Abstract
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.
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