Rapid determination of benzodiazepine derivative residues in alcoholic beverages by HPTLC

Worldwide studies indicate that the number of crimes related to drug use is increasing every day. These crimes are identified as "Drug-Facilitated Sexual Assault" (DFSA) and the combination of drugs with other crimes is termed "Drug-Facilitated Crime" (DFC). In DFSA and DFC cases, drug-facilitated beverages are known to be used for the commission of exploitation crimes and contain substances that are tasteless, colorless, and odorless, making them easily soluble in liquids. These beverages often contain sedative-hypnotic substances such as benzodiazepines and ketamine. Specifically, benzodiazepines (BZDs) prescribed for anxiety, insomnia, and seizures, such as diazepam and flunitrazepam derivatives, are commonly used. The forensic use of BZDs significantly contributes to the investigation of crimes and the apprehension of criminals. The presence or absence of these drugs is crucial in crime scene investigations, evaluating witness statements, and examining traffic safety incidents. This study aims to present a method that facilitates the identification of diazepam and flunitrazepam, both benzodiazepine derivatives frequently encountered in forensic cases, in order to be rapid, effective, and reliable. Unlike the commonly used MS systems in the literature, an HPTLC system was preferred for the method's speed, effectiveness, and reliability. The main plan of the study is based on the assumption that the lowest milligrams of commercially available diazepam and flunitrazepam-containing drugs are used and added to wine, beer and whiskey solutions of approximately 250-330 mL, considering that the easiest accessible forms may or may not be determined at the lowest concentrations. Diazepam is available in the market as Diaksi with 5 mg/2.5 mL and 10 mg/2.5 mL, and as Diapam with 10 mg/2 mL. Flunitrazepam is available as Rohypnol with 5 mg/2.5 mL. For HPTLC analysis, silica gel 60 F254 HPTLC glass plates (Merck Cat. No: 1.05649) sized 20 x 10 cm were chosen, providing highly sensitive results in the detection of benzodiazepines. The simultaneous analysis of the two active substances dissolved in methanol was tested with 8 different solutions or solution mixtures using classical TLC techniques. Method validation is the process of demonstrating that analytical procedures are suitable for their intended use and support the identity, quality, purity, and strength of drug substances and drug products. Our study has been tested according to validation procedures, and the method has been fully validated by demonstrating satisfactory data for all tested method validation parameters. This simple HPTLC method has been successfully developed to detect two sedative-hypnotic drugs (diazepam and flunitrazepam) suspected to be added to a liquid beverage in DFSA and DFC cases. The achieved high recoveries indicate the suitability of the method for examining evidence seized from suspected crime scenes.