Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines Under International Control (2012)

7/29/2019 Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines Under Internationa

1/78

Recommended methods for theIdentifcation and

Analysis oBarbiturates and Benzodiazepines

under International Control

Manual for use by national drug analysis laboratories


2/78

Photo credits:UNODC Photo Library; UNODC/Ioulia Kondratovitch; Alessandro Scotti.


3/78

Lby Sf S

United nationS oice on drUgS and crime

V

RecommendedM

ethods or theIdentifcation and Analysis o Barbiturates

and Benzodiazepines under International

Control

MANUAL FOR USE BYNATIONAL DRUG ANALYSIS LABORATORIES

UNITED NATIONS

New York, 2012


4/78

ii

Note

Operating and experimental conditions are reproduced rom the original reerence

materials, including unpublished methods, validated and used in selected nationallaboratories as per the list o reerences. A number o alternative conditions and

substitution o named commercial products may provide comparable results in many

cases, but any modication has to be validated beore it is integrated into laboratory

routines.

Mention o names o rms and commercial products does not imply the endorse-

ment o the United Nations.

United Nations, June 2012. All rights reserved.

The designations employed and the presentation o material in this publication do

not imply the expression o any opinion whatsoever on the part o the Secretariat

o the United Nations concerning the legal status o any country, territory, city or

area, or o its authorities, or concerning the delimitation o its rontiers or

boundaries.

This publication has not been ormally edited.

Publishing production: English, Publishing and Library Section, United Nations

Oce at Vienna.

ST/NAR/46


5/78

iii

Acknowledgements

The UNODC Laboratory and Scientic Section (LSS, headed by Dr. Justice Tettey),

wishes to express its appreciation and thanks to Proessor Alexander Gray, Strath-

clyde Institute o Pharmacy and Biomedical Sciences, University o Strathclyde,

United Kingdom, or the preparation o the rst drat o the present manual, and to

Dr Pirjo Lillisunde, Head o Drug Laboratory, National Institute or Health and

Welare, Finland, or the expert review o the document. The preparation o this

manual was coordinated by Dr. Iphigenia Naidis, sta member o LSS. The contri-

bution o other UNODC sta is grateully acknowledged.


6/78


7/78

v

Contents

Page

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Purpose and use o the manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

I. Barbiturates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1. Classications and denitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2. Description o pure compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3. Qualitative and quantitative analysis o materials containing

barbiturates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

3.1 Sampling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.2 Extraction and sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . 6

3.3 Presumptive tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

3.4 Thin-layer chromatography (TLC) . . . . . . . . . . . . . . . . . . . . . . . . . 9

3.5 Gas chromatography (GC) with fame ionization detector (FID) 11

3.6 Gas chromatography - mass spectrometry (GC-MS) . . . . . . . . . . 13

3.7 High perormance liquid chromatography (HPLC) . . . . . . . . . . . . 16

3.8 Inrared spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

II. Benzodiazepines and related substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

1. Classications and denitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2. Description o pure compounds (see also annex II). . . . . . . . . . . . . . . . 22

3. Qualitative and quantitative analysis o materials containing

benzodiazepines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

3.1 Sampling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

3.2 Extraction and sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . 23

3.3 Presumptive tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

3.4 Thin-layer chromatography (TLC) . . . . . . . . . . . . . . . . . . . . . . . . . 253.5 Gas chromatography (GC) with fame ionization detector (FID) 28

3.6 Gas chromatography - mass spectrometry (GC-MS) . . . . . . . . . . 30

3.7 High perormance liquid chromatography (HPLC) . . . . . . . . . . . 31

3.8 Inrared spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35


8/78

vi

Page

Reerences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Further reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Annex

I. Description o barbiturates under international control . . . . . . . . . . . . . . . . 42

II. Description o benzodiazepines under international control . . . . . . . . . . . . . 49


9/78

1

Introduction

Background

Barbiturates and benzodiazepines are used in medicine as anticonvulsants, anxioly-

tics, hypnotics, sedatives, skeletal muscle relaxants and tranquilizers. Currently,

twelve barbiturates and thirty-ve benzodiazepines are under international control.

Those that have ound their way into illicit use have mainly been diverted rom the

licit market.

Seizures o benzodiazepines and barbiturates increased by more than 50 per cent

between 2005 and 2009 according to the World Drug Report 2011 [1]. The use o

benzodiazepines is common among drug users, including substitution treatment

clients.

The non-medical use o barbiturates, benzodiazepines and other sedatives together

with opioids has been a commonly observed phenomenon in many regions. The

existence o parallel markets is observed, where prescription drugs are sold outside

the control o the health authorities as well as through illegally operating Internet

pharmacies. Benzodiazepines, specically aprazolam and diazepam, are among the

most oten diverted and abused psychotropic substances [2]. However, given the

absence o inormation on overall drug use patterns, it is dicult to estimate the

extent o non-medical prescription drug use worldwide.

Purpose and use o the manual

The present manual is one in a series o similar UNODC publications dealing with

the identication and analysis o various types o drugs under international control.

These manuals are the outcome o a programme pursued by UNODC since the early

1980s, aimed at the harmonization and establishment o recommended methods o

analysis or national drug analysis laboratories.

The present manual combines and updates the two existing manuals onRecommended

methods or testing barbiturate derivatives [3] and benzodiazepine derivatives [4]

under international control, published in 1989 and 1989 respectively. It has been

prepared taking into account developments in analytical technology with a view to


10/78

2 Recommended Methods or the Identifcation and Analysis o Barbiturates and Benzodiazepines

providing the basis or reliable orensic evidence on seized materials containing

barbiturate or benzodiazepine derivatives. It is divided into two parts which provide

analytical methodologies or the two types o substances with reerence to validated

techniques. Description o the individual barbiturates and benzodiazepines underinternational control is provided in two annexes.

In line with the overall objective o the series o UNODC publications, this manual

suggests approaches that may assist drug analysts in the selection o methods appro-

priate to the sample under examination and provide data suitable or the purpose at

hand, leaving room also or adaptation to the level o sophistication o dierent

laboratories and the various legal needs.

Thereore, the methods described here should be understood as practical guidance,

that is, minor modications to suit local circumstances should normally not change

the validity o the results. Not all methods described in this manual need to be

applied to all samples suspected to consist o or contain barbiturates or benzodiaze-

pines. The choice o the methodology and approach to analysis as well as the deci-

sion as to whether or not additional methods are required remain with the analyst

and may also be dependent on the availability o appropriate instrumentation and

the level o legally acceptable proo in the jurisdiction within which the analyst

works. The reader should be aware that there are a number o other methods, includ-

ing those published in orensic science literature, which may also produce acceptable

results. However, any new method that is about to be used in a laboratory must bevalidated and/or veried prior to routine use.

Attention is also drawn to the importance o the availability to drug analysts o

reerence materials and literature on drugs o abuse and analytical techniques. More-

over, the analyst must o necessity keep abreast o current trends in drug analysis,

consistently ollowing current analytical and orensic science literature.

UNODC Laboratory and Scientic Section welcomes observations on the contents and

useulness o the present manual. Comments and suggestions may be addressed to:

Laboratory and Scientic Section

United Nations Oce on Drugs and Crime

Vienna International Centre

P.O. Box 500

1400 Vienna

Austria

Fax: (+43-1) 26060-5967

E-mail: [email protected]

All manuals, as well as guidelines and other scientic-technical publications may

be requested by contacting the address above.


11/78

3

I. Barbiturates

The abuse o barbiturates is widespread and it has oten been ound mixed with

other substances o abuse such as heroin. The international nature o the illicit market

means that any orensic laboratory may encounter a range o these compounds.

However, virtually all o the barbiturates in the illicit market result rom diversionrom legitimate sources and there is no reported evidence o clandestine

manuacture.

The twelve barbiturate derivatives under international control (1971 Convention on

Psychotropic Substances) appear mainly as capsules and tablets. Some are marketed

in other pharmaceutical orms such as elixirs, injectable solutions and sterile powders

or injection. Pentobarbital sodium is available in some countries as rectal supposi-

tories and barbital sodium is commonly sold in powder orm. Barbiturates oten

occur as mixtures with other barbiturates (e.g. amobarbital/secobarbital), with otherdrugs (e.g. aspirin, caeine, codeine, ephedrine, theophylline) and with attendant

pharmaceutical excipients. This makes the isolation and identication o a specic

barbiturate a considerable analytical challenge.

Analysts should be aware o the particular barbiturates commonly available in their

area as well as the characteristics and methodologies or their identication and

analysis. As barbiturate derivatives end up on the illicit market rom diversion rom

legitimate sources, reerence should be made to national pharmacopoeias and drug

tablet and capsule identication guides or preliminary screening inormation.

1. Classications and denitions

Barbiturates are drugs that act as central nervous system depressants, and, by virtue

o this, they produce a wide spectrum o eects, rom mild sedation to total anes-

thesia. Barbiturates are therapeutically used as anaesthetics, anticonvulsants, anxio-

lytics, hypnotics and sedatives. They are usually classied according to the duration

o their clinical eects into long-, intermediate-, short- and ultrashort- acting

compounds.

They are synthetic drugs derived rom barbituric acid (gure 1 below), which is a

synthetic condensation product o malonic acid and urea. They dier mainly in the

substitution pattern at position-5 with some also including an N-methyl at N-1 (see


12/78


annex I). The most well known derivative, phenobarbitone (see annex I), has been

used medicinally since 1912, mainly in the treatment o epilepsy.

Over 2,500 barbiturates have reportedly been synthesized with more than 50 o these

presently marketed or clinical use throughout the world. Twelve o these are subject

to international control under the Convention on Psychotropic Substances 1971 as

ollows:

Schedule. II: Secobarbital

Schedule. III: Amobarbital, butalbital, cyclobarbital and pentobarbitalSchedule. IV: Allobarbital, barbital, butobarbital, methylphenobarbital, phenobar-

bital, secbutabarbital and vinylbital

In recent years, there has been a signicant downturn in prescribing and thereore

the general availability o barbiturates owing to their side-eects and associated

dependency problems. An exception is phenobarbitone which is still used as an

anticonvulsant/anti-epileptic. Through the years, alternatives to barbiturates have

been developed including methaqualone and mecloqualone (Recommended methods

or the identifcation and analysis o Methaqualone/Mecloqualone, ST/NAR/15/Rev.1), benzodiazepines and related compounds.

2. Description o pure compounds

The inormation related to each o the twelve barbiturates (allobarbital, amobarbital,

barbital, butalbital, butobarbital, cyclobarbital, methylphenobarbital, phenobarbital,

pentobarbital, secbutabarbital, secobarbital and vinylbital) currently under interna-

tional control, can be ound in the annex I. In addition, the Multilingual Dictionary

o Narcotic Drugs and Psychotropic Substances under International Control [5](MLD, ST/NAR/1/Rev.2) provides inormation on the nomenclature o the barbitu-

rate derivatives under international control. It also includes synonyms and dierent

trade names or the relevant substances and provides inormation on their control

status.

N

N OO

O

R1

R2R4

R3

1

35

(1) Barbituric acid, R1-R4 = H(1) Barbituric acid, R1-R4 = H


13/78

I. Barbiturates 5

3. Qualitative and quantitative analysis omaterials containing barbiturates

Generally, in attempting to establish the identity o a controlled substance in sus-

pected material, the analytical approach must entail the determination o at least

two uncorrelated parameters one o which should provide inormation on the chemi-

cal structure o the analyte (or example, IR, MS; or tandem methods such as

GC-MS). It is recognized that the selection o these parameters in any particular

case would take into account the drug involved and the laboratory resources avail-

able to the analyst. It is also accepted that unique requirements in dierent jurisdic-

tions may dictate the actual practices ollowed by a particular laboratory.

When possible, three entirely dierent analytical techniques should be used, orexample: colour tests, chromatography (e.g. TLC, GC or HPLC) and spectroscopy

(e.g. IR or UV). Hyphenated techniques, such as gas chromatography mass spec-

trometry (GC-MS), count as two parameters, provided the inormation rom both

techniques is used (i.e. retention time and mass spectral characteristics).

3.1 Sampling

Seizures o barbiturates and related substances may consist mainly o pharmaceuticaldrugs ormulated as tablets, capsules, oral liquids or injectables. Sampling depends

on the number and type o dosage units seized.

The principal reason or a sampling procedure is to permit an accurate and mean-

ingul chemical analysis. Because most methods, qualitative and quantitative, used

in orensic drug analysis laboratories require very small aliquots o material, it is

vital that these small aliquots be representative o the bulk rom which they have

been drawn. They should conorm to the principles o analytical chemistry, as laid

down, or example, in national pharmacopoeias or by regional or international

organizations. The use o an approved sampling system also helps to preserve valu-able resources and time by reducing the number o determinations needed. The

Guidelines on Representative Drug Sampling [6] provides the general aspects o

qualitative sampling o multi-unit samples. However, it is recognized that there may

be situations where, or legal reasons, the normal rules o sampling and homogeni-

zation cannot be ollowed.

Having chosen the materials to be examined, the analyst must identiy the specic

drug present and quantiy the amount o the drug present i this is required. The

ull physical description o the exhibit should be carried out and, i rom a licit

source, should be identied by reerence to the appropriate databases and the identityconrmed. The analyst should bear in mind that i the dosage orms appear to be

rom an illicit source, i.e. not recognizable as a pharmaceutical product, then a ull

chemical analysis o the material including extraction o the drug, presumptive tests,

screening and conrmatory tests will be required.


14/78


3.2 Extraction and sample preparation

Extraction techniques or substances in tablets, capsules, aqueous solutions (or

injection), syrups, residue rom syringes should be mentioned and reerence madeto relevant pharmacopoeias. General aspects, e.g. solubility or the relevant group

o substances, acid or salt concentration o the medicament in the licit preparation,

should be considered (see annex I).

Virtually all o the barbiturates encountered in illicit trac appear in the orm o

tablets, capsules and bulk powder diverted rom legitimate sources. They are present

as the ree acid or as the sodium or calcium salt (see annex I).

For qualitative analysis

Both the ree acids and the salts are soluble in methanol and this is the solvent o

choice or sample preparation or presumptive or qualitative analysis.

Method

Triturate a quantity o nely powdered tablet or capsule contents or bulk drugpowder with a small amount o methanol sucient to obtain a solution contain-

ing approximately 1 to 20 mg/ml o barbiturate. The extract may be used directlyor ltered and evaporated to dryness under a stream o nitrogen.

For quantitative analysis

(a) Capsules and tablets containing barbiturates in the ree acid orm

Method

Combine the contents o a representative number o capsules or tablets as deter-mined by the sampling procedure. Transer an accurately weighed amount o thecapsule or tablet contents, equal to the ull weight o one or more tablets orcapsules to a suitably sized volumetric fask and make up to volume with ethylacetate. The extract may be used directly or an aliquot removed, ltered andevaporated to dryness under a stream o nitrogen.

(b) Capsules and tablets containing barbiturates in the salt orm

Method

Dissolve or suspend an accurately weighed amount o the representative sampleo barbiturate as determined by the sampling procedure above, in an appropriate


15/78

I. Barbiturates 7

3.3 Presumptive tests

Tablet and capsule identication guides

As a rst test, analysts should reer to national identication guides or presumptiveidentication o the barbiturate products commonly available in their country. Some

useul guides [7, 8, 9, 10] include pictorial representations o legitimate capsule

and tablet orms to assist in identication.

Colour tests

It must be stressed that a positive result rom a colour test is only a presumptive

indication o the possible presence o a barbiturate derivative. Nevertheless, the

colour test suggested below [11, 12] is very useul because all compounds in the

barbiturate class react in a similar manner and very ew other drugs cross-react to

give the same colour with the test reagents. However, no inormation as to which

particular barbiturate is present can be obtained with this colour test.

(a) Dille-Koppanyi test

Solution 1: Dissolve 0.1g cobaltous acetate tetrahydrate in 100 mlabsolute methanol, then add 0.2 ml glacial acetic acid.

Solution 2: Mix 5 ml isopropylamine with 95 ml absolute methanol.

Method

Three drops o solution 1, ollowed by three drops o solution 2 are added toa small amount o the suspected sample on a test plate. It is recommended toseparately perorm negative controls and use reerence material o thesuspected barbiturate or positive results.

Results

A purple colour indicates the presence o a barbiturate but does not indicate aspecic barbiturate.

volume o water (10ml) in a separating unnel. Add 3N HCl dropwise to acidiythe solution. Extract with several 10ml portions o ethyl acetate. Combine theseethyl acetate extracts and lter through glass wool. Bring the ltered extract to

a suitable, known volume with ethyl acetate. The extract may be used directlyor an aliquot removed and evaporated to dryness under a stream o nitrogen.


16/78


(b) Koppanyi-Zwikker test

This is a modication o the Dille-Koppanyi test.

Solution 1: Prepare a 1 per cent w/v solution o cobalt nitrate in ethanol,then add 10l o pyrrolidine

Method

The sample to be tested is dissolved in 1ml o ethanol on a test plate to whichone drop o solution 1 is added and agitated.

Results

Barbiturates give a blue-violet colour. However, a number o other imide andsulphonamide-type drugs also react.

Note: The colours described or positive results o the above presumptive testsare subjective judgements due to individual perception o colours. Because o thissubjective aspect o colour evaluation, it is necessary or each analyst to testappropriate reerence standards in order to ensure that the colour o each testresult can be recognized. Similarly, it is advisable to carry out a blank test withoutthe target substance to ensure amiliarity with the colour o the reagent. Whenperormed properly, a negative colour test is generally quite reliable in establishingthe absence o a target compound. However, positive results are only presumptiveindications o the possible presence o a compound. Many other compounds,oten harmless and uncontrolled by national legislation or international treaties,may give similar colours with a given colour test reagent. Thereore, it is manda-

tory or the analyst to conrm a positive colour test or any legally controlledcompound by the use o additional laboratory analysis. To eliminate the possibilityo a alse positive result due to a contaminated spot plate, it may be advanta-geous to place the reagent onto the spot plate, and then add a small quantityo the sample to the reagent.

Salt determinationFor quantitative purposes, it is necessary to know whether the barbiturate is present

as a ree acid or in a salt orm. Test (a) is mainly applicable to bulk powder. As

excipients in tablets and capsules may interere with the observation, test (b) may

be more appropriate or those preparations.


17/78

I. Barbiturates 9

(a) Solubility

Place small amounts o the suspect material in each o two test tubes. Add several

drops o water to the rst test tube and several drops o ethyl acetate to the second.Observe in which solvent the material dissolves. Free acids are soluble in organic

solvents such as ethyl acetate, but are insoluble in water. The salt orms o the

barbiturates are readily soluble in water, but are insoluble in ethyl acetate. Other

organic solvents such as ether and chloroorm may substitute the ethyl acetate.

(b) pH determination

Place a small amount (ca.10-20 mg) o the suspected barbiturate in a test tube and

add 1 ml o water. Determine the pH. A pH greater than 8.0 indicates that the

barbiturate is present as the sodium or calcium salt.

3.4 Thin-layer chromatography (TLC)

TLC is a commonly used technique or the separation and identication o illicitly

manuactured drugs. It is inexpensive, rapid, sensitive (sub-milligram quantities o

analyte required), fexible in the selection o both the stationary and mobile phase

and amenable to a wide variety o substances, in base and salt orm, ranging rom

the most polar to non-polar materials. A variety o visualization techniques can beused. However, in many countries it is not accepted as a single technique or drug

identication.

TLC plates (stationary phases)

Coating: Silica gel G with a layer thickness o 0.25 mm and containing an inert

indicator, which fuoresces under UV light wavelength 254 nm (Silica gel GF254).

Typical plate sizes: 20 x 20 cm; 20 x 10 cm; 10 x 5 cm (the latter should be usedwith the 10 cm side vertical with the TLC tank).

Plates prepared by the analyst must be activated beore use by placing them into

an oven at 120C or at least 10 to 30 minutes. Plates are then stored in a grease-

ree desiccator over blue silica gel. Heat activation is not required or commercially

available coated plates.

Solvent systems (mobile phases, by volume)

System A: Ethyl acetate, methanol, 25 per cent ammonia solution (85 : 10 : 5 v/v/v)

System B*: Chloroorm, acetone (80 : 20 v/v)

* Note: Due to the carcinogenic potential o chloroorm this system should only be used with appropriatesaety precautions and ventilation.


18/78


Preparation o solutions to be applied to the TLC plate

Extract the material using the method outlined in section 3.2. Prepare a solution o

the sample in methanol at a concentration o approximately 5 mg/ml. All standard

solutions should be prepared at a concentration o 5 mg/ml in methanol. Apply 1

to 2 l o the sample and standard solutions to the plate.

Visualization

The plates must be dried prior to visualization. This can be done at 120C or 5

minutes in an oven or by using a hot air blower.

Visualization methods

A. UV light at 254 nm

First observe the plate under short wavelength UV light (254nm). Expose the plate

to concentrated ammonia vapours and observe again under UV light at the same

wavelength.

B. Mercuric chloride-diphenylcarbazone spray reagent

Dissolve 0.1g o diphenylcarbazone in 50 ml o ethanol to produce solution A.Dissolve 1g o mercuric chloride in 50 ml o ethanol to produce solution B. The

two solutions (A and B) should be reshly prepared and mixed just beore use.

Barbiturates give blue-violet spots on a pink background. The detection limit is

about 1-5g.

Note: Mercuric chloridediphenylcarbazone is the most sensitive spray reagent

among the many tested or the detection o barbiturates. However, the use omercury salts cannot be recommended because o environmental concerns. Detec-tion by visualization method 1 is sucient, but should the use o this reagentstill be required, the spraying procedure must be perormed with special care toguard against harmul mercury vapours.

Interpretation

Ater visualization, mark spots (e.g. with a pencil) and calculate retardation actor(R) values.

R = (Migration distance: rom origin to centre o spot)/(Development distance:

rom origin to solvent ront).


19/78

I. Barbiturates 11

Results (expressed as R x 100)

COMPOUNDS

Developing system

A B

Allobarbital 31 50

Amobarbital 40 52

Barbital 33 4

Butalbital 44 54

Butobarbital 39 50

Cyclobarbital 35 50

Methylphenobarbital 41 70

Pentobarbital 44 55

Phenobarbital 29 47

Secbutabarbital 44 50

Secobarbital 42 55

Vinybital 40 38

3.5 Gas chromatography (GC) with fame ionization detector(FID) [13, 14]

The GC instrument o choice or routine analytical work is the narrow bore capillary

gas chromatograph, using columns with an internal diameter o between 0.2 and

0.32 mm. It is recognized that there are laboratories that, or a variety o reasons,

may wish to maintain a packed column system. For those laboratories, a method

Analytical notes

R values are not always reproducible due to small changes in plate composition

and activation, in solvent systems, tank saturation or development distance. There-ore, the R values provided are indications o the chromatographic behaviour othe substances listed.


20/78


using packed columns was described in the earlier edition o this manual

(ST/NAR/18) and is not included in this revised version.

Capillary column technique without derivatization

Operating conditions

Column: 25m x 0.35mm: lm thickness 0.52m or narrower

Phase: Fused silica, chemically bonded and cross-linked methylsili-cone or methylphenylsilicone, such as OV-1, SE-30, SE-54

or equivalent

Carrier: Nitrogen at 1ml/min (or helium)

Injector: Split/splitless, 275C

Split ratio: 20:1

Oven: Isothermal at 200C or programmed rom 200-260C at4C/min

Detector: FID 275C, hydrogen 35 ml/min, air 350 ml/min

Internal standard: n-alkanes

Injection: 1l

Sample preparation

Prepare internal standard, drug standard and sample solution at concentrations o1 mg/ml. Use the sample extract obtained as described in section 3.2. Inject

successively 1l o the sample and standard solutions into the gas chromatograph.

Results

COMPOUND

Retention indices

SE-30 SE-54

Allobarbital 1575 1629

Amobarbital 1695 1751


21/78

I. Barbiturates 13

COMPOUND

Retention indices

SE-30 SE-54

Barbital 1465 1519

Butalbital 1642 1698

Butobarbital 1642 1695

Cyclobarbital 1946 2026

Methylphenobarbital 1875 1950

Pentobarbital 1719 1778

Phenobarbital 1934 2012

Secbutabarbital 1635 1692

Secobarbital 1770 1827

Vinylbital 1712 1774

Note: All o these methods should include the use o appropriate standards.Many analysts preer to derivatize barbiturates using some orm o methylatingagent, but many consider it unreliable or quantitative analysis.

3.6 Gas chromatography - mass spectrometry(GC-MS) [13, 15]

GC-MS is one o the most commonly used techniques or the identication and

quantitation o orensic drug samples. As a hyphenated technique, it combines the

separation power o a GC with the analyte specicity o a spectroscopic technique,

providing highly specic spectral data on individual compounds in a complex

mixture oten without prior separation.

Preparation o sample solutions

The same method o sample preparation described in section 3.5 can be adopted.


22/78


GC-MS operating conditions

Column: 30m x 0.25mm; lm thickness 0.25m

Phase: Fused silica, chemically bonded and cross-linked methylsili-cone or methylphenylsilicone, such as DB-1, OV-1, SE-30,SE-54 or equivalent

Carrier: Helium at 1ml/min; constant fow (or constant pressure)

Injector: Split/splitless, 250-300C (as appropriate)

Oven: Isothermal at 200C or programmed rom 200-260C at

4C/min (same as or capillary GC analysis described aboveor equivanlent)

Detector: Ionization mode: EI mode, 70 eVTranser line temp: 280C (or appropriate)Ion source temp: 230C (or appropriate)Scan parameters: TIC (SIM i required), scan range: to500amu

Results

Identication is accomplished by comparing the retention time and mass spectrum

o the analyte with that o a reerence standard. All compounds identied by GC-MS

and reported by the analyst must be compared to a current mass spectrum o the

appropriate reerence standard, preerably obtained on the same instrument, operated

under identical conditions.

Note: Most spectrometers have their own mass spectral reerence libraries thatmay be commercial or developed by the operator. However, these should be usedor reerence purposes only. Direct comparison o the GC-MS characteristics othe sample under test should be compared with those o an authentic samplederived under the same conditions.

Gas chromatography - mass spectrometry (GC-MS) or selected barbiturates

A GC-MS method or selected barbiturates including allobarbital, amobarbital, bar-

bital, pentobarbital, phenobarbital and secobarbital is presented below. The method

may be adapted or quantitative analysis o these barbiturates [16, 17, 18]


23/78

I. Barbiturates 15

Preparation o barbiturate standard and sample solutions

(a) Barbiturate standard solution

Weigh an appropriate amount o standard barbiturates into a volumetric fask toobtain a nal concentration o approximately 0.1mg/ml o each compound. Dilute

to volume with methanol. This stock solution is stable or at least three months

when stored at -20C.

(b) Barbiturate sample solution

Weigh an appropriate amount o sample into a volumetric fask so that the nal

barbiturate concentration is approximately that o the standard solution. Dilute to

volume with methanol. Depending on the provenance o the sample, any undissolved

solid particulates can be removed by ltration.


Column: HP-5MS, 0.25m lm, used silica capillary, 30m x0.25mm. i.d.

Column temperature: 100C or 1min, then 100-280 at 15C min-1, hold

280oC or 3min.

Injection temp: 250C

Ion source temp: 280C

Injection mode: Splitless

Mobile phase: Helium at 50kPa

Detector: EI mode 70eV; scanning range m/z50 500

Results

COMPOUND Approx. retention times of barbiturates (min)

Barbital 8.00

Allobarbital 8.98

Amobarbital 9.90Pentobarbital 10.15

Secobarbital 10.55

Phenobarbital 11.90


24/78


3.7 High perormance liquid chromatography (HPLC) [3]

HPLC is one o the major separation techniques commonly used in orensic drug

analysis. Reversed phase chromatography is recommended or the analysis o bar-biturates. There are a large variety o stationary and mobile phases available to the

analyst. The ollowing methods are provided as a guide. The most commonly

encountered and versatile column is a bonded octadecyl silica column (C18). Column

length, diameter, particle size, pore size and carbon load should be considered beore

nal selection o the column.

Preparation o barbiturate standard and sample solutions

(a) Barbiturate standard solution

Weigh an appropriate amount o standard barbiturates into a volumetric fask to

obtain a nal concentration o approximately 1 mg/ml o each compound. Dilute to

volume with methanol. This stock solution is stable or at least three months when

stored at -20C.

(b) Barbiturate sample solution

Weigh an appropriate amount o sample obtained by one o the extraction procedures

outlined in section 3.2 into a volumetric fask, dissolve in and make up to volumewith methanol to produce a nal barbiturate concentration o 1 mg/ml. Depending

on the provenance o the sample, any undissolved solid particulates can be removed

by ltration.

Method 1

Column: 250 mm x 4.6 mm ID

Packing material: Octadecyl-silica HPLC grade, 5m (Spherisorb 5 ODS-2 orequivalent)

Mobile phase: Acetonitrile: water (30:70 by volume)

Flow rate: 0.9 ml/min.

Detection: UV at 220 nm or diode array detection (DAD).

Injection volume: 1-5l

Quantitation: By peak area, external standard method


25/78

I. Barbiturates 17

Method 2

Column: 150 mm x 4.6 mm ID

Packing material: Octadecyl-silica HPLC grade, 5m

(ODS-Hypersil, or equivalent)

Mobile phase A: 0.1M sodium dihydrogen phosphate buer: methanol(60:40 by volume; adjust pH to 3.5 units with phosphoricacid)

Mobile phase B: 0.1M sodium dihydrogen phosphate buer: methanol

(60:40 by volume; adjust pH to 8.5 units with sodiumhydroxide solution)

Flow rate: 2.0 ml/min (low fow values should be considered withshorter columns and smaller particle sizes).

Detection: UV at 216 nm

Injection volume: 1-5l by syringe or loop injection.

Quantitation: By peak area, external standard method.

Results

COMPOUND

Capacity ratios (k values)

Method 1 Method 2

Mobile phase A Mobile phase B

Allobarbital 1.35 2.46 1.33

Amobarbital 4.86 10.91 7.05

Barbital 0.60 1.11 0.63

Butalbital 2.90 6.17 3.48

Butobarbital 2.56 5.43 3.42

Cyclobarbital 2.56 5.25 2.61

Methylphenobarbital 5.72 7.27 3.84

Pentobarbital 4.63 10.96 8.07


26/78


COMPOUND

Capacity ratios (k values)

Method 1 Method 2

Mobile phase A Mobile phase B

Phenobarbital 1.94 3.09 1.23

Secbutabarbital 2.24 4.89 3.32

Secobarbital 6.81 16.28 11.47

Vinylbital 4.86 10.40 7.05

The ollowing HPLC method [17, 19], involving the use o an internal standard,

may be adopted or the quantication o the selected barbiturates allobarbital, amo-

barbital, barbital, cyclobarbital, metharbital, pentobarbital, phenobarbital and

secobarbital.

Method

Column 1: 2m TSK gel Super-ODS C18

-reverse phase, 100 x4.6 mm.

Column 2: 5m Hypersil-ODS C18

-reverse phase, 100 x 4.6 mm.

Column temperature: 23C

Injection: 2l

Mobile phase: 30 per cent acetonitrile, 70 per cent (8 mM KH2O

3)

Flow rate: 0.4 ml/min

UV wavelength: 215 nm

Internal standard: 5-(4-methylphenyl)-5-phenylhydantoin (MPPH)

Quantitation: By peak area ratio, internal standard method


27/78

I. Barbiturates 19

Results

COMPOUND

Retention times of barbiturates (min)

Column 1 Column 2

Barbital 3.8 4.5

Allobarbital 5.1 5.7

Metharbital 5.9 7.2

Phenobarbital 6.1 8.8

Cyclobarbital 7.1 9.6

Pentobarbital 10.6 14.8

Amobarbital 10.9 15.8

Secobarbital 14.2 19.8

3.8 Inrared spectroscopy [12]

Theoretically, each substance has a unique inrared spectrum and this method wouldpermit the unequivocal identication o any barbiturate but would not distinguish

between enantiomers.

For powders, considered rom prior chromatographic analysis to be reasonably pure,

the inrared spectrum may be run directly in a KBr disk or comparison with the

spectra o the barbiturate ree acids or salts. However, with licit pharmaceutical

tablet and capsule samples separation o individual barbiturates rom excipients and

their isolation in pure orm is essential. For tablets, capsules and powders suspected

to be mixtures, the extraction procedures outlined in section 3.2. above may be used

to isolate the barbiturate as the ree acid.

An additional diculty in comparing inrared spectra arises rom the existence o

polymorphic orms o some barbiturates giving rise to dierences in the inrared

spectra. Interestingly, the very act o grinding a barbiturate sample to prepare, or

example a halide disk or nujol mull, may result in a change o crystalline orm. To

overcome this diculty, analysts should subject the standard barbiturate to the same

manipulations as the sample. This should convert the standard to the same crystal-

line orm as the sample and give good comparative inrared spectra.


28/78


29/78

21

II. Benzodiazepines and relatedsubstances

The abuse or misuse o benzodiazepines is internationally widespread which means

that any orensic laboratory may encounter a range o these compounds. In general,

benzodiazepines encountered in the illicit market are diverted rom legitimated

sources. The benzodiazepines, specically aprazolam and diazepam, are among the

most oten diverted and abused psychotropic substances [2]. In a ew cases,

combination products such as chlordiazepoxide-amitriptyline and chlordiazepoxide-

clidinium bromide also appear on the illicit market.

Analysts should be aware o the particular benzodiazepines commonly available in

their area as well as the characteristics and methodologies or their identication

and analysis. As benzodiazepine derivatives end up on the illicit market rom diver-

sion rom legitimate sources, reerence should be made to national pharmacopoeias

and drug tablet and capsule identication guides or preliminary screening inorma-

tion. Reerence can also be made to theMultilingual Dictionary o Narcotic Drugs

and Psychotropic Substances under International Control [5] (MLD, ST/NAR/1/

Rev.2) which provides inormation on the nomenclature o the substances under

international control, includes a listing o many brand names or the controlled

benzodiazepines and provides inormation on their control status.

1. Classications and denitions

Benzodiazepines enhance the eect o the neurotransmitter gamma-aminobutyric

and are subsequently used therapeutically as tranquilizers, hypnotics, anticonvulsantsand centrally acting muscle relaxants. Numerous benzodiazepines have reportedly

been synthesized and over ty o these are marketed or clinical use throughout

the world. Out o the marketed benzodiazepines, thirty-ve are subject to international

control under the 1971 Convention on Psychotropic Substances.


30/78


The classical benzodiazepines are based on a 5-aryl-1,4-benzodiazepine structure

(gure 2, below).

Some o the compounds that are classied alongside them are, strictly speaking, not

benzodiazepines. For example, brotizolam (gure 3) (see annex II) is a thienotria-

zolo-1,4-diazepine. Additionally, some compounds such as clobazam (gure 4) are

actually 1,5-benzodiazepines.

The benzodiazepines are ormulated mainly as capsules and tablets. However some

are available in other pharmaceutical orms such as injectable solutions. Diazepam,

the most traded and widely available benzodiazepine, can be ound as capsules,

tablets, aqueous or polyethyleneglycol solutions or injection, syrups and

suppositories.

2. Description o pure compounds [5, 21]

(see also annex II)

The inormation related to each o the thirty-ve benzodiazepines currently under

international control, can be ound in the annex II. In addition, the Multilingual

Dictionary o Narcotic Drugs and Psychotropic Substances under International

N

N

R1

R3R2

R4

R5

(2

) basic 5-phenyl-1,4-benzodiazepine skeleton

1

4

N

N

N

N

S

Br

Cl

N

N

Cl

O

O

(4) clobazam

1

5

(3) brotizolam

(2) basic 5-phenyl-1,4-benzodiazepine skeleton

(3) brotizolam (4) clobazam


31/78

II. Benzodiazepines and related substances 23

Control [5] (MLD, ST/NAR/1/Rev.2) provides inormation on the nomenclature o

the benzodiazepine derivatives under international control. It also includes synonyms

and dierent trade names or the relevant substances and provides inormation on

their control status.

3. Qualitative and quantitative analysis omaterials containing benzodiazepines

Generally, in attempting to establish the identity o a controlled drug in suspected

material, the analytical approach must entail the determination o at least two uncor-

related parameters. It is recognized that the selection o these parameters in anyparticular case would take into account the drug involved and the laboratory resources

available to the analyst. It is also accepted that unique requirements in dierent

jurisdictions may dictate the actual practices ollowed by a particular laboratory.

When possible, three entirely dierent analytical techniques should be used, or

example: colour tests, chromatography (e.g. TLC, GC or HPLC) and spectroscopy

(e.g. IR or UV). Hyphenated techniques, such as gas chromatography mass

spectrometry (GC-MS), count as two parameters, provided the inormation rom

both techniques is used (i.e. retention time and mass spectral characteristics).

3.1 Sampling

Similar to barbiturates, seizures o benzodiazepines may consist mainly o pharma-

ceutical substances ormulated as tablets, capsules, oral liquids or injectables and

should be sampled depending on the number and type o dosage units seized. The

Guidelines on Representative Drug Sampling [6] provides the general aspects o

qualitative sampling o multi-unit samples. Having chosen the materials to be exam-

ined, the analyst must identiy the specic drug present and quantiy the amount o

the drug present, i this is required. The ull physical description o the exhibit

should be carried out and, i rom a licit source, identied by reerence to the

appropriate databases and the identity conrmed. The analyst should bear in mind

that i the dosage orms appear to be rom an illicit source, i.e. not recognizable as

a pharmaceutical product, then a ull chemical analysis o the material including

extraction o the drug, presumptive tests, screening and some conrmatory test will

all be required.

3.2 Extraction and sample preparation

Virtually all o the benzodiazepines encountered in the illicit trac appear in the

orm o tablets, capsules, liquid preparation and bulk powder diverted rom


32/78


legitimate sources. They are usually present as the ree base or as the hydrochloride,

mesilate or other salt. However, some also have carboxylic acid unctionalities and

may be presented as potassium salts, e.g. chlorazepate. All the benzodiazepines are

generally soluble in methanol (see annex II) The analyst should reer to the relevantormularies or pharmacopoeias or the published content o the drug.

For qualitative analysis

Both the ree base/acid and the salts are soluble in methanol and this is the solvent

o choice or sample preparation or presumptive or qualitative analysis.

Method

Triturate a quantity o nely powdered tablet or capsule contents or bulk drugpowder with a small amount o methanol sucient to obtain a solution contain-ing approximately 1 to 20mg/ml o the benzodiazepine. The extract may be useddirectly or ltered and evaporated to dryness under a stream o nitrogen.

For quantitative analysis

Method

Combine the contents o a representative number o capsules or tablets as deter-mined by a sampling procedure. Transer an accurately weighed amount o thecapsule or tablet contents, equal to the ull weight o one or more tablets orcapsules to a suitably sized volumetric fask and dilute to volume with methanolto give a nal concentration o approximately 1 to 20mg/ml.

3.3. Presumptive tests

Tablet and capsule identication guides [7, 8, 9, 10]

As a rst test, analysts should reer to national identication guides or presumptive

identication o the benzodiazepine products commonly available in their country.Some useul guides include pictorial representations o legitimate capsule and tablet

orms to assist in identication. For example, capsules and tablets may be identied

using TICTAC [7] (www.tictac.org) or other available pharmaceutical identication

databases.


33/78


Colour tests [11, 22]

With such a large group o drugs containing diverse chemical unctionality, no one

colour test is specic or this class o drug. Subsequently, specic colour tests are

not recommended although the Zimmerman test is oten applied.

Zimmerman test

Solution 1: Prepare a solution containing 2,4-dinitrobenzene (1 per cent w/v) in

methanol.

Solution 2: Prepare a 15 per cent potassium hydroxide aqueous solution.

Method

On a micro-test plate, place sucient quantity o the sample to be tested, addone drop o solution 1 ollowed by one drop o solution 2, and mix. A red-purpleto pink colour indicates the probable presence o a benzodiazepine. A reerencematerial o the suspected drug should be tested at the same time.

This test is not specic to this class o compounds. Analysts are thereore advised

to use a combination o TLC and colour development ater spraying with selected

reagents as a presumptive test.

3.4 Thin-layer chromatography (TLC)

Benzodiazepines orm a diverse group o chemicals; however, the ollowing three

thin-layer chromatographic systems, when used in combination, give good separations

or a number o benzodiazepines. System C is a general system recommended alsoor the identication and analysis o cocaine, opium and amphetamine/

methamphetamine.

TLC plates (stationary phases)

Coating: Silica gel G with a layer thickness o 0.25 mm and containing an inert

indicator, which fuoresces under UV light wavelength 254 nm (Silica gel GF254).

Typical plate sizes: 20 x 20 cm; 20 x 10 cm; 10 x 5 cm (the latter should be usedwith the 10 cm side vertical with the TLC tank).

Plates prepared by the analyst must be activated beore use by placing them into

an oven at 120C or at least 10 to 30 minutes. Plates are then stored in a


34/78


grease-ree desiccator over blue silica gel. Heat activation is not required or

commercially available coated plates.

Solvent systems (mobile phases, by volume)

System A*: Chloroorm: acetone ( 80:20, v/v)

System B*: Chloroorm, methanol (90 : 10 v/v)

System C: Cyclohexane: toluene: diethylamine (75:15:10 v/v/v)

*Due to the carcinogenic potential o chloroorm, solvent systems A and B should only be used withappropriate saety precautions and ventilation.

Preparation o solutions to be applied to the TLC plates

Powder: Prepare a solution at a concentration o 5 mg/ml in

methanol.

Capsules: Remove the contents o a representative sample o capsules

and prepare a solution containing the equivalent o approxi-

mately 5 mg/ml o the drug in methanol.

Tablets: Grind a representative number o tablets to a ne powder and

prepare a solution containing the equivalent o approximately

5mg/ml o the drug in methanol.

Aqueous solutions: Spot directly or use a concentration o o 5mg/ml i the con-

centration o the drug is known.

Standard solutions: All solutions made at a concentration o 5mg/ml in

methanol.

Apply 2l o a 5mg/ml solution o the drug in methanol to the plate.

Visualization

The plates must be dried prior to visualization. This can be done at 120C or

5 minutes in an oven or using a hot air blower. It is important or proper colour

development that all traces o diethylamine be removed rom the plate.

Visualization methods

A. UV light at 254 nm

B. 2N H2SO

4/heat/observe under UV light at 366 nm

C. Acidied potassium iodoplatinate reagent


35/78


Spray reagent

Acidied potassium iodoplatinate reagent: Dissolve 0.25g o platinic chloride and

5g o potassium iodide in sucient water to produce 100 ml. This is potassium

iodoplatinate reagent; or the acidied version add 5ml o concentrated hydrochloric

acid to 100 ml o iodoplatinate solution.

Method

First observe the plate under short wave UV light. Spray with 2N H2SO

4and heat

in an oven at 80C or 5 minutes. Observe the fuorescent spots under long UV

light (366nm). The plate may then be oversprayed with the acidied iodoplatinate

reagent. All benzodiazepines give purple coloured spots when oversprayed with the

reagent (alkaloid positive reaction). Other alternative methods are described in theliterature [23, 24, 25, 26].

Results

COMPOUND

R x 100 values

System

A B C

Alprazolam 1 8 62Bromazepam 3 13 55

Camazepam 11 73 89

Chlordiazepoxide 2 15 58

Clobazam 11 68 83

Clonazepam 0 47 60

Clorazepic acid* 4 44 65

Clotiazepam 34 75 88

Cloxazolam 13 48 81

Delorazepam 7 46 69

Diazepam 30 70 87

Estazolam 1 11 50

Ethyl lofazepate 1 63 70

Fludiazepam 29 67 84

Flunitrazepam 16 67 86

Flurazepam 38 5 48

Halazepam 21 76 88

Haloxazolam 13 8 85

Ketazolam** 12,30 66,70 86


36/78


COMPOUND

R x 100 values

System

Loprazolam 2 3 49Lorazepam 1 31 48

Lormetazepam 9 63 81

Medazepam 45 72 89

Nimetazepam 12 45 87

Nitrazepam 1 42 64

Nordazepam* 5 44 63

Oxazepam 1 29 48

Oxazolam 4,15,19*** 69 63

Pinazepam 27 81 90

Prazepam 35 79 90

Temazepam 10 63 82

Tetrazepam 32 74 88

Triazolam 1 9 52

*Clorazepic acid converts to nordazepam.

**Ketazolam decomposes to diazepam.

***Multiple spots.

3.5 Gas chromatography (GC) with fame ionization detector(FID) [12, 28, 29, 30, 31]

Although gas chromatography can be recommended as a suitable method or the

analysis o most benzodiazepines, several o them, particularly the 3-hydroxyderiva-tives, undergo thermal degradation and rearrangements. Chlordiazepoxide, cloxa-

zolam, lormetazepam, haloxazolam, oxazolam, ethyl lofazepate and temazepam

yield multiple peaks. Similarly while clorazepic acid gives a single peak having a

retention index value corresponding to nordazepam, the commercial product which

is the dipotassium salt can not be analysed. Ketazolam gives a single peak corre-

sponding to diazepam. Loprazolam does not elute rom the GC columns under the

experimental conditions described in this manual.

Retention indices or benzodiazepines have been shown to be dependent on columntemperature. Values should be checked beore use by analysing ew reerence stand-

ards o known retention indices. As with the barbiturates, GC methods involving

the use o packed columns have not been included in this manual. Laboratories

using packed columns are reered to the previous manual on benzodiazepines [4].


37/78


Capillary column technique


Column: 25m x 0.25 mm: lm thickness 0.25m

Phase: Fused silica, chemically bonded and cross-linked methylsilicone,such as BP-1, DB-1 or equivalent

Carrier: Nitrogen at 1ml/min (or helium)

Injector: Split/splitless, 275C

Split ratio: 20:1

Oven: 250C

Detector: FID 275C, hydrogen 35 ml/min, air 350 ml/min

Injection: 1l

Preparation o solutionsSolutions o the standards and samples are prepared in methanol at a concentration

o 1 mg/ml.

Results

COMPOUND

Retention indices

BP-1 Capillary

Alprazolam 2936

Bromazepam 2626

Camazepam 2954

Chlordiazepoxide* 2815 (2531, 2646)

Clobazam 2582

Clonazepam 2833

Clorazepic acid* 2521 (2783)

Clotiazepam 2485

Cloxazolam* 2344 (2604)

Delorazepam 2537


38/78


COMPOUND

Retention indices

BP-1 Capillary

Diazepam 2477

Estazolam 2893

Ethyl lofazepate 2925 (2878)

Fludiazepam 2403

Flunitrazepam 2633

Flurazepam 2795

Halazepam 2314Haloxazolam* 2634 (2518, 2272)

Ketazolam* 2475

Loprazolam* Does not elute

Lorazepam 2448

Lormetazepam* 2703 (2946)

Medazepam 2287

Nimetazepam 2693

Nitrazepam 2755

Nordazepam 2522

Oxazepam 2374

Oxazolam 2514 (2534, 2212)

Pinazepam 2551

Prazepam 2676

Temazepam 2613 (2828)

Tetrazepam 2463

Triazolam 3025

* Thermally unstable.( ) Minor peaks in brackets

3.6 Gas chromatography - mass spectrometry (GC-MS)

GC-MS is one o the most commonly used techniques or the identication and

quantitation o orensic drug samples. As a hyphenated technique, it combines the


39/78


separation power o a GC with the analyte specicity o a spectroscopic technique,

providing highly specic spectral data on individual compounds in a complex mix-

ture o compounds oten without prior separation.

Preparation o solutions

Solutions o the standards and samples are prepared in methanol at a concentration

o 1 mg/ml

GC-MS operating conditions

Column: 10-15 m x 0.32 or 0.53 mm ID; lm thickness 1.5-3m

Phase: Fused silica, chemically bonded and cross-linked methylsilicone,e.g. DB-1 or equivalent

Oven Temp. prog., 4 min at 135, 13/min to 200, 6/min to 312,6 min nal hold (or similar programmes)

Carrier gas: Helium, 1 ml/min; constant fow (or constant pressure)

Injector: Split/splitless, 250-300C as appropriate

Detector: Ionization mode: EI mode, 70 eV

Transer line temp: 280C (or appropriate)

Ion source temp: 230C (or appropriate)

Scan parameters: TIC (SIM i required), scan range: to500amu

Results

Identication is accomplished by comparing the retention time and mass spectrum

o the analyte with that o a reerence standard. All compounds identied by GC-MS

and reported by the analyst must be compared to a current mass spectrum o the

appropriate reerence standard, preerably obtained on the same instrument, operated

under identical conditions.

3.7 High perormance liquid chromatography (HPLC)[31, 32, 33, 34, 35]

Because o the diversity o chemical structure among benzodiazepines, the use o

a single HPLC method to separate all possible compounds is dicult. Nevertheless,

the ollowing methods are recommended or the qualitative and quantitative analysis

o all benzodiazepine derivatives under international control. The selection o column


40/78


and solvent system by national laboratories will depend upon the particular benzo-

diazepines commonly available in that country.

Preparation o standard and sample solutions

Benzodiazepine standard solution

Weigh an appropriate amount o the benzodiazepine standard into a volumetric fask,

dissolve in and make up to volume with 50 per cent aqueous methanol to produce

a solution o concentration 1mg/ml.

Benzodiazepine sample solution

Weigh an appropriate amount o the sample obtained by one o the extraction meth-

ods outlined in section 3.2 into a volumetric fask, dissolve in and make up to

volume with 50 per cent v/v aqueous methanol to produce a nal benzodiazepine

concentration o approximately 1mg/ml. Depending on the provenance o the sample,

any undissolved particulates can be removed by ltration.

Method

Column: 250 mm by 5 mm ID

Packing material: Octadecyl-silica HPLC 5m diameter (ODS-Hypersil orequivalent)

Mobile phase: A. Methanol: water: phosphate buer (0.1M),(55:25:20, v/v/v), pH 7.25

B. Methanol: water: phosphate buer (0.1M),(70:10:20, v/v/v), pH 7.67

The phosphate buer (0.1M) is prepared by dissolving sodium dihydrogen-phosphate dehydrate (14.35g, 0.092 mol) and disodium hydrogen phosphate(1.14g, 0.008 mol) in 1000ml water.

Flow rate: 1.5 ml/min

Detector: UV at 240nm or DAD

Injection volume: 20l

Quantitation: By peak areas, external standard method


41/78


Results

Capacity ratios, k values or a selection o benzodiazepines in the two mobile phases

are provided below:

Capacity ratios, k values

COMPOUND Mobile Phases

A B

Alprazolam 3.35 1.28

Bromazepam 1.63 0.80

Camazepam 11.80 2.56

Chlordiazepoxide 6.68 1.65

Clobazam 4.14 1.09

Clonazepam 2.92 0.79

Clorazepic acid* 8.22 1.84

Clotiazepam 17.91 3.01

Cloxazolam 15.22 2.82

Delorazepam 7.20 1.53

Diazepam 10.41 2.29

Estazolam 4.22 1.08

Ethyl lofazepate 13.71 2.19

Fludiazepam 7.91 1.67

Flunitrazepam 3.34 0.86

Flurazepam 12.98 3.12

Halazepam 18.92 2.91

Haloxazolam 11.62 2.11

Ketazolam 10.78 2.22

Loprazolam 6.42 1.23

Lorazepam 5.16 1.14

Lormetazepam 7.19 1.46

Medazepam 41.46 6.31

Nimetazepam 3.91 1.01

Nitrazepam 3.22 0.87

Nordazepam 8.97 1.89


42/78


Capacity ratios, k values

COMPOUND Mobile Phases

A B

Oxazepam 5.42 1.25

Oxazolam** 22.43, 25.14 3.51,3. 87

Pinazepam 12.82 2.21

Prazepam 29.99 4.28

Temazepam 6.80 1.49

Tetrazepam 25.82 4.25

Triazolam 5.18 1.13

* Converts to Nordazepam during chromatography.** Two peaks due to equilibrium mixture o Z and E isomers [36]

Liquid chromatography - mass spectrometry (LC-MS)

This method represents a rapid, simple and highly sensitive procedure or the simul-

taneous analysis o ourteen benzodiazepines [37, 38, 39, 40, 41, 42] using a triple

quadrupole MS in ESI-mode. Samples o suitable concentration were prepared romthe ollowing deuterated internal standards, D5-diazepam, D5-temazepam, D5-alpra-

zolam, D4-clonazepam, as well as unlabelled standards: alprazolam, bromazepam,

chlordiazepoxide, clonazepam, diazepam, funitrazepam, furazepam, lorazepam,

midazolam, nitrazepam, nordiazepam, oxazepam, temazepam and triazolam. The

limit o quantication o most benzodiazepines uing this methid is approximately

0.5 ng/ml.

LC conditionsInstrument: Agilent 1200 Series RRLC; 6410 LC Triple Quadrupole

Masspectrometer

Column: ZORBAX Eclipse XDB C18

2.1 x 50 mm x 1.8 m

Column temperature: 35C

Injection volume: 5 L

Solvent fow rate: Time (minutes) Flow rate (ml/min)0 0.26.5 0.2

8 1.010 0.2Post time: 4.5 min

Mobile phase Acetonitrile: 20 mM ammonium ormate pH = 8.6(50:50 by vol.)


43/78


MS Conditions

Operation: Electrospray ESI positive mode

Gas temperature: 300C

Gas fow (N2): 6 L/min

Nebulizer pressure: 15 psi (pressure o 30 to 40 psi recommended)

Capillary voltage: 4,500 V

3.8 Inrared spectroscopy

In some countries, conrmation o identity by spectroscopic means may be required.

Theoretically each substance has a unique inrared spectrum and this method should

permit the unequivocable identication o any benzodiazepine. However, with licit

samples, the lack o solubility in chloroorm o certain benzodiazepine derivatives

and the isolation o the drug in a pure orm, ree rom pharmaceutical excipients,

place limitations on this method. The ollowing procedure is recommended. Othermethods utilizing column or preparative thin-layer chromatography may also be

employed. Because o solvate ormation and polymorphism, it is recommended that

an authentic pharmaceutical ormulation be carried through the same extraction

procedure and its spectrum compared with that o the sample.

Isolation o pure drugs rom sample

(a) Benzodiazepines as the hydrochloride salt

Chlordiazepoxide, furazepam and medazepam are marketed as hydrochloride salts.

Triturate the powders rom the capsule contents with 1 to 2ml o methanol. Filter,

collect the extract and evaporate to dryness. Induce crystallization and run the inra-

red spectrum o the resulting benzodiazepine salt by the KBr disk method.

(b) Benzodiazepine ree base

Suspend a portion o the powder or crushed tablet contents containing approximately

10mg o drug in 1ml o 0.1M NaOH solution and extract with CHCl3. Evaporate

the CHCl3 to dryness and induce crystallization.

Other standard methods can be used depending on instrumentation available. The

IR spectrum should be compared to a standard sample prepared/treated in the same

way as the test material.


44/78


45/78

37

References

1. World Drug Report 2011, United Nations Oce on Drugs and Crime, United

Nations publication, Sales No. E.10.XI.13

2. Report o the International Narcotics Control Board or 2010, E/INCB/2010/1,

United Nations Publication Sales No. E.11.XI.1.

3. Recommended Methods or Testing Barbiturate Derivatives under International

Control, ST/NAR/18, United Nations, 1989.

4. Recommended Methods or Testing Benzodiazepine Derivatives under

International Control, ST/NAR/16, United Nations, 1988

5. Multilingual Dictionary o Narcotic Drugs and Psychotropic substances under

International Control, ST/NAR/1/Rev. 2, 2006. Available at www.unodc.org

6. The Guidelines on Representative Drug Sampling, ST/NAR/38, United Nations,

2009

7. The Identication CD-ROM or Tablets And Capsules, TICTAC. Available at:

www.tictac.org

8. Physicians Desk Reerence, 63rd edition. Medical Economics Company,

Oradell, N.J., 2009

9. Compendium o Pharmaceuticals and Specialties, Canadian Pharmacists

Association, Ottawa, 2009. Available at www.e-CPS.ca

10. ITAKA 09, Identifering av Tabletter och Kapslar, Stockholm, 2009. Available

at www.ass.se/LIF/

11. Rapid Testing Methods o Drugs o Abuse, a manual or use by nationalnarcotics laboratories, ST/NAR/13 /Rev.1, United Nations, 1996

12. Clarkes Analysis o Drugs & Poisons, 3rd Edition. Moat A.C., Osselton M.D.

and Widdop B. (eds.) Pharmaceutical Press, 2004


46/78


13. Gill R., Stead A.H. and Moat A.C. Analytical aspects o barbiturate abuse:

identication o drugs by the eective combination o gas-liquid, high-

perormance liquid and thin-layer chromatographic techniques. Journal o

Chromatography, 204: 275-284, 1981

14. Christophersen A.S. and Rasmussen K.E. Glass capillary column gas chroma-

tography o barbiturates ater fash-heater derivatization with dimethyl-ormamide-

dimethylacetal. Journal o Chromatography A, 192(2): 363-374, 1980

15. Rop P.P., Spinazzola J., Zahra A., Bresson M., Quicke J. and Viala A. Column

liquid chromatographic analysis o barbiturates in biological fuids. Journal o

Chromatography, 427: 172-180, 1988. (modied).

16. Namera A., Yashiki M., Iwasaki Y., Ohtani M. and Kojima T. Automated pro-

cedure or determination o barbiturates in serum using the combined system

o PrepStation and gas chromatography-mass spectrometry.Journal o Chroma-

tography B, 716: 171-176, 1998

17. Cole M.D. The Analysis o Controlled Substances. Chapter 9. John Wiley and

Sons, 2003

18. Zhao H., Wang L., Qiu Y., Zhou Z., Zhong W., Li X. Multiwalled carbon

nanotubes as a solid-phase extraction adsorbent or the determination o threebarbiturates in pork by ion trap gas chromatography-tandem mass spectrometry

(GC/MS/MS) ollowing microwave assisted derivatization. Analytica Chimica

Acta, 586(1-2): 399-406, 2007

19. Tanaka E., Terada M., Tanno K., Misawa S. and Wakasugi C. Forensic analysis

o 10 barbiturates in human biological samples using a new reversed-phase

chromatographic column packed with 2-micrometre porous microspherical

silica-gel. Forensic Science International, 85(1): 73-82, 1997

20. Perrett D. Chapter 30. In: Clarkes Analysis o Drugs & Poisons, 3rd Edition.

Moat A.C., Osselton M.D. and Widdop, B. (eds.) Pharmaceutical Press, 2004

21. Hammersley R., Cassidy M.T. & Oliver J. Drugs associated with drug-related

deaths in Edinburgh and Glasgow, November 1990 to October 1992. Addiction,

90(7): 959965, 1995

22. The Identifcation and Analysis o Benzodiazepines under International Control:

I. Colour Tests and Chromatographic Methods, SCITEC/1, Kouidri C. and

Sackda S., United Nations, 1987 becomes 22

23. Snchez-Moyano E., Herrez M. and Pl-Delna J.M. A contribution to the

pharmaceutical analysis o benzodiazepines. Pharmaceutica Acta Helvetiae, 61:

167-176, 1986


47/78

Reerences 39

24. Wouters I., Roets E. and Hoogmartens J. Thin-layer chromatographic identica-

tion o nineteen benzodiazepine derivatives. Journal o Chromatography A,

179(2): 381-389, 1979

25. Roets E. and Hoogmartens J. Thin-layer chromatography o the acid-hydrolysis

products o 19 benzodiazepine derivatives.Journal o Chromatography, 194(2):

262-269, 1980

26. Chiarotti M., De Giovanni N. and Fiori A. Analysis o benzodiazepines: I.

Chromatographic identication. Journal o Chromatography A, 358: 169-178,

1986

27. Watson D.G. Chapter 26. In: Clarkes Analysis o Drugs & Poisons, 3rd Edition.

Moat A.C., Osselton M.D. and Widdop, B. (eds.) Pharmaceutical Press, 2004

28. Ardrey R.E, de Zeeuw R.A., Finkle B.S., Franke J.P., Moat A.C., Mller M.R.,

Mller R.K. Gas Chromatographic Retention Indices o Toxicologically Rele-

vant Substances on SE-30 or OV 1. 2nd Edition, Report II o the DFG Com-

mission or Clinical-Toxicological Analysis, Special Issue o the TIAFT Bulletin.

Aldermaston (GB) and Verlag Chemie Weinheim/Deereld Beach, 1985

29. Schtz H. (eds.) Benzodiazepines Handbook, Volume 2. Springer-Verlag,

Heidelberg, 1989

30. De Zeeuw, A. (Ed.) Gas Chromatographic Retention Indices o Toxicologically

Relevant Substances on Packed or Capillary Columns with Dimethylsilicone

Stationary Phases, 3rd Ed. Report XVII o the DFG Commission or Clinical-

Toxicological Analysis, Weinheim, The International Association o Forensic

Toxicologists & Deutsche Forschungsgemeinschat, 2002

31. Japp M., Garthwaite K., Geeson A.V. and Osselton M.D. Collection o analytical

data or benzodiazepines and benzophenones. Journal o Chromatography A,439(2): 317-339, 1988

32. Mura P., Piriou A., Fraillon P., Papet Y. and Reiss D. Screening procedure or

benzodiazepines in biological fuids by high-perormance liquid chromatography

using a rapid-scanning multichannel detector. Journal o Chromatography B,

416: 303-310, 1987

33. Theis D.L. and Bowman P.B. Development o a liquid chromatographic method

or the determination o triazolo-benzodiazepines. Journal o Chromatography

A, 268: 92-98, 1983

34. Flanagan R.J., Storey G.C.A., Bhamra R.K. and Jane I. High perormance liquid

chromatographic analysis o basic drugs on silica columns using non-aqueous

ionic eluents. Journal o Chromatography A, 247: 15-37, 1982


48/78


35. Chiarotti M., De Giovanni N. and Fiori A. Analysis o benzodiazepines : I.

Chromatographic identication. Journal o Chromatography A, 358: 169-178,

1986

36. Miyadera T., Terada A., Fukanaga M., Kowana Y., Kamioka T., Tamura C.,

Tagaki H. and Tachikawa R. Synthesis and pharmacology o benzo [6,7][1,4]

diazepino [5,4-b] oxazole derivatives and analogs. Journal o Medicinal

Chemistry, 14, 520-526, 1971

37. Moore, C., Coulter, C., Crompton, K. and Zumwalt, Tetrahydrocannabinol and

two o its metabolites in whole blood using liquid chromatography-tandem mass

spectrometry. Journal o Analytical Toxicology 32, 653-658, 2008

38. Smink B.E., Brandsma J.E., Dijkhuizen A., Lustho K.J., De Gier J.J., Egberts

A.C.G. and Uges D.R.A. Quantitative analysis o 33 benzodiazepines, metabo-

lites and benzodiazepine-like substances in whole blood by liquid chromato-

graphy-(tandem) mass spectrometry. Journal o Chromatography B, 811(1):

13-20, 2004

39. Dussy F.E., Hamberg C. and Briellmann T.A. Quantication o benzodiazepines

in whole blood and serum. International Journal o Legal Medicine, 120(6):

323-330, 2006

40. ElSohly M.A., Gul W., ElSohly K.M, Avula B., and Khan I.A. LC-MS-(TOF)

analysis method or benzodiazepines in urine samples rom alleged drug-

acilitated sexual assault victims. Journal o Analytical Toxicology, 30(8): 524-

538, 2006

41. ElSohly M.A., Gul W., Murphy T.P., Avula, B. and Khan I.A. LC-(TOF) MS

analysis o benzodiazepines in urine rom alleged victims o drug-acilitated

sexual assault. Journal o Analytical Toxicology, 31: 505-514, 2007

42. ElSohly M.A., Gul W., Avula B., Murphy T.P. and Khan I.A. Simultaneous

analysis o thirty-ve benzodiazepines in urine using liquid chromatography-

mass spectrometry-time o fight.Journal o Analytical Toxicology, 32(8): 547-

561, 2008

Further reading

The Analysis o Drugs o Abuse. Gough, T.A. (eds.) J. Wiley and Sons, Chichester,

New York, Brisbane, Toronto, Singapore, 1991.


49/78

Further reading 41

Saka K., Uemura K., Shintani-Ishida K. and Yoshida K-I. Determination o amo-

barbital and phenobarbital in serum by gas chromatography-mass spectrometry with

addition o ormic acid to the solvent. Journal o Chromatography B, 869(1-2):

9-15, 2008

Steijns L.S., Bouw J. and van der Weide J. Evaluation o fuorescence polarization

assays or measuring vaproic acid, phenytoin, carbamazepine and phenobarbital in

serum. Therapeutic Drug Monitoring, 24(3): 432-435, 2002

Jiang T-F., Wang Y-H., Lv Z-H. and Yue M-E. Direct determination o Barbiturates

in Urine by CE using a capillary coated dynamically with polycationic polymers.

Chromatographia, 65(9-10): 611-615, 2007.


50/78

42

Annex I. Description of barbiturates underinternational control

Allobarbital

5,5-diallylbarbituric acid; 5,5-di-2propenyl-2,4,6 (1H,3H, 5H) pyrimidinetrione

Sch. IV (1971)

Empirical ormula: C10

H12

N2O

3

CAS No.: 52-43-7

MWt: 208.2

MPt: 171 173 C

Physical appearance: White crystalline powder

Solubility: Soluble in aqueous alkali

Inrared data: Principal peaks at wavenumbers 1687, 1315, 925, 1219, 847,

1640 cm-1 (KBr disk) [3, 12]

NH

HN OO

O


51/78

Annex I. Description o barbiturates under international control 43

Amobarbital (amobarbital sodium)

5-ethyl-5-isopentylbarbituric acid; 5-ethyl-5-(3-methylbutyl)-2, 4,6(1H, 3H,

5H)-pyrimidinetrione

Sch. III (1971)

Empirical ormula: C11H18N2O3 (C11H17N2NaO3)CAS No.: 57-43-2 (64-43-7)

MWt: 226.3 (248.3)

MPt: 157 C (ater conversion to ree acid using method o Ph

Eur)

Physical appearance: Free acid is a white crystalline powder; sodium salt is a

white hygroscopic granular powder)

Solubility: Soluble in aqueous alkali

Inrared data: Principal peaks at wavenumbers 1725, 1696, 1758, 1317,

1240, 850 cm-1 (KBr disk) [3, 12]

Barbital (barbital sodium)

5,5-diethylbarbituric acid; 5,5-diethil-2, 4,6 (1H, 3H, 5H)-pyrimidinetrione

Sch. IV (1971)

Empirical ormula: C8H

12N

2O

3, (C

8H

11N

2NaO

3)

CAS No.: 57-44-3 (144-02-5)

MWt: 184.2 (206.2)

MPt: 188 192 C (247C)

Physical appearance: Free acid is a white crystalline powder (available as sodium

salt)

Solubility: Soluble in alcohol, boiling water and in aqueous alkali.

Inrared data: Principal peaks at wavenumbers 745, 1593, 725, 1495, 690,

1275 cm-1 (KBr disk) [3, 12]

NH

HN OO

O

N

HN ONaO

O

NH

HN OO

O


52/78


Butalbital

5-(2-methylpropyl)-5-(2-propenyl)-barbituric acid

Sch. III (1971)


H16

N2O

3

CAS No.: 77-26-9MWt: 224.3

MPt : 138.5 C

Physical appearance: Free acid is a white crystalline powder

Solubility: Soluble in alcohol, acetone, ether, chloroorm, boiling water

and in aqueous alkali.


1290, 1200 cm-1 (KBr disk) [3, 12]

Butobarbital

5-butyl-5-ethylbarbituric acid

Sch. IV (1971)


H16

N2O

3

CAS No.: 77-28-1

MWt: 212.3

MPt : 126 C

Physical appearance: Free acid is a white crystalline powder

Solubility: Soluble in alcohol, ether and in aqueous alkali

Inrared data: Principal peaks at wavenumbers 1696, 1727, 1760, 1242,850, 1215 cm-1 (KBr disk) [3, 12]

NH

HN OO

O

NH

HN OO

O


53/78

Annex I. Description o barbiturates under international control 45

Cyclobarbital (cyclobarbital calcium)

5-(1-cyclohexen-1-yl)-5-ethylbarbituric acid

Sch. III (1971)


H16

N2O

3([C

12H

15N

2O

3]

2Ca)

CAS No.: 52-31-3 (143-76-0)

MWt: 236.3 (510.6)

MPt : 171 174 C (>300 C)

Physical appearance: Free acid is white crystalline powder that slowly decom-

poses (calcium salt is slightly yellowish crystalline

powder)

Solubility: Soluble in 1 to 5 ethanol and 1 to 20 chloroorm and ether


1210, 830 cm-1

(KBr disk) [3, 12]

Methylphenobarbital

5-ethyl-1-methyl-5-phenylbarbituric acid

Sch. IV (1971)


H14

N2O

3

CAS No.: 115-38-8

MWt: 246.3

MPt : 178 C

Physical appearance: Colourless crystals or white crystalline powder,

NH

HN OO

O

NH

N OO

Oand enantiomer


54/78


Methylphenobarbital (continued)

Solubility: Soluble in hot water, soluble 1 in 40 in chloroorm, soluble

in aqueous alkaliInrared data: Principal peaks at wavenumbers 1707, 1684, 1754, 720,

1298, 1050 cm-1 (KBr disk) [3, 12]

Pentobarbital (pentobarbital calcium; pentobarbital

sodium)

5-ethyl-5-(1-methylbutyl)-barbituric acid

Sch. III (1971)


H18

N2O

3([C

11H

17N

2O

3]

2Ca; C

11H

17N

2NaO

3)

CAS No.: 76-74-4 (7563-42-0; 57-33-0)

MWt: 226.3 (490.6; 248.3)

MPt: 131 C (ater conversion to ree acid using method o Ph

Eur)

Physical appearance: Free acid is a white crystalline powder (sodium salt, white

hygroscopic powder)

Solubility: Soluble in acetone, alcohol, chloroorm, ether and methanol

and in aqueous alkali. (calcium salt sparingly soluble in

Documents

Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines Under International Control (2012)