6. AOACSPIFANMethods-2018Awards

259

AOAC Official Method 2017.03 Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis HPLC First Action 2017

Method is applicable to the determination of the total tryptophan content in infant, pediatric, and adult nutritional products as defined in SMPR 2014.013. See Tables 2017.03A - C for matrices for which SLV data has been generated, supporting acceptance of the method. See Figures 2017.03A - C for chromatograms. 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, tryptophan is quantitated by reverse phase, isocratic HPLC and fluorescence detection, which provides 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 and Materials

a) Micro-Analytical Balance b) Analytical Balance

c) Volumetric pipettes: 0.5, 1, 2, 5, 10 mL d) 50 mL polypropylene centrifuge tubes e) Rack capable of holding tubes from d) f) Volumetric flasks, 50 mL, 100 mL, 500 mL, 2000 mL