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

Journalof AnalyticalToxicology,Vol. 30, January/February2006 Determination of Benzodiazepinesin Human Urine usingSolid-PhaseExtractionand High-Performance Liquid Chromatography-ElectrosprayIonization Tandem Mass Spectrometry S. Hegstad*, E. I.. Oiestad, U. Johansen, and A.S. Christophersen Norwegian Instituteof Public Health, Division of Forensic Toxicologyand Drug Abuse, P.O. Box 4404 Nydalen, N0-0403 Oslo Abstract 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, ~hydroxyalprazolam, oxazepam, 3-OH-diazepam, and n-desmethyldiazepam. The method includes hydrolysis of urine samples (0.5 mL) with ~-glucuronidase at 60~ 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 analogueswere used as internal standards for all analytes except 7-aminonitrazepam and ~hydroxyalprazolam, which were quantified using 7-aminoclonazepam-d4and alprazolam-ds, respectively. The concentration range was 0.1-8.0pM for 7-aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, alprazolam, and ~hydroxyalprazolam and 0.5-40pM 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 0.01pM 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. Introduction Benzodiazepines (BZDs) belong to a group of drugs known for their sedative, hypnotic, and anticonvulsant properties and are among the most frequently prescribed drugs in the world for the therapy of anxiety and sleeping disorders (1-3). These 9Author to whom correspondence should be addressed. E-mail: . compounds are, however,also associated with misuse and have become a serious problem in many countries (4). In Norway, BZDs are some of the most frequently detected drugs in blood samples from suspected drugged drivers (5,6). The Norwegian Institute of Public Health receives about 25,000 urine samples each year from prison and probation services, social services, and workplace testing programs. Statistics based on the 5124 urine samples that tested positive after screening with immunological methods show that 50% of the samples contained benzodiazepines. Thus, a robust and specific quantitative method is needed. In urine, BZDs are predominantly excreted as glucuronide conjugates. Flunitrazepam, clonazepam, and nitrazepam are metabolized to 7-amino metabolites by reduction of the 7nitro group or by demethylation to n-desmethyl metabolites (7-9). The 7-amino metabolites are subsequently converted to N-glucuronides, and the n-desmethyl metabolites are further hydroxylated and glucuronidated. Diazepam is converted to 3-OH-diazepam, n-desmethyldiazepam, and oxazepam, which are subsequently conjugated to glucuronides. Alprazolam is metabolized to oc-hydroxyalprazolam and 4-OH-alprazolam (10), but about 10% is excreted in the urine as the parent drug. A number of studies have been reported on the analysis of BZDs and their metabolites in urine. Most of them used gas chromatography-mass spectrometry (GC-MS) techniques, which requires tedious derivatization (11-18). Liquid chromatography (LC)-MS is increasingly being used in forensic toxicologyto quantify a wide range of compounds in biological samples (4,19-25). Most of the studies mentioned used atmospheric pressure chemical ionization (APCI),but electrospray ionization (ESI) has also been used for the determination of BZDs in serum and urine (26-28). LC-MS-MSprovides greater sensitivity and selectivity than LC-MS and has recently been used for determination of BZDs in plasma (29), hair (30,31), larvae (32), and whole blood (33). The purpose of the present study was to developa robust and specific LC-MS-MS method for the simultaneous quantification of BZDs and their metabolites in urine that is suitable for Reproduction(photocopying)of editorialcontentof thisjournalis prohibitedwithoutpublisher'spermission. 31 Journal of Analytical Toxicology, Vol. 30, January/February 2006 routine analysis. The eight compounds included were: 7aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, alprazolam, (z-hydroxyalprazolam,oxazepam,3-OHdiazepam, and n-desmethyldiazepam. Such a method would increase the efficiency of our routine laboratory by replacing two chromatographic methods involving derivatization techniques [high-performance liquid chromatography (HPLC)-fluorescence and GC-MS]. Material and methods Chemicals and reagents Reference compounds were obtained from four pharmaceutical companies: 7-aminonitrazepam, 7-aminoflunitrazepam, o~-hydroxyalprazolam, and 3-OH-diazepam (Cerilliant Corp. Austin, TX); 7-aminonitrazepam, 7-aminoclonazepam, 7aminoflunitrazepam, alprazolam, and c~-hydroxyalprazolam (Lipomed, Arlsheim, Switzerland); alprazolam, 3-OH-diazepam, and oxazepam (Sigma-Aldrich, St. Louis, MO); oxazepam and n-desmethyldiazepam (Alltech,State College, PA). The internal standards 7-aminoclonazepam-d4, 7-aminoflunitrazepam-dT, alprazolam-ds, oxazepam-d5, 3-OH-diazepam-ds, and n-desmethyldiazepam-d5 were all purchased from Cerilliant Corp. (Austin, TX). The enzyme 13-glucuronidase(TypeLII, from Patella vulgata 1,000,000-3,000,000 units/g solid) was obtained from Sigma-Aldrich. Other chemicals were of HPLC or analytical grade from various commercial sources. The Oasis MCX (60 mg, 3 mL) extraction columns were purchased from Waters (Milford, MA). Standard solutions Benzodiazepine stock solutions (?-amino metabolites/alprazolam/c~-hydroxyalprazolam,50 IJM and oxazepam/3-OHdiazepam/n-desmethyldiazepam, 2501JM) and deuterated internal standards (30!JM)were dissolved in methanol. Standard and control solutions were prepared by appropriate dilution of a stock solution with water. Standard calibration solutions were prepared in blank urine in concentrations ranging from 0.1 to 8tim (7-amino metabolites, ot-hydroxyalprazolam, and alprazolam) or 0.5 to 401~M(oxazepam, 3OH-diazepam, and n-desmethyldiazepam). Quality control (QC) samples were prepared in blank urine at concentrations of 0.12-0.631aM and 1-51JM, respectively. The internal standard solution was diluted with water to concentrations in the range of 2-6t~M. column was washed with water (2 mL), 0.1M hydrochloric acid (1 mL), and methanol (...truncated)