SPIFAN Nutrients ERP Book_9-29-15

Amino-02 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

b) HPLC system with autosampler, column Oven to maintain 30°C, Fluorescence Detector, binary or quaternary HPLC Pump with degasser, HPLC peak area integrator or chromatography data processor.

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  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: 7.5 minutes, or as needed to fully collect the 5-methyl-DL-tryptophan peak.

E.

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 E (enzyme) Sigma Cat # P-5147, or equivalent.

3.

Solutions

a) Hydrochloric Acid, 1.0N Solution.

b) Trizma Buffer, 0.1M, pH 8.5.

page

2