Sign in →

Test Code 25HDN 25-Hydroxyvitamin D2 and D3, Serum

Reporting Name

25-Hydroxyvitamin D2 and D3, S

Useful For

Diagnosis of vitamin D deficiency


Differential diagnosis of causes of rickets and osteomalacia


Monitoring vitamin D replacement therapy


Diagnosis of hypervitaminosis D

Method Name

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Performing Laboratory

Mayo Medical Laboratories in Rochester

Specimen Type


Specimen Required


Preferred: Serum gel

Acceptable: Red top

Specimen Volume: 0.5 mL

Specimen Minimum Volume

0.25 mL

Specimen Stability Information

Specimen Type Temperature Time
Serum Refrigerated (preferred) 14 days
  Frozen  30 days
  Ambient  7 days

Reject Due To


Mild OK; Gross OK


Mild OK; Gross OK


Mild OK; Gross OK



Reference Values


<10 ng/mL (severe deficiency)*

10-19 ng/mL (mild to moderate deficiency)**

20-50 ng/mL (optimum levels)***

51-80 ng/mL (increased risk of hypercalciuria)****

>80 ng/mL (toxicity possible)*****

*Could be associated with osteomalacia or rickets

**May be associated with increased risk of osteoporosis or secondary hyperparathyroidism

***Optimum levels in the healthy population; patients with bone disease may benefit from higher levels within this range

****Sustained levels >50 ng/mL 25OH-VitD along with prolonged calcium supplementation may lead to hypercalciuria and decreased renal function

****80 ng/mL is the lowest reported level associated with toxicity in patients without primary hyperparathyroidism who have normal renal function. Most patients with toxicity have levels >150 ng/mL. Patients with renal failure can have very high 25-OH-VitD levels without any signs of toxicity, as renal conversion to the active hormone 1,25-OH-VitD is impaired or absent.


These reference ranges represent clinical decision values, based on the 2011 Institute of Medicine report, that apply to males and females of all ages, rather than population-based reference values. Population reference ranges for 25-OH-VitD vary widely depending on ethnic background, age, geographic location of the studied populations, and the sampling season. Population-based ranges correlate poorly with serum 25-OH-VitD concentrations that are associated with biologically and clinically relevant vitamin D effects and are therefore of limited clinical value.

Day(s) and Time(s) Performed

Monday through Friday; Continuous until 2:30 p.m.

Specimens on patients who are <1 year old are only performed on Tuesday and Friday.

CPT Code Information


LOINC Code Information

Test ID Test Order Name Order LOINC Value
25HDN 25-Hydroxyvitamin D2 and D3, S 49590-3


Result ID Test Result Name Result LOINC Value
2897 25-Hydroxy D2 49054-0
2898 25-Hydroxy D3 1989-3
83670 25-Hydroxy D Total 62292-8

Analytic Time

2 days


Long term use of anticonvulsant medications may result in vitamin D deficiency that could lead to bone disease; the anticonvulsants most implicated are phenytoin, phenobarbital, carbamazepine, and valproic acid. Newer antiseizure medications have not been studied or are not thought to contribute to vitamin D deficiency.

Method Description

Deuterated stable isotope (d3-25-hydroxyvitamin D) is added to a 0.1-mL serum sample as internal standard. 25-Hydroxyvitamin D2 (25-OH-VitD2), 25-hydroxyvitamin D3 (25-OH-VitD3), and the internal standard are extracted with acetone and ethyl acetate. The extracts are then derivatized before being analyzed by liquid chromatography-tandem mass spectrometry using multiple reaction monitoring. 25-OH-VitD2 and 25-OH-VitD3 are quantified and reported individually and as a sum with a clinical reference range attached to the sum. For children <1 year of age, a modified assay is performed to identify and eliminate interference due to C-3 epimers of 25-OH-VitD2 and 25-OH-VitD3.(Zimmer D, Pickard V, Czembor W, Muller C: Comparison of turbulent-flow chromatography with automated solid-phase extraction in 96-well plates and liquid-liquid extraction used as serum sample preparation techniques for liquid chromatography-tandem mass spectrometry. J Chromatogr A 1999 Aug 27;854[1-2]:23-35)


Based on animal studies and large human epidemiological studies, 25-hydroxyvitamin D2 and D3 (25-OH-VitD) <25 ng/mL are associated with an increased risk of secondary hyperparathyroidism, reduced bone mineral density, and fractures, particularly in the elderly. Intervention studies support this clinical cutoff, showing a reduction of fracture risk with 25-OH-VitD replacement.


Levels <10 ng/mL may be associated with more severe abnormalities and can lead to inadequate mineralization of newly formed osteoid, resulting in rickets in children and osteomalacia in adults. In these individuals, serum calcium levels may be marginally low, and parathyroid hormone (PTH) and serum alkaline phosphatase are usually elevated. Definitive diagnosis rests on the typical radiographic findings or bone biopsy/histomorphometry.


Baseline biochemical work-up of suspected cases of rickets and osteomalacia should include measurement of serum calcium, phosphorus, PTH, and 25-OH-VitD. In patients where testing is not completely consistent with the suspected diagnosis, in particular if serum 25-OH-VitD levels are >10 ng/mL, an alternative cause for impaired mineralization should be considered. Possible differential diagnosis includes: partly treated vitamin D deficiency, extremely poor calcium intake, vitamin D resistant rickets, renal failure, renal tubular mineral loss with or without renal tubular acidosis, hypophosphatemic disorders (eg, X-linked or autosomal dominant hypophosphatemic rickets), congenital hypoparathyroidism, activating calcium sensing receptor mutations, and osteopetrosis. Measurement of serum urea, creatinine, magnesium, and 1,25-OH-VitD is recommended as a minimal additional work-up for these patients.


25-OH-VitD replacement in the United States typically consists of VitD2. Lack of clinical improvement and no reduction in PTH or alkaline phosphatase may indicate patient noncompliance, malabsorption, resistance to 25-OH-VitD, or additional factors contributing to the clinical disease. Measurement of serum 25-OH-VitD levels can assist in further evaluation, in particular as the liquid chromatography-tandem mass spectrometry methodology allows separate measurement of 25-OH-VitD3 and of 25-OH-VitD2, which is derived entirely from dietary sources or supplements.


Patients who present with hypercalcemia, hyperphosphatemia, and low PTH may suffer either from ectopic, unregulated conversion of 25-OH-VitD to 1,25-OH-VitD, as can occur in granulomatous diseases, particular sarcoid, or from nutritionally-induced hypervitaminosis D. Serum 1,25-OH-VitD levels will be high in both groups, but only patients with hypervitaminosis D will have serum 25-OH-VitD concentrations of >80 ng/mL, typically >150 ng/mL.

Specimen Retention Time

2 weeks

Clinical Information

25-Hydroxyvitamin D2 and D3 (25-OH-VitD) are steroid hormones that require 1-alpha-hydroxylation before expressing biological activity. Vitamin D compounds are derived from dietary ergocalciferol (from plants, VitD2) or cholecalciferol (from animals, VitD3), or by conversion of 7-dihydrocholesterol to VitD3 in the skin upon ultraviolet exposure. VitD2 and VitD3 are subsequently 25-hydroxylated in the liver to 25-OH-VitD. 25-OH-VitD represents the main body reservoir and transport form of vitamin D, being stored in adipose tissue and tightly bound by a transport protein while in circulation. A fraction of circulating 25-OH-VitD is converted to its active metabolites 1,25-dihydroxy vitamin D2 and D3 (1,25-OH-VitD), mainly by the kidneys. This process is regulated by parathyroid hormone (PTH), which increases 1,25-OH-VitD synthesis at the expense of the alternative, biologically inactive hydroxylation product 24,25-OH-VitD. Like other steroid hormones, 1,25-OH-VitD binds to a nuclear receptor, influencing gene transcription patterns in target organs.


1,25-OH-VitD plays a primary role in the maintenance of calcium homeostasis. It promotes intestinal calcium absorption and, in concert with PTH, skeletal calcium deposition, or less commonly, calcium mobilization. Renal calcium and phosphate reabsorption are also promoted, while prepro-PTH mRNA expression in the parathyroid glands is down-regulated. The net result is a positive calcium balance, increasing serum calcium and phosphate levels, and falling PTH concentrations.


In addition to its effects on calcium and bone metabolism, 1,25-OH-VitD regulates the expression of a multitude of genes in many other tissues including immune cells, muscle, vasculature, and reproductive organs.


The exact 25-OH-VitD level reflecting optimal body stores remains unknown. Mild-to-modest deficiency can be associated with osteoporosis or secondary hyperparathyroidism. Severe deficiency may lead to failure to mineralize newly formed osteoid in bone, resulting in rickets in children and osteomalacia in adults. The consequences of vitamin D deficiency on organs other than bone are not fully known, but may include increased susceptibility to infections, muscular discomfort, and an increased risk of colon, breast, and prostate cancer.


Modest 25-OH-VitD deficiency is common; in institutionalized elderly, its prevalence may be >50%. Although much less common, severe deficiency is not rare either.


Reasons for suboptimal 25-OH-VitD levels include lack of sunshine exposure, a particular problem in Northern latitudes during winter; inadequate intake; malabsorption (eg, due to Celiac disease); depressed hepatic vitamin D 25-hydroxylase activity, secondary to advanced liver disease; and enzyme-inducing drugs, in particular many antiepileptic drugs, including phenytoin, phenobarbital, and carbamazepine, that increase 25-OH-VitD metabolism.


In contrast to the high prevalence of 25-OH-VitD deficiency, hypervitaminosis D is rare, and is only seen after prolonged exposure to extremely high doses of vitamin D. When it occurs, it can result in severe hypercalcemia and hyperphosphatemia.

Test Classification

This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration.


If not ordering electronically, complete, print, and send a General Request Form (T239) with the specimen (