Description: Aflatest Evaluation Protocol for Milk Related doc: GN-P0025

Doc.#: VP-1001-3 Effective date: 04/12/00

PROTOCOL FOR THE EVALUATION OF VICAMS AFLATEST IN MILK 1.0 BACKGROUND:

The objective of this study is to evaluate the performance of VICAMs AflaTest column for the determination of Aflatoxin M1 in milk.

2.0 PRINCIPLE:
The study will address the limit of detection, precision and accuracy of AflaTest columns. The limit of detection will be compared with aflatoxin M1 spiked nonfat dry (powder) milk samples. Each spike level (0.03 ppb, 0.05 ppb, 0.1 ppb, 0.2 ppb and 0.4 ppb) will be measured in duplicate. Precision will be compared by measuring spiked samples at low and high aflatoxin M1 levels (0.1 ppb and 2.0 ppb, respectively). Ten replicates of each level will be analyzed, the results averaged, the standard deviation calculated and the % coefficient of variance will be used to determine the precision. Accuracy will be compared using results obtained from the 0.05 ppb, 0.1 ppb, 0.2 ppb, 0.4 ppb, 0.5 ppb and 2.0 ppb sample determinations. All measurements will be done by HPLC using the procedure described below.

3.0 METHOD:
3.1 Sample Preparation: 3.1.1 Weigh 10g of milk powder into a 250 mL beaker. 3.1.2 Heat 100 mL water to 30-40C. 3.1.3 Add 80 mL of preheated water in small amounts to the milk powder. Mix continuously until a homogenous mixture is obtained. 3.1.4 Transfer to 100 mL measuring cylinder and make up to 10 mL with the remaining preheated water. 3.1.5 Filter through VWR 315 filter paper. Preparation of Spiked Samples & HPLC Standard: Milk samples were spiked and HPLC standard made with Aflatoxin M at 10ug/mL in acetonitrile from Supelco (1-800-247-6628) #4-6319. For example: For 2ppb milk spike: 2ppb = 2ng aflatoxin M/mL milk = 500ng aflatoxin M/250mL milk 10g/mL aflatoxin M standard = 10ng/L so 500ng 10ng/L standard = 50L spike into 250mL milk for 2ppb spike. Standard was diluted in acetonitrile to spike at lower levels.

3.2

________________________________________________________________________________________ VICAM L.P. Page 1 of 7 Date Printed: November 16, 2005

Description: Aflatest Evaluation Protocol for Milk Related doc: GN-P0025 For 2ppb HPLC standard:

Doc.#: VP-1001-3 Effective date: 04/12/00

2ppb = 2ng/mL milk = 100ng/50mL of milk passed over column column eluted with 1mL methanol =100ng aflatoxin M1 standard in 1mL methanol. 100ng 10ng/L aflatoxin M standard = 10L spike into 990L methanol. Then 1.5mL water added to give a total of 2.5mL as with samples. 100L injected onto HPLC 3.3 HPLC Setup: 3.3.1 Column: Reverse phase, C18 (5 mm X 12.5 cm C18 column, 5m) 3.3.2 Mobile phase: 45% methanol : H2O Isocratic 3.3.3 Flow rate: 0.8 mL/ min. (Beckman 110B Pump) 3.3.4 Detection Wavelength: 360nm excitation and 440 nm emission AflaTest Affinity Chromatography: 3.4.1 Set up syringe column assembly. 3.4.2 Attach an AflaTest column to the sample loading syringe barrel outlet. 3.4.3 Pour 50 mL of filtered milk into syringe barrel. Apply milk sample to the column at 2 drops/second flow rate. 3.4.4 Transfer column to a clean sample loading syringe barrel outlet. Wash column 2 times with 10 mL water. 3.4.5 Elute AflaTest column with 1 mL 100% methanol and collect eluate into a glass test tube. Add 1.5 mL of water to the eluate (final volume 2.5 mL) and inject 100 L into HPLC.

3.4

4.0 RESULTS
All measurements were performed by HPLC. The results are expressed as peak area and as aflatoxin concentration. The peak area was calculated by the HPLC integrator and the aflatoxin concentration was obtained using the regression analysis equations from the standard curves below. 4.1 Standard Curve #1 (For values from 0.2 ppb to 2.0 ppb)

Standards 0.05 ppb 0.2 ppb 2.0 ppb

Peak area 68460 127002 578292

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Description: Aflatest Evaluation Protocol for Milk Related doc: GN-P0025

Doc.#: VP-1001-3 Effective date: 04/12/00

y = 256939236x + 65213.573
600000

r = 0.999

500000

400000 Peak area

300000

200000

100000

0 0 0.5 1 1.5 2 2.5 Concentration

4.2

Standard Curve #2 (For values from 0.03 ppb to 0.2 ppb) Standards 0.05 ppb 0.2 ppb 0.5 ppb Peak area 56976 132417 346919

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Description: Aflatest Evaluation Protocol for Milk Related doc: GN-P0025

y = 654410.476x + 15169.048
400000 350000 300000 250000 Peak area 200000 150000 100000 50000 0 0 0.1 0.2 0.3 Concentration 0.4 0.5

Doc.#: VP-1001-3 Effective date: 04/12/00

r = 0.997

0.6

4.3

Limit of detection:

Aflatoxin M1 conc 0.03 ppb 0.05 ppb 0.1 ppb 0.2 ppb 0.4 ppb

AflaTest Peak area Conc. 26833 0.017 ppb 39745 0.037 ppb 44495 0.044 ppb 41027 0.039 ppb 49112 0.052 ppb 50309 0.053 ppb 108800 0.143 ppb 103628 0.135 ppb 190979 0.489 ppb 189274 0.483 ppb

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Description: Aflatest Evaluation Protocol for Milk Related doc: GN-P0025 4.4 Precision: AflaTest Peak area Conc. 49112 0.052 ppb 50309 0.053 ppb 49116 0.052 ppb 50626 0.054 ppb 76467 0.093 ppb 79756 0.098 ppb 63117 0.073 ppb 89791 0.114 ppb 90290 0.114 ppb 79864 0.098 ppb 67844.8 0.080 ppb 17237.3 0.026 25.40 32.63 Aflatoxin M1 conc 2.0 ppb

Doc.#: VP-1001-3 Effective date: 04/12/00

Aflatoxin M1 conc 0.1 ppb

Mean SD %CV 4.5 Accuracy

Mean SD %CV

AflaTest Peak area Conc. 415642 1.364 ppb 380603 1.227 ppb 376839 1.213 ppb 379244 1.222 ppb 433435 1.433 ppb 372826 1.197 ppb 381612 1.231 ppb 375267 1.206 ppb 388173 1.256 ppb 348905 1.104 ppb 385254.6 1.245 ppb 23500.9 0.0915 6.10 7.35

Aflatoxin M1 conc 0.05 ppb 0.1 ppb 0.2 ppb 0.4 ppb 0.5 ppb 2.0 ppb

AflaTest Peak area Conc. 44495 0.044 ppb 41027 0.039 ppb 49112 0.052 ppb 50309 0.053 ppb 108800 0.143 ppb 103628 0.135 ppb 190979 0.489 ppb 189274 0.483 ppb 215417 0.585 ppb 205061 0.544 ppb 415642 1.364 ppb 380603 1.227 ppb

________________________________________________________________________________________ VICAM L.P. Page 5 of 7 Date Printed: November 16, 2005

Description: Aflatest Evaluation Protocol for Milk Related doc: GN-P0025

Doc.#: VP-1001-3 Effective date: 04/12/00

5.0 SUMMARY OF RESULTS AND CONCLUSIONS:

5.1 Limit of Detection:

Aflatoxin M1 conc 0.03 ppb 0.05 ppb 0.1 ppb 0.2 ppb 0.4 ppb

AflaTest Conc. (mean) 0.027 ppb 0.041 ppb 0.052 ppb 0.143 ppb 0.486 ppb

The results from the experiment to determine the ability of AflaTest columns to detect aflatoxin M1 showed that AflaTest can detect at least 0.03 ppb levels of aflatoxin M1. 5.2 Precision:

Mean SD %CV

2ppb 1.245 ppb 0.091 7.35

0.1ppb 0.080 ppb 0.026 32.63

Samples spiked with high (2.0 ppb) or low (0.1 ppb) levels of aflatoxin M1 were used to measure the reproducibility of the results obtained with AflaTest. Results from 10 replicate samples containing 2.0 ppb aflatoxin M1 showed a percentage coefficient of variation of 7.3. At 0.1 ppb contaminating level, the results were 32.6%.

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Description: Aflatest Evaluation Protocol for Milk Related doc: GN-P0025 5.3 Accuracy:

Doc.#: VP-1001-3 Effective date: 04/12/00

Aflatoxin M1 conc. 0.05 ppb 0.1 ppb 0.2 ppb 0.4 ppb 0.5 ppb 2.0 ppb

AflaTest conc. (mean) 0.041 ppb 0.053 ppb 0.139 ppb 0.486 ppb 0.564 ppb 1.295 ppb

For the purpose of this experiment, accuracy was defined as the percentage recovered, i.e., the amount of aflatoxin M1 recovered from each spike level. 100% recovery was achieved when the amount recovered equaled the amount spiked into the sample. The table below shows the percentage recovery calculated based on the results shown in the table above.

Aflatoxin M1 conc. 0.05 ppb 0.1 ppb 0.2 ppb 0.4 ppb 0.5 ppb 2.0 ppb Mean

AflaTest % recovery 82 53 69.5 121.5 113 65 84

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