A UPLC-MS/MS method for simultaneous determination of six bioactive compounds in rat plasma, and its application to pharmacokinetic studies of Naoshuantong Granule in rats

Background: It is urgently needed to clarify the pharmacokinetic mechanism for the multi-bioactive constituents in Traditional Chinese Medicines for its clinical applications. A rapid, sensitive and reliable ultra-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of Danshensu, Ferulic acid, Astragaloside IV, Naringin, Neohesperidin and Puerarin after oral administration of Naoshuantong Granule using Carba-mazepine as internal standard (IS). Methods: The plasma samples were pretreated by liquid-liquid extraction method using ethyl acetate after acidification, and separated on a Waters ACQUITY UPLC (R) BEH C-18 column (50x2.1 mm, i d, 1.7 mu m) by gradient elution with a mobile phase composing of water (containing 0.1% formic acid) and acetonitrile at a flow rate of 0.2 mL/min. Multiple reaction monitoring (MRM) mode with both positive and negative ion mode was operated using an electrospray ionization (ESI) to detect the six compounds. Result: All calibration curves showed good linearity (r>0.99) over a wide concentration range. The intra- and inter-day precision (RSD%) was below 8.4% and the accuracy (RE%) ranged from 91.1% to 107.5%. The extraction recoveries of the six analytes and IS in the plasma were more than 77.9% and no severe matrix effect was observed. Conclusion: The fully validated method was successfully applied to the pharmacokinetics of Naoshuantong Granule.