The analgesic paracetamol/acetaminophen (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 in humans. To perform internal exposure-based risk assessment, the use of accurate and optimized biomonitoring methods is critical. 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 issue of potential underestimation of APAP use using current standard analytical methods based on urinary analyses of free APAP and its phase II conjugates. The question we address is whether investigating additional metabolites than direct phase II conjugates could improve the monitoring of APAP. Using non-targeted analyses based on high-resolution mass spectrometry, we identified, in a controlled longitudinal exposure study with male volunteers, overlooked 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, these 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 therefore provides an option to decrease potential underestimation of APAP use. Moreover, our data challenge 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 a non-persistent chemical such as APAP.

扑热息痛/对乙酰氨基酚镇痛药（N-乙酰基-4-氨基苯酚，APAP）通常用于缓解疼痛，发烧和不适。尽管全球范围内的销售额都有所增长，但是越来越多的实验和流行病学证据表明，APAP是人类各种健康疾病的潜在危险因素。为了进行基于内部暴露的风险评估，使用准确，优化的生物监测方法至关重要。然而，由于其半衰期短，因此回顾性评估人类对APAP的药物使用具有挑战性。这项研究的目的是使用基于游离APAP及其II期结合物尿液分析的当前标准分析方法，解决可能低估APAP使用的关键问题。我们要解决的问题是，研究除直接II期结合物以外的其他代谢物是否可以改善对APAP的监测。使用基于高分辨率质谱的非目标分析，我们在一项针对男性志愿者的纵向对照对照研究中发现，APAP代谢物被忽略，形成和排泄速率延迟。我们假定这些代谢产物是通过肝肠循环后通过硫代甲基分流形成的，正如在啮齿动物中已经观察到的那样。重要的是，这些共轭的硫代甲基代谢产物具有以下特征：（i）具有与目前的方法检测到的游离APAP及其II期共轭物相当的诊断敏感性；（ii）与其他APAP代谢物相比，血液和尿液的峰值水平有所延迟，因此有可能扩大暴露评估的范围；（iii）从毒理学的角度提供有关目标代谢途径的相关信息。因此，将这些代谢物纳入未来的APAP生物监测方法中，可提供一种减少对APAP使用的潜在低估的选择。此外，我们的数据挑战了这样一种观念，即仅基于母体化合物及其II期代谢产物进行生物监测的标准方法足以对非持久性化学品（如APAP）进行人类生物监测。