A Comparative Study on Flunitrazepam and 7-aminoflunitrazepam Isolation from Human Plasma by Liquid-liquid Extraction and Solid Phase Extraction

The aim of this work was to establish the best experimental conditions for the isolation of flunitrazepam and its main metabolite 7-aminoflunitrazepam from human plasma. Several mixtures of solvents (ethyl acetate, tert-butylmethyl ether, hexane, chloroform/ ethyl ether (20/80), dichloromethane / ethyl ether (20/80) and extraction cartridges (Oasis MCX , SepPak C18, Chromafix C18, Oasis HLB) were tested at different pH values on the samples. In order to assay flunitrazepam and its major metabolite, an ultra performance liquid chromatography with UV detection (UPLC-UV) method was applied. The best results for liquid-liquid extraction (LLE) of flunitrazepam and 7-aminoflunitrazepam from human plasma were obtained using as extraction solvent ethyl acetate after a previous sample alkalization with ammonia hydroxide. Recovery rates were 82.37% for flunitrazepam and 63.70% for 7-aminoflunitrazepam and the interferences at the retention time of these two compounds were minimal. In the case of solid phase extraction (SPE), the best recovery rates were obtained using SepPak and HLB cartridges (108.38% and 81.46% for flunitrazepam and 82.53% and 76.81% for 7-amino flunitrazepam, respectively), these results being correlated with minimal interferences at the retention time of flunitrazepam and 7-aminoflunitrazepam. LLE proved to be as efficient as SPE for the isolation of flunitrazepam and aminoflunitrazepam from human plasma. Liquid phase extraction using ethyl acetate as solvent in basic conditions and solid phase extraction using SepPak and HLB cartriges can be used in toxicological studies in order to eficiently isolate flunitrazepam and 7-aminoflunitrazepam from human plasma.