

are substituted structures of amphetamine. Mephedrone

(4-methyl-methcathinone) and methylone (3,4-methylenedioxy-

N-methylcathinone) are modified forms of cathinone. Many ana-

lytical methods for the analysis and identification of bath salts

have been studied and published, but there has been little

reported on the impact of these drugs on existing GC–MS

amine procedures.

Derivatization can offer a number of advantages for qualitative

and quantitative analysis of sympathomimetic amines to help al-

leviate complications. One advantage in forming a derivative is

the reduction of peak tailing, which can help with resolution

and make quantitative analysis more accurate. Derivatization

commonly reduces the polarity and alters other physical and

chemical properties of the stimulant amines, increasing their

retention time and enhancing their separation from potentially

interfering endogenous compounds or designer drugs. Sensitivity

or detectability may also improve when the compound is deriva-

tized. For example, low-molecular-weight compounds can demon-

strate better fragmentation when derivatization is used to increase

the mass-to-charge ratio (m/z; 8–17). The larger m/z fragments

are more distinctive in helping discern subtle difference in

chemical structures. This is especially helpful when dealing

with designer compounds because the larger m/z fragment

can identify where the substitution has occurred. Amine com-

pounds with a hydroxyl group, such as phenylpropanolamine,

ephedrine and PE, may form a di-derivative at the hydroxyl and

amine locations.

New designer amphetamines/cathinones are constantly being

synthesized by illicit drug chemists because of their structural

similarities to amphetamine, methamphetamine and other sym-

pathomimetic amines. The potential exists that these com-

pounds might interfere with current analytical methods for

this class of compounds due to their structural similarities to

the compounds involved. Within the Department of Defense

Forensic Toxicology Drug Testing Laboratories (DoD FTDTL),

there have been a number of cases where interferences with am-

phetamine, methamphetamine, methylenedioxyamphetamine

and methylenedioxymethamphetamine confirmation have been

traced to designer amphetamines/cathinones. The following

# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Journal of Analytical Toxicology 2014;38:295–303

doi:10.1093/jat/bku017 Advance Access publication March 30, 2014 Technical Note

report is an investigation of potential interference of four design-

er amphetamines with three in-house derivatization assays for

amphetamine and methamphetamine. In addition, two service

member specimens containing one of the designer ampheta-

mines were further investigated after initial interference was

observed at the drug testing laboratory.

Experimental

Chemicals and reagents

All solvents were of HPLC grade and were purchased from Fisher

Scientific (Pittsburgh, PA, USA). Potassium hydroxide pellets

were also purchased from Fisher Scientific. Sodium phosphate

monobasic and dibasic, triethylamine, heptafluorobutyric anhydride

(HFBA) and chlorodifluoroacetic anhydride (ClF2AA)were purchased

from Sigma-Aldrich (St. Louis, MO, USA). 4-MA, D, L-amphetamine

(AMP), D, L-amphetamine-d11, D, L-methamphetamine (METH), D,

L-methamphetamine-d14, 4-FA and 4-fluoromethamphetamine

(4-FMA) were purchased from Cerilliant (Round Rock, TX, USA).

Methiopropamine was purchased from Cayman Chemical (Ann

Arbor,MI, USA).R(-)-a-methoxy-a-trifluoromethylphenylacetyl chlor-

ide (R-MTPAC; .99.5% purity) was purchased from Fluka

(Switzerland). CEREXw POLYCROMTM CLIN II (691–0506)

solid-phase extraction columns were obtained from SPEware

Corporation (San Pedro, CA, USA). It should be noted that AMP

and METHwere found as impurities in 4-FA and 4-FMA standards,

respectively. The impurities were identified by the vendor

as well.

Sample preparation, extraction and analysis

All extraction procedures used were previously validated (17–19),

and the selected-ion-monitoring (SIM) ions are listed in Table I.

Three different derivatives (R-MTPAC, HFBA, ClF2AA) were used

to examine potential interference of designer amphetamines in

current assays (Fig. 1). Urine samples were analyzed using an

Agilent Technologies 6890 GC coupled to a 5973 mass spectrom-

eter (MS; Palo Alto, CA, USA). The separation column was a J&W

DB-5MS (Rancho Cordova, CA, USA) 20 m � 0.18 mm i.d. �0.18 mm with helium carrier gas maintained at a constant flow of

1.0 mL/min. The results were processed for quantitation using the

Target DB software (Marietta, GA, USA).

Urine specimens were also analyzed by immunoassay on a

Roche/Hitachi Modular P (Indianapolis, IN, USA). The reagent

kits analyzed were Syva Emitw II Plus Amphetamines (Newark,

DE, USA), Roche KIMS Amphetamines II and Microgenics DRIw

MDMA (Fremont, CA, USA).

Initial service member specimens results from DoD FTDTL

Two service member specimens caused a positive screen result

by immunoassay for both the Syva and Roche Amphetamines

assays. A positive immunoassay screen initiates an R-MTPAC iso-

meric GC–MS confirmation analysis. The specimens are

extracted by solid-phase extraction, derivatized with R-MTPAC

and analyzed by GC–MS monitoring the SIM ions: m/z 274,

275 and 200. The results for both member specimens suggested

the presence of methamphetamine; the retention time was with-

in 0.5% of the calibrator and all the three ions monitored were

present. The compound identified was racemic which is unusual

but not uncommon for methamphetamine; however, the mass

ion ratio for the second qualifier (m/z 200) was outside the

acceptable +20% range (28.2 and 30.6%) for both specimens.

The specimens were reported negative but there was concern

as to what drug was present.

Service member specimens reanalysis at Divisionof Forensic toxicology

The specimens were sent to the Division of Forensic Toxicology

(DFT) for further analysis. A comprehensive alkaline drug screen

identified the interferent compound as methiopropamine. Upon

further review of the original DoD FTDTL GC–MS data, it was ap-

parent that the peak representing L-methiopropamine was the

peak integrated at the expected retention time of D-METH

(Fig. 2). The samples were extracted using the same method as

the original analysis performed at the DoD FTDTL and deriva-

tized with R-MTPAC. GC–MS analysis was performed using

three SIM ions: m/z 274, 275 and 176, with the m/z 176 ion

being monitored instead of the m/z 200 ion as in the DoD

FTDTL method. The specimens were analyzed neat and with a

1:10 dilution. Peak integration was observed for D-METH ions,

though an ion ratio failure was observed due to the absence of

qualifier ion m/z 176. Analysis using the alternative DFT amines

confirmation method using a ClF2AA derivative also revealed

integrated METH peaks. However, using the ClF2AA derivative,

there were ion ratio failures for both qualifier ions. Overall, the

results seen were similar to those observed at by the DoD FTDTL.

Experiment design

The initial positive immunoassay screen results on the service

member specimens were produced by the Syva and Roche

Amphetamines immunoassay kits. With no methamphetamine

or amphetamine present, it was concluded that methiopropa-

mine caused the positive result, and therefore, it was imperative

to determine the cross-reactivity of these designer compounds

for the immunoassay kits used by the DoD laboratories. Three im-

munoassay kits were examined for their cross-activity: Syva

Emitw II Plus Amphetamines, Roche KIMS Amphetamines II

and Microgenics DRIw MDMA . Each drug was spiked at increas-

ing concentrations until a positive result was achieved for each

assay.

Three derivatization methods commonly used for GC–MS

amine confirmation analysis were challenged with spiked speci-

mens containing methiopropamine, 4-FA, 4-FMA and 4-MA to

determine whether they could be misidentified as AMP or

METH. Methiopropamine was chosen because of its presence

Table I.Ions (m/z) Monitored by Method for Amphetamine, Amphetamine-d11, Methamphetamine and

Methamphetamine-d14

R-MTPAC HFBA ClF2AA

Amphetamine-d11 264, 130 244, 128 160, 128Amphetamine 260, 162, 118 240, 118, 91 156, 118, 91Methamphetamine-d14 281, 98 261, 213 177, 179Methamphetamine 274, 275, 176, 200a 254, 210, 118 170, 118, 91

am/z 200 is monitored by several DoD drug testing laboratories and was evaluated in this study.

296 Holler et al.

in the member specimen, and 4-FA, 4-FMA and 4-MA were

selected because of their availability and structural similarities

with AMP and METH. Negative urine (no AMP/METH) and a

low control (containing AMP/METH) were spiked with individ-

ual designer drugs at 5,000 and 10,000 ng/mL. The specimens

were processed as normal samples using the standard forensic ac-

ceptance criteria for retention times (+2%), ion ratio (+20%) and

quantitation (+20% of theoretical).

Results

Immunoassay

The two service member specimens containing methiopropa-

mine showed no presence of AMP or METH when employing

GC–MS but resulted in two positive results when subjected to

immunoassay. Since the positive results were most likely caused

by methiopropamine, the cross-reactivity of all the four designer

amphetamines were assessed by immunoassay. The designer

amphetamines were spiked individually into negative urine and

analyzed by three different assays. Each drug was spiked at in-

creasing concentrations until a positive result was achieved for

each assay. Significant cross-reactivity was observed using both

AMP kits. 4-MA spiked at 250 ng/mL produced a positive re-

sponse for the Roche AMP kit as did 4-FMA at 750 ng/mL.

Methiopropamine showed significant response yielding positive

responses at 900 and 4,000 ng/mL for the Syva and Roche AMP,

respectively. The MDMA kit evaluated showed very little cross-

reactivity with the four designer amphetamine compounds.

The complete immunoassay results are listed in Table II.

GC–MS confirmation analysis

Full scan mass spectra of the AMP, METH, methiopropamine,

4-FA, 4-FMA and 4-MA derivatized with R-MTPAC, HFBA and

ClF2AA are illustrated in Figure 3. Common ions are observed

with all the three derivatives for all the four designer compounds

with AMP and METH. Any co-elution of the compounds will

result in inaccurate quantitation and ion ratio failures, from con-

tribution to either the quantitation and qualifier ions. A summary

of the observed interference and subsequent impact is exhibited

in Table III.

R(-)-a-Methoxy-a-trifluoromethylphenylacetyl chloride

R-MTPAC derivatization of 4-FA produced a retention timewithin

1% of D-AMP but with only one mass fragment (m/z 260) in com-

mon. The negative urine spiked with 4-FA at 5,000 ng/mL does

not result in peak integration; however, at 10,000 ng/mL the

abundance of 4-FA quantitation ion erroneously results in peak

integration for D-AMP when using this method. Even though

the quantitation ion is present for 4-FA, the two qualifier ions

are almost completely absent causing ion ratio failures. When

spiked into the low control, the D-AMP quantitation value is ex-

tremely high caused by the contribution of 4-FA to the quantita-

tion ion. The integration does result in significant ion ratio

failures since 4-FA does not contain the qualifying ions. Due to

structural differences between the two compounds, there was

no effect on the METH analysis.

4-FMA derivatization with R-MTPAC results in a retention time

within 1% of both isomers of METH in the calibrator, but it is the

D-isomer of 4-FMA that co-elutes with L-METH. 4-FMA derivatiza-

tion with R-MTPAC results in three of the four ions monitored for

METH to be present. The negative urine spiked with 4-FMA

results in integration for L-METH with a significant ratio failure

for the second qualifier (m/z 176) but acceptable ratios when

monitoring m/z 200. When spiked into the low control, the

L-METH quantitation value is extremely high, caused by contribu-

tion of 4-FMA and ion ratios were within acceptable parameters

when using m/z 200. As expected, due to structural differences

with AMP, there were no analytical complications.

Derivatization of 4-MA using R-MTPAC produced retention

times within 2% of D-METH in the calibrator but did not contain

any common ions. 4-MA did have an m/z 260 ion present as

would be expected due to inclusion of a methyl group on the

Figure 1. Structures of (A) AMP, (B) METH, (C)methiopropamine, (D) 4-FA, (E) 4-FMA, (F) 4-MA, (G) HFBA, (H) ClF2AA and (I) R-MTPAC.

Evaluation of Designer Amphetamine Interference 297

phenyl ring. Negative urine and low controls spiked with 4-MA

did not result in any qualitative or quantitative issues for AMP

or METH.

Methiopropamine analysis using R-MTPAC produced retention

times within 2% of D-METH. The fragmentation of methiopropa-

mine also resulted in three fragmentation ions used for METH:

m/z 274, 275 and 200. Analysis of negative urine spiked with

methiopropamine produced apparent integration for D-METH

with ion ratio failures form/z 176 and 200. The results observed

with the service member specimens were reproduced. Even

though methiopropamine fragmentation produces m/z 200,

the resulting ratio using the quantitation ion is outside the foren-

sic standard of 20%. The same ion ratio failures were observed

when spiked into the low control for D-METH.

Heptafluorobutyric anhydride

Analytical results of the designer amphetamines when using

HFBA derivatization were better than those observed with

R-MTPAC. The retention times of the designer amphetamines

were within 2% of AMP and METH except 4-MA when using

this derivative. The fragmentation patterns were similar to

those of R-MTPAC but did not cause any qualitative or quantita-

tive issues. Designer amphetamines which share one or two

common ions did not result in any observed integration for

AMP or METH when spiked into the negative urine or any

observed increased quantitations for the low control.

Figure 2. Total ion chromatogram and SIM ions (METH) for service member specimen containing methiopropamine with R-MTPAC derivatization.

Table II.Designer Amphetamine Concentrations (ng/mL) Producing a Positive Immunoassay Response

Roche Amps Syva Amps Microgenics MDMA

4-Fluoroamphetamine 3,900 2,950 37,5004-FMA 750 2,500 11,0004-MA 250 7,000 100,000Methiopropamine 4,000 900 None

298 Holler et al.

Chlorodifluoroacetic anhydride

Analysis using ClF2AA derivatization produced results similar to

those of R-MTPAC. All designer amphetamines produced reten-

tion times within 2% of either AMP or METH. 4-FA spiked into

negative urine produced a measurable AMP quantitation, both

ion ratios failed, however, as only the quantitation ion m/z 156

was present. When 4-FA was spiked into the low control, there

were no observed qualitative or quantitative issues, and the

AMP peak was correctly identified even in the presence of

4-FA. 4-FMA spiked into both the negative urine and low control

produced no issues for METH identification and quantitation.

4-FMA does produce the quantitation ion m/z 170 in significant

abundance but the absence of the qualifying ions allows proper

identification of METH. As observed with the other assays, 4-MA

has a retention time similar to that of METH though fragmenta-

tion common to AMP, but no issues were encountered.

Methiopropamine derivatization with ClF2AA produced a reten-

tion time within 2% of METH with fragmentation generating

the METH quantitation ion m/z 170. Analysis of negative urine

spiked with methiopropamine resulted in this peak being inte-

grated as METH but significant ratio failures for both qualifying

ions, m/z 118 and 91. When spiked into the low control, no

qualitative or quantitative issues were noted for METH as proper

peak identification was observed.

Discussion

The effect of the designer amphetamines interference using

common analytical procedures indicates the importance of

method validation as well as revalidation. The drugs used in

this investigation were not available when the methods were ini-

tially developed and validated; this analysis shows that it is

Figure 3. Full scan MS of each derivatized compound.

Evaluation of Designer Amphetamine Interference 299

imperative that laboratories continue to monitor and challenge

existing assays as new drugs emerge and become readily avail-

able. The data presented also emphasize the importance of

some fundamentals of forensic toxicology and mass spectrom-

etry: using three ions when performing SIM analysis to produce

ion ratios (+20%) as well as retention time requirements (+2%)

ensures accuracy and certainty in analyte identification and

quantitation. With the exception of 4-MA, the structural similar-

ities of these drugs enable the retention times to be within ac-

ceptable limits. Monitoring mass fragments that represent the

structures of AMP/METH allows the designer drugs to be distin-

guished from these intended targets. When using R-MTPAC deri-

vatization, some methods monitor m/z 274, 275 and 200 which

ignores the structurally important phenyl ring moiety of the drug

(20). By monitoring these fragments, methiopropamine could

conceivably be misidentified as methamphetamine. Although

METH and methiopropamine share m/z 200, they contain

enough structural difference to produce ion ratios outside the

analytical forensic standard of +20%. This structural difference

from METH is accomplished by replacement of the phenyl ring

with a thiophene ring, if mass fragments from this part of the

compound are not monitored, misidentification may result.

Fragments generated from a similar part of the molecule may pro-

duce identical fragments and ratios that meet forensic standards.

The results of this study indicate a potential weakness in

GC–MS SIM analysis of common sympathomimetic amines.

Even with derivatization, molecular fragmentation encountered

using GC–MS may not yield ideal fragmentation patterns which

are diagnostic for these compounds. Absence or low abundance

of the molecular ion reduces the ability to distinguish specific

Figure 3. Continued

300 Holler et al.

compounds based on molecular weight as can be done with soft

ionization techniques. GC–MS fragments can be small such as

m/z 91 and 118, which are common in many sympathomimetic

amines. This study also shows that chromatographic separation is

an integral part of GC–MS identification process. As technology

improves and workloads increase, the push for faster run times

may appear practical and prudent. It must be stressed, however,

that proper identification should always take precedence.

The designer amphetamines showed cross-reactivity with am-

phetamine immunoassays resulting in positive responses at rela-

tively low concentrations. This could be viewed as beneficial for

postmortem/investigative laboratories as it allows further testing

and the potential identification of new designer drugs. However,

cross-reactivity may be viewed as detrimental to those laborator-

ies with set reporting panels, such as urine drug testing labora-

tories where it could increase the number of presumptive

positives, decrease the confirmation rate and functionally

increase the cost of analysis.

Conclusion

Two service member specimens containing a compound struc-

turally similar to that of methamphetamine led to conflicting ana-

lytical results, costly additional analysis and subsequent

identification of methiopropamine as the interferent. Discovery

of this interference, caused by a new designer drug, led to the

reevaluation of previously validated amine methods using four

novel designer amphetamine compounds not available during

initial validation. The presented data show the need to continu-

ally challenge and validate existing methods as new structurally

similar designer drugs become available. The study also shows

the importance of monitoring ions that are representative of

Figure 3. Continued

Evaluation of Designer Amphetamine Interference 301

distinct molecule targets when performing SIM analysis. This in-

formation also reemphasizes the importance of forensic accept-

ability standards and why it is important to monitor and review

both retention time and the mass ion ratio acceptability criteria.

Also evident from the result of this study, the forensically accept-

able criteria of +2% for retention times may not be sufficient.

More stringent requirement may be necessary in order to avoid

possible interference from new emerging designer drugs.

Acknowledgment

The authors thank the Fort Meade Forensic Toxicology Drug

Testing Laboratory for their assistance in providing the submitted

samples.

Conflict of Interest statement

The opinions or assertions presented hereafter are the private

views of the authors and should not be construed as official or

as reflecting the views of the Department of Defense, its branches,

the US Army Medical Research and Materiel Command or the

Armed Forces Medical Examiner System.

Funding

This work was funded in part by the American Registry of

Pathology.

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Table III.Summary of Interfering Effects Observed with 4-FA, 4-FMA, 4-MA and Methiopropamine When Derivatized with R-MTPAC, HFBA and ClF2AA

Designer drug Derivative Compound

AMP METH

Commonion(s)

Retentiontime (+2%)

Ion ratio (+20%) Quantitation (+20%) Commonion(s)

Retention time(+2%)

Ion ratio (+20%) Quantitation (+20%)

4-FA R-MTPAC Yes Pass Fail Fail High No No interferenceobserved

HFBA Yes Pass Peaks Resolved NoInterference

Pass No No interferenceobserved

ClF2AA Yes Pass Pass if AMP present;fail if no AMP present

Pass if AMP present;fail if no AMP present

No No interferenceobserved

4-FMA R-MTPAC No No interference observed Yes Pass Integrates as L-METH Pass(with m/z 200); Fail(with m/z 176)

Fail high L-METH

HFBA No No interference observed Yes Pass Peaks resolved nointerference

Pass

ClF2AA No No interference observed Yes Pass Peaks resolved nointerference

Pass

4-MA R-MTPAC Yes No interference observed No No interferenceobserved

HFBA Yes No interference observed No No interferenceobserved

ClF2 Yes No interference observed No No interferenceobserved

Methiopropamine R-MTPAC No No interference observed Yes Pass Fail (with m/z 200); fail(with m/z 176)

Fail high

HFBA No No interference observed Yes Pass Peaks resolved nointerference

Pass

ClF2AA No No interference observed Yes Pass Pass if METH present; fail ifno METH present

Pass if METH present;fail if no METH present

302 Holler et al.

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Evaluation of Designer Amphetamine Interference 303