Abstract
The objective was the development and validation of a method for simultaneous mycotoxins determination (aflatoxins B1 — AFB1, G1 — AFG1, G2 — AFG2 and M1 — AFM1, ochratoxin — OTA, and zearalenone — ZEA) in milk by high-performance liquid chromatography with fluorescence detector. A mixture planning was used to define the mobile phase and a fractional factorial design to define a flow rate of 1.4 mL min−1, allowing the subsequent application of a rotational central composite design (CCRD). Through the CCRD, it was possible to obtain the desired resolution of 1.86 and 2.95 between the peaks of AFM1-AFG2 and ZEA-OTA, respectively, using acidification of the mobile phase 0.85%, column temperature 38 °C, and injection volume of 40 µL. In parallel, an extraction method based on the QuEChERS method was developed. The limits of quantification were 0.16, 1.08, 0.01, 0.12, 0.33, and 8.93 µg·kg−1 for AFM1, AFB1, AFG1, AFG2, OTA, and ZEA, respectively. Recovery varied between 73 and 114%. The method was applied to evaluate multi-mycotoxins’ occurrence in fluid milk (n = 30). The presence of AFB1, AFG1, AFG2, and AFM1 was detected in 26%, 30%, 93%, and 80% of the samples, respectively; however, the presence of AFM1 was below the maximum limit allowed by Brazilian legislation (0.5 µg·kg−1). OTA and ZEA were below the limit of detection. Thus, the method was capable and efficient in quantifying multi-mycotoxins in fluid milk and can also be applied as a routine method to monitor milk quality.
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Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.
References
Abdallah MF, Girgin G, Baydar T (2019) Mycotoxin detection in maize, commercial feed, and raw dairy milk samples from Assiut City, Egypt. Vet Sci 6. https://doi.org/10.3390/VETSCI6020057
Altafini A, Roncada P, Guerrini A et al (2021) Occurrence of ochratoxin A in different types of cheese offered for sale in Italy. Toxins (Basel) 13:1–15. https://doi.org/10.3390/toxins13080540
Andrade PD, da Silva JLG, Caldas ED (2013) Simultaneous analysis of aflatoxins B1, B2, G1, G2, M1 and ochratoxin A in breast milk by high-performance liquid chromatography/fluorescence after liquid-liquid extraction with low temperature purification (LLE-LTP). J Chromatogr A 1304:61–68. https://doi.org/10.1016/j.chroma.2013.06.049
ANVISA - Agência Nacional de Vigilância Sanitária (2022) In no 160, de 01 de Julho de 2022. Estabelece os limites máximos tolerados (LMT) de contaminantes em alimentos. https://www.in.gov.br/en/web/dou/-/instrucao-normativa-in-n-160-de-1-de-julho-de-2022-413367081
AOAC. Association Of Official Analytical Chemists (2005) Official methods of analysis of aoac international, v 18th ed, chapt 49. Official Methods of Analysis of the AOAC International, Gailhersburg, Gailhersburg
Becker-Algeri TA, Castagnaro D, de Bortoli K et al (2016) Mycotoxins in bovine milk and dairy products: a review. J Food Sci 81:R544–R552. https://doi.org/10.1111/1750-3841.13204
Bhat R, Rai RV, Karim AA (2010) Mycotoxins in food and feed: present status and future concerns. Compr Rev Food Sci Food Saf 9:57–81
Brasil (2017) Resolução da Diretoria Colegiada - RDC n° 166, de 24 de Julho de 2017. Dispõe sobre a validação de métodos analíticos e dá outras providências. Ministério Da Saúde 2017:1–21
Breitkreitz MC, Jardim ICSF, Bruns RE (2009) Combined column-mobile phase mixture statistical design optimization of high-performance liquid chromatographic analysis of multicomponent systems. J Chromatogr A 1216:1439–1449. https://doi.org/10.1016/j.chroma.2008.12.093
Commission E (2006) Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Off J Eur Communitites L364:5–24. https://doi.org/10.2203/dose-response.06-012.Hanekamp
CONAB - Companhia Nacional de Abastecimento (2022) Análise mensal - Leite e Derivados Junho de 2022. CONAB
Derringer G, Suich R (1980) Simultaneous optimization of several response variables. J Qual Technol 12:214–219. https://doi.org/10.1080/00224065.1980.11980968
Dionísio AGG, Batistão MB, Santos VB et al (2010) Novas Tendências em Preparo de Amostras para Cromatografia Líquida. Sci Chromatogr 2:19–29
dos Santos Neto ÁJ (2010) Problemas com o formato dos picos em cromatografia líquida. Sci Chromatogr 2:71–81
Fink-Gremmels J (2008) Mycotoxins in cattle feeds and carry-over to dairy milk: a review. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 25:172–180. https://doi.org/10.1080/02652030701823142
Foerster C, Monsalve L, Gajardo GR (2023) Occurrence of aflatoxin M1 in milk and exposure estimation for its consumption in the Chilean population Title. Food Control 148. https://doi.org/10.1016/j.foodcont.2023.109677
Garcia I, Valente D, Carolino N et al (2023) Occurrence of zearalenone in dairy farms — a study on the determinants of exposure and risk assessment. Toxicon 225:718. https://doi.org/10.1016/j.toxicon.2023.107051
Gonçalves KDM, Sibaja KVM, Feltrin ACP et al (2018) Occurrence of aflatoxins B1 and M1 in milk powder and UHT consumed in the city of Assomada (Cape Verde Islands) and southern Brazil. Food Control 93:260–264. https://doi.org/10.1016/j.foodcont.2018.06.010
González-Jartín JM, Rodríguez-Cañás I, Alfonso A et al (2021) Multianalyte method for the determination of regulated, emerging and modified mycotoxins in milk: QuEChERS extraction followed by UHPLC–MS/MS analysis. Food Chem 356. https://doi.org/10.1016/j.foodchem.2021.129647
Hamed AM, Moreno-González D, García-Campaña AM, Gámiz-Gracia L (2017) Determination of aflatoxins in yogurt by dispersive liquid–liquid microextraction and HPLC with photo-induced fluorescence detection. Food Anal Methods 10:516–521. https://doi.org/10.1007/s12161-016-0611-6
IARC (2012) Agents classified by the IARC monographs, Volumes 1–104. IARC Monogr 7:1–25. https://doi.org/10.1007/s13398-014-0173-7.2
Jia W, Chu X, Ling Y et al (2014) Multi-mycotoxin analysis in dairy products by liquid chromatography coupled to quadrupole orbitrap mass spectrometry. J Chromatogr A 1345:107–114. https://doi.org/10.1016/j.chroma.2014.04.021
Juan C, Berrada H, Mañes J, Oueslati S (2017) Multi-mycotoxin determination in barley and derived products from Tunisia and estimation of their dietary intake. Food Chem Toxicol 103:148–156. https://doi.org/10.1016/j.fct.2017.02.037
Kumar P, Mahato DK, Kamle M et al (2017) Aflatoxins: a global concern for food safety, human health and their management. Front Microbiol 7:1–10. https://doi.org/10.3389/fmicb.2016.02170
Lanças FM (2009) Como economizar (ou eliminar o uso de) acetonitrila em tempos de “crise”? Sci Chromatogr 1:51–60
MarimónSibaja KV, Gonçalves KDM, Garcia SDO et al (2019) Aflatoxin M1 and B1 in Colombian milk powder and estimated risk exposure. Food Addit Contam Part B Surveill 12:97–104. https://doi.org/10.1080/19393210.2019.1567611
Martins CR, Lopes WA, De Andrade JB (2013) Solubilidade das substâncias orgânicas. Quim Nova 36:1248–1255. https://doi.org/10.1590/S0100-40422013000800026
Meinhart AD, Ballus CA, Bruns RE et al (2011) Chemometrics optimization of carbohydrate separations in six food matrices by micellar electrokinetic chromatography with anionic surfactant. Talanta 85:237–244. https://doi.org/10.1016/j.talanta.2011.03.056
Mogollón NGS, De Lima PF, Gama MR et al (2014) O estado da arte da cromatografia líquida bidimensional: Conceitos fundamentais, instrumentac¸ão e aplicac¸ões. Quim Nova 37:1680–1691. https://doi.org/10.5935/0100-4042.20140261
Murshed S (2020) Evaluation and assessment of aflatoxin M1 in milk and milk products in Yemen using high-performance liquid chromatography. J Food Qual 2020. https://doi.org/10.1155/2020/8839060
Ostry V, Malir F, Toman J, Grosse Y (2017) Mycotoxins as human carcinogens—the IARC monographs classification. Mycotoxin Res 33:65–73. https://doi.org/10.1007/s12550-016-0265-7
Patyal A, Singh Gill JP, Bedi JS, Aulakh RS (2020) Occurrence of aflatoxin M1 in raw, pasteurized and UHT milk from Punjab, India. Curr Sci 118:79–86. https://doi.org/10.18520/cs/v118/i1/79-86
Prestes OD, Adaime MB, Zanella R (2011) QuEChERS: possibilidades e tendências no preparo de amostra para determinação multirresíduo de pesticidas em alimentos. Sci Chromatogr 3:51–64. https://doi.org/10.4322/sc.2011.004
Rodríguez-Cañás I, González-Jartín JM, Alvariño R et al (2023) Detection of mycotoxins in cheese using an optimized analytical method based on a QuEChERS extraction and UHPLC-MS/MS quantification. Food Chem 408. https://doi.org/10.1016/j.foodchem.2022.135182
Rodríguez-Carrasco Y, Izzo L, Gaspari A et al (2018) Simultaneous determination of AFB1 and AFM1 in milk samples by ultra high performance liquid chromatography coupled to quadrupole orbitrap. Beverages 43:1–9. https://doi.org/10.3390/beverages4020043
Sainz MJ, González-Jartín JM, Aguín O, Pedro Mansilla J, Botana LM (2018) Isolation, characterization, and identification of mycotoxin-producing fungi. In: Botana LM (ed) Environmental Toxicology. De Gruyter, Berlin, Boston. https://doi.org/10.1515/9783110442045
SANTE 11813 (2017) Guidance document on analytical quality control and method validation procedures for pesticide residues and analysis in food and feed. https://www.eurlpesticides.eu/userfiles/file/EurlALL/SANTE_11813_2017-fin.pdf
Sarmast E, Fallah AA, Jafari T, MousaviKhaneghah A (2021) Impacts of unit operation of cheese manufacturing on the aflatoxin M1 level: a global systematic review and meta-analysis. LWT 148:111772. https://doi.org/10.1016/j.lwt.2021.111772
Sartori AV, Mattos JS de, Moraes MHP de, Nóbrega AW da (2015) Determination of aflatoxins M1, M2, B1, B2, G1, G2 and ochratoxin A in infant formulas from Brazil using a modified QuEChERS method and UHPLC-MS/MS. Food Anal. Methods
Seid A, Mama A (2019) Aflatoxicosis and occurrence of aflatoxin M1 (AFM1) in milk and dairy products: a review. Austin J Vet Sci Anim Husb 1:1–12
Smith NW, Fletcher AJ, Hill JP, McNabb WC (2022) Modeling the contribution of milk to global nutrition. Front Nutr 8:1–7. https://doi.org/10.3389/fnut.2021.716100
Ushkalov V, Danchuk V, Midyk S et al (2020) Mycotoxins in milk and dairy products. Food Sci Technol 14:137–150. https://doi.org/10.15673/fst.v14i3.1786
von Mühlen C, Lanças FM (2004) Cromatografia Unificada. Quim Nova 27:747–753
Xiong J, Zhang X, Zhou H et al (2021) Aflatoxin M1 in pasteurized, ESL and UHT milk products from central China during summer and winter seasons: prevalence and risk assessment of exposure in different age groups. Food Control 125:107908. https://doi.org/10.1016/j.foodcont.2021.107908
Yunus AW, Imtiaz N, Khan H et al (2019) Aflatoxin contamination of milk marketed in Pakistan: a longitudinal study. Toxins (Basel) 11:1–9. https://doi.org/10.3390/toxins11020110
Zentai A, Jóźwiak Á, Süth M, Farkas Z (2023) Carry-over of aflatoxin B1 from feed to cow milk—a review. Toxins (Basel) 15:195. https://doi.org/10.3390/toxins15030195
Zhang Z, Song Y, Ma L et al (2022) Co-occurrence of Staphylococcus aureus and ochratoxin A in pasteurized milk. Toxins (Basel) 14. https://doi.org/10.3390/toxins14100718
Zhao Y, Yuan YC, Bai XL et al (2020) Multi-mycotoxins analysis in liquid milk by UHPLC-Q-Exactive HRMS after magnetic solid-phase extraction based on PEGylated multi-walled carbon nanotubes. Food Chem 305:125429. https://doi.org/10.1016/j.foodchem.2019.125429
Zheng B, Yu Y, Wang M et al (2022) Qualitative-quantitative analysis of multi-mycotoxin in milk using the high-performance liquid chromatography-tandem mass spectrometry coupled with the quick, easy, cheap, effective, rugged and safe method. J Sep Sci 45:432–440. https://doi.org/10.1002/jssc.202100641
Funding
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior — Brazil (CAPES) — Finance Code 001 and CNPq (scholarship). The authors acknowledge the Programa de Pós-graduação em Tecnologia de Alimentos (PPGTA-UTFPR), Central Analítica Multiusuário de Medianeira, and Universidade Tecnológica Federal do Paraná (UTFPR).
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All authors contributed to the study’s conception and design. Material preparation, data collection, and analysis were performed by DNM and DAD. The first draft of the manuscript was written by KCM and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Daniélly N. Morais declares that she has no conflict of interest. Kelly C. Massarolo declares that she has no conflict of interest. Elizandra N. G. Ardohain declares that she has no conflict of interest. Juliana F. Lima declares that she has no conflict of interest. Flavio D. Ferreira declares that he has no conflict of interest. Deisy A. Drunkler declares that she has no conflict of interest.
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Morais, D.N., Massarolo, K.C., Ardohain, E.N.G. et al. Method for Determination of Multi-mycotoxins in Milk: QuEChERS Extraction Modified Followed by HPLC-FL Analysis. Food Anal. Methods 17, 47–60 (2024). https://doi.org/10.1007/s12161-023-02550-0
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DOI: https://doi.org/10.1007/s12161-023-02550-0