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Monitoring of aflatoxin M1 in raw milk during four seasons in Croatia
Accepted Manuscript Monitoring of aflatoxin M1 in raw milk during four seasons in Croatia Nina Bilandžić, Ivana Varenina, Božica Solomun Kolanović, Đurđica Božić, Maja Đokić, Marija Sedak, Sanin Tanković, Dalibor Potočnjak, Željko Cvetnić PII:
S0956-7135(15)00097-3
DOI:
10.1016/j.foodcont.2015.02.015
Reference:
JFCO 4305
To appear in:
Food Control
Received Date: 13 November 2014 Revised Date:
2 February 2015
Accepted Date: 10 February 2015
Please cite this article as: Bilandžić N., Varenina I., Kolanović B.S., Božić Đ., Đokić M., Sedak M., Tanković S., Potočnjak D. & Cvetnić Ž., Monitoring of aflatoxin M1 in raw milk during four seasons in Croatia, Food Control (2015), doi: 10.1016/j.foodcont.2015.02.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Monitoring of aflatoxin M1 in raw milk during four
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seasons in Croatia
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Nina Bilandžića,∗∗, Ivana Vareninaa, Božica Solomun Kolanovića, Đurđica Božića, Maja
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Đokića, Marija Sedaka, Sanin Tankovićb, Dalibor Potočnjakc, Željko Cvetnićd
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a
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Department of Veterinary Public Health, Laboratory for Residue Control, Croatian Veterinary Institute,
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Savska cesta 143, HR-10000 Zagreb, Croatia
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Veterinary Office of Bosnia and Herzegovina, Radićeva 8, 71000 Sarajevo, Bosnia and Herzegovina
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Department of Internal Medicine, Faculty of Veterinary Medicine, University of Zagreb, Heinzlova 55,
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HR-10000 Zagreb, Croatia d
General Department, Croatian Veterinary Institute, Savska cesta 143, HR-10000 Zagreb, Croatia
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ABSTRACT
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A total of 3543 raw cow milk samples were collected in three regions of Croatia: western,
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eastern and other regions during four seasons. Samples were measured for aflatoxin M1
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(AFM1) concentrations using the enzyme immunoassay method. Elevated levels (>50 ng/kg)
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of AFM1 were analysed by validated liquid chromatography with triple quadruple mass
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spectrometry (LC-MS/MS). The limits of detection (LOD) and quantification (LOQ) of the
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LC-MS/MS method were 7.3 and 28 ng/kg, respectively. The mean AFM1 levels measured in
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the three regions over four seasons were in the ranges (ng/kg): eastern Croatia 7.25–26.6;
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western Croatia 5.91–9.26; other regions of Croatia 7.17–13.6. The highest incidence of
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samples exceeding the EU MRL (50 ng/kg) of 9.32% was measured in autumn (October–
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December) in the eastern region. Only eight samples were found to exceed the EU MRL in
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winter. The highest AFM1 levels were measured in December (764.4 ng/kg) and January
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ACCEPTED MANUSCRIPT (383.3 ng/kg). Elevated AFM1 levels were found in summer in only four samples from the
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western and other regions, and two samples in the eastern region. This can be attributed to
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localized and random usage of contaminated feed for dairy cows in those regions. The much
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lower incidence of elevated AFM1 in comparison to a previous study showed that the
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outbreak of the crisis due to elevated AFM1 levels in 2013 resulted in a more careful
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approach to the control of supplementary feedstuff for lactating cows.
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Key words: Aflatoxin M1; Cow milk; ELISA; LC-MS/MS; Croatian regions
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∗
Corresponding author. Tel.: +385 1 612 3601, fax: +385 1 612 3636
E-mail address: [email protected]
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1. Introduction
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Due to their high nutritional properties and high intake by all age groups, milk and
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dairy products hold one of the most important roles in the human diet and, consequently, are
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of great economic importance for every country. The presence of aflatoxin M1 (AFM1), as a
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known xenobiotic, may have negative health implications for consumers and for agricultural
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production, as its appearance in milk can incur economic damages due to production losses
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(Tsakiris et al., 2013). This is emphasized by the fact that the AFM1 molecule cannot be
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inactivated by thermal processing used in the dairy industry, i.e. pasteurization and ultra-high-
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temperature treatment (Fallah, Rahnama, Jafari, & Saei-Dehkordi, 2011; Oruc, Cibik, Yilmaz,
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& Kalkanli, 2006). AFM1 is the main hydroxyl-metabolite of the aflatoxin B1 (AFB1) that is
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formed in the liver of lactating animals following consumption of contaminated feedstuffs
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ACCEPTED MANUSCRIPT (Prandini, Tansini, Sigolo, Filippi, Laporta, & Piva, 2009). AFB1 demonstrates teratogenic,
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mutagenic and carcinogenic effects in mammals and has been classified as a Group 1 toxin.
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Recently, AFM1 has been also classified in Group 1 as a possible hepatotoxic substance and
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carcinogen for humans (IARC, 2002). AFM1 may cause DNA damage, which ultimately
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leads to gene mutation, chromosomal anomalies and cell transformation in mammalians cells
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(Prandini, Tansini, Sigolo, Filippi, Laporta, & Piva, 2009).
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When grazing is decreased in winter, significantly higher amounts of concentrated
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feed, especially corn silage and grass, are given to meet the energy needs of high-yielding
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cows. In the case of contaminated feed usage, AFM1 will be present in the milk for 2–3 days
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following ingestion (Prandini et al., 2009). Therefore, the risk is largely related to grain-based
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feed (Fink-Gremmels, 2008; Duarte et al., 2013). The transmission of AFB1 from foodstuffs
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to milk (carry-over) in dairy cows is influenced by various nutritional and physiological
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factors, including feeding, the degree of digestion, animal health, biotransformation capacity
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of the liver, and milk production (Duarte et al., 2013).
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Contamination of milk and dairy products and variations in concentrations are
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associated with geographic location and climate, the degree of development of the region and
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the season (Rahimi, Bonyadian, Rafei, & Kazemeini, 2010). Climatic conditions in tropical
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and subtropical regions with high temperatures and drought, constant warmth and humidity
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favour the growth of the toxigenic mould species Aspergillus (Picinin, Cerqueira, Vargas,
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Lana, Toaldo, & Bordignon-Luiz, 2013). However, long periods of high temperatures and
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long-lasting drought in summer during the maize-growing and maize-harvesting periods also
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favour the development of these moulds in feed in other climatic regions (Bilandžić et al.,
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2014a; Decastelli et al., 2007). With regard to the fact that the level of AFB1 contamination is
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cumulative, harvesting time and conditions of drying and storage of foodstuffs can actually
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have the same role in aflatoxin production. During grain storage, the drying process is the
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primary factor required to maintain a low moisture level, as irregular moisture of the mass
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favours fungal development (Prandini et al., 2009). In recent years, elevated concentrations of AFM1 have been measured in different
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countries around the world: Lebanon (Assem, Mohamad, & Oula, 2011), Iran (Fallah et al.,
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2011; Nemati, Mehran, Hamed, & Masoud, 2010; Rahimi et al., 2010), Syria (Ghanem &
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Orfi, 2009), Turkey (Golge, 2014), Pakistan (Asi, Iqbal, Ariño, & Hussain, 2012; Hussain &
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Anwar, 2008; Iqbal & Asi, 2013; Sadia et al. 2012), South Africa (Dutton, Mwanza, de Kock,
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& Khilosia, 2012), Sudan (Elzupir & Elhussein, 2010), Thailand (Ruangwises & Ruangwises,
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2009), Indonesia (Nuryono et al., 2009), Brazil (Picinin, Cerqueira, Vargas, Lana, Toaldo, &
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Bordignon-Luiz, 2013), China (Xiong, Wang, Ma, & Liu, 2013), Serbia (Škrbić, Živančev,
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Antić, & Godula, 2014) and Croatia (Bilandžić, Varenina, & Solomun, 2010; Bilandžić et al.,
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2014a,b).
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Extremely elevated concentrations of AFM1 reported in 2013 in cow milk from
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eastern Croatia raised concerns and resulted in increased additional measures to control
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AFM1 in milk and AFB1 contamination in feedstuffs (Bilandzic et al., 2014a; Pleadin, Vulić,
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Perši, Škrivanko, Capek, & Cvetnić, 2014). Following this, the aim of this study was to
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evaluate the concentrations of AFM1 in raw cow milk collected during a one-year period in
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three regions of Croatia: western, eastern and other regions. For the purpose of quantifying
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increased concentrations of AFM1, an additional aim of this study was to implement and
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validate a confirmatory method of liquid chromatography–tandem mass spectrometry (LC-
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MS/MS).
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2. Materials and methods
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2.1. Sample collection 4
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A total of 3198 raw cow milk samples were collected in the period from October 2013
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to September 2014 from dairy farms in eastern (total n=1589) and western Croatia (total
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n=1609). Milk samples were also collected on small farms across other Croatian regions, i.e.
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from southern, southwestern, central and northern Croatia. However, as the number of
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samples from these areas was significantly lower than the number of samples collected in
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eastern and western Croatia, this group of samples was pooled into the group other regions
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(total n=345). The volume of the collected milk samples was approximately 0.5 litres.
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Samples were stored at 2–8°C or frozen at -20°C until further analysis of AFM1.
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2.2. Chemicals and reagents
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AFM1 concentrations were measured using competitive enzyme immunoassay
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(ELISA) of Ridascreen "Enzyme immunoassay for the quantitative analysis of aflatoxin M1"
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(R1121, R-Biopharm AG, Darmstadt, Germany). The reagents for the test kit contain: AFM1
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standard solutions in milk buffer for the calibration curve (0, 5, 10, 20, 40 and 80 ng/L), anti-
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aflatoxin M1 antibody (concentrate), conjugate (peroxidase conjugated aflatoxin M1,
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concentrate), substrate /chromogen (tetramethylbenzidine), stop solution (1 N H2SO4), sample
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dilution buffer, conjugate, antibody dilution and washing buffer for the preparation of 10 mM
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phosphate buffer (PBS, pH 7.4) containing 0.05% Tween 20. Conjugate and antibody
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concentrates were diluted to 1:11 by the dilution buffer before analysis. Buffer salt was
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dissolved in one litre of distilled water and was ready for use for 4–6 weeks.
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Milk samples for LC-MS/MS analysis were prepared using immunoaffinity columns
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(IAC) VICAM Afla M1TM HPLC purchased from VICAM (Milford, USA). LC grade
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acetonitrile was purchased from Merck (Darmstadt, Germany). Ammonium formate (97%)
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ACCEPTED MANUSCRIPT and formic acid (≥ 96%) used for mobile phase were purchased from Sigma Aldrich Chemie
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GmbH (Germany). Nitrogen 5.0 and 5.5 were purchased from SOL spa (Monza, Italy). Ultra
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pure water was obtained by the Direct-Q® 5 UV Remote Water Purification System (Merck
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KGaA, Darmstadt, Germany). Aflatoxin M1 and internal standard aflatoxin B1 was obtained
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from Sigma-Aldrich (St. Louis, MO, USA). Mobile phase A consisted of 5 mM ammonium
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formate in water with the addition of 0.1% formic acid; mobile phase B was 0.1% formic acid
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in acetonitrile.
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The standard stock solutions of AFM1 and AFB1 (1000 µg/L) were prepared by
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dissolving certified reference material aflatoxin M1 solution (Product number: CRM46319,
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Supelco, Sigma-Aldrich, St. Louis, USA) in acetonitrile (LC grade). Working solutions were
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prepared at 10 and 100 ng/mL by further dilution of the stock solution and were used for
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preparation of the calibration curve and for spiking samples. Stock solutions were stored at
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4°C for no longer than 6 months, and working solutions were used within 3 months.
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Standards for the calibration curve were prepared in ultra pure water and acetonitrile
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(1:1) at concentrations of 0.2, 1, 2.5, 10 and 20 ng/mL of aflatoxin M1, where each calibration
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level included the internal standard aflatoxin B1 at 2.5 ng/mL.
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2.3. Instrumentation
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Milk samples for the ELISA method were prepared using the Vortex Genius 3 (IKA®
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-Werke GmbH & CO.KG, Germany) and centrifuge Rotanta 460R (Hettich GmbH & Co.KG,
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Tuttlingen, Germany). The Sunrise Absorbance Reader (Tecan Austria GmbH, Salzburg,
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Austria) was used to measure the optical density at 450 nm.
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The following equipment was used in sample preparation for LC-MS/MS analysis:
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IKA® Vortex model MS2 Minishaker (Staufen, Germany), Iskra ultrasonic bath (Slovenia), 6
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Supelco vacuum manifold (Bellefonte, PA), centrifuge Rotanta 460R (Hettich Zentrifugen,
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Tuttlingen, Germany) and Nitrogen evaporation system N-EVAP® model 112 (Orgamonation
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Associates Inc., USA). Analysis by high performance liquid chromatography with tandem mass spectrometry
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was carried out with the LC-MS/MS system, consisting of HPLC 1260 and Triple Quad
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LC/MS 6410 mass spectrometer (Agilent, Palo Alto, USA).
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2.3. ELISA test procedure
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The ELISA test procedure for the detection of AFM1 in raw milk was performed
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according to the manufacturer's instructions. The method was validated according the
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European Commission guidelines (European Commission, 2002) as previously described
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(Bilandžić et al., 2014a). The validation parameters were (ng/kg): detection capacity (CCβ)
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33.0, limit of detection (LOD) 22.2, limit of quantification (LOQ) 34.2.
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The quality of the results was tested in a proficiency test of milk powder organized by
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FAPAS (Food and Environmental Research Agency, York, UK), as Proficiency Test 04213 in
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2013. The proficiency test results were satisfactory, with a calculated z-score value of 0.9
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(acceptable range -2 ≤ z ≤ 2).
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Prior to analysis using the ELISA test, milk samples were centrifuged for 10 minutes
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at 3500×g at 10°C. After centrifugation, the upper cream layer was completely removed by
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aspirating through a Pasteur pipette. Skimmed milk was used directly in the test (100 µl per
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well). In the case where the AFM1 concentration exceeded 80 ng/mL, samples were diluted
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with sample dilution buffer from the test kit and reanalyzed.
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2.4. LC-MS-MS method
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2.4.1. Extraction of samples for LC-MS/MS
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Prior to analysis, 100 mL milk was defatted by centrifugation at 4000xg for 15
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minutes. IAC columns and reservoirs for application of the sample were attached to the
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vacuum manifold for solid phase extraction. A 50 g sample of defatted milk was passed
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through the column at a rate of 2.5 mL per minute. Columns were then washed twice with 10
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mL distilled water. Aflatoxin M1 was eluted with 2.5 mL acetonitrile at a rate of 0.5 mL per
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minute. The sample eluate was collected in the tube and in this step, the internal standard was
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added. The eluate was evaporated to dryness with nitrogen at 50±5°C and dissolved with 100
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µL ultra pure water and 100 µL acetonitrile, vortexed and left in an ultrasonic bath for 5
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minutes. Samples were further centrifuged at room temperature, for 15 minutes at 4500xg and
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filtered through 0.45 µm regenerated cellulose membrane filters prior to injection in the LC-
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MS/MS.
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2.4.2. Standard solution preparation
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Standard stock solutions of AFM1 and AFB1 (1000 µg/L) were prepared by
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dissolving the certified reference material Aflatoxin M1 solution (product number:
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CRM46319, Supelco, Sigma-Aldrich, St. Louis, USA) in acetonitrile (LC grade). Working
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solutions were prepared at 10 and 100 ng/mL by further dilution of the stock solution and
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were used for the preparation of the calibration curve and for the spiking of samples. Stock
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solutions were stored at 4°C for no longer than 6 months, and working solutions were used
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within 3 months.
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Standards for the calibration curve were prepared in ultrapure water and acetonitrile
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(1:1) at concentrations of 0.2, 1, 2.5, 10 and 20 ng/mL aflatoxin M1, where each calibration
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level included the internal standard aflatoxin B1 at 2.5 ng/mL.
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2.4.3. Chromatographic and MS parameters
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Chromatographic separation was achieved by isocratic elution on the Poroshell 120
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EC C18, 3x50 mm, 2.7 µm (Agilent, USA) with 60% mobile phase A and 40% mobile phase
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B. Mobile phase A consisted of 5 mM ammonium formate in water with the addition of 0.1%
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formic acid; mobile phase B was 0.1% formic acid in acetonitrile. The injection volume was 7
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µL and mobile phase flow was 0.3 mL/min. One chromatographic run was recorded in 2.5
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minutes. Column temperature was 30°C. The triplequad mass spectrometer consisted of an
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ESI ion source and was operated in positive mode, gas temperature 350°C, gas flow 5 L/min,
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nebulizer 35 psi, capillary voltage 4500 V. Data were acquired according to the multiple
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reaction monitoring approach (MRM), by selecting the two most intense ion transitions of the
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analytes, which is reported in Table 1.
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2.4.4. Method validation
The method was validated according to the criteria set by Commission Decision
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2002/657/EC (European Commission, 2002). Parameters determined were specificity
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(selectivity), matrix effect (ruggedness), linearity, repeatability (precision) and within-
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laboratory reproducibility, trueness, decision limit (CCα) and detection capability (CCβ).
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Linearity was calculated from the five point standard calibration curve at the following
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concentrations: 0.2, 1, 2.5, 10, 20 ng/mL. The concentration for the internal standard was set
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at 2.5 ng/mL for each level. The regression curve was prepared by plotting the ratio of the
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analyte area and internal standard area of the first transition (An1/An IS) versus the
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concentration of the analyte. Specificity was tested by analysing 20 representative blank cow
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milk samples in order to verify the absence of potential interfering compounds. Trueness was expressed in terms of recovery and precision and intra laboratory
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reproducibility as a relative standard deviation (RSD%). Samples were spiked at 0.5 times
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MRL, MRL and 1.5 times MRL (i.e., 0.025, 0.05, 0.075 µg/kg) in eight replicates and
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analysed on three different days by different analysts. The decision limit and detection
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capability were calculated by applying the calibration curve procedure. CCα was expressed as
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the sum of the average concentration of samples spiked at the MRL level and 1.64 times the
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reproducibility standard deviation at MRL. CCβ was calculated as the CCα value plus 1.64
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times the reproducibility standard deviation at CCα. Limit of detection (LOD) was calculated
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by multiplying the standard deviation of 21 samples spiked at 0.025 µg/kg with the Student t-
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value of 2.53. Limit of quantification (LOQ) was expressed as 10 times the standard deviation
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of 21 samples spiked at the lowest level.
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2.5. Statistical analysis
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Statistical analyses were performed using the Statistica 10 software package (StatSoft
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Inc., Tulsa, USA). Levels of AFM1 were expressed as mean ± SD, minimum and maximum
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value, percentages of samples exceeding the LOD and EU MRL.
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3. Results and discussion
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3.1. Method validation
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laboratory reproducibility of the LC-MS-MS method validated for the quantification of milk
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samples with elevated concentrations (>50 ng/kg) are summarized in Table 2. In the
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specificity testing, no naturally occurring substances were found in the elution region of the
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analyte. The linearity of the standard calibration curve was evaluated by calculating the
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coefficient of the regression curve (R2), which was not below 0.998. Recovery was calculated
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from the standard calibration curve and ranged between 60.1 and 118.2% for the three
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concentration levels. These are within the limits laid down by Commission Regulation
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401/2006 (European Commission, 2006a).
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Satisfactory values for precision and intra-laboratory reproducibility were achieved.
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Relative standard deviations (RSD%) of the intra-laboratory reproducibility were lower than
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14.9%. The chromatogram of spiked cow milk is shown in Fig. 1. The results indicate that the
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LC-MS/MS method used was reliable for the quantification of AFM1 in milk and met the
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criteria for detecting residues of AFM1.
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3.2. AFM1 concentrations in raw milk
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The AFM1 concentrations in raw milk from eastern, western and other regions of Croatia in
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the period from October 2013 to September 2014 are presented in Table 3. The results are
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shown by month, but are also pooled as seasons (e.g., January to March: spring). A
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chromatogram of an AFM1 negative uncontaminated sample is presented in Fig. 2. The mean
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AFM1 levels determined during four seasons in the three regions were in the ranges (ng/kg):
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eastern Croatia 7.25–26.6; western Croatia 5.91–9.26; other regions of Croatia 7.17–13.6. The
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highest total AFM1 mean concentrations (26.6 ng/kg) were measured in autumn (October–
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December) in the eastern region. An incidence of 58 samples (9.32%) was found to exceed
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the highest mean of 30.5 ng/kg and 5.94% samples exceeding the EU MRL in November.
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During autumn, there were no elevated AFM1 levels in the western and other regions. In
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winter, samples exceeding the EU MRL value were found in the regions (%): eastern 2.37,
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western 0.24, other 2.1. The highest concentrations of AFM1 were measured in December
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(764.4 ng/kg) and January (383.3 ng/kg). The LC-MS/MS chromatogram of the raw milk
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sample contaminated with AFM1 (383.3 ng/kg) is shown in Fig. 3.
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During spring, no samples exceeded the EU MRL in any of the regions. In summer,
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two samples with elevated concentrations were found in the eastern region, and one in the
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other regions. It can be concluded that the elevated concentrations found in four samples in
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the western and other regions, and two samples in the eastern region during the summer was a
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consequence of localized and random usage of contaminated feed for dairy cows on small
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farms, likely following storage in poor and inadequate conditions.
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Estimation of the AFM1 concentration between the LOD and EU MRL values (22.2–
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49.9 ng/kg) in three regions during autumn showed the following percentages (%): eastern
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28.9, western 4.45, other 6.67. In winter, outbreaks of these concentrations were determined
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in percentages by regions (%): eastern 4.45, western 1.18, other 12.6. In spring and summer,
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this incidence was even lower and was below 2.22%. Therefore, compared to the incidences
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found in a previous survey in the eastern region during winter and spring 2013 (13.9–23.6%),
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the incidences obtained in this study were significantly lower (Bilandžić et al., 2014a).
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In comparison to the range of 35.4–45.9% of raw milk samples exceeding the EU
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MRL during February and March 2013 (Bilandžić et al., 2014a), only a small proportion of
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such samples were found in eastern Croatia in the present study during autumn 2013 and
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winter 2014. In fact, only 4.21% of the total milk samples analyzed in a one-year period in the
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present study had AFM1 concentrations exceeding the permitted EU MRL value, which is
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significantly less than in the previous study, where elevated levels were found in 27.8% of
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samples. It is well known that supplementary feedstuffs, e.g. dry hay, corn and concentrated
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feed, are used in much greater amounts for cows during autumn and winter. If contaminated
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with AFB1, this results in an increased AFM1 content in milk (Prandini et al., 2009; Bilandžić
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et al., 2014a). Previously reported elevated levels of AFM1 in milk in eastern Croatia were
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confirmed with elevated levels of AFB1 measured in 36.5% of maize samples collected in
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three Croatian regions in 2013, with levels exceeding the maximal permitted level of 20 µg/kg
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(Pleadin et al., 2014).
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The AFM1 levels obtained in raw milk samples in different seasons from different
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countries are presented in Table 4. In recent years, global studies have shown seasonal
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variations of AFM1 concentrations and elevated levels (> 50 ng/L) in milk in winter in
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Pakistan, Croatia, Iran, Turkey, Morocco, Thailand, Serbia and China (Asi et al., 2012;
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Bilandžić et al., 2014a; Fallah et al., 2011; Golge, 2014; Marnissi, Belkhou, Morgavi,
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Bennani, & Boudra, 2012; Rahimi et al., 2010; Ruangwises & Ruangwises, 2009; Škrbić
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et al., 2014; Xiong et al., 2013). In the most recent studies conducted in Turkey and China,
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40.4% and 72.2% of milk samples respectively exceeded the EU MRL value in winter, with
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maximal concentrations of 1101 and 420 ng/kg (Golge, 2014; Xiong et al., 2013). Elevated
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levels of AFM1 (540–1440 ng/L) were also reported in February 2013 in Serbia (Škrbić et al.,
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2014).
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These studies confirmed that the occurrence of AFM1 poses a problem in countries
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with a dry climate or with seasons of long drought periods that favour the development of
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mould and elevated AFB1 levels in feed (Asi et al., 2012; Bilandžić et al., 2014b; Fallah et
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al., 2011; Prandini et al., 2009). Therefore, in tropical and subtropical climate zones, high
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AFM1 concentrations were measured throughout the year, with maximum levels in excess of
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Elzupir & Elhussein, 2010; Ghanem & Orfi, 2009; Hussain & Anwar, 2008; Iqbal & Asi,
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2013; Sadia et al. 2012). The maximum AFM1 levels measured in this study (December
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764.4 ng/kg) are comparable to the above maximum concentrations. For example, in recent
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studies in Pakistan, AFM1 levels exceeding the EU MRL were found in 71% and 41% of
331
milk samples (Iqbal & Asi, 2013; Sadia et al. 2012). Furthermore, in Sudan, 100% of samples
332
showed AFM1 levels exceeding 50 ng/kg, with maximal AFM1 concentrations of 6900 ng/L
333
(Elzupir & Elhussein, 2010).
SC
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326
334
4. Conclusions
M AN U
335 336
A previous study in Croatia reported the occurrence of elevated AFM1 concentrations
338
in eastern Croatia in 2013. The present study is a continuation of the monitoring of AFM1
339
concentrations in milk from farms in eastern Croatia over a one-year period, which also
340
included monitoring concentrations in raw milk from the western region and in other Croatian
341
regions. Therefore, this study is a large-scale effective control of AFM1 levels in raw milk in
342
accordance with the defined maximum residue levels of 50 ng/kg.
EP
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337
Markedly improved results were determined in this survey, with a much lower number
344
of samples with increased AFM1 concentrations in milk during autumn and winter. This can
345
be explained due to better weather conditions during the growth of grain used for
346
supplementary feeding of cows in 2013 and 2014, and proper and controlled conditions
347
during grain storage. Therefore, these results showed that the outbreak of elevated
348
concentrations of AFM1 in 2013 led to a more careful approach towards the control of
349
supplementary feedstuff for lactating cows.
AC C
343
14
ACCEPTED MANUSCRIPT Furthermore, it can be concluded that elevated concentrations were found in only four
351
samples in the western and other regions, and in two samples in the eastern region during
352
summer. This was the result of sporadic and localized usage of contaminated feed for dairy
353
cows on small farms. The results further support the conclusion that continuous inspection
354
and control of AFM1 in milk and dairy products, together with the control of AFB1 in raw
355
material and supplementary feedstuffs for dairy cattle is necessary.
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350
356
References
SC
357 358
Asi, R. M., Iqbal, S. Z., Ariño, A., & Hussain, A. (2012). Effect of seasonal variations and
360
lactation times on aflatoxin M1 contamination in milk of different species from Punjab,
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Pakistan. Food Control, 25, 34–38.
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Assem, E., Mohamad, A., & Oula, E. A. (2011). A survey on the occurrence of aflatoxin M1
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in raw and processed milk samples marketed in Lebanon. Food Control, 22, 1856-1858.
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Bilandžić, N., Varenina, I., & Solomun, B. (2010). Aflatoxin M1 in raw milk in Croatia. Food
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Control, 21, 1279-1281.
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Bilandžić, N., Božić, Đ., Đokić, M., Sedak, M., Solomun Kolanović, B., Varenina, I., et al.
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(2014a). Seasonal effect on aflatoxin M1 contamination in raw and UHT milk from Croatia.
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Bilandžić, N., Božić, Đ., Đokić, M., Sedak, M., Solomun Kolanović, B., Varenina, I., et al.
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Iqbal, S. Z., & Asi, M. R. (2013). Assessment of aflatoxin M1 in milk and milk products from
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Punjab, Pakistan. Food Control, 30, 235–239.
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Iqbal, S. Z., Asi, M. R., & Jinap S. (2013). Variation of aflatoxin M1 contamination in milk
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Marnissi, B. E., Belkhou, R., Morgavi, P.D., Bennani, L., & Boudra, H. (2012). Occurrence
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Toxicology, 50, 2819–2821.
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Nemati, M., Mehran, M. A., Hamed, P. K., & Masoud, A. (2010). A survey on the occurrence
432
of aflatoxin M1 in milk samples in Ardabil, Iran. Food Control, 21, 1022–1024.
433
Nuryono, N., Agus, A., Wedhastri, S., Maryudani, Y. B., Sigit Setyabudi, F. M. C., Böhm J.,
435
et al. (2009). A limited survey of aflatoxin M1 in milk from Indonesia by ELISA. Food
436
Control, 20, 721–724.
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Oruc, H. H., Cibik, R., Yilmaz, E., & Kalkanli, O. (2006). Distribution and stability of
439
aflatoxin M1 during processing and ripening of traditional white pickled cheese. Food
440
Additives and Contaminants, 23, 190–195.
441
AC C
438
442
Picinin, L. C. A., Cerqueira, M. M. O. P., Vargas, E. A., Lana, A. M. O., Toaldo, I. M., &
443
Bordignon-Luiz, M. T. (2013). Influence of climate conditions on aflatoxin M1 contamination
444
in raw milk from Minas Gerais State, Brazil. Food Control, 31, 419–424.
445
18
ACCEPTED MANUSCRIPT 446
Pleadin, J., Vulić, A., Perši, N., Škrivanko, M., Capek, B., & Cvetnić, Ž. (2014). Aflatoxin B1
447
occurrence in maize sampled from Croatian farms and feed factories during 2013. Food
448
Control, 40, 286-291.
449
Prandini, A., Tansini, G., Sigolo, S., Filippi, L., Laporta, M., & Piva, G. (2009). On the
451
occurrence of aflatoxin M1 in milk and dairy products. Food and Chemical Toxicology, 47,
452
984–991.
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450
SC
453
Rahimi, E., Bonyadian, M., Rafei, M., & Kazemeini, H. R. (2010). Occurrence of aflatoxin
455
M1 in raw milk of five dairy species in Ahvaz, Iran. Food and Chemical Toxicology, 48, 129–
456
131.
M AN U
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457
Ruangwises, S., & Ruangwises, N. (2009). Occurrence of aflatoxin M1 in pasteurized milk of
459
the school milk project in Thailand. Journal of Food Protection, 72, 1761–1763.
TE D
458
460
Sadia, A., Jabbar, M. A., Deng, J., Hussain, E. A., Riffat, S., Naveed, S., et al. (2012). A
462
survey of aflatoxin M1 in milk and sweets of Punjab, Pakistan. Food Control, 26, 235-240.
EP
461
AC C
463 464
Škrbić, B., Živančev, J., Antić, I., & Godula, M. (2014). Levels of aflatoxin M1 in different
465
types of milk collected in Serbia: Assessment of human and animal exposure. Food Control,
466
40, 113-119.
467 468
Tsakiris, I. N., Tzatzarakis, M. N., Alegakis, A. K., Vlachou, M. I., Renieri, E. A., &
469
Tsatsakis, A. M. (2013). Risk assessment scenarios of children’s exposure to aflatoxin M1
19
ACCEPTED MANUSCRIPT 470
residues in different milk types from the Greek market. Food and Chemical Toxicology 56,
471
261–265.
472
Xiong, J. L., Wang, Y. M., Ma, M. R., & Liu, J. X. (2013). Seasonal variation of aflatoxin M1
474
in raw milk from the Yangtze River Delta region of China. Food Control, 34, 703–706.
RI PT
473
AC C
EP
TE D
M AN U
SC
475
20
ACCEPTED MANUSCRIPT Table 1 MS/MS conditions for MRM analysis of sulphonamides. RT (min)
Precursor ion
Aflatoxin M1
16.8 ± 0.5
329 313
273 259 285 241
Fragmentor (V) 95 115
AC C
EP
TE D
M AN U
SC
Aflatoxin B1
Product ion
Collision energy 24 26 24 10
RI PT
Analyte
ACCEPTED MANUSCRIPT Table 2 Validation parameters of the LC-MS/MS method used for quantification of AFM1.
7.06 3.02
Recovery CCα (%) (ng/kg)
CCβ (ng/kg)
LOD (ng/kg)
LOQ (ng/kg)
96.0
64.64
7.26
28.71
87.6 87.7
M AN U TE D
75
6.23
EP
50
RSD (%)
AC C
25
Mean ±SD (ng/kg) 25.72 ± 1.60 49.42 ± 3.49 75.15 ± 2.27
56.49
SC
Spiking level (ng/kg)
Intra laboratory reproducibility (n = 24) Mean RSD ±SD (%) (ng/kg) 25.81 ± 14.9 2.87 49.47 ± 8.7 3.95 75.56 ± 12.6 4.92
RI PT
Precision (n = 8)
ACCEPTED MANUSCRIPT
Table 3
Western Croatia
Other regions
Month
n
Range (ng/kg)
Mean (ng/kg±SD)
n> LOD a
n exc. EU b
n
Range (ng/kg)
Mean (ng/kg±SD)
October
186
3.51-212.4
25.1 ± 21.1
64
15
147
3.08-45.1
10.0 ± 7.10
November
236
3.27-262.6
30.5 ± 23.9
90
37
188
2.89-45.2
10.2 ± 7.36
December
200
4.02-764.4
23.9 ± 73.4
26
6
193
2.48-33.6
7.79 ± 3.82
Total autumn
622
3.27-764.4
26.6 ± 44.7
180
58
528
2.48-45.2
January
140
4.08-383.3
16.2 ± 33.1
6
3
153
February
100
3.72-104.4
14.4 ± 13.9
6
3
148
March
97
3.44-101.7
12.3 ± 12.4
3
2
123
Total winter
337
3.44 - 379.6
14.7 ± 24.8
15
8
424
April
104
2.70-23.6
8.62 ± 4.26
2
0
119
May
89
3.84-48.1
9.88 ± 7.71
5
0
June
117
3.49-20.2
8.04 ± 3.04
0
0
Total spring
310
2.70-48.1
8.76 ± 5.19
7
July
100
2.93- 75.4
8.17 ± 7.34
1
August
106
2.37-53.0
8.25 ± 5.28
1
September
114
2.39-13.51
5.52 ± 2.11
Total
320
2.37-75.4
7.25 ± 5.40
n> LOD a
n exc. EU b
SC
Eastern Croatia
RI PT
Distribution of AFM1 concentrations in raw milk from eastern, western and other regions of Croatia during seasons 2013/2014.
n
Range (ng/kg)
Mean (ng/kg±SD)
n> LOD a
n exc. EU b
0
15
3.26-30.3
8.76 ± 7.20
0
0
13
0
18
1.65-48.6
12.5 ± 16.2
2
0
2
0
12
2.29-34.5
7.17 ± 8.93
1
0
9.26 ± 6.31
24
0
45
1.65-48.6
9.83 ± 11.9
3
0
3.88-21.2
8.59 ± 3.25
0
0
35
3.52-109.1
16.8 ± 23.4
5
2
4.05-68.6
8.97 ± 7.16
5
1
24
3.93-41.9
15.3 ± 9.86
5
0
TE D
M AN U
9
7.81 ± 2.52
0
0
36
2.57-39.7
9.33 ± 7.09
2
0
2.81 - 68.6
8.49 ± 4.86
5
1
95
2.57-109.1
13.6 ± 15.9
12
2
3.02-17.2
7.06 ± 2.16
0
0
55
4.32-42.0
7.86±5.83
2
0
EP
2.81-15.3
3.55-24.8
6.67 ± 2.95
1
0
33
2.40-42.1
6.97±6.91
0
0
119
3.83-17.9
7.17 ± 2.94
0
0
15
2.64-8.43
5.08±1.79
0
0
AC C
88
0
326
3.02-24.8
6.99 ± 2.68
1
0
103
2.40-42.1
7.17 ± 5.86
2
0
1
113
3.68- 14.2
6.37 ± 1.77
0
0
25
2.91-134.3
11.2±26.8
2
1
1
111
2.48-13.2
6.13 ± 1.83
0
0
36
1.81-8.13
7.04±8.45
0
0
0
0
107
0.11-13.9
5.20 ± 2.21
0
0
41
2.19-12.3
5.29±2.68
0
0
2
2
331
0.11-14.2
5.91 ± 2.00
0
0
102
1.81-224.4
8.23±22.5
2
1
ACCEPTED MANUSCRIPT
summer
TE D
M AN U
SC
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Number of samples above LOD and below EU MRL: 22.2 – 49.9 ng/kg Number of samples exceeding EU MRL
EP
b
AC C
a
ACCEPTED MANUSCRIPT
Table 4 Incidence of aflatoxin M1 in raw cow milk in different seasones measured worlwide.
Pakistan Pakistan Brazil
Pakistan Pakistan Lebanon Sudan Iran
RI PT
Samples exceeding EU MRL (%) 31.8 40.4 33.3 9.1 45.9 35.4 29.9 10.7 4.64 0 72.2 5.6 11.1 5.6 36 40
SC
n.s. n.s. n.s. n.s. 69.5 ± 114.6 55.3 ± 83.5 44.8 ± 50.0 26.1 ± 53.2 17.7 ± 13.5 14.1 ± 7.57 123.6 ± 101 29.1 ± 22.6 31.9 ± 26.7 31.6 ± 25.3 28 ± 2 73 ± 6
Maximal measured value (ng/L) 552 1101 150 102 1105.2 1135.0 398.6 470.5 85.9 36.4 420 98 82 76 n.s. n.s.
M AN U
China
Autumn Winter Spring Summer February March April May June July Winter Spring Summer Autumn Summer Winter
TE D
Croatia
63 47 January 2012 – December 2012 33 33 749 990 969 2013 393 280 355 n.s. November 2011 – n.s. September 2012 n.s. n.s. November 2011– 56 September 2012 48 November 2010 – 107 April 2011 43 August 2009 – 43 February 2010 43 27 2010 27 n.s. 17 2010 38 2009 44 24 2008 21
Seasone
EP
Turkey
Year
Mean (ng/L)
Autumn-Winter
150.7 ± 11.9
845.4
71
Dry period Transition period Rainy period Summer Winter n.s. Spring-summer n.s. Winter Spring
35.9 ± 4.4 17.1 ± 3.0 5.5 ± 9.4 22 ± 6 89 ± 2 110 ± 195 60.4 2070 93 ± 19 31 ± 5
105.7 70.9 24.9 95 150 794 126 6900
30.2 11.6 n.s. n.s. n.s. 41 73.6 100
394
35.2
AC C
Country
No of samples
Reference
Golge, 2014
Bilandžić et al. 2014
Xiong, Wang, Ma, & Liu, 2013
Iqbal, Asi, & Jinap, 2013 Iqbal & Asi, 2013 Picinin, Cerqueira, Vargas, Lana, Toaldo, & Bordignon-Luiz, 2013 Asi, Iqbal, Ariño, & Hussain, 2012 Sadia et al. 2012 Assem, Mohamad, & Oula, 2011 Elzupir & Elhussein, 2010 Fallah, Rahnama, Jafari, & Saei-Dehkordi, 2011
ACCEPTED MANUSCRIPT
75
2006
23 22 22 23
Thailand
2006–2007
Syria
2005–2006
74
2005
14 14 14 14 14 14 14 14 14 14 14 14
Pakistan
n.s. - not specified
28 ± 5 51 ± 6 60.1 ± 57.4
n.s.
Spring Summer Autumn Winter Sumer Rain seasone Winter April 2005 -April 2006 January February March April May June July August September October November December
52.9 ± 4.4 17.4 ± 3.1 22.3 ± 0.9 56.3 ± 6.6 50 ± 21 71 ± 28 89 ± 34
81.9 55.9 28.9 85.0
36
RI PT
Iran
2007–2008
Summer Autumn November 2007December 2008
Rahimi, Bonyadian, Rafei, & Kazemeini, 2010
33
Nemati, Mehran, Hamed, & Masoud, 2010
114
47.5 66.3 80
Ruangwises & Ruangwises, 2009
143 ± 53.22
690
95
Ghanem & Orfi, 2009
503 ± 92 466 ± 47 404 ± 74 398 ± 61 323 ± 44 351 ± 61 329 ± 75 199 ± 99 328 ± 71 345 ± 71 403 ± 57 403 ± 60
700 570 470 500 390 490 390 420 400 450 470 470
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
Hussain & Anwar, 2008
SC
Iran
Maximal Samples measured value exceeding EU Reference (ng/L) MRL (%)
M AN U
21 22
Mean (ng/L)
Seasone
TE D
No of samples
EP
Year
AC C
Country
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
Fig. 1. Bovine milk containing 25 ng/kg AFM1 and 10 ng/kg AFB1 as internal standard.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 2. Total ion chromatogram (TIC) and extracted ion chromatogram for AFM1 from the uncontaminated sample.
AC C
EP
TE D
M AN U
SC
RI PT
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Fig. 3. Total ion chromatogram (TIC) and extracted ion chromatogram for AFM1 from the contaminated raw milk sample at concentration of 383.3 ng/kg.
ACCEPTED MANUSCRIPT Highlights
► AFM1 concentrations in milk were monitored during one year period in Croatia. ► During autumn 9.32% samples exceeding the EU MRL in the eastern region.
RI PT
► In other seasons there were no differences in AFM1 levels between three regions.
AC C
EP
TE D
M AN U
SC
►Only a few samples with elevated concentrations were found during the summer.
S0956-7135(15)00097-3
DOI:
10.1016/j.foodcont.2015.02.015
Reference:
JFCO 4305
To appear in:
Food Control
Received Date: 13 November 2014 Revised Date:
2 February 2015
Accepted Date: 10 February 2015
Please cite this article as: Bilandžić N., Varenina I., Kolanović B.S., Božić Đ., Đokić M., Sedak M., Tanković S., Potočnjak D. & Cvetnić Ž., Monitoring of aflatoxin M1 in raw milk during four seasons in Croatia, Food Control (2015), doi: 10.1016/j.foodcont.2015.02.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Monitoring of aflatoxin M1 in raw milk during four
2
seasons in Croatia
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3
4
Nina Bilandžića,∗∗, Ivana Vareninaa, Božica Solomun Kolanovića, Đurđica Božića, Maja
5
Đokića, Marija Sedaka, Sanin Tankovićb, Dalibor Potočnjakc, Željko Cvetnićd
7
a
SC
6
Department of Veterinary Public Health, Laboratory for Residue Control, Croatian Veterinary Institute,
8
Savska cesta 143, HR-10000 Zagreb, Croatia
10
c
Veterinary Office of Bosnia and Herzegovina, Radićeva 8, 71000 Sarajevo, Bosnia and Herzegovina
M AN U
b
9
Department of Internal Medicine, Faculty of Veterinary Medicine, University of Zagreb, Heinzlova 55,
11 12
HR-10000 Zagreb, Croatia d
General Department, Croatian Veterinary Institute, Savska cesta 143, HR-10000 Zagreb, Croatia
13
ABSTRACT
15
A total of 3543 raw cow milk samples were collected in three regions of Croatia: western,
16
eastern and other regions during four seasons. Samples were measured for aflatoxin M1
17
(AFM1) concentrations using the enzyme immunoassay method. Elevated levels (>50 ng/kg)
18
of AFM1 were analysed by validated liquid chromatography with triple quadruple mass
19
spectrometry (LC-MS/MS). The limits of detection (LOD) and quantification (LOQ) of the
20
LC-MS/MS method were 7.3 and 28 ng/kg, respectively. The mean AFM1 levels measured in
21
the three regions over four seasons were in the ranges (ng/kg): eastern Croatia 7.25–26.6;
22
western Croatia 5.91–9.26; other regions of Croatia 7.17–13.6. The highest incidence of
23
samples exceeding the EU MRL (50 ng/kg) of 9.32% was measured in autumn (October–
24
December) in the eastern region. Only eight samples were found to exceed the EU MRL in
25
winter. The highest AFM1 levels were measured in December (764.4 ng/kg) and January
AC C
EP
TE D
14
1
ACCEPTED MANUSCRIPT (383.3 ng/kg). Elevated AFM1 levels were found in summer in only four samples from the
27
western and other regions, and two samples in the eastern region. This can be attributed to
28
localized and random usage of contaminated feed for dairy cows in those regions. The much
29
lower incidence of elevated AFM1 in comparison to a previous study showed that the
30
outbreak of the crisis due to elevated AFM1 levels in 2013 resulted in a more careful
31
approach to the control of supplementary feedstuff for lactating cows.
RI PT
26
32
Key words: Aflatoxin M1; Cow milk; ELISA; LC-MS/MS; Croatian regions
34
36
∗
Corresponding author. Tel.: +385 1 612 3601, fax: +385 1 612 3636
E-mail address: [email protected]
37
42
1. Introduction
TE D
38 39 40 41
M AN U
35
SC
33
Due to their high nutritional properties and high intake by all age groups, milk and
44
dairy products hold one of the most important roles in the human diet and, consequently, are
45
of great economic importance for every country. The presence of aflatoxin M1 (AFM1), as a
46
known xenobiotic, may have negative health implications for consumers and for agricultural
47
production, as its appearance in milk can incur economic damages due to production losses
48
(Tsakiris et al., 2013). This is emphasized by the fact that the AFM1 molecule cannot be
49
inactivated by thermal processing used in the dairy industry, i.e. pasteurization and ultra-high-
50
temperature treatment (Fallah, Rahnama, Jafari, & Saei-Dehkordi, 2011; Oruc, Cibik, Yilmaz,
51
& Kalkanli, 2006). AFM1 is the main hydroxyl-metabolite of the aflatoxin B1 (AFB1) that is
52
formed in the liver of lactating animals following consumption of contaminated feedstuffs
AC C
EP
43
2
ACCEPTED MANUSCRIPT (Prandini, Tansini, Sigolo, Filippi, Laporta, & Piva, 2009). AFB1 demonstrates teratogenic,
54
mutagenic and carcinogenic effects in mammals and has been classified as a Group 1 toxin.
55
Recently, AFM1 has been also classified in Group 1 as a possible hepatotoxic substance and
56
carcinogen for humans (IARC, 2002). AFM1 may cause DNA damage, which ultimately
57
leads to gene mutation, chromosomal anomalies and cell transformation in mammalians cells
58
(Prandini, Tansini, Sigolo, Filippi, Laporta, & Piva, 2009).
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When grazing is decreased in winter, significantly higher amounts of concentrated
60
feed, especially corn silage and grass, are given to meet the energy needs of high-yielding
61
cows. In the case of contaminated feed usage, AFM1 will be present in the milk for 2–3 days
62
following ingestion (Prandini et al., 2009). Therefore, the risk is largely related to grain-based
63
feed (Fink-Gremmels, 2008; Duarte et al., 2013). The transmission of AFB1 from foodstuffs
64
to milk (carry-over) in dairy cows is influenced by various nutritional and physiological
65
factors, including feeding, the degree of digestion, animal health, biotransformation capacity
66
of the liver, and milk production (Duarte et al., 2013).
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Contamination of milk and dairy products and variations in concentrations are
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associated with geographic location and climate, the degree of development of the region and
69
the season (Rahimi, Bonyadian, Rafei, & Kazemeini, 2010). Climatic conditions in tropical
70
and subtropical regions with high temperatures and drought, constant warmth and humidity
71
favour the growth of the toxigenic mould species Aspergillus (Picinin, Cerqueira, Vargas,
72
Lana, Toaldo, & Bordignon-Luiz, 2013). However, long periods of high temperatures and
73
long-lasting drought in summer during the maize-growing and maize-harvesting periods also
74
favour the development of these moulds in feed in other climatic regions (Bilandžić et al.,
75
2014a; Decastelli et al., 2007). With regard to the fact that the level of AFB1 contamination is
76
cumulative, harvesting time and conditions of drying and storage of foodstuffs can actually
77
have the same role in aflatoxin production. During grain storage, the drying process is the
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primary factor required to maintain a low moisture level, as irregular moisture of the mass
79
favours fungal development (Prandini et al., 2009). In recent years, elevated concentrations of AFM1 have been measured in different
81
countries around the world: Lebanon (Assem, Mohamad, & Oula, 2011), Iran (Fallah et al.,
82
2011; Nemati, Mehran, Hamed, & Masoud, 2010; Rahimi et al., 2010), Syria (Ghanem &
83
Orfi, 2009), Turkey (Golge, 2014), Pakistan (Asi, Iqbal, Ariño, & Hussain, 2012; Hussain &
84
Anwar, 2008; Iqbal & Asi, 2013; Sadia et al. 2012), South Africa (Dutton, Mwanza, de Kock,
85
& Khilosia, 2012), Sudan (Elzupir & Elhussein, 2010), Thailand (Ruangwises & Ruangwises,
86
2009), Indonesia (Nuryono et al., 2009), Brazil (Picinin, Cerqueira, Vargas, Lana, Toaldo, &
87
Bordignon-Luiz, 2013), China (Xiong, Wang, Ma, & Liu, 2013), Serbia (Škrbić, Živančev,
88
Antić, & Godula, 2014) and Croatia (Bilandžić, Varenina, & Solomun, 2010; Bilandžić et al.,
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2014a,b).
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Extremely elevated concentrations of AFM1 reported in 2013 in cow milk from
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eastern Croatia raised concerns and resulted in increased additional measures to control
92
AFM1 in milk and AFB1 contamination in feedstuffs (Bilandzic et al., 2014a; Pleadin, Vulić,
93
Perši, Škrivanko, Capek, & Cvetnić, 2014). Following this, the aim of this study was to
94
evaluate the concentrations of AFM1 in raw cow milk collected during a one-year period in
95
three regions of Croatia: western, eastern and other regions. For the purpose of quantifying
96
increased concentrations of AFM1, an additional aim of this study was to implement and
97
validate a confirmatory method of liquid chromatography–tandem mass spectrometry (LC-
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MS/MS).
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2. Materials and methods
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2.1. Sample collection 4
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A total of 3198 raw cow milk samples were collected in the period from October 2013
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to September 2014 from dairy farms in eastern (total n=1589) and western Croatia (total
106
n=1609). Milk samples were also collected on small farms across other Croatian regions, i.e.
107
from southern, southwestern, central and northern Croatia. However, as the number of
108
samples from these areas was significantly lower than the number of samples collected in
109
eastern and western Croatia, this group of samples was pooled into the group other regions
110
(total n=345). The volume of the collected milk samples was approximately 0.5 litres.
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Samples were stored at 2–8°C or frozen at -20°C until further analysis of AFM1.
112 113
2.2. Chemicals and reagents
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AFM1 concentrations were measured using competitive enzyme immunoassay
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(ELISA) of Ridascreen "Enzyme immunoassay for the quantitative analysis of aflatoxin M1"
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(R1121, R-Biopharm AG, Darmstadt, Germany). The reagents for the test kit contain: AFM1
118
standard solutions in milk buffer for the calibration curve (0, 5, 10, 20, 40 and 80 ng/L), anti-
119
aflatoxin M1 antibody (concentrate), conjugate (peroxidase conjugated aflatoxin M1,
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concentrate), substrate /chromogen (tetramethylbenzidine), stop solution (1 N H2SO4), sample
121
dilution buffer, conjugate, antibody dilution and washing buffer for the preparation of 10 mM
122
phosphate buffer (PBS, pH 7.4) containing 0.05% Tween 20. Conjugate and antibody
123
concentrates were diluted to 1:11 by the dilution buffer before analysis. Buffer salt was
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dissolved in one litre of distilled water and was ready for use for 4–6 weeks.
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Milk samples for LC-MS/MS analysis were prepared using immunoaffinity columns
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(IAC) VICAM Afla M1TM HPLC purchased from VICAM (Milford, USA). LC grade
127
acetonitrile was purchased from Merck (Darmstadt, Germany). Ammonium formate (97%)
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129
GmbH (Germany). Nitrogen 5.0 and 5.5 were purchased from SOL spa (Monza, Italy). Ultra
130
pure water was obtained by the Direct-Q® 5 UV Remote Water Purification System (Merck
131
KGaA, Darmstadt, Germany). Aflatoxin M1 and internal standard aflatoxin B1 was obtained
132
from Sigma-Aldrich (St. Louis, MO, USA). Mobile phase A consisted of 5 mM ammonium
133
formate in water with the addition of 0.1% formic acid; mobile phase B was 0.1% formic acid
134
in acetonitrile.
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The standard stock solutions of AFM1 and AFB1 (1000 µg/L) were prepared by
136
dissolving certified reference material aflatoxin M1 solution (Product number: CRM46319,
137
Supelco, Sigma-Aldrich, St. Louis, USA) in acetonitrile (LC grade). Working solutions were
138
prepared at 10 and 100 ng/mL by further dilution of the stock solution and were used for
139
preparation of the calibration curve and for spiking samples. Stock solutions were stored at
140
4°C for no longer than 6 months, and working solutions were used within 3 months.
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Standards for the calibration curve were prepared in ultra pure water and acetonitrile
142
(1:1) at concentrations of 0.2, 1, 2.5, 10 and 20 ng/mL of aflatoxin M1, where each calibration
143
level included the internal standard aflatoxin B1 at 2.5 ng/mL.
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2.3. Instrumentation
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Milk samples for the ELISA method were prepared using the Vortex Genius 3 (IKA®
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-Werke GmbH & CO.KG, Germany) and centrifuge Rotanta 460R (Hettich GmbH & Co.KG,
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Tuttlingen, Germany). The Sunrise Absorbance Reader (Tecan Austria GmbH, Salzburg,
150
Austria) was used to measure the optical density at 450 nm.
151
The following equipment was used in sample preparation for LC-MS/MS analysis:
152
IKA® Vortex model MS2 Minishaker (Staufen, Germany), Iskra ultrasonic bath (Slovenia), 6
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Supelco vacuum manifold (Bellefonte, PA), centrifuge Rotanta 460R (Hettich Zentrifugen,
154
Tuttlingen, Germany) and Nitrogen evaporation system N-EVAP® model 112 (Orgamonation
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Associates Inc., USA). Analysis by high performance liquid chromatography with tandem mass spectrometry
157
was carried out with the LC-MS/MS system, consisting of HPLC 1260 and Triple Quad
158
LC/MS 6410 mass spectrometer (Agilent, Palo Alto, USA).
159
2.3. ELISA test procedure
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The ELISA test procedure for the detection of AFM1 in raw milk was performed
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according to the manufacturer's instructions. The method was validated according the
164
European Commission guidelines (European Commission, 2002) as previously described
165
(Bilandžić et al., 2014a). The validation parameters were (ng/kg): detection capacity (CCβ)
166
33.0, limit of detection (LOD) 22.2, limit of quantification (LOQ) 34.2.
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The quality of the results was tested in a proficiency test of milk powder organized by
168
FAPAS (Food and Environmental Research Agency, York, UK), as Proficiency Test 04213 in
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2013. The proficiency test results were satisfactory, with a calculated z-score value of 0.9
170
(acceptable range -2 ≤ z ≤ 2).
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Prior to analysis using the ELISA test, milk samples were centrifuged for 10 minutes
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at 3500×g at 10°C. After centrifugation, the upper cream layer was completely removed by
173
aspirating through a Pasteur pipette. Skimmed milk was used directly in the test (100 µl per
174
well). In the case where the AFM1 concentration exceeded 80 ng/mL, samples were diluted
175
with sample dilution buffer from the test kit and reanalyzed.
176 177
2.4. LC-MS-MS method
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2.4.1. Extraction of samples for LC-MS/MS
179
Prior to analysis, 100 mL milk was defatted by centrifugation at 4000xg for 15
181
minutes. IAC columns and reservoirs for application of the sample were attached to the
182
vacuum manifold for solid phase extraction. A 50 g sample of defatted milk was passed
183
through the column at a rate of 2.5 mL per minute. Columns were then washed twice with 10
184
mL distilled water. Aflatoxin M1 was eluted with 2.5 mL acetonitrile at a rate of 0.5 mL per
185
minute. The sample eluate was collected in the tube and in this step, the internal standard was
186
added. The eluate was evaporated to dryness with nitrogen at 50±5°C and dissolved with 100
187
µL ultra pure water and 100 µL acetonitrile, vortexed and left in an ultrasonic bath for 5
188
minutes. Samples were further centrifuged at room temperature, for 15 minutes at 4500xg and
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filtered through 0.45 µm regenerated cellulose membrane filters prior to injection in the LC-
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MS/MS.
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2.4.2. Standard solution preparation
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Standard stock solutions of AFM1 and AFB1 (1000 µg/L) were prepared by
195
dissolving the certified reference material Aflatoxin M1 solution (product number:
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CRM46319, Supelco, Sigma-Aldrich, St. Louis, USA) in acetonitrile (LC grade). Working
197
solutions were prepared at 10 and 100 ng/mL by further dilution of the stock solution and
198
were used for the preparation of the calibration curve and for the spiking of samples. Stock
199
solutions were stored at 4°C for no longer than 6 months, and working solutions were used
200
within 3 months.
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Standards for the calibration curve were prepared in ultrapure water and acetonitrile
202
(1:1) at concentrations of 0.2, 1, 2.5, 10 and 20 ng/mL aflatoxin M1, where each calibration
203
level included the internal standard aflatoxin B1 at 2.5 ng/mL.
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2.4.3. Chromatographic and MS parameters
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Chromatographic separation was achieved by isocratic elution on the Poroshell 120
208
EC C18, 3x50 mm, 2.7 µm (Agilent, USA) with 60% mobile phase A and 40% mobile phase
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B. Mobile phase A consisted of 5 mM ammonium formate in water with the addition of 0.1%
210
formic acid; mobile phase B was 0.1% formic acid in acetonitrile. The injection volume was 7
211
µL and mobile phase flow was 0.3 mL/min. One chromatographic run was recorded in 2.5
212
minutes. Column temperature was 30°C. The triplequad mass spectrometer consisted of an
213
ESI ion source and was operated in positive mode, gas temperature 350°C, gas flow 5 L/min,
214
nebulizer 35 psi, capillary voltage 4500 V. Data were acquired according to the multiple
215
reaction monitoring approach (MRM), by selecting the two most intense ion transitions of the
216
analytes, which is reported in Table 1.
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2.4.4. Method validation
The method was validated according to the criteria set by Commission Decision
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2002/657/EC (European Commission, 2002). Parameters determined were specificity
221
(selectivity), matrix effect (ruggedness), linearity, repeatability (precision) and within-
222
laboratory reproducibility, trueness, decision limit (CCα) and detection capability (CCβ).
223
Linearity was calculated from the five point standard calibration curve at the following
224
concentrations: 0.2, 1, 2.5, 10, 20 ng/mL. The concentration for the internal standard was set
225
at 2.5 ng/mL for each level. The regression curve was prepared by plotting the ratio of the
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analyte area and internal standard area of the first transition (An1/An IS) versus the
227
concentration of the analyte. Specificity was tested by analysing 20 representative blank cow
228
milk samples in order to verify the absence of potential interfering compounds. Trueness was expressed in terms of recovery and precision and intra laboratory
230
reproducibility as a relative standard deviation (RSD%). Samples were spiked at 0.5 times
231
MRL, MRL and 1.5 times MRL (i.e., 0.025, 0.05, 0.075 µg/kg) in eight replicates and
232
analysed on three different days by different analysts. The decision limit and detection
233
capability were calculated by applying the calibration curve procedure. CCα was expressed as
234
the sum of the average concentration of samples spiked at the MRL level and 1.64 times the
235
reproducibility standard deviation at MRL. CCβ was calculated as the CCα value plus 1.64
236
times the reproducibility standard deviation at CCα. Limit of detection (LOD) was calculated
237
by multiplying the standard deviation of 21 samples spiked at 0.025 µg/kg with the Student t-
238
value of 2.53. Limit of quantification (LOQ) was expressed as 10 times the standard deviation
239
of 21 samples spiked at the lowest level.
240
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2.5. Statistical analysis
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Statistical analyses were performed using the Statistica 10 software package (StatSoft
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Inc., Tulsa, USA). Levels of AFM1 were expressed as mean ± SD, minimum and maximum
245
value, percentages of samples exceeding the LOD and EU MRL.
246 247
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3. Results and discussion
248 249
3.1. Method validation
250
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252
laboratory reproducibility of the LC-MS-MS method validated for the quantification of milk
253
samples with elevated concentrations (>50 ng/kg) are summarized in Table 2. In the
254
specificity testing, no naturally occurring substances were found in the elution region of the
255
analyte. The linearity of the standard calibration curve was evaluated by calculating the
256
coefficient of the regression curve (R2), which was not below 0.998. Recovery was calculated
257
from the standard calibration curve and ranged between 60.1 and 118.2% for the three
258
concentration levels. These are within the limits laid down by Commission Regulation
259
401/2006 (European Commission, 2006a).
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Satisfactory values for precision and intra-laboratory reproducibility were achieved.
261
Relative standard deviations (RSD%) of the intra-laboratory reproducibility were lower than
262
14.9%. The chromatogram of spiked cow milk is shown in Fig. 1. The results indicate that the
263
LC-MS/MS method used was reliable for the quantification of AFM1 in milk and met the
264
criteria for detecting residues of AFM1.
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The AFM1 concentrations in raw milk from eastern, western and other regions of Croatia in
269
the period from October 2013 to September 2014 are presented in Table 3. The results are
270
shown by month, but are also pooled as seasons (e.g., January to March: spring). A
271
chromatogram of an AFM1 negative uncontaminated sample is presented in Fig. 2. The mean
272
AFM1 levels determined during four seasons in the three regions were in the ranges (ng/kg):
273
eastern Croatia 7.25–26.6; western Croatia 5.91–9.26; other regions of Croatia 7.17–13.6. The
274
highest total AFM1 mean concentrations (26.6 ng/kg) were measured in autumn (October–
275
December) in the eastern region. An incidence of 58 samples (9.32%) was found to exceed
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277
the highest mean of 30.5 ng/kg and 5.94% samples exceeding the EU MRL in November.
278
During autumn, there were no elevated AFM1 levels in the western and other regions. In
279
winter, samples exceeding the EU MRL value were found in the regions (%): eastern 2.37,
280
western 0.24, other 2.1. The highest concentrations of AFM1 were measured in December
281
(764.4 ng/kg) and January (383.3 ng/kg). The LC-MS/MS chromatogram of the raw milk
282
sample contaminated with AFM1 (383.3 ng/kg) is shown in Fig. 3.
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During spring, no samples exceeded the EU MRL in any of the regions. In summer,
284
two samples with elevated concentrations were found in the eastern region, and one in the
285
other regions. It can be concluded that the elevated concentrations found in four samples in
286
the western and other regions, and two samples in the eastern region during the summer was a
287
consequence of localized and random usage of contaminated feed for dairy cows on small
288
farms, likely following storage in poor and inadequate conditions.
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Estimation of the AFM1 concentration between the LOD and EU MRL values (22.2–
290
49.9 ng/kg) in three regions during autumn showed the following percentages (%): eastern
291
28.9, western 4.45, other 6.67. In winter, outbreaks of these concentrations were determined
292
in percentages by regions (%): eastern 4.45, western 1.18, other 12.6. In spring and summer,
293
this incidence was even lower and was below 2.22%. Therefore, compared to the incidences
294
found in a previous survey in the eastern region during winter and spring 2013 (13.9–23.6%),
295
the incidences obtained in this study were significantly lower (Bilandžić et al., 2014a).
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In comparison to the range of 35.4–45.9% of raw milk samples exceeding the EU
297
MRL during February and March 2013 (Bilandžić et al., 2014a), only a small proportion of
298
such samples were found in eastern Croatia in the present study during autumn 2013 and
299
winter 2014. In fact, only 4.21% of the total milk samples analyzed in a one-year period in the
300
present study had AFM1 concentrations exceeding the permitted EU MRL value, which is
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ACCEPTED MANUSCRIPT 301
significantly less than in the previous study, where elevated levels were found in 27.8% of
302
samples. It is well known that supplementary feedstuffs, e.g. dry hay, corn and concentrated
304
feed, are used in much greater amounts for cows during autumn and winter. If contaminated
305
with AFB1, this results in an increased AFM1 content in milk (Prandini et al., 2009; Bilandžić
306
et al., 2014a). Previously reported elevated levels of AFM1 in milk in eastern Croatia were
307
confirmed with elevated levels of AFB1 measured in 36.5% of maize samples collected in
308
three Croatian regions in 2013, with levels exceeding the maximal permitted level of 20 µg/kg
309
(Pleadin et al., 2014).
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The AFM1 levels obtained in raw milk samples in different seasons from different
311
countries are presented in Table 4. In recent years, global studies have shown seasonal
312
variations of AFM1 concentrations and elevated levels (> 50 ng/L) in milk in winter in
313
Pakistan, Croatia, Iran, Turkey, Morocco, Thailand, Serbia and China (Asi et al., 2012;
314
Bilandžić et al., 2014a; Fallah et al., 2011; Golge, 2014; Marnissi, Belkhou, Morgavi,
315
Bennani, & Boudra, 2012; Rahimi et al., 2010; Ruangwises & Ruangwises, 2009; Škrbić
316
et al., 2014; Xiong et al., 2013). In the most recent studies conducted in Turkey and China,
317
40.4% and 72.2% of milk samples respectively exceeded the EU MRL value in winter, with
318
maximal concentrations of 1101 and 420 ng/kg (Golge, 2014; Xiong et al., 2013). Elevated
319
levels of AFM1 (540–1440 ng/L) were also reported in February 2013 in Serbia (Škrbić et al.,
320
2014).
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These studies confirmed that the occurrence of AFM1 poses a problem in countries
322
with a dry climate or with seasons of long drought periods that favour the development of
323
mould and elevated AFB1 levels in feed (Asi et al., 2012; Bilandžić et al., 2014b; Fallah et
324
al., 2011; Prandini et al., 2009). Therefore, in tropical and subtropical climate zones, high
325
AFM1 concentrations were measured throughout the year, with maximum levels in excess of
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327
Elzupir & Elhussein, 2010; Ghanem & Orfi, 2009; Hussain & Anwar, 2008; Iqbal & Asi,
328
2013; Sadia et al. 2012). The maximum AFM1 levels measured in this study (December
329
764.4 ng/kg) are comparable to the above maximum concentrations. For example, in recent
330
studies in Pakistan, AFM1 levels exceeding the EU MRL were found in 71% and 41% of
331
milk samples (Iqbal & Asi, 2013; Sadia et al. 2012). Furthermore, in Sudan, 100% of samples
332
showed AFM1 levels exceeding 50 ng/kg, with maximal AFM1 concentrations of 6900 ng/L
333
(Elzupir & Elhussein, 2010).
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4. Conclusions
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A previous study in Croatia reported the occurrence of elevated AFM1 concentrations
338
in eastern Croatia in 2013. The present study is a continuation of the monitoring of AFM1
339
concentrations in milk from farms in eastern Croatia over a one-year period, which also
340
included monitoring concentrations in raw milk from the western region and in other Croatian
341
regions. Therefore, this study is a large-scale effective control of AFM1 levels in raw milk in
342
accordance with the defined maximum residue levels of 50 ng/kg.
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Markedly improved results were determined in this survey, with a much lower number
344
of samples with increased AFM1 concentrations in milk during autumn and winter. This can
345
be explained due to better weather conditions during the growth of grain used for
346
supplementary feeding of cows in 2013 and 2014, and proper and controlled conditions
347
during grain storage. Therefore, these results showed that the outbreak of elevated
348
concentrations of AFM1 in 2013 led to a more careful approach towards the control of
349
supplementary feedstuff for lactating cows.
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351
samples in the western and other regions, and in two samples in the eastern region during
352
summer. This was the result of sporadic and localized usage of contaminated feed for dairy
353
cows on small farms. The results further support the conclusion that continuous inspection
354
and control of AFM1 in milk and dairy products, together with the control of AFB1 in raw
355
material and supplementary feedstuffs for dairy cattle is necessary.
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References
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lactation times on aflatoxin M1 contamination in milk of different species from Punjab,
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Fallah, A. A., Rahnama, M., Jafari, T., & Saei-Dehkordi, S. S. (2011). Seasonal variation of
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407 408
Ghanem, I., & Orfi, M. (2009). Aflatoxin M1 in raw, pasteurized and powdered milk
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available in the Syrian market. Food Control, 20, 603–605.
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410
Golge, O. (2014). A survey on the occurrence of aflatoxin M1 in raw milk produced in Adana
412
province of Turkey. Food Control, 45, 150-155.
413
Hussain, I., & Anwar, J. (2008). A study on contamination of aflatoxin M1 in raw milk in the
414
Punjab province of Pakistan. Food Control, 19, 393–395.
AC C
415
EP
411
416
IARC, International Agency for Research on Cancer. (2002). Some traditional herbal
417
medicines, some mycotoxins, naphthalene and styrene. In IARC monograph on the evaluation
418
of carcinogenic risk to humans, Vol. 82 (pp. 171-175). Lyon, France: IARC Scientific
419
Publication.
420
17
ACCEPTED MANUSCRIPT 421
Iqbal, S. Z., & Asi, M. R. (2013). Assessment of aflatoxin M1 in milk and milk products from
422
Punjab, Pakistan. Food Control, 30, 235–239.
423
Iqbal, S. Z., Asi, M. R., & Jinap S. (2013). Variation of aflatoxin M1 contamination in milk
425
and milk products collected during winter and summer seasons. Food Control , 34, 714–718
RI PT
424
426
Marnissi, B. E., Belkhou, R., Morgavi, P.D., Bennani, L., & Boudra, H. (2012). Occurrence
428
of aflatoxin M1 in raw milk collected from traditional dairies in Morocco. Food and Chemical
429
Toxicology, 50, 2819–2821.
M AN U
SC
427
430 431
Nemati, M., Mehran, M. A., Hamed, P. K., & Masoud, A. (2010). A survey on the occurrence
432
of aflatoxin M1 in milk samples in Ardabil, Iran. Food Control, 21, 1022–1024.
433
Nuryono, N., Agus, A., Wedhastri, S., Maryudani, Y. B., Sigit Setyabudi, F. M. C., Böhm J.,
435
et al. (2009). A limited survey of aflatoxin M1 in milk from Indonesia by ELISA. Food
436
Control, 20, 721–724.
EP
437
TE D
434
Oruc, H. H., Cibik, R., Yilmaz, E., & Kalkanli, O. (2006). Distribution and stability of
439
aflatoxin M1 during processing and ripening of traditional white pickled cheese. Food
440
Additives and Contaminants, 23, 190–195.
441
AC C
438
442
Picinin, L. C. A., Cerqueira, M. M. O. P., Vargas, E. A., Lana, A. M. O., Toaldo, I. M., &
443
Bordignon-Luiz, M. T. (2013). Influence of climate conditions on aflatoxin M1 contamination
444
in raw milk from Minas Gerais State, Brazil. Food Control, 31, 419–424.
445
18
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Pleadin, J., Vulić, A., Perši, N., Škrivanko, M., Capek, B., & Cvetnić, Ž. (2014). Aflatoxin B1
447
occurrence in maize sampled from Croatian farms and feed factories during 2013. Food
448
Control, 40, 286-291.
449
Prandini, A., Tansini, G., Sigolo, S., Filippi, L., Laporta, M., & Piva, G. (2009). On the
451
occurrence of aflatoxin M1 in milk and dairy products. Food and Chemical Toxicology, 47,
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984–991.
RI PT
450
SC
453
Rahimi, E., Bonyadian, M., Rafei, M., & Kazemeini, H. R. (2010). Occurrence of aflatoxin
455
M1 in raw milk of five dairy species in Ahvaz, Iran. Food and Chemical Toxicology, 48, 129–
456
131.
M AN U
454
457
Ruangwises, S., & Ruangwises, N. (2009). Occurrence of aflatoxin M1 in pasteurized milk of
459
the school milk project in Thailand. Journal of Food Protection, 72, 1761–1763.
TE D
458
460
Sadia, A., Jabbar, M. A., Deng, J., Hussain, E. A., Riffat, S., Naveed, S., et al. (2012). A
462
survey of aflatoxin M1 in milk and sweets of Punjab, Pakistan. Food Control, 26, 235-240.
EP
461
AC C
463 464
Škrbić, B., Živančev, J., Antić, I., & Godula, M. (2014). Levels of aflatoxin M1 in different
465
types of milk collected in Serbia: Assessment of human and animal exposure. Food Control,
466
40, 113-119.
467 468
Tsakiris, I. N., Tzatzarakis, M. N., Alegakis, A. K., Vlachou, M. I., Renieri, E. A., &
469
Tsatsakis, A. M. (2013). Risk assessment scenarios of children’s exposure to aflatoxin M1
19
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residues in different milk types from the Greek market. Food and Chemical Toxicology 56,
471
261–265.
472
Xiong, J. L., Wang, Y. M., Ma, M. R., & Liu, J. X. (2013). Seasonal variation of aflatoxin M1
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in raw milk from the Yangtze River Delta region of China. Food Control, 34, 703–706.
RI PT
473
AC C
EP
TE D
M AN U
SC
475
20
ACCEPTED MANUSCRIPT Table 1 MS/MS conditions for MRM analysis of sulphonamides. RT (min)
Precursor ion
Aflatoxin M1
16.8 ± 0.5
329 313
273 259 285 241
Fragmentor (V) 95 115
AC C
EP
TE D
M AN U
SC
Aflatoxin B1
Product ion
Collision energy 24 26 24 10
RI PT
Analyte
ACCEPTED MANUSCRIPT Table 2 Validation parameters of the LC-MS/MS method used for quantification of AFM1.
7.06 3.02
Recovery CCα (%) (ng/kg)
CCβ (ng/kg)
LOD (ng/kg)
LOQ (ng/kg)
96.0
64.64
7.26
28.71
87.6 87.7
M AN U TE D
75
6.23
EP
50
RSD (%)
AC C
25
Mean ±SD (ng/kg) 25.72 ± 1.60 49.42 ± 3.49 75.15 ± 2.27
56.49
SC
Spiking level (ng/kg)
Intra laboratory reproducibility (n = 24) Mean RSD ±SD (%) (ng/kg) 25.81 ± 14.9 2.87 49.47 ± 8.7 3.95 75.56 ± 12.6 4.92
RI PT
Precision (n = 8)
ACCEPTED MANUSCRIPT
Table 3
Western Croatia
Other regions
Month
n
Range (ng/kg)
Mean (ng/kg±SD)
n> LOD a
n exc. EU b
n
Range (ng/kg)
Mean (ng/kg±SD)
October
186
3.51-212.4
25.1 ± 21.1
64
15
147
3.08-45.1
10.0 ± 7.10
November
236
3.27-262.6
30.5 ± 23.9
90
37
188
2.89-45.2
10.2 ± 7.36
December
200
4.02-764.4
23.9 ± 73.4
26
6
193
2.48-33.6
7.79 ± 3.82
Total autumn
622
3.27-764.4
26.6 ± 44.7
180
58
528
2.48-45.2
January
140
4.08-383.3
16.2 ± 33.1
6
3
153
February
100
3.72-104.4
14.4 ± 13.9
6
3
148
March
97
3.44-101.7
12.3 ± 12.4
3
2
123
Total winter
337
3.44 - 379.6
14.7 ± 24.8
15
8
424
April
104
2.70-23.6
8.62 ± 4.26
2
0
119
May
89
3.84-48.1
9.88 ± 7.71
5
0
June
117
3.49-20.2
8.04 ± 3.04
0
0
Total spring
310
2.70-48.1
8.76 ± 5.19
7
July
100
2.93- 75.4
8.17 ± 7.34
1
August
106
2.37-53.0
8.25 ± 5.28
1
September
114
2.39-13.51
5.52 ± 2.11
Total
320
2.37-75.4
7.25 ± 5.40
n> LOD a
n exc. EU b
SC
Eastern Croatia
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Distribution of AFM1 concentrations in raw milk from eastern, western and other regions of Croatia during seasons 2013/2014.
n
Range (ng/kg)
Mean (ng/kg±SD)
n> LOD a
n exc. EU b
0
15
3.26-30.3
8.76 ± 7.20
0
0
13
0
18
1.65-48.6
12.5 ± 16.2
2
0
2
0
12
2.29-34.5
7.17 ± 8.93
1
0
9.26 ± 6.31
24
0
45
1.65-48.6
9.83 ± 11.9
3
0
3.88-21.2
8.59 ± 3.25
0
0
35
3.52-109.1
16.8 ± 23.4
5
2
4.05-68.6
8.97 ± 7.16
5
1
24
3.93-41.9
15.3 ± 9.86
5
0
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M AN U
9
7.81 ± 2.52
0
0
36
2.57-39.7
9.33 ± 7.09
2
0
2.81 - 68.6
8.49 ± 4.86
5
1
95
2.57-109.1
13.6 ± 15.9
12
2
3.02-17.2
7.06 ± 2.16
0
0
55
4.32-42.0
7.86±5.83
2
0
EP
2.81-15.3
3.55-24.8
6.67 ± 2.95
1
0
33
2.40-42.1
6.97±6.91
0
0
119
3.83-17.9
7.17 ± 2.94
0
0
15
2.64-8.43
5.08±1.79
0
0
AC C
88
0
326
3.02-24.8
6.99 ± 2.68
1
0
103
2.40-42.1
7.17 ± 5.86
2
0
1
113
3.68- 14.2
6.37 ± 1.77
0
0
25
2.91-134.3
11.2±26.8
2
1
1
111
2.48-13.2
6.13 ± 1.83
0
0
36
1.81-8.13
7.04±8.45
0
0
0
0
107
0.11-13.9
5.20 ± 2.21
0
0
41
2.19-12.3
5.29±2.68
0
0
2
2
331
0.11-14.2
5.91 ± 2.00
0
0
102
1.81-224.4
8.23±22.5
2
1
ACCEPTED MANUSCRIPT
summer
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Number of samples above LOD and below EU MRL: 22.2 – 49.9 ng/kg Number of samples exceeding EU MRL
EP
b
AC C
a
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Table 4 Incidence of aflatoxin M1 in raw cow milk in different seasones measured worlwide.
Pakistan Pakistan Brazil
Pakistan Pakistan Lebanon Sudan Iran
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Samples exceeding EU MRL (%) 31.8 40.4 33.3 9.1 45.9 35.4 29.9 10.7 4.64 0 72.2 5.6 11.1 5.6 36 40
SC
n.s. n.s. n.s. n.s. 69.5 ± 114.6 55.3 ± 83.5 44.8 ± 50.0 26.1 ± 53.2 17.7 ± 13.5 14.1 ± 7.57 123.6 ± 101 29.1 ± 22.6 31.9 ± 26.7 31.6 ± 25.3 28 ± 2 73 ± 6
Maximal measured value (ng/L) 552 1101 150 102 1105.2 1135.0 398.6 470.5 85.9 36.4 420 98 82 76 n.s. n.s.
M AN U
China
Autumn Winter Spring Summer February March April May June July Winter Spring Summer Autumn Summer Winter
TE D
Croatia
63 47 January 2012 – December 2012 33 33 749 990 969 2013 393 280 355 n.s. November 2011 – n.s. September 2012 n.s. n.s. November 2011– 56 September 2012 48 November 2010 – 107 April 2011 43 August 2009 – 43 February 2010 43 27 2010 27 n.s. 17 2010 38 2009 44 24 2008 21
Seasone
EP
Turkey
Year
Mean (ng/L)
Autumn-Winter
150.7 ± 11.9
845.4
71
Dry period Transition period Rainy period Summer Winter n.s. Spring-summer n.s. Winter Spring
35.9 ± 4.4 17.1 ± 3.0 5.5 ± 9.4 22 ± 6 89 ± 2 110 ± 195 60.4 2070 93 ± 19 31 ± 5
105.7 70.9 24.9 95 150 794 126 6900
30.2 11.6 n.s. n.s. n.s. 41 73.6 100
394
35.2
AC C
Country
No of samples
Reference
Golge, 2014
Bilandžić et al. 2014
Xiong, Wang, Ma, & Liu, 2013
Iqbal, Asi, & Jinap, 2013 Iqbal & Asi, 2013 Picinin, Cerqueira, Vargas, Lana, Toaldo, & Bordignon-Luiz, 2013 Asi, Iqbal, Ariño, & Hussain, 2012 Sadia et al. 2012 Assem, Mohamad, & Oula, 2011 Elzupir & Elhussein, 2010 Fallah, Rahnama, Jafari, & Saei-Dehkordi, 2011
ACCEPTED MANUSCRIPT
75
2006
23 22 22 23
Thailand
2006–2007
Syria
2005–2006
74
2005
14 14 14 14 14 14 14 14 14 14 14 14
Pakistan
n.s. - not specified
28 ± 5 51 ± 6 60.1 ± 57.4
n.s.
Spring Summer Autumn Winter Sumer Rain seasone Winter April 2005 -April 2006 January February March April May June July August September October November December
52.9 ± 4.4 17.4 ± 3.1 22.3 ± 0.9 56.3 ± 6.6 50 ± 21 71 ± 28 89 ± 34
81.9 55.9 28.9 85.0
36
RI PT
Iran
2007–2008
Summer Autumn November 2007December 2008
Rahimi, Bonyadian, Rafei, & Kazemeini, 2010
33
Nemati, Mehran, Hamed, & Masoud, 2010
114
47.5 66.3 80
Ruangwises & Ruangwises, 2009
143 ± 53.22
690
95
Ghanem & Orfi, 2009
503 ± 92 466 ± 47 404 ± 74 398 ± 61 323 ± 44 351 ± 61 329 ± 75 199 ± 99 328 ± 71 345 ± 71 403 ± 57 403 ± 60
700 570 470 500 390 490 390 420 400 450 470 470
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
Hussain & Anwar, 2008
SC
Iran
Maximal Samples measured value exceeding EU Reference (ng/L) MRL (%)
M AN U
21 22
Mean (ng/L)
Seasone
TE D
No of samples
EP
Year
AC C
Country
SC
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EP
TE D
M AN U
Fig. 1. Bovine milk containing 25 ng/kg AFM1 and 10 ng/kg AFB1 as internal standard.
AC C
EP
TE D
M AN U
SC
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ACCEPTED MANUSCRIPT
Fig. 2. Total ion chromatogram (TIC) and extracted ion chromatogram for AFM1 from the uncontaminated sample.
AC C
EP
TE D
M AN U
SC
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Fig. 3. Total ion chromatogram (TIC) and extracted ion chromatogram for AFM1 from the contaminated raw milk sample at concentration of 383.3 ng/kg.
ACCEPTED MANUSCRIPT Highlights
► AFM1 concentrations in milk were monitored during one year period in Croatia. ► During autumn 9.32% samples exceeding the EU MRL in the eastern region.
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► In other seasons there were no differences in AFM1 levels between three regions.
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SC
►Only a few samples with elevated concentrations were found during the summer.