{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T18:05:32Z","timestamp":1787162732546,"version":"build-2736575974"},"reference-count":23,"publisher":"Oxford University Press (OUP)","issue":"1","license":[{"start":{"date-parts":[[2025,7,16]],"date-time":"2025-07-16T00:00:00Z","timestamp":1752624000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/pages\/standard-publication-reuse-rights"}],"funder":[{"DOI":"10.13039\/501100003196","name":"Italian Ministry of Health","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100003196","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,4,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Diuretics are commonly used in doping because they can conceal the presence of performance-enhancing substances in an athlete\u2019s urine through dilution and promote rapid weight loss. As a result, these substances are prohibited in sports by the World Anti-Doping Agency (WADA) under the S5 category (\u201cDiuretics and Masking Agents\u201d). Chlorthalidone, a thiazide-like diuretic, is medically used as an antihypertensive agent and is prescribed for conditions such as heart failure and liver cirrhosis. However, it is also misused in doping. The detection of chlorthalidone or its metabolite markers in an athlete\u2019s urine is essential to prove consumption. Therefore, the aim of the study was to assess the metabolism of the substance in humans. For this purpose, chlorthalidone metabolites were predicted with GLORYx (Hamburg University, Germany) to identify the transformations that may occur with higher probability; the compound was incubated with 10-donor-pooled human hepatocytes to simulate hepatic metabolism; and the incubates were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS\/MS) and software-aided data mining. In silico simulations predicted 11 Phase II metabolites, with N-acetylation at the sulfonamide group being the predominant transformation (88% probability score); other major reactions included O-glucuronidation, O-sulfation, and glutathione conjugation, with probability scores lower than 70%. Two metabolites were identified in in vitro hepatocyte incubates and presented a reduction or a hydroxylation at the phthalimidine moiety. To the best of the authors\u2019 knowledge, these metabolites are specific to chlorthalidone and can be targeted as markers for analytical screening in anti-doping controls.<\/jats:p>","DOI":"10.1093\/jat\/bkaf072","type":"journal-article","created":{"date-parts":[[2025,7,16]],"date-time":"2025-07-16T14:52:27Z","timestamp":1752677547000},"source":"Crossref","is-referenced-by-count":2,"title":["Chlorthalidone in vitro metabolite identification for documenting exposure in doping"],"prefix":"10.1093","volume":"50","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2997-221X","authenticated-orcid":false,"given":"Alessandro","family":"Di Giorgi","sequence":"first","affiliation":[{"name":"Unit of Forensic Toxicology, Section of Legal Medicine, Department of Biomedical Sciences and Public Health, Marche Polytechnic University , Ancona, 60126,","place":["Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gloria","family":"Daziani","sequence":"additional","affiliation":[{"name":"Unit of Forensic Toxicology, Section of Legal Medicine, Department of Biomedical Sciences and Public Health, Marche Polytechnic University , Ancona, 60126,","place":["Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anastasio","family":"Tini","sequence":"additional","affiliation":[{"name":"Unit of Forensic Toxicology, Section of Legal Medicine, Department of Biomedical Sciences and Public Health, Marche Polytechnic University , Ancona, 60126,","place":["Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Livio","family":"Tronconi","sequence":"additional","affiliation":[{"name":"Unit of Forensic Medicine, Department of Public Health, Experimental and Forensic Medicine, University of Pavia , Pavia, 27100,","place":["Italy"]},{"name":"Maria Cecilia Hospital , Cotignola, 48033,","place":["Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3332-9518","authenticated-orcid":false,"given":"Jeremy","family":"Carlier","sequence":"additional","affiliation":[{"name":"Unit of Forensic Toxicology, Section of Legal Medicine, Department of Biomedical Sciences and Public Health, Marche Polytechnic University , Ancona, 60126,","place":["Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Omayema","family":"Taoussi","sequence":"additional","affiliation":[{"name":"Unit of Forensic Toxicology, Section of Legal Medicine, Department of Biomedical Sciences and Public Health, Marche Polytechnic University , Ancona, 60126,","place":["Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2025,7,16]]},"reference":[{"key":"2026041500165458700_bkaf072-B1","doi-asserted-by":"publisher","first-page":"3021","DOI":"10.1093\/EURHEARTJ\/EHY339","article-title":"2018 ESC\/ESH Guidelines for the management of arterial hypertension","volume":"39","author":"Williams","year":"2018","journal-title":"Eur Heart J"},{"key":"2026041500165458700_bkaf072-B2","doi-asserted-by":"publisher","first-page":"4","DOI":"10.1161\/01.HYP.0000103632.19915.0E\/FORMAT\/EPUB","article-title":"Hydrochlorothiazide versus chlorthalidone: evidence supporting their interchangeability","volume":"43","author":"Carter","year":"2004","journal-title":"Hypertension"},{"key":"2026041500165458700_bkaf072-B3","doi-asserted-by":"publisher","first-page":"1378","DOI":"10.1002\/DTA.3661","article-title":"Elimination profile of low-dose chlortalidone and its detection in hair for doping analysis-Implication for unintentional non-therapeutic exposure","volume":"16","author":"Thieme","year":"2024","journal-title":"Drug Test Anal"},{"key":"2026041500165458700_bkaf072-B4","author":"World Anti-Doping Agency (WADA)"},{"key":"2026041500165458700_bkaf072-B5","doi-asserted-by":"publisher","first-page":"1074","DOI":"10.1002\/DTA.3634","article-title":"Does a hair test allow discriminating a tail end of a doping regimen from a contamination in case of challenging an anti-doping rule violation? II. Case report involving chlortalidone","volume":"16","author":"Kintz","year":"2024","journal-title":"Drug Test Anal"},{"key":"2026041500165458700_bkaf072-B6","doi-asserted-by":"publisher","first-page":"119890","DOI":"10.1016\/j.cca.2024.119890","article-title":"Knowing the minimal detectable dose can facilitate the interpretation of a hair test result: case example with chlortalidone","volume":"562","author":"Kintz","year":"2024","journal-title":"Clin Chim Acta"},{"key":"2026041500165458700_bkaf072-B7","author":"World Anti-Doping Agency (WADA)"},{"key":"2026041500165458700_bkaf072-B8","author":"World Anti-Doping Agency (WADA)"},{"key":"2026041500165458700_bkaf072-B9","volume-title":"StatPearls","author":"Kerndt","year":"2024"},{"key":"2026041500165458700_bkaf072-B10","doi-asserted-by":"publisher","first-page":"1291","DOI":"10.1093\/BIOINFORMATICS\/BTZ695","article-title":"NERDD: a web portal providing access to in silico tools for drug discovery","volume":"36","author":"Stork","year":"2020","journal-title":"Bioinformatics"},{"key":"2026041500165458700_bkaf072-B11","doi-asserted-by":"publisher","first-page":"286","DOI":"10.1021\/ACS.CHEMRESTOX.0C00224\/ASSET\/IMAGES\/LARGE\/TX0C00224_0004.JPEG","article-title":"GLORYx: prediction of the metabolites resulting from phase 1 and phase 2 biotransformations of xenobiotics","volume":"34","author":"De Bruyn Kops","year":"2021","journal-title":"Chem Res Toxicol"},{"key":"2026041500165458700_bkaf072-B12","doi-asserted-by":"publisher","first-page":"122740","DOI":"10.1016\/J.TALANTA.2021.122740","article-title":"In silico prediction, LC-HRMS\/MS analysis, and targeted\/untargeted data-mining workflow for the profiling of phenylfentanyl in vitro metabolites","volume":"235","author":"Di Trana","year":"2021","journal-title":"Talanta"},{"key":"2026041500165458700_bkaf072-B13","doi-asserted-by":"publisher","first-page":"1989","DOI":"10.1007\/S00204-022-03289-Z","article-title":"In silico, in vitro, and in vivo human metabolism of acetazolamide, a carbonic anhydrase inhibitor and common \u201cdiuretic and masking agent\u201d in doping","volume":"96","author":"Busard\u00f2","year":"2022","journal-title":"Arch Toxicol"},{"key":"2026041500165458700_bkaf072-B14","doi-asserted-by":"publisher","first-page":"5483","DOI":"10.3390\/MOLECULES28145483","article-title":"A review of recent progress in drug doping and gene doping control analysis","volume":"28","author":"Lu","year":"2023","journal-title":"Molecules"},{"key":"2026041500165458700_bkaf072-B15","doi-asserted-by":"publisher","first-page":"164","DOI":"10.1007\/S11419-018-0448-3","article-title":"Sensitive quantification of BB-22 and its metabolite BB-22 3-carboxyindole, and characterization of new metabolites in authentic urine and\/or serum specimens obtained from three individuals by LC-QTRAP-MS\/MS and high-resolution LC-Orbitrap-MS\/MS","volume":"37","author":"Minakata","year":"2019","journal-title":"Forensic Toxicol"},{"key":"2026041500165458700_bkaf072-B16","doi-asserted-by":"publisher","first-page":"27","DOI":"10.1016\/J.JPBA.2018.05.007","article-title":"Synthetic cannabinoid BB-22 (QUCHIC): human hepatocytes metabolism with liquid chromatography-high resolution mass spectrometry detection","volume":"157","author":"Carlier","year":"2018","journal-title":"J Pharm Biomed Anal"},{"key":"2026041500165458700_bkaf072-B17","doi-asserted-by":"publisher","first-page":"230","DOI":"10.3390\/IJMS22010230","article-title":"Pyrrolidinyl synthetic cathinones \u03b1-PHP and 4F-\u03b1-PVP metabolite profiling using human hepatocyte incubations","volume":"22","author":"Carlier","year":"2020","journal-title":"Int J Mol Sci"},{"key":"2026041500165458700_bkaf072-B18","doi-asserted-by":"publisher","first-page":"162","DOI":"10.1093\/JAT\/BKAC057","article-title":"Toxicological investigation of a case series involving the synthetic cathinone \u03b1-pyrrolidinohexiophenone (\u03b1-PHP) and identification of phase I and II metabolites in human urine","volume":"47","author":"Grapp","year":"2023","journal-title":"J Anal Toxicol"},{"key":"2026041500165458700_bkaf072-B19","doi-asserted-by":"publisher","first-page":"2466","DOI":"10.1016\/J.CHROMA.2009.01.032","article-title":"Detection of urinary markers for thiazide diuretics after oral administration of hydrochlorothiazide and altizide-relevance to doping control analysis","volume":"1216","author":"Deventer","year":"2009","journal-title":"J Chromatogr A"},{"key":"2026041500165458700_bkaf072-B20","doi-asserted-by":"publisher","first-page":"123162","DOI":"10.1016\/J.JCHROMB.2022.123162","article-title":"3F-\u03b1-pyrrolydinovalerophenone (3F-\u03b1-PVP) in vitro human metabolism: multiple in silico predictions to assist in LC-HRMS\/MS analysis and targeted\/untargeted data mining","volume":"1193","author":"Carlier","year":"2022","journal-title":"J Chromatogr B Analyt Technol Biomed Life Sci"},{"key":"2026041500165458700_bkaf072-B21","author":"World Anti-Doping Agency (WADA)"},{"key":"2026041500165458700_bkaf072-B22","author":"World Anti-Doping Agency (WADA)"},{"key":"2026041500165458700_bkaf072-B23","doi-asserted-by":"publisher","first-page":"166","DOI":"10.1016\/J.FORSCIINT.2016.08.029","article-title":"Detection of the diuretic hydrochlorothiazide in a doping control urine sample as the result of a non-steroidal anti-inflammatory drug (NSAID) tablet contamination","volume":"267","author":"Helmlin","year":"2016","journal-title":"Forensic Sci Int"}],"container-title":["Journal of Analytical Toxicology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/jat\/advance-article-pdf\/doi\/10.1093\/jat\/bkaf072\/63777437\/bkaf072.pdf","content-type":"application\/pdf","content-version":"am","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/jat\/article-pdf\/50\/1\/bkaf072\/63777437\/bkaf072.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/jat\/article-pdf\/50\/1\/bkaf072\/63777437\/bkaf072.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,15]],"date-time":"2026-04-15T04:17:01Z","timestamp":1776226621000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/jat\/article\/doi\/10.1093\/jat\/bkaf072\/8203393"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,16]]},"references-count":23,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2026,4,10]]}},"URL":"https:\/\/doi.org\/10.1093\/jat\/bkaf072","relation":{},"ISSN":["0146-4760","1945-2403"],"issn-type":[{"value":"0146-4760","type":"print"},{"value":"1945-2403","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2026,1]]},"published":{"date-parts":[[2025,7,16]]},"article-number":"bkaf072"}}