Benzodiazepines, sometimes called "benzos", are a class of psychoactive drugs whose core chemical structure is the fusion of a benzene ring and a diazepine ring. As depressants—drugs which lower brain activity—they are prescribed to treat conditions such as anxiety, insomnia, and seizures. The most common side effects associated with benzodiazepines are: sedation, dizziness, weakness, and unsteadiness. Other side effects include: transient drowsiness commonly experienced during the first few days of treatment, a feeling of depression, loss of orientation, headache, sleep disturbance, confusion, irritability, aggression, excitement, and memory impairment.

1. METABOLIC FATE OF
BENZODIAZEPINES AND
THEIR IDENTIFICATION
PRESENTED BY:
NEHA BADHWAR
M.Sc.(P) FORENSIC SCIENCE
2nd Semester
ROLL NO.: 7
SUBMITTED TO:
DEPARTMENT OF
ZOOLOGY,KURUKSHETRA
UNIVERSITY

2. CONTENTS:
Introduction
The GABA Receptor Complex and Benzodiazepine Receptors
Metabolic Fate of Benzodiazepines
Benzodiazepines that undergo Phase I Metabolism
Benzodiazepines that undergo Phase II Metabolism
Examples of the Metabolism of Benzodiazepines
Presumptive Test for identification of BDZ
Analytical Methods for identification of BDZ
Benzodiazepines detection time
Benzodiazepines Alternatives- Antidepressants, Other drugs
Medico-Legal Aspects of BDZ
Case Study
Conclusion
References

3. INTRODUCTION
It is a Psychoactive drug whose core chemical structure is fusion of a benzene ring
and a diazepine ring.
First such drug- chlordiazepoxide (Librium), Discovered by
Leo Sternbach in 1950 and made available in 1960 by
Hoffmann-La Roche
Family of drugs- Minor Tranquilizers
Enhance the effect of GABA receptor resulting in:
Sedative, hypnotic (sleep-inducing), anxiolytic (anti-anxiety),
anti-covulsant and muscle relaxant properties

4. Benzodiazepines Stereochemistry: BDZ’S do not have a chiral
centre but 7 membered ring adopt one of two energetically preferred
boat conformations (I and II) which are enantiomeric relative to each
other.
Benzodiazepines Lipophilicity: Due to their high
hydrocarbon content and presence of halogen atoms.
Basicity and reactivity of the
Benzodiazepines: Strong acid salts
are unstable and readily undergo
sequential hydrolyses, first at the imine
bond and then at the amide to yield
inactive products. The first hydrolysis
reaction (imine hydrolysis) is
reversible, however the second (amide
hydrolysis) eliminates GABA receptor
activity.

5. THE GABA RECEPTOR COMPLEX AND BENZODIAZEPINE
RECEPTORS
• Cell-surface receptors: GABA-A and GABA-B and GABA-C
receptors
• bind to the to γ-aminobutyric acid type A receptor (GABA-A) at the
alpha-subunit
• Increases conductance of the chloride channel and inhibits neuronal
excitability
• Consists of combinations of α, β, γ subunits- two α1 subunits, two β2
subunits, and one γ2 subunit
• BDZ’s bind to α2, α3, and α5 subunits
• BDZ’s are allosteric modulators

6. BZR
BZR-1
cerebellum
BZR-2
cortex,
hippocampus,
and spinal cord
BZR-3
peripheral
tissues,
mitochondrial
• result in new subtype-selective agents for the treatment of
anxiety, sleep disorders, convulsions, or memory deficits
• in recent years, the use of benzodiazepines has declined, due
to the increasing unacceptability of their side effects, such as
sedation, dizziness, interaction with alcohol, and the risk of
dependence with long-term use

7. METABOLIC FATE OF BENZODIAZEPINES
• metabolized in liver and excreted in the urine as glucuronide conjunctions
• to some extent, they are eliminated through the digestive system
Metabolism of benzodiazepines decreases with age-
• characterized by shifted metabolism through oxidation rather than conjugation.
• may cause increase compound’s half-life and consequently- sleepiness
• under reduced hepatic blood flow, for example in liver diseases, metabolism of benzodiazepines is
slower
• recommended to use drugs (oxazepam, lorazepam, temazepam), which do not form active metabolite
• may decrease when they are taken in parallel with drugs inhibiting the activity of cytochrome P-450

8. BENZODIAZEPINES UNDERGO PHASE I AND PHASE II
METABOLIC PATHWAYS
PHASE I
By enzyme CYP450
Hepatic oxidation and Reduction
By enzymes CYP2C19 and CYP3A4
Hydroxylation-Alprazolam, triazolam,
midazolam, and diazepam
Nitroreduction-clonazepam
PHASE II
Glucuronidation- include lorazepam,
oxazepam, temazepam (LOT)

9. modification of metabolism
and/or removal of that group,
active metabolites are formed
metabolic hydroxylation at
position 3, resulting in the
formation of an active derivative.
conjugation of the 3-hydroxy
compounds mainly with
glucuronic acid
Metabolism of benzodiazepines is characterized by three basic steps:
• First phase has the highest efficiency
• have longer duration time in comparison to parent drug
• Eg: Demetylodiazepam (nordiazepam), which is a common metabolite of
diazepam, clorazepate, halazepam and prazepam. Its half-life of
approximately 60 hours determines the accumulation of this compound in
the body and its prolonged activity
• Metabolite compounds may also have a biological half-life similar to
parent compound, e.g. α-hydroksyalprazolam
• Second phase: This process occurs slower than first phase, without
problems associated with the accumulation of hydroxylated products from
unmodified groups at position 1
• Last step involves in the conjugation of the 3-hydroxy compounds mainly
with glucuronic acid, with pharmacologically inactive product as a result.

10. BENZODIAZEPINES THAT UNDERGO PHASE I METABOLISM

11. BENZODIAZEPINES THAT UNDERGO PHASE II METABOLISM

12. EXAMPLES OF METABOLISM OF THE BENZODIAZEPINES
 Metabolism of Chlordiazepoxide/Amidine
BDZ: (Interim Onset, Long Duration)
• absorbed after oral administration, but slowly and
erratically absorbed from IM injection sites.
• drug is metabolized to active benzodiazepine
metabolites, beginning with CYP-mediated OND-
demethylation of the amidine group
• "desmethyl" metabolite is slowly hydrolysed to
demoxepam which can undergo three different
reactions:
1). hydrolysis to an inactive ring opened form
2). aromatic hydroxylation to phenol metabolites that
retain some activity and
3). Reduction to an active "nordiazepam" metabolite.
The nordiazepam metabolite may be further oxidized to
an active "oxazepam" metabolite that can be conjugated
as a glucuronide which is inactive and eliminated.

13.  Metabolism of Chlorazepate / 3-Carboxy-BDZ (Fast Onset, Long Duration):
• benzodiazepines have a 3C-acidic group from
which salts can be formed
• undergo spontaneous decarboxylation in the GI
tract (and other tissues) to yield "nordiazepam"
intermediates
• nordiazepam formed is active as a BZ-ligand and
is oxidized by CYP isozymes to an "oxazepam"
metabolite that is also active
• oxazepam metabolite may be conjugated as a
glucuronide which is inactive and eliminated
• this compound gives rise to several active,
relatively long-lived metabolites, it has a long
duration of action

14.  Metabolism of the C-3-Hydroxy-Substituted BDZ(“Oxazepams”): Slow to intermediate
onset, Intermediate duration of action
• benzodiazepines are more polar and generally
have a slower onset
• since they all contain a 3-hydroxyl group in
their structure, they can be directly conjugated
as inactive glucuronides and eliminated
• safer to use in the elderly and other patients
with compromised oxidative metabolic capacity

15. PRESUMPTIVE TEST FOR IDENTIFICATION OF BDZ
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.
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.

16. ANALYTICAL METHODS FOR IDENTIFICATION OF BDZ
Analytical
methods
Chromatography
TLC
(Thin Layer
Chromatography)
HPLC
(High Performance
Liquid
Chromatography)
GC
(Gas
Chromatography)
Mass Spectroscopy

17. THIN-LAYER CHROMATOGRAPHY:
 used for the determination of several benzodiazepines like
diazepam, nitrazepam, flunitrazepam, chlordiazepoxide,
clonazepam, tetrazepam, oxazepam and temazepam
 used for the determination of benzodiazepines in biological
material, primarily in serum, plasma, whole blood, urine, and less
commonly in stomach content
 EXTRACTION METHODS : Liquid-liquid extraction and solid-
liquid extraction
 SOLVENT SYSTEMS :
System A: Chloroform: acetone (80:20, v/v)
System B: Chloroform, methanol (90: 10 v/v)
System C: Cyclohexane: toluene: diethylamine (75:15:10 v/v/v)
 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 Sulphuric Acid/heat/observe under UV light at 366 nm
C. Acidified potassium iodoplatinate reagent

18. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
 applicable to both qualitative and quantitative analysis
 used not only in the pharmaceutical industry, but also in toxicological
analysis, control of doping in forensic medicine, agriculture and food
industry derivatives mainly in biological fluids such as urine , hair,
plasma, serum , whole blood, nails, gastric fluids , tissue fragments and
oral secretion fluids
 in pharmaceutical preparations i.e., tablets, capsules and injection
solutions
 stationary phase : polyvinyl alcohol, phenyl groups, cyano groups or β-
cyclodextrin
GAS CHROMATOGRAPHY
 used in the analysis of benzodiazepine derivatives extracted from urine or
blood using for example n-butyl chloride or a mixture of
chloroform:isopropanol(9:1)
 Undergoes Derivatization process
 Detection of compounds is carried out using (ECD),(GC-MS) or GC-MS-
MS with (EI), (PCI) and (NCI)

19. MASS SPECTROSCOPY
 sensitive and selective HPLC/APCI-MS/MS method
 utilises butyl chloride extraction at alkaline pH followed by reversed-phase
liquid chromatography
 suitable for screening analyses and confirmation of identity of the
benzodiazepines at their lowest reported therapeutic concentrations using 500
microL of blood
 triple quadrupole mass analyzer is frequently utilized
 Also single quadrupole and ion trap mass analyzer
 applicable for most benzodiazepines, except for ketazolam

20. Benzodiazepines Detection Time
 The length of time depends on the drug’s half-life or the time it takes for half the drug to be metabolized and
eliminated from a person’s body
 also determines how soon, and for how long, the drug can be detected:
 Blood tests -shortest window of detection
 Saliva (oral fluid screening) and urine (urinalysis)-intermediate testing window
 Hair tests- longest
 Factors influencing: individual’s age, metabolism, physiology, and health or medical concerns

21. Benzodiazepines alternatives
Antidepressants:
 Sertraline (zoloft)- for the long-term treatment of panic disorder and generalized anxiety
Other drugs:
 Buspirone (BuSpar)- treatment of chronic anxiety, enhances the effect of the neurotransmitter serotonin
 Ramelteon- introduced treatment for insomnia, acts at receptors for melatonin, a hormone that helps to regulate
the body's circadian (24-hour) cycles
MEDICO LEGALASPECTS OF BDZ
Forensic approach should consider:
 Firstly the reality of the benzodiazepines absorption and implication in committing violence (urine test,
chronology, amnesia);
 Secondly, the association of unusual behaviour and converging circumstances (pharmacological,
pharmacokinetic, psychopathology, external conditions);
 Thirdly the consumer's knowledge of the disinhibition effect.

22. CASE STUDY
Some people have found new tricks to deprive trains and bus passengers of their cash and valuables. These crooks are using any
form of eatable including buy offerings (Prasad), ladoo, biscuits or soft drinks as a bait to rob passengers. On Jan 19 Kunal,
resident of Rohini West, New Delhi was drugged through biscuit in a train and then robbed. He was on from Akshardham Mandir,
New Delhi. He told the police that, the stranger seated next to him offered biscuits. Few minutes after eating biscuits he became
drowsy and lost consciousness. He do not remember anything. Next day, he found himself in hospital with a glucose drip on. The
crook escaped with victim’s bag containing Rs. 5000, laptop and some official files. Hospital authorities diagnosed Kunal illness
as consumption of sedatives. Police found opened biscuit packet from the place of crime with still two cream biscuits left which
they forwarded to FSL in sealed condition for further opinion on whether it contain any sedative or not. Cream biscuit which was
the suspected sample found at the crime scene at New Delhi was used as the sample and sedatives were extracted using drug
extraction procedure and analysed using TLC and Fourier Transform Infrared Spectrometry (FTIR).

23. Ethylacetate: Methanol: Ammonia (15:5:0.5)
Showing TLC Plate under UV Light Showing TLC Plate after spraying Dragendroff

24. CONCLUSION
Metabolism is an integral part of drug elimination. Facilitating excretion of a
drug affect the pharmacological response of a drug by altering its potency or
duration of action. Metabolites of drugs are more polar and are therefore
likely to be excreted from the body. Metabolites may be pharmacologically
inactive or may be active. Formation of active metabolites changes the profile
of drug action. In some cases reabsorption occurs, it reverses the secretion
process. Drugs may get destroyed by the acid in the stomach, by enzymes in
the gastro-intestinal tract or may be hardly absorbed at all because of their
chemical nature. These factors reduce the drug’s bioavailability. The
confirmation of BZDs consumption theoretically includes a screening test,
followed, in case of positivity, by a confirmation test.

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