Review Team (February 2016)

Bio-02 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

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, mix well. 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, collect the filtrate. 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 the sample filtrate onto the column as per Table 1 and initialise the flow with the help of a vacuum pump. 10. Let the solution pass through the column by gravity at the rate of one drop per second. 11. Wash the column by passing 10mL of PBS through the column followed by 10mL of water (initialise the flow with the help of vacuum at every step and leave it for gravity). 12. Remove any residual liquid from the column by introducing gentle vacuum 13. 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. 14. Evaporate the eluent to dryness using a heating block set at 85±5 0 C, under a gentle nitrogen blow down.

15. Cool down to room temperature by keeping it outside for about 15 minutes 16. Re-dissolve with 1mL water; cap the reacti-vials and vortex for 30 seconds. 17. Filter using syringe filter into a clean glass insert 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 0.5 1.0 5.0

0.5 1.0 5.0

20 10 10

100 100 100 100 100 100

50 20 10 10 10

1 mL 1 mL 1 mL 1 mL 1 mL 1 mL

1 1 1 1 5

5 2 5

50.0

2.0 (Slurry 16g) 1.0 (Slurry 8g) 0.5 (Slurry 4g)

10 10 10

50.0

100.0 400.0

100.0

5

2.5

7. STANDARD PREPARATION Stock Standard (100μg/mL): Weigh 25mg of biotin reference material into a 250mL amber volumetric flask. Add 150mL of water and sonicate at room temperature for 90 minutes with occasional shaking. Make up to volume with water. Intermediate Standard (100μg/100mL): Dilute 1mL of the stock standard to 100mL with water. Standard 1 (1.0μg/100mL): Dilute 100μL of intermediate standard to 10mL with water. Standard 2 (2.5μg/100mL): Dilute 250μL of intermediate standard to 10mL with water. Standard 3 (5.0μg/100mL): Dilute 500μL of intermediate standard to 10mL with water. Standard 4 (7.5μg/100mL): Dilute 750μL of intermediate standard to 10mL with water. Standard 5 (10μg/100mL): Dilute 1mL of intermediate standard to 10mL with water. Standard 6 (20μg/100mL): Dilute 2mL of intermediate standard to 10mL with water.

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