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

Order No.: 38038, for 100 tests
25-OH-Vitamin D2, 25-OH-Vitamin D3

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

CE-IVD validated product ready for IVDR within timeframes and transition periods specified by the IVDR-2022 regulation

25-OH-Vitamin D2

25-OH-Vitamin D3

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 advantage

  • 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).

More Information
Method of Analysis HPLC
Number of Tests 100
Limit of quantification

1.1 µg/l - 1.4 µg/l

Linearity up to at least 250 µg/l
Recovery 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 %

Analysis Time 10 min
Specimen Serum/Plasma
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.
Injection Volume 25 µl (10-50 µl)
Flow rate 0.7 ml/min
Column temperature approx. 25 °C
Wavelength 265 nm
Additional Info Isocratic HPLC system with UV detector.
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.
Parameter 25-OH-Vitamin D2, 25-OH-Vitamin D3
Injection interval 12 min
Autosampler rinsing solution Water:acetonitrile = 50:50
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