Comprehensive vitamin D status assessment including supply, bioavailability, and supplementation efficacy. Performed by high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).
4 metabolites
DEQAS Participant
This method provides a comprehensive assessment of the body's vitamin D supply, enabling clinicians and researchers to evaluate status, supplementation efficacy, and metabolic bioavailability through a multi-metabolite profile.
Clinical indications include:
| Analyte | Also Known As | Notes |
|---|---|---|
| 25(OH)D3 | Calcifediol, Calcidiol, 25-Hydroxy Vitamin D3 | Primary marker of vitamin D3 status |
| 25(OH)D2 | 25-Hydroxy Vitamin D2 | Marker of vitamin D2 (plant-derived) intake |
| 24,25(OH)₂D3 | 24,25-Dihydroxy Vitamin D3 | Catabolic metabolite used to calculate bioavailability ratio |
| Epi-25(OH)D3 | 3-epi-25-Hydroxy Vitamin D3 | Epimeric form with reduced biological activity. Measured but excluded from clinical interpretation to avoid false elevation of results |
The result is expressed as the sum of 25(OH)D2 and 25(OH)D3, providing the total 25(OH)D concentration. Additionally, the bioavailability coefficient is calculated from the ratio of 25(OH)D3 to its catabolite 24,25(OH)₂D3, indicating the balance between absorption/production and excretion processes.
Vitamin D, discovered over 100 years ago, was initially associated primarily with the body's calcium-phosphate balance — rickets in children, and osteomalacia and osteoporosis in adults. The discovery of the vitamin D receptor (VDR) in the 1970s in most extraskeletal human cells initiated extensive research into its role across numerous tissues.
Cholecalciferol — produced by animals via UV-B skin synthesis
Ergocalciferol — produced by plants, algae, and fungi
Months per year with negligible cutaneous synthesis at higher latitudes
The primary sources of vitamin D for humans are skin synthesis (UV-B exposure) and intestinal absorption. However, these sources are inherently limited. Geographical latitude significantly affects cutaneous synthesis — in many regions, effective vitamin D production is negligible for 4–6 months of the year. Even a healthy, varied diet cannot fully satisfy the body's demand, raising the need for targeted supplementation across all age groups.
Vitamin D is involved in a broad range of physiological processes. It regulates calcium metabolism and adequate bone mineralisation, influences cell proliferation and differentiation in the immune system, modulates the renin-angiotensin-aldosterone system and insulin secretion, and has anti-inflammatory and antioxidant effects.
Deficiency can be associated with the development of numerous conditions including osteoporosis, dental caries, depression, atherosclerosis, muscle pain, decreased concentration, and increased susceptibility to seasonal infections. Vitamin D deficiency during pregnancy can increase the risk of complications and directly impacts supply to the foetus.
Overdose, although extremely rare, is a real risk in individuals taking very high doses. It leads primarily to hypercalcaemia and may manifest as nausea, vomiting, abdominal pain, dehydration, weight loss, and renal dysfunction.
While routine screening is not indicated for the general healthy population (where established supplementation regimens are recommended), an increasing number of clinical situations require vitamin D monitoring and optimisation.
Equally important are cases of genetic hypersensitivity to vitamin D, resulting from mutations in genes encoding vitamin D metabolising enzymes. These mutations can cause disproportionately strong responses to standard supplementation, potentially leading to toxicity.
Total 25(OH)D concentration is used to assess vitamin D status. The following reference ranges are based on international endocrine society guidelines.
| 25(OH)D (ng/mL) | Status | Recommendation |
|---|---|---|
| 0 – 20 | Deficiency | Start supplementation per current guidelines. Repeat test after 3 months. If below 10 ng/mL, consult physician for treatment of vitamin D deficiency. |
| 20 – 30 | Suboptimal | Start or increase supplementation dose by 50%. Repeat test in 6 months. Note: while >20 ng/mL is considered sufficient for rickets prevention, maintaining 30–50 ng/mL is recommended for broader health benefits. |
| 30 – 50 | Optimal | Continue current management. Considered optimal by most endocrine societies worldwide. |
| 50 – 100 | High | Generally recommended to reduce dose by 50% or pause supplementation for 1–2 months. In some clinical situations, concentrations above 50 ng/mL may be advisable. Consult physician to discuss strategy. |
| > 100 | Toxic | Discontinue supplementation immediately. Retest 25(OH)D at monthly intervals. Toxic effects are associated with accompanying hypercalcaemia and hypercalciuria. Physician consultation required. |
The metabolite 24,25(OH)₂D3 is formed from 25(OH)D3 by the enzyme 24-hydroxylase. The ratio of 25(OH)D3 to 24,25(OH)₂D3 provides insight into supplementation efficacy and potential genetic variations in vitamin D metabolism.
| 25(OH)D3 : 24,25(OH)₂D3 | Interpretation | Recommendation |
|---|---|---|
| < 6.98 | Resistance to supplementation | Supplement only under medical supervision. Indicates increased 24-hydroxylase enzyme activity, which may contribute to resistance to vitamin D supplementation. In situations of low 25(OH)D concentrations, intake may be increased but must be supervised. |
| 6.98 – 23.64 | Optimal supplementation | Continue supplementation per current guidelines. Indicates normal 24-hydroxylase activity, characteristic of the majority of the population. No action required if total 25(OH)D is normal. If deficient, follow standard supplementation guidelines. |
| > 23.64 | Insufficient supplementation | Supplement only under medical supervision. Most commonly caused by insufficient 25(OH)D concentration. May also indicate bone disease, renal dysfunction, or calcium homeostasis disorders. A small group of individuals carry mutations in CYP24A1 (impaired 24-hydroxylase) or SLC34A1 (excessive calcitriol synthesis), both of which increase the risk of hypervitaminosis D. |
An increase in the concentration of 24,25(OH)₂D3 primarily indicates an increased concentration of the active form of vitamin D (calcitriol), but may also suggest ongoing bone regeneration processes (e.g., after a fracture).
The test is performed using the isotopic dilution method and high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).
The use of LC-MS/MS provides analytical capabilities not achievable with other techniques. It delivers high repeatability and accuracy of measurements and ensures that results are not influenced by interfering substances such as 3-epi-25(OH)D3 or biotin — a known source of interference in immunoassay-based methods.
This method can be performed on three sample types: serum, plasma, or dried blood spot (DBS). DBS collection enables remote sampling without venepuncture, making it ideal for home testing kits, clinical trials with decentralised logistics, and paediatric applications.
Masdiag Laboratory participates in the Vitamin D External Quality Assessment Scheme (DEQAS), an international proficiency testing programme that evaluates the accuracy and reliability of 25(OH)D measurements across participating laboratories worldwide.
Whether you need testing services, method transfer, or white-label kit development — we'd love to hear from you.