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

FOR EXPERT REVIEW PANEL USE ONLY NOT FOR DISTRIBUTION

Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method AMINO-04 Title: Determination of free and/or total proteinogenic amino acids and taurine by pre-column derivatization and UHPLC Author: George Joseph and Asha Varughese Reviewer Name: Reviewer 2 Summary of Method: The method can be able to determinate free and / or proteinogenic amino acids from Infant Formula and Adult and Pediatric Nutritional Formulas by different sample preparation. 1- Method for determination of total proteinogenic amino acids and taurine The analytical method is capable of quantitative determination for 22 standard amino acids except tryptophan which is destroyed during the acid hydrolysis. Asparagine is determined as aspartic acid and glutamine as glutamic acid. The cystine and cysteine are converted to S-2-carboxyethylthiocysteine (CYSx) and the derivative is separated from the other amino acids The samples are hydrolysed in 6M HCl for 24 hours at 110±5°C in presence of phenol, 3-3’- dithiodipropionic acid (DTDPA) and internal standard norvaline. Phenol is added to prevent halogenation of tyrosine. DTDPA is added to convert cystine and cysteine to S-2-carboxyethylthiocysteine (CYSx) and quantified against derivatised calibration standard. After neutralization, amino acids and CYSx are derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and are separated using reverse phase UHPLC with UV detection at 260nm. The analytical method is capable of quantitative determination for 24 standard amino acids including tryptophan. The amino acids cystine and cysteine are not separated and are quantified together as total. Citrulline which is present in some matrices is separated from other amino acids and included in the method performance evaluation Samples are dissolved in 0.5 M HCl along with added internal standard. The sample is then derivatized with 6-aminoquinolyl N hydroxyl succinimidyl carbamate. Amino acids are separated using quaternary pump Dionex UHPLC and detection by UV absorbance at 260nm. Norvaline is used as internal standard in the quantification of amino acids. 2- Method for determination of free aminoacids The method is suitable for the determination of free L-α-amino acids and taurine.

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