Simultaneous determination of gallic acid and gentisic acid in organic anion transporter expressing cells by liquid chromatography–tandem mass spectrometry

• Improved bioanalysis of gallic acid and gentisic acid with LC–MS/MS.
• The established method increased speed, sensitivity and linear dynamic range.
• Application in a novel cell lysate-derived biological matrix was optimized.
• Showed utility of method in cellular uptake study in transporter-expressing cells.
• Gallic acid was identified as an actual substrate of murine Oat1 using the method.

In order to elucidate the role of organic anion transporters (OATs) in the renal elimination of gallic acid and gentisic acid, a new, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the simultaneous determination of gallic acid and gentisic acid in cell lysate, using Danshensu as the internal standard (IS). After a simple liquid–liquid extraction, the analytes were detected in negative ESI mode using selected reaction monitoring. The precursor-to-product ion transitions (m/z) were 169.0 → 125.0, 153.1 → 108.0, and 196.8 → 135.2 for gallic acid, gentisic acid, and the IS, respectively. Chromatographic separation was achieved on a C18 column using mobile phases consisting of water with 0.1% acetic acid (A) and acetonitrile with 0.05% formic acid. (B) The total run time was 3 min and calibration curves were linear over the concentrations of 0.33–2400 ng/mL for both compounds (r2 > 0.995). Good precision (between 3.11% and 14.1% RSD) and accuracy (between −12.7% and 11% bias) was observed for quality controls at concentrations of 0.33 (lower limit of quantification), 1, 50, and 2000 ng/mL. The mean extraction recovery of gallic acid and gentisic acid was 80.7% and 83.5%, respectively. Results from post-column infusion and post-extraction methods indicated that the analytical method exhibited negligible matrix effects. Finally, this validated assay was successfully applied in a cellular uptake study to determine the intracellular concentrations of gallic acid and gentisic acid in OAT expressing cells.