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Vitamin D, 25-Hydroxy, LC/MS/MS

Vitamin D, 25-Hydroxy, LC/MS/MS

Test Summary

Vitamin D, 25-Hydroxy, LC/MS/MS


Clinical Use

  • Diagnose vitamin D deficiency or toxicity

  • Monitor response to vitamin D2 or vitamin D3 supplementation

Clinical Background

Vitamin D helps to promote bone development and maintain bone and skeletal health by enhancing the intestinal absorption of calcium and phosphorus.1,2 Vitamin D deficiency is associated with bone diseases such as rickets, osteomalacia, and osteoporosis; emerging evidence also suggests links to illnesses such as infectious diseases, autoimmune diseases, heart disease, and cancer.1,2 Vitamin D toxicity, which manifests as hypercalcemia, hypercalciuria, or kidney stones, is rare but can result from taking vitamin D at doses 10 times the daily recommended amount.2,3,4 The Institute of Medicine and the American Association of Clinical Endocrinologists recommend maintaining adequate vitamin D levels, with vitamin D supplementation if necessary, to prevent bone and skeletal disease.1,5 Accurate determination of vitamin D levels can help assess vitamin D status and diagnose vitamin D deficiency and toxicity.

Vitamin D occurs in 2 forms, vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Vitamin D3 is the main form and is produced in the skin in response to sunlight. However, both forms can be obtained in relatively small amounts through normal diet. Both are also available as over-the-counter supplements, but only vitamin D2 is available in high-dose prescription form in the United States. Vitamins D3 and D2 are metabolized to produce different 25-hydroxyvitamin D [25(OH)D] forms, 25(OH)D3 and 25(OH)D2, which are in turn metabolized to produce their corresponding active forms.2 The sum of 25(OH)D3 and 25(OH)D2 concentrations yields the total 25(OH)D level, which is the accepted standard for determining vitamin D status; the active form of vitamin D (1,25-dihydroxyvitamin D) is not useful for assessing vitamin D status because its levels are often normal in patients with vitamin D deficiency.2,3

Separate 25(OH)D3 and 25(OH)D2 measurements may help to differentiate the contributions of vitamin D3 and vitamin D2 to vitamin D status.6 The LC/MS/MS method accurately and precisely measures 25(OH)D3 and 25(OH)D2, and sums these 2 analytes to obtain the total 25(OH)D concentration; in contrast, immunoassays tend to underestimate 25(OH)D2 levels.7 LC/MS/MS provides an analytical sensitivity greater than that of an electrochemiluminescence immunoassay and comparable to that of a radioimmunoassay method, without the need for radioisotopes.8 The Vitamin D, 25-Hydroxy, LC/MS/MS test is useful for diagnosing vitamin D deficiency, monitoring response to vitamin D2 or D3 supplementation, and differentiating between vitamin D toxicity and other causes of hypercalcemia.

Individuals Suitable for Testing

  • Individuals with suspected vitamin D deficiency (eg, those with persistent, nonspecific musculoskeletal pain) or at increased risk for vitamin D deficiency (eg, the elderly, housebound individuals, those with dark skin pigmentation, and those with conditions or taking medications associated with reduced 25(OH)D levels [see Table])

  • Individuals being treated with vitamin D2 or vitamin D3 supplementation

  • Individuals with suspected toxicity (eg, those with hypercalcemia of obscure origin)

Table. Effect of Various Disorders and Medications on
25-Hydroxyvitamin D Concentration2,9-13

Total 25(OH)D


Vitamin D toxicity  ↑
Nutritional rickets  ↓
Osteomalacia  ↓
Obesity  ↓
Fat malabsorption disorders, short bowel syndrome  ↓
Intestinal diseases causing excessive loss of vitamins D2 and D3  ↓
Anticonvulsant, antiretrovirals, or antituberculosis medications (increased metabolism)  ↓
Severe cholestatic or parenchymal liver disease (impaired 25-hydroxylation of vitamin D)  ↓
Hyperphosphatemic tumoral calcinosis  ↓
Tuberculosis  ↓
Secondary hyperparathyroidism  ↓
Sarcoidosis N or
Severe renal disease N or
Vitamin D-dependent rickets, type I N or
Vitamin D-dependent rickets, type II N or
25(OH)D, 25-hydroxyvitamin D; N, normal.


  • Liquid chromatography, tandem mass spectrometry (LC/MS/MS) 

   Extraction via protein precipitation

   Separation via high-performance liquid chromatography (HPLC)

   Detection and quantitation via tandem mass spectrometry

   25(OH)D2 and 25(OH)D3 concentrations used to calculate total 25(OH)D levels

  • Report includes concentration of total 25(OH)D, 25(OH)D2, and 25(OH)D3

  • Analytical sensitivity: 4 ng/mL for 25(OH)D2 and for 25(OH)D3

  • Analytical specificity: no cross-reactivity with vitamin D2 or D3; 1-hydroxy and 1,25-dihydroxy forms of vitamin D2 or D3; 24,25(OH)2D3; and 25,26(OH)2D3

  • Reportable range: 4-512 ng/mL for 25(OH)D2 and for 25(OH)D3; 4-1024 ng/mL for total 25(OH)D

  • Aliases: 25(OH)D, 25-hydroxycholecalciferol, 25-hydroxyergocalciferol

Interpretive Information

Abnormal 25(OH)D levels are associated with a range of conditions and medications (Table).

Levels of 25(OH)D3 reflect both endogenous production and exogenous sources such as diet or supplementation, whereas levels of 25(OH)D2 reflect only exogenous sources and are detected in significant amounts only in response to intake of vitamin D2 supplements.6,8 There is no consensus about the optimal total 25(OH)D level, which may vary with the assay used and functional outcome measured. However, many experts accept a range of 30-60 ng/mL as optimal.2,5 Total 25(OH)D levels <20 ng/mL suggest vitamin D deficiency, while levels between 20 and 30 ng/mL suggest insufficiency.2 Expert opinions also vary about what constitutes 25(OH)D toxicity, with reported thresholds ranging from 50 ng/mL1 to 150 ng/mL (374 nmol/L).2


  1. Ross AC, Manson JE, Abrams SA, et al. The 2011 Report on Dietary Reference Intakes for Calcium and Vitamin D from the Institute of Medicine: What Clinicians Need to Know. J Clin Endocrinol Metab. 2011;96:53-58.

  2. Holick MF. Vitamin D deficiency. N Engl J Med. 2007;357:266-281.

  3. Cranney A, Horsley T, O’Donnell S, et al. Effectiveness and Safety of Vitamin D in Relation to Bone Health. Rockville, MD: Agency for Healthcare Research and Quality (US); 2007. Available at: http://www.ncbi.nlm.nih.
    gov/books/NBK38416. Accessed January 28, 2011.

  4. Vieth R. Vitamin D supplementation, 25-hydroxyvitamin D concentrations, and safety. Am J Clin Nutr. 1999;69:842-856.

  5. Watts NB, Bilezikian JP, Camacho PM, et al. American Association of Clinical Endocrinologists medical guidelines for clinical practice for the diagnosis and treatment of postmenopausal osteoporosis. Endocr Pract. 2010;16(Suppl 3):1-37.

  6. Maunsell Z, Wright DJ, Rainbow SJ. Routine isotope-dilution liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of the 25-hydroxy metabolites of vitamins D2 and D3. Clin Chem. 2005;51:1683-1690.

  7. Glendenning P, Taranto M, Noble JM, et al. Current assays overestimate 25-hydroxyvitamin D3 and underestimate 25-hydroxyvitamin D2 compared with HPLC: Need for assay-specific decision limits and metabolite-specific assays. Ann Clin Biochem. 2006;43:23-30.

  8. Herrmann M, Harwood T, Gaston-Parry O, et al. A new quantitative LC tandem mass spectrometry assay for serum 25-hydroxy vitamin D. Steroids. 2010;75:1106-1112.

  9. Bringhurst FR, Demay MB, Kronenberg HM. Hormones and disorders of mineral metabolism. In: Larsen PR, Kronenberg HM, Melmed S, Polonsky KS, eds. Williams Textbook of Endocrinology. 10th ed. Philadelphia, PA: WB Saunders; 2003:1303-1371.

  10. St-Arnaud R, Glorieux FH. Hereditary defects in vitamin D metabolism and action. In: DeGroot LJ, Jameson JL, et al, eds. Endocrinology. 4th ed. Philadelphia, PA: WB Saunders; 2001:1154-1168.

  11. Goldring SR, Krane SM, Avioli LV. Disorders of calcification: osteomalacia and rickets. In: DeGroot LJ, Jameson JL, et al, eds. Endocrinology. 4th ed. Philadelphia, PA: WB Saunders; 2001:1223-1243.

  12. Jacobs DS, Garg U, Oxley DK, et al. Chemistry: Vitamin D, Serum. In: Jacobs DS, Oxley DK, Demott WR, eds. Jacobs & Demott Laboratory Test Handbook. 5th ed. Cleveland, Ohio: Lexi-Comp, Inc; 2001:300-301.

  13. Holick MF, Binkley NC, Heike A, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society Clinical Practice Guideline. J Clin Endocrin Metab. 2011;96:1911-1930.

Content reviewed 09/2013
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