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Consequently, human exposure occurs through multiple pathways, including dietary intake and environmental contact. Previous analytical methods have primarily focused on parent compounds, often excluding key metabolites due to matrix effects and poor quantification in complex biological samples. This gap severely limits the ability to accurately assess total NEO exposure and internal dose, highlighting the urgent need for more comprehensive and reliable biomonitoring approaches. This study developed a comprehensive LC\u2013MS\/MS method to quantify 21 NEOs and metabolites in human urine, uniquely including key transformation products such as clothianidin-n-desmethyl, clothianidin-urea, imidacloprid-urea, imidacloprid-olefin, 5-hydroxy-imidacloprid, thiamethoxam-urea, and thiamethoxam-n-desmethyl, thereby enabling a more thorough assessment of human exposure to NEOs. The method was validated using both synthetic and pooled human urine, providing a critical assessment of matrix-dependent variations in detection limits, accuracy, and precision. Method performance demonstrated high sensitivity, specificity, accuracy, and precision, with limits of quantification (LOQ) from 0.017 to 0.46\u00a0ng\/mL and limits of detection (LOD) from 0.0050 to 0.14\u00a0ng\/mL. The method was applied to urine samples from 246 pregnant women and 47 farmers in eastern Iowa to assess exposure levels. Results indicated widespread NEO exposure with detection frequencies of 99% of pregnant women and 100% in farmers, with 18 different NEOs and metabolites detected across both populations. Metabolite monitoring revealed that imidacloprid metabolites (IMI-O, 5-OH-IMI) were stronger predictors of total neonicotinoid burden than parent compounds alone (\n                    <jats:italic>r<\/jats:italic>\n                    <jats:sub>s<\/jats:sub>\n                    \u2009=\u20090.735 vs\u2009&lt;\u20090.6 for parents), with some metabolites showing higher concentrations in pregnant women than farmers despite farmers having higher overall exposure. These findings demonstrate that parent-only biomonitoring substantially underestimates internal neonicotinoid exposure and fails to capture critical exposure pathways, particularly for vulnerable populations where metabolite patterns may differ significantly from occupational exposure profiles.\n                  <\/jats:p>","DOI":"10.1007\/s11356-026-37779-9","type":"journal-article","created":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T04:28:13Z","timestamp":1777609693000},"page":"7576-7593","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Development and validation of matrix-validated LC\u2013MS\/MS method for simultaneous quantification of 21 neonicotinoids and their metabolites in human urine"],"prefix":"10.1007","volume":"33","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1284-2056","authenticated-orcid":false,"given":"Wenjing","family":"Xi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah","family":"Onysio","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mary C.","family":"Rhodes","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexis J.","family":"Faudel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John C.","family":"Flunker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark K.","family":"Santillan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Donna A.","family":"Santillan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David M.","family":"Cwiertny","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Darrin A.","family":"Thompson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,5,1]]},"reference":[{"key":"37779_CR1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijheh.2021.113797","volume":"236","author":"A Anai","year":"2021","unstructured":"Anai A, Hisada A, Yunohara T, Iwasaki M, Arizono K, Katoh T (2021) Urinary neonicotinoids level among pregnant women in Japan. 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