Use of Benzodiazepine in Forensic Analysis

BY SIFS India | August 10, 2020

Use of Benzodiazepine in Forensic Analysis


Drug abuse, however, does not only include the consumption of illegal drugs such as cocaine, cannabis, opiates but also includes prescription medications such as painkillers and sleeping pills.

According to the latest news reports Benzodiazepines prescriptions have skyrocketed since the pandemic began.

Several studies reveal a special link between the accomplishment of a crime and benzodiazepine use, abuse and dependence.

There is a wide range of problems related to Benzodiazepine that includes diversion, misuse, dependency, driving impairment, morbidity and mortality related to overdose and withdrawal. 

On July 11, every year World Benzodiazepine Awareness Day (W-BAD) is held to raise global awareness about benzodiazepines dependence and the dangers of its adverse effects on consumption.

This article mainly summaries misuse of benzodiazepines along with techniques followed for their analysis in forensic laboratories. 

What are Benzodiazepines (BZDs)?

Benzodiazepines (BZDs) are a class of psychoactive drugs which has hypnotic, tranquilizing, and anticonvulsant properties.

BZDs are one of the most commonly prescribed medications in the world used therapeutically as anxiolytics, tranquilizers, hypnotics, and anticonvulsants in epilepsy to induce sleep and in the treatment of many psychiatric disorders.

This drug mainly works by producing a calming effect which enhances the effect of the neurotransmitter gamma-aminobutyric acid (GABA) present in the brain. Benzodiazepines are categorized according to the duration of action as either-

Short acting (<1-12 hrs): Triazolam, Oxazepam, Medazolam

Intermediary (12-24 hrs): Alprazolam, Lorazepam, Clonezepam,Temazepam

Longer acting (>24 hrs): Dizepam, Chlordiazepoxide, flunitrazepam.

BZDs Misuse/Abuse

These drugs are frequently abused and are encountered in clinical and forensic casework samples involving road traffic offenses, sexual assaults or drug overdose, an overdose of BZDs can cause respiratory depression, coma, and death.

Numerous benzodiazepines have reportedly been synthesized and over fifty of these are marketed for clinical use throughout the world. Out of the marketed benzodiazepines, thirty-five are subject to international control under the 1971 Convention on Psychotropic Substances.

BZDs abuse or misuse is globally 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 legitimate sources.

The benzodiazepines, specifically alprazolam and diazepam, are among the most often diverted and abused psychotropic substances.

Analysis of Benzodiazepine in Forensics

In order to establish the identity of a specific drug in any substance, proper analytical techniques are needed to be applied. Generally, three analytical techniques to be used are color tests, chromatography (eg. TLC), and spectroscopy (eg. UV or IR). Hyphenated techniques such as GC-MS can also be used.

BDUs are mainly formulated as tablets, powders, oral liquids, or injectable and should be sampled depending on the number and type of dosage units seized. To examine for the presence of any specific drug in material the analyst should perform a full chemical analysis of the material including extraction of the drug, presumptive tests, screening, and some confirmatory test is also required.

Extraction and Sample Preparation

BDUs are usually present as the free base or as hydrochloride, mesylate, or as potassium salts and some have carboxylic acid functionalities. All the BDZs are generally soluble in methanol.

These drugs are encountered in a number of suspected samples such as in drinks, food items etc.

Therefore, these drugs are extracted from suspected samples using drug extraction procedures. Also, the samples are prepared with different procedures for both qualitative and quantitative analysis.

Color Test

There is no specific color test for this class of drug, although the Zimmerman test is often applied which gives red-purple to pink color for the presence of a BDZs on the addition of two different solutions.

This test is not specific to this class of compounds. Therefore analysts are should use a combination of TLC and color development after spraying with selected reagents as a presumptive test. 

Thin-Layer Chromatographyy (TLC)

The TLC is the simplest of the chromatographic methods. The spots are placed on the sheet (Silica gel G) called as stationary phase and let to elute in required solvent system called eluent.

The eluent diffuses along with the plate on the basis of interactions. Then for visualization certain spraying reagents are used and further Rf values can be calculated and compared with the standard values.

This technique is quite fast (about half an hour) but lacks precision and sensitivity.

Gas Chromatography-Mass Spectrometry (GC-MS) 

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 analyte specific of a spectroscopic technique, providing highly specific spectral data on individual compounds in a complex mixture of compounds often without prior separation.

This technique is mostly applied for identification of BZDs in biological matrices. GC-MS technique relies on detection of specific high mass ions of the substance that is to be analyzed and then to search for the specific ions obtained comparison with the reference library is performed. Also, GC poses a number of thermo ability problems with most of the BZDs. 

Infrared (FTIR) Spectroscopy

A quality control procedure was developed for the determination of benzodiazepines in samples using Fourier Transform Infrared Spectroscopy (FTIR). KBr pellets are prepared using KBr powder mixed with the extracted sample and are scanned in FTIR .

The peak values obtained are compared with the standard ones.

IR techniques are best to be used for quantitative estimation of drugs.

The widespread use of this class of drugs has occasionally raised concerns about recreational benzodiazepine abuse.

These drugs are misused because of their wide range and easy availability. Considering the increased potential of BZDs addiction and abuse, their proper sampling, separation, and identification techniques are needed to be applied.


Schmitz, Allison. “Benzodiazepine use, misuse, and abuse: A review.” The mental health clinician vol. 6,3 120-126. 6 May. 2016, doi:10.9740/mhc.2016.05.120

Qriouet, Zidane, et al. "Analytical methods used for the detection and quantification of benzodiazepines." Journal of analytical methods in chemistry 2019 (2019).

Malik, Ritu,et al.  “Forensic Examination of Benzodiazepines: A Case Study.” Journal of Forensic Sciences & Criminal Investigation (2018) 8. 10.19080/JFSCI.2018.08.555727.