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They also play a crucial role in cellular oxidative stress responses, following cell membrane depolarization, making accurate quantification of FFAs essential. This study presents a novel supercritical fluid chromatography-mass spectrometry (SFC-MS) method using selected ion recording in negative electrospray ionization mode, enabling rapid quantification of 31 FFAs within 6\u00a0min without derivatization. FFAs are identified and quantified using an HSS C18 SB column and a secondary mobile phase consisting of methanol with formic acid by detecting their [M\u2009\u2212\u2009H]<jats:sup>\u2212<\/jats:sup> ions. Calibration curves showed strong linearity (<jats:italic>R<\/jats:italic>\n            <jats:sup>2<\/jats:sup>\u2009\u2265\u20090.9910), spanning 1000\u201312,000\u00a0ng\/mL for short-chain FFAs and 50\u20131200\u00a0ng\/mL for medium- and long-chain FFAs. The method achieves detection limits as low as 1\u00a0ng\/\u00b5L for short-chain FFAs and 0.05\u00a0pg\/\u00b5L for other FFAs per on-column injection. The method demonstrated high accuracy and precision, with bias and coefficients of variation maintained below 15% across five quality control levels. Freeze\u2013thaw and autosampler stability studies confirmed the behavior of matrix-matched standards under optimal storage conditions. The validated method was applied to the analysis of pharmaceutical-grade egg yolk powders, using 13 deuterated FFAs as internal standards (IS) in comparison with heptadecanoic acid (C17:0). Significant variations in FFA quantification using two different IS approaches underscore the importance of selecting an appropriate IS. In summary, this study introduces a reliable and validated SFC-MS method for analyzing FFAs ranging from C4 to C26, requiring minimal sample preparation.<\/jats:p>\n          <jats:p>\n            <jats:bold>Graphical Abstract<\/jats:bold>\n          <\/jats:p>","DOI":"10.1007\/s00216-025-05732-3","type":"journal-article","created":{"date-parts":[[2025,1,23]],"date-time":"2025-01-23T18:16:39Z","timestamp":1737656199000},"page":"1461-1478","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Comprehensive quantification of C4 to C26 free fatty acids using a supercritical fluid chromatography-mass spectrometry method in pharmaceutical-grade egg yolk powders intended for total parenteral nutrition use"],"prefix":"10.1007","volume":"417","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9682-6840","authenticated-orcid":false,"given":"Mark Dennis Chico","family":"Retrato","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anh Vu","family":"Nguyen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5722-4908","authenticated-orcid":false,"given":"S. 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