Simultaneous determination of GFA and its active metabolites in human plasma by liquid chromatography electrospray ionization mass spectrometry and its application to pharmacokinetic studies.

A sensitive and rapid liquid chromatography electrospray ionization mass spectrometry (LC–ESI–MS) method has been developed and validated for simultaneous quantification of guanfu base A (GFA) and its metabolites guanfu base I (GFI) and guanfu alcohol-amine (AA) in human plasma with phenoprolamine hydrochloride (DDPH) as the internal standard. The analytes were extracted from human plasma by using liquid–liquid extraction with ethyl acetate and the LC separation was performed on a Diamonsil C18 analytical column (150mm×2.1mm i.d., 5μm). The MS acquisition was performed in selected ion monitoring (SIM) mode of positive ions. Analysis was carried out in SIM mode at m/z 430.25 for GFA [M+H]+, m/z 388.25 for GFI [M+H]+, m/z 346.25 for AA [M+H]+ and m/z 344.20 for the IS DDPH [M+H]+. The calibration curves were linear over the range of 50–5000ng/mL for GFA and 5–1000ng/mL for GFI and AA, with coefficients of correlation above 0.999. The lower limit of quantification for GFA was 1ng/mL, while for GFI and AA were both 5ng/mL. The intra- and inter-day precisions (CV) of analysis were within 9%, and the accuracy ranged from 91% to 108%. The overall recoveries for GFA, GFI and AA were about 94.2%, 87.8% and 80.6%, respectively. The total LC–MS run-time was only 5.5min. This quantitation method was successfully applied to the simultaneous determination of GFA and its metabolites in human plasma for the metabolic study and pharmacokinetic evaluation.