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Vitamin D comprises a group of secosteroids, and mainly refers to two physiologically inactive fatsoluble prohormones: vitamin
D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Vitamin D3 is photochemically synthesized from 7dehydrocholesterol in the epidermal layer of the skin of vertebrates under ultraviolet
(UV) B light (270–290 nm) radiation. Vitamin D2 is derived from fungal and plant sterol when ergosterol is exposed to UV radiation. Vitamin D2 is not produced by the human body and is much less effective than vitamin D3 in humans.1The 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
Fifteen experts from universities, research institutes and university hospitals worldwide called for international agencies,
such as the Food and Nutrition Board (WA, USA) and the European Commission's Health and Consumer Protection DirectorateGeneral
(Belgium), to reassess dietary recommendations for vitamin D as a matter of high priority because the formal advice from health
agencies is outdated and putting the public at risk.6 Currently, the FDAapproved prescription oral tablet drug FOSAMAX PLUS D (70 mg alendronate sodium/5600 IU cholecalciferol)
has been widely used for people diagnosed with osteoporosis. Alendronate slows bone loss and increases bone mass, which helps
prevent bone fractures, while cholecalciferol aids bone absorption of calcium. 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
