Absolute quantitation of acetaminophen‐modified human serum albumin in acute liver failure patients by liquid chromatography/tandem mass spectrometry

Rationale Acetaminophen (APAP) is a well‐known analgesic, deemed a very safe over‐the‐counter medication. However, it is also the main cause of acute liver failure (ALF) in the Western world, via the formation of its reactive metabolite, N ‐acetyl p ‐benzoquinone imine (NAPQI), and its covalent attachment to liver proteins. The aim of this study was to develop a sensitive and robust quantitative assay to monitor APAP‐protein binding to human serum albumin (HSA) in patient samples. Methods A combination of isotope dilution, peptic digestion and solid‐phase extraction coupled to liquid chromatography/multiple reaction monitoring (LC/MRM) was employed. An external calibration curve with surrogate modified protein spiked into blank serum was used for absolute quantitation. Samples were analyzed by LC/MRM to measure the modified active site peptide of HSA. The LC/MRM assay was validated and successfully applied to serum samples from patients suffering from APAP‐induced ALF. Results Accuracy ranged from 83.8–113.3%, within‐run coefficient of variation (CV) ranged from 0.3–6.9%, and total CVs from 1.6–10.6%. Patient samples ranged from 0.12–3.91 nmol/mL NAPQI‐HSA; in‐between the assay dynamic range of 0.11–50.13 nmol/mL serum. In vivo median concentrations were found to be 0.62 nmol/mL and 0.91 nmol/mL for non‐spontaneous survivors ( n  = 25) and individuals with irreversible liver damage ( n  = 10), respectively ( p ‐value = 0.028), demonstrating significant potential as a biomarker for ALF outcome. Conclusions A fast and sensitive assay was developed to accurately quantify NAPQI‐HSA as a biomarker for APAP‐related covalent binding in human serum.