Acetaminophen Metabolism Revisited Using Non-Targeted Analyses: Implications for Human Biomonitoring

The analgesic paracetamol (N-acetyl-4-aminophenol, APAP) is commonly used to relieve pain, fever and malaise. While sales have increased worldwide, a growing body of experimental and epidemiological evidence has suggested APAP as a possible risk factor for various health disorders. To perform internal exposure-based risk assessment, the use of accurate and optimized biomonitoring methods is criticical. However, retrospectively assessing pharmaceutical use of APAP in humans is challenging because of its short half-life. The objective of this study was to address the key biomonitoring issues with APAP using current standard analytical methods based on urinary analyses of free APAP and its phase II conjugates. Using non-targeted analyses based on high-resolution mass spectrometry, we identified in a controlled longitudinal exposure study with male volunteers, unrecognized APAP metabolites with delayed formation and excretion rates. We postulate that these metabolites are formed via the thiomethyl shunt after the enterohepatic circulation as already observed in rodents. Importantly, the conjugated thiomethyl metabolites were (i) of comparable diagnostic sensitivity as the free APAP and its phase II conjugates detected by current methods; (ii) had delayed peak levels in blood and urine compared to other APAP metabolites and therefore potentially extend the window of exposure assessment; and (iii) provide relevant information regarding metabolic pathways of interest from a toxicological point of view. Including these metabolites in future APAP biomonitoring methods provide an option to decrease potential underestimation of APAP use and challenges the notion that the standard methods in biomonitoring based exclusively on the parent compound and its phase II metabolites are adequate for human biomonitoring of non-persistant chemical such as APAP.