Simultaneous quantification of urine flunitrazepam, nimetazepam and nitrazepam by using liquid chromatography tandem mass spectrometry.

Background: Benzodiazepines are used in hypnotics, sedation, and anti-anxiety. Recently liquid chromatography tandem mass spectrometry (LC-MS/MS) has been vastly developed for drug analysis in biological samples.

Methods: We developed and validated a LC-MS/MS method for simultaneous quantification of flunitrazepam (FM2), nimetazepam and nitrazepam levels in 87 benzodiazepine positive human urine specimens by enzyme immunoassay. Deuterated analogues were used as internal standard.

Results: The limits of quantification were found to be 0.25, 2.5, 5, 5 and 1ng/ml for FM2, 7-aminoFM2, nimetazepam, 7-amino-nimetazepam and nitrazepam, respectively. The intraday and inter-day CVs ranged from 0.6 to 4.6% and 1.2-9.4%, respectively. The within-day accuracy ranged from 80.8 to 108.7% and the between-day accuracy ranged from 80.5 to 118.0%. The recovery rate ranged from 70.5 to 96.7% for five different analytes. A group of 34 urine samples previously gas chromatography-mass spectrometry determined to contain 7-aminoFM2 was analyzed by this new LC-MS/MS approach. Quantitative data produced by both methods agreed well.

Conclusions: The LC-MS/MS method has proved to be robust and specific for the quantification of FM2, nimetazepam and nitrazepam in urine samples. This study also confirmed that nitrazepam and 7-aminonimetazepam are the metabolic products of nimetazepam.