Simultaneous determination of caffeic acid and its major pharmacologically active metabolites in rat plasma by LC-MS/MS and its application in pharmacokinetic study.

A simple, sensitive and selective high-performance liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination and pharmacokinetic study of caffeic acid (CA) and its active metabolites. The separation with isocratic elution used a mobile phase composed of methanol and water (containing 0.1% formic acid) at a flow rate of 0.2 mL/min. The detection of target compounds was done in selected reaction monitoring (SRM) mode. The SRM detection was operated in the negative electrospray ionization mode using the transitions m/z 179 ([M - H](-) ) → 135 for CA, m/z 193 ([M - H](-) ) → 134.8 for ferulic acid and isoferulic acid and m/z 153 ([M - H](-) ) → 108 for protocatechuic acid. The method was linear for all analytes over the investigated range with all correlation coefficients 0.9931. The lower limits of quantification were 5.0 ng/mL for analytes. The intra- and inter-day precisions (relative standard deviation) were <5.86 and <6.52%, and accuracy (relative error) was between -5.95 and 0.35% (n = 6). The developed method was applied to study the pharmacokinetics of CA and its major active metabolites in rat plasma after oral and intravenous administration of CA.