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Herein, a LC-MS\/MS method was developed and validated for accurate quantification of milk allergens in foods using a novel dual-target approach. This method simultaneously quantifies whey and casein fractions using high-purity protein standards of \u03b2-lactoglobulin and \u03b2-casein through marker peptides LIVTQTMK and VLPVPQK, respectively, coupled with stable isotope-labeled peptide internal standards containing flanking sequences. The dual-target strategy overcomes limitations of conventional single-target methods by providing accurate total milk protein determination regardless of processing-induced alterations in protein ratios. Method validation demonstrated excellent performance with detection limits of 0.1 mg\/kg for \u03b2-lactoglobulin and \u03b2-casein, recovery rates of 80.6%\u201392.4%, and precision below 12.7% RSD. This dual-target approach provides a robust analytical tool for accurate milk allergens determination in processed foods, significantly advancing allergen control and regulatory compliance.<\/jats:p>","DOI":"10.1021\/acs.jafc.6c08928","type":"journal-article","created":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T13:01:44Z","timestamp":1789477304000},"page":"30358-30371","source":"Crossref","is-referenced-by-count":0,"title":["LC-MS\/MS Quantification of Total Milk Protein Allergens:\nA Dual-Target Approach Using \u03b2-Lactoglobulin\/\u03b2-Casein\nStandards and Stable Isotope-Labeled Peptide Calibration"],"prefix":"10.1021","volume":"74","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7879-403X","authenticated-orcid":true,"given":"Shupeng","family":"Yang","sequence":"first","affiliation":[{"name":"Chinese Academy of Agricultural Sciences , , ,","place":["Beijing, People\u2019s Republic of China, 100193"]},{"name":"Agro-product & Food Inspection and Testing Center (Beijing), 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100193"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li","family":"Siyi","sequence":"additional","affiliation":[{"name":"Chinese Academy of Agricultural Sciences , , ,","place":["Beijing, People\u2019s Republic of China, 100193"]},{"name":"Agro-product & Food Inspection and Testing Center (Beijing), Ministry of Agriculture and Rural Affairs , ,","place":["Beijing, People\u2019s Republic of China, 100193"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingnan","family":"Shencheng","sequence":"additional","affiliation":[{"name":"Chinese Academy of Agricultural Sciences , , ,","place":["Beijing, People\u2019s Republic of China, 100193"]},{"name":"Agro-product & Food Inspection and Testing Center (Beijing), Ministry of Agriculture and Rural Affairs , ,","place":["Beijing, People\u2019s Republic of China, 100193"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoxue","family":"Jia","sequence":"additional","affiliation":[{"name":"Chinese Academy of Agricultural Sciences , , ,","place":["Beijing, People\u2019s Republic of China, 100193"]},{"name":"Agro-product & Food Inspection and Testing Center (Beijing), Ministry of Agriculture and Rural Affairs , ,","place":["Beijing, People\u2019s Republic of China, 100193"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-3533-9974","authenticated-orcid":true,"given":"Shuyan","family":"Liu","sequence":"additional","affiliation":[{"name":"Chinese Academy of Agricultural Sciences , , ,","place":["Beijing, People\u2019s Republic of China, 100193"]},{"name":"Agro-product & Food Inspection and Testing Center (Beijing), Ministry of Agriculture and Rural Affairs , ,","place":["Beijing, People\u2019s Republic of China, 100193"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanan","family":"Gao","sequence":"additional","affiliation":[{"name":"Chinese Academy of Agricultural Sciences , , ,","place":["Beijing, People\u2019s Republic of China, 100193"]},{"name":"Agro-product & Food Inspection and Testing Center (Beijing), Ministry of Agriculture and Rural Affairs , ,","place":["Beijing, People\u2019s Republic of China, 100193"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min","family":"Gao","sequence":"additional","affiliation":[{"name":"Chinese Academy of Agricultural Sciences , , ,","place":["Beijing, People\u2019s Republic of China, 100193"]},{"name":"Agro-product & Food Inspection and Testing Center (Beijing), Ministry of Agriculture and Rural Affairs , ,","place":["Beijing, People\u2019s Republic of China, 100193"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Li","sequence":"additional","affiliation":[{"name":"Chinese Academy of Agricultural Sciences , , ,","place":["Beijing, People\u2019s Republic of China, 100193"]},{"name":"Agro-product & Food Inspection and Testing Center (Beijing), Ministry of Agriculture and Rural Affairs , ,","place":["Beijing, People\u2019s Republic of China, 100193"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,15]]},"reference":[{"key":"2026093009081731900_cit1","doi-asserted-by":"crossref","first-page":"100687","DOI":"10.1016\/j.waojou.2022.100687","article-title":"Managing food\nallergy: GA(2)LEN guideline 2022","volume":"15","author":"Muraro","year":"2022","journal-title":"World Allergy\nOrgan. 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Food Allergen Detect.\nControl"},{"key":"2026093009081731900_cit27","doi-asserted-by":"publisher","first-page":"2088","DOI":"10.1111\/all.16080","article-title":"Quality control\nof allergen products with mass spectrometry\npart I: Positioning within the EU regulatory framework","volume":"79","author":"Spiric","year":"2024","journal-title":"Allergy"},{"key":"2026093009081731900_cit28","doi-asserted-by":"publisher","first-page":"138170","DOI":"10.1016\/j.foodchem.2023.138170","article-title":"A SILAC-based accurate\nquantification of shrimp allergen tropomyosin in complex food matrices\nusing UPLC-MS\/MS","volume":"439","author":"Wu","year":"2024","journal-title":"Food Chem."},{"key":"2026093009081731900_cit29","doi-asserted-by":"publisher","first-page":"150775","DOI":"10.1016\/j.foodchem.2026.150775","article-title":"Addressing\nfruit allergen detection gaps: A validated LC-MS\/MS method for eight\nallergenic fruits in foods","volume":"526","author":"Shencheng","year":"2026","journal-title":"Food Chem."},{"key":"2026093009081731900_cit30","doi-asserted-by":"publisher","first-page":"110444","DOI":"10.1016\/j.foodcont.2024.110444","article-title":"Development and\nvalidation of a targeted mass spectrometry\nmethod for the quantification of milk protein allergens in multiple\ncomplex food matrices using matrix-independent calibration","volume":"162","author":"Ramachandran","year":"2024","journal-title":"Food Control"},{"key":"2026093009081731900_cit31","doi-asserted-by":"publisher","first-page":"128533","DOI":"10.1016\/j.foodchem.2020.128533","article-title":"Discovery\nbased high resolution MS\/MS analysis for selection of allergen markers\nin chocolate and broth powder matrices","volume":"343","author":"Pilolli","year":"2021","journal-title":"Food\nChem."},{"key":"2026093009081731900_cit32","unstructured":"CLSI-C64 Quantitative Measurement of Proteins and\nPeptides by Mass\nSpectrometry (1st Edition). 2021. https:\/\/clsi.org\/standards\/products\/clinical-chemistry-and-toxicology\/documents\/c64\/. 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AOAC Int."},{"key":"2026093009081731900_cit36","doi-asserted-by":"publisher","first-page":"728","DOI":"10.1016\/j.fshw.2022.09.006","article-title":"Determination\nof egg and milk allergen in food products\nby liquid chromatography-tandem mass spectrometry based on signature\npeptides and isotope-labeled internal standard","volume":"12","author":"Fan","year":"2023","journal-title":"Food Sci. Hum. Well."},{"key":"2026093009081731900_cit37","doi-asserted-by":"publisher","first-page":"132","DOI":"10.5740\/jaoacint.17-0403","article-title":"Simultaneous analysis\nof multiple allergens in food products by LC-MS\/MS","volume":"101","author":"New","year":"2018","journal-title":"J. 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AOAC Int."},{"key":"2026093009081731900_cit39","doi-asserted-by":"publisher","first-page":"79","DOI":"10.1016\/j.foodcont.2016.11.030","article-title":"Development of a\nliquid chromatography-tandem mass spectrometry method for simultaneous\ndetection of the main milk allergens","volume":"74","author":"Ji","year":"2017","journal-title":"Food Control"},{"key":"2026093009081731900_cit40","doi-asserted-by":"publisher","first-page":"136337","DOI":"10.1016\/j.foodchem.2023.136337","article-title":"Detection of beta-lactoglobulin\nunder different thermal-processing conditions by immunoassay based\non nanobody and monoclonal antibody","volume":"424","author":"Li","year":"2023","journal-title":"Food Chem."},{"key":"2026093009081731900_cit41","doi-asserted-by":"publisher","first-page":"6625","DOI":"10.1021\/acs.jafc.3c08362","article-title":"Advancing\nhigh-throughput MS-based protein quantification: A case study on quantifying\n10 major food allergens by LC-MS\/MS using a one-sample multipoint\nexternal calibration curve","volume":"72","author":"Yang","year":"2024","journal-title":"J. Agr. Food Chem."},{"key":"2026093009081731900_cit42","doi-asserted-by":"publisher","first-page":"5755","DOI":"10.1007\/s00216-021-03550-x","article-title":"Comparison\nof allergen\nquantification strategies for egg, milk, and peanut in food using\ntargeted LC-MS\/MS","volume":"413","author":"Xiong","year":"2021","journal-title":"Anal. Bioanal. Chem."},{"key":"2026093009081731900_cit43","doi-asserted-by":"publisher","first-page":"133126","DOI":"10.1016\/j.foodchem.2022.133126","article-title":"Comprehensive\nquantitation\nof multi-signature peptides originating from casein for the discrimination\nof milk from eight different animal species using LC-HRMS with stable\nisotope labeled peptides","volume":"390","author":"Zhang","year":"2022","journal-title":"Food Chem."},{"key":"2026093009081731900_cit44","doi-asserted-by":"publisher","first-page":"139768","DOI":"10.1016\/j.foodchem.2024.139768","article-title":"How processing affects marker peptide\nquantification - A comprehensive estimation on bovine material relevant\nfor food and feed control","volume":"454","author":"Stobernack","year":"2024","journal-title":"Food Chem."},{"key":"2026093009081731900_cit45","doi-asserted-by":"publisher","first-page":"1097","DOI":"10.1021\/ac503697j","article-title":"Natural flanking\nsequences for peptides included in a quantification concatamer internal\nstandard","volume":"87","author":"Cheung","year":"2015","journal-title":"Anal. Chem."}],"container-title":["Journal of Agricultural and Food Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jafc.6c08928","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jafcau\/article-pdf\/74\/38\/30358\/68510039\/acs.jafc.6c08928.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jafcau\/article-pdf\/74\/38\/30358\/68510039\/acs.jafc.6c08928.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T13:31:01Z","timestamp":1790775061000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jafcau\/article\/74\/38\/30358\/5429921\/LC-MS-MS-Quantification-of-Total-Milk-Protein"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,15]]},"references-count":45,"journal-issue":{"issue":"38","published-online":{"date-parts":[[2026,9,15]]},"published-print":{"date-parts":[[2026,9,30]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jafc.6c08928","relation":{},"ISSN":["0021-8561","1520-5118"],"issn-type":[{"value":"0021-8561","type":"print"},{"value":"1520-5118","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,15]]}}}