Health risk assessment of aflatoxin M1 in infant formula milk in IR Iran
Graphical abstract
Introduction
The aflatoxins are a group of mycotoxins produced by certain Aspergillus species, in particular, Aspergillus flavus and Aspergillus parasiticus that contaminate plants and their products (Yazdanpanah and Eslamizad, 2015; Yazdanpanah, 2006). Four major aflatoxins including aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1, and aflatoxin G2, and two metabolic products including aflatoxin M1 (AFM1) and aflatoxin M2 are recognized as direct contaminants of foods and feeds. Aflatoxins are known hepatotoxic, mutagenic, carcinogenic, teratogenic, and immunosuppressive toxins (EFSA 2005; Herzallah, 2009). AFB1 is the most potent hepatocarcinogen, which is classified by International Agency for Research on Cancer (IARC) as a Group 1 carcinogen (IARC, 2002; Cavaliere et al., 2005). Aflatoxin M1 (AFM1) is the monohydroxylated derivative of AFB1 that is excreted in the human and animal milk following consumption of AFB1 contaminated food. AFM1 is relatively stable either to thermal processes like sterilization and pasteurization or during preparation and storage of different dairy products (Cavaliere et al., 2005). Even though the toxicity of AFM1 is less than AFB1, it has been classified as probable human carcinogens Group 2B by IARC (IARC, 2002).
AFM1 is found in milk and dairy products including yogurt, cheese, milk powder, ice cream, and regular cream. In IR Iran, there are a lot of information about contamination of milk with AFM1 (Ghaffarian Bahraman et al., 2019; Abyaneh et al., 2019; Fooladi Moghaddam et al., 2019; Khaneghahi Abyaneh et al., 2020; Rahmani et al., 2018; Khaneghahi Abyaneh et al., 2019; Kamkar et al., 2014; Yazdanpanah, 2006). In addition, infant formula milk which is one of the important products manufactured from milk is contaminated with AFM1 (Quevedo-Garza et al., 2020; Meucci et al., 2010; Kim et al., 2000; Srivastava et al., 2001).
High milk consumption by all age groups and especially by children seems to be one of the most important sources of dietary exposure for AFM1 (Tsakiris et al., 2013). After birth, human milk is the first choice for the newborn, but the role of infant formula milks is considerable, when breastfeeding is not possible, not recommended or not sufficient (Meucci et al., 2010). Therefore, many countries have established maximum limits for AFM1 in infant formula. For example, the maximum limits for AFM1 in infant formula in the European Union and USA are 25 pg/g and 50 pg/g, respectively (Commission Regulation, 2006; FDA, 2005; Jawaid et al., 2015). In IR Iran, the National Standard Organization (NSO) has established the maximum limit of AFM1 at 25 pg/g for infant formula milks (ISIRI, 2002). There are several HPLC methods were published for the determination of AFM1 in infant formula (Quevedo-Garza et al., 2020; Ivastava et al., 2001; Kim et al., 2000; Meucci et al., 2010). Considering the probable human carcinogenic effect of AFM1 on one hand and high infant formula consumption by infants on the other hand, determination of this contaminant in infant formula using sensitive, accurate, precise, and selective method is necessary.
In this study, two selective, sensitive, accurate, and precise HPLC methods for determining of AFM1 in infant formula milk were developed and validated, and performance characteristics of the methods compared in terms of matrix effect, accuracy, precision, and limits of detection and quantification. In these methods, HPLC monolithic column with a fluorescence detector, and immunoaffinity column (IAC) as a clean-up procedure were used and matrix effect was investigated in infant formula samples. To establish the capability and the suitability of these methods, a variety of infant formula milk samples were analyzed for the presence of AFM1, and risk of AFM1 was assessed for infants 0–6 months old by calculation of Hazard Index (HI), Margin of Exposure (MOE) and cancer risk considering the prevalence of Hepatitis B Virus (HBV).
Section snippets
Chemicals
All reagents were of analytical grade. Organic solvents were HPLC grade from Merck (Darmstadt, Germany). Ultra-pure water was produced from distilled water by a Water Purification system (Millipore, Bedford, MA, USA). Stock solutions of AFM1 and AFB2 (as internal standard, IS) were purchased from Sigma (St. Louis, MO, USA). The IACs was purchased from Vicam (Watertown, MA, USA).
Sample collection
Twenty nine samples of 6 infant formula milk brands were purchased from different drugstores in Tehran and Mashhad
Selectivity
In Fig. 1, Fig. 2, data on the selectivity of the two methods are presented. No matrix interfering substances were observed at the retention times of AFM1 and AFB2 in blank samples analyzed. The selectivity may result from using antibodies in clean up procedure. In method 2, AFM1 and AFB2 peaks were well resolved.
Linearity and matrix effect
In method 1, spiked calibration curve for AFM1 was linear in the range of 5–100 pg/ml (n = 15) with the regression coefficients calculated as R2>0.995. Standard calibration curve for
Method validation
Accuracy and precision of the analytical methods 1 and 2 developed for determination of AFM1 were close to 100% and were in the acceptable range of European Commission regulation (Commission Regulation, 2006). Comparison of the RSDr of 2 methods showed that the method 2 was more precise than the method 1 because of using AFB2 as IS in analysis of AFM1.
The sensitivity of AFM1 analytical methods characterize a current challenge in developing new analytical methods (Ketney et al., 2017). Table 5
Conclusion
Because of high toxicity and carcinogenic properties of AFM1, its presence in milk and its products is a real health concern. AFM1 is resistant to pasteurization, thermal inactivation, autoclaving and other variations of food processing procedures (Oveisi et al., 2007). In the present study, in addition to developing two selective, sensitive, accurate and precise HPLC methods for determining AFM1 in infant formula milk, for the first time, spiked calibration curve approach for AFM1
CRediT authorship contribution statement
Shirin Hooshfar: Investigation, Validation, Resources. Roya Khosrokhavar: Conceptualization, Supervision. Hassan Yazdanpanah: Conceptualization, Supervision, Writing - review & editing. Samira Eslamizad: Data curation, Methodology, Writing - original draft, Writing - review & editing. Farzad Kobarfard: Conceptualization, Supervision, Methodology, Validation. Firouzeh Nazari: Investigation, Validation. Venetia Kokaraki: Writing - review & editing. Manolis Kokkinakis: Writing - review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This study was financially supported by Center for Food and Drug Control Reference Laboratories, IR Iran. FDA, Ministry of Health and Medical Education, IR Iran. The authors are grateful to staffs of Toxicology Lab, School of Pharmacy, Shahid Beheshti University of Medical Sciences for their helpful cooperation during this project.
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These authors contributed equally to this work.