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Despite advances in analytical methods, sensing and quantifying steroid hormones remains challenging. Immunoassays offer excellent sensitivity but are inherently labour-intensive, costly, and prone to false positives. Mass spectrometry (MS) has been increasingly utilised, with the main hurdle being the isobaric tendencies of similar analytes, which complicates their separation and accurate quantification. This study compares ultrahigh-performance supercritical fluid chromatography separation (UHPSFC) and ultra-high-performance liquid chromatography (UHPLC) for MS detection. It optimises the column chemistry, temperature, and pressure to provide an operational protocol for the resolution and quantification of analytes. It presents the systematic characterisation of UHPSFC-MS performance by investigating spiked blood samples using Solid-Phase Extraction (SPE) and describes the matrix effects associated with MS measurements. Although both separation methods showed adequate resolution, specificity, and retention time, UHPSFC-MS was superior for five out of seven columns tested. With added high-throughput capacities, UHPSFC-MS, thus, offers an optimal solution for the analysis of steroid hormones for research, medical chemistry, and clinical diagnostics.<\/jats:p>","DOI":"10.1007\/s00702-024-02862-3","type":"journal-article","created":{"date-parts":[[2024,11,15]],"date-time":"2024-11-15T09:42:05Z","timestamp":1731663725000},"page":"1815-1823","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["An orthogonal approach for analysis of underivatized steroid hormones using ultrahigh performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS)"],"prefix":"10.1007","volume":"132","author":[{"given":"Perry","family":"Devo","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Victoria","family":"Cretu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Harsha","family":"Radhakrishnan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Darren","family":"Hamilton-Pink","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Stergios","family":"Boussios","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9522-4159","authenticated-orcid":false,"given":"Saak V.","family":"Ovsepian","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2024,11,15]]},"reference":[{"issue":"2","key":"2862_CR1","doi-asserted-by":"publisher","first-page":"645","DOI":"10.1042\/BST20150234","volume":"44","author":"R Andrew","year":"2016","unstructured":"Andrew R, Homer NZ (2016) Mass spectrometry and its evolving role in assessing tissue specific steroid metabolism. 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