Quantitation of Benzodiazepines and Metabolites in Urine by Liquid Chromatography–Tandem Mass Spectrometry

Background Benzodiazepines (BZDs) are central nervous system depressants that are prescribed to prevent seizures, manage anxiety, or help sleep. When misused, BZDs can lead to addiction and sometimes cause death. Measurement of BZDs in urine is used to identify their use, especially in pain management settings. LC-MS/MS is preferred for these measurements because of its high sensitivity and specificity. Here, we report an LC-MS/MS assay for measuring 7 BZDs and metabolites in urine. Methods Urine sample was incubated at 60 °C for 30 min after addition of internal standards and a β-glucuronidase solution. After centrifugation, the supernatant was diluted with methanol and water before being injected onto a C18 analytical column in an LC-MS/MS system for quantification. The analytical time between injections was 4.35 min. The analytes included 7-aminoclonazepam, α-hydroxyalprazolam, α-hydroxytriazolam, oxazepam, lorazepam, nordiazepam, and temazepam. Results The lower limit of quantification ranged from 30 ng/mL to 50 ng/mL with an analytical recovery >80% for all 7 analytes. Total CV was <10% for all analytes (3 concentration levels of 100, 2500, and 5000 ng/mL; n = 30 each). This method had 100% agreement with a GC-MS method offered by an independent laboratory for negative urine samples. For the positive urine samples, this method showed a strong correlation (R > 0.96) with the GC-MS method. Conclusions The LC-MS/MS assay allows accurate and precise measurement of 7 BZDs and metabolites in a single analytical run with a short analytical run time and broad measuring ranges.