Metabolites of lorazepam: Relevance of past findings to present day use of LC‐MS/MS in analytical toxicology

Abstract

The advent of liquid chromatography-tandem mass spectrometry (LC-MS/MS), with the sensitivity it confers, permits the analysis of both phase I and II drug metabolites that in the past would have been difficult to target using other techniques. These metabolites may have relevance to current analytical toxicology employing LC-MS/MS, and lorazepam was chosen as a model drug for investigation, as only the parent compound has been targeted for screening purposes. Following lorazepam administration (2 mg, p.o.) to 6 volunteers, metabolites were identified in urine by electrospray ionization LC-MS/MS, aided by the use of deuterated analogues generated by microsomal incubation for use as internal chromatographic and mass spectrometric markers. Metabolites present were lorazepam glucuronide, a quinazolinone, a quinazoline carboxylic acid, and two hydroxylorazepam isomers, one of which is novel, having the hydroxyl group located on the fused chlorobenzene ring. The quinazolinone, and particularly the quinazoline carboxylic acid metabolite, provided longer detection windows than lorazepam in urine extracts not subjected to enzymatic hydrolysis, a finding that is highly relevant to toxicology laboratories that omit hydrolysis in order to rapidly reduce the time spent on gas chromatography-mass spectrometry (GC-MS) analysis. With hydrolysis, the longest windows of detection were achieved by monitoring lorazepam, supporting the targeting of the aglycone with free drug for those incorporating hydrolysis in their analytical toxicology procedures.

Signal enhancement of glucuronide conjugates in LC‐MS/MS by derivatization with the phosphonium propylamine cation tris(trimethoxyphenyl) phosphonium propylamine, for forensic purposes

“…Good windows of detection have been obtained for underivatized glucuronides in urine, such as lorazepam glucuronide, [20] but further improvements could be envisaged with the use of precharged derivatives. Good windows of detection have been obtained for underivatized glucuronides in urine, such as lorazepam glucuronide, [20] but further improvements could be envisaged with the use of precharged derivatives.…”

“…This work indicates that the concept of charge derivatization, such as with the phosphonium-containing TMPP-C 3 H 6 NH 2 derivatization reagent can improve the sensitivity of detection of glucuronide metabolites. Good windows of detection have been obtained for underivatized glucuronides in urine, such as lorazepam glucuronide, [20] but further improvements could be envisaged with the use of precharged derivatives. The derivatization reagent may have application in targeted analysis of low concentrations of analyte if a suitable purification method is developed first to eliminate interferences from other urinary constituents.…”