Validation of Methods for Determination of Vitamins B1, D2, and D3 in Commercial Animal Feed Matrixes by LC-MS/MS

Vitamin B1 (thiamine), D2 (ergocalciferol), and D3 (cholecalciferol) are essential nutrients in animal diets, critical for health and the maintenance of physiological processes. Deficiencies or excesses can lead to severe health issues, necessitating reliable quantification methods for QC, label claim compliance, and safety assurance in the pet food industry.

The aim of this study was to extend and validate AOAC Official Methods  2015.14 (for vitamin B1) and 2016.05 (for vitamins D2 and D3) to animal feed matrixes using LC-MS/MS in a single-laboratory study.

Vitamin B1 was extracted via enzymatic digestion with acid phosphatase, while vitamins D2 and D3 were extracted using saponification, followed by liquid-liquid extraction.

The methods were validated for cat kibble, dog kibble, and wet dog feed, demonstrating high sensitivity (LODs: 0.018 ng/mL for B1 and 0.005 ng/mL for D2 and D3. LOQs: 0.06 ng/mL for B1, 0.016 ng/mL for D2, and 0.018 ng/mL for D3), excellent linearity (R2 = 0.997-0.999), and acceptable recoveries (88-115%). Quantification revealed vitamin B1 concentrations exceeding the Association of American Feed Control Officials minimum recommendations, with the lowest at 8.99 mg/kg on a dry matter basis (DMB) in wet dog food. Vitamin D levels were within recommended ranges, with the lowest in cat kibble at 18.13 μg/kg DMB for vitamin D3, while vitamin D2 contributed minimally to total vitamin D, at 0.27 μg/kg DMB.

LC-MS/MS methods based AOAC 2015.14 and 2016.05 were single-laboratory validated and applied to the analysis of vitamin B1 and vitamins D2 and D3, respectively, in cat kibble, dog kibble, and wet dog food.

These results support the utility of the methods for routine analysis of animal feeds to verify label claims, nutritional adequacy, and safety.