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

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.

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