SPSFAM ERP

11. Pipet 1 mL of this filtrate into a 4 dram vial and evaporate the solution to complete dryness under a flow of nitrogen at 55 ± 3°C. This may be achieved using an N-Evap 112 Nitrogen Evaporator or a heating block. 12. Once vials are completely dry, add 10 mL water to each. Cap and vortex for at least 15 seconds. Allow vials to sit for at least 15 minutes and repeat vortexing procedure. 13. Dilute samples as necessary so that all analytes are within the calibration curve. It may be necessary to perform multiple dilutions on a given sample to accomplish this. 14. Filter the diluted sample using a disposable syringe and 0.45 μm GHP filter into an injection vial. G-2. Sample Preparation (for high protein matrices using Carrez extraction, >35% total protein on DMB or unknown protein content) 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. Transfer 25 mL of the filtrate to an additional cup using a serological pipet. 9. Add 5 mL of Carrez I reagent and allow to stir on a stir plate for 5 minutes. 10. Add 5 mL of Carrez II reagent and a stir bar, and allow to stir on a stir plate for 10 minutes. Note that the solution with begin to turn cloudy. 11. Using a pH meter, bring the pH of the sample to a value between 7.5 and 8.0 using 1N NaOH. Note that a precipitate will begin to form. 12. Quantitatively transfer the solution to a 50 mL volumetric flask using water. 13. Bring the flask to volume using water. Cap and invert to mix. 14. Filter then entire volume of the 50 mL flask through qualitative filter paper and funnel. Collect the filtrate into a plastic cup or other suitable container.