Development and validation of a UPLC-MS/MS method for the quantification of acetaminophen in human plasma and its application to pharmacokinetic studies

We developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of acetaminophen concentration in human plasma. Following protein precipitated extraction, the analytes were separated and analyzed using an UPLC-MS/MS in the multiple reaction monitoring (MRM) mode with the respective [M+H]⁺ ions, m/z 152.06 → 110.16 for acetaminophen and m/z 180.18 → 138.12 for phenacetin (internal standard, IS). The method showed a linear response from 1 to 100 µg/mL (r > 0.9982). The limit of quantitation for acetaminophen in plasma was 1 µg/mL. The intra- and inter-day accuracy ranged in the ranges of 94.40-99.56% and 90.00-99.20%, respectively. The intra- and inter-day precision ranged in the ranges of 2.64-10.76% and 6.84-15.83%, respectively. This method was simple, reliable, precise and accurate and can be used to determine the concentration of acetaminophen in human plasma. Finally, this fully validated method was successfully applied to a pharmacokinetic study of acetaminophen in healthy volunteers following oral administration.