AOAC SPIFAN Nutrients ERP-Final Review (Jan. 2019)

D RAHER & W HITE : J OURNAL OF AOAC I NTERNATIONAL V OL . 101, N O . 3, 2018 827

Figure 2017.03A. Typical medium standard chromatogram.

(h) Column rinse solution. — Prepare a maintenance solution of approximately 25% methanol in water to use as a system rinse following each analysis run.

standard to all tubes, including the enzyme blanks. Add 3.0 mL of 0.1 M pH 8.5 Trizma buffer and 200 µL methanol to all tubes, including the enzyme blanks. Cap the tubes and mix on a vortex mixer lightly to mix contents. It is important to be sure that powder samples are completely dissolved, but do not overagitate the solution to avoid foaming. Incubate the samples for 16 – 24 h in a heating unit previously set to 50°C. (b) Remove the samples from the incubation unit after a minimum of 16 h. Allow samples to cool to room temperature. Remove the caps and add 12mLmethanol to each tube. Dilute each tube to a 50 mL volume with laboratory water. (c) Cap the tubes and mix thoroughly by inversion. Attach a 0.45 µm filter to a 3 cc syringe and transfer several milliliter of each sample to the syringe. Filter the samples into an autosampler vial, cap, and analyze the samples.

E. Sample Preparation

(a) Set the heating unit to 50°C prior to the experiment. Weigh liquid samples and reconstituted powders directly into tarred 50 mL centrifuge tubes using a disposable transfer pipet. For nonreconstituted powder samples, weigh into a tarred tube with a spatula. Add 500 µL protease enzyme solution to all samples. Prepare no less than two blank enzyme solutions per run. Blank solutions contain enzyme, internal standard, and buffer only. Add 0.5 mL 5-methyl- DL -tryptophan internal

Figure 2017.03B. Typical blank chromatogram.