Determination of paracetamol and its metabolites via LC-MS/MS in dried blood volumetric absorptive microsamples: A tool for pharmacokinetic studies

Abstract

Paracetamol (acetaminophen, APAP) is the most frequently used analgesic and antipyretic worldwide. Nonetheless, APAP induced hepatotoxicity is the most common cause of acute liver failure in the western world. This hepatotoxicity is related to the metabolism of APAP, via the formation of the electrophilic oxidation product N-acetyl-para-benzoquinone imine. To investigate differences in APAP metabolism in specific patient populations and to optimize dosing regimens, quantification of metabolites from the different metabolic pathways is needed to perform pharmacokinetic (PK) studies. For this purpose, sensitive and short liquid chromatography-tandem mass spectrometry methods were developed for the quantitation of APAP and four of its metabolites (APAP-glucuronide, APAP-sulfate, APAP-mercapturate, and APAP-cysteine) in plasma, whole blood and dried blood microsamples collected via 10 µL volumetric absorptive microsampling (VAMS) devices. The methods were successfully validated based on internationally accepted guidelines (EMA, FDA), encompassing selectivity, evaluation of the calibration model, matrix effect and recovery, accuracy and precision, stability, and dilution integrity. In addition, for the VAMS samples, the effect of the hematocrit on the recovery was evaluated. Successful application on whole blood and plasma, as well as on VAMS samples prepared from venous or capillary blood of patients, demonstrated that the methods were fit-for-purpose and can be used for future PK studies.