Development and application of a LC-MS/MS assay for simultaneous analysis of 25-hydroxyvitamin-D and 3-epi-25-hydroxyvitamin-D metabolites in canine serum

Abstract

Hypovitaminosis D and hypervitaminosis D are well recognised disorders in dogs. Hypovitaminosis D can occur following consumption of a diet inadequately supplemented with vitamin D or as a sequelae of severe intestinal disease. Hypervitaminosis D may occur as a result of consuming proprietary dog foods over-supplemented with vitamin D or through ingestion of vitamin D containing medicinal products or rodenticides. Consequently, there is a clear need to establish a methodology that can accurately quantify vitamin D metabolites across a broad dynamic range in dogs. The existence of C3-epimers of vitamin D metabolites has yet to be elucidated in dogs, yet are known to interfere with the analysis of vitamin D and have unknown biological activity in other species. Here, we describe the development and validation of a sensitive, specific and robust analytical liquid chromatography tandem mass spectrometry (LC-MS/MS) assay capable of separating and accurately measuring 25-hydroxyvitamin-D_2/3 (25(OH)D_2/3) and 3-epi-25-hydroxyvitamin-D_2/3 (3-epi-25(OH)D_2/3). We describe a simplified workflow utilising supported liquid extraction (SLE) without derivatization that provides good linearity (mean r > 0.996) and accuracy across a broad dynamic range of 4-500 nmol/L for D₃ metabolites and 7.8-500 nmol/L for D₂ metabolites. Upon application of this assay to 117 canine serum samples, 25(OH)D₃ was detectable in all samples with a median concentration of 82.1 nmol/L (inter-quartile range (IQR) 59.7-101.8 nmol/L). 3-epi-25(OH)D₃ could be detected in 87.2 % of the study population, with a median concentration of 5.2 nmol/L (2.4-8.1 nmol/L). However, 3-epi-25(OH)D₃ was quantified below the LLOQ in 40.2 % of these samples. 3-epi-25(OH)D₃ contributed on average 6.3 % to 25(OH)D₃ status (contribution ranges from 0 to 23.8%) and a positive correlation was detected between 25(OH)D₃ and 3-epi-25(OH)D₃ concentrations. Free 25(OH)D was also measured using an immunoassay with a median concentration of 15.2 pmol/L (12.5-23.2 pmol/L), and this metabolite was also positively correlated to both 3-epi-25(OH)D₃ and 25(OH)D₃ concentrations. D₂ metabolites were not detected in canine serum as expected. Vitamin D metabolite concentrations were variable between individuals, and research into the causes of this variation should include factors such as breed, age, sex and neuter status to determine the impact of genetic and hormonal factors. Given the clinical importance of vitamin D in dogs, and the immense potential for utilising this species as a model for human disease, further elucidation of the vitamin D pathway in this species would provide immense clinical and research benefit.

Keywords: 25-Hydroxyvitamin-D; C3-epi-25-hydroxyvitamin-D; Canine; Free 25-hydroxyvitamin-D; Liquid chromatography tandem mass spectrometry.