Quantitative assay of lorazepam and its metabolite glucuronide by reverse-phase liquid chromatography-tandem mass spectrometry in human plasma and urine samples

A LC/MS/MS method for the quantitative determination of lorazepam in human plasma and urine samples was developed and validated. The enantioselective assay allowed to separate the enantiomers and to verify the stereochemical instability of lorazepam. The linearity assessed for lorazepam unchanged was 0.2–20 ng of each enantiomer/ml plasma and 0.2–15 ng of each enantiomer/ml urine. The linearity assessed for total lorazepam (after enzymatic hydrolysis) was 1–30 ng of each enantiomer/ml plasma and 10–150 ng of each enantiomer/ml urine. The coefficients of variation obtained for the intra- and interassay precision were less than 15%. The method was applied to the investigation of the kinetic disposition and metabolism of racemic lorazepam administered as a single oral dose of 2 mg to a parturient. The occurrence of racemization required the calculation of the pharmacokinetic parameters as enantiomeric mixtures of lorazepam (t1/2a 3.5 h; Ka 0.198 ng h−1; t1/2 11.5 h; β 0.060 h−1; AUC0−∞ 192.1 ng h/ml; CLt/f 2.41 ml/min kg; Vd/f 173.5 l; Fel 0.41%, and ClR 0.0099 ml/min kg) and its metabolite lorazepam–glucuronide (t1/2f 1.2 h; Kf 0.578 h−1; t1/2 16.6 h; β 0.042 h−1; AUC0−∞ 207.6 ng h/ml; Fel 51.80%, and ClR 98.32 ml/min kg). However, the determined confidence limits make the method suitable for application to clinical pharmacokinetic studies, even if the quantification of both the enantiomers is required.