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

2017.03 (JULY 2018) Amino-02 Tryptophan by enz hyd_Abbott Labs_Method

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

b) Set column oven to 30°C. c) The instrument method should designate an excitation wavelength of 295 nm and emission wavelength of 345 nm. The Agilent G1321A fluorescence detector lamp may be designated to be on during analysis only, due to the near instant warm up of the lamp. d) Load standards, enzyme blanks, and samples onto the autosampler tray. e) Sequence should include a minimum of four initial injections for equilibration. The first of the four injections should be a high standard to check that the TRP peak response does not exceed approximately 50% of full scale. If significantly over 50%, lower the PMT gain by one unit. This will decrease the signal by about ½. Results are best with high standard between 20% and 50% of full scale. f) The remaining three precision injections should be the 5.0/50 standard injections to provide for check of system equilibration by calculating the 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.

G. System Suitability Check

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

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