AOAC SPIFAN ERP Meeting Book-March 16, 2016

Bio-02 w/SLV FOR ERP USE ONLY DO NOT DISTRIBUTE

3. 0.1% Phosphoric acid: In a 1L volumetric flask, add 500 mL water. Add 1.2 mL of ortho- phosphoric acid. Mix and make up to mark with water.

5. APPARATUS 1.Whatman Glass Microfiber Filters, CAT No. 1820-125 2.Biopharm Immunoaffinity column Pack P82/ P82B 3.SPE Manifold with accessories 4.Autoclave set at 121 0 C

6. SAMPLE PREPARATION Note: Please see Table 1 for weight and loading volume for different product range. Slurry may be used wherever product heterogeneity is expected. Slurry: Reconstitute the 25g powder with warm water (~50ºC) to a total weight of 200g. Mix thoroughly on a horizontal shaker for 15 minutes and then sonicate at 50 0 C for 10 minutes. Cool to room temperature. 1. Weigh sample/ slurry into a 100mL amber glass screw cap bottle. Refer to Table 1. 2. Add 0.15 M sodium phosphate buffer to a volume of 50mL. 3. Swirl gently to mix. 4. Autoclave the sample preparation at 121 0 C for 25 minutes. 5. Cool the sample to room temperature. Quantitatively transfer the extracts into a 100mL volumetric flask and make up to mark with 0.15 M sodium phosphate buffer. 6. Transfer extracts into centrifuge tubes and centrifuge the samples at 4000rpm for 15min. 7. Filter the samples using the Whatman glass microfiber filter paper (re-filter the samples if the filtrate looks cloudy). 8. Set up the SPE manifold. Attach the immunoaffinity column connected to a 10mL reservoir. Drain off the buffer just above the gel. 9. Load filtrate onto the column and let the solution pass through the column by gravity. 10. Wash the column by passing 10mL of PBS through the column followed by 10mL of water. 11. Introduce a reacti-vial and elute the analyte under gravity with 2mL methanol. Elute further with additional 1mL of methanol. Back flush at least 3 times when eluting. 12. Evaporate the eluent to dryness using a heating block set at 85 + 5 0 C, under a gentle nitrogen blow down. 13. Re-dissolve with 1mL water, cap the reacti-vials and vortex for 30 seconds. Filter and vial for HPLC analysis. Table 1: Sample Preparation Product (µg/100g) Sample Preparation Conc (µg/100mL)

Volume (mL)

Weight (g)

Load (mL)

Final

Min

Max

Min

Max

0.1 1.0 5.0

1.0 5.0

20 10

100 100 100 100 100

50 10 10 10

1 mL 1 mL 1 mL 1 mL 1 mL

1 1 1 5

10

5

50.0

2 (Slurry 16g) 1 (Slurry 8g)

10 10

50.0

100.0 250.0

100.0

0.5

5

2.5

6.25

5841 v2

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