Validated LC-MS/MS method for the simultaneous determination of hyperoside and 2''-O-galloylhyperin in rat plasma: application to a pharmacokinetic study in rats.

An LC-MS/MS method was developed for the first time to simultaneously determine hyperoside and 2''-O-galloylhyperin, two major components in Pyrola calliantha extract, in rat plasma. Following extraction by one-step protein precipitation with methanol, the analytes were separated on a Venusil MP-C18 column within 2 min, using methanol-water-formic acid (50:50:0.1, v/v/v) as the mobile phase at a flow rate of 0.4 mL/min. Detection was performed on electrospray negative ionization mass spectrometry by multiple-reaction monitoring of the transitions of 2''-O-galloylhyperin at m/z 615.1 → 301.0, of hyperoside at m/z 463.1 → 300.1, and of internal standard at m/z 415.1 → 295.1. The limits of quantification were 2 ng/mL for both hyperoside and 2''-O-galloylhyperin. The precisions were <13.1%, and the accuracies were between -9.1 and 5.5% for both compounds. The method was successfully applied in pharmacokinetic studies following intravenous administration of the total flavonoids of P. calliantha extract in rats.