{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,16]],"date-time":"2026-02-16T11:07:35Z","timestamp":1771240055112,"version":"3.50.1"},"reference-count":22,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,2,14]],"date-time":"2026-02-14T00:00:00Z","timestamp":1771027200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>In recent years, xylazine has emerged as a cutting agent combined with illicit drugs to extend their effects. The present study aimed to discover drug use patterns associated with xylazine-positive and -negative urine toxicology drug screens and to assess whether xylazine can be used as a marker for exposure to designer drugs\/new psychoactive substances in our study population. This is a retrospective analysis of urine toxicology results from two different analytical platforms: a targeted, structurally confirmatory, high-performance liquid chromatography\u2013tandem mass spectrometry (LC-MS\/MS) assay that quantifies 136 drugs and metabolites including xylazine; and a non-targeted ThermoFisher Orbitrap Tribrid mass spectrometry system (Thermo ScientificTM, Bremen, Germany) in combination with database searches for the identification of drugs not captured by the targeted assay. All participants were patients receiving care through the Addiction Research and Treatment Services (ARTS), with documented substance misuse, undergoing routine urine drug toxicology testing at the iC42 Clinical Toxicology. Data analysis was performed using Sciex OS version 2.2.0.5738 after extraction using the targeted, structurally confirmatory and quantitative LC-MS\/MS platform (SCIEX, Framingham, MA, USA). The drug patterns found in xylazine-positive and -negative urine samples were statistically significantly different (p &lt; 0.001), indicating different consumption patterns associated with xylazine. Moreover, the overall concentrations of drugs (normalized to creatinine) were also statistically significantly different with higher concentrations in the urine samples that tested negative for xylazine. In contrast, samples that were positive for xylazine contained significantly higher concentrations of various designer drugs\/new psychoactive substances as detected by the untargeted platform (p &lt; 0.0001). The results indicated that xylazine has become increasingly common in Denver\u2019s drug circulation and that xylazine may be used as a marker to prompt reflex testing with non-targeted high-resolution mass spectrometry assays in combination with database searches to test for the exposure to designer drugs\/new psychoactive substances in our patient population.<\/jats:p>","DOI":"10.3390\/ijms27041822","type":"journal-article","created":{"date-parts":[[2026,2,16]],"date-time":"2026-02-16T10:14:32Z","timestamp":1771236872000},"page":"1822","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Retrospective Toxicology Study of Polysubstance Use Patterns Associated with Xylazine"],"prefix":"10.3390","volume":"27","author":[{"given":"Wanzhu","family":"Zhao","sequence":"first","affiliation":[{"name":"iC42 Toxicology, Department of Anesthesiology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carlos","family":"Goncalves","sequence":"additional","affiliation":[{"name":"iC42 Toxicology, Department of Anesthesiology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Emily","family":"Ruggiano","sequence":"additional","affiliation":[{"name":"iC42 Toxicology, Department of Anesthesiology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Trenton","family":"Deanna","sequence":"additional","affiliation":[{"name":"iC42 Toxicology, Department of Anesthesiology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elnaz","family":"Navid","sequence":"additional","affiliation":[{"name":"iC42 Toxicology, Department of Anesthesiology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fabiola","family":"Estrada","sequence":"additional","affiliation":[{"name":"iC42 Toxicology, Department of Anesthesiology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Austin","family":"Rawlings","sequence":"additional","affiliation":[{"name":"iC42 Toxicology, Department of Anesthesiology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Monte","family":"Thompson","sequence":"additional","affiliation":[{"name":"iC42 Toxicology, Department of Anesthesiology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6210-8418","authenticated-orcid":false,"given":"Andrew","family":"Monte","sequence":"additional","affiliation":[{"name":"Rocky Mountain Poison & Drug Safety, Denver, CO 80045, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7297-147X","authenticated-orcid":false,"given":"Uwe","family":"Christians","sequence":"additional","affiliation":[{"name":"iC42 Toxicology, Department of Anesthesiology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,14]]},"reference":[{"key":"ref_1","unstructured":"(2025, September 29). Xylazine, National Institute on Drug Abuse, Available online: https:\/\/nida.nih.gov\/research-topics\/xylazine#overdose-crisis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1097\/PAF.0000000000000622","article-title":"Xylazine, a veterinary tranquilizer, detected in 42 accidental fentanyl intoxication deaths","volume":"42","author":"Nunez","year":"2021","journal-title":"Am. J. Forensic Med. Pathol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"294","DOI":"10.1111\/j.1751-0813.1994.tb03448.x","article-title":"Effects of xylazine on humans: A review","volume":"71","author":"Fyffe","year":"1994","journal-title":"Aust. Vet. J."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1093\/jat\/25.4.245","article-title":"Severe intoxication with the veterinary tranquilizer xylazine in humans","volume":"25","author":"Hoffmann","year":"2001","journal-title":"J. Anal. Toxicol."},{"key":"ref_5","unstructured":"(2025, September 29). Xylazine Drug Enforcement Administration, Available online: https:\/\/www.deadiversion.usdoj.gov\/drug_chem_info\/Xylazine.pdf."},{"key":"ref_6","first-page":"89","article-title":"Severe cutaneous ulcerations secondary to xylazine (tranq): A case series","volume":"36","author":"Wei","year":"2023","journal-title":"JAAD"},{"key":"ref_7","first-page":"113","article-title":"Xylazine-induced acute skin necrosis in two patients who inject fentanyl","volume":"36","author":"Rose","year":"2023","journal-title":"JAAD"},{"key":"ref_8","first-page":"e36864","article-title":"Xylazine in the opioid epidemic: A systematic review of case reports and clinical implications","volume":"15","author":"Ayub","year":"2023","journal-title":"Cureus"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"109380","DOI":"10.1016\/j.drugalcdep.2022.109380","article-title":"Xylazine spreads across the US: A growing component of the increasingly synthetic and polysubstance overdose crisis","volume":"233","author":"Friedman","year":"2022","journal-title":"Drug Alcohol Depend."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"519","DOI":"10.1007\/s11524-011-9662-6","article-title":"The emerging of xylazine as a new drug of abuse and its health consequences among drug users in Puerto Rico","volume":"89","author":"Reyes","year":"2012","journal-title":"J. Urban Health"},{"key":"ref_11","unstructured":"(2025, September 29). Xylazine. Colorado Department of Public Health and Environment, Available online: https:\/\/cdphe.colorado.gov\/overdose-prevention\/xylazine."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"565","DOI":"10.1097\/FTD.0000000000000429","article-title":"A high-throughput HPLC-MS\/MS assay for the detection, quantification and simultaneous structural confirmation of 136 drugs and metabolites in human urine","volume":"39","author":"Klepacki","year":"2017","journal-title":"Ther. Drug Monit."},{"key":"ref_13","unstructured":"U.S. Department of Health and Human Services, Food and Drug Administration (2026, January 31). M10 Bioanalytical Method Validation and Study Sample Analysis. Guidance for Industry, November 2022, Available online: https:\/\/www.fda.gov\/media\/162903\/download."},{"key":"ref_14","unstructured":"(2022). Liquid Chromatography-Mass Spectrometry Methods. 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Open"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/4\/1822\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,16]],"date-time":"2026-02-16T10:16:01Z","timestamp":1771236961000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/4\/1822"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,14]]},"references-count":22,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["ijms27041822"],"URL":"https:\/\/doi.org\/10.3390\/ijms27041822","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,14]]}}}