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

c) Constant temperature oven capable of maintaining 50 (+/- 1) degree Centigrade 2. Instrument Parameters and Conditions a) Mobile Phase Flow Rate : 0.5 mL /minute b) Column Temperature: 30 degrees Centigrade c) Injection volume: 10 µ L. d) Detector settings: Excitation wavelength to 295 nm and emission wavelength to 345 nm e) Typical PMT setting: 7 for Agilent G1321A (see F.3.a) f) Run time: 8.5 minutes, or as needed to fully collect the 5-methyl-DL-tryptophan peak.

D. D. REAGENTS 1. Standards L-Tryptophan USP Standard Cat #1700501, or equivalent. 2. Chemicals a) Methanol, HPLC grade b) Sodium Phosphate Monobasic, HPLC grade. c) Phosphoric Acid, (85%) d) Trizma Base e) Hydrochloric Acid, 6N.

f) Laboratory Water – minimum of 18 mega ohm conductivity g) 5-Methyl-DL-tryptophan Sigma Cat # M-0534, or equivalent. h) Protease from Streptomyces griseus : Type XIV (enzyme): Sigma Cat # P- 5147, or equivalent. 3. Solutions a) Hydrochloric Acid, 1.0N Solution.

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