Measurement of benzodiazepines in urine by liquid chromatography-tandem mass spectrometry: confirmation of samples screened by immunoassay

First published on 7 December 2009
Ann Clin Biochem
© 2009 Association for Clinical Biochemistry


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Original Article

Sarah J Glover and
Keith R Allen

Department of Clinical Biochemistry, Leeds Teaching Hospitals NHS Trust, Britannia House, Morley, Leeds LS27 0DQ, UK

Corresponding author: Dr Sarah J Glover, Department of Clinical Biochemistry, Old Medical School, Great George Street, Leeds General Infirmary, Leeds LS1 3EX, UK. Email: Sarah.Glover{at}

Background: Liquid chromatography linked to tandem mass spectrometry (LC/MS/MS)provides the ability to identify a range of benzodiazepinesin accordance with European Union criteria and is an attractivemethod for the confirmation of benzodiazepines following immunoassayscreening.

Methods: An LC/MS/MS method to detect and quantitate the six most common benzodiazepines/metabolites (diazepam, nitrazepam, nordiazepam, oxazepam, temazepam and 7-aminonitrazepam) was developed together with a qualitative screening method for a further 11 benzodiazepines/metabolites. These methods were used for confirmation of 250 urine samples submitted for routine drug screening by immunoassay for benzodiazepines (100 samples positive for a benzodiazepine, assay cut-off >200 µsg/L).

Results: The lower limits of detection and quantitation were less than 2.5 and 5 µg/L for the six most common benzodiazepines. Recoveries ranged between 97% and 102% and calibration curves were linear to at least 4000 µg/L (r = 0.99). Intra and inter-assay imprecision were <10% (n = 10) and <20% (n = 15), respectively. Confirmation of benzodiazepines usingLC/MS/MS was achieved for 89% of the immunoassay-positive urinesamples. Of the immunoassay-negative urine samples, 31% of thesedemonstrated a benzodiazepine using LC/MS/MS.

Conclusion: The validated LC/MS/MS method developed is effective for theconfirmation of immunoassay screening results for benzodiazepines.The lower limit of detection and assay specificity offers alonger window of detection and more detailed clinical informationcompared with immunoassay screening.

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