SIMULTANEOUS DETERMINATION OF FLUNITRAZEPAM AND ITS METABOLITES IN PLASMA AND URINE BY HPLC DAD AFTER SOLID-PHASE EXTRACTION/

A high performance liquid chromatography (HPLC) assay was developed fo r the determination of flunitrazepam (FNZ) and its metabolites in urin e and plasma. The analytes and the internal standard (triazolam, TRZ) were extracted by Sep-Pak C18 SPE-cartridge and separated utilizing a 5 mu m ChromSpher C8 glass column with a gradient mobile phase contain ing methanol and 0.125% (v/v) of isopropylamine in water. Diode array detection (DAD) was carried out at a monitoring wavelength of 240 nm a nd a reference wavelength of 550 nm. Standard curves were linear from their quantitation limits until 200 ng ml(-1) urine or 250 ng ml(-1) p lasma for 7-amino-desmethyl-flunitrazepam (ADF), 7-amino-flunitrazepam (AF), 7-acetamino-flunitrazepam (ACF) and until 400 ng ml(-1) urine o r 500 ng ml(-1) plasma for FNZ, 1-desmethyl-flunitrazepam (DF), and 3- hydroxyl-flunitrazepam (HF). The intraday and interday coefficients of variation ranged from 2.04 to 9.07% and from 2.64 to 14.10%, respecti vely in urine and from 5.13 to 8.60% and from 7.27 to 10.46%, respecti vely in plasma. The developed method is used in forensic toxicology an d is also applicable to pharmacokinetic studies in man. (C) 1997 Elsev ier Science B.V.