Upgrade for 3-epi-25-OH-Vitamin D3/D2 - LC-MS/MS

Upgrade for 3-epi-25-OH-Vitamin D3/D2 - LC-MS/MS

  • 25-OH-vitamin D2
  • 25-OH-vitamin D3
  • 3-epi-25-OH-vitamin D2
  • 3-epi-25-OH-vitamin D3

Upgrade for order no. 62000

MassChrom® 25-OH-Vitamin D3/D2 in Serum/Plasma

Product Name Qty
Internal Standard Epimers-Mix
Order no.: 62044
Analytical column (epimers)
Order no.: 62120
Trap column (epimers)
Order no.: 62110/Epi
Tuning Mix (epimers)
Order no.: 62016
3PLUS1® Multilevel Serum Calibrator Set (including both epimeric forms)
Order no.: 62029
MassCheck® 3-epi-25-OH-D3/D2 and 25-OH- D3/D2 Serum Control, Bi-Level (I + II)
Order no.: 0310


Clinical relevance

A significant part of 25-OH-Vitamin D3/D2 may occur in infants under the age of one year in the form of a so called C3-epimer. In the epimers, only the hydroxyl function in position 3 of the secosteroid skeleton is changed in its spatial orientation. As the biological activity of the epimeric forms differs from that of the non-epimeric forms, it is of considerable importance to distinguish between the concentrations of the various 25-OH-vitamin D forms. Thus, a significantly reduced calcaemic effect in bone mineralisation is expected for the epimeric forms.


Product advantages

  • Simultaneous measurement of vitamin D3, vitamin D2 and its C3-epimers
  • Isotopically labelled internal standard
  • 3PLUS1® Multilevel Calibrator Set with blank calibrator and MassCheck® controls


This upgrade to MassChrom® 25-OH-vitamin D3/D2 analysis allows the separation of the primary metabolites of vitamins D3 and D2, 25-hydroxycholecalciferol and 25-hydroxyergocalciferol, in combination with their epimeric forms 3-epi-25-OH-vitamin D3 and 3-epi-25-OH-vitamin D2, enabling their separate quantification using LC-MS/MS.

The manual sample preparation is limited to a simple and effective protein precipitation step. As in the determination of 25-OH-vitamin D3/D2, the analytes are concentrated using an online trap column and interfering matrix components are separated. The trap column is attached by an easy valve to a special high-resolution analytical column, which provides chromatographic separation and further purification of all analytes. This method is characterised by the use of atmospheric pressure chemical ionisation (APCI) and isotopically labelled internal standard modified for the epimeric forms. Matrix effects (“ion suppression”) are minimised and the precision and robustness of the method are optimised. The 3PLUS1® Multilevel Calibrator Set and the MassCheck® Controls also contain both epimeric forms of 25-OH-vitamin D3/D2, guaranteeing highly accurate results for all vitamin D-epimers.

This upgrade set is an enhancement of the standard method for the determination of 25-OH-vitamin D3/D2 (order no. 62000). Furthermore, a kit for automated sample preparation in 96 well filter plates (62000/1000/F) is also available for laboratories with higher sample throughput.


Technical Data

Method of Analysis



25-OH-vitamin D2, 25-OH-vitamin D3, 3-epi-25-OH-vitamin D2, 3-epi-25-OH-vitamin D3

Analysis Time

8.5–10 min

Limit of quantification

(3-epi-)25-OH-Vitamin D2: 1.0 μg/l (3-epi-)25-OH-Vitamin D3: 2.0 μg/l


up to 250 μg/l


CV < 5%


CV < 6%



Pre-analytic Treatment

Patient samples are stable up to 3 days at ambient temperature and up to 1 week at +2 to +8 °C. For longer storage (maximum 1 month) deep-freeze the samples below -18 °C.

Sample Preparation

  • Place 100 μl sample/calibrator/MassCheck® control into a reaction vial.
  • Add 25 μl Precipitation Reagent.
  • Add 200 μl Internal Standard and mix 20 s (vortex).
  • Incubate 10 min at +4 °C.
  • Centrifuge 5 min at 15000 x g.
  • Transfer 200 μl supernatant into an autosampler vial.

Sample Stability

Samples are stable up to 3 days at ambient temperature, or up to one week at +2 to +8°C. For longer storage period (up to one month) keep samples frozen below -18°C.

Injection Volume

10–50 μl

Column temperature

40 °C


APCI positive



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.


Calibrators available separately