Highly Sensitive and Quantitative Method for Measuring Aflatoxin M1

Chapter 18

Highly Sensitive and Quantitative Method for Measuring Aflatoxin M in Milk 1

Jianmin Liu* and Stephen Powers

Downloaded by COLUMBIA UNIV on July 1, 2012 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch018

VICAM Group of Waters Technology Corporation, 313 Pleasant Street, Watertown, MA 02472 *Corresponding author: [email protected]

Aflatoxin M (AFM ) is a metabolite of aflatoxin B found in milk of dairy cattle which have consumed feed contaminated with aflatoxin B . A F M has been reported to be carcinogenic and hepatotoxic. The European Commission has established maximum permissible limits of 50 parts per trillion (ppt) for A F M in milk. We have developed an assay for A F M which meets the E U sensitivity requirements and can be performed using a simple single-wellfluorometer.The method utilizes a novel "mobile bead" configuration immunoaffinity column and requires less than 40 min including sample preparation. The assay has excellent linearity (R = 0.998) over a concentration range of 0-200 ppt. Precision at 25 ppt and 50 ppt was excellent with % CV below 10%. The limit of detection was 12.5 ppt. 1

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306

© 2008 American Chemical Society

In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

307 Aflatoxin B (AFB ) is one of the most toxic and common natural toxins found in grains, oilseeds, spices, and tree nuts. It has significant impact on food/feed quality, human health and agricultural economics. It has been wellknown that AFB is a carcinogen, and that high level exposure produces an acute necrosis, cirrhosis and carcinoma of the liver in mammals (1). Aflatoxin M (AFM ) is one of the metabolites of AFB in human and animals that consume commodities contaminated with AFBi. It has been found in urine, milk and meats (7, 4). The estimated conversion rate from AFBi to A F M in vivo is about 1-3% (2, 5). A F M is a hydroxylated form of AFB , also with carcinogenic properties. Both compounds are heat-stable and react with DNA and albumin. A F M and its DNA/albumin adducts have been used as biomarkers to determine the degree of human exposure (4, 7). The U.S. Food and Drug Administration (FDA) has established maximum limits for aflatoxin levels in human foods and animal feeds. The current FDA guidelines allow 20 ppb of total aflatoxins in feed fed to lactating dairy cows and 0.5 ppb of A F M in fluid milk (5). However, the regulatory levels of the European Union for aflatoxins in food and feed are much stricter. The action levels for A F M in milk are 50 ppt for adults and 25 ppt for infants (5). There are several methods commercially available for analysis of milk for A F M , , including liquid chromatography (LC), enzyme linked immunosorbent assay (ELISA), a lateral flow based strip test, and affinity column/fluorometric measurement. However, there is no affinity column/fluorometric method that can detect A F M below 25 ppt in milk, although the LC method can determine as low as 5 ppt in liquid milk (8). VICAM recently has developed a simple, rapid, and quantitative method for A F M test in fluid milk. This special A F M test is highly sensitive, and can detect as low as 12.5 ppt A F M in milk. It does not require high technical skill and can be used in the field. 1

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Materials and Methods Reagents and Affinity Column Aflatoxin M i standard and HPLC grade methanol were purchased from Sigma. Aflatest developer concentrate (0.03% bromine), AflaMi mobile resin affinity column, Aflatest-M™ fluorometer calibration standard, glass cuvette, Afla M i 4-position pump stand and fluorometer Series 4 were obtained from VICAM in this study. The Afla Mi column is a 3 mL plastic column packed with 250 nL of resin containing covalently bound antibody of anti-AFBi. FL+

F L +

Sample Preparation and Cleanup If the milk is homogenized, for example purchased from a grocery store, it can be used directly for analysis. However, raw milk from a dairy farm is In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

308 usually not clear and centrifugation is needed. Centrifuge approximately 50 mL milk at 2000 x g for 10 min. Carefully take the skim portion (bottom layer) of the milk for analysis without disturbing the top (fat) layer. Forty mL of skim milk is needed for each affinity column.

Column Preparation F L +


The Afla M i column is a mobile resin column. Make sure that the level of resin inside the column is horizontal. If the resin is not horizontal, gently tap the lower side of the column several times with afingerto level the resin.

Column Chromatography F L +

Pass 40 mL skim milk completely through the Afla M i affinity column at a rate of about 1-2 drops/sec until air comes through column. Remove the Afla M i columnfromthe loading syringe barrel. Remove the top frit from the Afla M i column using the Frit-picker. Fill the Afla M i column headspace with methanol-water (10:90, v/v). Fit the Afla M i column to a clean glass syringe barrel. Fill the glass syringe barrel with 10 mL methanol-water (10:90, v/v), which is passed through the column at a rate of about 2-3 drops/sec until the level of solvent is about 3 cm above the top of the resin bed. Remove the column from the syringe barrel and cap it with the cap that comes with the column. Invert the column 10 times until resin beads are completely washed from the bottom frit. Remove the top cap from the column. Fill the column headspace with methanol-water (10:90, v/v) and place the column back onto the syringe barrel. Fill the glass syringe barrel with 10 ml methanol-water (10:90, v/v) and pass it through the column at a rate of about 2-3 drops/sec. Repeat this step once more until air comes through the column. Elute the AFMi from the affinity column with 1 mL methanol-water (80:20, v/v) at a rate of about 1 drop/sec until air comes through the column, collecting the eluate in a cuvette. Add 1.0 mL of AflaTest developer concentrate diluted 1:10 with water to the eluate in the cuvette. Mix well, place the cuvette in a calibrated fluorometer and read it. F L +

F L +

F L +

F L +

Results and Discussion Limit of Quantitation In this study, the limit of quantitation was defined as the smallest amount of AFMi which is reproducibly and accurately detected. Raw milk samples were


309 collected from two different dairy farms, and were determined to be AFMpfree by LC analysis. The AFM free milk samples were spiked with AFMi (Supelco, PA) at 0, 12.5, and 25 ppt. Two independent experiments were done on two different days. The results are presented in Table I. Two A F M Î spiking levels, 12.5 and 25 ppt in raw milk, were examined; the average fluorometer readings were 12.4 and 28.3 ppt, respectively. The percent coefficients of variation (CV) for 12.5 ppt and 25 ppt levels were 34.7% and 9.2% respectively, suggesting that the limit of quantitation for this test is at or below 12.5ppt


r

Linearity Linearity was determined using AFM spiked raw milk samples ranging from 0 ppt to 200 ppt. Individual data points are shown in Table II. The percent coefficient of variation was less than 10% in all spiked levels except for the 50 ppt level. The graph below shows the linear regression analysis equation for the amount of AFM! measured by fluorometer vs the amount spiked in raw milk. The linear regression equation is Y = 1.037x + 0.246. The correlation coefficient (r) of 0.999 from the above linear regression equation indicates excellent linearity between the A F M ! spiked level and flurometer reading. r

Table I. Fluorometer reading in AFM free and -spiked raw milk r

25 ppt spike

0

12.5 ppt spike 14

0

20

28

0

15

31

5.5 0

8

30

10 12

29 29

8

24

0 ppt spike

0

28

32 30 25 25 Average reading

0.9

12.4

28.3

SD

2.2

4.3

%CV

-

34.7

2.6 9.2



200

200

210

100

110

100 220

98

59

53

50

65

23

25

27

12

10

12

12.5 25

0

0

0

3

2

1

Spike level (PPt) 0

t

210

95

208

100.6

57.6

25.4

27 61

11.5

0

0 11

Mean

4

8.4

4.0

5.6

10.5

6.1 5.6

6.6

8.7

0

CV (%)

1.7

1.0

0.0

SD

Table II. A F M Spiking Level vs Fluorometer Reading in Milk



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Spike levels of A F M ! in raw milk (ppt) Figure 1. Dose response of AFM¡ spiking vs fluorometer reading

Correlation Between LC and Fluorometer Results

Naturally contaminated milk samples were collected and analyzed by LC and the VICAM fluorometer method. As shown in Figure 2, the correlation coefficient of r = 0.96 indicates a strong positive correlation between LC and VICAM fluorometer results, suggesting that VICAM fluorometer AFM! test is an excellent predictor of measuring AFMi in milk.

Conclusion The VICAM AflaMi test in milk involves specific antibody-based affinity column isolation, AFMi derivatization with bromine and fluorometer reading. The intensity offluorescentsignal directly reflects the amount of AFMi in the milk sample. The test is straightforward, easy to run, highly sensitive, and



312

quantitative. The result is accurate and comparable to that for LC. It does not require high technical skill and can be used in the field.

Acknowledgement Partial data (Figure 2) from this study were generated by Drs. S. Tenti and P. Berzaghi in the Dept. of Animal Science, University of Padova, Italy.

References 1. 2.

The Toxicology of Aflatoxins; Eaton, D. L.; Groopman, J. D., Eds.; Academic Press: New York, 1994; pp 383-426. Price, R. L.; Paulson, J. H.; Lough, O. G.; Gingg, C.; Kurtz, A.G. J. Food Prot. 1985, 48, 11-15.


313 Applebaum, R. S.; Brackett, R. E.; Wiseman, D. W.; Marth, E. H. J. Food Prot. 1982, 45, 752. 4. Sudakin, D.L. J. Toxicol. Clin. Toxicol. 2003, 41, 195-204. 5. FDA. Compliance Policy Guide. CPG 527 400 (CPG 7106.10). 6. European Commission Regulation. Off. J. Eur. Commun. 1998, EC No. 1525/98, L201, 43-46. 7. Wild, C. P.; Hasegawa, R.; Barraud, L.; Chutimataewin, S.; Chapot, B.; Ito, N.; Montesano,R. Cancer Epidemiol. Biomark. Prevent. 1996, 5, 179189. 8. Dragacci, S.; Grosso, F.; Gilbert, J. JAOAC Int. 2001, 84, 437-443. Downloaded by COLUMBIA UNIV on July 1, 2012 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch018

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Highly Sensitive and Quantitative Method for Measuring Aflatoxin M1

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