Bioanalysis of propylparaben and p-hydroxybenzoic acid, and their sulfate conjugates in rat plasma by liquid chromatography–tandem mass spectrometry

• New bioanalytical assays for propylparaben and its three major metabolites were reported.
• It was discovered that the esterase inhibitor (PMSF) reacted with analyte of interest.
• Citric acid was used to inhibit esterase activity to stabilize propylparaben in rat plasma.
• Strategies were reported to overcome challenges for analyte stability and interferences.
• The two bioanalytical assays were validated and applied to a toxicological study in rats.

Two rugged liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods for the determination of propylparaben, its major metabolite, p-hydroxybenzoic acid (pHBA), and their sulfate conjugates have been developed and validated in citric acid-treated rat plasma. To prevent propylparaben being hydrolyzed to pHBA ex vivo, rat plasma was first treated with citric acid; then collected and processed at a reduced temperature (ice bath). Stable isotope labeled internal standards, d4-propylparaben, 13C6-pHBA, and the d4-labeled internal standards of their sulfate conjugates were used in the methods. The analytes were extracted from the matrix using protein precipitation, followed by chromatographic separation on a Waters ACQUITY UPLC HSS T3 column. Quantification using negative ion electrospray was performed on a Sciex API 4000 mass spectrometer. The analytical ranges were established from 2.00 to 200 ng/mL for propylparaben, 50.0–5000 ng/mL for pHBA, 50.0–10,000 ng/mL for the sulfate conjugate of propylparaben (SPP) and 200–40,000 ng/mL for the sulfate conjugate of pHBA (SHBA). Inter- and intra-run precision for the quality control samples were less than 5.3% and 4.4% for all analytes; and the overall accuracy was within ±5.7% of the nominal values. The validated bioanalytical methods demonstrated excellent sensitivity, specificity, accuracy and precision and were successfully applied to a rat toxicology study under the regulations of Good Laboratory Practices (GLP). Strategies have been developed and applied toward overcoming the challenges related to analyte stability, and environmental and endogenous background.