AOAC OMA# 2017.03 (Final Action)-OMB

1244  D raher & W hite : J ournal of AOAC I nternational V ol . 101, N o . 4, 2018

HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis: Single-Laboratory Validation, First Action 2017.03 J onathan D raher and N orman W hite Abbott Nutrition, 3300 Stelzer Rd, Building RP4-3, Columbus, OH 43219 OFFICIAL METHODS

Stakeholder Panel on Infant Formula and Adult Nutrtionals Expert Review Panel for Nutrient Methods

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

Darryl Sullivan (Chair), Covance Labs John Austad, Covance Labs Sean Austin, Nestlé Sneh Bhandari, Mérieux NutriSciences Esther Campos-Giménez, Nestlé Scott Christiansen, Perrigo Nutritionals Hans Cruijsen, FrieslandCampina Jon DeVries, DeVries & Associates Brendon Gill, Fonterra Cooperative Don Gilliland, Abbott Nutrition Harvey Indyk, Fonterra Cooperative Estela Kneeteman, INTI Adrienne McMahon, Wyeth/Nestlé Maria Ofitserova, Pickering Labs, Inc. Melissa Phillips, NIST Shay Phillips, Mead Johnson Nutrition Günther Raffler, Eurofins/CLF Kate Rimmer, NIST Karen Schimpf, Abbott Nutrition Martine van Gool, FrieslandCampina David Woollard, Eurofins Jinchuan Yang, Waters Corp. Jie Zhang, Mead Johnson Nutrition

[Applicable to the determination of the total tryptophan (Trp) content in infant, pediatric, and adult nutritional products as defined in Standard Method Performance Requirement (SMPR ® ) 2014.013 (1).] See Tables 2017.03A – C for matrixes for which single- laboratory validation data has been generated, supporting acceptance of the method. A. Principle Trp is released (hydrolyzed) from intact protein using a combination of proteolytic enzymes found in pronase, which was isolated from Streptomyces griseus for this purpose. The pronase enzyme powder contains at least 10 proteolytic enzymes (depending on the source, supplier, etc.) that hydrolyze peptide bonds internally (endoproteases) and externally (exopeptidases) either at the N-terminal end (amino peptidases) or at the C terminus (carboxypeptidases; 2, 3). The protein is thus attacked on different sites simultaneously, releasing Trp in a relatively short period of time. Following proteolysis, Trp is quantitated by reversed-phase isocratic HPLC and fluorescence detection, which provides for a selective and specific determination of Trp in nutritional products. The enzymes in the pronase self-digest to produce background Trp in the absence of a sample. Consequently, the enzyme system is nonspecific for the Trp sample, and a blank subtraction is mandatory. Using this approach, recoveries of free Trp spikes as well as Trp from Bovine Serum Albumin (BSA) spikes are found to be essentially equivalent, 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 16 h (overnight) to ensure complete hydrolysis of all sample types. (Although many samples are fully hydrolyzed within 6 h, some have been found to require longer; thus, a 16 h minimum is recommended for full applicability.) After hydrolysis, the sample–enzyme mixture is diluted to 50 mL with methanol–water and filtered. The sample is injected into a C8 column with reference standards and enzyme blank

Submitted for publication April 2018. Adopted as a First Action Official Method by the Expert Review Panel on Nutrient Methods . Corresponding author’s e-mail: jonathan.draher@abbott.com DOI: 10.5740/jaoacint.2017.03