LC‐ESI‐MS/MS analysis and pharmacokinetics of heterophyllin B, a cyclic octapeptide from Pseudostellaria heterophylla in rat plasma

Heterophyllin B (HB) is a cyclic octapeptide isolated from Pseudostellaria heterophylla. HB is used as the quality control index for evaluating P. heterophylla in the Chinese Pharmacopoeia. A rapid and sensitive LC-ESI-MS/MS method was developed and validated for the analysis of HB in rat plasma. Sample preparation consisted of a solid-phase extraction step for the removal of interference and preconcentration of the target analyte HB and the internal standard N-acetylcysteine before chromatographic analysis by MS/MS detection. The separation of HB and N-acetylcysteine was performed using a Hypersil GOLD™ C18 column and a mixture of methanol-water (60:40, v/v) containing 10 mmol/L ammonium formate and 0.1% formic acid as the mobile phase. The determination step was optimized in the selected reaction monitoring mode for the highly selective and sensitive quantitation of HB in rat plasma. Intra- and inter-assay precision (as relative standard deviation) was ≤9.1%, and accuracy was between 92.6 and 102.7%. The validated method was successfully applied to quantify HB concentrations up to 7 h after tail intravenous injections of 2.08, 4.16 and 8.32 mg/kg HB in rats. The LC-MS/MS method identified the relevant pharmacokinetic parameters of HB and its studied analog.