A sensitive UPLC–ESI–MS/MS method for the quantification of cinnamic acid in vivo and in vitro: application to pharmacokinetic and protein binding study in human plasma

Abstract

The purpose of this study was to develop a sensitive method for quantifying cinnamic acid in human plasma using UPLC–ESI–MS/MS. Cinnamic acid was separated using water containing 0.005% (v/v) formic acid and acetonitrile as a mobile phase by gradient elution at a flow rate 0.2 mL/min, equipped with a HALO-C18 column (2.1 × 100 mm, 2.7 μm). Quantitation of this analysis was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring negative ion mode. The mass transitions were m/z 146.8 → 103.0 for cinnamic acid, and 432.9 → 225.0 for geniposide as internal standard. Liquid–liquid extraction and protein precipitation with ethyl acetate–methanol-1.2% acetic acid (4/16/1, v/v/v) were used in the sample extraction. The chromatograms showed high resolution, sensitivity, and selectivity with no interference with plasma constituents. The calibration curves for cinnamic acid in human plasma were 0.1–500 ng/mL and displayed excellent linearity with correlation coefficients (r) greater than 0.99. Both the intra- and inter-day precisions (CV%) were within 3.88% for human plasma. The accuracy was 99.34–106.69% for human plasma. In vitro plasma protein binding of cinnamic acid was 64.26 ± 1.89 and 65.50 ± 1.78% for the spiked human plasma concentrations of 100 and 1000 ng/mL, respectively. The developed analytical method satisfied the criteria of international guidance and could be successfully applied to the pharmacokinetic study of cinnamic acid after oral administration of Socheongryong-tang tablets to humans.