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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

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4. Method Precision

For each of the matrices, prepare the same sample 6 times from a single slurry and analyze in one analytical run. Calculated RSD should be ≤ 5% for each analyte. Table 12A: Product Matrix 1: Stage 3 Growing up milk formula powder Result (mcg/100g)

Thiamine HCl

Nicotinic acid

Pantothenic acid

Analyte

Riboflavin

Nicotinamide

Niacin

Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 Replicate 6

863.34 878.20 890.34 886.56 881.60 887.07

1179.51 1203.63 1199.37 1241.75 1186.54 1154.56

261.59 254.25 267.08 268.85 264.78 258.05

8370.41 8898.23 8733.89 8670.04 8755.92 8737.74

8632.00 9152.48 9000.97 8938.89 9020.69 8995.79

3354.80 3435.52 3381.34 3386.93 3374.63 3426.97

%RSD

1.11

2.43

2.12

2.02

1.94

0.93

Acceptance Criteria:

RSD ≤ 5%

RSD ≤ 5%

RSD ≤ 5%

RSD ≤ 5%

RSD ≤ 5%

RSD ≤ 5%

Acceptable (Y/N) Y Remark: Niacin is sum of Nicotinic acid and Nicotinamide, and reported as Niacin. Y Y Y Y Y

Table 12B: Product Matrix 1: Stage 3 Growing up milk formula powder Result (mcg/100g) Analyte Pyridoxine Pyridoxal Pyridoxamine Vit B6 (as Pyridoxine)

Biotin 13.50 14.05 13.94 14.12 13.80 13.97

Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 Replicate 6

1126.72 1185.61 1155.33 1172.37 1192.93 1146.62

107.17 118.72 120.09 115.92 115.14 116.67

13.87 14.14 14.82 14.70 13.07 14.26

1249.14 1319.99 1291.77 1304.47 1322.60 1279.04

%RSD

2.15

3.91

4.48

2.14

1.60

Acceptance Criteria:

RSD ≤ 5%

RSD ≤ 5%

RSD ≤ 5%

RSD ≤ 5%

RSD ≤ 5%

Acceptable (Y/N) Y Remark: Vitamin B6 (as Pyridoxine) is sum of Pyridoxine, Pyridoxal and Pyridoxamine. All data have been mass corrected and expressed as Pyridoxine. Y Y Y Y

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