2017.03 Amino Acids (Tryptophan) - Amino-02

2017.03 (JULY 2018) Amino-02 TRP_AOAC_2017.03_MLT_Corrected 080218

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B. 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. C. APPARATUS 1. Equipment a) Analytical Column: YMC PAC C8 50 × 3 mm column with 3 micron particle size – Part #OC12S03-053030 – or equivalent. b) HPLC system with autosampler, column oven to maintain 30°C, Fluorescence Detector, binary or quaternary HPLC Pump with degasser, HPLC peak area integrator or chromatography data processor. c) Constant temperature oven capable of maintaining 50 (+/- 1) degree Centigrade 2. Instrument Parameters and Conditions a) Mobile Phase Flow Rate : 0.5 mL /minute b) Column Temperature: 30 degrees Centigrade c) Injection volume: 10 µ L. d) Detector settings: Excitation wavelength to 295 nm and emission wavelength to 345 nm