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

f) Hydrochloric acid, 35% - AR grade . g) Ortho-Phosphoric acid, 85%- AR grade. h) Hydrogen peroxide, 30% v/v. i) Formic acid, 99% - AR & HPLC grade. j) Ethanolamine – HPLC grade. k) 1,1,1-trichloro-2-methyl-2-propanol – AR grade. l) Phenol – AR grade. m) Sodium Hydroxide – AR grade. D. Standards:

a) Amino acids standard, Hydrolysate, 2.5 mM concentration of each amino acid except Cystine(1.25mM) : Waters, Part No.: WAT 088122, (contains Aspartic acid, Serine, Glutamic acid, Glycine, Histidine, Arginine, Threonine, Alanine, Proline, Cystine, Tyrosine, Lysine, Valine,

Methionine, Isoleucine, Leucine and Phenyl Alanine). b) Tryptophan : Sigma, product No.: T0254 or equivalent). c) Taurine : Sigma, product No.: T0625 or equivalent). d) Cysteic acid: Sigma, product No.: 30170 or equivalent). e) Methionone Sulfone : Sigma, product No.: M0876 or equivalent).

f) Norvaline: Sigma, product No.: N7627 or equivalent). g) Standard Reference Material – NIST SRM 1849a. E. Solutions Preparation: a) Hydrochloric acid, 6M : Add 53 mL of Hydrochloric acid to 47 mL of water in a glass beaker. Cover the beaker with a watch glass and boil for 2 minutes in a fume hood. b) Hydrochloric acid, 20 mM: Add 1.7 ml of 6M Hydrochloric acid to 500 mL laboratory water. c) Mobile Phase A1: Quantitatively transfer 25 ml of Eluent A concentrate into a 500 mL volumetric flask, add approximately 200 mL laboratory water to dissolve and fill to volume with laboratory water. d) Mobile Phase B1 : Add 6.5(±0.1) mL Formic acid (HPLC grade) to 500 mL Acetonitrile and dissolve. e) Mobile Phase A2: Dissolve 2.85 mL acetic acid in 900 ml laboratory water and add 50 mL methanol containing 0.5g of 1,1,1-trichloro-2-methyl-2-propanol. Adjust to pH 5.0 with Ethanolamine and makeup to 1000 ml. f) Performic acid solution: Dissolve 0.24g of phenol in 45 mL of formic acid. Add 5 mL of 30% v/v Hydrogen peroxide and mix gently. Incubate the solution between 20 - 30 ºC for 1 hour to form performic acid, then cool in an ice bath for 15 minutes before adding to the samples. g) Reconstitution of Derivatizing agent from Derivatization kit: Transfer 1 mL of diluent from vial 2B to vial 2A. Cap the vial tightly and vortex for 2 minutes. Heat on a preheated water bath at 55°C for 5 minutes. h) Sodium Hydroxide solution, 4M: Dissolve 16g sodium hydroxide pellets in 80 mL laboratory water. Cool and dilute to 100 mL with laboratory water. i) Orthophosphoric acid, 0.5M: Add 3.4 mL of orthophosphoric acid to 80 mL laboratory water and make upto 100 mL. j) Hydrochloric acid, 1.0M: Add 16.4 mL of hydrochloric acid (6M) to laboratory water and make upto 100 mL with water. k) Standards Preparation: 1) Preparation of Stock Standards: i) Amino acids standard, Hydrolysate, 2.5mM: Transfer the content from the ampoule into a new HPLC vial, close tightly and store at -20°C±2°C for 3 months. ii) Taurine Stock standard preparation, 2.5 mM: Accurately weigh 15.64 mg of Taurine into a 50 mL standard volumetric flask. Dissolve and makeup to 50 mL with laboratory water. Store at -20°C±2°C for 2 months.

2