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

5) Raise the hot air oven temperature to 110°C±5°C and hydrolyze the samples for 20 hours. 6) Cool the vials to room temperature and quantitatively transfer the contents into a 50 ml polypropylene centrifuge tubes. 7) Add 5 mL of 0.5M Orthophosphoric acid and adjust the pH of the solution to 3.0 with 1M Hydrochloric acid 8) Quantitatively transfer the contents to 50 mL standard volumetric flask. 9) Add 10 mL of methanol to the flask and makeup to the mark with laboratory water. 10) Mix well and let it stand for 10 minutes at room temperature. 11) Take 1 mL of the supernatant into a 10 mL standard volumetric flask and makeup to the mark with water. 12) Filter through 0.22 μm membrane filter and inject 2μL into UHPLC-FLD. e) Chromatographic Conditions: 1) Chromatographic conditions for Acid Hydrolysis method: Table 6: Column AccQ.Tag Ultra C18, 1.7 μM, 2.1 x 100 mm Mobile phase A Mobile Phase A1 Mobile phase B Mobile Phase B1 Flow rate 0.7 mL/minute Injection Volume 1.0 μL Column Temperature 55°C Detector Wavelength 260 nm Total Run Time 12.5 minutes Gradient Method Time (minutes) A % B% 0.00 99.90 0.10 0.54 99.90 0.10 2.50 98.70 1.30 3.00 98.00 2.00 4.03 96.00 4.00 4.83 93.90 6.10 5.13 93.10 6.90 5.48 92.00 8.00 5.74 90.90 9.10 8.74 78.80 21.20 9.04 40.40 59.60 9.05 10.00 90.00 9.64 10.00 90.00 9.73 99.90 0.10 12.5 99.90 0.10

2) Chromatographic conditions for Oxidative Acid Hydrolysis method: Table 7:

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