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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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Applicable to the determination of total tryptophan in infant formula and adult/pediatric
nutritional formulas. Add sample amount targeting 30 – 50 mg of protein added to the
sample preparation. The limit of quantitation of a typical ready to feed formula is about
0.02 mg/100g.
A. PRINCIPLE
Tryptophan is released (hydrolyzed) from intact using a combination of proteolytic
enzymes found in pronase, isolated from Streptomyces griseus, for this purpose. The
pronase enzyme powder contains at least 10 proteolytic enzymes (depending on the
source, supplier etc.), which hydrolyze peptide bonds internally (endoproteases) and
externally (exopeptidases), either at the N-terminal end (amino peptidases) or at the
carboxy terminus (carboxypeptidases). The protein is thus "attacked" on different sites
simultaneously releasing tryptophan in a relatively short period of time. Following
proteolysis, free tryptophan is quantitated by reverse phase (isocratic) HPLC and
fluorescence detection, which provide for a selective and specific for the determination of
tryptophan in nutritional products.
The enzymes in pronase self-digest to produce background tryptophan in the absence of
sample. Consequently, the enzyme system is non-specific for the sample tryptophan, and
a blank subtraction is mandatory. Using this approach, recoveries of free tryptophan
spikes as well as tryptophan from BSA spikes are found essentially quantitative,
indicating near comparable self-digestion rates with and without sample.
Sample preparation consists of adding a weighed sample, the enzyme solution, internal
standard (5-methyl-DL-tryptophan) and Trizma buffer into a tube. A small amount of
methanol is added as a bactericidal agent. The preparation is mixed and incubated at
50°C for sixteen hours (overnight), to assure complete hydrolysis of all sample types.
(While many samples are fully hydrolyzed within 6 hours, some have been found to
require longer, thus 16 hours for full applicability). After hydrolysis, the sample-enzyme
mixture is diluted to 50 mL with methanol/water and filtered. The sample is injected onto
a C-8 column with reference standards and enzyme blank preparations and the analytes of
interest detected and quantified fluorometrically.
B. APPARATUS
1. Equipment
a)
Analytical Column: Betasil C8 50
3 mm column with 3 micron particle size –
Part #70203-053030 – or equivalent.
Amino-02
FOR ERP USE ONLY
DO NOT DISTRIBUTE