Simultaneous determination of baicalin and chlorogenic acid in human plasma by UPLC-MS/MS

To develop and validate an ultra performance liquid chromatography tandem mass spectrometric (UPLC-MS/MS) method for the simultaneous quantification of baicalin and chlorogenic acid in human plasma after iv infusion of Yinhuang injection, the analytes were isolated from plasma by protein precipitation with methanol. Then they were chromatographied on an Acquity UPLC BEH C₁₈ column (50 mm×2.1 mm ID, 1.7 μm) at 40 ℃. The mobile phase A consisted of water and 0.1% formic acid. The mobile phase B consisted of methanol and 0.1% formic acid. The analytes were eluted from the column with a linear gradient from 5% B to 80% B in 5 min, then hold for 0.5 min before returning to initial condition. The flow rate was 0.35 mL·min^-1. A tandem mass spectrometer equipped with electrospray ionization source was used as detector. Multiple reaction monitoring (MRM) using the precursor to product ion pairs of m/z 447→271 (for baicalin), m/z 353→191 (for chlorogenic acid) and m/z 287→287 (for internal standard) were used to quantification. The linear concentration ranges of the calibration curves for baicalin and chlorogenic acid ranged from 9.6 to 1 540 ng·mL^-1 and from 7.5 to 1 200 ng·mL^-1, respectively. The intra- and inter-day relative standard deviation (RSD) across three validations run over the entire concentration range was less than 10.2% for both baicalin and chlorogenic acid. After iv infusion of Yinhuang injection to the volunteers, the concentration-time curves of baicalin and chlorogenic acid fitted the two-compartment and three-compartment model. T_1/2α were (4.47±0.89) and (7.65±4.42) min, T_1/2β were (46.22±10.03) and (34.40±19.16) min, respectively. The method was proved to be highly sensitive, selective, and suitable for pharmacokinetic investigations of both baicalin and chlorogenic acid.