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The serum level of 25-hydroxyvitamin D [25(OH)D] is the primary biomarker for assessing vitamin D status, and its precise quantification is critical for clinical diagnosis and treatment. Chemiluminescence immunoassay (CLIA) and liquid chromatography\u2013tandem mass spectrometry (LC-MS\/MS) are widely used analytical methods; however, methodological discrepancies often lead to inconsistent results that may be further influenced by demographic factors such as age and gender. In this study, we evaluated the consistency and correlation between CLIA and LC-MS\/MS measurements using a Generative Fuzzy Inference System (GENFIS). Analysis of 138 serum samples showed that LC-MS\/MS produced significantly higher 25(OH)D concentrations than CLIA (\n                    <jats:italic>p<\/jats:italic>\n                    \u2009&lt;\u20090.01; mean difference\u2009=\u20091.33\u2009\u00b1\u20093.71; 95% CI: \u2212\u20095.95 to 8.61), though the two methods exhibited strong linear correlation and agreement (Cohen\u2019s Kappa\u2009=\u20090.8257; R\u00b2 = 0.9075; intraclass correlation coefficient\u2009=\u20090.93). GENFIS analysis indicated a possible 30\u201340-year female pattern of larger between the detection differences of two methods. To verify this finding, an additional 59 samples were analyzed, revealing a relative risk (RR) of 3.18 (95% CI: 1.71\u20135.93;\n                    <jats:italic>p<\/jats:italic>\n                    \u2009&lt;\u20090.05) for this subgroup compared with other populations, supporting the GENFIS inference. These results highlight age- and gender-related differences in 25(OH)D measurement between CLIA and LC-MS\/MS, providing valuable insights for improving the standardization and clinical interpretation of vitamin D testing.\n                  <\/jats:p>","DOI":"10.1038\/s41598-026-41793-9","type":"journal-article","created":{"date-parts":[[2026,3,3]],"date-time":"2026-03-03T15:44:42Z","timestamp":1772552682000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Analysis of chemiluminescence and liquid chromatography-mass spectrometry in 25-hydroxyvitamin D detection using fuzzy logic"],"prefix":"10.1038","volume":"16","author":[{"given":"Hongkun","family":"Liu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sirui","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kok Wai","family":"Wong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yujie","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinyi","family":"He","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuhao","family":"Liang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hui","family":"Deng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Linlin","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianli","family":"Cui","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,3,3]]},"reference":[{"key":"41793_CR1","doi-asserted-by":"publisher","first-page":"928","DOI":"10.1017\/S0007114518000491","volume":"119","author":"X Liu","year":"2018","unstructured":"Liu, X., Baylin, A. & Levy, P. D. Vitamin D deficiency and insufficiency among US adults: Prevalence, predictors and clinical implications. Br. J. Nutr. 119, 928\u2013936 (2018).","journal-title":"Br. J. Nutr."},{"key":"41793_CR2","doi-asserted-by":"publisher","first-page":"219","DOI":"10.1093\/labmed\/lmaa066","volume":"52","author":"E Ertilav","year":"2021","unstructured":"Ertilav, E., Barcin, N. E. & Ozdem, S. Comparison of serum free and bioavailable 25-hydroxyvitamin D levels in Alzheimer\u2019s disease and healthy control patients. Lab. Med. 52, 219\u2013225 (2021).","journal-title":"Lab. Med."},{"key":"41793_CR3","doi-asserted-by":"publisher","first-page":"e180","DOI":"10.1542\/peds.2010-0442","volume":"127","author":"CA Camargo Jr","year":"2011","unstructured":"Camargo, C. A. Jr et al. Cord-blood 25-hydroxyvitamin D levels and risk of respiratory infection, wheezing, and asthma. 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This research was conducted as a retrospective study using laboratory testing data from Sichuan Taikang Hospital. The Ethics Committee granted a waiver of informed consent in accordance with the Declaration of Helsinki and relevant national guidelines, as the study involved the analysis of anonymized data and posed no risk to participants. All methods were performed in accordance with the relevant guidelines and regulations.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethic statement"}}],"article-number":"11886"}}