Targeted high-throughput UHPLC-MS/MS methodology to quantify cholecalciferol and its major metabolites calcidiol and calcitriol in serum, tissues and egg yolk of broiler chickens and laying hens

This study aimed to develop and validate a high-throughput UHPLC-MS/MS method for quantifying cholecalciferol (VitD3) and metabolites calcidiol (25(OH)VitD3) and calcitriol (1,25(OH)2VitD3), in chicken serum, egg yolk and tissues. Sample preparation involved protein precipitation for serum and liquid extraction for egg yolk and tissues, followed by clean-up using Oasis® Ostro™ or PRiME HLB. The method was validated in-house, demonstrating calibration range (lower limit of quantification (LLOQ) – 100 ng mL −1 or ng g −1 , r ≥ 0.99), accuracy and precision within VICH GL49 acceptance criteria, and limits of detection (LOD) (serum: 0.05–2.86 ng mL −1 ; liver, kidney and muscle: 0.18–0.34 ng g −1 ; egg yolk and skin+fat: 0.23–0.42 ng g −1 ). The LLOQ values for VitD3 and 25(OH)VitD3 (0.5–1 ng mL −1 or ng g −1 ) were sufficiently low to quantify these components in all tested matrices. The method's applicability was tested using samples from laying hens and broiler chickens in field trials. Results showed that feed supplementation with VitD3 increased VitD3 concentrations in serum and egg yolk but did not affect 25(OH)VitD3 levels. Conversely, supplementation with 25(OH)VitD3 increased only 25(OH)VitD3 concentrations. Supplementation with 1,25(OH)2VitD3 did not result in an increase in concentration. In tissue samples, highest concentrations of VitD3 and 25(OH)VitD3 were found in the liver, kidney, and skin+fat. This validated method is suitable for evaluating the VitD3 status in animal-derived foods and assessing the nutritional status and health of chickens. • Quantitative analysis of cholecalciferol, calcidiol and calcitriol. • Cost-efficient, high-throughput UHPLC-MS/MS method. • Validation in chicken serum, egg yolk and tissues according to VICH GL49 guidelines. • Applicability for evaluating vitamin D status in animal-derived foods. • Assessing nutritional status and health of chickens.