25-OH Vitamin D HPLC Assay


The 25-OH Vitamin D3 HPLC Assay kit is intended for the quantitative determination of 25-OH Vitamin D3 in plasma and serum. The 25-OH Vitamin D3 HPLC Assay kit is for research use only and not to be used in diagnostic procedures.

25-OH Vitamin D HPLC Assay

The 25-OH Vitamin D HPLC Assay is For Research Use Only

Size: 100 tests
Incubation Time: 15 minutes
Sample Type: Plasma, Serum
Sample Size: 400 µL
Controls Included

Reference Values (Normal): 
Summer    50-300 nmol/l (20-120 µg/l)
Winter       25-150 nmol/l (10-60 µg/l)

It is suggested that each laboratory establish its own normal ranges

Product Developed and Manufactured in Germany
Product Support in the USA

Assay Background

Vitamin D is one of the most important essential bioregulators of the Ca2+- and phosphate metabolism in higher animals. Together with the peptide hormones parathormone and calcitonin it is responsible to obtain the Ca2+- and phosphate homoeostasis.  The most important physiological functions of vitamin D are the stimulation of the intestinal resorption of Ca2+ and phosphate and their incorporation in the bone matrix.

D-vitamins or calciferols arise from provitamins by the UV radiation of sunlight catalyzed splitting of the B-ring of the steran backbone. In the skin, formed vitamin D3 is bound to a vitamin D binding-protein in the plasma, transported into the liver and hydroxylated in position 25 to form 25-OH vitamin D3. Another hydroxylation is performed in the kidney to yield 1,25 (OH)2 vitamin D3 and 24,25 (OH)2 vitamin D3.   1,25 (OH)2 vitamin D3 is the metabolic most active form of vitamin D.  Nevertheless, more than 95% of 25-OH vitamin D is 25-OH vitamin D3.  A lack of vitamin D leads in growing humans and animals can lead to rickets, hypocalcaemia and secondary hyperparathyroidism and in adults to osteomalacia.

The Eagle Biosciences 25-OH Vitamin D3 HPLC Assay kit makes it possible to determine the vitamin in an easy, fast and precise method. The kit includes all reagents in ready to use form for preparation and separation of the samples with exception of the column (IC3401rp) and the controls (IC3401ko). Both can be supplied by Eagle Biosciences.  Beside the complete test kit, it is possible to order all components separately. Please request our single component price list.

Related Products

25-OH Vitamin D HPLC Assay Control Set
25-OH Vitamin D HPLC Assay Column
25-OH Vitamin D ELISA Assay Kit

Additional Information

Assay Principle

For the determination of 25-OH vitamin D3 a sample preparation has to be performed. Therefore a precipitation step is combined with an extraction. After centrifugation the upper layer is injected into the HPLC system.  The HPLC separation works with an isocratic method at 30 °C with a “reversed phase” column. Chromatograms are detected by an UV-detector. The separation takes 15 minutes for each run depending on the column used. Results are quantified by the delivered serum calibrator and calculated by the “internal standard-method” by integration of the peak areas or heights.

Assay Procedure

  1. Pipette into 1.5 ml reaction tubes:  400 µl sample, CAL or CTRL + 400 µl IS ice cold  (Important: for better precipitation use ice cold IS)
  2. Mix briefly on a vortex mixer.
  3. Add 500 µl PREC
  4. Mix for 2 min on a vortex mixer. Leave the tubes for 15 minutes at 2-8°C and centrifuge afterwards at 10.000g for 5 minutes. Crucial step: After centrifugation the sample shows 2 liquid phases and a solid disc (precipitated proteins). For injection only use the upper liquid phase.
  5. Inject 50 µl of the supernatant for chromatography into the HPLC-system

Limitations to the Assay

  • Whole blood is not suited for the 25-OH Vitamin D3 HPLC Assay kit.
  • Using gel vials for blood collection might cause disturbed chromatograms caused by ingredients of the gel. We recommend using EDTA plasma or serum vials without gel.

Package Inserts


Product Citations