{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T04:06:43Z","timestamp":1790827603693,"version":"4.1.0"},"reference-count":36,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2025,4,15]],"date-time":"2025-04-15T00:00:00Z","timestamp":1744675200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Italian Ministry of Health (MOH)","award":["IZSLT 01\/21 RC \u201cSTRATEGICA\u201d"],"award-info":[{"award-number":["IZSLT 01\/21 RC \u201cSTRATEGICA\u201d"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>The presence of mycotoxins in cheese is a significant concern due to their potential health risks. Mycotoxins can contaminate cheese through two main routes: indirectly via contaminated animal feed, and\/or directly, because of mold growth on dairy products. It has been reported that cheese may contain metabolites of aflatoxin B1 such as aflatoxin M1 (AFM1), aflatoxicol (AFL), and, its precursor, sterigmatocystin (STC). This study presents a reliable method for the simultaneous determination of AFM1, AFL, and STC in cheeses made from ovine, goat, or buffalo milk. The method was developed using single liquid extraction, clean-up by an immunoaffinity column (IAC), and liquid chromatography-tandem mass spectrometry (LC-MS\/MS) determination. The method was subjected to initial validation according to EU regulations, which outline the required performance parameters and criteria of analytical methods for official food control. The limits of quantification (LOQs) of the method for AFM1, AFL, and STC are 2.0 ng\/kg, 5.0 ng\/kg, and 1.0 ng\/kg, respectively. The method was applied in a study for the assessment of mycotoxin transfer from milk to cheeses and also their growth.<\/jats:p>","DOI":"10.3390\/molecules30081774","type":"journal-article","created":{"date-parts":[[2025,4,15]],"date-time":"2025-04-15T09:41:11Z","timestamp":1744710071000},"page":"1774","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Emerging Mycotoxins in Cheese: Simultaneous Analysis of Aflatoxin M1, Aflatoxicol, and Sterigmatocystin by LC-MS\/MS"],"prefix":"10.3390","volume":"30","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9564-1112","authenticated-orcid":false,"given":"Maurizio","family":"Cossu","sequence":"first","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale della Sardegna \u201cG.Pegreffi\u201d, Via Vienna, n.2, 07100 Sassari, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3024-4527","authenticated-orcid":false,"given":"Andrea","family":"Sanna","sequence":"additional","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale della Sardegna \u201cG.Pegreffi\u201d, Via Vienna, n.2, 07100 Sassari, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giuseppe","family":"Mangano","sequence":"additional","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale della Sardegna \u201cG.Pegreffi\u201d, Via Vienna, n.2, 07100 Sassari, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giuseppe","family":"Ledda","sequence":"additional","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale della Sardegna \u201cG.Pegreffi\u201d, Via Vienna, n.2, 07100 Sassari, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giannina","family":"Chessa","sequence":"additional","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale della Sardegna \u201cG.Pegreffi\u201d, Via Vienna, n.2, 07100 Sassari, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9676-2290","authenticated-orcid":false,"given":"Pasquale","family":"Gallo","sequence":"additional","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via Salute, n.2, 80055 Portici, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Antonio","family":"Vella","sequence":"additional","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale della Sicilia, Via Gino Marinuzzi, 3, 90129 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2889-1071","authenticated-orcid":false,"given":"Ivan","family":"Pecorelli","sequence":"additional","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale dell\u2019Umbria e delle Marche, Via G. Salvemini, 1, 06126 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9214-8082","authenticated-orcid":false,"given":"Stefano","family":"Sdogati","sequence":"additional","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale dell\u2019Umbria e delle Marche, Via G. Salvemini, 1, 06126 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marilena","family":"Gili","sequence":"additional","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d\u2019Aosta, Via Bologna, 148, 10154 Torino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3990-1063","authenticated-orcid":false,"given":"Carlo","family":"Boselli","sequence":"additional","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale del Lazio E Della Toscana \u201cM. Aleandri\u201d\u2014National Reference Centre for Ovine and Caprine Milk and Dairy Products Quality (C.Re.L.D.O.C.), 00178 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,4,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"291","DOI":"10.3920\/WMJ2008.x041","article-title":"Mycotoxins and Mould Contamination in Cheese: A Review","volume":"1","author":"Sengun","year":"2008","journal-title":"World Mycotoxin J."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"437","DOI":"10.1111\/1541-4337.12069","article-title":"Filamentous fungi and mycotoxins in cheese: A review","volume":"13","author":"Hymery","year":"2014","journal-title":"Compr. Rev. Food Sci. Food Saf."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/S0168-1605(98)00112-3","article-title":"Mycotoxin production by Aspergillus, Fusarium and Penicillium species","volume":"43","author":"Sweeney","year":"1998","journal-title":"Int. J. Food Microbiol."},{"key":"ref_4","first-page":"99","article-title":"Contamination of dairy products by fungal metabolites","volume":"10","author":"Lafont","year":"1990","journal-title":"J. Environ. Pathol. Toxicol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1077","DOI":"10.1080\/02652030310001594478","article-title":"Aflatoxin B1 and its interconverting metabolite aflatoxicol in milk: The situation in Mexico","volume":"20","author":"Carvajal","year":"2003","journal-title":"Food Addit. Contam."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/S0378-4274(01)00479-9","article-title":"Update of survey, regulation and toxic effects of mycotoxins in Europe","volume":"127","author":"Creppy","year":"2002","journal-title":"Toxicol. Lett."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Sdogati, S., Pacini, T., Bibi, R., Caporali, A., Verdini, E., Orsini, S., Ortenzi, R., and Pecorelli, I. (2024). Co-Occurrence of Aflatoxin B1, Zearalenone and Ochratoxin A in Feed and Feed Materials in Central Italy from 2018 to 2022. Foods, 13.","DOI":"10.3390\/foods13020313"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.foodcont.2012.05.058","article-title":"Aflatoxin M1 in milk and distribution and stability of aflatoxin M1 during production and storage of yoghurt and cheese","volume":"29","author":"Iha","year":"2013","journal-title":"Food Control"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1080\/15287397709529459","article-title":"Production of aflatoxicol from aflatoxin B1 by postmitochondrial liver fractions","volume":"2","author":"Salhab","year":"1977","journal-title":"J. Toxicol. Environ. Health"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1016\/j.toxlet.2018.02.007","article-title":"Genotoxicity of aflatoxins and their precursors in human cells","volume":"287","author":"Theumer","year":"2018","journal-title":"Toxicol. Lett."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1016\/j.fct.2018.11.046","article-title":"Presence of unreported carcinogens, aflatoxins and their hydroxylated metabolites, in industrialized Oaxaca cheese from Mexico City","volume":"124","year":"2019","journal-title":"Food Chem. Toxicol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"460","DOI":"10.1016\/j.fct.2018.05.057","article-title":"Sterigmatocystin: A Mycotoxin to Be Seriously Considered","volume":"118","author":"Nieto","year":"2018","journal-title":"Food Chem. Toxicol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Pietri, A., Leni, G., Mulazzi, A., and Bertuzzi, T. (2022). Ochratoxin A and sterigmatocystin in long-ripened Grana cheese: Occurrence, wheel rind contamination and effectiveness of cleaning techniques on grated products. Toxins, 14.","DOI":"10.3390\/toxins14050306"},{"key":"ref_14","unstructured":"Italian Ministry of Health (2025, February 24). Micotossine non Regolamentate: Metaboliti Dell\u2019aflatossina B1 (Aflatossina M1 e Aflatossicolo) e Sterigmatocistina in Prodotti Lattiero-Caseari, Available online: https:\/\/www.salute.gov.it\/imgs\/C_17_pubblicazioni_3065_allegato.pdf."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1513","DOI":"10.1093\/jaoac\/94.5.1513","article-title":"Rosa Maria Duarte Favaro Chromatographic Method for the Determination of Aflatoxin M1 in Cheese, Yogurt, and Dairy Beverages","volume":"94","author":"Iha","year":"2011","journal-title":"J. AOAC Int."},{"key":"ref_16","unstructured":"Hird, S., Martin, K., Collette Zabe, N., and Toth, D. (2021). Determination of Aflatoxins in a Wide Range of Food and Agricultural Commodities Using Immunoaffinity Chromatography Column Clean-Up Coupled with UPLC or HPLC with Fluorescence Detection, VICAM. Application Notes Waters Corporation."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/j.foodchem.2015.07.006","article-title":"Enzyme-assisted extraction for the HPLC determination of aflatoxin M1 in cheese","volume":"192","author":"Pietri","year":"2016","journal-title":"Food Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1016\/j.aca.2015.10.013","article-title":"Analytical methods for determination of mycotoxins: An update (2009\u20132014)","volume":"901","author":"Turner","year":"2015","journal-title":"Anal. Chim. Acta"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1080\/02652030802342497","article-title":"Determination of sterigmatocystin in cheese by high-performance liquid chromatography-tandem mass spectrometry","volume":"26","year":"2009","journal-title":"Food Addit. Contam. A"},{"key":"ref_20","first-page":"766","article-title":"Application of molecularly imprinted polymers in food analysis: Clean-up and chromatographic improvements","volume":"10","author":"Regal","year":"2012","journal-title":"Cent. Eur. J. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"606","DOI":"10.1016\/j.bios.2017.01.018","article-title":"Molecularly imprinted polymers for sample preparation and biosensing in food analysis: Progress and perspectives","volume":"91","author":"Ashley","year":"2017","journal-title":"Biosens. Bioelectron."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Vaz, A., Cabral Silva, A.C., Rodrigues, P., and Ven\u00e2ncio, A. (2020). Detection Methods for Aflatoxin M1 in Dairy Products. Microorganisms, 8.","DOI":"10.3390\/microorganisms8020246"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"129647","DOI":"10.1016\/j.foodchem.2021.129647","article-title":"Multianalyte Method for the Determination of Regulated, Emerging and Modified Mycotoxins in Milk: QuEChERS Extraction Followed by UHPLC\u2014MS\/MS Analysis Es Rodr\u00edguez-Ca N","volume":"356","author":"Alfonso","year":"2021","journal-title":"Food Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"719","DOI":"10.1093\/jaoacint\/qsaa164","article-title":"Determination of Aflatoxin M1 in Liquid Milk, Cheese, and Selected Milk Proteins by Automated Online Immunoaffinity Cleanup with Liquid Chromatography\u2014Fluorescence Detection","volume":"104","author":"Wood","year":"2021","journal-title":"J. AOAC Int."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1273","DOI":"10.5740\/jaoacint.16-0098","article-title":"Determination of Sterigmatocystin in Infant Cereals from Hangzhou, China","volume":"99","author":"Liu","year":"2016","journal-title":"J. AOAC Int."},{"key":"ref_26","unstructured":"McSweeney, P.L.H., Fox, P.F., Cotter, P., and Everett, D.W. (2017). Mycotoxins in cheese. Cheese: Chemistry, Physics and Microbiology, Elsevier. [4th ed.]."},{"key":"ref_27","unstructured":"European Commission (2025, January 17). Regulation (EC) 2023\/915 of 25 April 2023. Official Journal of the European Union L 119\/103 2023, Available online: https:\/\/eur-lex.europa.eu\/eli\/reg\/2023\/915\/oj."},{"key":"ref_28","unstructured":"European Commission (2025, January 15). Regulation No. 2023\/2782 of 14 December 2023 Laying Down the Methods of Sampling and Analysis for the Control of the Levels of Mycotoxins in Food and Repealing EC Regulation No. 401\/2006. Official Journal of the European Union, Available online: https:\/\/data.europa.eu\/eli\/reg_impl\/2023\/2782\/oj."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"135182","DOI":"10.1016\/j.foodchem.2022.135182","article-title":"Detection of mycotoxins in cheese using an optimized analytical method based on a QuEChERS extraction and UHPLC-MS\/MS quantification","volume":"408","author":"Alfonso","year":"2023","journal-title":"Food Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"681","DOI":"10.1111\/1471-0307.12784","article-title":"Optimisation, validation and comparison of methods for aflatoxin M1 determination in cheese","volume":"74","author":"Popov","year":"2021","journal-title":"Int. J. Dairy Technol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"107676","DOI":"10.1016\/j.foodcont.2020.107676","article-title":"Natural contaminants in infant food: The case of regulated and emerging mycotoxins","volume":"123","author":"Braun","year":"2021","journal-title":"Food Control"},{"key":"ref_32","unstructured":"Akinbami, L.J., Chen, T.-C., Davy, O., Ogden, C.L., Fink, S., Clark, J., Riddles, M.K., and Mohadjer, L.K. (2025, February 24). National Health and Nutrition Examination Survey, 2017\u2013March 2020 Prepandemic File: Sample Design, Estimation, and Analytic Guidelines. 2(190), Available online: https:\/\/stacks.cdc.gov\/view\/cdc\/115434."},{"key":"ref_33","unstructured":"CLAL (2025, February 24). Per Capita Cheese Consumption\u2014Year 2023. Available online: https:\/\/www.clal.it\/en\/?section=tabs_consumi_procapite#C."},{"key":"ref_34","unstructured":"European Commission (2025, April 07). Regulation (EU) 2022\/2379 of the European Parliament and of the Council of 23 November 2022 on Statistics on Agricultural Input and Output, Amending Commission Regulation (EC) No 617\/2008 and Repealing Regulations (EC) No 1165\/2008, (EC) No 543\/2009 and (EC) No 1185\/2009 of the European Parliament and of the Council and Council Directive 96\/16\/EC. Available online: https:\/\/eur-lex.europa.eu\/eli\/reg\/2022\/2379\/oj\/eng."},{"key":"ref_35","unstructured":"European Commission (2025, January 15). Regulation (EU) 2017\/625 of the European Parliament and of the Council of 15 March 2017 on Official Controls and Other Official Activities Performed to Ensure the Application of Food and Feed Law, Rules on Animal Health and Welfare, Plant Health and Plant Protection Products, Amending Regulations (EC) No 999\/2001, (EC) No 396\/2005, (EC) No 1069\/2009, (EC) No 1107\/2009, (EU) No 1151\/2012, (EU) No 652\/2014, (EU) 2016\/429 and (EU) 2016\/2031 of the European Parliament and of the Council, Council Regulations (EC) No 1\/2005 and (EC) No 1099\/2009 and Council Directives 98\/58\/EC, 1999\/74\/EC, 2007\/43\/EC, 2008\/119\/EC and 2008\/120\/EC, and Repealing Regulations (EC) No 854\/2004 and (EC) No 882\/2004 of the European Parliament and of the Council, Council Directives 89\/608\/EEC, 89\/662\/EEC, 90\/425\/EEC, 91\/496\/EEC, 96\/23\/EC, 96\/93\/EC and 97\/78\/EC and Council Decision 92\/438\/EEC (Official Controls Regulation). Official Journal of the European Union, Available online: https:\/\/eur-lex.europa.eu\/legal-content\/PL\/TXT\/?uri=CELEX%3A32017R0625."},{"key":"ref_36","unstructured":"European Commission (2025, February 24). Directorate-General for Health and Food Safety (DG SANTE). Guidance Document SANTE 11312\/2021 v2\u2014Analytical Quality Control and Method Validation Procedures for Pesticide Residues Analysis in Food and Feed. Version 2 Implemented by 01\/01\/2024. Available online: https:\/\/food.ec.europa.eu\/document\/download\/d4786faf-c574-4222-a5c6-45086b3920b8_en?filename=pesticides_mrl_guidelines_wrkdoc_2021-11312.pdf."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/30\/8\/1774\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:15:03Z","timestamp":1760030103000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/30\/8\/1774"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4,15]]},"references-count":36,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2025,4]]}},"alternative-id":["molecules30081774"],"URL":"https:\/\/doi.org\/10.3390\/molecules30081774","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,4,15]]}}}