Determination of hypnotic benzodiazepines (alprazolam, estazolam, and midazolam) and their metabolites in rat hair and plasma by reversed-phase liquid-chromatography with electrospray ionization mass spectrometry.

Sensitive determination of benzodiazepines i.e., alprazolam (ALP), estazolam (EST), and midazolam (MDZ), and their metabolites, was carried out by reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS). The chromatography separations were achieved using a semi-micro HPLC column (3 microm particle size; 100 x 2.0 mm, i.d.) with acetonitrile-water containing 1% acetic acid as eluent. The mass spectrometer was operated in selected-ion monitoring mode at protonated-molecular ions [M+H](+) of parent drugs and the metabolites. The proposed procedure was applied to the determination in hair shaft of Dark Agouti rats after intraperitoneal (i.p.) administration of benzodiazepines twice a day for 5 days. Various metabolites together with parent drugs were identified in the hair shaft, 1-hydroxyalprazolam (1-HA) and 4-hydroxyalprazolam (4-HA) from ALP administration; 8-chloro-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine-4-one (K-EST) from EST administration; 1-hydroxymidazolam (1-HM) and 4-hydroxymidazolam (4-HM) from MDZ administration. A few unknown metabolites were also detected in the hair samples. These structures were elucidated with acetylation using acetic anhydride and pyridine. The time course studies of parent drugs and the metabolites in both hair root and plasma were also carried out after single i.p. administration of benzodiazepines. The results suggested that the concentrations of parent drugs and the metabolites in the hair samples were mainly dependent upon those in the plasma.