AOAC SPIFAN Nutrients ERP Method Reviews (September 7, 2019)

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Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method Amino-04 (Sep 2019)

Title: Determination of free and/or Total Proteinogenic Amino Acids and Taurine by pre-column derivatization and UHPLC Author: George Joseph, Asha Varughese (AsureQuality Limited) Reviewer Name: Reviewer 1 Summary of Method: The method has 2 parts –determination of total and determination of free amino acids. Since sample preparation procedures are different these parts should be considered as 2 deferent methods. For total amino acids: The samples are hydrolyzed in 6N HCl for 24 H in presence of phenol, 3,3’ – dithiodipropionic acid (DTDPA) and internal standard Norvaline. DTDPA converts Cysteine and Cystine to S- 2-carboxyethylthiocysteine (CYSx) derivative. After hydrolysis the amino acids and CYSx are derivatized using 6-amino-N-hydroxysuccinimidyl carbamate (AQC), separated on reversed-phase UHPLC column and detected at 260 nm. This method is not applicable to determination of Tryptophan as this amino acid is destroyed during the hydrolysis. Asparagine is converted to Aspartic acid and Glutamine to Glutamic acid during the hydrolysis. For free amino acids: Amino acids are extracted using 0.5 N HCl with Norvaline as internal standard, derivatized using 6-amino-N-hydroxysuccinimidyl carbamate (AQC), separated on reversed-phase UHPLC column and detected at 260 nm. Method Scope/Applicability: Method is applicable to determination of proteinogenic amino acids and taurine in infant, pediatric and adult formulas (powders, ready-to-feed and concentrates).

General comments about the method:

The method is clearly written and is easy to follow. Method utilizes well-established methodology and uses common analytical equipment. No proprietary reagents are used. For SLV of determination of total amino acids all SPIFAN matrixes, NIST SRM 1849a, SRM 1869 and 2 additional matrices (goat milk infant formula and whey protein concentrate) were used.

For SLV of free amino acids analysis spikes of NIST SRM 1869 at 3 different levels were used.

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