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Amino Acids in Infant Formula and Adult/Pediatric Formulas

Ultra High Performance Liquid Chromatography

6

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:

Amino-03 (February 2016)

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