A sensitive HPLC-MS method for simultaneous determination of thirteen components in rat plasma and its application to...

A rapid, sensitive and selective method based on high-performance liquid chromatography coupled with Q-Exactive mass spectrometry was developed and validated for simultaneous quantification of thirteen components in rat plasma, including chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, luteoloside, isochlorogenic acid A, B and C, scutellarin, baicalin, wogonin, baicalein, phillyrin and forsythoside A. After precipitating proteins from the plasma samples with methanol, chromatographic separation of the thirteen components was achieved by using an XBridge™ C₁₈ column (2.1mm×150mm, 5μm) with the mobile phase consisting of methanol and 0.1% formic acid in water at a flow rate of 0.3mL/min. High-resolution MS quantification was adopted with detection on a Q-Exactive mass spectrometer in full-scan mode, and the results were obtained using a mass extraction window of 10ppm at a mass resolution of 70, 000. All the calibration curves exhibited good linearity (r²>0.991) over the measured ranges. The lower limit of quantitation (LLOQ) was in the range of 1.05-8.13ng/mL. The intra- and inter-day precision (RSD) was less than 11.70% and the accuracy (RE) ranged from -5.58% to 12.29%. No significant matrix effect was observed and the extraction recoveries of all the analytes were more than 79.36%. The developed method was applied to a pharmacokinetic study of the thirteen ingredients in rats after intravenous administration of Tanreqing at three doses of 3, 6 and 12mL/kg. The results indicated that 8 of the 13 components, isochlorogenic acid A, B and C, chlorogenic acid, baicalin, wogonin, luteoloside and forsythoside A, had linear pharmacokinetic properties in the tested dosage range.