Test Center

My Recent Searches

  • No Recent Search.

My Tests Viewed

  • No Test Viewed.

QuestAssureD 25-Hydroxyvitamin D (D2, D3), LC/MS/MS

QuestAssureD 25-Hydroxyvitamin D (D2, D3), LC/MS/MS

Test Summary

QuestAssureD™ 25-Hydroxyvitamin D (D2, D3), LC/MS/MS


Clinical Use

  • Diagnose vitamin D deficiency or toxicity

  • Monitor response to vitamin D2 or vitamin D3 supplementation

Clinical Background

As much as 68% to 77% of the population is estimated to have suboptimal (<30 ng/mL) levels of vitamin D.1,2 This important nutrient promotes skeletal health by enhancing the intestinal absorption of calcium and phosphorus. Deficiency is associated with bone diseases such as rickets, osteomalacia, and osteoporosis. Emerging evidence also suggests links to nonskeletal illnesses such as cancer (especially colorectal and breast), cardiovascular disease, and infectious and autoimmune diseases.3,4 Vitamin D toxicity, which manifests as hypercalcemia, hypercalciuria, or kidney stones, is rare but can result at serum levels above 150 ng/mL.4 Current guidelines recommend maintaining adequate vitamin D levels (with vitamin D supplementation if necessary) to maximize bone health and prevent bone disease.4-6 Accurate determination of vitamin D levels can help diagnose vitamin D deficiency and toxicity, and inform clinical management.

The term "vitamin D" typically refers to 2 molecular forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D3 is the main form and is produced in the skin in response to sunlight.4 However, both forms can be obtained in relatively small amounts through normal diet. Both are also available as over-the-counter supplements and as high-dose prescription formulations in the United States.3 Vitamin D3 is also available as a high-dose supplement.

Vitamins D2 and D3 are metabolized in the liver to their respective 25-hydroxyvitamin D (25[OH]D) metabolites, 25(OH)D2 and 25(OH)D3, which are converted in the kidneys to their corresponding active forms.4 The sum of 25(OH)D2 and 25(OH)D3 concentrations yields the total 25(OH)D level, which is the accepted standard for determining vitamin D status.4 The active metabolite of vitamin D (1,25-dihydroxyvitamin D) is not useful for assessing vitamin D status because its levels are often normal or even elevated in patients with vitamin D deficiency.4

The QuestAssureD™ 25-Hydroxyvitamin D (D2, D3), LC/MS/MS test (test code: 92888), 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. The test uses liquid chromatography-tandem mass spectrometry (LC/MS/MS), which accurately and precisely measures 25(OH)D2 and 25(OH)D3 and sums these 2 analytes to obtain the total 25(OH)D concentration.7,8 Separate 25(OH)D2 and 25(OH)D3 measurements may help differentiate the contributions of vitamin D2 and vitamin D3 to vitamin D status if this information is required. In contrast, most immunoassay techniques do not differentiate the 2 forms.

Individuals Suitable for Testing

  • Individuals with suspected vitamin D deficiency (eg, those with osteoporosis, osteomalacia, or persistent, nonspecific musculoskeletal pain)

  • Individuals at increased risk for vitamin D deficiency (eg, pregnant, lactating, dark-skinned, elderly, obese, or housebound individuals; those dwelling in latitudes higher than 33° north or 33° south; and infants who are breastfeeding without vitamin D supplementation or from vitamin D-deficient mothers)

  • Individuals with disorders associated with reduced 25[OH]D levels (see Table)

  • Individuals taking certain 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)

For infants/toddlers <36 months old, use QuestAssureD for Infants, 25-Hydroxyvitamin D, LC/MS/MS (test code 91935).


  • 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 concentrations 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; or 25,26(OH)2D3
  • Reportable range: 4 to 512 ng/mL for 25(OH)D2 and for 25(OH)D3; 4 to 1,024 ng/mL for total 25(OH)D

Interpretive Information

Abnormal 25(OH)D levels are associated with a range of conditions and medications (Table).3,4,9-18 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.7,8 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 minimum of 30 ng/mL with a range of 40 to 60 ng/mL as optimal.3 A level up to 100 ng/mL is considered to be safe.3,4 Total 25(OH)D levels ≤20 ng/mL suggest vitamin D deficiency, while levels between 21 and 29 ng/mL suggest insufficiency.4 Expert opinions also vary about what constitutes 25(OH)D toxicity, with reported thresholds ranging from 50 ng/mL for outcome measures such as increased risk for mortality not related to calcium and bone metabolism (125 nmol/L)1 to 150 ng/mL (374 nmol/L) for mortality related to calcium and bone metabolism.4

Table. Effect of Various Disorders and Medications on 25-Hydroxyvitamin D Concentration3,4,9-18


 25(OH)D Concentration

Chronic kidney disease3,9

↓ or N

Fat malabsorption disorders, short bowel syndrome, inflammatory bowel disease, Crohn disease4,10

Hypercalcemia of cancera,11

↓ or N or ↑

Hypophosphatemic ricketsb,3


Lymphoma, granulomatous disorders3,4

Medications that increase vitamin D metabolism (eg, anticonvulsants, antiretrovirals, and glucocorticoids)3,4

Nephrotic syndrome12

Nutritional rickets or osteomalacia13



↓ or N

Primary hyperparathyroidism3,4

↓ or N

Secondary hyperparathyroidism15

Severe parenchymal liver disease (impaired 25-hydroxylation of vitamin D)12,16

Tumor-induced osteomalacia (oncogenic osteomalacia)3,17


Vitamin D-dependent rickets, type Ic,18


Vitamin D-dependent rickets, type IId,18


Vitamin D (25[OH]D) toxicity (uncommon)12

25(OH)D, 25-hydroxyvitamin D; N, normal; ↑, elevated; ↓, reduced.


PTHrP (parathyroid hormone–related peptide)-mediated


Autosomal dominant or X-linked hypophosphatemic rickets


Also called pseudo-vitamin D-deficiency rickets.


Also called hereditary vitamin D-resistant rickets.


  1. Lai JK, Lucas RM, Clements MS, et al. Assessing vitamin D status: pitfalls for the unwary. Mol Nutr Food Res. 2010;54:1062-1071.

  2. Ginde AA, Liu MC, Camargo CA, Jr. Demographic differences and trends of vitamin D insufficiency in the US population, 1988-2004. Arch Intern Med. 2009;169:626-632.

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

  4. Holick MF, Binkley NC, Bischoff-Ferrari HA, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011;96:1911-1930.

  5. 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.

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

  7. 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.

  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. National Kidney Foundation. K/DOQI clinical practice guidelines for bone metabolism and disease in chronic kidney disease. Am J Kidney Dis. 2003;42(suppl 3):S1-S201.

  10. Margulies SL, Kurian D, Elliott MS, et al. Vitamin D deficiency in patients with intestinal malabsorption syndromes—think in and outside the gut. J Dig Dis. 2015;16:617-633.

  11. Goldner W. Cancer-related hypercalcemia. J Oncol Pract. 2016;12:426-432.

  12. Bouillon R. Vitamin D: from photosynthesis, metabolism, and action to clinical applications. In: Jameson JL, De Groot LJ, eds. Endocrinology. 6th ed. Philadelphia, PA: Saunders; 2010:1089-1110.

  13. Pettifor JM. Calcium and vitamin D metabolism in children in developing countries. Ann Nutr Metab. 2014;64(suppl 2):15-22.

  14. Lips P. Relative value of 25(OH)D and 1,25(OH)2D measurements. J Bone Miner Res. 2007;22:1668-1671.

  15. Lips P. Vitamin D deficiency and secondary hyperparathyroidism in the elderly: consequences for bone loss and fractures and therapeutic implications. Endocr Rev. 2001;22:477-501.

  16. Stokes CS, Volmer DA, Grunhage F, et al. Vitamin D in chronic liver disease. Liver Int. 2013;33:338-352.

  17. Hautmann AH, Hautmann MG, Kölbl O, et al. Tumor-induced osteomalacia: an up-to-date review. Curr Rheumatol Rep. 2015;17:512.

  18. St-Arnaud R, Glorieux FH. Genetic defects in vitamin D metabolism and action. In: Jameson JL, De Groot LJ, eds. Endocrinology. 6th ed. Philadelphia, PA: Saunders; 2010:1236-1249.

Content reviewed 07/2018

top of page

* The tests listed by specialist are a select group of tests offered. For a complete list of Quest Diagnostics tests, please refer to our Directory of Services.