Analysis of Vitamins B1, D2, and D3 in Commercial Animal Feed Matrices by LC-MS/MS: Validation of AOAC Official Methods

BACKGROUND: 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 quality control, label claim compliance, and safety assurance in the pet food industry. OBJECTIVE: 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 matrices using LC-MS/MS in a single-laboratory study. METHODS: Thiamine was extracted via enzymatic digestion with acid phosphatase, while vitamins D2 and D3 were extracted using saponification, followed by liquid-liquid extraction. RESULTS: The methods were validated for cat kibble, dog kibble, and wet dog feed, demonstrating high sensitivity (limits of detection: 0.018 ng/mL for B1, 0.005 ng/mL for D2 and D3; limits of quantification: 0.06 ng/mL for B1, 0.016 ng/mL for D2, 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. CONCLUSION: The AOAC Official Methods 2015.14 and 2016.05 were suitable for the analysis of vitamins B1 and vitamins D2/D3, respectively, in cat kibble, dog kibble, and wet dog food. HIGHLIGHTS: These results support the utility of the methods for routine analysis of animal feeds to verify label claims, nutritional adequacy, and safety.