Monitoring of aflatoxin M1 in raw milk during four ...

Food Control 54 (2015) 331e337

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Monitoring of aflatoxin M1 in raw milk during four seasons in Croatia Nina Biland zi c a, *, Ivana Varenina a, Bo zica Solomun Kolanovi c a, ÐurCica Bo zi c a, a a b c c , Dalibor Poto cnjak , Zeljko Cvetni cd Maja Ðoki c , Marija Sedak , Sanin Tankovi a

Department of Veterinary Public Health, Laboratory for Residue Control, Croatian Veterinary Institute, Savska Cesta 143, HR-10000 Zagreb, Croatia Veterinary Office of Bosnia and Herzegovina, Radiceva 8, 71000 Sarajevo, Bosnia and Herzegovina c Department of Internal Medicine, Faculty of Veterinary Medicine, University of Zagreb, Heinzlova 55, HR-10000 Zagreb, Croatia d General Department, Croatian Veterinary Institute, Savska Cesta 143, HR-10000 Zagreb, Croatia b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 November 2014 Received in revised form 2 February 2015 Accepted 10 February 2015 Available online 20 February 2015

A total of 3543 raw cow milk samples were collected in three regions of Croatia: western, eastern and other regions during four seasons. Samples were measured for aflatoxin M1 (AFM1) concentrations using the enzyme immunoassay method. Elevated levels (>50 ng/kg) of AFM1 were analysed by validated liquid chromatography with triple quadruple mass spectrometry (LC-MS/MS). The limits of detection (LOD) and quantification (LOQ) of the LC-MS/MS method were 7.3 and 28 ng/kg, respectively. The mean AFM1 levels measured in the three regions over four seasons were in the ranges (ng/kg): eastern Croatia 7.25e26.6; western Croatia 5.91e9.26; other regions of Croatia 7.17e13.6. The highest incidence of samples exceeding the EU MRL (50 ng/kg) of 9.32% was measured in autumn (OctobereDecember) in the eastern region. Only eight samples were found to exceed the EU MRL in winter. The highest AFM1 levels were measured in December (764.4 ng/kg) and January (383.3 ng/kg). Elevated AFM1 levels were found in summer in only four samples from the western and other regions, and two samples in the eastern region. This can be attributed to localized and random usage of contaminated feed for dairy cows in those regions. The much lower incidence of elevated AFM1 in comparison to a previous study showed that the outbreak of the crisis due to elevated AFM1 levels in 2013 resulted in a more careful approach to the control of supplementary feedstuff for lactating cows. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Aflatoxin M1 Cow milk ELISA LC-MS/MS Croatian regions

1. Introduction Due to their high nutritional properties and high intake by all age groups, milk and dairy products hold one of the most important roles in the human diet and, consequently, are of great economic importance for every country. The presence of aflatoxin M1 (AFM1), as a known xenobiotic, may have negative health implications for consumers and for agricultural production, as its appearance in milk can incur economic damages due to production losses (Tsakiris et al., 2013). This is emphasized by the fact that the AFM1 molecule cannot be inactivated by thermal processing used in the dairy industry, i.e. pasteurization and ultra-high-temperature treatment (Fallah, Rahnama, Jafari, & Saei-Dehkordi, 2011; Oruc, Cibik, Yilmaz, & Kalkanli, 2006). AFM1 is the main hydroxylmetabolite of the aflatoxin B1 (AFB1) that is formed in the liver of

* Corresponding author. Tel.: þ385 1 612 3601; fax: þ385 1 612 3636. E-mail address: [email protected] (N. Biland zi c). http://dx.doi.org/10.1016/j.foodcont.2015.02.015 0956-7135/© 2015 Elsevier Ltd. All rights reserved.

lactating animals following consumption of contaminated feedstuffs (Prandini et al., 2009). AFB1 demonstrates teratogenic, mutagenic and carcinogenic effects in mammals and has been classified as a Group 1 toxin. Recently, AFM1 has been also classified in Group 1 as a possible hepatotoxic substance and carcinogen for humans (IARC, 2002). AFM1 may cause DNA damage, which ultimately leads to gene mutation, chromosomal anomalies and cell transformation in mammalians cells (Prandini et al., 2009). When grazing is decreased in winter, significantly higher amounts of concentrated feed, especially corn silage and grass, are given to meet the energy needs of high-yielding cows. In the case of contaminated feed usage, AFM1 will be present in the milk for 2e3 days following ingestion (Prandini et al., 2009). Therefore, the risk is largely related to grain-based feed (Duarte et al., 2013; FinkGremmels, 2008). The transmission of AFB1 from foodstuffs to milk (carry-over) in dairy cows is influenced by various nutritional and physiological factors, including feeding, the degree of digestion, animal health, biotransformation capacity 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 associated with geographic location and climate, the degree of development of the region and the season (Rahimi, Bonyadian, Rafei, & Kazemeini, 2010). Climatic conditions in tropical and subtropical regions with high temperatures and drought, constant warmth and humidity favour the growth of the toxigenic mould species Aspergillus (Picinin et al., 2013). However, long periods of high temperatures and long-lasting drought in summer during the maize-growing and maize-harvesting periods also favour the development of these moulds in feed in other climatic regions (Biland zic et al., 2014a; Decastelli et al., 2007). With regard to the fact that the level of AFB1 contamination is cumulative, harvesting time and conditions of drying and storage of foodstuffs can actually have the same role in aflatoxin production. During grain storage, the drying process is the primary factor required to maintain a low moisture level, as irregular moisture of the mass favours fungal development (http://www.sciencedirect. com/science/article/pii/S0956713512004501Prandini et al., 2009). In recent years, elevated concentrations of AFM1 have been measured in different countries around the world: Lebanon (Assem, Mohamad, & Oula, 2011), Iran (Fallah et al., 2011; Nemati, Mehran, Hamed, & Masoud, 2010; Rahimi et al., 2010), Syria (Ghanem & Orfi, 2009), Turkey (Golge, 2014), Pakistan (Asi, Iqbal, ~ o, & Hussain, 2012; Hussain & Anwar, 2008; Iqbal & Asi, Arin 2013; Sadia et al. 2012), South Africa (Dutton, Mwanza, de Kock, & Khilosia, 2012), Sudan (Elzupir & Elhussein, 2010), Thailand (Ruangwises & Ruangwises, 2009), Indonesia (Nuryono et al., 2009), Brazil (Picinin et al., 2013), China (Xiong, Wang, Ma, & Liu, 2013), Serbia (Skrbi c, Zivan cev, Anti c, & Godula, 2014) and Croatia (Biland zi c, Varenina, & Solomun, 2010; Biland zi c et al., 2014a, b). Extremely elevated concentrations of AFM1 reported in 2013 in cow milk from eastern Croatia raised concerns and resulted in increased additional measures to control AFM1 in milk and AFB1 contamination in feedstuffs (Biland zi c et al., 2014a; Pleadin et al., 2014). Following this, the aim of this study was to evaluate the concentrations of AFM1 in raw cow milk collected during a oneyear period in three regions of Croatia: western, eastern and other regions. For the purpose of quantifying increased concentrations of AFM1, an additional aim of this study was to implement and validate a confirmatory method of liquid chromatographyetandem mass spectrometry (LC-MS/MS). 2. Materials and methods 2.1. Sample collection A total of 3198 raw cow milk samples were collected in the period from October 2013 to September 2014 from dairy farms in eastern (total n ¼ 1589) and western Croatia (total n ¼ 1609). Milk samples were also collected on small farms across other Croatian regions, i.e. from southern, southwestern, central and northern Croatia. However, as the number of samples from these areas was significantly lower than the number of samples collected in eastern and western Croatia, this group of samples was pooled into the group other regions (total n ¼ 345). The volume of the collected milk samples was approximately 0.5 L. Samples were stored at 2e8 C or frozen at 20 C until further analysis of AFM1. 2.2. Chemicals and reagents AFM1 concentrations were measured using competitive enzyme immunoassay (ELISA) of Ridascreen “Enzyme immunoassay for the quantitative analysis of aflatoxin M1” (R1121, R-Biopharm AG, Darmstadt, Germany). The reagents for the test kit contain: AFM1 standard solutions in milk buffer for the calibration curve (0, 5, 10,

20, 40 and 80 ng/L), anti-aflatoxin M1 antibody (concentrate), conjugate (peroxidase conjugated aflatoxin M1, concentrate), substrate/chromogen (tetramethylbenzidine), stop solution (1 N H2SO4), sample dilution buffer, conjugate, antibody dilution and washing buffer for the preparation of 10 mM phosphate buffer (PBS, pH 7.4) containing 0.05% Tween 20. Conjugate and antibody concentrates were diluted to 1:11 by the dilution buffer before analysis. Buffer salt was dissolved in 1 L of distilled water and was ready for use for 4e6 weeks. Milk samples for LC-MS/MS analysis were prepared using immunoaffinity columns (IAC) VICAM Afla M1™ HPLC purchased from VICAM (Milford, USA). LC grade acetonitrile was purchased from Merck (Darmstadt, Germany). Ammonium formate (97%) and formic acid (96%) used for mobile phase were purchased from Sigma Aldrich Chemie GmbH (Germany). Nitrogen 5.0 and 5.5 were purchased from SOL spa (Monza, Italy). Ultra pure water was obtained by the Direct-Q® 5 UV Remote Water Purification System (Merck KGaA, Darmstadt, Germany). Aflatoxin M1 and internal standard aflatoxin B1 was obtained from SigmaeAldrich (St. Louis, MO, USA). Mobile phase A consisted of 5 mM ammonium formate in water with the addition of 0.1% formic acid; mobile phase B was 0.1% formic acid in acetonitrile. The standard stock solutions of AFM1 and AFB1 (1000 mg/L) were prepared by dissolving certified reference material aflatoxin M1 solution (Product number: CRM46319, Supelco, SigmaeAldrich, St. Louis, USA) in acetonitrile (LC grade). Working solutions were prepared at 10 and 100 ng/mL by further dilution of the stock solution and were used for preparation of the calibration curve and for spiking samples. Stock solutions were stored at 4 C for no longer than 6 months, and working solutions were used within 3 months. Standards for the calibration curve were prepared in ultra pure water and acetonitrile (1:1) at concentrations of 0.2, 1, 2.5, 10 and 20 ng/mL of aflatoxin M1, where each calibration level included the internal standard aflatoxin B1 at 2.5 ng/mL. 2.3. Instrumentation Milk samples for the ELISA method were prepared using the Vortex Genius 3 (IKA® -Werke GmbH & CO.KG, Germany) and centrifuge Rotanta 460R (Hettich GmbH & Co.KG, Tuttlingen, Germany). The Sunrise Absorbance Reader (Tecan Austria GmbH, Salzburg, Austria) was used to measure the optical density at 450 nm. The following equipment was used in sample preparation for LC-MS/MS analysis: IKA® Vortex model MS2 Minishaker (Staufen, Germany), Iskra ultrasonic bath (Slovenia), Supelco vacuum manifold (Bellefonte, PA), centrifuge Rotanta 460R (Hettich Zentrifugen, Tuttlingen, Germany) and Nitrogen evaporation system N-EVAP® model 112 (Orgamonation Associates Inc., USA). Analysis by high performance liquid chromatography with tandem mass spectrometry was carried out with the LC-MS/MS system, consisting of HPLC 1260 and Triple Quad LC/MS 6410 mass spectrometer (Agilent, Palo Alto, USA). 2.4. ELISA test procedure The ELISA test procedure for the detection of AFM1 in raw milk was performed according to the manufacturer's instructions. The method was validated according the European Commission guidelines (European Commission, 2002) as previously described (Biland zi c et al., 2014a). The validation parameters were (ng/kg): detection capacity (CCb) 33.0, limit of detection (LOD) 22.2, limit of quantification (LOQ) 34.2. The quality of the results was tested in a proficiency test of milk powder organized by FAPAS (Food and Environmental Research


Agency, York, UK), as Proficiency Test 04213 in 2013. The proficiency test results were satisfactory, with a calculated z-score value of 0.9 (acceptable range 2 z 2). Prior to analysis using the ELISA test, milk samples were centrifuged for 10 min at 3500 g at 10 C. After centrifugation, the upper cream layer was completely removed by aspirating through a Pasteur pipette. Skimmed milk was used directly in the test (100 mL per well). In the case where the AFM1 concentration exceeded 80 ng/mL, samples were diluted with sample dilution buffer from the test kit and reanalysed. 2.5. LC-MS-MS method 2.5.1. Extraction of samples for LC-MS/MS Prior to analysis, 100 mL milk was defatted by centrifugation at 4000x g for 15 min. IAC columns and reservoirs for application of the sample were attached to the vacuum manifold for solid phase extraction. A 50 g sample of defatted milk was passed through the column at a rate of 2.5 mL per minute. Columns were then washed twice with 10 mL distilled water. Aflatoxin M1 was eluted with 2.5 mL acetonitrile at a rate of 0.5 mL per minute. The sample eluate was collected in the tube and in this step, the internal standard was added. The eluate was evaporated to dryness with nitrogen at 50 ± 5 C and dissolved with 100 mL ultra pure water and 100 mL acetonitrile, vortexed and left in an ultrasonic bath for 5 min. Samples were further centrifuged at room temperature, for 15 min at 4500x g and filtered through 0.45 mm regenerated cellulose membrane filters prior to injection in the LC-MS/MS. 2.5.2. Standard solution preparation Standard stock solutions of AFM1 and AFB1 (1000 mg/L) were prepared by dissolving the certified reference material Aflatoxin M1 solution (product number: CRM46319, Supelco, SigmaeAldrich, St. Louis, USA) in acetonitrile (LC grade). Working solutions were prepared at 10 and 100 ng/mL by further dilution of the stock solution and were used for the preparation of the calibration curve and for the spiking of samples. Stock solutions were stored at 4 C for no longer than 6 months, and working solutions were used within 3 months. Standards for the calibration curve were prepared in ultrapure water and acetonitrile (1:1) at concentrations of 0.2, 1, 2.5, 10 and 20 ng/mL aflatoxin M1, where each calibration level included the internal standard aflatoxin B1 at 2.5 ng/mL. 2.5.3. Chromatographic and MS parameters Chromatographic separation was achieved by isocratic elution on the Poroshell 120 EC C18, 3 50 mm, 2.7 mm (Agilent, USA) with 60% mobile phase A and 40% mobile phase B. Mobile phase A consisted of 5 mM ammonium formate in water with the addition of 0.1% formic acid; mobile phase B was 0.1% formic acid in acetonitrile. The injection volume was 7 mL and mobile phase flow was 0.3 mL/min. One chromatographic run was recorded in 2.5 min. Column temperature was 30 C. The triplequad mass spectrometer consisted of an ESI ion source and was operated in positive mode, gas temperature 350 C, gas flow 5 L/min, nebulizer 35 psi, capillary voltage 4500 V. Data were acquired according to the multiple reaction monitoring approach (MRM), by selecting the two most intense ion transitions of the analytes, which is reported in Table 1. 2.5.4. Method validation The method was validated according to the criteria set by Commission Decision 2002/657/EC (European Commission, 2002). Parameters determined were specificity (selectivity), matrix effect (ruggedness), linearity, repeatability (precision) and within-laboratory reproducibility, trueness, decision limit (CCa)

333

Table 1 MS/MS conditions for MRM analysis of sulphonamides. Analyte

RT (min)

Precursor ion

Product ion

Aflatoxin M1 Aflatoxin B1

16.8 ± 0.5

329

273 259 285 241

313

Fragmentor (V) 95 115

Collision energy 24 26 24 10

Underlined product ion: the most common transition.

and detection capability (CCb). Linearity was calculated from the five point standard calibration curve at the following concentrations: 0.2, 1, 2.5, 10, 20 ng/mL. The concentration for the internal standard was set at 2.5 ng/mL for each level. The regression curve was prepared by plotting the ratio of the analyte area and internal standard area of the first transition (An1/An IS) versus the concentration of the analyte. Specificity was tested by analysing 20 representative blank cow milk samples in order to verify the absence of potential interfering compounds. Trueness was expressed in terms of recovery and precision and intra laboratory reproducibility as a relative standard deviation (RSD%). Samples were spiked at 0.5 times MRL, MRL and 1.5 times MRL (i.e., 0.025, 0.05, 0.075 mg/kg) in eight replicates and analysed on three different days by different analysts. The decision limit and detection capability were calculated by applying the calibration curve procedure. CCa was expressed as the sum of the average concentration of samples spiked at the MRL level and 1.64 times the reproducibility standard deviation at MRL. CCb was calculated as the CCa value plus 1.64 times the reproducibility standard deviation at CCa. Limit of detection (LOD) was calculated by multiplying the standard deviation of 21 samples spiked at 0.025 mg/kg with the Student t-value of 2.53. Limit of quantification (LOQ) was expressed as 10 times the standard deviation of 21 samples spiked at the lowest level. 2.6. Statistical analysis Statistical analyses were performed using the Statistica 10 software package (StatSoft® Inc., Tulsa, USA). Levels of AFM1 were expressed as mean ± SD, minimum and maximum value, percentages of samples exceeding the LOD and EU MRL. 3. Results and discussion 3.1. Method validation The performance parameters LOD, LOQ, CCb, CCa, recovery, precision and intra-laboratory reproducibility of the LC-MS-MS method validated for the quantification of milk samples with elevated concentrations (>50 ng/kg) are summarized in Table 2. In the specificity testing, no naturally occurring substances were found in the elution region of the analyte. The linearity of the standard calibration curve was evaluated by calculating the coefficient of the regression curve (R2), which was not below 0.998. Recovery was calculated from the standard calibration curve and ranged between 60.1 and 118.2% for the three concentration levels. These are within the limits laid down by Commission Regulation 401/2006 (European Commission, 2006a). Satisfactory values for precision and intra-laboratory reproducibility were achieved. Relative standard deviations (RSD%) of the intra-laboratory reproducibility were lower than 14.9%. The chromatogram of spiked cow milk is shown in Fig. 1. The results indicate that the LC-MS/MS method used was reliable for the quantification of AFM1 in milk and met the criteria for detecting residues of AFM1.


334

Table 2 Validation parameters of the LC-MS/MS method used for quantification of AFM1. Precision (n ¼ 8)

Intra laboratory reproducibility (n ¼ 24)

Spiking level (ng/kg)

Mean ± SD (ng/kg)

RSD (%)

Mean ± SD (ng/kg)

RSD (%)

Recovery (%)

CCa (ng/kg)

CCb (ng/kg)

LOD (ng/kg)

LOQ (ng/kg)

25 50 75

25.72 ± 1.60 49.42 ± 3.49 75.15 ± 2.27

6.23 7.06 3.02

25.81 ± 2.87 49.47 ± 3.95 75.56 ± 4.92

14.9 8.7 12.6

96.0 87.6 87.7

56.49

64.64

7.26

28.71

3.2. AFM1 concentrations in raw milk The AFM1 concentrations in raw milk from eastern, western and other regions of Croatia in the period from October 2013 to September 2014 are presented in Table 3. The results are shown by month, but are also pooled as seasons (e.g., January to March: spring). A chromatogram of an AFM1 negative uncontaminated sample is presented in Fig. 2. The mean AFM1 levels determined during four seasons in the three regions were in the ranges (ng/kg): eastern Croatia 7.25e26.6; western Croatia 5.91e9.26; other regions of Croatia 7.17e13.6. The highest total AFM1 mean concentrations (26.6 ng/kg) were measured in autumn (OctobereDecember) in the eastern region. An incidence of 58 samples (9.32%) was found to exceed the EU MRL (50 ng/kg; European Commission, 2006b). Individual results by month showed the highest mean of 30.5 ng/kg and 5.94% samples exceeding the EU MRL in November. During autumn, there were no elevated AFM1 levels in the western and other regions. In winter, samples exceeding the EU MRL value were found in the regions (%): eastern 2.37, western 0.24, other 2.1. The highest concentrations of AFM1 were measured in December (764.4 ng/kg) and January (383.3 ng/kg). The LC-MS/MS chromatogram of the raw milk sample contaminated with AFM1 (383.3 ng/kg) is shown in Fig. 3. During spring, no samples exceeded the EU MRL in any of the regions. In summer, two samples with elevated concentrations were found in the eastern region, and one in the other regions. It

can be concluded that the elevated concentrations found in four samples in the western and other regions, and two samples in the eastern region during the summer was a consequence of localized and random usage of contaminated feed for dairy cows on small farms, likely following storage in poor and inadequate conditions. Estimation of the AFM1 concentration between the LOD and EU MRL values (22.2e49.9 ng/kg) in three regions during autumn showed the following percentages (%): eastern 28.9, western 4.45, other 6.67. In winter, outbreaks of these concentrations were determined in percentages by regions (%): eastern 4.45, western 1.18, other 12.6. In spring and summer, this incidence was even lower and was below 2.22%. Therefore, compared to the incidences found in a previous survey in the eastern region during winter and spring 2013 (13.9e23.6%), the incidences obtained in this study were significantly lower (Biland zic et al., 2014a). In comparison to the range of 35.4e45.9% of raw milk samples exceeding the EU MRL during February and March 2013 (Biland zic et al., 2014a), only a small proportion of such samples were found in eastern Croatia in the present study during autumn 2013 and winter 2014. In fact, only 4.21% of the total milk samples analysed in a one-year period in the present study had AFM1 concentrations exceeding the permitted EU MRL value, which is significantly less than in the previous study, where elevated levels were found in 27.8% of samples. It is well known that supplementary feedstuffs, e.g. dry hay, corn and concentrated feed, are used in much greater amounts for cows

Fig. 1. Bovine milk containing 25 ng/kg AFM1 and 10 ng/kg AFB1 as internal standard.


335

Table 3 Distribution of AFM1 concentrations in raw milk from eastern, western and other regions of Croatia during seasons 2013/2014. Month

October November December Total autumn January February March Total winter April May June Total spring July August September Total summer a b

Eastern Croatia

Western Croatia

Other regions

n

Range (ng/kg) Mean n > LODa n exc. n (ng/kg ± SD) EUb

186 236 200 622

3.51e212.4 3.27e262.6 4.02e764.4 3.27e764.4

25.1 30.5 23.9 26.6

± 21.1 ± 23.9 ± 73.4 ± 44.7

64 90 26 180

15 37 6 58

147 188 193 528

3.08e45.1 2.89e45.2 2.48e33.6 2.48e45.2

10.0 10.2 7.79 9.26

± 7.10 ± 7.36 ± 3.82 ± 6.31

9 13 2 24

0 0 0 0

15 18 12 45

3.26e30.3 1.65e48.6 2.29e34.5 1.65e48.6

8.76 12.5 7.17 9.83

± 7.20 ± 16.2 ± 8.93 ± 11.9

0 2 1 3

0 0 0 0

140 100 97 337

4.08e383.3 3.72e104.4 3.44e101.7 3.44e379.6

16.2 14.4 12.3 14.7

± 33.1 ± 13.9 ± 12.4 ± 24.8

6 6 3 15

3 3 2 8

153 148 123 424

3.88e21.2 4.05e68.6 2.81e15.3 2.81e68.6

8.59 8.97 7.81 8.49

± 3.25 ± 7.16 ± 2.52 ± 4.86

0 5 0 5

0 1 0 1

35 24 36 95

3.52e109.1 3.93e41.9 2.57e39.7 2.57e109.1

16.8 15.3 9.33 13.6

± 23.4 ± 9.86 ± 7.09 ± 15.9

5 5 2 12

2 0 0 2

104 89 117 310

2.70e23.6 3.84e48.1 3.49e20.2 2.70e48.1

8.62 9.88 8.04 8.76

± 4.26 ± 7.71 ± 3.04 ± 5.19

2 5 0 7

0 0 0 0

119 88 119 326

3.02e17.2 3.55e24.8 3.83e17.9 3.02e24.8

7.06 6.67 7.17 6.99

± 2.16 ± 2.95 ± 2.94 ± 2.68

0 1 0 1

0 0 0 0

55 33 15 103

4.32e42.0 2.40e42.1 2.64e8.43 2.40e42.1

7.86 6.97 5.08 7.17

± 5.83 ± 6.91 ± 1.79 ± 5.86

2 0 0 2

0 0 0 0

100 106 114 320

2.93e75.4 2.37e53.0 2.39e13.51 2.37e75.4

8.17 8.25 5.52 7.25

± 7.34 ± 5.28 ± 2.11 ± 5.40

1 1 0 2

1 1 0 2

113 111 107 331

3.68e14.2 2.48e13.2 0.11e13.9 0.11e14.2

6.37 6.13 5.20 5.91

± 1.77 ± 1.83 ± 2.21 ± 2.00

0 0 0 0

0 0 0 0

25 36 41 102

2.91e134.3 1.81e8.13 2.19e12.3 1.81e224.4

11.2 7.04 5.29 8.23

± 26.8 ± 8.45 ± 2.68 ± 22.5

2 0 0 2

1 0 0 1

Range (ng/kg)

Mean (ng/kg ± SD)

n > LODa n exc. n EUb

Range (ng/kg) Mean n > LODa n exc. (ng/kg ± SD) EUb

Number of samples above LOD and below EU MRL: 22.2e49.9 ng/kg. Number of samples exceeding EU MRL.

during autumn and winter. If contaminated with AFB1, this results in an increased AFM1 content in milk (Biland zi c et al., 2014a; Prandini et al., 2009). Previously reported elevated levels of AFM1 in milk in eastern Croatia were confirmed with elevated levels of AFB1 measured in 36.5% of maize samples collected in three

Croatian regions in 2013, with levels exceeding the maximal permitted level of 20 mg/kg (Pleadin et al., 2014). The AFM1 levels obtained in raw milk samples in different seasons from different countries are presented in Table 4. In recent years, global studies have shown seasonal variations of AFM1

Fig. 2. Total ion chromatogram (TIC) and extracted ion chromatogram for AFM1 from the uncontaminated sample.

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.


336

Table 4 Incidence of aflatoxin M1 in raw cow milk in different seasons measured worldwide. Country

Year

No of samples

Season

Mean (ng/L)

Maximal measured value (ng/L)

Samples exceeding EU MRL (%)

Reference

Turkey

January 2012e December 2012

2013

China

November 2011e September 2012

Pakistan

November 2011e September 2012 November 2010e April 2011 August 2009e February 2010

Autumn Winter Spring Summer February March April May June July Winter Spring Summer Autumn Summer Winter Autumn-Winter

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 ± 6 150.7 ± 11.9

552 1101 150 102 1105.2 1135.0 398.6 470.5 85.9 36.4 420 98 82 76 n.s. n.s. 845.4

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 71

Golge, 2014

Croatia

63 47 33 33 749 990 969 393 280 355 n.s. n.s. n.s. n.s. 56 48 107 43 43 43 27 27 17 38 44

Dry period Transition period Rainy period Summer Winter n.s. Spring-summer n.s.

35.9 ± 4.4 17.1 ± 3.0 5.5 ± 9.4 22 ± 6 89 ± 2 110 ± 195 60.4 2070

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

24 21 21 22 75

Winter Spring Summer Autumn November 2007e December 2008 Spring Summer Autumn Winter Sumer Rain season Winter April 2005eApril 2006 January February March April May June July August September October November December

93 ± 19 31 ± 5 28 ± 5 51 ± 6 60.1 ± 57.4

394

35.2

Sadia et al. 2012. Assem et al., 2011 Elzupir & Elhussein, 2010 Fallah et al., 2011

n.s.

36

Rahimi et al., 2010

81.9 55.9 28.9 85.0 114

33

Nemati et al., 2010

47.5 66.3 80 95 n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

Ruangwises & Ruangwises, 2009

Pakistan Brazil

Pakistan

2010

Pakistan Lebanon Sudan

n.s. 2010 2009

Iran

2008

Iran

2007e2008

Iran

2006

Thailand

2006e2007

Syria Pakistan

2005e2006 2005

23 22 22 23

74 14 14 14 14 14 14 14 14 14 14 14 14

52.9 ± 4.4 17.4 ± 3.1 22.3 ± 0.9 56.3 ± 6.6 50 ± 21 71 ± 28 89 ± 34 143 ± 53.22 503 ± 92 466 ± 47 404 ± 74 398 ± 61 323 ± 44 351 ± 61 329 ± 75 199 ± 99 328 ± 71 345 ± 71 403 ± 57 403 ± 60

690 700 570 470 500 390 490 390 420 400 450 470 470

Biland zi c et al., 2014a

Xiong et al., 2013

Iqbal, Asi, & Jinap, 2013 Iqbal & Asi, 2013 Picinin et al., 2013

Asi et al., 2012

Ghanem & Orfi, 2009 Hussain & Anwar, 2008

n.s. e not specified.

concentrations and elevated levels (>50 ng/L) in milk in winter in Pakistan, Croatia, Iran, Turkey, Morocco, Thailand, Serbia and China (Asi et al., 2012; Biland zi c et al., 2014a; Fallah et al., 2011; Golge, 2014; Marnissi, Belkhou, Morgavi, Bennani, & Boudra, 2012; c Rahimi et al., 2010; Ruangwises & Ruangwises, 2009; Skrbi et al., 2014; Xiong et al., 2013). In the most recent studies conducted in Turkey and China, 40.4% and 72.2% of milk samples respectively exceeded the EU MRL value in winter, with maximal concentrations of 1101 and 420 ng/kg (Golge, 2014; Xiong et al., 2013). Elevated levels of AFM1 (540e1440 ng/L) were also re c et al., 2014). ported in February 2013 in Serbia (Skrbi These studies confirmed that the occurrence of AFM1 poses a problem in countries with a dry climate or with seasons of long

drought periods that favour the development of mould and elevated AFB1 levels in feed (Asi et al., 2012; Biland zic et al., 2014b; Fallah et al., 2011; Prandini et al., 2009). Therefore, in tropical and subtropical climate zones, high AFM1 concentrations were measured throughout the year, with maximum levels in excess of 650 ng/L in countries such as South Africa, Sudan, Syria and Pakistan (Dutton et al., 2012; Elzupir& Elhussein, 2010; Ghanem & Orfi, 2009; Hussain & Anwar, 2008; Iqbal & Asi, 2013; Sadia et al. 2012). The maximum AFM1 levels measured in this study (December 764.4 ng/kg) are comparable to the above maximum concentrations. For example, in recent studies in Pakistan, AFM1 levels exceeding the EU MRL were found in 71% and 41% of milk samples (Iqbal & Asi, 2013; Sadia et al. 2012). Furthermore, in


Sudan, 100% of samples showed AFM1 levels exceeding 50 ng/kg, with maximal AFM1 concentrations of 6900 ng/L (Elzupir & Elhussein, 2010). 4. Conclusions A previous study in Croatia reported the occurrence of elevated AFM1 concentrations in eastern Croatia in 2013. The present study is a continuation of the monitoring of AFM1 concentrations in milk from farms in eastern Croatia over a one-year period, which also included monitoring concentrations in raw milk from the western region and in other Croatian regions. Therefore, this study is a large-scale effective control of AFM1 levels in raw milk in accordance with the defined maximum residue levels of 50 ng/kg. Markedly improved results were determined in this survey, with a much lower number of samples with increased AFM1 concentrations in milk during autumn and winter. This can be explained due to better weather conditions during the growth of grain used for supplementary feeding of cows in 2013 and 2014, and proper and controlled conditions during grain storage. Therefore, these results showed that the outbreak of elevated concentrations of AFM1 in 2013 led to a more careful approach towards the control of supplementary feedstuff for lactating cows. Furthermore, it can be concluded that elevated concentrations were found in only four samples in the western and other regions, and in two samples in the eastern region during summer. This was the result of sporadic and localized usage of contaminated feed for dairy cows on small farms. The results further support the conclusion that continuous inspection and control of AFM1 in milk and dairy products, together with the control of AFB1 in raw material and supplementary feedstuffs for dairy cattle is necessary. References ~ o, A., & Hussain, A. (2012). Effect of seasonal variations and Asi, R. M., Iqbal, S. Z., Arin lactation times on aflatoxin M1 contamination in milk of different species from Punjab, Pakistan. Food Control, 25, 34e38. Assem, E., Mohamad, A., & Oula, E. A. (2011). A survey on the occurrence of aflatoxin M1 in raw and processed milk samples marketed in Lebanon. Food Control, 22, 1856e1858. Biland zi c, N., Bo zi c, Ð., Ðoki c, M., Sedak, M., Solomun Kolanovi c, B., Varenina, I., et al. (2014a). Seasonal effect on aflatoxin M1 contamination in raw and UHT milk from Croatia. Food Control, 40, 260e264. Biland zi c, N., Bo zi c, Ð., Ðoki c, M., Sedak, M., Solomun Kolanovi c, B., Varenina, I., et al. (2014b). Assessment of aflatoxin M1 contamination in milk of four dairy species in Croatia. Food Control, 43, 18e21. Biland zi c, N., Varenina, I., & Solomun, B. (2010). Aflatoxin M1 in raw milk in Croatia. Food Control, 21, 1279e1281. Decastelli, L., Lai, J., Gramaglia, M., Monaco, A., Nachtmann, C., Oldano, F., et al. (2007). Aflatoxins occurrence in milk and feed in Northern Italy during 2004e2005. Food Control, 18, 1263e1266. Duarte, S. C., Almeida, A. M., Teixeira, A. S., Pereira, A. L., Falc~ ao, A. C., Pena, A., et al. (2013). Aflatoxin M1 in marketed milk in Portugal: assessment of human and animal exposure. Food Control, 30, 411e417. Dutton, M. F., Mwanza, M., de Kock, S., & Khilosia, L. D. (2012). Mycotoxins in South African foods: a case study on aflatoxin M1 in milk. Mycotoxin Research, 28(1), 17e23. Elzupir, O. A., & Elhussein, A. M. (2010). Determination of aflatoxin M1 in dairy cattle milk in Khartoum State, Sudan. Food Control, 21, 945e946. European Commission. (2002). European Commission decision no. 2002/657/EC (2002) of 14 August 2002, implementing council directive 96/23/EC concerning

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