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

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.

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.

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.

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.