Simultaneous Method for the Separation and Identification of Certain Benzodiazepine Drugs in Pharmaceutical Formulations by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Abstract

Background: The Benzodiazepine (BZD) drugs are frequently encountered in clinical and forensic casework samples involving road traffic offences and/or drug overdoses. An overdose of BZD can cause respiratory depression, coma and death. A sensitive and selective method for the simultaneous method for the separation and identification of certain abused BZDs viz. diazepam, lorazepam, nitrazepam, chlordiazepoxide, flurazepam, alprazolam, etizolam, clobazam, clonazepam, and midazolam drugs was developed together with a qualitative screening in pharmaceutical formulations. Methodology: The instrument was operated using an electrospray ionization mode (ESI) in combination with the C18 reverse phase column (120 Å, 2.1 x 150 mm Acclaim 120) 4.5 minutes resulted in the best separation and was selected. Chromatographic separation was performed with a different solvent combinations were tested to achieve the best separation. The mobile phase consisted of a mixture of methanol and 20 mM ammonium formate (50: 50, v/v) adjusted pH 8.6. A run of 25 min, including a re-equilibration time of 10 min was selected. Results: The most implicated drug class was BZDs, either alone or in combination with alcohol. In this report, a procedure was developed for the screening of ten BZDs in pharmaceutical formulations by LC-MS/MS. The limit of detection ranged from 25 - 250 μg/mL for all BZDs. Our results demonstrate the suitability of the LC-MS/MS method for the simultaneous separation and identification of BZDs, and new psychoactive substances in forensic samples. Recommendations: The results of the study showed that the proposed LC-MS/MS method is simple, rapid, precise and accurate, which is useful for the separation and identification of BZD drugs in forensic, clinical analytic toxicology and might be useful for the analyses of suspected drug-facilitated sexual assault (DFSA) cases.