38900-1000 Online Method HPLC Kit Vitamin D3 Vitamin D2 Serum Plasma

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

Order no.: 38900/1000
1000 Tests
  • 25-OH-vitamin D2
  • 25-OH-vitamin D3

Small sample volume: 100 µl
Online sample preparation
Reliable separation


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 D2 and D3
  • 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).



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.


Technical Data

Method of Analysis


Number of Tests



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

Analysis Time

10 min

Limit of quantification

5 µg/l


5 - 250 µg/l


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 %


The HPLC analysis of 25-OH-vitamin D3/D2 (online method) requires two HPLC pumps, a 6-port-valve and a UV detector.



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)


265 nm

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 A

Order no.: 38901

Mobile Phase B

Order no.: 38902

Internal Standard

Order no.: 38904

HPLC column

Order no.: 38910

Trap column

Order no.: 38920

PEEK Prefilter Housing

Order no.: 15010



Controls available separately