Determination of Benzodiazepines in Human Urine using Solid-Phase Extraction and High-Performance Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for the determination of benzodiazepines, on the market in Norway, and/or their metabolites in human urine. The following compounds were included: 7-aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, alprazolam, α-hydroxyalprazolam, oxazepam, 3-OH-diazepam, and n-desmethyldiazepam. The method includes hydrolysis of urine samples (0.5 mL) with β-glucuronidase at 60°C for 2 h before solid-phase extraction with a polymer-based mixed.mode column. The analytes were quantified in multiple reaction monitoring mode using two transitions. Deuterated analogues were used as internal standards for all analytes except 7-aminonitrazepam and α-hydroxyalprazolam, which were quantified using 7-aminoclonazepam-d₄ and alprazolam-d₅, respectively. The concentration range was 0.1–8.0µM for 7-aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, alprazolam, and α-hydroxyalprazolam and 0.5–40µM for the other compounds. The average recovery for the different analytes ranged from 56% to 83%. The between-day precision of the method was in the range of 3–12%. The limits of quantification were found to be between 0.002 and 0.01µM for the different compounds. Comparison with other analytical methods was performed for method validation, using approximately 500 samples provided by the routine laboratory at the Norwegian Institute of Public Health. The LC-MS-MS method has proven to be robust and specific for the determination of benzodiazepines in urine. It has been routinely used for approximately 1800 samples in the past 7 months.