Professional Documents
Culture Documents
Barbiturates and Benzodiazepines
Barbiturates and Benzodiazepines
UNITED NATIONS
New York, 2012
Note
Operating and experimental conditions are reproduced from the original reference
materials, including unpublished methods, validated and used in selected national
laboratories as per the list of references. A number of alternative conditions and
substitution of named commercial products may provide comparable results in many
cases, but any modification has to be validated before it is integrated into laboratory
routines.
Mention of names of firms and commercial products does not imply the endorse-
ment of the United Nations.
ST/NAR/46
ii
Acknowledgements
The UNODC Laboratory and Scientific Section (LSS, headed by Dr. Justice Tettey),
wishes to express its appreciation and thanks to Professor Alexander Gray, Strath-
clyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde,
United Kingdom, for the preparation of the first draft of the present manual, and to
Dr Pirjo Lillisunde, Head of Drug Laboratory, National Institute for Health and
Welfare, Finland, for the expert review of the document. The preparation of this
manual was coordinated by Dr. Iphigenia Naidis, staff member of LSS. The contri-
bution of other UNODC staff is gratefully acknowledged.
iii
Contents
Page
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Purpose and use of the manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
I. Barbiturates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1. Classifications and definitions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2. Description of pure compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3. Qualitative and quantitative analysis of materials containing
barbiturates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.1 Sampling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.2 Extraction and sample preparation. . . . . . . . . . . . . . . . . . . . . . . . . 6
3.3 Presumptive tests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.4 Thin-layer chromatography (TLC). . . . . . . . . . . . . . . . . . . . . . . . . 9
3.5 Gas chromatography (GC) with flame ionization detector (FID) . 11
3.6 Gas chromatography - mass spectrometry (GC-MS) . . . . . . . . . . 13
3.7 High performance liquid chromatography (HPLC). . . . . . . . . . . . 16
3.8 Infrared spectroscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
v
Page
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Further reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Annex
I. Description of barbiturates under international control . . . . . . . . . . . . . . . . 42
II. Description of benzodiazepines under international control. . . . . . . . . . . . . 49
vi
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-five benzodiazepines are under international control.
Those that have found their way into illicit use have mainly been diverted from the
licit market.
The present manual combines and updates the two existing manuals on R
ecommended
methods for 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
1
2 Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines
providing the basis for reliable forensic evidence on seized materials containing
barbiturate or benzodiazepine derivatives. It is divided into two parts which provide
analytical methodologies for the two types of substances with reference to validated
techniques. Description of the individual barbiturates and benzodiazepines under
international control is provided in two annexes.
In line with the overall objective of the series of UNODC publications, this manual
suggests approaches that may assist drug analysts in the selection of methods appro-
priate to the sample under examination and provide data suitable for the purpose at
hand, leaving room also for adaptation to the level of sophistication of different
laboratories and the various legal needs.
UNODC Laboratory and Scientific Section welcomes observations on the contents and
usefulness of the present manual. Comments and suggestions may be addressed to:
They are synthetic drugs derived from barbituric acid (figure 1 below), which is a
synthetic condensation product of malonic acid and urea. They differ mainly in the
substitution pattern at position-5 with some also including an N-methyl at N-1 (see
3
4 Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines
annex I). The most well known derivative, phenobarbitone (see annex I), has been
used medicinally since 1912, mainly in the treatment of epilepsy.
R1
O N O
1
5 3
R4 N
R2
R3
O
(1) Barbituric
(1) Barbituric acid, R1=-RH 4 = H
acid, R -R 1 4
Over 2,500 barbiturates have reportedly been synthesized with more than 50 of these
presently marketed for clinical use throughout the world. Twelve of these are subject
to international control under the Convention on Psychotropic Substances 1971 as
follows:
In recent years, there has been a significant downturn in prescribing and therefore
the general availability of barbiturates owing to their side-effects 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
for the identification and analysis of Methaqualone/Mecloqualone, ST/NAR/15/
Rev.1), benzodiazepines and related compounds.
When possible, three entirely different analytical techniques should be used, for
example: 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 information from both
techniques is used (i.e. retention time and mass spectral characteristics).
3.1 Sampling
The principal reason for a sampling procedure is to permit an accurate and mean-
ingful chemical analysis. Because most methods, qualitative and quantitative, used
in forensic drug analysis laboratories require very small aliquots of material, it is
vital that these small aliquots be representative of the bulk from which they have
been drawn. They should conform to the principles of analytical chemistry, as laid
down, for example, in national pharmacopoeias or by regional or international
organizations. The use of an approved sampling system also helps to preserve valu-
able resources and time by reducing the number of determinations needed. The
Guidelines on Representative Drug Sampling [6] provides the general aspects of
qualitative sampling of multi-unit samples. However, it is recognized that there may
be situations where, for legal reasons, the normal rules of sampling and homogeni-
zation cannot be followed.
Having chosen the materials to be examined, the analyst must identify the specific
drug present and quantify the amount of the drug present if this is required. The
full physical description of the exhibit should be carried out and, if from a licit
source, should be identified by reference to the appropriate databases and the identity
confirmed. The analyst should bear in mind that if the dosage forms appear to be
from an illicit source, i.e. not recognizable as a pharmaceutical product, then a full
chemical analysis of the material including extraction of the drug, presumptive tests,
screening and confirmatory tests will be required.
6 Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines
Virtually all of the barbiturates encountered in illicit traffic appear in the form of
tablets, capsules and bulk powder diverted from legitimate sources. They are present
as the free acid or as the sodium or calcium salt (see annex I).
Method
Method
Method
Colour tests
It must be stressed that a positive result from a colour test is only a presumptive
indication of the possible presence of a barbiturate derivative. Nevertheless, the
colour test suggested below [11, 12] is very useful because all compounds in the
barbiturate class react in a similar manner and very few other drugs cross-react to
give the same colour with the test reagents. However, no information as to which
particular barbiturate is present can be obtained with this colour test.
(a) Dille-Koppanyi test
Method
Results
A purple colour indicates the presence of a barbiturate but does not indicate a
specific barbiturate.
8 Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines
(b) Koppanyi-Zwikker test
Method
Results
Note: The colours described for positive results of the above presumptive tests
are subjective judgements due to individual perception of colours. Because of this
subjective aspect of colour evaluation, it is necessary for each analyst to test
appropriate reference standards in order to ensure that the colour of each test
result can be recognized. Similarly, it is advisable to carry out a blank test without
the target substance to ensure familiarity with the colour of the reagent. When
performed properly, a negative colour test is generally quite reliable in establishing
the absence of a target compound. However, positive results are only presumptive
indications of the possible presence of a compound. Many other compounds,
often harmless and uncontrolled by national legislation or international treaties,
may give similar colours with a given colour test reagent. Therefore, it is manda-
tory for the analyst to confirm a positive colour test for any legally controlled
compound by the use of additional laboratory analysis. To eliminate the possibility
of a false 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 quantity
of the sample to the reagent.
Salt determination
For quantitative purposes, it is necessary to know whether the barbiturate is present
as a free acid or in a salt form. Test (a) is mainly applicable to bulk powder. As
excipients in tablets and capsules may interfere with the observation, test (b) may
be more appropriate for those preparations.
I. Barbiturates 9
(a) Solubility
Place small amounts of the suspect material in each of two test tubes. Add several
drops of water to the first test tube and several drops of 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 forms of the
barbiturates are readily soluble in water, but are insoluble in ethyl acetate. Other
organic solvents such as ether and chloroform may substitute the ethyl acetate.
(b) pH determination
Place a small amount (ca.10-20 mg) of the suspected barbiturate in a test tube and
add 1 ml of water. Determine the pH. A pH greater than 8.0 indicates that the
barbiturate is present as the sodium or calcium salt.
TLC is a commonly used technique for the separation and identification of illicitly
manufactured drugs. It is inexpensive, rapid, sensitive (sub-milligram quantities of
analyte required), flexible in the selection of both the stationary and mobile phase
and amenable to a wide variety of substances, in base and salt form, ranging from
the most polar to non-polar materials. A variety of visualization techniques can be
used. However, in many countries it is not accepted as a single technique for drug
identification.
Plates prepared by the analyst must be activated before use by placing them into
an oven at 120°C for at least 10 to 30 minutes. Plates are then stored in a grease-
free desiccator over blue silica gel. Heat activation is not required for commercially
available coated plates.
Visualization
The plates must be dried prior to visualization. This can be done at 120°C for 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.
Interpretation
After visualization, mark spots (e.g. with a pencil) and calculate retardation factor
(Rf) values.
Developing system
COMPOUNDS
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
Analytical notes
Rf values are not always reproducible due to small changes in plate composition
and activation, in solvent systems, tank saturation or development distance. There-
fore, the Rf values provided are indications of the chromatographic behaviour of
the substances listed.
The GC instrument of choice for routine analytical work is the narrow bore capillary
gas chromatograph, using columns with an internal diameter of between 0.2 and
0.32 mm. It is recognized that there are laboratories that, for a variety of reasons,
may wish to maintain a packed column system. For those laboratories, a method
12 Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines
using packed columns was described in the earlier edition of this manual
(ST/NAR/18) and is not included in this revised version.
Operating conditions
Injection: 1µl
Sample preparation
Prepare internal standard, drug standard and sample solution at concentrations of
1 mg/ml. Use the sample extract obtained as described in section 3.2. Inject
successively 1µl of the sample and standard solutions into the gas chromatograph.
Results
Retention indices
Retention indices
Note: All of these methods should include the use of appropriate standards.
Many analysts prefer to derivatize barbiturates using some form of methylating
agent, but many consider it unreliable for quantitative analysis.
GC-MS is one of the most commonly used techniques for the identification and
quantitation of forensic drug samples. As a “hyphenated” technique, it combines the
separation power of a GC with the analyte specificity of a spectroscopic technique,
providing highly specific spectral data on individual compounds in a complex
mixture often without prior separation.
Results
Note: Most spectrometers have their own mass spectral reference libraries that
may be commercial or developed by the operator. However, these should be used
for reference purposes only. Direct comparison of the GC-MS characteristics of
the sample under test should be compared with those of an authentic sample
derived under the same conditions.
Weigh an appropriate amount of sample into a volumetric flask so that the final
barbiturate concentration is approximately that of the standard solution. Dilute to
volume with methanol. Depending on the provenance of the sample, any undissolved
solid particulates can be removed by filtration.
Operating conditions
Column temperature: 100°C for 1min, then 100-280° at 15°C min-1, hold
280oC for 3min.
Results
HPLC is one of the major separation techniques commonly used in forensic drug
analysis. Reversed phase chromatography is recommended for the analysis of bar-
biturates. There are a large variety of stationary and mobile phases available to the
analyst. The following 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 before
final selection of the column.
Method 1
Method 2
(ODS-Hypersil, or equivalent)
Flow rate: 2.0 ml/min (low flow values should be considered with
shorter columns and smaller particle sizes).
Detection: UV at 216 nm
Results
The following HPLC method [17, 19], involving the use of an internal standard,
may be adopted for the quantification of the selected barbiturates allobarbital, amo-
barbital, barbital, cyclobarbital, metharbital, pentobarbital, phenobarbital and
secobarbital.
Method
Injection: 2µl
UV wavelength: 215 nm
Results
Theoretically, each substance has a unique infrared spectrum and this method would
permit the unequivocal identification of any barbiturate but would not distinguish
between enantiomers.
21
22 Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines
R2
1 R3
N
R4
R1 N4
R5
(2 ) (2)
basic 5-phenyl-1,4-benzodiazepine
basic skeleton
5-phenyl-1,4-benzodiazepine skeleton
Some of the compounds that are classified alongside them are, strictly speaking, not
benzodiazepines. For example, brotizolam (figure 3) (see annex II) is a thienotria-
zolo-1,4-diazepine. Additionally, some compounds such as clobazam (figure 4) are
actually 1,5-benzodiazepines.
N
N 1 O
N N
S
Br
N Cl N
5 O
Cl
(3)
(3) brotizolam
brotizolam (4)clobazam
(4) clobazam
The benzodiazepines are formulated mainly as capsules and tablets. However some
are available in other pharmaceutical forms such as injectable solutions. Diazepam,
the most traded and widely available benzodiazepine, can be found as capsules,
tablets, aqueous or polyethyleneglycol solutions for injection, syrups and
suppositories.
When possible, three entirely different analytical techniques should be used, for
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 information from
both techniques is used (i.e. retention time and mass spectral characteristics).
3.1 Sampling
Virtually all of the benzodiazepines encountered in the illicit traffic appear in the
form of tablets, capsules, liquid preparation and bulk powder diverted from
24 Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines
legitimate sources. They are usually present as the free base or as the hydrochloride,
mesilate or other salt. However, some also have carboxylic acid functionalities and
may be presented as potassium salts, e.g. chlorazepate. All the benzodiazepines are
generally soluble in methanol (see annex II) The analyst should refer to the relevant
formularies or pharmacopoeias for the published content of the drug.
Method
Method
Zimmerman test
Method
This test is not specific to this class of compounds. Analysts are therefore advised
to use a combination of TLC and colour development after spraying with selected
reagents as a presumptive test.
Plates prepared by the analyst must be activated before use by placing them into
an oven at 120°C for at least 10 to 30 minutes. Plates are then stored in a
26 Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines
grease-free desiccator over blue silica gel. Heat activation is not required for
commercially available coated plates.
*Due to the carcinogenic potential of chloroform, solvent systems A and B should only be used with
appropriate safety precautions and ventilation.
Standard solutions:
All solutions made at a concentration of 5mg/ml in
methanol.
Visualization
The plates must be dried prior to visualization. This can be done at 120°C for
5 minutes in an oven or using a hot air blower. It is important for proper colour
development that all traces of diethylamine be removed from the plate.
Visualization methods
A. UV light at 254 nm
B. 2N H2SO4/heat/observe under UV light at 366 nm
C. Acidified potassium iodoplatinate reagent
II. Benzodiazepines and related substances 27
Spray reagent
Acidified potassium iodoplatinate reagent: Dissolve 0.25g of platinic chloride and
5g of potassium iodide in sufficient water to produce 100 ml. This is potassium
iodoplatinate reagent; for the acidified version add 5ml of concentrated hydrochloric
acid to 100 ml of iodoplatinate solution.
Method
First observe the plate under short wave UV light. Spray with 2N H2SO4 and heat
in an oven at 80°C for 5 minutes. Observe the fluorescent spots under long UV
light (366nm). The plate may then be oversprayed with the acidified iodoplatinate
reagent. All benzodiazepines give purple coloured spots when oversprayed with the
reagent (alkaloid positive reaction). Other alternative methods are described in the
literature [23, 24, 25, 26].
Results
Rf x 100 values
COMPOUND System
A B C
Alprazolam 1 8 62
Bromazepam 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 loflazepate 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
28 Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines
Rf x 100 values
COMPOUND System
Loprazolam 2 3 49
Lorazepam 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.
Operating conditions
Oven: 250°C
Injection: 1µl
Preparation of solutions
Solutions of the standards and samples are prepared in methanol at a concentration
of 1 mg/ml.
Results
Retention indices
COMPOUND 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
30 Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines
Retention indices
COMPOUND BP-1 Capillary
Diazepam 2477
Estazolam 2893
Ethyl loflazepate 2925 (2878)
Fludiazepam 2403
Flunitrazepam 2633
Flurazepam 2795
Halazepam 2314
Haloxazolam* 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
GC-MS is one of the most commonly used techniques for the identification and
quantitation of forensic drug samples. As a “hyphenated” technique, it combines the
II. Benzodiazepines and related substances 31
Preparation of solutions
Solutions of the standards and samples are prepared in methanol at a concentration
of 1 mg/ml
Results
Identification is accomplished by comparing the retention time and mass spectrum
of the analyte with that of a reference standard. All compounds identified by GC-MS
and reported by the analyst must be compared to a current mass spectrum of the
appropriate reference standard, preferably obtained on the same instrument, operated
under identical conditions.
and solvent system by national laboratories will depend upon the particular benzo-
diazepines commonly available in that country.
Method
Column: 250 mm by 5 mm ID
Results
Capacity ratios, k’ values for a selection of benzodiazepines in the two mobile phases
are provided below:
LC conditions
Instrument: Agilent 1200 Series RRLC; 6410 LC Triple Quadrupole
Masspectrometer
Column: ZORBAX Eclipse XDB C18 2.1 x 50 mm x 1.8 μm
Column temperature: 35°C
Injection volume: 5 μL
Solvent flow rate: Time (minutes) Flow rate (ml/min)
0 0.2
6.5 0.2
8 1.0
10 0.2
Post time: 4.5 min
Mobile phase Acetonitrile: 20 mM ammonium formate pH = 8.6
(50:50 by vol.)
II. Benzodiazepines and related substances 35
MS Conditions
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for the determination of triazolo-benzodiazepines. Journal of Chromatography
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34. Flanagan R.J., Storey G.C.A., Bhamra R.K. and Jane I. High performance liquid
chromatographic analysis of basic drugs on silica columns using non-aqueous
ionic eluents. Journal of Chromatography A, 247: 15-37, 1982
40 Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines
36. Miyadera T., Terada A., Fukanaga M., Kowana Y., Kamioka T., Tamura C.,
Tagaki H. and Tachikawa R. Synthesis and pharmacology of benzo [6,7][1,4]
diazepino [5,4-b] oxazole derivatives and analogs. Journal of Medicinal
Chemistry, 14, 520-526, 1971
37. Moore, C., Coulter, C., Crompton, K. and Zumwalt, Tetrahydrocannabinol and
two of its metabolites in whole blood using liquid chromatography-tandem mass
spectrometry. Journal of Analytical Toxicology 32, 653-658, 2008
38. Smink B.E., Brandsma J.E., Dijkhuizen A., Lusthof K.J., De Gier J.J., Egberts
A.C.G. and Uges D.R.A. Quantitative analysis of 33 benzodiazepines, metabo-
lites and benzodiazepine-like substances in whole blood by liquid chromato
graphy-(tandem) mass spectrometry. Journal of Chromatography B, 811(1):
13-20, 2004
40. ElSohly M.A., Gul W., ElSohly K.M, Avula B., and Khan I.A. LC-MS-(TOF)
analysis method for benzodiazepines in urine samples from alleged drug-
facilitated sexual assault victims. Journal of 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 of benzodiazepines in urine from alleged victims of drug-facilitated
sexual assault. Journal of Analytical Toxicology, 31: 505-514, 2007
42. ElSohly M.A., Gul W., Avula B., Murphy T.P. and Khan I.A. Simultaneous
analysis of thirty-five benzodiazepines in urine using liquid chromatography-
mass spectrometry-time of flight. Journal of Analytical Toxicology, 32(8): 547-
561, 2008
Further reading
The Analysis of Drugs of Abuse. Gough, T.A. (eds.) J. Wiley and Sons, Chichester,
New York, Brisbane, Toronto, Singapore, 1991.
Further reading 41
Saka K., Uemura K., Shintani-Ishida K. and Yoshida K-I. Determination of amo-
barbital and phenobarbital in serum by gas chromatography-mass spectrometry with
addition of formic acid to the solvent. Journal of Chromatography B, 869(1-2):
9-15, 2008
Steijns L.S., Bouw J. and van der Weide J. Evaluation of fluorescence polarization
assays for 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 of Barbiturates
in Urine by CE using a capillary coated dynamically with polycationic polymers.
Chromatographia, 65(9-10): 611-615, 2007.
Annex I. Description of barbiturates under
international control
Allobarbital
5,5-diallylbarbituric acid; 5,5-di-2propenyl-2,4,6 (1H,3H, 5H) pyrimidinetrione
Sch. IV (1971)
H
O N O
NH
42
Annex I. Description of barbiturates under international control 43
NH N
O O
H
O N O
NH
Butalbital
5-(2-methylpropyl)-5-(2-propenyl)-barbituric acid
Sch. III (1971)
H
O N O
NH
Butobarbital
5-butyl-5-ethylbarbituric acid
Sch. IV (1971)
H
O N O
NH
NH
Methylphenobarbital
5-ethyl-1-methyl-5-phenylbarbituric acid
Sch. IV (1971)
O N O
NH
and enantiomer
O
Methylphenobarbital (continued)
H
O N O
NH
and enantiomer
H O
NH
NH
and enantiomer
H O
Empirical formula: C10H16N2O3 (C10H15N2NaO3)
CAS No.: 125-40-6 (143-81-7)
MWt: 212.2 (234.2)
MPt : 165 – 168º C
Physical appearance: Free acid is a white microcrystalline powder (sodium salt is
a white powder)
Solubility: Soluble in alcohol, chloroform, ether and in aqueous alkali
(sodium salt is soluble in alcohol and water, almost insoluble
in ether)
Infrared data: Principal peaks at wavenumbers 1675, 1760, 1317, 1303,
1230, 853 cm-1 (KBr disk) [3, 12]
48 Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines
NH
and enantiomer
H O
Empirical formula: C12H18N2O3 (C12H17N2O3Na)
CAS No.: 76-73-3 (309-43-3)
MWt: 238.3 (260.3)
MPt: 100º C
Physical appearance: Free acid is a white microcrystalline powder (sodium salt is
a white powder)
Solubility: Soluble in alcohol, chloroform, ether and in aqueous alkali
(sodium salt is soluble in alcohol and water, almost insoluble
in ether)
Infrared data: Principal peaks at wavenumbers 1559, 1648, 1690, 1298,
1270, 925 cm-1 KBr disk) [3, 12]
Vinylbital
5-ethenyl-5-(1-methylbutyl)-barbituric acid
Sch. IV (1971)
H
O N O
NH
and enantiomer
H O
Alprazolam
8-chloro-1-methyl-6-phenyl-4H-s-[1,2,4]triazolo[4,3-a][1,4] benzodiazepine
Sch. IV (1971)
N
N
N
Cl N
49
50 Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines
Bromazepam
7-bromo-1,3-dihydro-5-(2-pyridyl)-2H-1,4-benzodiazepin-2-one
Sch. IV (1971)
H O
N
Br N
Brotizolam
2-bromo-4-(o-chlorophenyl)-9-methyl-6H-thieno[3,2-f]-s-triazolo[4,3-a][1,4]
diazepine
Sch. IV (1971)
N
N
S N
Br
N
Cl
Camazepam
7‑chloro‑1,3‑dihydro‑3‑hydroxy‑1‑methyl‑5‑phenyl‑2H‑1,4‑benzodiazepin‑2‑one
dimethylcarbamate (ester)
Sch. IV (1971)
O
N
O
Cl N N
O
H
N
N
Cl N
O
Clobazam
7-chloro-1-methyl-5-phenyl-1H-1,5-benzodiazepine-2,4 (3H,5H)-dione
Sch. IV (1971)
O
N
Cl N
O
Clonazepam
5-(o-chlorophenyl)-1,3-dihydro-7-nitro-2H-1,4-benzodiazepin-2-one
Sch. IV (1971)
H O
N
O 2N N
Cl
7‑chloro‑2,3‑dihydro‑2‑oxo‑5‑phenyl‑1H‑1,4‑benzodiazepine‑3‑carboxylic acid
(Potassium (3RS)-7-chloro-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepine-3-
carboxylate with potassium hydroxide (1:1)
Sch. IV (1971)
H H O
N O N
H
COOH
O2K
Cl N Cl N
andenantiomer
and enantiomer . KOH
. KOH
Clotiazepam
5‑(o‑chlorophenyl)‑7‑ethyl‑1,3‑dihydro‑1‑methyl‑2H‑thieno[2,3‑e]‑1,4‑diaz-
epin‑2‑one
Sch. IV (1971)
O
S N
Cl
Cloxazolam
10-chloro-11b-(o-chlorophenyl)-2,3,7,11b-tetrahydrooxazolo[3,2-d][1,4]
benzodiazepin-6(5H)-one
Sch. IV (1971)
H O
N
Cl N
O
Cl
and enantiomer
Annex II. Description of benzodiazepines under international control 55
Delorazepam
7-chloro-5-(o-chlorophenyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one
Sch. IV (1971)
H O
N
Cl N
Cl
Diazepam
7-chloro-1,3-dihydro-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one
Sch. IV (1971)
O
N
Cl N
56 Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines
Diazepam (continued)
Estazolam
Sch. IV (1971)
N
N
N
Cl N
Ethyl loflazepate
Ethyl 7-chloro-5-(o-fluorophenyl)-2,3-dihydro-2-oxo-1H-1,4-benzodiazepine-3-
carboxylate
Sch. IV (1971)
H O
N
H
O
Cl N
O
F and enantiomer
Fludiazepam
7-chloro-5-(o-fluorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one
Sch. IV (1971)
O
N
Cl N
Flunitrazepam
5-(2-fluorophenyl)-1,3-dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepin-2-one
Sch. IV (1971)
O
N
O 2N N
O
N
Cl N
Halazepam
7-chloro-1,3-dihydro-5-phenyl-1-(2,2,2-trifluoroethyl)- 2H-1,4-benzodiazepin-2-one
Sch. IV (1971)
F
F
F O
N
Cl N
Haloxazolam
10-bromo-11b-(o-fluorophenyl)-2,3,7,11b-tetrahydrooxazolo[3,2d][1,4]
benzodiazepin-6(5H)-one
Sch. IV (1971)
H O
N
Br N
O
F and enantiomer
Empirical formula: C17H14BrFN2O2
CAS No.: 59128-97-1
MWt: 377.2
MPt: 185º C
Physical appearance: White crystalline powder
Solubility: Slightly soluble in chloroform, ethanol and ethyl acetate,
sparingly soluble in water
Infrared data: Principal peaks at wavenumbers 1688, 1484, 764, 1417,
2887, 3201 cm-1 (KBr disk) [4]
Ketazolam
11-chloro-8,12b-dihydro-2,8-dimethyl-12b-phenyl)-4H-[1,3]oxazino[3,2d][1,4]
benzodiazepin-4,7 (6H)-dione
Sch. IV (1971)
O
N
Cl N O and enantiomer
O
H N
N
O
O 2N N
Cl
Lorazepam
7-chloro-5-(o-chlorophenyl)-1,3-dihydro-3-hydroxy- -2H-1,4-benzodiazepin-2-one
Sch. IV (1971)
H O
N
OH
H
Cl N
and enantiomer
Cl
62 Recommended Methods for the Identification and Analysis of Barbiturates and Benzodiazepines
Lorazepam (continued)
O
N
OH
H
Cl N
and enantiomer
Cl
Medazepam
7-chloro-2,3-dihydro-1-methyl-5-phenyl-1H-1,4-benzodiazepine
Sch. IV (1971)
N
Cl N
Midazolam
8-chloro-6-(o-fluorophenyl)-1-methyl- -4H-imidazo[1,5-a][1,4]benzodiazepine
Sch. IV (1971)
N
Cl N
Nimetazepam
Sch. IV (1971)
O
N
O 2N N
Nitrazepam
1,3-dihydro-7-nitro-5-phenyl-2H-1,4-benzodiazepin-2-one
Sch. IV (1971)
H O
N
O 2N N
Nordazepam
7-chloro-1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one
Sch. IV (1971)
H O
N
Cl N
Oxazolam
10-chloro-2,3,7,11b-tetrahydro-2-methyl-11b-phenyloxazolo[3,2-d][1,4]benzodiaz-
epin-6(5H)-one oxazolazepam
Sch. IV (1971)
H O
N
Cl N
and diastereomers
O
H
Pinazepam
7-chloro-1,3-dihydro-5-phenyl-1-(2-propynyl)-2H-1,4-benzodiazepin-2-one.
Sch. IV (1971)
O
N
Cl N
Prazepam
7-chloro-1-cyclopropylmethyl-1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one
Sch. IV (1971)
O
N
Cl N
Tetrazepam
7-chloro-5-(cyclohexen-1-yl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one
Sch. IV (1971)
O
N
Cl N
Triazolam
8-chloro-6-(o-chlorophenyl)-1-methyl-4H-[1,2,4]-s-triazolo[4,3-a][1,4]
benzodiazepine
Sch. IV (1971)
N
N
N
Cl N
Cl
*1252222*
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