SUBJECT FORENSIC SCIENCE

Paper No. and Title PAPER No. 9: Drugs of Abuse

Module No. and Title MODULE No. 10: Common Depressants: Benzodiazepines

Module Tag FSC_P9_M10

FORENSIC SCIENCE PAPER No.9 : Drugs of Abuse

MODULE No. 10: Common Depressants: Benzodiazepines
 TABLE OF CONTENTS

1. Learning Outcomes
2. Introduction to Benzodiazepines
3. Forensic Issues
4. Chemistry of Benzodiazepines
5. Classification of Benzodiazepines
6. Fatal Dose
7. Mechanism of Action of Benzodiazepines
8. Pharmacokinetics of Benzodiazepines
9. Effects of Benzodiazepines
10. Uses of Benzodiazepines
11. Signs and Symptoms
12. Withdrawal Symptoms of Benzodiazepines
13. Extraction of Benzodiazepines for Forensic Analysis
14. Presumptive Analysis for Benzodiazepines
15. Instrumental Analysis for Benzodiazepines
16. Medico- legal aspect of Benzodiazepines
17. Summary

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MODULE No. 10: Common Depressants: Benzodiazepines
 1. Learning Outcomes

After studying this module, you will be able to know about-

 Benzodiazepines and their types

 Mode of action and pharmacokinetics of Benzodiazepines
 Forensic Examination of Benzodiazepines

2. Introduction to Benzodiazepines

Benzodiazepines are one of the most extensively prescribed groups of drugs and are
recurrently found in toxicological cases. Benzodiazepines are one of the most significant
groups of psychotropic drugs. They are extensively used as tranquilizers, sedatives and
hypnotics.
Ever since the introduction of benzodiazepines in the 1960s by Leo Sternback, they have
developed progressively more popular as anxiolytic agents and sedatives, displacing the
barbiturates from their held top spot hitherto. In spite of widespread use worldwide, there
have been only a few cases reported involving fatalities, demonstrating the wide margin
of safety of benzodiazepines. But self-satisfaction must be avoided and the safety profile
of these drugs should not be taken for granted, as deaths have been reported in some
recent cases even from unexpectedly low doses of certain benzodiazepines. There are
also indications that some of the newer benzodiazepines have a slightly smaller threshold
of safety. Another important issue is with reference to the use of benzodiazepines to
deliberately induce amnesia in certain individuals in order to accomplish an immoral act
(e.g. date rape). Many of these drugs, mainly flunitrazepam, are capable of causing
retrograde amnesia. Flunitrazepam (Rohypnol or “Roofies”) has become popular as a
drug of abuse, often combined with alcohol, marijuana, or cocaine to produce an intense
“high”. It has been used as a “date rape” drug, both for its properties of lowering
inhibitions and because it can cause retrograde amnesia.

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MODULE No. 10: Common Depressants: Benzodiazepines
 While addiction to benzodiazepines is an undeniable possibility among patients on long-
term therapy, the abuse potential is much less when compared to most other sedative-
hypnotics such as the barbiturates. Withdrawal reactions are also generally less severe
and more easily managed.

3. Appearance of Forensic Exhibits

Confiscation of benzodiazepines may involve mainly of pharmaceutical substances

formulated as tablets, capsules, oral liquids or injectable and should be sampled
depending on the number and type of dosage units seized.

4. Chemistry of Benzodiazepines

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

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MODULE No. 10: Common Depressants: Benzodiazepines
 Molecular Structure of some common Benzodiazepines:

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MODULE No. 10: Common Depressants: Benzodiazepines
 The following are important Benzodiazepines generally encountered in Forensic
arena:
Alprazolam, Brotizolam, Chlordiazepoxide, Chlorazepate, Clobazam, Clonazepam,
Diazepam, Estazolam, Flunitrazepam, Flurazepam, Halazepam, Lorazepam,
Lormetazepam, Medazepam, Midazolam, Nitrazepam, Oxazepam, Pinazepam, Prazepam,
Quazepam, Temazepam, Triazolam, Zolazepam, etc.

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MODULE No. 10: Common Depressants: Benzodiazepines
 Benzodiazepines are formulated largely as capsules and tablets. However, some are
available in other pharmaceutical form 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.

They are commonly 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. clorazepate. All the benzodiazepines are generally soluble in
methanol.

5. Classification of Benzodiazepines
Benzodiazepines are characterized on the basis of their action and are as follows:

 Short Acting Benzodiazepines:

Short-acting compounds have an average half-life of 1–12 hours. They have few residual
effects if taken before sleep time, rebound insomnia may occur upon discontinuation,
and they might cause daytime withdrawal symptoms such as next day rebound anxiety
with prolonged usage. Examples are Brotizolam, midazolam, and Triazolam.

 Intermediate Acting Benzodiazepines:

Intermediate-acting compounds have an average half-life of 12–40 hours. They may
have some residual effects in the first half of the day if used as a hypnotic. Rebound
insomnia, however, is more common upon withdrawal of intermediate-acting
benzodiazepines than longer-acting benzodiazepines. Examples are alprazolam,
estazolam, flunitrazepam, clonazepam, lormetazepam, lorazepam, nitrazepam, and
temazepam.

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MODULE No. 10: Common Depressants: Benzodiazepines
  Long Acting Benzodiazepines:

Long-acting compounds have a half-life of 40–250 hours. They have a risk of

accumulation in the elderly and in individuals with severely impaired liver function, but
they have a reduced severity of rebound effects and withdrawal. Examples are diazepam,
clorazepate, chlordiazepoxide, and flurazepam.

List of some Benzodiazepines and their action duration:

Lorazepam Intermediate
Flurazepam Long-acting
Quazepam Long-acting
Triazolam Short-Acting
Clonazepam Long-acting
Chlordiazepoxide Long-acting
Clobazam Long-acting
Estazolam Intermediate
Temazepam Intermediate
Oxazepam Intermediate
Clorazepate Long-acting
Diazepam Long-acting
Midazolam Short-acting
Alprazolam Intermediate

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MODULE No. 10: Common Depressants: Benzodiazepines
 6. Fatal Dose
It is ambiguous for most benzodiazepines. Even consumption of up to 2000 mg diazepam
has not resulted in death, or for that matter, even serious morbidity. However, several
cases of fatality due to triazolam and flunitrazepam overdose have been stated. In
general, benzodiazepine metabolism appears to be inhibited by ethanol when given
concurrently. Clinically, associated administration of high doses of ethanol and
benzodiazepines act to synergistically reduce respiration.

7. Mechanism of Action of Benzodiazepines

Benzodiazepines express their pharmacological activity by binding to the so-called
GABAA receptor which mediates the effect of gamma-aminobutyric acid (GABA), the
inhibitory neurotransmitter in the brain. After binding to the receptor, the benzodiazepine
locks the GABAA receptor into a conformation in which the neurotransmitter GABA has
higher affinity for the receptor. This increases the frequency of opening of the associated
chloride ion channel and causes hyperpolarization of the membrane. As a result, the
inhibitory effect of the available GABA is increased, leading to sedation and other
symptoms. In addition, benzodiazepines cause hypnotic, anticonvulsant, muscle relaxant
and amnesic As a result, the inhibitory effect of the available GABA is increased, leading
to sedation and other symptoms. In addition, benzodiazepines cause hypnotic,
anticonvulsant, muscle relaxant and amnesic effects. Although most benzodiazepines
trigger the same physical effects, their dosage and absorption time into the bloodstream
can vary extensively.

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MODULE No. 10: Common Depressants: Benzodiazepines
 8. Pharmacokinetics of Benzodiazepines
Most benzodiazepines are administered orally or by Intra venous injection. Intramuscular
injection may lead to irregular absorption. However, lorazepam and midazolam are
exceptions to this and can be given Intra Muscular. Following absorption, all
benzodiazepines are bound to plasma proteins to the extent of 70 to 99%, and are
metabolised broadly by different microsomal enzyme systems in the liver. Metabolites
are invariably as active as the parent compound. Some benzodiazepines are eliminated
from the body gradually. Thus, ingesting multiple doses over long periods of time can
lead to substantial accumulation in fatty tissues. Hydroxylation and demethylation of the
benzodiazepine diazepam gives the metabolites temazepam and oxazepam, both of which
are also available as drugs.

9. Effects of Benzodiazepines
Weakness, headache, amnesia, vertigo, diplopia, nausea, diarrhea, and rarely chest pain
are the effects of Benzodiazepine consumption. Besides, Paradoxical effects
(disinhibition or dyscontrol reaction) may sometimes occur characterized by restlessness,
agitation, and hallucinations. Flurazepam has been associated with nightmares and
hallucinations. Allergic, hepatotoxic and hematological reactions are nearly rare.

Drug Interactions of Benzodiazepine:

Ethanol has a synergistic effect with benzodiazepines and increases both the rate of
absorption as well as associated CNS depression. Similar effect is also seen with
associated administration of phenothiazines and barbiturates. Sodium Valproate may
cause psychotic reactions when given along with benzodiazepines.

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MODULE No. 10: Common Depressants: Benzodiazepines
 10. Uses of Benzodiazepines
Benzodiazepines are suggested for relaxation, calmness, and relief from anxiety and
tension. Medically, Benzodiazepines are used to treat following ailments:
 Seizure disorders
 Anxiety disorders
 Movement disorders
 Insomnia
 Mania
 For inducing skeletal muscle relaxation
 Pre-anaesthetic medication
 Treatment of alcohol withdrawal.

11. Signs and Symptoms

Benzodiazepines are one of the harmless drugs when their over-dosage occurs alone.
Cases have been reported when as many as 70 tablets of any of them are unlikely to
produce anything more than mild effect in most adults.

Various benzodiazepines have active metabolites that justifies for their prolonged
sedative effects. Benzodiazepines potentiate the effects of other CNS depressants,
particularly alcohol, tricyclic antidepressants and barbiturates. Flurazepam is most likely
to produce significant CNS depression. The signs and symptoms that are produced are:
(i) Drowsiness
(ii) Dizziness
(iii) Ataxia and slurred speech
(iv) Respiratory depression
(v) Hypotension and coma
Long-term use of benzodiazepines is associated with the development of tolerance.
Tolerance to certain benzodiazepines occurs most often in those who have used for 6
months or more.

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MODULE No. 10: Common Depressants: Benzodiazepines
 12. Withdrawal Symptoms of Benzodiazepines
Symptoms of Withdrawal from benzodiazepines are mainly:
 Insomnia
 Gastric problems
 Tremors
 Agitation
 Fearfulness
 Muscle spasms
However, fewer recurrent effects comprises of irritability, excessive perspiration,
depersonalization, derealization, allergic reaction to stimuli, depression, suicidal
behavior, psychosis, seizures, and delirium tremens. Severe symptoms typically occur as
a result of abrupt or over-rapid withdrawal. Abrupt withdrawal can be dangerous;
therefore, a gradual reduction procedure is suggested.

13. Extraction of Benzodiazepines for Forensic Analysis

Benzodiazepines may conveniently be extracted into methanol for both qualitative and
quantitative analyses. The dose form should be triturated in methanol and any solid
material may be removed by centrifugation or filtration prior to analysis of the drug in
solution. Subsequently, presumptive tests, TLC and confirmatory analysis are then
carried out on the prepared extract.

14. Presumptive Analysis for Benzodiazepines

 ZIMMERMAN TEST
Preparation of reagent:
 Solution 1: 2,4-Dinitrobenzene (1 % w/v) in Methanol
 Solution 2: 15 % Potassium Hydroxide aqueous solution

Procedure:
On a micro-test plate, place appropriate quantity of the sample to be tested, add one drop
of Solution 1 followed by one drop of Solution 2, and mix.

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MODULE No. 10: Common Depressants: Benzodiazepines
 Result:
Red-purple to pink colour indicates the probable presence of a benzodiazepine.

 VITALI – MORIN’S TEST

Preparation of reagent:
 Solution 1: Conc. Nitric Acid
 Solution 2: Acetone
 Solution 3: 0.1 N Ethanolic Potassium Hydroxide

Procedure:
On a micro-test plate, place adequate quantity of the sample to be tested, add Solution 1
followed by Solution 2 and further followed by Solution 3, and mix.

Result:
Yellow- Orange indicates the possible presence of benzodiazepines.

 MARQUIS TEST

Preparation of reagent:
1 volume of Formalin is added to 9 volume of concentrated Sulphuric Acid

Procedure:
On a micro-test plate, place adequate quantity of the sample to be tested, add the reagent
and agitate.

Result:
Yellow- Orange indicates the possible presence of benzodiazepines.

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  FORMALDEHYDE- SULPHURIC ACID TEST
Preparation of reagent:
4 parts of Sulphuric Acid is added with 6 parts of Formalin.

Procedure:
As mentioned in the above tests

Result:
Red/ Pink/ Blue/ violet/ Red-violet/ Blue- violet indicates the possible presence of
benzodiazepines

 HYDROCHLORIC ACID TEST

Preparation of reagent:
0.2 N Hydrochloric Acid

Procedure:
As mentioned in the above tests

Result:
Appearance of Yellow Color points toward the possible existence of Benzodiazepines.

15. Instrumental Analysis for Benzodiazepines

 THIN LAYER CHROMATOGRAPHY:

Stationary Phase: Activated Silica Gel (with or without fluorescing substance)

TLC plates of 0.25/ 0.20 mm thickness.
Mobile Phase: Solvent System A Chloroform : Acetone
(80 : 20)
Solvent System B Chloroform : Methanol
(90 : 10)
Solvent System C Cyclohexane : Toluene : Diethylamine
(75 : 15 : 10)

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MODULE No. 10: Common Depressants: Benzodiazepines
 Sample preparation: The solutions of exhibit powder/ tablet/ capsules and
standard may be prepared in methanol
Spray Reagents: Dragendorff’s Reagent Orange Spots
Acidified Iodoplatinate Reagent Purple colour
Visualization: The plates must be dried prior to visualization at 120°C for 5
minutes in an oven or, by using a hot air blower to remove
all traces of diethylamine from the plate

Benzodiazepines form a mixed group of chemicals; however, the above mentioned three
Thin-Layer Chromatographic systems, when used in combination, give good separations
for a number of benzodiazepines. Solvent System C is a general system suggested also
for the identification and analysis of cocaine, opium and amphetamine or
methamphetamine.

Preparation of Spray Reagents:

 Dragendorff’s Reagent: 1 gm of Bismuth Subnitrate is dissolved in 3 ml. of 10M

of Hydrochloric Acid. It is diluted to 20 ml. 1 gm of Potassium Iodide is dissolved
in it. If black precipitate of Bismuth Tri-Iodide separates, it is dissolved in 2M
Hydrochloric Acid.

 Acidified Iodoplatinate Reagent: 0.25g of Platinic Chloride and 5g of Potassium

Iodide is dissolved in sufficient water to produce 100 ml solution. This is
Potassium Iodoplatinate Reagent. For the acidified version 5ml of concentrated
Hydrochloric Acid added to 100 ml of Iodoplatinate solution.

 GAS CHROMATOGRAPHY:

It is a procedure whereby volatile components of a mixture may be separated by partition

between a solid or liquid stationary phase and a gaseous mobile phase. The efficiency in
the separation is attained by controlling several factors viz., Column Type (capillary or
wide bore), Column Length, Column Diameter, nature of liquid phase, Carrier Gas, flow
rate and temperature. This technique has now been hyphenated with other techniques viz.
SFC-GC, GC-MS.

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MODULE No. 10: Common Depressants: Benzodiazepines
 Although Gas Chromatography can be recommended as a suitable method for the
analysis of most benzodiazepines, several of them, particularly the 3-hydroxyderivatives,
undergo thermal degradation and rearrangements. Chlordiazepoxide, cloxazolam,
lormetazepam, haloxazolam, oxazolam, ethyl loflazepate and temazepam yield multiple
peaks.

 UV-VIS SPECTROSCOPY

Benzodiazepine derivatives may be studied by scanning the samples in appropriate

organic solvents with the help of UV-Vis spectrophotometry. The value of lmax of the
spectrum can be compared with the standard value given in the literatures or with using
the standard sample of benzodiazepine derivatives.

Absorption Maxima (nm) in

Compound
0.5N H2SO4 0.5N NaOH Ethanol
Alprazolam 260 - -
Chlordiazepoxide 246,308 262 -
Clonazepam 273 - 245, 309
Diazepam 242, 284, 368 - -
Flunitrazepam - - 252, 308
Flurazepam 236, 284 231, 312 -
Ketazolam - - 242
Lorazepam - - 230, 316
Nitrazepam 289 - 280
Temazepam 237, 284,358 231, 313 230, 314

 GAS CHROMATOGRAPHY- MASS SPECTROSCOPY

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 of
compounds often without prior separation.

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MODULE No. 10: Common Depressants: Benzodiazepines
 Benzodiazepine derivatives may be studied by analyzing the samples with the help of
Mass spectrometry (GC-MS). The value of principal peaks at m/z can be compared
either with the standard value given in the literatures or with using the standard sample of
Benzodiazepine derivatives. Identification is accomplished by comparing the Retention
Time and Mass Spectrum of the analyte with that of a reference standard.

GC operating conditions and parameters used for the GC–MS analysis of Benzodiazepine
derivatives:

SYSTEM CONDITIONS

Column BP-1: 25 m × 0.22 mm i.d.; df, 0.25 μm

Injection temperature 275°C

Column oven temperature 250°Ca

Carrier gas He, at a flow rate of 1 ml/ min

Split ratio 20:1

Mass spectrometric, temperature and settings as
Detector
required

 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

The analysis of forensic samples by HPLC has become popular due to development of
HPLC in attaining performance by using high performance column, new detector,
optimum methods etc. The HPLC method allows quantitative determination of non-
volatile and thermally labile compounds without derivatization and much clean-up of
samples.

HPLC operating conditions and parameters used for the analysis of benzodiazepines

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MODULE No. 10: Common Depressants: Benzodiazepines
 SYSTEM CONDITIONS

Column Spherisorb ODS-2: 25 cm × 4.6 mm i.d.; 5 μm

particle size
Mobile phase MeOH/water/0.1 M phosphate buffer at pH 7.25
(55:25:20), or (70:10:20)a
Flow rate 1.5 ml/ min

Injection volume 5–10 μlb

Detection UV, at 240 nm

16. Medico- legal aspect of Benzodiazepines

Benzodiazepines are remarkably safe drugs and rarely produce serious toxic effects even
with substantial ingestion. Death is exceptional unless other synergistic drugs have also
been ingested. However, newer benzodiazepines such as alprazolam, triazolam, and
temazepam are associated with fatalities.

Benzodiazepine tranquillizers are prescribed widely and therefore occur more frequently
than any other type of drug in overdose cases. The effects of these drugs in overdose are
usually mild, although they may have a synergistic effect when taken with alcohol or
other drugs. The anticonvulsive benzodiazepine clonazepam (Rivotril) is also used to
detoxify patients with very severe (other) benzodiazepine dependence. Although these
drugs do not seem to cause lethal intoxications, reports of deaths from benzodiazepines
have been published, most of which refer to elderly people or cases of combined
overdose of flunitrazepam and opiates.

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MODULE No. 10: Common Depressants: Benzodiazepines
 17. Summary

 The abuse or misuse of benzodiazepines is internationally widespread which means

that any forensic laboratory may encounter a range of these compounds. In general,
benzodiazepines encountered in the illicit market are diverted from legitimated
sources.

 Benzodiazepines enhance the effect of the neurotransmitter gamma-aminobutyric

acid and are subsequently used therapeutically as tranquilizers, hypnotics,
anticonvulsants and centrally acting muscle relaxants.

 Over 30 benzodiazepines are available; some of these are both the parent compound
and a metabolite of other benzodiazepines. All benzodiazepines and their
unconjugated metabolites (except the parent drug Potassium Clorazepate) are
extractable from body fluids into an organic solvent and can be quantified in serum
or plasma by normal-phase HPLC with UV detection. GC with ECD can also be
used.

 Long-term use of benzodiazepines is associated with the development of tolerance.

Abrupt cessation provokes a mild withdrawal reaction characterized by anxiety,
insomnia, headache, tremor, and paraesthesia. Restlessness, encephalopathy, and
hallucinations may occur after abrupt withdrawal from high daily doses. Convulsions
may occur after a lapse of 3 to 10 days.

 Withdrawal reactions of Benzodiazepines are also generally less severe and more
easily managed. However, abrupt cessation takes place after prolonged use.

 Benzodiazepines, specifically alprazolam and diazepam, are among the most often
diverted and abused psychotropic substances.

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MODULE No. 10: Common Depressants: Benzodiazepines