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Method Validation Report

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Validation Report for the Determination of Thiamin HCl, Riboflavin, Niacin (Nicotinic Acid and Nicotinamide), Pantothenic acid, Vitamin B6 (Pyridoxine, Pyridoxal and Pyridoxamine) and Biotin in Infant Formula and Adult Nutritionals by LC-MS/MS

Document Title

Table 18: Standard Ranges of Pyridoxine, Pyridoxal, Pyridoxamine and Biotin Linearity range: Pyridoxine Pyridoxal Pyridoxamine Biotin

min (mcg/100g)

27.86

0.32

1.12

1.13

Analyte

max (mcg/100g)

501.47

69.73

20.25

20.37

Acceptance Criteria

Linearity, Accuracy and Precision satisfied

8. Ruggedness Prepare and analyze the same lot of test samples on 6 different days. Calculated RSD should be < 5%.

Table 18A: Ruggedness of Stage 3 Growing up milk formula powder Concentration (mcg/100g) Thiamine HCl Riboflavin Nicotinic acid Nicotinamide

Pantothenic acid

Niacin

Day 1 Day 2

895.15

1175.98

251.43

8473.14

8724.57

3381.43

876.14

1195.91

259.06

8440.51

8699.56

3225.25

Day 3

877.99

1186.05

250.08

8562.13

8812.21

3449.38

Day 4

886.88

1196.86

247.62

8338.39

8586.01

3231.22

Day 5 Day 6 Mean

883.28 845.79 877.54

1160.40 1141.60 1176.13

265.08 250.63 253.98

9012.18 8546.75 8562.18

9277.27 8797.39 8816.17

3537.07 3454.21 3379.76

% RSD

1.93

1.85

2.63

2.74

2.72

3.77

Acetance Criteria Accetable (Y/N)

≤ 5%

≤5%

≤5%

≤5%

≤5%

≤5%

Y Y Remark: Niacin is sum of Nicotinic acid and Nicotinamide, and reported as Niacin. Y Y Y Y

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