AOAC 2019 First Action Methods

AOAC Official Method 2019.09 Total Proteinogenic Amino Acids and Taurine in Infant Formula and Adult/Pediatric Formula Precolumn Derivatization and UHPLC First Action 2019

[Method is applicable to the determination of free and/or total proteinogenic L- α -amino acids and taurine in all forms of infant, adult, and/or pediatric formulas (powders, ready-to-feed liquids, and liquid concentrates).] Caution : Analysts using this procedure should be familiar with good laboratory practice. It is the responsibility of the analyst to ensure they are familiar with the appropriate SDSs and safety practices for all the chemicals and equipment utilized. A. Principle/Methodology The samples are hydrolyzed 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 cysteine and cystine to S-2-carboxyethylthiocysteine (CYSx) and quantified against derivatized 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. During acid hydrolysis, glutamine and asparagine are converted to glutamic acid and aspartic acid, respectively. Therefore, glutamic acid values represent the combined values of glutamic acid and glutamine and aspartic acid values represent the combined values of aspartic acid and asparagine. Cystine values represent the combined values of cysteine and cystine since both are converted to CYSx by DTDPA. Cystine is a derived amino acid that is formed from the oxidative linkage of two cysteine residues by a disulfide covalent bond. Cystine is formed in many proteins after incorporation of free cysteines into the primary structure to stabilize their folded conformation. Cystine is the form in which cysteine exists in many biological samples. B. Chemicals Note : Use only reagents and water of recognized analytical grade unless otherwise stated. (a) Laboratory reagent grade water (b) Phenol (Sigma Aldrich P5566) or equivalent (c) Disodium tetraborate, decahydrate (d) 3,3’-Dithiodipropionic acid (DTDPA) (Sigma Aldrich 109010) or equivalent (e) Sodium hydroxide pellets (f) Methanol HPLC grade (g) Formic acid HPLC grade (h) Acetonitrile HPLC grade (i) Hydrochloric acid concentrated 37% (~12M) (j) 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (Pure Science N1001113-100) or equivalent (k) L-alanine (Sigma Aldrich A7469) or equivalent (l) L-arginine monohydrochloride (Sigma Aldrich A6969) or equivalent (m) L-aspartic acid (Sigma Aldrich A8949) or equivalent