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Background: A multi-laboratory study was completed with AOAC First Action Method 2017.03, HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis. Objective: Ten laboratories from seven countries participated in the multi-laboratory study. Each laboratory analyzed 14 infant, pediatric, and adult nutritionals in duplicate. Product matrices analyzed included milk, soy, partially hydrolyzed milk, partially hydrolyzed soy, and elemental-based infant formula powders, milk-based infant formula ready-to-feed (RTF) liquids, adult low-fat powders, and adult high-fat and high-protein ready-to-drink nutritionals. Methods: Tryptophan was released from the intact protein in product matrices with a combination of proteolytic enzymes found in pronase. Following proteolysis, tryptophan was quantitated by reverse-phase isocratic HPLC and fluorescence detection. Prepared samples were injected onto a C8 HPLC column with a methanol/0.05 M phosphate buffer mobile phase. Results: Overall for tryptophan, repeatability averaged 2.1% relative SD (RSD) with a range of 0.9–3.6% RSD, and reproducibility averaged 4.2% RSD with a range of 3.0–9.9% RSD. Conclusions: Repeatability Standard Method Performance Requirements (SMPRs®) were met for 13 of the 14 matrices and reproducibility SMPRs were met for 11 of the 14 product matrices analyzed.

journal of aoac international

HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis, Multilaboratory Testing Study: Final Action 2017.03