# Studying APAP metabolism in a cohort of intoxicated patients using HRMS-based profiling: detection of catechol and delayed thiomethyl metabolites

Thomas Gicquel, Romain Pelletier, Eva Gorrochategui, Margaux Heurté, Diane Le Bouëdec, Jade Chaker, Isabelle Morel, Brendan Le Daré, Arthur David

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## ► To cite this version:

Thomas Gicquel, Romain Pelletier, Eva Gorrochategui, Margaux Heurté, Diane Le Bouëdec, et al. Studying APAP metabolism in a cohort of intoxicated patients using HRMS-based profiling: detection of catechol and delayed thiomethyl metabolites. Toxicology Letters, 2025, 413, pp.111727. 10.1016/j.toxlet.2025.111727. hal-05274003

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Submitted on 15 Nov 2025

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# Studying APAP metabolism in a cohort of intoxicated patients using HRMS-based profiling: Detection of catechol and delayed thiomethyl metabolites

Thomas Gicquel<sup>a,b,*</sup>, Romain Pelletier<sup>a,b</sup>, Eva Gorrochategui<sup>c</sup>, Margaux Heurté<sup>c</sup>, Diane Le Bouedec<sup>a</sup>, Jade Chaker<sup>c</sup>, Isabelle Morel<sup>a,b</sup>, Brendan Le Daré<sup>a,d</sup>, Arthur David<sup>c</sup>

## ARTICLE INFO

**Keywords:**

Acetaminophen/paracetamol

Metabolism

Intoxication

Thiomethylshunt pathway

HRMS

Catechol pathway

## ABSTRACT

Acetaminophen (APAP) overdose is one of the most important causes of drug-induced liver injury worldwide. Hepatotoxicity induced by APAP is mainly caused by the production of N-acetyl-p-benzoquinone imine (NAPQI), a highly reactive intermediate. Although, the medical management of APAP intoxication is well known, research is still ongoing to identify markers that could help to predict adverse issues after APAP intoxication. In this study, we aimed to study APAP biotransformation pathways in a cohort of patients with proven acute APAP intoxication to identify new biomarkers using state-of-the-art high-resolution mass spectrometry (HRMS) methodologies that could help improve the diagnosis of intoxication as well as patient follow-up. We used a cohort of 37 patients whom blood plasma samples were stratified according to the collection time after APAP intoxication. Our results showed that direct phase II metabolites from glucuronidation and sulfation pathways remain the main markers of APAP consumption. Our study also revealed that several oxidative pathways produce significant metabolites (including catechol ones) that could also help to monitor the intoxication and the elimination of the hepatotoxic NAPQI. In particular, significant levels of thiomethyl metabolites derived from the glutathione-NAPQI conjugates could be detected with a delay in their kinetics of appearance.

## 1. Introduction

Acetaminophen (APAP, N-acetyl-4-aminophenol or paracetamol) is one of the most prescribed analgesic and antipyretic drug in the world (Brune et al., 2015). Medical benefits of APAP are widely recognized and the current maximum recommended dosage is 4 g/day in adults and 60 mg/kg/day in children (Freo et al., 2021). APAP is thus listed on the WHO's List of Essential Medicines for the treatment of pain (WHO). Nevertheless, products containing APAP alone is the first leading cause of substance poisoning fatalities according to the 2023 National Poison Data System report (Gummin et al., 2024). Between 2017 and 2022, intentional APAP poisonings accounted for 11.8 % (range 1.8–37.3 %) of all intentional poisonings, and APAP was involved in an average of 5.4 intentional poisonings per 100,000 population per year in the world (Chidiac et al. 2023).

APAP metabolism was first described in the 1970's, and current knowledge is often based on this early literature. APAP is known to be extensively metabolized in the liver, mainly by phase II enzymes (uridine diphosphate glucuronosyltransferases [UGTs]) and sulfotransferase [SULTs]). A smaller proportion of APAP is also metabolized by phase I cytochrome P450 (CYP) 2E1 (and CYP3A4, CYP2D6 and CYP1A2 to a lesser extent) enzymes to produce the hepatotoxic N-acetyl-p-benzoquinone imine (NAPQI) (Manyike et al., 2000, Sarich et al., 1997, McGill and Jaeschke 2013, Prescott et al., 1980). Consequently, the main metabolic pathways historically described are glucuronidation (50–60 %) and sulfation (30–40 %), while only less than 5 % of APAP is considered to be excreted unchanged and around 5–15 % is thought to be oxidized by CYP450 to NAPQI (Prescott, 1980, Bessems and Vermeulen, 2001, Mazaleuskaya et al. 2015, Gicquel et al. 2013, Chidiac et al. 2023). Other minor pathways that have been already described

<footnote><sup>a</sup> Inserm, Univ Rennes, INRAE, Institut NUMECAN (Nutrition, Métabolismes et Cancer) UMR_A 1341, UMR_S 1317, Rennes F-35000, France</footnote>

<footnote><sup>b</sup> Laboratoire de toxicologie biologique et médico-légale, CHU Rennes, Rennes, France</footnote>

<footnote><sup>c</sup> Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) - UMR_S, Rennes 1085, France</footnote>

<footnote><sup>d</sup> Pôle Pharmacie, CHU Rennes, Rennes, France</footnote>

<footnote>* Correspondence to: Laboratoire de toxicologie biologique et medico-légale, CHU Pontchaillou, 2 Rue Henri Le Guilloux, Rennes 35000, France.
E-mail address: thomas.gicquel@chu-rennes.fr (T. Gicquel).</footnote>
include the oxidative pathway that produce catechol APAP metabolites such as hydroxy-APAP and methoxy-APAP. However these metabolites were considered to be minor in human when first studied.

The hepatotoxicity of APAP is mainly caused by the production of NAPQI, an electrophile that covalently binds to intracellular proteins, most commonly to cysteine residues, to form APAP protein adducts (Curry et al., 2019). At therapeutic doses, NAPQI is inactivated by conjugation to glutathione (GSH), the major endogenous antioxidant, to give the neutral APAP glutathione metabolite (APAP-SG). This non-toxic conjugate is known to be eliminated as cysteine (APAP-CYS) or mercapturic acid (APAP-NAC) conjugates (mercapturate pathway) by renal excretion (Davis et al. 1974, Bessems and Vermeulen, 2001, Mazaleuskaya et al. 2015, Chidiac et al. 2023). Above therapeutic doses, APAP overdose can cause life-threatening hepatotoxicity due to massive production of NAPQI from APAP, and the subsequent production of protein adducts and depletion of hepatic glutathione (GSH) (Saito et al. 2010). Symptoms begin with nausea, vomiting and abdominal pain, and can progress to acute liver failure, sometimes leading to liver transplantation or death (Bouvet et al., 2020; Dart et al. 2023). The medical management of APAP intoxication is well known and includes rapid treatment by N-acetylcysteine (NAC) as a glutathione precursor antidote, with the aim of detoxifying NAPQI. However, research is still ongoing to identify potential relevant biomarkers that could assess the risk of adverse issues after APAP intoxication.

Recently, APAP metabolism has been revisited at therapeutic doses, revealing the existence of other relevant but overlooked APAP metabolites derived from the CYP450 oxidative pathways (Fig. 1) (David et al., 2021, Gorrochategui et al. 2023). NAPQI have been shown to produce significant signals of conjugated thiomethyl metabolites such as S-methyl-3-thioacetaminophen sulfate (S-CH₃-APAP-S) via the thiomethyl shunt pathway through the activity of cysteine conjugate β-lyases that cleave mercapturate and cysteine conjugates (Cooper et al. 2010, David et al. 2021) (Fig. 1). So far, the production of these thiomethyl metabolites (initially detected as non-conjugated in human) from glutathione-derived metabolites has been largely considered to be negligible, which could be explained by their delayed peak concentrations, probably due to delayed reabsorption after enterohepatic circulation (David et al. 2021, Gorrochategui et al., 2023). These metabolites (mainly S-CH₃-APAP-S and SO-CH₃-APAP-S), which are linked to NAPQI detoxification, could potentially help for the diagnosis of APAP intoxication, or the monitoring of NAPQI elimination. However, little data has been available in humans on the importance of glutathione-derived metabolites (APAP-SG) and mercapturate derivatives (APAP-Cys and APAP-NAC) after APAP intoxication, which justifies the need for further studies in this area.

In this recent study (Gorrochategui et al. 2023), high signals were also observed for catechol APAP derivatives such as 3-Hydroxy-APAP-Sulfate, 3-Hydroxy-APAP, 3-Methoxy-APAP-Glucuronide (respectively 3-OH-APAP-S, 3-OH-APAP and 3-O-CH₃-APAP-Glu) through CYP450

oxidation. These phase I metabolites are poorly described and still considered as minor or non-existent in humans, despite new evidence suggesting a significant contribution from this pathway even at therapeutic doses (Fig. 1) (David et al. 2021, Gorrochategui et al. 2023). The contribution of these different pathways after APAP overdose has not been studied comprehensively so far.

Based on our preliminary data, we hypothesised that the metabolism of APAP in overdosed patients is more complex than previously thought, in particular for the elimination of the hepatotoxic metabolite NAPQI. Using state-of-the-art methodologies enabling the broad and simultaneous measurement of thousands of small molecules, we aimed to study APAP biotransformation pathways in a larger cohort of patients with proven acute APAP intoxication to provide new data for APAP and 14 of its metabolites (including phase II conjugates and metabolites from the different oxidative pathways) that could help to improve the diagnosis of intoxication as well as patient follow-up. Therefore, this study builds on our previous work by including a larger number of patients and additional time points to monitor the appearance and elimination of additional APAP metabolites. We also present additional data for a specific case study of a 20-year-old man who consumed 20 g of APAP.

## 2. Materials and methods

### 2.1. Chemicals and reagents

All chemicals, reagents and solvents were of LC-MS grade quality and purchased from Biosolve Chimie (Dieuze, France). The list of standards are provided in Suppl Table S1.

### 2.2. Design and participants

Plasma samples from APAP intoxicated patients were collected from 06/23/2022–11/15/2022 and stored at –80°C before extraction and analysis. This observational study (APAPTHIO) was approved by the Scientific Ethical Committee of Rennes Hospital in France (advice n° 22.185) and involved 25 patients admitted to Rennes University Hospital for suspicion of acute APAP intoxication with a positive para-cetamolemia ([APAP] > 5 mg/L) and correspond to 37 plasma samples. The following information was collected anonymously and reported in supplementary Table S2: sex, age of patients, APAP concentration, estimated time after intoxication, co-consumed drugs, treatment with NAC and liver markers (aspartate aminotransferase (AST), alanine aminotransferase (ALT), Prothrombin time and factor V). This heterogeneous cohort of patients was stratified according to the time taken to collect samples after taking APAP (4 h, between 6 and 8 h, between 10 and 16 h and between 24 and 48 h).

<figure><img src="image_5.png" /><figcaption>Fig. 1. APAP metabolism overview including the metabolites formed by the thiomethyl shunt and the catechol pathway.</figcaption></figure>
## 2.3. Intoxication case

We report the case of a 20-year-old man whose only history was untreated depression. He reported to consumed alcohol, 20 g of APAP and tramadol two hours before arriving comatose at the emergency department with a Glasgow score at 11, miosis and no other clinical abnormalities. Given the massive APAP dose ingested, treatment with NAC was initiated according to French recommandations (Mégarbane et al., 2020). APAP levels were respectively 72.3 mg/L and 61.1 mg/L 2 and 4 h after intoxication. As the APAP concentration was below 150 mg/L at 4 h, NAC treatment was stopped. Blood alcohol level was 0.97 g/L and the hepatic markers (AST/ALT/prothrombin time and factor V) were normal. APAP level was < 5 mg/L 42 h after ingestion.

## 2.4. APAP plasma level measurement

APAP levels were measured on Roche Cobas® 8000 on a G6PDH-based Immunoenzymatic absorption spectrophotometry method in Rennes University Hospital as already published (Lefèvre et al. 2023). Limit of quantification was 5 mg/L. Quality controls were carried out every day before and after each batch.

## 2.5. UHPLC-ESI-QTOFMS non-targeted analyses

Plasma samples (80 µL) spiked with the 10 µL of internal standard (APAP-D4, 10 µg/mL) were extracted using the protein precipitation method described in Gorrochategui et al. (2023) in order to be less selective and to avoid impacting the recoveries of APAP metabolites during the sample preparation step. As Briefly, Protein precipitation was

carried out using a 4:1 (v/v) cold methanol to matrix ratio. Samples were left at -20°C for 1 h to improve protein removal. Centrifugation was performed at 4°C and 17,000 g for 20 min, after which the supernatants were collected and evaporated to dryness under vacuum. Samples were recovered in 90:10 (v/v) ultrapure water to acetonitrile mixture. Extracts were profiled using an Exon UHPLC system (AB SCIEX, USA) coupled to an AB SCIEX X500R Q-TOF-MS system (SCIEX, Canada), equipped with a DuoSpray ion source as described in David et al. (David et al. 2021). All the samples were analyzed in a full-scan experiment (50-1100 Da) in negative ESI mode (5 ppm mass tolerance). Molecules were identified according to their retention time and *m/z* [M-H'] exact mass (Table 1), and confirmation of identification was done using MS2 data after injections in DDA mode as already described (David et al. 2021). MS2 fragments and confidence levels for the identification of all metabolites are presented in the Supp Table S3 (Schymanski et al. 2014).

## 2.6. Quality control

To evaluate and control potential background contaminants, one workup sample (i.e., extraction with HPLC grade water instead of sample) per analytical batch was prepared and injected. Quality control (QC) samples, consisting of pools of all biological samples, were injected all along the analytical batch in order to monitor for UHPLC-ESI-TOF-MS repeatability and sensitivity. To ensure there was no presence of remaining traces in the UHPLC system from previous batches, that could affect subsequent analytical runs, solvent blank samples (acetonitrile/ H₂O (20:80)) were also injected within the analytical batch. Sample batch order of injection was as follows: the first injections corresponded

<table><caption>**Table 1**

Analytical method parameters (RT= retention time).</caption><thead><tr><th></th><th>Formula</th><th>Name</th><th>m/z [M-H']</th><th>RT (min)</th></tr></thead><tbody><tr><td>Internal standard</td><td>C<sub>8</sub>H<sub>5</sub>D<sub>4</sub>NO<sub>2</sub></td><td>APAP-D4 (acetaminophen-D4)</td><td>154.0868</td><td>4.1</td></tr><tr><td>Parent</td><td>C<sub>8</sub>H<sub>9</sub>NO<sub>2</sub></td><td>APAP (acetaminophen)</td><td>150.0560</td><td>4.2</td></tr><tr><td rowspan="2">Conventional metabolites</td><td>C<sub>8</sub>H<sub>9</sub>NO<sub>5</sub>S</td><td>APAP-S (acetaminophen sulfate)</td><td>230.0129</td><td>4.1</td></tr><tr><td>C<sub>14</sub>H<sub>17</sub>NO<sub>8</sub></td><td>APAP-Glu (acetaminophen glucuronide)</td><td>326.0881</td><td>3.5</td></tr><tr><td rowspan="4">Catechol (pathway)</td><td>C<sub>8</sub>H<sub>9</sub>NO<sub>3</sub></td><td>3-OH-APAP (3-hydroxyacetaminophen)</td><td>166.051</td><td>4.0</td></tr><tr><td>C<sub>8</sub>H<sub>9</sub>NO<sub>6</sub>S</td><td>3-OH-APAP-S (3-hydroxyacetaminophen sulfate)</td><td>246.0078</td><td>4.0</td></tr><tr><td>C<sub>15</sub>H<sub>19</sub>NO<sub>9</sub></td><td>3-OCH<sub>3</sub>-APAP-Glu (3-methoxyacetaminophen glucuronide)</td><td>356.0987</td><td>3.8</td></tr><tr><td>C<sub>9</sub>H<sub>11</sub>NO<sub>3</sub></td><td>O-CH<sub>3</sub>-APAP (3-methoxyacetaminophen)</td><td>180.0663</td><td>4.4</td></tr><tr><td>APAP-Glutathion derivated</td><td>C<sub>18</sub>H<sub>26</sub>N<sub>4</sub>O<sub>8</sub>S</td><td>APAP-SG (acetaminophen glutathione)</td><td>455.1236</td><td>3.8</td></tr><tr><td rowspan="2">Mercapturate (pathway)</td><td>C<sub>13</sub>H<sub>16</sub>N<sub>2</sub>O<sub>5</sub>S</td><td>NAC-APAP (Acetaminophen mercapturate)</td><td>311.0707</td><td>4.5</td></tr><tr><td>C<sub>11</sub>H<sub>14</sub>N<sub>2</sub>O<sub>4</sub>S</td><td>APAP-Cys (3-(Cystein-S-yl)acetaminophen)</td><td>269.0602</td><td>3.6</td></tr><tr><td rowspan="4">Thiomethylshunt (pathway)</td><td>C<sub>14</sub>H<sub>17</sub>NO<sub>8</sub>S</td><td>SH-APAP-Glu (3-thioacetaminophen glucuronide)</td><td>358.0602</td><td>4.1</td></tr><tr><td>C<sub>9</sub>H<sub>11</sub>NO<sub>5</sub>S<sub>2</sub></td><td>S-CH<sub>3</sub>-APAP-S (S-methyl-3-thioacetaminophen sulfate)</td><td>276.0006</td><td>5.1</td></tr><tr><td>C<sub>15</sub>H<sub>19</sub>NO<sub>8</sub>S</td><td>S-CH<sub>3</sub>-APAP-Glu (S-methyl-3-thioacetaminophen glucuronide)</td><td>372.0759</td><td>4.2</td></tr><tr><td>C<sub>9</sub>H<sub>11</sub>NO<sub>6</sub>S<sub>2</sub></td><td>SO-CH<sub>3</sub>-APAP-S (S-methyl-3-thioacetaminophen sulphoxide sulfate)</td><td>291.9955</td><td>4.4</td></tr><tr><td>C<sub>15</sub>H<sub>19</sub>NO<sub>9</sub>S</td><td>C<sub>9</sub>H<sub>11</sub>NO<sub>6</sub>S<sub>2</sub></td><td>SO-CH<sub>3</sub>-APAP-Glu (S-methyl-3-thioacetaminophen sulphoxide glucuronide)</td><td>388.0708</td><td>3.5</td></tr></tbody></table>
to blank samples (workup and solvent), followed by the injection of a QC sample six times consecutively. Then all samples from the batch, randomly distributed in sets of 5, interspersed each time with a periodic QC to monitor the analytical sensitivity and repeatability, as already performed in Gorrochategui et al. (2023).

## 2.7. Data retreatment

Peak integration of all APAP metabolites was manually performed using the SCIEX OS Analytics tool to ensure accuracy in the study of the excretion rates of APAP metabolites for each individual. Figures were created using Graphpad (version 8.0.2, La Jolla, CA, USA).

# 3. Results

## 3.1. Characteristics of the patients

Overall, the cohort of intoxicated patients was predominantly female (80 %) with a median age of 20 years (Table 2). The mean plasma level was 97.5 mg/L 4 h after APAP ingestion (N = 15). We also identified that half of intoxicated patients also consumed other drugs at the time of overdose (codeine, ibuprofen, opium powder, fluoxetine, tramadol or zopiclone) or alcohol. Patients did not develop liver acute failure (Suppl. Table S2). The antidote, NAC, was administrated to a quarter of patients (7/25). The time between APAP uptake and blood sample collection was known for 31 of the 37 samples, with a median time of 4 h (ranging from 2 to 60 h). NAC presence was confirmed in treated patients using LC-HRMS non-targeted method, by searching the ion *m/z* ([M-H]* = 162.023, RT= 1.0 min).

## 3.2. Kinetics of appearance of metabolites in plasma samples

In intoxicated patients, plasma levels of APAP and its metabolites were measured and grouped into four time intervals after APAP consumption: 4 h (N = 15), 6–8 h (N = 4), 10–16 h (N = 4) and 24–48 h (N = 4) (Fig. 2A). Samples taken before 4 h post-ingestion were

<caption>**Table 2**</caption>

Characteristics of the cohort of paracetamol-intoxicated patients (37 plasma samples corresponding to 25 patients) NAC = N-acetyl-cysteine.

<table><thead><tr><th></th><th></th></tr></thead><tbody><tr><td>Sex</td><td>20 Female (80 %) (30 plasma samples)<br>5 Male (20 %) (7 plasma samples)</td></tr><tr><td>Age in years (mean, median, [range])</td><td></td></tr><tr><td>Total</td><td>26.6, 20, [12–84]</td></tr><tr><td>Female</td><td>22.8, 18, [12–50]</td></tr><tr><td>[APAP] in mg/L (mean, median, [range])</td><td></td></tr><tr><td>Total</td><td>63.7, 46.5, [< 5–284.4]<br>(N = 37)</td></tr><tr><td>h</td><td>97.5, 75.7, [20.3–284.4]<br>(N = 15)</td></tr><tr><td>6–8h</td><td>70.1, 52.5, [44.4–136.5]<br>(N = 4)</td></tr><tr><td>10–16h</td><td>23.7, 18.7, [8.7–48.9]<br>(N = 4)</td></tr><tr><td>24–48h</td><td>3.8, 2.7, [< 5–9.8] (N = 4)<br>50.4, 40.5 [12.1 – 100.4]<br>(N = 6)</td></tr><tr><td>unknown</td><td>9.0, 4.0, [2; 42]</td></tr><tr><td>Delay between APAP uptake and blood collection in<br>hours (mean, median, [range])<br>(known for 31 of 37 samples)</td><td></td></tr><tr><td>NAC administration</td><td>9 samples / 37 (24.3 %)<br>7 patients / 25 (28.0 %)</td></tr><tr><td>Co-intoxication (Yes / No / unknown), n (%)</td><td></td></tr><tr><td>Patients (n = 25)</td><td>13 (52 %), 11 (44 %), 1<br>(4 %)</td></tr><tr><td>Samples (n = 37)</td><td>18 (48.6 %), 18 (48.6 %), 1<br>(2.7 %)</td></tr></tbody></table>

excluded (N = 4) and for 6 samples the exact time between APAP consumption and collected could not be established.

Peak area was measured in plasma samples for APAP, APAP-S, APAP-Glu, catechol, mercapturate, thiomethyl shunt metabolites and APAP-SG as described in Table 1. As expected, APAP intensity, as APAP concentration, decreased over time (Fig. 2A-B). The conventional metabolites derived from glucuronidation and sulfation (respectively APAP-GLU and APAP-S) were already intensely detected between 4 and 8 h post-ingestion, before decreasing over time (Fig. 2A).

APAP metabolites derived from the catechol pathway were less intensely detected but showed similar kinetics. The most intense catechol derivative was 3-O-CH₃-APAP-GLU, but 3-OH-APAP-GLU and 3-OH-APAP-S were also detected at each time interval (Fig. 2C). APAP-CYS and APAP-NAC, i.e. the conjugates from the mercapturate pathway, were weakly detected in these plasma samples (Fig. 2D). Metabolites from the thiomethyl shunt pathway were increasingly detected between 4 h and 24–48 h, i.e. with a delay compared to the metabolites described previously. Among these metabolites, S-CH₃-APAP-S was the most intensively detected (Fig. 2E).

Only 3 patients (corresponding to 7 samples) were positive for APAP-SG, including two young women aged 14 and 17 (Suppl. Data S2) and one 20-year-old man. In this context, and due to the inconstant identification of APAP-SG in patient samples, we then focused on a case of intoxication that presented a late sample which showed the disappearance of conventional metabolites, APAP-SG and the appearance of metabolites from the thiomethyl shunt pathway (see 3.3 below).

## 3.3. Focus on a case of intoxication

In the case of the intoxicated 20 year-old man, APAP concentration measured by immunoassay 4 h and 42 h after APAP ingestion was 61.1 mg/L and < 5 mg/L, respectively. Four hours after consumption, APAP-S and APAP-Glu were the most intense metabolites detected, followed by APAP-SG (Fig. 3A). The intensities of catechol-derived metabolites was low, while mercapturate and thiomethyl were almost undetectable (Fig. 3A). At 42 h post-ingestion, thiomethyl metabolites were intensely detected, while APAP, APAP-S, APAP-Glu, APAP-SG and catechol metabolites decreased compared with 4 h post-ingestion (Fig. 3B). Among the thiomethyl metabolites, S-CH₃-APAP-S was the most intensely detected metabolite followed by S-CH₃-APAP-Glu and SO-CH₃-APAP-S (Fig. 3B).

In the chromatogram obtained by LC-HRMS on the sample collected 42 h post-ingestion, S-CH₃-APAP-S (RT=5.1 min) was the most intensely detected metabolite (Fig. 4). In addition to APAP-Glu (RT=3.5 min) O-CH₃-APAP-Glu and APAP-SG were also well detected in plasma samples.

# 4. Discussion

In this study, we aimed to characterize APAP biotransformation pathways in an authentic cohort of patients with proven acute APAP intoxication to provide new data that could help improve the diagnosis of intoxication as well as patients follow-up.

## 4.1. A relevant real-life cohort

In this retrospective cohort of patients intoxicated with APAP, plasma samples were collected at different times, the absorbed dose of APAP and concentrations were variable and patients sometimes presented co-intoxications. The median age of intoxicated patients was 20 years, and 80 % were women, which is consistent with literature data. APAP is the most frequently found in intentional intoxication among young women under the age of 20, as reported in England (Cook et al. 2008; Prescott et al. 2009; Casey et al. 2020), and in Asia-Pacific regions such as Singapore (Tan and Sklar, 2017), Malaysia (Marzilawati et al. 2012), Thailand (Pholmoo and Bunchorntavakul, 2019) and Australia
<figure><img src="image_6.png" /><figcaption>Fig. 2. A) Analytical responses (represented as peak areas) (mean +/- SEM) of the main metabolism pathways for 27 plasma samples according to 4 time intervals: 4 h, 6–8 h, 10–16 h and 24–48 h. B) APAP patient concentrations measured by immunoenzymatic absorption spectrophotometry according to time intervals. Intensities of 27 patient plasma according to time for C) catechol pathway metabolites D) mercapturate pathway metabolites E) thiomethylshunt pathway metabolites.</figcaption></figure>

(Singer et al. 1995). Hence, the population observed in this cohort and the associated data reported are representative of previous studies on APAP intoxication.

The median time from APAP ingestion to blood sampling was 4 h in our cohort, in line with Rumack and Matthew's revised nomogram reporting that APAP plasma concentrations are uninterpretable before 4 h post-intoxication (Dart et al. 2023). Here, the mean concentration 4 h post-intoxication was 97.5 mg/L, close to the known toxic concentration of 100 mg/L (Schulz et al. 2020). In addition, APAP concentration > 150 mg/L 4 h post-ingestion is criteria requiring treatment with the antidote NAC (Dart et al. 2023). In our study, only 2 patients were in this case (188.7 and 284.4 mg/L at 4 h post-ingestion), although 7 patients received NAC. Given the relative safety of NAC in cases of co-intoxication or when a very large dose is assumed to have been ingested, it is common practice to administer NAC to patients (Akakpo et al. 2022). In terms of follow-up, our cohort mainly consists of intoxicated patients with minor consequences of their intoxication, since no liver acute failure were reported. These observations are in line with literature data reporting that patients presenting with an acute APAP overdose with APAP level < 150 µg/mL are at low risk of hepatotoxicity (Alhammad et al., 2024).

## 4.2. Direct phase II APAP conjugates are important for diagnosis of acute intoxication

At therapeutic dose, peak plasma concentrations of APAP are generally reached one hour after oral administration, then decline and become undetectable less than 24 h after consumption (Court et al., 2017). In case of intoxication, it has been reported that half-lives of APAP and its main direct phase II metabolites (APAP-GLU and APAP-SUL) are increased (McGill and Jaeschke, 2013; Xie et al. 2015; Vliegenthart et al., 2017). Here, we observed that before 24 h, these phase II metabolites remain the main markers of APAP consumption, even when free APAP has disappeared (<5 mg/L) (Figs. 2–3). Direct phase II APAP conjugates could therefore be appropriate to confirm recent exposure to APAP in case of intoxication when free APAP cannot be detected anymore. In case of APAP overdose, literature data suggest that the large amount of APAP would saturate glucuronidation and sulfation pathways. This would contribute to increased production of NAPQI, and depletion of glutathione stock due to production of APAP-glutathione conjugate (APAP-SG) (Davis et al. 1974; Jaeschke and Bajt, 2006; James et al. 2003).
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Analytical responses (represented as peak areas) in plasma samples for each metabolic pathway in a 20 year-old man at A) 2 h and B) 42 h after APAP intoxication.</figcaption></figure>

### 4.3. The catechol pathway is not a minor pathway after APAP intoxication as observed at therapeutic doses

The catechol pathway produced metabolites (3-O-CH₃-APAP, 3-O-CH₃-APAP-Glu and 3-OH-APAP-S) with a moderate but significant intensity and a kinetics close to APAP-Glu and APAP-S. These results confirm previous observations after consumption of therapeutic doses of APAP (David et al. 2021; Gorrochategui et al., 2023). Interestingly, while 3-OH-APAP was described as a non-toxic catechol metabolite produced *in vitro* via human CYPs 2A6 and 2E1 (Chen et al. 1998; Hinson et al. 1980), 3-O-CH₃-APAP was presented as hepatotoxic as APAP (Forte et al. 1984). This oxidative metabolism of APAP to 3-O-CH₃-APAP could therefore contribute to the toxicity of consumed APAP. These data suggest that the study of the APAP catechol pathway is of interest for a better understanding of APAP-induced hepatotoxicity.

### 4.4. APAP-glutathione derivative (APAP-SG) is an inconstant but intense APAP plasma metabolite

In the literature, circulating APAP metabolites formed by CYP450 enzymes (APAP-SG, APAP-Cys and NAC-APAP) have been proposed as toxicokinetic biomarkers that stratify patient by their risk of subsequent acute liver injury (ALI) (Vliegenthart et al. 2017). In our study, mercapturate pathway-derived metabolites (APAP-Cys and APAP-NAC) were poorly detected in plasma samples whatever the time after APAP consumption, and no cases of severe liver damage were observed, which did not allow us to corroborate literature data. This could be partly due to the use of a negative ionisation detection method, which is probably not optimal for detecting these compounds. At the same time, APAP-SG was identified with significant intensity in 7 of 37 plasma samples in our study, corresponding to 3 patients. Here, the presence of APAP-SG did not appear to be associated with more severe liver damage (Suppl. Data S2), although further studies are needed on a large cohort of patients to establish a link between liver toxicity and APAP-SG concentration. This
<figure><img src="image_8.png" /><figcaption>Fig. 4. Chromatogram obtained by LC-HRMS after analysis of a sample taken 42 h post-ingestion of 20 g of paracetamol in a 20-year-old man. APAP-Glu (m/z 326.0881), O-CH₃-APAP-Glu (m/z 356.0987), APAP-SG (m/z 455.1236) and S-CH₃-APAP-S (m/z 276.0006).</figcaption></figure>

need is all the more relevant in view of the findings of Roušar et al. (2012) reporting that APAP-SG is a toxic conjugate capable of inducing oxidative stress in rat liver (Roušar et al., 2012).

### 4.5. Thiomethyl shunt pathway allow to monitor the elimination of the toxic NAPQI

Production of conjugated thiomethyl metabolites from the mercapturate pathway-derived metabolites was poorly known and for a long time considered to be negligible (i.e. < 1 % of the initial dose (Klutch et al. 1978)). Recently, NAPQI has been shown to produce significant signals of conjugated thiomethyl metabolites via the thiomethyl shunt pathway, thus providing a new insight into the oxidative metabolism of APAP (Cooper et al. 2010; David et al. 2021). In particular, S-CH₃-APAP-S and SO-CH₃-APAP-S were found to be stable biomarkers consistently detected in urine from pregnant women after APAP intake and in blood samples collected after APAP acute intoxication, with a delayed excretion compared to conventional metabolites (David et al. 2021, Gorrochategui et al., 2023).

Based on these findings, we postulated that thiomethyl metabolites could provide a more reliable history of APAP intoxication due to their delayed excretion compared to conventional APAP metabolites. Here, we first confirm in a cohort of 37 intoxicated patient plasmas that the metabolite S-CH3-APAP-S is the most intensely detected among those of the thiomethyl pathway, and that its kinetics of appearance was delayed compared to more conventional metabolites. This suggests that it could be proposed as a clinically relevant marker in the event of APAP intoxication with delayed medical attention (> 24 h), and when free APAP plasma concentrations are undetectable. This is particularly relevant as more than half of patients presenting to hospital with APAP-induced acute liver injury have undetectable levels of APAP (Leventhal et al. 2019). As sulfo- and glucuroconjugated metabolites are still intensely detectable by mass spectrometry 42 h after intake, thiomethyl metabolites could constitute complementary late biomarkers. Their usefulness in this context could make it possible to monitor the elimination kinetics of NAPQI over late periods, yet to be defined, by analysing samples from intoxicated patients > 48 h. This case highlights the value of monitoring metabolites as a function of time, with in particular i) the identification of unconventional metabolites easily detectable by chromatography, such as APAP-SG (Fig. 4), and ii) the disappearance of conventional metabolites associated with the delayed appearance of

S-CH3-APAP-S. A single analytical approach for APAP and its metabolites could be more practical than monitoring APAP-protein adducts, which requires a distinct analytical setup despite its demonstrated utility (Khandelwal et al. 2011; James et al. 2009). Additional inhibition studies of thiomethyl metabolite formation, either *in vitro* or *in vivo*, would be valuable to further clarify the role of these metabolites as potential biomarkers of APAP intoxication.

In addition, the Rumack-Matthew nomogram is not useful to predict risk for late presenters (>24 h) (Chidiac et al. 2023). This justifies the need for new decision criteria for antidote administration in the event of late intoxication. In this context, the measurement of these thiomethyl metabolites could also serve to determine metabolite ratios predictive of a risk of hepatotoxicity by studying the ratio of the short-term metabolites over the long-term ones (Gorrochategui et al., 2023). This would require the study of these parameters on a larger cohort, with more sampling time for each patient. Lastly, metabolites would have to be quantified, which implies chemical synthesis of these molecules.

The current study presents limitations due to the production of HRMS-based semi-quantitative data. Hence, these data cannot be used to extrapolate the accurate percentage of excretion of thiomethyl metabolites or any other metabolites in APAP metabolism. Nevertheless, our quality control data provide confidence for the sample-to-sample comparison to study the kinetics of the appearance or elimination of individual metabolites at different time points. Comparing the relative contributions of APAP metabolites on the basis of their analytical responses is therefore relevant for application in human biomonitoring and clinical toxicology given that similar platforms (LC-MS) and ion sources (i.e. electrospray ion sources) are usually used for APAP monitoring.

### 5. Conclusion

In this study we revised APAP metabolism in an intoxicated patient cohort using LC-HRMS. Data produced from authentic cases of APAP poisoning reveal that i) metabolites from the catechol pathway are not minor after intoxication ii) APAP-glutathione derivative (APAP-SG) is an inconstant but intense APAP plasma metabolite detected in some patients iii) measurement of conjugated thiomethyl metabolites could serve to provide a more reliable history of APAP ingestion and in particular that S-CH₃-APAP-S is a relevant delayed marker after APAP intoxication as observed at therapeutic doses. Future steps include the
synthesis of pure standards for the conjugated catechol and thiomethyl
metabolites that are not currently commercially available and the
development of high-throughput targeted methods.

CRediT authorship contribution statement

**Thomas Gicquel:** Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Resources, Project administration, Methodology, Formal analysis, Data curation, Conceptualization. **Romain Pelletier:** Writing – review & editing, Methodology, Investigation, Conceptualization. **Eva Gorrochategui:** Writing – review & editing, Methodology, Data curation, Conceptualization. **Margaux Heurté:** Software, Data curation. **Brendan Le Daré:** Writing – review & editing, Visualization, Validation, Supervision, Software, Data curation. **Arthur David:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Diane Le Bouedec:** Investigation, Conceptualization. **Jade Chaker:** Supervision, Software. **Isabelle Morel:** Supervision.

Ethical approval

The study was conducted according to the guidelines of the Decla-
ration of Helsinki, and approved by the Ethics Committee of University
Hospital of Rennes.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgements

We thank the research infrastructure France Exposé.

**Appendix A. Supporting information**

Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.toxlet.2025.111727.

Data Availability

The data that has been used is confidential.

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