Review Team (February 2016)

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Amino Acids in Infant Formula and Adult/Pediatric Formulas Ultra High Performance Liquid Chromatography

Applicable for the determination of amino acids (including Taurine) in all forms of infant, adult, and/or pediatric formula (powders, ready to feed liquids and liquid concentrates). The LOQs for the method ranged from 2.0 to 10000 mg/100g. Caution: Refer to Material Safety Data Sheet (MSDS) of chemicals prior to use, and follow the suggested personal protective equipment. A. Principle: This procedure involves the conversion of proteins present in the foods to amino acids by different hydrolysis methods (see table 1) based on the amino acids chemical nature. The resulting amino acids are derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reagent (except Tryptophan) to form stable urea derivatives of each amino acid. The derivatized forms of amino acids are separated by UHPLC and quantitated by Diode Array Detector (Tryptophan by Fluorescence detector) using Norvaline as internal standard. Table 1: Different Hydrolysis Procedures and amino acids to be determined in each method Procedure Amino acids Acid Hydrolysis Histidine, Taurine, Serine, Arginine, Glycine, Aspartic acid (sum of Aspartic acid and Asparagine), Glutamic acid (sum of Glutamic acid and Glutamine), Threonine, Alanine, Proline, Lysine, Tyrosine, Valine, Isoleucine, Leucine, Phenyl alanine. Oxidative Acid Hydrolysis Cysteine a (sum of cysteine and cystine), Methionine b Alkaline Hydrolysis Tryptophan c a, b Cystine and Methionine are converted into Cysteic acid and Methionine sulfone by performic B. Apparatus: a) Liquid Chromatograph: Agilent UHPLC Infinity series with Binary Solvent Manager capable of 1200 bar pressure. b) Column: AccQ.Tag TM Ultra, C18, 1.7 μm, 2.1 x 100 mm (Waters Part No. 205000343). c) Detector: Agilent Infinity series UV/DAD detector with 1 μL flow cell and Fluorescence detector. d) Injector: Agilent Infinity Series Auto sampler with column oven. e) Data System: Agilent Open lab CDS. f) Centrifuge. g) Hot air oven: Capable of 110 ± 2°C h) Turbo-vap evaporator: Concentration evaporator workstation (Biotage) i) pH meter. j) Digestion Vessels: 20 mL glass vials with relse cap (crimp caps and Teflon lined septas). k) Vortex mixer l) Syringe filters: 0.22 μm, PTFE. m) Total Recovery Vials: Waters (Part No.: 186000384C) C. Reagents: a) AccQ-Tag Eluent A concentrate: Waters, Part No: 186003838 b) AccQ-Tag Ultra Derivatization kit: Waters, Part No: 186003836. This kit contains 5 sets of vials and each set contains 3 vials labeled with 1 (Borate Buffer), 2A (Reagent Powder), 2B (Reagent Diluent). c) De-ionized laboratory water - ≥ 18 Mohm/cm acid oxidation prior to acid hydrolysis. c Determined by Fluorescence detector

d) Acetonitrile – HPLC grade. e) Methanol – HPLC grade.

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