Simultaneous LC-MS/MS quantification and pharmacokinetics of baicalin, chlorogenic acid and forsythin after intravenous administration of Shuang-huang-lian powder to dogs

Abstract

A sensitive and specific high-performance liquid chromatography-tandem mass method (LC-MS/MS) has been developed and validated for the simultaneous determination of baicalin, chlorogenic acid and forsythin in dog plasma using a chemical synthetic compound buspirone as the internal standard (IS). Chromatographic separation was accomplished on a Phenomenex Gemini C18 column (2.0 × 50 mm, I.D., 3.5 μm) with a gradient elution system composed of 0.1% formic acid in water and 0.1% formic acid in methanol at a flow rate of 0.40 mL min⁻¹. Detection was based on a triple quadrupole mass spectrometer using a multiple reaction monitoring (MRM) mode with an electrospray ionization source (ESI). The precursor/product transitions (m/z) in the positive ion mode were 447.3 → 271.1, 355.3 → 163.3, 556.9 → 309.3, and 386.5 → 122.1 for baicalin, chlorogenic acid, forsythin and IS, respectively. All of the calibration curves showed good linearity (r > 0.995) over the concentration range of 1.0 to 1000 ng mL⁻¹ and the lower limits of quantification (LLOQs) were all 1.0 ng mL⁻¹ for three analytes. The intra-day and inter-day precisions (% RSD) of low-level QC samples were all less than 20% and the precisions (% RSD) of high- and medium-level were all less than 15%. The accuracies at three QC levels ranged from 80 to 120%. The mean recoveries were all above 80% for three analytes. All the three analytes in dog plasma were stable at three different conditions. The well-established and fully validated method was firstly applied to the pharmacokinetics of baicalin, chlorogenic acid and forsythin in dogs after intravenous administration of Shuang-huang-lian freeze-dried powder. Significant double and/or multiple-peak phenomena in plasma concentration–time profiles of three analytes were observed in all six dogs, which are thought to be the results of the enterohepatic cycle.