Determination of flunitrazepam in human plasma and urine by HPLC with mass spectrometry detection

A new, sensitive, and selective liquid chromatography coupled with mass spectrometry (LC/MS/MS) method for quantification of flunitrazepam in human plasma and urine was validated. The detection of flunitrazepam was in multiple reaction monitoring mode using an ion trap mass spectrometer equipped with an atmospheric pressure chemical ionisation ion source. The method was validated and proved to be linear, accurate, and precise over the range of 0.7-49.4ng/mL in plasma samples and 0.5-33ng/mL in urine. This is the first reported method for analysis of flunitrazepam in human plasma and urine that uses protein precipitation for plasma/direct injection for urine as a sample processing procedure. The total run time of the analytical method is less than 2 minutes. Another advantage of the method, besides its simplicity, is the very good recovery of the analyte. The validated LC/MS/MS method has been successfully applied to a pharmacotoxicological study of flunitrazepam.