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

2017.03 (JAN 2019) Amino-02-(ERP Review for Pubs) Tryptophan by enz hyd_Abbott Labs_Method_10172018

FOR ERP USE ONLY DO NOT DISTRIBUTE

HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis Jonathan Draher and Norman White Abbott Nutrition, Division of Abbott Laboratories

Add 12.0 mL of methanol to each of the flasks containing the standard/internal standard mixtures, and bring to volume with Laboratory Water. Transfers diluted standards to autosampler vials as needed and freeze the balance in plastic bottles

for later use. Storage: keep frozen. Expiration date: 6 months. g) Mobile Phase: (18:82, Methanol: 0.05 M Buffer, pH 2.3)

Dissolve 6.9 ± 0.1 grams of sodium phosphate monobasic and quantitatively transfer with water to a beaker containing approximately 900 mL of Milli-Q water. Add 2.0 mL of concentrated phosphoric acid (85%) with a class “A” volumetric pipet while stirring. This should adjust the pH to 2.3. Transfer to a 1 liter volumetric flask and dilute to volume with water. Filter the buffer solution through a 0.45nm filter. Transfer about 500 mL of the filtered buffer into a 1 liter volumetric flask. Add 180 mL of Methanol and swirl to mix. Allow to degas, then dilute to volume with more filtered buffer. Discard excess buffer. Mix flask contents thoroughly and transfer to a 1 liter HPLC system reagent reservoir. Storage: Room temperature. Expiration date: Six (6) weeks (in capped bottle). Volumes and weight may be varied proportionately for larger yield. h) Column Rinse Solution: Prepare a maintenance solution of approximately 25% methanol in water to use as a system rinse following each analysis run. Change maintenance solutions a minimum of every 3 months. E. PROCEDURE 1. Sample Preparation a) Set heating unit to 50°C prior to experiment. b) Using an analytical balance, tare a 50 mL centrifuge tube. c) Weigh liquid samples and reconstituted powders directly into tarred centrifuge tubes using a disposable transfer pipette. For non-reconstituted powder samples weigh into a tarred tube with a spatula. Record weights. d) Add 0.5 mL (500 µ L) of protease enzyme solution to all samples. e) Prepare no fewer than two "blank" enzyme solutions per run. Blank solutions will contain enzyme, internal standard, and buffer only. Otherwise follow sample preparation. f) Add 0.5 mL of 5-methyl-DL-tryptophan internal standard to all tubes including the enzyme blanks.

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