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Determination of Aflatoxin M1 and Heavy Metals in Infant Formula Milk Brands Available in Pakistani Markets

Aflatoxin M1 (AFM1) after its bioconversion from aflatoxin B1 in animal liver becomes the part of milk while heavy metals get entry into milk and milk products during handling in the supply chain. Aflatoxin M1 and heavy metals being toxic compounds are needed to be monitored continuously to avoid any ailments among consumers of foods contaminated with such toxicants. Thirteen commercially available infant formula milk (IFM) brands available in Pakistani markets were analyzed for the quantitative determination of AFM1 and heavy metals through ELISA and atomic absorption spectrophotometer, respectively. AFM1 was found positive in 53.84% samples while 30.76% samples were found exceeding the maximum EU limit i.e. 0.025 μg/kg for AFM1 in IFM. Heavy metals lead (Pb) and cadmium (Cd) were found below the detection limits in any of the sample, whereas the concentrations of iron (Fe), zinc (Zn) and nickel (Ni) ranged between 45.40-97.10, 29.72-113.50 and <0.001-50.90 μg/kg, respectively. The concentration of Fe in all the tested brands was found in normal ranges while the concentrations of Zn and Ni were found exceeding the standard norms. Elevated levels of AFM1, Zn and Ni in some of the tested IFM brands indicated that a diet completely based on these IFM brands might pose sever health implications in the most vulnerable community i.e., infants.

and heavy metals being toxic compounds are needed to be monitored continuously to avoid any ailments among consumers of foods contaminated with such toxicants. Thirteen commercially available infant formula milk (IFM) brands available in Pakistani markets were analyzed for the quantitative determination of AFM 1 and heavy metals through ELISA and atomic absorption spectrophotometer, respectively. AFM 1 was found positive in 53.84% samples while 30.76% samples were found exceeding the maximum EU limit i.e. 0.025 µg/kg for AFM 1 in IFM. Heavy metals lead (Pb) and cadmium (Cd) were found below the detection limits in any of the sample, whereas the concentrations of iron (Fe), zinc (Zn) and nickel (Ni) ranged between 45.40-97.10, 29.72-113. 50 and <0.001-50.90 µg/ kg, respectively. The concentration of Fe in all the tested brands was found in normal ranges while the concentrations of Zn and Ni were found exceeding the standard norms. Elevated levels of AFM 1

Introduction
Mother milk being a rich source of all the basic nutrients is the only food for infants during the early few months of their lives. Infants up to 6 mon of age undergo some vulnerable changes in growth and development. Infants require mother milk for at least 2 years as it helps establish healthy immune system, nervous system, digestive system, reproductive system and strong physical structure of body (Kazi et al., 2010). World Health Organization (WHO) recommends breastfeeding as best and sole source of infant feeding (WHO, 2009). However, rapid urbanization and advanced life style of recent days has led to concentrate on readymade foods for infants in the form of infant formula milk (IFM). IFM has become popular all around the globe. If mother faces health issues or an infant denies mother's milk, the usage of infant formula becomes right choice as an alternative to breast feeding.
A number of food commodities available in the markets is specifically designed and manufactured for infants. Care is always taken to protect infant foods from any of the harmful compounds due to the fact that these food items are consumed by one of the most vulnerable age groups of people i.e., infants. Mycotoxin contamination in food chain is considered as one of the brazen food safety issues worldwide (Bouaziz et (Liu and Wu, 2010). There are different types of aflatoxins and their toxicities ranked in decreasing order as follows; aflatoxin B1 (AFB1) > aflatoxin B2 (AFB2) > aflatoxin G1 (AFG1) > aflatoxin G2 (AFG2), respectively. AFB1 if ingested by the animals is converted into aflatoxin M1 (AFM1) inside the liver it appears in the milk of dairy animals and is also reported in human milk. AFM1, although ten times less carcinogenic as compared to AFB1, it is still categorized as probable human carcinogen (group 2B) by the International Agency for Research on Cancer (IARC, 2002). The permissible limit for AFM 1 in animal milk (0.05 µg/L) set by European Commission (2006) is 40 times stricter than the total aflatoxins limit in food and feed items (20 µg/L) while more stringent permissible limits are adopted for AFM 1 in IFM i.e., 0.025 µg/L. Infants health may also be adversely affected due to the prolonged feeding on aflatoxin M 1 contaminated food i.e., milk, infant formulas and related products From last few decades, heavy metals appeared in different food commodities as a matter of serious concern and threat for human health. The penetration of heavy metals in food chain is a result of escalating industrialization and agricultural processes throughout the world. Heavy met-als are the elements having densities higher than 5 g/cm 3 and among the most toxic ones are Arsenic (As), Pb, Mercury (Hg) and Cd. Although, Zinc (Zn) and Iron (Fe) are among the essential micronutrients having wide range of biochemical functions in human body, these elements may cause toxicity when consumed in higher concentrations. Heavy metals may enter human body by inhalation and ingestion (Tripathi et al., 1999). The intake of metals in infants mainly depends on the bioavailability of metal content in milk and milk based foods ( The envisaged project was designed to investigate the concentrations of two major chemical toxicants i.e., AFM 1 and metals (Pb, Cd, Fe, Zn and Ni) in thirteen IFM brands available in Pakistani markets.

Materials and Method
The analysis for AFM 1 and heavy metals in IFM samples were performed in the Food Analysis Laboratory of Institute of Food Science & Nutrition, Bahauddin Zakariya University, Multan-Pakistan.

Collection of samples
IFM of thirteen different brands used for infants aged 1-6 mon were purchased from pharmaceutical stores and local markets. Ten samples of each IFM brand were collected and the analyses were performed in triplicate. The samples were stored in refrigerator until analyzed.

Sample preparation
Powdered IFM (1 g) was taken and mixed with 10 mL of distilled or deionized water. Samples were defatted thr-ough centrifugation (Germany) for 10 min at 2000×g. The upper layer of fat was removed carefully by using a spatula. Defatted milk samples were used for the determination of AFM 1 through ELISA kits.

ELISA assay protocol
ELISA kits were purchased from Euro Proxima, (catalogue No. 5121AFM 1 , Netherlands) and the analysis for AFM 1 was performed according to the guidelines provided in the kit manual. Defatted samples, standard solutions and blanks (100 µL each) were added to their respective wells and were incubated in dark for a period of 1 hr. Specific antibody binding sites in each well bounded free AFM 1 available in samples or standards. After incubation the remaining solution in each well was discarded and the wells were washed three times with rinsing buffer. Now, conjugate solution (AFM 1 -horseradish peroxidase) was added to each well in the quantity of 100 µL. The plates were again incubated in dark at room temperature for a period of 30 min. The remaining solution was discarded and the microtiter plate wells were again washed three times with rinsing buffer. After drying, 100 µL of substrate solution was added in each well and the plate was incubated at room temperature. After 30 min, 100 µL of stop solution was added to each well. The absorbance of each well at 450 nm was read through ELISA reader (Bio-Tek ELx800, Indonesia). The limit of detection (LOD) for AFM 1 was 0.006 ng/mL. The concentration of AFM 1 in each sample was calculated through standard calibration curve.

Quality control
For validation of results, a standard solution of AFM 1 was purchased from Sigma Aldrich Chemicals (A6428). AFM 1 free milk samples were spiked with standard solution of AFM 1 at the concentrations of 0.01, 0.05, 0.1 and 0.2 µg/L. AFM 1 recovery percentages were recorded in the range of 96.3-98.4% (Table 1).

Heavy Metals Assessment in IFM Brands
Apparatus and chemicals All the chemicals and reagents used were of analytical grade purchased from Merck Chemicals, USA. Hot plate (Lab Tec; EH 35A plus) was used for digestion of the samples. Flame atomic absorption spectrophotometer (Thermo Scientific; iCE-3000 series) was used for the analysis of heavy metals. The standards for heavy metals were purchased from CPA chemicals limited. Samples and standards were diluted with double distilled water to make final volumes.

Quantification of heavy metals
Metals included in current study were Pb, Cd, Fe, Zn and Ni. For the estimation of heavy metals in IFM samples, wet digestion method of Weldegebriel et al. (2012) was adopted. Briefly, 0.5 g sample was weighed by using Digital Weighing Balance (Precisa XB 120A) followed by addition of 10 mL of nitric acid (HNO 3 ) and 5 mL perchloric acid (HClO 4 ), and kept at room temperature for one night. Next day, the sample was heated on a hot plate until the volume of solution dropped down to 2-3 mL and the color became transparent. The sample was diluted with double distilled water up to convenient volumes and filtered through Whatman's filter paper No. 42. Finally, the samples and blanks were loaded on flame atomic absorp-  Table 2.

Statistical analysis
Statistical evaluation of data obtained from each parameter was done through Statistix 8.1 software (Statistix Inc., USA). For comparison purpose, the data were subjected to one-way analysis of variance (ANOVA) followed by LSD (least significant difference) test. The differences were considered statistically significant at the probability level of p<0.05. Mean values and the measurement uncertainty (standard deviations) were computed through Microsoft Excel 2013.

Aflatoxin M 1 in different IFM brands
The results of mean AFM 1 level in different brands of IFM (n=13) are presented in Table 3. Statistical analysis revealed significant differences in the concentration of AFM 1 in various brands of IFM (p<0.05). AFM 1 was found positive in seven brands (53.84%) while the range of AFM 1 in all the tested brands was <0.006-0.108 µg/kg. European Union permissible limit for AFM 1 in IFM samples is 0.025 µg/kg (European Commission, 2010). The current study revealed that 30.76% of the samples of IFM brands were found exceeding the maximum permissible limits set by European Commission.
The presence of AFM 1 beyond permissible limit in such a high percentage of IFM samples is a serious health issue particularly for infants who rely on this food during very early stage of their life with developing immunity. Higher levels of AFM 1 in animal milk samples from Pakistan are also reported by Ismail et al. (2016), according to which 93% out of a total of 520 milk samples were found positive for AFM 1, while 53% samples were reported exceeding the EU maximum limit for AFM 1 in milk (0.05 µg/L). Kanungo and Bhand (2015) reported the level of AFM 1 in IFM samples from India and found that all samples had AFM 1 above the maximum EU limits. The range of AFM 1 in IFM samples was 0.501-0.713 µg/kg. The level of AFM 1 in IFM samples from India is much higher as compared to our findings. In a study conducted in Spain by Beltran et al. (2011), AFM 1 level was measured in 14 baby food samples, 7% of which were found positive for AFM 1 while the reported mean concentration for AFM 1 was 0.006 µg/ kg, however none of the samples was found exceeding the maximum EU limit. Meucci et al. as compared to our results indicating a better control of aflatoxins in these countries as compared to Pakistan. The availability of IFM brands contaminated with AFM 1 beyond set standards might be associated with favorable environment for the growth of fungus responsible for the production of aflatoxins, lack of surveillance system and lack of control of law enforcement agencies on manufactures and suppliers.

Determination of heavy metals in IFM brands
The results of minerals elements in IFM samples of various brands are presented in Table 4. Statistical analysis showed significant differences in the concentration of mineral elements among various brands however the differences were non-significant for Pb and Cd (p<0.05). Mineral elements concentrations in various brands were found in the order of Fe > Zn > Ni. Heavy metals like Pb and Cd were found below detection limits in all of the tested brands, indicating the adoption of good manufac- turing practices by the IFM manufacturers to control these highly toxic heavy metals. Fe is an essential element for the normal growth and development of human body. The results of current study showed the presence of iron in all the IFM brands ranged from 45.40-97.10 mg/kg. The statistical analysis showed significant differences in the level of iron among various brands of IFM (Table 4). The level of iron found in our study showed 5-10% (±) variations as compared to the labeled values. Lesniewicz et al. (2010) quantified the concentration of Fe in 12 different types of IFM brands available in the markets of Poland The mean level of Fe in different brands ranged 35-74 mg/kg which is almost in agreement with our findings. Pandelova et al. (2012) measured the level of Fe in baby milk samples available in the markets of Germany and found mean Fe value as 47.7 mg/kg that is almost in line with our study. These results showed that the concentration of Fe found in current study was in normal range.
Zn is a minor inorganic element necessary for the growth and development of infants. It is believed to be involved in cellular metabolism. Zn is also needed for the synthesis of DNA, division of cells and for catalytic activity of more than 100 enzymes (Beigi and Maverakis, 2015;Tariba et al., 2016). The results of present study revealed that the concentration of Zn in different infant formula samples ranged between 29.72-113.50 mg/kg (Table 4). A difference of about 2-3% was observed among the calculated and labeled values. According to Polish standards the Zn content in IFM samples must not exceed 55 mg/kg (PN-A-94015). Comparing this limit with our results the IFM of two brands were found exceeding the permissible limit. Level of Zn reported from Poland in 12 differ-ent brands of IFM samples ranged between 16-56 mg/kg and these results are lower as compared to our findings.

Conclusion
The results of current study indicated that the concentrations of toxic metals Pb and Cd in IFM samples were detected within safe limits. The level of Fe was also found in normal ranges but the levels of Zn and Ni in some of the IFM brands were found above the normal ranges. The analysis of AFM 1 in IFM samples revealed that 30.76% infant formula samples exceeded the EU maximum permissible limit which might result in severe toxicity in infants being immunity compromised group of age. The elevated levels of AFM 1 , Zn and Ni in some of the IFM brands demand surveillance and implementation of regulations to avoid any severe and irrecoverable health implications as a result of bioaccumulation of these toxic compounds in infants.