Overview
The analysis of benzodiazepines in biological fluids is important to clinicians and forensic toxicologists because of their sedative, hypnotic, antianxiety, antiepileptic and muscle relaxant properties. It is challenging to analyse a mixture of etizolam, triazolam, and their metabolites because their chemical structures, molecular weights and fragmentation patterns during mass spectrometry are very similar.

This application note, developed by Shimadzu (Japan)[1], shows a high-resolution separation using the robust YMC-Triart C18 for the simultaneous analysis of etizolam, triazolam, and their metabolites 8-ethylhydroxyetizolam, alpha-hydroxyetizolam, 4-hydroxytriazolam and alpha-hydroxytriazolam.
Three samples were analysed: a mixture of standards including etizolam, triazolam, alpha-hydroxytriazolam and 4-hydroxytriazolam (A), metabolised etizolam and triazolam using human liver S9 (mix of liver enzymes) (B) and blank metabolised matrix (C).
