Standardization of a Protocol to Quantify Benzodiazepines in Human Urine Samples Employing Gas Chromatography–Mass Spectrometry─A Green Analytical Chemistry Approach

Abstract

Benzodiazepines (BZDs) are central nervous system depressants, prescribed to slow brain activity during anxiety, seizures, insomnia, etc. However, their long-term usage is associated with health risks such as physical and physiological dependence and cognitive decline. Poisoning due to BZDs is frequently encountered in forensic toxicology, which requires their rapid and robust detection. However, their low availability in biological samples and the lack of sensitive and rapid detection methods pose challenges. In this study, we standardized a protocol for the detection and quantification of lorazepam (LZ), clonazepam (CZ), and alprazolam (AZ), the short-to-intermediate and long-acting BZDs, without employing any derivatization steps and using gas chromatography–mass spectrometry (GC–MS). The detection limit (LOD) of BZDs by GC–MS was 5 ng/mL, and the detection was linear between 5 and 50 ng/mL, with coefficients of determination (R2) of 0.9984, 0.9968, and 0.9936 for LZ, CZ, and AZ, respectively. Further, rapid dispersive liquid–liquid microextraction allowed enriching BZDs from human urine samples for following GC–MS analysis. Interday and intraday detection precisions were analyzed, which were within agreeable limits. Together, we have developed a green analytical chemistry protocol for the rapid detection of BZDs that can be employed in forensic toxicology and analytical chemistry laboratories for criminal cases, drug overdoses, or drug monitoring.