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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
4
d) Add 0.5 mL (500
L) of protease enzyme solution to all samples.
e)
Prepare no fewer than two "blank" enzyme solutions per run. Blank solutions
will contain enzyme, internal standard, and buffer only. Otherwise follow
sample preparation.
f)
Add 0.5 mL of 5-methyl-DL-tryptophan internal standard to all tubes
including the enzyme blanks.
g) Add 3.0 mL of 0.1M, pH 8.5 Trizma buffer and 200
L methanol to all tubes
including enzyme blanks. Cap tubes and vortex lightly (about 2 seconds) to
mix contents. Be sure powder samples are completely dissolved but do not
over agitate solution to avoid foaming.
h)
Incubate samples for 16 to 24 hours in heating unit previously set to 50°C.
Remove samples from incubation unit after a minimum of 16 hours. Samples
may be prepared for overnight incubation if removed within 24 hours.
i)
Let samples cool to room temperature. Remove caps and add 12 mL of
methanol to each tube using a bottle re-pipettor.
j)
Dilute each tube to the 50 mL mark with Laboratory Water.
k) Cap tubes and mix thoroughly by inversion.
l)
Attach a 0.45
m filter to a 3 or 5 cc syringe and transfer several mL of a
sample to the syringe. Filter sample into an autosampler vial, and cap. Repeat
with fresh syringe and filter for each sample and blank.
m) Samples are ready for analysis or may be frozen for future analysis within 72
hours.
G.
CALCULATIONS
1. Tryptophan Concentration in Samples:
TRP (g/100g sample) = (CTS – CTB) x (volume / SW) x (100g/1000mg/g)
Where:
CTS = Concentration of Tryptophan in Sample (mg/L)
CTB = Concentration of Tryptophan in Blank (mg/L)
Volume = Final volume in Liters i.e. 50 mL = 0.05L
SW = Sample Weight in grams
AMINO-02 (FEBRUARY 2017) - METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE
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