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In this study, a new method was developed for the simultaneous analysis of caffeine, paraxanthine, theobromine and theophylline in horse urine using gas chromatography\u2013mass spectrometry (GC\u2013MS). Urine samples were treated using solid\u2010phase extraction followed by the elution with dichloromethane\u2013isopropanol (90:10) after the pH was adjusted to 6, and then derivatization with <jats:italic>N<\/jats:italic>\u2010methyl\u2010<jats:italic>N<\/jats:italic>\u2010trimethylsilyl\u2010trifluoroacetamide\u20131% trimethylchlorosilane before analysis with GC\u2013MS. Sample preparation and derivatization steps were optimized and the method permitted elution all of these analytes within 13\u2009min. The method was fully validated according to Commission Decision, 2002\/657\/EC guidelines. The calibration curves were linear with a correlation coefficient of &gt;0.99. Precision and accuracy were well within the 15% acceptance range and the method was robust. The validation results demonstrated that the method is highly reproducible, easily applicable and selective. The method was applied to urine samples collected from racehorses to demonstrate its applicability.<\/jats:p>","DOI":"10.1002\/bmc.5445","type":"journal-article","created":{"date-parts":[[2022,7,7]],"date-time":"2022-07-07T12:30:33Z","timestamp":1657197033000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Simultaneous quantification of caffeine and its main metabolites by gas chromatography mass spectrometry in horse urine"],"prefix":"10.1002","volume":"36","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1813-9188","authenticated-orcid":false,"given":"Eylem Funda","family":"G\u00f6kta\u015f","sequence":"first","affiliation":[{"name":"Doping Control Laboratory Istanbul Pendik Veterinary Control Institude  Istanbul Turkey"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0339-0353","authenticated-orcid":false,"given":"Erol","family":"Kabil","sequence":"additional","affiliation":[{"name":"Doping Control Laboratory Istanbul Pendik Veterinary Control Institude  Istanbul Turkey"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7605-7877","authenticated-orcid":false,"given":"Levent","family":"Yatanaslan","sequence":"additional","affiliation":[{"name":"Doping Control Laboratory Istanbul Pendik Veterinary Control Institude  Istanbul Turkey"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9872-1882","authenticated-orcid":false,"given":"Ertu\u011frul","family":"G\u00fcne\u015f","sequence":"additional","affiliation":[{"name":"Doping Control Laboratory Istanbul Pendik Veterinary Control Institude  Istanbul Turkey"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1792-8273","authenticated-orcid":false,"given":"Levent","family":"Dirikolu","sequence":"additional","affiliation":[{"name":"Department of Comparative Biomedical Sciences, School of Veterinary Medicine Louisiana State University  Baton Rouge LA USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2022,7,15]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.foodchem.2004.11.013"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.2741\/1367"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/0009-9236(95)90025-X"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1093\/chromsci\/40.1.45"},{"key":"e_1_2_8_6_1","unstructured":"Budhraja A. 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