38038 HPLC Kit Vitamin D3 Vitamin D2 Serum Plasma

25-OH-Vitamin D3/D2 in Serum/Plasma - HPLC

Order no.: 38038
100 Tests
  • 25-OH-vitamin D2
  • 25-OH-vitamin D3

Vitamins D3 and D2 with one analysis
Improved separation
Quality controls and calibrator available


Clinical relevance

Vitamin D (cholecalciferol) is a recognised clinical chemical determinant associated with osteoporosis and osteopenia (reduction in bone density). It also plays a role in the body's immune response, cardiovascular diseases and some types of cancer. Therefore, vitamin D is highly important from a number of diagnostic perspectives.

25-OH-vitamin D3 is the primary metabolite in the human body and the direct precursor of the physiologically active form 1.25-(OH)2-vitamin D3. First-line treatment for vitamin D deficiency is nutritional supplements with vitamin D3 or vitamin D2. Vitamin D2 is easier to synthesise and is widely used as a supplement outside of the EU, and especially in the United States. Therefore, its determination in serum/plasma is also relevant.


Product advantages

  • Combined analysis of vitamins D3 and D2
  • Reliable separation
  • Reliable quantification with a stable internal standard


This Chromsystems kit allows the simultaneous and reliable chromatographic determination of 25-OH-Vitamin D3 and 25-OH-Vitamin D2 on an isocratic HPLC system with UV detection. By means of efficient protein precipitation and selective solid phase extraction interfering components are removed. A non-physiological derivative of vitamin D is used as a stable internal standard to produce reliable quantification. This way, reproducible and reliable results can be produced at a low expenditure of cost and time.

Alternatively, for high sample throughput Chromsystems offers an online method with selective online solid phase extraction (order no. 38900/1000) as well as methods for analysis using mass spectrometry (order no. 62000).


Technical Data

Method of Analysis


Number of Tests



25-OH-vitamin D2, 25-OH-vitamin D3

Analysis Time

10 min

Limit of quantification

1.1 µg/l - 1.4 µg/l


up to at least 250 µg/l


25-OH-Vitamin D3: 86 %; 25-OH-Vitamin D2: 87 %


25-OH-Vitamin D3: CV 3.0 %
25-OH-Vitamin D2: CV 1.8 %


25-OH-Vitamin D3: CV 3.3 %
25-OH-Vitamin D2: CV 4.6 %



Sample Preparation

  • Mix 500 µl serum/plasma with 50 µl Internal Standard in a light-protected reaction vial.
  • Add 500 µl Precipitation Reagent, vortex-mix for 20 s.
  • Incubate 10 min to +4 °C.
  • Centrifuge 5 min at 15 000 x g.
  • Apply complete supernatant to the extraction column and draw through by centrifugation or suction, discard effluent.
  • Draw 2 x 1 ml Wash Buffer 1 through the column by centrifugation or suction, discard effluents.
  • Draw 75 µl Wash Buffer 2 through column by centrifugation or suction, discard effluent.
  • Elute 25-OH-Vitamin D3 and D2 with 200 µl Elution Buffer and dilute with 20 µl dist. water.
  • Inject 25 µl eluate into the HPLC system.

Sample Stability

At ambient temperature up to 3 days, at +2 to +8 °C up to 1 week. It is recommended to keep samples cool for transport. Freeze for longer storage (up to 1 year) at -20 °C.

Additional Info

Isocratic HPLC system with UV detector.

Injection Volume

25 µl (10-50 µl)

Flow rate

0.7 ml/min

Column temperature

approx. 25 °C


265 nm

Injection interval

12 min

Autosampler rinsing solution

Water:acetonitrile = 50:50

Please note

The freely available information on this website, in particular on the sample preparation, are not sufficient to work with our products. Please read instructions and warning notices on products and/or instruction manuals.

Kit Components

Components available separately

Mobile Phase

Order no.: 38031

Internal Standard

Order no.: 38004

Precipitation Reagent

Order no.: 38005

Wash Buffer 1

Order no.: 38006

Wash Buffer 2

Order no.: 38007

Elution Buffer

Order no.: 38009

Sample Clean Up Columns

Order no.: 38008

Reaction vials

Order no.: 33005



Controls available separately