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/RUO

Simultaneous analysis of
– 25-OH-D3, 3-epi-25-OH-D3
– 25-OH-D2, 3-epi-25-OH-D2
2 isotopically labelled internal standards
3PLUS1® Multilevel Calibrator Set and MassCheck® Controls
Online sample preparation

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


The upgrade to MassChrom® 25-OH-vitamin D3/D2 analysis allows for the separation of 25-Hydroxycholecalciferol and 25-Hydroxyergocalciferol from their epimeric forms 3-epi-25-OH-vitamin D3 and 3-epi-25-OH-vitamin D2, hence allowing for separate quantification by LC MS/MS. Manual sample preparation is still simple and effective protein precipitation. As with the 25-OH-vitamin D3/D2 determination, the analytes are enriched with the aid of a trap column ONLINE and thus, interfering matrix components are isolated. Chromatographic separation and further purification of all analytes is then performed by a special high resolution analytical column connected to the trap column by a valve. The method features the use of APCI (atmospheric pressure chemical ionisation) and the isotopically labelled internal standards adjusted to the epimeric forms. Matrix effects (“ion suppression”) are minimised and the performance of the method is optimised. The 3PLUS1® multilevel calibrators also include both epimeric forms of 25-OH-vitamin D3/D2 and ensure high performance of results.


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 min



Sample Preparation

  • Pipette 100 μl research sample/calibrator/MassCheck® control into a 1.5 ml 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.
  • Inject 10–50 μl of the supernatant into the HPLC-MS/MS system.

Sample Stability

The prepared research samples are stable up to 4 days at ambient temperature and up to 1 week at +2 to +8 °C. For longer storage (maximum 2 weeks) deep-freeze the research samples below -18 °C.

Additional Info

The reserach sample extracts are purified through a trap column and the analytes are subsequently separated on a high resolution analytical column. A binary gradient pump, a 2-position 6-way switching valve and a column oven are required. Through the use of an additional isocratic pump, the analysis time can be reduced to 8.5 min.

Injection Volume

10–50 μl (mass spectrometer dependent)

Column temperature

40 °C


APCI positive



Additional Info

LC-MS/MS parameter

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