Liquid chromatography-tandem mass spectrometry for detection of low concentrations of 21 benzodiazepines, metabolites, and analogs in urine: method with forensic applications.

Background: Commonly used methods for detecting benzodiazepines (BZPs) and BZP-like substances, such as zolpidem and zopiclone, may not detect low concentrations of these drugs. We developed a liquid chromatographic-tandem mass spectrometric method for identifying these drugs and their relevant metabolites.

Methods: We extracted BZPs from urine by solid-phase extraction with a mixed-mode phase (OASIS HLB cartridges). Chromatographic separation was performed with a Waters XTerra MS C18 [150 x 2.1 mm (i.d.); bead size, 5 microm] reversed-phase column with deuterated analogs of the analytes as internal standards (IS). Detection was performed with a triple-quadruple mass spectrometer that monitored 2 specific transitions per compound in the electrospray, positive-ion selected-reaction monitoring mode. We tested this technique on urine samples from 12 healthy volunteers and 1 forensic sample obtained in a case of alleged drug-facilitated sexual assault.

Results: Chromatographic separation was achieved within 18 min. The linear dynamic ranges extended from 0.02 or 0.1 microg/L (depending on the drug or metabolite) to 50 microg/L. Extraction recovery (range) was 77%-110%. Limits of detection were < or = 0.05 microg/L. No ion suppression was seen except for alprazolam, for which baseline decreased by almost 20%. In the forensic urine sample, the method detected alprazolam (3.5 microg/L) and its characteristic metabolite, alpha-hydroxyalprazolam (0.17 microg/L).

Conclusion: This method measured low concentrations of BZPs and BZP-like substances and might be useful for analyses of urine in suspected drug-facilitated sexual assault cases.