Determination of Clozapine and Its N Demethylated Metabolite in Serum by Gas Chromatography Mass Spectrometry with Selected Ion Monitoring
气相色谱-质谱法测定氯氮平及其去甲基代谢物

This article reports a highly sensitive method specific for determination of clozapine(CLP) and its N-demethylated metabolite (DCLP) in serum by gas chromatography-mass spectrometry with selected ion monitoring. A 0.5 mL serum was alkalized with 0.5 mL 1.0 mol/L sodium hydroxide and was extracted with 5 mL ether, then the organic phase was transferred to another tube containing 1 mL 0.1 mol/L hydrochloric acid. After being shaken and centrifuged, the organic phase was discarded, and 10 microL 100 micrograms/L diazepam (DAP) was added to the water phase as internal standard, then 0.5 mL 1.0 mol/L sodium hydroxide and 5 mL ether were added. The extract was evaporated to dryness and then was acylated with 50 microL trifluoroacetic anhydride under 50 degrees C for 45 min. The excess of the reagent was evaporated and the residue was disolved in 20 microL acetone. Take 2 microL for gas chromatography-mass spectrometry analysis. Chromatographic conditions were: column DB-1 (30 m x 0.25 mm x 0.25 microns), injector 260 degrees C, interface 250 degrees C, column oven with temperature program: equation: see text] Helium carrier gas flow rate was 1.8 mL/min. The mass spectrometer was adjustd to record the ion m/z352 for CLP and DCLP, and m/z 256 for DAP in quantitation. The minimum detection limits were 0.1 microgram/L for CLP and 0.2 microgram/L for DCLP. The standard curves of CLP and DCLP were linear between 1-128 micrograms/L (rCLP = 0.998, rDCLP = 0.999). Recoveries of two substances were better than 83%, and the RSD values were less than 10%. This method has been successfully used in clinical pharmacokinetic study of CLP before and after CYP1A2 inhibitor used. The results indicate that the metabolism of clozapine is strongly affected by CYP1A2 activity.