2017.03 Amino Acids (Tryptophan) - Amino-02

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 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.

E.

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 : Sigma Cat # P-5147, or equivalent. 3. Solutions a) Hydrochloric Acid, 1.0N Solution. b) Trizma Buffer, 0.1M, pH 8.5. c) Protease Solution - Weigh 0.125 ± 0.010 grams to 25 mL with Trizma buffer. Scale volume as needed with proportional weight. Expiration: 6 hours.

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