A sensitive LC-MS/MS method for the quantitative determination of biflorin in rat plasma and its application to...

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric method (LC-MS/MS) was developed for the quantification of biflorin in rat plasma. Using naringin as an internal standard, plasma samples were subjected to a direct protein precipitation process using methanol. Chromatographic separation was achieved on a Gemini C18 column with an isocratic mobile phase consisting of 0.1% formic acid and methanol (50:50, v/v) at a flow rate of 0.5mL/min. Biflorin was analyzed in the multiple reaction monitoring mode with negative electrospray ionization. The precursor/product ion pairs were m/z 353.0/205.0 and m/z 579.0/271.0 for biflorin and the IS, respectively. The calibration curve was linear over the concentration range of 5-2000ng/mL. The intra- and inter-day precision was less than 7.3% and the accuracy ranged from 96.5 to 103.3%. No significant variation was observed in the stability tests. This method was successfully applied to a pharmacokinetic study of biflorin after the intravenous and oral administration of biflorin to rats. The half-life and oral bioavailability of biflorin were determined as 3.4h and 43%, respectively. This is the first report on the quantitative determination of biflorin in rat plasma as well as the pharmacokinetic characterization of biflorin, which should provide a meaningful foundation for further preclinical and clinical applications of biflorin.