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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

I. BACKGROUND Method 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.

II. TEST SAMPLES:

Matrix/Test Sample

No.

1 2 3 4

Reference Material SRM 1849a Infant formula Stage 3 Growing up milk formula powder Stage 1 Infant formula powder(milk-based) Partially hydrolyzed protein infant formula powder

III. VALIDATION DATA: 1. Linearity Table 1: Linearity of Thiamine HCl Cncentration

Area Response 1

Area Response 2

Area Response 3

(ng/ml)

Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Standard 7 Standard 8 Standard 9 Standard 10 Y-Intercept Correlation Coefficient Acceptance Criteria Slope

16.3 32.6 65.2

0.1819 0.3343 0.6785 1.0010 1.3670 1.6950 2.0360 2.4280 2.7740 3.1000 0.01049 0.0027

0.1845 0.3334 0.6860 1.0080 1.3580 1.6890 2.0610 2.4170 2.7830 3.0850 0.0105 0.00331

0.1844 0.3325 0.6762 1.0170 1.3650 1.6880 2.0250 2.3980 2.7530 3.1010 0.01045 0.00401

97.81

130.41 163.01 195.61 228.21 260.82 293.42

(weighting: 1/x)

0.99983

0.99984

0.99987

Correlation Coefficient (r) ≥ 0.995

Acceptable Y/N

Y

Y

Y

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