SPSFAM ERP

Reference Standard Stock Solution Volume (mL)

Target concentration ( µ g/mL)

Final Volume (mL)

Calibration Working Standard

Intermediate Internal Standard Solution Volume (mL)

Amount of NaN 3 (mg)

WS1 WS2 WS3 WS4 WS5

1 1 1 1 1

1 4

20 20 20 20 20

200 200 200 200 200

0.5

2 5

10 20 30

10 15

F. Safety See AOAC Official Methods of Analysis (2005), Appendix B: Laboratory Safety. Use appropriate personal protective equipment such as lab coat, safety glasses, rubber gloves, and a fume hood. Dispose of solvents and solutions according to federal, state, and local regulations. G-1. Sample Preparation (for low protein matrices, <35% total protein on DMB) 1. Weigh 2.5 ± 0.1 g of sample into a suitable container such as a 500-mL Erlenmeyer flask. Record weight to at least the nearest 0.001 g. 2. Add 200 mL of Extraction Solution with a bottle top dispenser. 3. Add 0.250 mL of Internal Standard Stock Solution with a positive displacement pipet or electronic repeater pipet with equivalent accuracy. The same Internal Standard Stock Solution must be used for the preparation of both standards and samples. 4. Add a magnetic stir bar, cap with stopper, and mix vigorously on a stir plate for approximately 30 minutes. 5. Add another 100 mL of Extraction Solution using a bottle top dispenser. Cap the flask and continue to stir on a stir plate for another 30 minutes. 6. Add another 200 mL of Extraction Solution to achieve a final volume of 500 mL. Cap again and mix well by shaking and inverting the flask. Allow sufficient time for the sample to settle before filtering. 7. Pour the solution through qualitative filter paper and a funnel, and collect filtrate into a suitable container, such as a plastic cup. If needed, add Celite onto filter paper to aid filtration. Filtration may be further aided by transferring a portion of the sample from the Erlenmeyer flask to an appropriately sized centrifuge tube and centrifuging until some of the solids have spun down, approximately 5 minutes. 8. For each sample, pipet 3 mL of Extraction Solution into separate molecular weight filters (MWCO). Cap and centrifuge at 1500 g for approximately 20 minutes. 9. After 20 minutes, discard the filtered solution and any remaining liquid in the filter. 10. Reassemble the MWF and fill with approximately 4 mL of sample filtrate. Centrifuge at 1500 g for at least 30 minutes or until more than 1 mL of filtrate has been collected beneath the filter.