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Analysis of aflatoxin M1 and M2 in commercial dairy products using high-performance liquid chromatography with a fluorescence detector
Food Control 50 (2015) 467e471
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Analysis of aflatoxin M1 and M2 in commercial dairy products using high-performance liquid chromatography with a fluorescence detector Donghun Lee, Kwang-Geun Lee* Department of Food Science and Biotechnology, Dongguk University-Seoul, 3-26 Pil-dong, Jung-gu, Seoul 100-715, Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 June 2014 Received in revised form 11 September 2014 Accepted 16 September 2014 Available online 20 September 2014
Aflatoxin M1 (AFM1) and M2 (AFM2) in commercial dairy products were analyzed by high-performance liquid chromatography (HPLC) with a fluorescence detector (FLD). To ensure an accurate analysis, two derivatization methods, bromination and aflatoxinetrifluoroacetic acid derivatization (ATD), were compared. The limits of detection (LODs) of the bromination method were 124.42e151.73 ng/kg, and the recovery rates were between 64 and 102%. The detection rates and concentration levels of AFM1 were 6 e74% and 14.48e270.94 ng/kg, respectively. AFM1 was detected in 74% of milk powder samples and 36% of ice cream samples. The mean values of AFM1 in milk powder and ice cream samples were 270.94 and 33.16 ng/kg, respectively. In the case of AFM2, the detection rates were 2e10%, and the concentration levels were 20.62e55.67 ng/kg in milk and milk powder. Among milk and milk powder samples, ultra heat-treated (UHT) milk had lower AFM1 contamination levels than pasteurized milk. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Aflatoxins Dairy products Immunoaffinity column Bromination
1. Introduction Aflatoxins are secondary metabolites of Aspergillus parasiticus and Aspergillus flavus (Hwang & Lee, 2006). They have carcinogenic, teratogenic, and mutagenic properties (Ali et al., 2005; Cho et al., 2007). Aflatoxin M1 (AFM1) is produced by the metabolizing systems of humans and dairy cows with aflatoxin B1 ingestion (Diaz & Espitia, 2006; EFSA, 2007). The International Agency for Research on Cancer (IARC, 1993) classified AFM1 as a possible human carcinogen (Group 2B). For this reason, many countries have set limits for AFM1 in milk, cheese, and baby food (e.g., 500 ng/kg) (Anfossi, Baggiani, Giovannoli, & Giraudi, 2011). According to the previous studies, TLC (thin layer chromatography), high-performance liquid chromatography (HPLC), and ELISA are widely used for the analysis of aflatoxins (Awad, Ghareeb, & Bohm, 2012; Manetta et al., 2005). Although the TLC method is the AOAC's official method, it is limited in its ability to quantitate aflatoxins accurately (Awad et al., 2012). The ELISA method is a quick AOAC method; however, it has a 20% false-positive rate (Awad et al., 2012). HPLC with a fluorescence detector (FLD) and mass spectrometry (MS) detector is suitable for aflatoxin quantification. However, few studies have used an MS for aflatoxin analysis by
* Corresponding author. Tel.: þ82 2 2260 3370; fax: þ82 2 2285 3370. E-mail addresses: [email protected], [email protected] (K.-G. Lee). http://dx.doi.org/10.1016/j.foodcont.2014.09.020 0956-7135/© 2014 Elsevier Ltd. All rights reserved.
estimating particular samples, such as milk and herbal medicines. Recently, LC/MS was used for the qualitative analysis of aflatoxins, and HPLC-FLD was used for the quantitative analysis of aflatoxins (Bognanno et al., 2006; Jang et al., 2007). Many previous studies have analyzed aflatoxins by HPLC-FLD with immunoaffinity extraction. Analytical methods of aflatoxins are usually carried out by HPLC-FLD either with trifluoroacetic acid (TFA) or bromination derivatization (Reiter, Zentek, & Razzazi, 2009). Though AFM1 is known as a stable material (Iha, Barbosa, Okada, & Trucksess, 2012), many previous studies have reported that AFM1 levels are altered during processing and storage. Ultra heat-treated (UHT) milk has been shown to have lower AFM1 contamination levels than pasteurized milk (Rahimi & Behzadnia, 2012; Zheng et al., 2012). In yogurt, cheese, ice cream, and sherbet, processing and storage have been shown to affect AFM1 levels in products (Iha et al., 2012; Wiseman & Marth, 1983). On the other hand, the processing and storage of milk probably did not affect aflatoxins, because milk has a very short distribution period from production to consumption. In this study, various sample preparation methods such as derivatization method and immunoaffinity columns were compared and validated to determine the optimum analytical method for AFM1 and AFM2 in various dairy products. In addition, the levels of AFM1 and AFM2 in the dairy products (e.g., milk,
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yogurt, milk powder, ice cream, and sherbet) were analyzed by the optimized method.
2. Materials and methods 2.1. Reagents and materials AFM1 and AFM2 standard powder (Enzo Life Sciences, Lausen, Switzerland) with 70% methanol (J.T. Baker, Phillipsburg, NJ, USA) was prepared for the stock solution. Working solutions were prepared as 500, 1,000, 2,000, 5,000, and 10,000 ng/kg for calibration curves. Water and acetonitrile, and methanol were HPLC grade (J.T. Baker). TFA (SigmaeAldrich, MO, USA) and Kobra cell (K01, Biopharm Rhone, Glasgow, Scotland) were prepared for derivatization.
2.2. Sampling Milk sampling (including flavored milk) in Seoul, Korea was conducted from June to August 2012. Samples were collected from commercial markets. Yogurt samples were of the drinking yogurt type. Ice cream samples having more than 6% milk lipids were selected, and sherbet samples having less than 3% milk lipids were selected.
2.5. Aflatoxin extraction for milk powder samples First, 10 g of sample with 40 ml of 70% (v/v) methanol was put into a 50-ml conical tube. It was added to 0.5 g of NaCl and shaken at 250 rpm for 1 h. It was centrifuged at 6500 rpm (10 min) and filtrated using a 45-mm syringe filter. Next, 10 ml of filtered solution was diluted by 10 ml of water, and 20 ml of the diluted aflatoxin extracted solution was loaded into an immunoaffinity column. After that, 10 ml of water was loaded for cleaning. The immunoaffinity column was air-dried for 10 min. Finally, 1 ml of methanol was loaded into the column for aflatoxin elution. 2.6. Aflatoxin extraction for ice cream samples Ice cream samples were first melted and filtered by a sieve. Then, 20 g of ice cream filtered of toppings and fruit was put in a 50ml conical tube with 20 ml of methanol. It was added to 0.5 g of NaCl and shaken at 250 rpm for 1 h. Before shaking, 5 ml of hexane was added, and the results were compared with those when 1 drop of Tween 80 was added. It was centrifuged at 6500 rpm (10 min) and filtrated using a 45-mm syringe filter. Next, 10 ml of filtered solution was diluted by 10 ml of water, and 20 ml of the diluted aflatoxin extracted solution was loaded into an immunoaffinity column. After that, 10 ml of water was loaded for cleaning. The immunoaffinity column was air-dried for 10 min. Finally, 1 ml of methanol was loaded into the column for aflatoxin elution.
2.3. Determination of validation values for the analytical method Tests to determine recoveries, coefficient of variation (CV, %) values, Z-scores, limits of detection (LODs), limits of quantitation (LOQs), linearity, and R-squared (R2) values were conducted. Intraand inter-day recovery and CV tests were performed with each representative sample. Tests to determine LODs of aflatoxins were carried out by 3.3 sigma (s)/slope factor of calibration curve. Sigma was obtained by determining the standard deviation of the y-intercept of seven specific calibration curves. Each specific calibration curve was constituted by three-point concentrations. Linearity and R2 values were calculated using each aflatoxin standard curve for quantification. Method detection limits (MDLs) and method detection quantitation (MDQ) values were calculated by LOD/(dilution rate sample weight) and 3 MDL, respectively. 2.4. Aflatoxin extraction for milk, yogurt, and sherbet samples First, 20 g of sample with 20 ml of methanol was put into a 50ml conical tube. In the case of sherbet, it was melted and filtered by a sieve. It was then added to 0.5 g of NaCl and shaken at 250 rpm for 1 h. It was centrifuged at 6500 rpm (10 min) and filtrated using a 45-mm syringe filter. Next, 10 ml of filtered solution was diluted by 10 ml of water, and 20 ml of the diluted aflatoxin extracted solution was loaded into an immunoaffinity column. After that, 10 ml of water was loaded for cleaning. The immunoaffinity column was then air-dried for 10 min. Finally, 1 ml of methanol was loaded into the column for aflatoxin elution.
2.7. AflatoxineTFA derivatization (ATD) method Eluted aflatoxin extracts were dried by nitrogen gas at 40 C. For the derivatization, 0.2 ml of TFA with 1 ml of hexane was added, and it was then stored for 20 min in a darkroom. After that, 50% (v/ v) methanol was mixed to 1 ml using a vortex, and the bottom layer was then injected for analysis. 2.8. Analytical conditions of HPLC-FLD analysis A Waters 1525 system (Milford, MA, USA) and 474 FLD (Milford, MA, USA) were used. For the ATD method, the mobile phase was acetonitrile:water (7:3, v/v), and the column was an Agilent XDBC18 (250 mm 4.6 mm and 5 mm: Palo Alto, CA, USA). Wavelengths for excitation and emission were 360 nm and 450 nm, respectively. For bromination, the mobile phase was a 0.001 M KBr mixture of water, methanol, and acetonitrile (6:2:2, v/v) with 350 ml of 4 M nitric acid for the 1 L mobile phase, and the column was an Agilent Sb-Aq (250 mm 4.6 mm and 5 mm: Palo Alto, CA, USA). Wavelengths for excitation and emission were 365 nm and 435 nm, respectively. In both conditions, the column temperature was 40 C, and the flow rate was 1 ml/min. The injection volume was 20 ml. 2.9. Internal quality control To ensure an accurate analysis, internal quality control was performed. When the analysis of 10 samples was done, 10 ng/g
Table 1 Limit of detection (LOD), Limit of quantification (LOQ), Linearity, and R-squared (R2) values of aflatoxinetrifluoroacetic acid derivatization (ATD) and bromination methods.
Bromination Aflatoxinetrifluoroacetic acid derivatization (ATD)
Type of aflatoxin
LOD (ng/kg)
LOQ (ng/kg)
Linearity
AFM1 AFM2 AFM1 AFM2
125.42 151.73 57.78 189.27
418.05 505.77 263.91 879.71
y y y y
¼ ¼ ¼ ¼
5820.5x þ 635.34 4224.1x þ 137.16 5048.7x þ 4297.4 4770x þ 5362
R2 0.9988 0.9951 0.9906 0.9819
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standard solutions were analyzed to check the sensitivity variation of HPLC-FLD. In addition, when the analysis of 20 samples was done, a recoveries test was performed as an intra-day test. Recovery tests were performed such that AFM1 and AFM2 were spiked with 0.5 ng in samples.
Table 3 Limit of detection (LOD), limit of quantification (LOQ), method detection limits (MDLs) and method detection quantitation (MDQ) of each dairy sample.
LOD
3. Results and discussion LOQ
3.1. Comparing analytical methods' validation values and optimization of sample preparation
MDL MQL
The method validation values of aflatoxins are shown in Table 1. The LOD of bromination method for AFM1 and AFM2 were 125.42 and 151.73 ng/kg, respectively. Though the LOD of the bromination method for AFM1 was higher than that of the ATD method, the Rsquared (R2) values of the bromination method were higher than the ATD method. The repeatability of the bromination method was more stable than the ATD method. Due to these factors, the bromination method was selected for the optimum derivatization method. For the optimized aflatoxin extraction, four immunoaffinity columns were prepared based on the study of S¸enyuva and Gilbert (2010) and compared with recovery tests in milk samples. For the substantive analysis, 0.5 ml of 10 ng/g AFM1 and AFM2 mixture was spiked in 20 g of milk sample, and Afla Prep M was selected for the analysis of this study. Recoveries of each immunoaffinity column are shown in Table 2. The recoveries of each immunoaffinity column were slightly lower than reported in the previous study. According to S¸enyuva and Gilbert (2010), recoveries of immunoaffinity columns were over 80e90% when 2e20 ng of AFM1 and AFM2 were spiked. In this study, recoveries of immunoaffinity columns were 20.4e104.4% when 5 ng of AFM1 and AFM2 were spiked. The different thing of this study compared with that of S¸enyuva & Gilbert was a use of commercial milk instead of a skimmed milk. In the study of S¸enyuva & Gilbert, they used a skimmed milk as a sample for recovery test. Usually recovery of AFMs increases with low fat samples. Previous studies reported 70% methanol as an effective condition for methanol extraction (S¸enyuva & Gilbert, 2010). In this study, almost all samples were prepared in liquid state such as milk and yogurt. With the liquid samples dilution effect occurred while the extraction of AFs. To compensate this, the adequate amount of methanol was measured by freeze-dried, and the water contents of the samples were estimated. With this measurement, 20 ml of methanol was added to 20 g of sample such as milk, yogurt, and sherbet, to make a 70% methanol concentration. Since ice cream samples needed reagents for clear chromatograms, Tween 80 and hexane were compared for their ability to minimize matrix effects, and hexane was selected. Methanol extraction with hexane had higher recoveries for AFM2 than that with Tween 80. Aflatoxins are scarcely extracted by hexane (Cho et al., 2007), and it is an effective method for removing lipids. In addition, several studies have used emulsifiers for aflatoxin analysis (Sadia et al., 2012; Zhang et al., 2012). Table 2 Recoveries of each immunoaffinity column in milk samples. Company R-bio pharm Rhone LTD. NEOGEN Europe LTD. VICAM® R-bio pharm Rhone LTD.
Product Afla-Rhone wide Neo Column Afla M1™ HPLC Afla Prep M
AFM1 (recovery, %) 20.41 104.40 26.32 82.59
Type of AFM
Milk (ng/kg)
Yogurt (ng/kg)
Milk powder (ng/kg)
Ice cream (ng/kg)
Sherbet (ng/kg)
AFM1 AFM2 AFM1 AFM2 AFM1 AFM2 AFM1 AFM2
125.42 151.73 418.05 505.77 25.08 83.61 30.35 101.15
125.42 151.73 418.05 505.77 25.08 83.61 30.35 101.15
125.42 151.73 418.05 505.77 50.16 167.22 60.69 202.31
125.42 151.73 418.05 505.77 25.08 83.61 30.35 101.15
125.42 151.73 418.05 505.77 25.08 83.61 30.35 101.15
LOD, LOQ, MDL and method quantitative limits (MQLs) are shown in Table 3 using the optimized method. Each specific calibration curve was comprised of three concentrations. The MDL and MDQ ranges were 25.08e167.22 and 83.61e202.31 ng/kg, respectively. Recovery tests were conducted for each sample with an intra- and inter-day test (shown in Table 4). Recoveries and CV values of AFM were 64.17e101.86% and 1.83e14.35%, respectively. 3.2. AFM1 and AFM2 contamination levels in milk, yogurt, milk powder, ice cream, and sherbet Contamination levels of AFM1 and AFM2 are shown in Table 5. Each of the 50 samples was analyzed, and the detection rates (%) of AFM1 and AFM2 were 6e74% and 2e10%, respectively. Milk powder samples had the highest contamination rate and AFM1 level. It is assumed that the milk powder underwent an evaporation process, and the level of AFM1 is concentrated through the evaporation process. Although many countries have maximum residue limits (MRLs) of AFM1 in dairy products (e.g., 500 ng/kg or 250 ng/kg), risk-assessment methods have not yet been clearly prepared. This is because AFM1 has no accurate bench mark dose limit (BMDL) or provisional tolerable weekly intake (PTWI) from the official organization. However, milk powder is mainly consumed by infants and toddlers, and the ingestion amount of milk powder per kilogram of body weight is much higher in infants and toddlers than in adults. This could lead to serious health problems in infants and toddlers, though the toxicity of AFM1 has been evaluated as being one tenth that of aflatoxin B1 (Boon, Bakker, van Klaveren, & van Rossum, 2009). In the case of AFM2, yogurt has the highest detection rate, maximum values, and 95 percentile and 99 percentile levels. Iha et al. (2012) reported that the level of AFM1 is not affected by yogurt fermentation. In this study, milk and yogurt samples had similar AFM1 concentrations. However, yogurt samples had higher AFM2 concentrations than milk samples. In dairy products, lactic acids react with AFB1 and AFG1, AFB2 and AFG2 are produced (Ram, Ramtej, & Daxa, 2002). Thus, it is likely that lactic acid affects whether AFM1 converts to AFM2. The detection rate and Table 4 Recoveries and coefficient variation (CV) of aflatoxin M1 and M2 (AFM1 and AFM2) in milk, yogurt, milk powder, ice cream, and sherbet samples. Sample type
AFM2 (recovery, %) 104.43 42.44 72.19 93.02
Note: Commercial milk samples were used and spiked with 0.5 ml of 100 mg/kg aflatoxin M1 and M2 mixture for the recoveries test.
469
Recoveries (% ± CV) Inter-day
Milk Yogurt Milk powder Ice cream Sherbet
84.62 77.60 64.70 69.26 82.44
Intra-day AFM2
AFM1 ± ± ± ± ±
5.28 7.46 5.22 10.22 7.21
93.13 64.17 83.32 82.90 83.85
AFM1 ± ± ± ± ±
5.71 1.83 7.82 1.85 2.67
85.56 70.96 65.45 73.83 69.30
AFM2 ± ± ± ± ±
7.20 8.10 7.79 5.43 12.18
101.86 70.72 78.95 96.94 90.92
± ± ± ± ±
7.74 8.36 4.99 14.35 11.11
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Table 5 Mean, geomean, median, 95 and 99 percentile, and maximum values of aflatoxin M1 and M2 (AFM1 and AFM2) in samples. Samples
Type of aflatoxin Detectionrate (%, n ¼ 50) Mean (ng/kg) Geomean (ng/kg) Median (ng/kg) 95 percentile (ng/kg) 99 percentile (ng/kg) Maximum (ng/kg)
Milk
AFM1 AFM2 Yogurt AFM1 AFM2 Milk powder AFM1 AFM2 Ice cream AFM1 AFM2 Sherbet AFM1 AFM2
6 2 14 10 74 2 36 8 6 4
20.81 20.62 22.54 55.67 270.94 30.91 33.16 25.62 14.48 67.53
14.40 16.09 16.05 19.97 147.61 30.75 22.13 18.10 13.49 17.83
12.54 15.17 12.54 15.17 171.93 30.35 12.54 15.17 12.54 15.17
33.78 15.17 73.78 252.17 680.45 30.35 112.36 111.12 19.96 15.17
200.55 154.11 141.22 797.65 795.78 44.68 120.81 183.90 54.30 1344.42
228.60 287.60 191.29 850.01 835.57 58.45 121.31 214.54 61.22 2336.63
Table 6 Mean, geomean, median, 95 and 99 percentile, and maximum values of aflatoxin M1 and M2 (AFM1 and AFM2) in milk and milk powder samples determined by sterilization method. Samples
Type of AFM
Sample number (n)
Mean (ng/kg)
Geomean (ng/kg)
Median (ng/kg)
95 percentile (ng/kg)
99 percentile (ng/kg)
Maximum (ng/kg)
UHT milk
AFM1 AFM2 AFM1 AFM2 AFM1 AFM2 AFM1 AFM2
36
20.81 20.62 22.54 55.67 270.94 30.91 33.16 25.62
14.40 16.09 16.05 19.97 147.61 30.75 22.13 18.10
12.54 15.17 12.54 15.17 171.93 30.35 12.54 15.17
33.78 15.17 73.78 252.17 680.45 30.35 112.36 111.12
200.55 154.11 141.22 797.65 795.78 44.68 120.81 183.90
228.60 287.60 191.29 850.01 835.57 58.45 121.31 214.54
Pasteurized milk UHT milk powder Pasteurized milk powder
14 43 7
contamination level of AFM1 in ice cream were 36% and 0.03 ng/g, respectively. In addition, ice cream and sherbet samples had higher detection rates and contamination levels than milk samples. Wiseman & Marth reported an increase of AFM1 during ice cream and sherbet manufacture and storage by improving the recovery of bound AFM1. In this study, ice cream samples had higher concentrations of AFM1 than milk samples, but sherbet samples did not. Khoshnevis, Azizi, Shateri, and Mousavizadeh (2012) reported that 22% of ice cream samples were contaminated by AFM1, with mean values of 33.98 ng/kg. Among the samples, milk samples had lower detection rates and contamination levels than the other samples. Kocasari, Tasci, and Mor (2012) analyzed dairy products in which higher AFM1 levels were detected in milk powder, white cheese, butter, and yogurt. AFM1 concentrations in milk and milk powders determined by the sterilization method are shown in Table 6. According to previous studies, the milk sterilization method affects AFM1 concentration in milk. AFM1 concentrations in pasteurized milk have been shown to be higher than those of raw and UHT milk (Hwang, Hwang, Kim, & Oh, 2012; Kocasari et al., 2012; Zheng et al., 2012). In this study, pasteurized milk had higher mean values of AFM1 and AFM2 concentrations than UHT milk. However, pasteurized milk powder had lower AFM1 levels than UHT milk powder. AFM2 in pasteurized milk powder had higher 95 and 99 percentiles and maximum values than in UHT milk powder. 4. Conclusion In conclusion, the HPLC-FLD analytical method using bromination was successfully applied to quantify AFM1 and AFM2 in dairy products. In this study, concentrations of AFM1 and AFM2 were 20.62e270.94 ng/kg. Among the samples, milk powder samples had a detection rate of 74% and an AFM1 concentration of 270.94 ng/kg. Pasteurized milk had higher AFM1 levels than UHT milk. Conversely, UHT milk powder had higher AFM1 levels than pasteurized milk powder. In addition, pasteurized milk powder had higher AFM2 concentrations, 95 and 99 percentiles, and maximum values.
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Analysis of aflatoxin M1 and M2 in commercial dairy products using high-performance liquid chromatography with a fluorescence detector Donghun Lee, Kwang-Geun Lee* Department of Food Science and Biotechnology, Dongguk University-Seoul, 3-26 Pil-dong, Jung-gu, Seoul 100-715, Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 June 2014 Received in revised form 11 September 2014 Accepted 16 September 2014 Available online 20 September 2014
Aflatoxin M1 (AFM1) and M2 (AFM2) in commercial dairy products were analyzed by high-performance liquid chromatography (HPLC) with a fluorescence detector (FLD). To ensure an accurate analysis, two derivatization methods, bromination and aflatoxinetrifluoroacetic acid derivatization (ATD), were compared. The limits of detection (LODs) of the bromination method were 124.42e151.73 ng/kg, and the recovery rates were between 64 and 102%. The detection rates and concentration levels of AFM1 were 6 e74% and 14.48e270.94 ng/kg, respectively. AFM1 was detected in 74% of milk powder samples and 36% of ice cream samples. The mean values of AFM1 in milk powder and ice cream samples were 270.94 and 33.16 ng/kg, respectively. In the case of AFM2, the detection rates were 2e10%, and the concentration levels were 20.62e55.67 ng/kg in milk and milk powder. Among milk and milk powder samples, ultra heat-treated (UHT) milk had lower AFM1 contamination levels than pasteurized milk. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Aflatoxins Dairy products Immunoaffinity column Bromination
1. Introduction Aflatoxins are secondary metabolites of Aspergillus parasiticus and Aspergillus flavus (Hwang & Lee, 2006). They have carcinogenic, teratogenic, and mutagenic properties (Ali et al., 2005; Cho et al., 2007). Aflatoxin M1 (AFM1) is produced by the metabolizing systems of humans and dairy cows with aflatoxin B1 ingestion (Diaz & Espitia, 2006; EFSA, 2007). The International Agency for Research on Cancer (IARC, 1993) classified AFM1 as a possible human carcinogen (Group 2B). For this reason, many countries have set limits for AFM1 in milk, cheese, and baby food (e.g., 500 ng/kg) (Anfossi, Baggiani, Giovannoli, & Giraudi, 2011). According to the previous studies, TLC (thin layer chromatography), high-performance liquid chromatography (HPLC), and ELISA are widely used for the analysis of aflatoxins (Awad, Ghareeb, & Bohm, 2012; Manetta et al., 2005). Although the TLC method is the AOAC's official method, it is limited in its ability to quantitate aflatoxins accurately (Awad et al., 2012). The ELISA method is a quick AOAC method; however, it has a 20% false-positive rate (Awad et al., 2012). HPLC with a fluorescence detector (FLD) and mass spectrometry (MS) detector is suitable for aflatoxin quantification. However, few studies have used an MS for aflatoxin analysis by
* Corresponding author. Tel.: þ82 2 2260 3370; fax: þ82 2 2285 3370. E-mail addresses: [email protected], [email protected] (K.-G. Lee). http://dx.doi.org/10.1016/j.foodcont.2014.09.020 0956-7135/© 2014 Elsevier Ltd. All rights reserved.
estimating particular samples, such as milk and herbal medicines. Recently, LC/MS was used for the qualitative analysis of aflatoxins, and HPLC-FLD was used for the quantitative analysis of aflatoxins (Bognanno et al., 2006; Jang et al., 2007). Many previous studies have analyzed aflatoxins by HPLC-FLD with immunoaffinity extraction. Analytical methods of aflatoxins are usually carried out by HPLC-FLD either with trifluoroacetic acid (TFA) or bromination derivatization (Reiter, Zentek, & Razzazi, 2009). Though AFM1 is known as a stable material (Iha, Barbosa, Okada, & Trucksess, 2012), many previous studies have reported that AFM1 levels are altered during processing and storage. Ultra heat-treated (UHT) milk has been shown to have lower AFM1 contamination levels than pasteurized milk (Rahimi & Behzadnia, 2012; Zheng et al., 2012). In yogurt, cheese, ice cream, and sherbet, processing and storage have been shown to affect AFM1 levels in products (Iha et al., 2012; Wiseman & Marth, 1983). On the other hand, the processing and storage of milk probably did not affect aflatoxins, because milk has a very short distribution period from production to consumption. In this study, various sample preparation methods such as derivatization method and immunoaffinity columns were compared and validated to determine the optimum analytical method for AFM1 and AFM2 in various dairy products. In addition, the levels of AFM1 and AFM2 in the dairy products (e.g., milk,
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yogurt, milk powder, ice cream, and sherbet) were analyzed by the optimized method.
2. Materials and methods 2.1. Reagents and materials AFM1 and AFM2 standard powder (Enzo Life Sciences, Lausen, Switzerland) with 70% methanol (J.T. Baker, Phillipsburg, NJ, USA) was prepared for the stock solution. Working solutions were prepared as 500, 1,000, 2,000, 5,000, and 10,000 ng/kg for calibration curves. Water and acetonitrile, and methanol were HPLC grade (J.T. Baker). TFA (SigmaeAldrich, MO, USA) and Kobra cell (K01, Biopharm Rhone, Glasgow, Scotland) were prepared for derivatization.
2.2. Sampling Milk sampling (including flavored milk) in Seoul, Korea was conducted from June to August 2012. Samples were collected from commercial markets. Yogurt samples were of the drinking yogurt type. Ice cream samples having more than 6% milk lipids were selected, and sherbet samples having less than 3% milk lipids were selected.
2.5. Aflatoxin extraction for milk powder samples First, 10 g of sample with 40 ml of 70% (v/v) methanol was put into a 50-ml conical tube. It was added to 0.5 g of NaCl and shaken at 250 rpm for 1 h. It was centrifuged at 6500 rpm (10 min) and filtrated using a 45-mm syringe filter. Next, 10 ml of filtered solution was diluted by 10 ml of water, and 20 ml of the diluted aflatoxin extracted solution was loaded into an immunoaffinity column. After that, 10 ml of water was loaded for cleaning. The immunoaffinity column was air-dried for 10 min. Finally, 1 ml of methanol was loaded into the column for aflatoxin elution. 2.6. Aflatoxin extraction for ice cream samples Ice cream samples were first melted and filtered by a sieve. Then, 20 g of ice cream filtered of toppings and fruit was put in a 50ml conical tube with 20 ml of methanol. It was added to 0.5 g of NaCl and shaken at 250 rpm for 1 h. Before shaking, 5 ml of hexane was added, and the results were compared with those when 1 drop of Tween 80 was added. It was centrifuged at 6500 rpm (10 min) and filtrated using a 45-mm syringe filter. Next, 10 ml of filtered solution was diluted by 10 ml of water, and 20 ml of the diluted aflatoxin extracted solution was loaded into an immunoaffinity column. After that, 10 ml of water was loaded for cleaning. The immunoaffinity column was air-dried for 10 min. Finally, 1 ml of methanol was loaded into the column for aflatoxin elution.
2.3. Determination of validation values for the analytical method Tests to determine recoveries, coefficient of variation (CV, %) values, Z-scores, limits of detection (LODs), limits of quantitation (LOQs), linearity, and R-squared (R2) values were conducted. Intraand inter-day recovery and CV tests were performed with each representative sample. Tests to determine LODs of aflatoxins were carried out by 3.3 sigma (s)/slope factor of calibration curve. Sigma was obtained by determining the standard deviation of the y-intercept of seven specific calibration curves. Each specific calibration curve was constituted by three-point concentrations. Linearity and R2 values were calculated using each aflatoxin standard curve for quantification. Method detection limits (MDLs) and method detection quantitation (MDQ) values were calculated by LOD/(dilution rate sample weight) and 3 MDL, respectively. 2.4. Aflatoxin extraction for milk, yogurt, and sherbet samples First, 20 g of sample with 20 ml of methanol was put into a 50ml conical tube. In the case of sherbet, it was melted and filtered by a sieve. It was then added to 0.5 g of NaCl and shaken at 250 rpm for 1 h. It was centrifuged at 6500 rpm (10 min) and filtrated using a 45-mm syringe filter. Next, 10 ml of filtered solution was diluted by 10 ml of water, and 20 ml of the diluted aflatoxin extracted solution was loaded into an immunoaffinity column. After that, 10 ml of water was loaded for cleaning. The immunoaffinity column was then air-dried for 10 min. Finally, 1 ml of methanol was loaded into the column for aflatoxin elution.
2.7. AflatoxineTFA derivatization (ATD) method Eluted aflatoxin extracts were dried by nitrogen gas at 40 C. For the derivatization, 0.2 ml of TFA with 1 ml of hexane was added, and it was then stored for 20 min in a darkroom. After that, 50% (v/ v) methanol was mixed to 1 ml using a vortex, and the bottom layer was then injected for analysis. 2.8. Analytical conditions of HPLC-FLD analysis A Waters 1525 system (Milford, MA, USA) and 474 FLD (Milford, MA, USA) were used. For the ATD method, the mobile phase was acetonitrile:water (7:3, v/v), and the column was an Agilent XDBC18 (250 mm 4.6 mm and 5 mm: Palo Alto, CA, USA). Wavelengths for excitation and emission were 360 nm and 450 nm, respectively. For bromination, the mobile phase was a 0.001 M KBr mixture of water, methanol, and acetonitrile (6:2:2, v/v) with 350 ml of 4 M nitric acid for the 1 L mobile phase, and the column was an Agilent Sb-Aq (250 mm 4.6 mm and 5 mm: Palo Alto, CA, USA). Wavelengths for excitation and emission were 365 nm and 435 nm, respectively. In both conditions, the column temperature was 40 C, and the flow rate was 1 ml/min. The injection volume was 20 ml. 2.9. Internal quality control To ensure an accurate analysis, internal quality control was performed. When the analysis of 10 samples was done, 10 ng/g
Table 1 Limit of detection (LOD), Limit of quantification (LOQ), Linearity, and R-squared (R2) values of aflatoxinetrifluoroacetic acid derivatization (ATD) and bromination methods.
Bromination Aflatoxinetrifluoroacetic acid derivatization (ATD)
Type of aflatoxin
LOD (ng/kg)
LOQ (ng/kg)
Linearity
AFM1 AFM2 AFM1 AFM2
125.42 151.73 57.78 189.27
418.05 505.77 263.91 879.71
y y y y
¼ ¼ ¼ ¼
5820.5x þ 635.34 4224.1x þ 137.16 5048.7x þ 4297.4 4770x þ 5362
R2 0.9988 0.9951 0.9906 0.9819
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standard solutions were analyzed to check the sensitivity variation of HPLC-FLD. In addition, when the analysis of 20 samples was done, a recoveries test was performed as an intra-day test. Recovery tests were performed such that AFM1 and AFM2 were spiked with 0.5 ng in samples.
Table 3 Limit of detection (LOD), limit of quantification (LOQ), method detection limits (MDLs) and method detection quantitation (MDQ) of each dairy sample.
LOD
3. Results and discussion LOQ
3.1. Comparing analytical methods' validation values and optimization of sample preparation
MDL MQL
The method validation values of aflatoxins are shown in Table 1. The LOD of bromination method for AFM1 and AFM2 were 125.42 and 151.73 ng/kg, respectively. Though the LOD of the bromination method for AFM1 was higher than that of the ATD method, the Rsquared (R2) values of the bromination method were higher than the ATD method. The repeatability of the bromination method was more stable than the ATD method. Due to these factors, the bromination method was selected for the optimum derivatization method. For the optimized aflatoxin extraction, four immunoaffinity columns were prepared based on the study of S¸enyuva and Gilbert (2010) and compared with recovery tests in milk samples. For the substantive analysis, 0.5 ml of 10 ng/g AFM1 and AFM2 mixture was spiked in 20 g of milk sample, and Afla Prep M was selected for the analysis of this study. Recoveries of each immunoaffinity column are shown in Table 2. The recoveries of each immunoaffinity column were slightly lower than reported in the previous study. According to S¸enyuva and Gilbert (2010), recoveries of immunoaffinity columns were over 80e90% when 2e20 ng of AFM1 and AFM2 were spiked. In this study, recoveries of immunoaffinity columns were 20.4e104.4% when 5 ng of AFM1 and AFM2 were spiked. The different thing of this study compared with that of S¸enyuva & Gilbert was a use of commercial milk instead of a skimmed milk. In the study of S¸enyuva & Gilbert, they used a skimmed milk as a sample for recovery test. Usually recovery of AFMs increases with low fat samples. Previous studies reported 70% methanol as an effective condition for methanol extraction (S¸enyuva & Gilbert, 2010). In this study, almost all samples were prepared in liquid state such as milk and yogurt. With the liquid samples dilution effect occurred while the extraction of AFs. To compensate this, the adequate amount of methanol was measured by freeze-dried, and the water contents of the samples were estimated. With this measurement, 20 ml of methanol was added to 20 g of sample such as milk, yogurt, and sherbet, to make a 70% methanol concentration. Since ice cream samples needed reagents for clear chromatograms, Tween 80 and hexane were compared for their ability to minimize matrix effects, and hexane was selected. Methanol extraction with hexane had higher recoveries for AFM2 than that with Tween 80. Aflatoxins are scarcely extracted by hexane (Cho et al., 2007), and it is an effective method for removing lipids. In addition, several studies have used emulsifiers for aflatoxin analysis (Sadia et al., 2012; Zhang et al., 2012). Table 2 Recoveries of each immunoaffinity column in milk samples. Company R-bio pharm Rhone LTD. NEOGEN Europe LTD. VICAM® R-bio pharm Rhone LTD.
Product Afla-Rhone wide Neo Column Afla M1™ HPLC Afla Prep M
AFM1 (recovery, %) 20.41 104.40 26.32 82.59
Type of AFM
Milk (ng/kg)
Yogurt (ng/kg)
Milk powder (ng/kg)
Ice cream (ng/kg)
Sherbet (ng/kg)
AFM1 AFM2 AFM1 AFM2 AFM1 AFM2 AFM1 AFM2
125.42 151.73 418.05 505.77 25.08 83.61 30.35 101.15
125.42 151.73 418.05 505.77 25.08 83.61 30.35 101.15
125.42 151.73 418.05 505.77 50.16 167.22 60.69 202.31
125.42 151.73 418.05 505.77 25.08 83.61 30.35 101.15
125.42 151.73 418.05 505.77 25.08 83.61 30.35 101.15
LOD, LOQ, MDL and method quantitative limits (MQLs) are shown in Table 3 using the optimized method. Each specific calibration curve was comprised of three concentrations. The MDL and MDQ ranges were 25.08e167.22 and 83.61e202.31 ng/kg, respectively. Recovery tests were conducted for each sample with an intra- and inter-day test (shown in Table 4). Recoveries and CV values of AFM were 64.17e101.86% and 1.83e14.35%, respectively. 3.2. AFM1 and AFM2 contamination levels in milk, yogurt, milk powder, ice cream, and sherbet Contamination levels of AFM1 and AFM2 are shown in Table 5. Each of the 50 samples was analyzed, and the detection rates (%) of AFM1 and AFM2 were 6e74% and 2e10%, respectively. Milk powder samples had the highest contamination rate and AFM1 level. It is assumed that the milk powder underwent an evaporation process, and the level of AFM1 is concentrated through the evaporation process. Although many countries have maximum residue limits (MRLs) of AFM1 in dairy products (e.g., 500 ng/kg or 250 ng/kg), risk-assessment methods have not yet been clearly prepared. This is because AFM1 has no accurate bench mark dose limit (BMDL) or provisional tolerable weekly intake (PTWI) from the official organization. However, milk powder is mainly consumed by infants and toddlers, and the ingestion amount of milk powder per kilogram of body weight is much higher in infants and toddlers than in adults. This could lead to serious health problems in infants and toddlers, though the toxicity of AFM1 has been evaluated as being one tenth that of aflatoxin B1 (Boon, Bakker, van Klaveren, & van Rossum, 2009). In the case of AFM2, yogurt has the highest detection rate, maximum values, and 95 percentile and 99 percentile levels. Iha et al. (2012) reported that the level of AFM1 is not affected by yogurt fermentation. In this study, milk and yogurt samples had similar AFM1 concentrations. However, yogurt samples had higher AFM2 concentrations than milk samples. In dairy products, lactic acids react with AFB1 and AFG1, AFB2 and AFG2 are produced (Ram, Ramtej, & Daxa, 2002). Thus, it is likely that lactic acid affects whether AFM1 converts to AFM2. The detection rate and Table 4 Recoveries and coefficient variation (CV) of aflatoxin M1 and M2 (AFM1 and AFM2) in milk, yogurt, milk powder, ice cream, and sherbet samples. Sample type
AFM2 (recovery, %) 104.43 42.44 72.19 93.02
Note: Commercial milk samples were used and spiked with 0.5 ml of 100 mg/kg aflatoxin M1 and M2 mixture for the recoveries test.
469
Recoveries (% ± CV) Inter-day
Milk Yogurt Milk powder Ice cream Sherbet
84.62 77.60 64.70 69.26 82.44
Intra-day AFM2
AFM1 ± ± ± ± ±
5.28 7.46 5.22 10.22 7.21
93.13 64.17 83.32 82.90 83.85
AFM1 ± ± ± ± ±
5.71 1.83 7.82 1.85 2.67
85.56 70.96 65.45 73.83 69.30
AFM2 ± ± ± ± ±
7.20 8.10 7.79 5.43 12.18
101.86 70.72 78.95 96.94 90.92
± ± ± ± ±
7.74 8.36 4.99 14.35 11.11
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Table 5 Mean, geomean, median, 95 and 99 percentile, and maximum values of aflatoxin M1 and M2 (AFM1 and AFM2) in samples. Samples
Type of aflatoxin Detectionrate (%, n ¼ 50) Mean (ng/kg) Geomean (ng/kg) Median (ng/kg) 95 percentile (ng/kg) 99 percentile (ng/kg) Maximum (ng/kg)
Milk
AFM1 AFM2 Yogurt AFM1 AFM2 Milk powder AFM1 AFM2 Ice cream AFM1 AFM2 Sherbet AFM1 AFM2
6 2 14 10 74 2 36 8 6 4
20.81 20.62 22.54 55.67 270.94 30.91 33.16 25.62 14.48 67.53
14.40 16.09 16.05 19.97 147.61 30.75 22.13 18.10 13.49 17.83
12.54 15.17 12.54 15.17 171.93 30.35 12.54 15.17 12.54 15.17
33.78 15.17 73.78 252.17 680.45 30.35 112.36 111.12 19.96 15.17
200.55 154.11 141.22 797.65 795.78 44.68 120.81 183.90 54.30 1344.42
228.60 287.60 191.29 850.01 835.57 58.45 121.31 214.54 61.22 2336.63
Table 6 Mean, geomean, median, 95 and 99 percentile, and maximum values of aflatoxin M1 and M2 (AFM1 and AFM2) in milk and milk powder samples determined by sterilization method. Samples
Type of AFM
Sample number (n)
Mean (ng/kg)
Geomean (ng/kg)
Median (ng/kg)
95 percentile (ng/kg)
99 percentile (ng/kg)
Maximum (ng/kg)
UHT milk
AFM1 AFM2 AFM1 AFM2 AFM1 AFM2 AFM1 AFM2
36
20.81 20.62 22.54 55.67 270.94 30.91 33.16 25.62
14.40 16.09 16.05 19.97 147.61 30.75 22.13 18.10
12.54 15.17 12.54 15.17 171.93 30.35 12.54 15.17
33.78 15.17 73.78 252.17 680.45 30.35 112.36 111.12
200.55 154.11 141.22 797.65 795.78 44.68 120.81 183.90
228.60 287.60 191.29 850.01 835.57 58.45 121.31 214.54
Pasteurized milk UHT milk powder Pasteurized milk powder
14 43 7
contamination level of AFM1 in ice cream were 36% and 0.03 ng/g, respectively. In addition, ice cream and sherbet samples had higher detection rates and contamination levels than milk samples. Wiseman & Marth reported an increase of AFM1 during ice cream and sherbet manufacture and storage by improving the recovery of bound AFM1. In this study, ice cream samples had higher concentrations of AFM1 than milk samples, but sherbet samples did not. Khoshnevis, Azizi, Shateri, and Mousavizadeh (2012) reported that 22% of ice cream samples were contaminated by AFM1, with mean values of 33.98 ng/kg. Among the samples, milk samples had lower detection rates and contamination levels than the other samples. Kocasari, Tasci, and Mor (2012) analyzed dairy products in which higher AFM1 levels were detected in milk powder, white cheese, butter, and yogurt. AFM1 concentrations in milk and milk powders determined by the sterilization method are shown in Table 6. According to previous studies, the milk sterilization method affects AFM1 concentration in milk. AFM1 concentrations in pasteurized milk have been shown to be higher than those of raw and UHT milk (Hwang, Hwang, Kim, & Oh, 2012; Kocasari et al., 2012; Zheng et al., 2012). In this study, pasteurized milk had higher mean values of AFM1 and AFM2 concentrations than UHT milk. However, pasteurized milk powder had lower AFM1 levels than UHT milk powder. AFM2 in pasteurized milk powder had higher 95 and 99 percentiles and maximum values than in UHT milk powder. 4. Conclusion In conclusion, the HPLC-FLD analytical method using bromination was successfully applied to quantify AFM1 and AFM2 in dairy products. In this study, concentrations of AFM1 and AFM2 were 20.62e270.94 ng/kg. Among the samples, milk powder samples had a detection rate of 74% and an AFM1 concentration of 270.94 ng/kg. Pasteurized milk had higher AFM1 levels than UHT milk. Conversely, UHT milk powder had higher AFM1 levels than pasteurized milk powder. In addition, pasteurized milk powder had higher AFM2 concentrations, 95 and 99 percentiles, and maximum values.
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