A survey of AFM1 contamination in row cow milk produced in region of Kumanovo

Aflatoxins (AFs) are type of mycotoxins produced by certain Aspergillus species. In the livers of cows, the aflatoxin В1 (AFB1) ingested with contaminated food is metabolized in carcinogenic aflatoxin М1 (AFM1) which is then excreted in the milk and poses a serious risk to the health of the consumers. In January of 2019, twenty five (25) samples of milk were collected from local farms in the surrounding area of Kumanovo, Republic of North Macedonia. The samples were analysed in the Centre for Public Health, Kumanovo. A method MKC EN ISO 14501:2007 for analysis of AFM1 by HPLC with fluorescence detection was applied. This method is selective, precise and applicative. The final analytical result has shown AFM1 below the LOD in any of the analyzed samples. Due to the wellknown fact that AFM1 is toxic, i.e. carcinogenic and the fact that milk is widely used as staple food, continuous development as well as introduction of new, more sensitive methods for detection of AFM1 are required.


Introduction
Mycotoxins are secondary metabolites of certain fungi that can be found as contaminants in food and animal feed. Aflatoxins (AFs) are a type of mycotoxins. They are the main secondary metabolites of certain Aspergillus species, such as Aspergillus flavus, Aspergillus parasiticus and rarely Aspergillus nomius (Bennett and Klich, 2003). There are four types of AFs: B 1 , B 2 , G 1 and G 2 , of which AFB 1 is the most toxic and carcinogenic one (Gourama and Bullerman, 1995). When cows ingest animal feed contaminated with AFB 1 , 4hydroxy derivative of AFB 1 (AFBM 1 ) is produced in the liver via CYP450 enzymes which is then eliminated through milk (Kuilman et al., 2000). Many researchers have proven that AFBM 1 is less toxic and carcinogenic than AFB 1 (Hsieh et al., 1984). Due to this, the International Agency for Research on Cancer (IARC) reclassified it in Group 2B (possibly carcinogenic to humans) hepatic carcinogens (IARC Monographs Vol. 82, 2002). The aflatoxin М 1 is relatively stable during the process of pasteurization, preparation and storage of dairy products. The presence of AFВM 1 in milk poses a serious __________________ * ivafurunovik@hotmail.com Maced. pharm. bull., 65 (2) 49 -55 (2019) risk to human health, given that milk is used as staple food. According to the European Commission, the maximum allowed concentration of AFВM 1 in milk is 0.05 µg/kg (Commission Regulations (EC) No. 1881, 2006.
The purpose of this research was to analyse the presence and concentration of AFBM 1 in milk samples taken from local farms in the surrounding area of Kumanovo, Republic of North Macedonia. HPLC technique was used according to the ISO method recommended for determination of AFBM 1 in milk.

Materials and methods
Chemicals HPLC grade reagents (acetonitrile, water) and chemicals were purchased from Merck (Darmstadt, Germany). For clean-up/purification, immune-affinity columns Aflaprep M1 Glasgow, Scotland were used. Stock solution aflatoxin M 1 in acetonitrile with a concentration of 0.5 µg/mL, (Romer Labs Diagnostic GmgH Austria) was used as a standard.

Methods
Qualitative and quantitative analyses of AFM 1 was carried out using the method MKC EN ISO 14501:007 (Milk and milk powder -determination of aflatoxin M 1 content -clean up by immune-affinity chromatography and determination by high-performance liquid chromatography).

Milk samples
Twenty five (25) milk samples (500 mL each sample) were collected in five consecutive days (five samples per day) from different ten (10) farms that supply the dairy factory Buchen Kozjak, Kumanovo with milk. The samples were collected in accordance with regulations, without exposure to sunlight and in sterile containers (ISO 707/IDF 50, 2008). The samples were delivered to the Centre for Public Health, Kumanovo where they were immediately analysed according to the MKC EN ISO 14501:2007 method, at room temperature and without exposure to sunlight.

Sample preparation
The samples were defatted when 100 mL of each sample of milk was heated in a water bath at temperatures from 35 to 37 °C and centrifuged at 2000 x g for 15 minutes. The fat accumulated onto the surface of the test tube was mechanically removed and the milk sample was filtered through filter paper. 50 mL of the filtered sample was passed through an aflatoxin immune-affinity column М 1 . After the entire milk volume has been passed through, the column was washed with 10 mL water (GRADE 1). The aflatoxin М 1 was eluted using 2 mL 10% acetonitrile solution. This eluate was collected in vials and immediately analysed using HPLC.
In each lot of samples prepared for analysis, one sample was spiked with 500 µL 0.005 µg/mL standard working solution for AFM 1 .

HPLC conditions
HPLC analysis was performed with Agilent Technologies 1260 Series chromatographic system equipped with vacuum degasser G4225A, Binary Pump G1312B, Autosampler G1329E, Column Compartment G1316C. Aflatoxin M 1 was separated on ZORBAX Eclipse Plus C18 Column 4.6 x 100 mm, 3.5 µm at room temperature. The mobile phase was a mixture of water and acetonitrile (25:75, V/V). The flow rate was 1 mL/min and the injection volume was 50 µL. The detection was carried out at λ ex = 365nm and λ em = 435nm. Data were acquired using Agilent life Sciences OpenLAB CDS ChemStation software.

Identification of AFM 1 based on retention time
To test for carryover, a mobile phase blank was injected. As shown in Fig. 1 the chromatogram of this blank showed no detectable peak. Fig. 2 shows the chromatogram of aflatoxin working standard solution with concentration of 0.2 ng/mL and retention time 5.257 min).
Chromatographic repeatability was confirmed via replicate injection of working standard solution with concentration of 0.1 ng/mL of AFM1, demonstrating exceptional reproducibility. Analyses of working standard solutions were performed in a concentration range of 0.05-0.4 ng/mL and aflatoxin М 1 was identified based on retention time. Concentrations of working standard solutions and the corresponding retention time for aflatoxin М 1 are shown in Table 1. The relative standard deviation %RSD of the retention time for M 1 was 0.13% demonstrating exceptional reproducibility. The limit of detection (LOD) based upon signal to noise S/N was 0.02 ng/mL and the limit of quantitation (LOQ) based S/N was calculated to be 0.05 ng/mL.

Standard solution preparation and calibration
With several dilutions of stock standard solution, the working standard solutions of AFM 1 with concentrations of 0.05, 0.10, 0.20, 0.3, 0.4 ng/mL were prepared and subsequently were used to obtain the calibration/standard curve as described by the following regression equation: Y=0.64042297X+0.003654, where Y is the peak area and X is the corresponding concentration of AFM 1 (Fig. 3). The linearity of the standard curve over the range studied is presented in Fig. 3. The coefficient of regression (R 2 ) was 0.99975.

Results and discussion
The analysis of all twenty five samples has shown that AFM 1 was not detected in any of the milk samples. Because there is no significant difference between chromatograms from each sample (it was not detected M 1 peak) on Fig. 4 (a-e), is represented one chromatogram from daily analysis.
The analyses were performed in January when the highest concentration of aflatoxin M 1 was expected as a result of positive conditions for mold spreading and feeding circumstances (Tajkarimi et al., 2017). In order to prove the accuracy of the method and the results, one milk sample was spiked with known amounts of working standard solution of AFM 1 . The obtained peak was identified as AFМ 1 based on retention time (Fig. 5). Overall the recovery of 124% is very good considering the rater complexed matrix for sample extractions and very low concentration.
The applied method for determination of aflatoxin M 1 is fast, simple, selective and suitable for routine application (MKC EN ISO 14501:2007).
The obtained results are encouraging, but continued monitoring is suggested. In addition, several studies have been conducted to determine the contamination of milk with AFM 1 ( Table 2). The incidence of AFM 1 observed in the presented study by other authors was higher (Bilandzic et al., 2015;Bilandzic et al., 2016;Dimitrieska-Stojkovic et al., 2013;Dimitreska et al., 2014;Godic Tarkar et al., 2008;Jajic et al., 2018;Markaki et al., 1997;Polovinski et al., 2009;Polovnski-Horvatovic et al., 2016;Skrbic et al., 2014). Presented results may be attributed to the differences in the regions, the origin of animal feed, season of milk collection and the different AFM1 analytical methods for determination.

Conclusion
The method applied to determine the AFМ 1 using HPLC with fluorescence detection is sensitive, specific and accurate. However, it is of general interest to continuously develop new methods for determination of AFM 1 in milk. It was also revealed that in any of analyzed samples AFM 1 was lower than LOD. Even though the results from the survey are encouraging, when it comes to the mycotoxicological quality of the milk available on our market, a legal obligation of continuing monitoring is obvious, in order to prevent possible poisonings, especially in children as the most vulnerable group in society whose diet consists mostly of milk.