105 / 164
HPLC Determination of Total Tryptophan in Infant Formula and
Adult/Pediatric Nutritional Formula Following Enzymatic
Hydrolysis
Abbott Nutrition, Division of Abbott Laboratories
Page
1
This method is applicable to the determination of total tryptophan in infant formula and
adult/pediatric nutritional formulas. A sample amount targeting about 30 – 50 mg of
protein is added to the sample preparation. The limit of quantitation of a typical ready to
feed formula is approximately 0.18 mg/100g.
A. PRINCIPLE
Tryptophan is released (hydrolyzed) from intact protein 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
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 minimum 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: YMC PAC C8 50
3 mm column with 3 micron particle size
– Part #OC12S03-053030 – or equivalent.
AMINO-02 (FEBRUARY 2017) - METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE
11