25-OH-Vitamin D3/D2 in Serum/Plasma (Online Method) - HPLC

Order No.: 38900/1000, for 1000 tests
25-OH-Vitamin D2, 25-OH-Vitamin D3

Small sample volume: 100 µl
Online sample preparation
Reliable separation

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 advantages

  • Combined analysis of vitamins D3 and D2
  • 100 µl sample volume is sufficient
  • Selective online sample preparation
  • Up to 100 samples per day


This Chromsystems kit allows simple and fast determination of 25-OH-Vitamin D3 and 25-OH-Vitamin D2 in a single HPLC run with UV detection. Following an efficient protein precipitation step, selective online solid phase extraction enables up to 100 analyses per day. Requiring only small sample volume, this analysis is also suitable for measuring paediatric samples. A stable internal standard ensures reliable quantification. This allows reproducible and reliable results while keeping costs and required laboratory time low.

Alternatively, there is also a standard method for HPLC (order no. 38038) as well as methods for determination using mass spectrometry (order no. 62000).


Content for 1000 tests
Mobile Phase A (online method), 10 x 1000 ml
Mobile Phase B (online method), 10 x 1000 ml
Internal Standard (online method), 10 x 25 ml
25-OH-Vitamin D3/D2 Serum Calibration Standard (lyoph.), 2 x 2 ml
25-OH-Vitamin D3/D2 Serum Control, Level I, 2 x 2 ml
25-OH-Vitamin D3/D2 Serum Control, Level II, 2 x 2 ml
HPLC Column, 1 pc.
Trap Column, 1 pc.
PEEK-encased Prefilter, 2 μm, 5 pcs.
Reaction Vials, transparent, 100 pcs.

More Information
Method of Analysis HPLC
Number of Tests 1000
Limit of quantification 5 µg/l
Linearity 5 - 250 µg/l
Recovery 25-OH-Vitamin D3: 99 %; 25-OH-Vitamin D2: 93 %

25-OH-Vitamin D3: CV ≤ 3.2 %
25-OH-Vitamin D2: CV ≤ 4.1 %


25-OH-Vitamin D3: CV ≤ 5.6 %
25-OH-Vitamin D2: CV ≤ 5.7 %

Analysis Time 10 min
Specimen Serum/Plasma
Sample Preparation
  • Mix 100 µl serum/plasma with 250 µl Internal Standard in a reaction vial for 20 s (vortex).
  • Cool down 10 min at +4 °C.
  • Centrifuge 5 min at 15 000 x g.
  • Inject 75 µl of the supernatant into the HPLC system.
Sample Stability At ambient temperature: 3 days, at +2 to +8 °C: 1 week and below -18 °C: 1 year. Keep samples light protected and cool for transport.
Injection Volume 75 µl
Flow rate Mobile Phase A (trap column): 1.0 ml/min; Mobile Phase B (analytical column): 0.7 ml/min
Column temperature ambient (~25 °C)
Wavelength 265 nm

The HPLC analysis of 25-OH-vitamin D3/D2 (online method) requires two HPLC pumps, a 6-port-valve and a 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
As a customer please login or register to gain full access.