Simultaneous determination of six bioactive constituents of Guizhi Fuling Capsule in rat plasma by UHPLC–MS/MS: Application to a pharmacokinetic study

• Six bioactive constituents were simultaneously analyzed by UPLC-MS/MS.
• The sample preparation was acidified by 1% formic acid and then pretreated by PPT.
• The method was used successfully for the pharmacokinetic study of GFC.

A rapid and selective ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed for simultaneous determination of gallic acid, amygdalin, albiflorin, paeoniflorin, paeonol and cinnamic acid, the major bioactive constituents of Guizhi Fuling Capsule in rat plasma using phenacetin as internal standard (IS). The plasma samples were pretreated by protein precipitation with acetonitrile after acidification and separated on a Waters BEH C18 column (50 mm × 2.1 mm, 1.7 μm) using gradient elution with a mobile phase consisting of water (containing 0.1% formic acid) and acetonitrile at a flow rate of 0.2 mL/min. Mass spectrometric detection was performed on Micromass Quattromicro API mass spectrometer equipped with electrospray ionization source in multiple reaction monitoring (MRM) mode. The intra- and inter-day precisions (as relative standard deviation) were below 14.6% for all analytes, and the accuracies (as relative error) were within ±5.0%. The lower limits of quantification (LLOQ) were 10, 10, 5, 5, 25, 25 ng/mL for gallic acid, amygdalin, albiflorin, paeoniflorin, paeonol and cinnamic acid, respectively. Extraction recovery, matrix effect and stability were satisfactory in rat plasma. This method was fully validated and applied to a pharmacokinetic study of the six bioactive constituents after oral administration of Guizhi Fuling Capsule to rats.