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The metabolic pathways of vitamins D₃ and D₂ in humans are similar. Vitamin D₃ is hydroxylated in the liver and converted to a nonactive storage form of 25hydroxycholecalciferol, which is further metabolized in the kidneys into the physiologically major active hormone, 1,25dihydroxycholecalciferol. This hormone is the main circulating form of vitamin D, which enhances the ability of the small intestine to absorb calcium and retain phosphate from the diet, and prevents metabolic bone diseases such as rickets, osteomalacia, osteoporosis and agerelated macular degeneration,^2,3 which are caused by vitamin D deficiency.

In 2007, the Canadian Cancer Society recommended that adults living in Canada should consider taking vitamin D supplements of 1000 IU/day during the autumn and winter, and those at higher risk of lower vitamin D levels should take 1000 IU/day all year round.⁴ Meanwhile, the US Dietary Reference Intake for Adequate Intake of vitamin D₃ lists: 200 IU/day as adequate for children and adults aged ≤50 years, 400 IU/day for adults aged 51–70 years, and 600 IU/day for adults aged >71 years.⁵

Levels of vitamin D₃ and its metabolites, 25hydroxyvitamin D₃ and 1,25dihydroxyvitamin D₃, are usually clinical indicators of nutritional vitamin D deficiency in humans.^7,8 Based on FDA guidance for bioavailability and bioequivalence studies for orally administered drug products, the moieties to be measured in biological fluids collected are either the API or its active moiety in the administered dosage form (parent drug) and, when appropriate, its active metabolites.

For bioequivalence studies, measurement of the concentration–time profile of the parent drug released from the dosage form, rather than the metabolite, is generally recommended because it is more sensitive to changes in formulation performance than metabolites.

Measurement of a metabolite may be preferred when parent drug levels are too low to allow reliable analytical measurement in blood, plasma or serum for an adequate length of time, or when a metabolite may be formed as a result of gut wall or other presystemic metabolism.⁹ Therefore, it is of more clinical importance to directly monitor vitamin D₃ levels in the human body when a vitamin D drug or a supplement is taken.

Previous determination of endogenous vitamin D has mainly been conducted by high performance liquid chromatography/ultraviolet (HPLC/UV) and gas chromatography/mass spectrometry (GC/MS) methods. These methods were considered challenging because their low sensitivities for low vitamin D levels in circulation required highblood volumes. The similarity between vitamin D and its analogs and metabolites meant that HPLC methods suffered from inadequate chromatographic separation and lacked specificity of interference. The dynamic endogenous concentration of vitamin D in the presence of UV light, and complex labourintensive and timeconsuming sample extraction procedures from ligand binding assays and low recovery from derivatization methods were also problematic.^10–12