A Novel UHPLC–MS/MS Method for the Measurement of 25-Hydroxyvitamin D3 in Canine Serum and Its Application to Healthy Dogs

Several studies have shown the importance of vitamin D3 supplementation in small animals. In dogs, a low vitamin D3 status is associated not only with bone metabolism but also with different kinds of disorders, such as congestive heart failure, gastrointestinal diseases, chronic kidney diseases, and some types of cancer. However, it is crucial to maintain balance and monitor the introduction of this essential nutrient through the diet because over-supplementation can result in toxicity. Due to the clinical importance of assessing the vitamin D3 status in small animal patients, a quick, simple, and highly performing analytical method for its measurement is needed. In this study, we describe the development of a novel liquid chromatography–tandem mass spectrometry method for 25-hydroxyvitamin D3 quantification in canine serum. The approach was successfully validated following current European guidelines, proving excellent linearity (R2 always ≥0.996), accuracy (always within ±13%) and precision (always <10%). The application of the validated approach to samples collected from 40 healthy dogs made possible the definition of a reliable reference interval for 25-hydroxyvitamin D3, the main biomarker of vitamin D3. In addition, variations below 5% in the results obtained quantifying the same samples using a water-based calibration curve demonstrated that a surrogate matrix may be used without affecting data accuracy. Thanks to its simplicity, the proposed technique represents a useful tool for supporting clinical routine and investigating correlations between serum concentrations of this metabolite and multiple diseases. Additionally, it could enable the monitoring of supplementation in small animal patients in veterinary clinical practice.