25-Hydroxy Vitamin D (D2 + D3 Fractionated) Test
The fractionated 25-hydroxyvitamin D test measures total 25-OH-D by LC-MS/MS and reports the D2 (ergocalciferol) and D3 (cholecalciferol) fractions separately, identifying which vitamer drives the total result. It is used to confirm absorption and adherence in patients on prescription D2, interpret D2-to-D3 regimen changes, and resolve totals discordant with expected supplementation response.
2
Biomarkers
Blood (serum)
Sample
3–5 business days
Turnaround
No
Fasting
What is the 25-OH Vitamin D (Fractionated) test?
The 25-Hydroxy Vitamin D (D2 + D3 Fractionated) Test measures total 25-hydroxyvitamin D and reports the D2 (ergocalciferol) and D3 (cholecalciferol) fractions separately by liquid chromatography–tandem mass spectrometry. The fractionation is the point of the test: it resolves which vitamer is driving the total result, information a standard total 25-OH-D immunoassay cannot provide. Routine sufficiency screening is served adequately by a total 25-OH-D, so fractionation is ordered where the source of vitamin D matters clinically — confirming absorption and adherence in patients on prescription D2, interpreting results in patients switching between D2 and D3 regimens, and investigating a total result that is discordant with the expected response to supplementation. Because LC-MS/MS measures D2 and D3 individually, it also avoids the variable D2 cross-reactivity seen with some immunoassays.
What does the 25-OH Vitamin D (Fractionated) test measure?
25-Hydroxyvitamin D2 is the hepatic 25-hydroxylation product of ergocalciferol, the plant- and fungus-derived vitamer supplied almost exclusively by prescription-strength oral vitamin D2 in most territories. It circulates bound to vitamin D-binding protein and, like D3, requires a second renal 1-alpha-hydroxylation to the active 1,25-dihydroxy form. D2 has a shorter serum half-life and lower binding-protein affinity than D3, so its contribution to measured total 25-OH-D falls more quickly between doses.
Fractionation isolates the D2 contribution, letting the practitioner confirm absorption and adherence in patients prescribed high-dose ergocalciferol and attribute a rising total 25-OH-D to the prescribed vitamer rather than endogenous or dietary D3. A total result that is adequate only because of a large D2 fraction, with a low D3 fraction, carries a different monitoring implication than a D3-driven result.
25-Hydroxyvitamin D3 is the 25-hydroxylation product of cholecalciferol, generated in the skin from 7-dehydrocholesterol under UV-B exposure and supplied by diet and most non-prescription supplements. It is the principal circulating vitamer in people not taking D2, has a longer half-life (roughly two to three weeks) than D2, and is the fraction that best reflects cumulative endogenous and supplemental D3 status.
The D3 fraction reflects sun exposure, dietary intake, and D3 supplementation. In a patient not on ergocalciferol it approximates the total, and a low D3 fraction despite an adequate total flags reliance on D2 dosing. Tracking the D3 fraction is useful where the source of repletion must be distinguished.
Why is the 25-OH Vitamin D (Fractionated) test ordered?
- Patients on high-dose prescription ergocalciferol (D2) where fractionation confirms absorption and adherence by demonstrating a rising D2 fraction
- Patients transitioning between D2 and D3 regimens, where attributing the total result to the correct vitamer guides dose adjustment
- Investigation of an unexpectedly low or non-responsive total 25-OH-D despite documented supplementation, to distinguish malabsorption from non-adherence or wrong-vitamer dosing
- Patients in whom a prior immunoassay total 25-OH-D is suspected of under- or over-reporting D2, where an LC-MS/MS fractionated measurement resolves the discrepancy
- Malabsorption states (bariatric surgery, coeliac disease, cystic fibrosis, inflammatory bowel disease) where documenting which vitamer is absorbed informs the repletion strategy
- Specialist metabolic bone or endocrinology work-up where the vitamer source must be characterised, not just total status
Sample collection and turnaround
Sample type
Blood (serum)
Fasting required
No
Collection method
Venipuncture at patient service centre
Turnaround
3–5 business days
Collection notes
Phlebotomy at a LabCorp draw site; no special preparation. Record the current vitamin D regimen (D2 vs D3, dose, and time since last dose) on the requisition, as this is essential to interpreting the fractions.
Specimen requirements
Minimum 0.5 mL serum. Protect from prolonged light exposure; 25-OH-D is stable refrigerated for several days. Note the LC-MS/MS method on the requisition for serial comparability.
What can affect 25-OH Vitamin D (Fractionated) results?
Prescription D2 raises the measured D2 fraction and the total; this is the expected pharmacodynamic effect, but it must be recognised as D2-driven rather than interpreted as improved endogenous status.
Some automated total 25-OH-D immunoassays under-recover D2, giving falsely low totals in D2-treated patients. LC-MS/MS fractionation does not share this limitation and may legitimately disagree with an earlier immunoassay.
Pregnancy, oestrogen therapy, nephrotic syndrome, and hepatic disease alter binding-protein concentration and can shift total 25-OH-D independent of true bioavailable vitamin D status.
A large recent oral bolus transiently elevates the corresponding fraction; timing of the draw relative to the last dose should be recorded for serial interpretation.
Impaired absorption of the fat-soluble vitamin lowers the fraction corresponding to the oral vitamer, which fractionation can demonstrate directly.
Sequestration of vitamin D in adipose tissue lowers circulating 25-OH-D for a given intake, so higher or more sustained dosing may be required to reach target.
What do 25-OH Vitamin D (Fractionated) results mean?
Elevated
A high total with a large D2 fraction reflects ergocalciferol dosing; a high total with a dominant D3 fraction reflects sun exposure, diet, or D3 supplementation. Markedly elevated total 25-OH-D (approaching or exceeding ~100 ng/mL) warrants review of dosing and assessment for hypercalcaemia.
Normal
Total 25-OH-D within target with a vitamer pattern consistent with the documented regimen confirms adequate status. A D2-dependent adequate total with a low D3 fraction should prompt a plan for maintenance once D2 is stopped, as the D2 contribution falls quickly.
Reduced
A low total with a low D3 fraction in a patient not on D2 indicates deficiency from inadequate synthesis or intake. A low total despite prescribed D2 — demonstrated by a low D2 fraction — points to malabsorption or non-adherence rather than insufficient prescribing.
Note: 25-OH-D is the correct marker of status; 1,25-dihydroxyvitamin D is not and should not be used for screening. The D2 and D3 fractions are interpreted together as a total, with the fractionation used to attribute source. Sufficiency thresholds differ between guidelines and must be applied consistently.
Frequently asked questions
A standard test reports a single total 25-hydroxyvitamin D value. The fractionated LC-MS/MS test additionally separates the D2 and D3 contributions, so the practitioner can see which vitamer is responsible for the total — information a total-only result cannot provide.
How to access the 25-OH Vitamin D (Fractionated) test
The 25-OH Vitamin D (Fractionated) reference page is free and open to all practitioners. Create a free practitioner account to see pricing, save tests to your list, and generate requisition forms. Verification takes 1–2 business days.
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Published: October 7, 2026
Last reviewed: October 7, 2026
- Holick MF, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. (2011)
- Ross AC, et al. The 2011 report on dietary reference intakes for calcium and vitamin D from the Institute of Medicine. J Clin Endocrinol Metab. (2011)
- Sempos CT, et al. Vitamin D assays and the definition of hypovitaminosis D: results from the First International Conference on Controversies in Vitamin D. Br J Clin Pharmacol. (2018)
- Tsugawa N, et al. Determination of 25-hydroxyvitamin D in human plasma using LC–MS/MS. Anal Chem. (2005)
This content is for licensed healthcare practitioners only and does not constitute medical advice. Clinical decisions should be based on the full clinical picture and not on any single test result.
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