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HPLC Determination of Total Tryptophan in Infant Formula and
Adult/Pediatric Nutritional Formula Following Enzymatic
Hydrolysis
Abbott Nutrition, Division of Abbott Laboratories
Page
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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: 8.5 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 from
Streptomyces griceus
: Sigma Cat # P-5147, or equivalent.
3.
Solutions
a)
Hydrochloric Acid, 1.0N Solution.
b)
Trizma Buffer, 0.1M, pH 8.5.
AMINO-02 (FEBRUARY 2017) - METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE
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