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Detection of single drug exposure from DFSA requires a systematic toxicological analysis strategy including sensitive methods covering a broad spectrum of substances. The aim of this study was to develop and validate an UHPLC\u2013MS\/MS screening method for analysis of samples from DFSA cases and incorporate an automated enzymatic pre\u2010treatment of urine samples into a robotic sample preparation for an efficient laboratory workflow. The screening method included 144 drugs of abuse, pharmaceuticals, and metabolites relevant to DFSA. The use of a recombinant enzyme showed an efficient glucuronide hydrolysis with an average parent drug recovery of 97%. Investigation of matrix effect showed no pronounced ion enhancement or suppression for most analytes (96%), and extraction recovery was above 80% for 97% of analytes. Process efficiency ranged from 50% to 138% for most analytes. The LODs ranged from 0.0001\u2009mg\/L to 2 mg\/L depending on analyte, and most analytes met the SOFT recommended minimum performance limits. The validated method was applied to authentic suspected DFSA cases (<jats:italic>n<\/jats:italic>\u2009=\u200938). Results showed that drugs of abuse, benzodiazepines, and antidepressants were most commonly found in suspected DFSA cases. Incorporation of an automated enzymatic hydrolysis step during sample preparation enables a fast and simple workflow for simultaneous analysis of blood and urine samples for an improved systematic toxicological analysis strategy for DFSA cases.<\/jats:p>","DOI":"10.1002\/dta.3640","type":"journal-article","created":{"date-parts":[[2024,1,24]],"date-time":"2024-01-24T00:40:43Z","timestamp":1706056843000},"page":"1254-1270","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Automated enzymatic hydrolysis of urine samples for improved systematic toxicological analysis of drug\u2010facilitated sexual assault cases"],"prefix":"10.1002","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1350-9300","authenticated-orcid":false,"given":"Kathrine","family":"Skov","sequence":"first","affiliation":[{"name":"Section of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health and Medical Sciences University of Copenhagen  Copenhagen Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9555-5134","authenticated-orcid":false,"given":"Sys Stybe","family":"Johansen","sequence":"additional","affiliation":[{"name":"Section of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health and Medical Sciences University of Copenhagen  Copenhagen Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristian","family":"Linnet","sequence":"additional","affiliation":[{"name":"Section of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health and Medical Sciences University of Copenhagen  Copenhagen Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5159-8549","authenticated-orcid":false,"given":"Marie Katrine Klose","family":"Nielsen","sequence":"additional","affiliation":[{"name":"Section of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health and Medical Sciences University of Copenhagen  Copenhagen Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2024,1,23]]},"reference":[{"key":"e_1_2_5_2_1","doi-asserted-by":"publisher","DOI":"10.26355\/eurrev_202201_27767"},{"key":"e_1_2_5_3_1","doi-asserted-by":"publisher","DOI":"10.1093\/jat\/bkz110"},{"key":"e_1_2_5_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jflm.2021.102151"},{"key":"e_1_2_5_5_1","doi-asserted-by":"publisher","DOI":"10.3390\/ph14050432"},{"key":"e_1_2_5_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jflm.2017.02.005"},{"key":"e_1_2_5_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jcfm.2005.03.009"},{"key":"e_1_2_5_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.legalmed.2023.102325"},{"key":"e_1_2_5_9_1","unstructured":"Society of Forensic Toxicologists.Recommended minimum performance limits for common DFC drugs and metabolites in urine samples. 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