Measurement of benzodiazepines in urine by liquid chromatography-tandem mass spectrometry: confirmation of samples screened by immunoassay.
BACKGROUND
Liquid chromatography linked to tandem mass spectrometry (LC/MS/MS) provides the ability to identify a range of benzodiazepines in accordance with European Union criteria and is an attractive method for the confirmation of benzodiazepines following immunoassay screening.


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 microsg/L).


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


CONCLUSIONS
The validated LC/MS/MS method developed is effective for the confirmation of immunoassay screening results for benzodiazepines. The lower limit of detection and assay specificity offers a longer window of detection and more detailed clinical information compared with immunoassay screening.
Glover
Sarah J
SJ
Department of Clinical Biochemistry, Leeds Teaching Hospitals NHS Trust, Britannia House, Morley, Leeds LS27 0DQ, UK. Sarah.Glover@leedsth.nhs.uk
Allen
Keith R
KR
D001569:Benzodiazepines / Q000032:analysis* / Q000652:urine; D001681:Biological Assay / Q000379:methods; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods; D005062:European Union; D007118:Immunoassay / Q000379:methods; D057230:Limit of Detection; D009567:Nitrazepam / Q000031:analogs & derivatives / Q000032:analysis / Q000652:urine; D003708:Nordazepam / Q000032:analysis / Q000652:urine; D010076:Oxazepam / Q000032:analysis / Q000652:urine; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013693:Temazepam / Q000032:analysis / Q000652:urine
D001569:Benzodiazepines; C043577:7-aminonitrazepam; D003708:Nordazepam; D010076:Oxazepam; D009567:Nitrazepam; D013693:Temazepam