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Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Aflatoxin M1 in buffalo and cow milk in Afyonkarahisar, Turkey a
Recep Kara & Sinan İnce
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Department of Food Hygiene and Technology , Afyon Kocatepe University , Afyonkarahisar , Turkey b
Department of Pharmacology and Toxicology , Afyon Kocatepe University , Afyonkarahisar , Turkey Accepted author version posted online: 17 Jul 2013.
To cite this article: Food Additives & Contaminants: Part B (2013): Aflatoxin M1 in buffalo and cow milk in Afyonkarahisar, Turkey, Food Additives & Contaminants: Part B: Surveillance, DOI: 10.1080/19393210.2013.825646 To link to this article: http://dx.doi.org/10.1080/19393210.2013.825646
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Aflatoxin M1 in Buffalo and Cow Milk in Afyonkarahisar, Turkey Recep KARA1*, Sinan İNCE2 1
Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Food Hygiene and Technology, Afyonkarahisar, Turkey.
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Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Afyonkarahisar, Turkey.
*Corresponding author
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Abstract Potential hazardous human exposure to aflatoxin M1 (AFM1) via consumption of milk and milk products has been demonstrated by many researchers. The aim of the present study was to investigate the presence of this mycotoxin in buffalo and cow milk products in the city of Afyonkarahisar, Turkey. For this purpose, 126 buffalo and 124 cow milk samples were collected from dairy farms in Afyonkarahisar province. AFM1 levels were determined by high-performance liquid chromatography (HPLC) with tandem mass spectrometric detection (LC/MS/MS). Although AFM1 was not detected in cow milk samples, AFM1 was found above the limit of detection (< 0.008-0.032 µg/L) in 27% (34 out of 126) of the buffalo milk samples. The results of this study indicated the importance of continuous surveillance of commonly consumed milk or milk product samples for AFM1 contamination in Turkey.
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Keywords: Buffalo Milk, Cow Milk, Aflatoxin M1, LC/MS/MS
Introduction Aflatoxins (AFs) are toxic metabolites produced mainly by the moulds Aspergillus flavus and Aspergillus parasiticus (Zheng et al. 2013). These are produced at pre-harvest and during storage of cereal grains particularly maize, rice, wheat, barley, oats, and sorghum (Khoury et al. 2011). AFs, the most studied mycotoxins, mainly consist of 4 naturally occurring compounds including AFB 1, B2, G1, and G2, which are found in various food commodities (Fallah 2010). The hydroxylation process in the rumen of dairy mammals as the direct intake by these animals of AFB 1 contaminated feed leads to accumulation of AFM1 in milk (Creppy 2002). The formation of AFM1 occurs in the liver and it is secreted into the milk (Cathey et al. 1994). About 0.36.2 % of AFB1 in animal feed is transformed to AFM1 in milk (Creppy 2002). AFM1 has lower toxicity than AFB1 and is part of the secondary groups of carcinogenic compounds which have been classified by the International Agency on the Research on Cancer (Bakirci 2001). Milk is a balanced diet which has been provided by nature (Hussain et al. 2010). Dairy products including milk are important sources of animal protein, vitamins, and essential fatty acids for infants and young adults (Jensen and Nielsen, 1982). Milk is a major commodity for introducing AFM1 in the human diet and evidence of hazardous human exposure to AFM1 through dairy products has been shown by several investigators (Kamhar 2005; Unusan 2006; Oliveira et al. 2011; Rohani et al. 2011). AFM1 is relatively stable during pasteurization, sterilization, preparation, and storage of various dairy products (Gurbay et al. 2006a). Presence of AFM1 in milk and milk products may have negative health implications for consumers, particularly for infants and children (Filazi et al. 2010; El Marnissi et al. 2012). It is an acute toxic compound and has shown to be immunosuppressive, mutagen, teratogen and carcinogen (Liu and Wu, 2010). AFM1 level in milk and milk products is regulated in many countries and the European Union (EC 2010) and Turkey (TFC 2011) fixed the limit of AFM1 in milk at 0.05 µg/L. The aim of this study was to determine AFM 1 content in raw buffalo and cow milk in Afyonkarahisar province in Turkey. Also the present paper focused on the suitability of LC/MS/MS with the electro spray ionization (ESI) technique for the determination of AFs in milk.
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Recep Kara, Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Food Hygiene and Technology, Afyonkarahisar, Turkey.
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Materials and Methods Chemicals The AFM1 standard, ammonium acetate and HPLC-grade acetonitrile were obtained from Sigma Aldrich (Interlab A.S., Istanbul, Turkey). Water was purified with a PureLab-Q system with a LC 140 cartridge column, purchased from ELGA (Marlow, UK). Milk samples and extraction A total of 250 raw milk samples including 126 buffalo (Anatolian) and 124 cow (Holstein) milk samples were randomly selected from Afyonkarahisar province and different districts during August 2012 and January 2013. Milk samples of at least 1 liter were taken in a sterile bottle from the mammary of cows and buffalos. The samples were transported to the laboratory in ice packets in an icebox and stored at -20 ºC until analysis of AFM1. Milk sample extractions were carried out according to validated methods reported by Tanaka (2002). Briefly, 1 mL sample was put into a 10 mL centrifuge tube, followed by adding 4 mL acetonitrile. After shaking for 10 min, the mixed solution was centrifuged at 1,650 g for 5 min and then 1 mL supernatant was transferred into auto sampler vials. Standard preparation and calibration The AFM1 standard was dissolved in acetonitrile at 1 mg/L and stored at 4 °C in the dark until use. To prepare the working standard for LC-MS/MS analysis, AF stock solution was pipetted and transferred into a vial and diluted with the mobile phase. The final AFM1 concentration was 1 µg/L. The linearity of the calibration curve was evaluated from peak area calculations after five injections of standard solutions prepared in acetonitrile at 0.01, 0.05, 0.1, 0.5, and 1 µg/L. The limit of detection (LOD) was defined as the minimum concentration of an analyte that can be identified, measured and reported with 99 % confidence. It was calculated from the relative standard deviation (RSD) of average detector responses of ten replicate injections, via the formula LOD = 3x RSD x concentration (provided that the response of the blank was zero). From these calculated values, a best estimated, rounded LOD value was established. The estimated LOQ was defined as the minimum concentration of an analyte that can be identified and quantified with 99 % confidence. It was calculated by LOQ = 10x RSD x concentration. The analytical methods used in this survey were evaluated using milk samples spiked with AFM1 at levels of 0.01 - 1 µg/L (n = 5 per spiking level). Instrumental conditions The LC-MS/MS system used in this work consisted of an Agilent 1200 series (Agilent Technologies, Waldbronn, Germany), including a vacuum solvent degassing unit, a binary high-pressure gradient pump, an automatic sample injector, a column thermostat and a photodiode array detector. LC separation was performed on a 4.6 x 100 mm i.d. column packed with 3.5 μm Zorbax Hilic Plus C18 (Agilent Technologies, Santa Clara, CA, USA) at 40 °C. The LC mobile phase was aqueous 0.4 mM ammonium formamide and 0.2 mL formic acid in water (A) and acetonitrile (B). The initial gradient condition was 10 % A and 90 % B changing linearly to 100 % in 10 min. After analysis the column was equilibrated for 15 min. The flow rate was 0.8 mL/min and the injection volume was 10 μL. Mass spectrometry was performed using an Agilent 6460 LC-MS Triple Quadrupole instrument equipped with an electrospray ionization (ESI) source (Agilent Technologies, Waldbronn, Germany). The nebulizer gas as well as drying gas (350 °C) was nitrogen, generated from pressurized air by a Balston model 75–72 nitrogen generator (Balston, Haverhill, MA, USA). The capillary voltages for ion transmission, fragmentor voltage for insource-fragmentation and vaporizer temperature were all optimized using the analytical column with mycotoxin standard at 0.01 µg/L. Nebulizer gas, drying gas, capillary voltage and vaporizer temperature were set at 45 psi, 11 L/min, 3000 V and 400 °C, respectively. Quantitative analysis was carried out using SRM mode. Retention time was 1.2 minutes. Moleculer weight, precursor ion (m/z), and product ion (m/z) were 328, 350 and 200, respectively.
Results and Discussion Calibration curves for quantification of the standard showed good linearity with correlation coefficients (r2 > 0.999) over the range studied by ESI mode (table 1). LOD, LOQ and RSD of AFM1 in milk using LCMS/MS were 0.008 and 0.05 µg/L and 2.56 %, respectively. Samples with AFM1 levels below 0.008 µg/L were considered negative. Recovery for AFM1 was 96 %. The occurrence and levels of AFM1 in raw buffalo and cow milk samples from Afyonkarahisar province are presented in Table 2. AFM1 was found above LOD in 27 % (34/126) of buffalo milk samples and not detected AFM 1 in cow milk samples. Considering the European Commission (EC, 2010) and Turkish Food Codex (TFC, 2011) limit, no buffalo and cow milk sample contained AFM1 in concentrations above the maximum limit of 0.050 µg/L. Many researchers have investigated AFM1 in milk. In 81% of the LC-MS analyzed bovine milk samples AFM1 was detected ranging from 2 to 108 ng/L (Bognanno et al. 2006). In another study, Rohani et al. (2011) reported AFM1 in 72 cow milk samples ranging from LOD
n>0.05µg/L
Mean (µg/L)
Range (µg/L)
Buffalo
126
34
-
0.003