Determination of four pyridine alkaloids from Tripterygium wilfordii Hook. f. in human plasma by high-performance liquid chromatography coupled with mass spectrometry.

A novel liquid chromatography-atmospheric-pressure chemical ionization-mass spectrometry (LC-APCI/MS) method was developed and validated for the simultaneous determination of four sesquiterpene pyridine alkaloids (wilfortrine, wilfordine, wilforgine and wilforine) in human plasma. The chromatographic separation was performed on a Shim-pack XR-ODS column using an ammonium acetate buffer solution-acetonitrile in a gradient program. The detection was achieved by an ion trap mass spectrometry in the positive selected ion monitoring (SIM) mode. The method utilized acetonitrile as protein precipitation solvent and followed by solid-phase extraction (SPE). Calibration curves were linear for the four alkaloids over the range of 0.5-100.0 μg/L with the limits of quantification of 0.5 μg/L, while the method exhibited the recovery of 86.5-98.6%, intra- and inter-day RSDs of less than 8.2% and 12.8%, respectively. Methodology was validated in line with the EU requirements (Commission Decision 2002/657/EC). Results of incurred samples demonstrated excellent reproducibility. To our knowledge, this is the first analytical method for simultaneous determination of the four sesquiterpene pyridine alkaloids in plasma. The method was applicable to clinical pharmaceutical research of alkaloids in rheumatoid arthritis volunteer patients after oral administrations.