SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

F21
DETERMINA TION OF BENZODIAZEPINES IN HUMAN URINE USING SOLID-PHASE
EXTRACTION AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
ELECTROSPRA Y IONISATION TANDEM MASS SPECTROMETRY
S Hegstad*, E L 0iestad, U Johansen and A S Christophersen
Division of Forensic Toxicology and Drug abuse, Norwegian Institute of Public Health, P.O. Box 4404
Nydalen, NO-0403 Oslo.
Aims: A liquid chromatography-tandem mass spectrometry (LCIMSIMS) method has been developed and
validated for the determination of benzodiazepines registered in Norway and/or their metabolites in human
urine. These compounds are frequently associated with misuse. The following compounds are included: 7­
aminonitrazepam, 7-aminoc)onazepam, 7-aminoflunitrazepam, alprazol am, alphahydroxyalprazolam,
oxazepam, 3-0H-diazepam and N-desmethyldiazepam. The method was evaluated by analysing urine
specimens from prison inmates, suspected of drug abuse.
Methods: Urine samples (0.5 ml) were hydrolysed with l3-glucuronidase (from Patella vulgata) at 60°C for
2 hours before solid-phase extraction with a polymer-based mixed-mode column (Oasis MCX).
Chromatographic separation of extracts was achieved using a Waters Symmetry CI8 (2.IXlOO mm, 3.5
/-1m) column with a flow rate at 0.3 mllmin with gradient elution. The analyses were performed on a Waters
Alliance 2695 system in combination with a Waters Quattro Ultima Pt tandem-quadropole mass
spectrometer equipped with a Z-spray electrospray interface. Positive ionization was performed in the
MRM (multiple reaction monitoring) mode. Two transitions were monitored for the analytes, and one for
the internal standards. Deuterated analogues were used as internal standard for all analytes except for 7­
aminonitrazepam and alphahydroxyalprazolam, which were quantified using 7-aminoclonazepam-d4 and
alprazolam-d5, respectively.
Results: The concentration range was 0.1-8.0 /-1M for 7-aminonitrazepam, 7-aminoclonazepam, 7­
aminoflunitrazepam, alprazolam, alphahydroxyalprazolam and 0.5-40 /-1M for the other compounds. The
average recovery of the analytes ranged from 56-83 %. The between-day relative standard derivation of the
method ranged from 3 -12 %. The limits ofquantification were found to be between 0.002 and 0.01 /-1M.
Conclusion: The LC/MSIMS method proved to be robust and specific for the determination of
benzodiazepines in urine. The method developed offers significant efficiency advantages in our routine
laboratory replacing two chromatographic methods, which involved time-consuming derivatisation
techniques. (HPLC-fluorescence, GC-MS). In addition, inclusion of new benzodiazepines is more
convenient due to high specificity and flexibility ofthe new method.
Keywords: Benzodiazepines, urine, LC/MS/MS
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