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Herein, we describe a newly developed, highly accurate, and precise isotope-dilution liquid chromatography-mass spectrometry (LC-MS\/MS) method capable of simultaneously measuring eight clinically relevant polar and non-polar steroid hormones in 200 \u00b5L of serum. This steroid hormone panel method uses sequential liquid-liquid extractions for the isolation of total testosterone (TT), estradiol (E2), progesterone (P4), 17-hydroxyprogesterone (17-OHP), androstenedione (AD), estrone (E1), estrone sulfate (E1S), and dehydroepiandrosterone sulfate (DHEAS) without derivatization or hydrolysis. The method demonstrated a broad analytical measurement range for all eight hormones as a result of improved selectivity and sensitivity, making it suitable for the analysis of general population serum samples, including postmenopausal women and children. The total imprecision, expressed as coefficients of variation, was evaluated at three levels for each analyte and ranged from 3.5 to 10.7%. The mean bias to well-established secondary reference materials was \u22121.00% for TT and 1.91% for E2. When applied to 268 commercially sourced serum samples from men, women, and children, only 2% of measurement results were below the limits of detection; therefore, this method was deemed suitable for measurement of eight steroid hormones across the general population. The establishment of this new steroid hormone panel LC-MS\/MS method will facilitate investigations of associations between health disorders and individualized steroid profiles, which can be utilized for evaluations at the individual or population levels.<\/jats:p>","DOI":"10.1007\/s00216-026-06504-3","type":"journal-article","created":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T21:34:37Z","timestamp":1777498477000},"page":"4153-4168","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Highly specific ID-UHPLC-MS\/MS method for analyzing polar and non-polar steroid hormones"],"prefix":"10.1007","volume":"418","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-1060-9356","authenticated-orcid":false,"given":"Lumi","family":"Duke","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-5409-2081","authenticated-orcid":false,"given":"Paul H.","family":"Kim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7527-9200","authenticated-orcid":false,"given":"Alicia N.","family":"Lyle","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6740-7169","authenticated-orcid":false,"given":"Julianne C.","family":"Botelho","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0847-9170","authenticated-orcid":false,"given":"Natalie D.","family":"Shaw","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9649-9015","authenticated-orcid":false,"given":"Hubert W.","family":"Vesper","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,4,29]]},"reference":[{"key":"6504_CR1","doi-asserted-by":"publisher","first-page":"213","DOI":"10.1016\/j.jsbmb.2014.06.003","volume":"145","author":"MO Goodarzi","year":"2015","unstructured":"Goodarzi MO, Carmina E, Azziz R. 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Use of blood samples by CDC was consistent with the IRB approval and donor consent. No personal identifiers were provided to CDC. This activity using commercially sourced donor samples was reviewed by CDC, deemed not research, and was conducted consistent with applicable federal law and CDC policy. The NIEHS donor samples used in this study were reviewed and approved by the NIEHS IRB.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethical statements and sources for biological materials"}},{"value":"N\/A","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Animal welfare"}},{"value":"The authors declare no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Conflict of interest"}},{"value":"The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention\/the Agency for Toxic Substances and Disease Registry. Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, and the US Department of Health and Human Services. This research was supported, in part, by the Intramural Research Program of the National Institutes of Health (NIH) (Z01-ES103315 and ZIC ES103363 to NDS). NDS is also supported as a Lasker Clinical Research Scholar (1SI2ES025429-01). The contributions of the NIH author(s) were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services.","order":5,"name":"Ethics","group":{"name":"EthicsHeading","label":"Disclaimer"}}]}}