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 iii) L-Cysteic acid, 2.5mM: Accurately weigh 23.40 mg of L-Cysteic acid monohydrate into a 50 ml standard volumetric flask. Dissolve and makeup to 50 ml with water. Store at -20°C±2°C for 2 months. iv) L-Methionine Sulfone, 2.5 mM: Accurately weigh 22.65 mg of L-Methionine Sulfone into a 50 ml standard volumetric flask. Dissolve and makeup to 50 ml with water. Store at -20°C±2°C for 2 months. v) Tryptophan stock standard, 500 mg/L: Accurately weigh 25.0 mg of tryptophan standard into a 50 ml standard volumetric flask and dissolve in 0.1 M HCl and makeup to the mark. vi) Norvaline, stock Internal Standard Preparation, 2.5 mM: Accurately weigh 14.64 mg of Norvaline into a 50 ml standard volumetric flask. Dissolve and makeup to 50 ml with water. Store at -20 °C±2°C for 2 months. (Note: Apply appropriate Purity, Salt and Moisture corrections to the stock standards concentrations) 2) Preparation of Intermediate, working standards and derivatization procedure for Acid

Hydrolysis and Oxidative Acid Hydrolysis methods: i) Table 2: Intermediate standards (IS) preparation:

Stock std. Conc. (mM)

Volume of Stock std. (ml)

Volume of diluent c (ml)

Intemediate std. Conc. (mM)

Intermediate std code

Stock Std. Name

Amino acids, Hydrolysate

2.5

0.40

0.20

1.0

IS Mix - 1

Taurine

2.5

0.40

L-Cysteic acid L-Methionine Sulfone

2.5

0.40

1.0

IS Mix - 2

0.20

2.5

0.40

Norvaline

2.5

0.10

0.90

0.25

--

Norvaline

0.25

0.20

0.80

0.05

--

c Diluent – Laboratory water Storage and Validity: - 20 ± 2˚C for 2 m onths ii) Table 3: Working standards preparation:

Vol. of working Std. (mL)

Final Volume (mL)

Working std. Conc. (mM)

IS Mix std. d Conc. (mM)

Vol. of 2.5 mM Norvaline (mL)

Vol. of Diluent* (mL)

1.00 1.00 1.00 0.50 0.10

0.50 0.25 0.10 0.10 0.10

0.10 0.10 0.10 0.10 0.10

0.40 0.65 0.80 0.80 0.80

1.00 1.00 1.00 1.00 1.00

0.50 0.25 0.10 0.05 0.01

d Choose appropriate IS Mix standard based on the hydrolysis procedure. iii) Preparation of Calibration standards by derivatization of working standards: Quantitatively transfer 10 μL of each working standard into total recovery vials. Add 70 μL of borate buffer to each vial and vortex for 30 seconds. Add 20 μL of reconstituted AccQ.Tag Ultra reagent powder and vortex for 30 seconds. Let it stand for 1 minute at

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