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 percent difference in the last two middle standard injection TRP peak areas. The system is considered equilibrated if the areas agree within approximately 1%. If not equilibrated, add an additional middle standard injection and recheck for area agreement. This is a pre-run suitability check only. When evaluating after an analysis run, defer to 3.c), below. When the system is equilibrated, order the sequence with the four standard levels (high to low recommended), blanks, samples, and then bracketing standard injections. Repeat calibration after approximately 20 sample injections. g) Following the sample analysis, rinse the analytical column with a solution of approximately 25% methanol in water. For longer storage, the column is best left in 60% to 80% organic, preferably acetonitrile. a) Resolution: In the Laboratory Control sample, the resolution of the Tryptophan peak from the small peak following shall be > 1.25. b) USP Tailing: The USP tailing factor may be calculated within the chromatography software and should be < 1.59 for Tryptophan and < 1.26 for 5-methyl-tryptophan. Excessive tailing indicates the need for a new column to restore expected performance. c) Residual Error of Linear Regression: The residual error for any one point of the three point linear regression is not to exceed 5%. In the event of excessive error, repeat the analysis with fresh standard solutions

G. System Suitability Check

H. CALCULATIONS

1. Manual Calculations a) Calculate concentrations of the tryptophan standards (mg/L) using the following equations:

(1) Tryptophan Stock Standard Solutions:

TRP (mg/L) = Weight TRP (g) x 1000 mg Volume (L) g Example: Tryptophan Weight = 0.0100 grams.

Page

8