25-OH-Vitamin D3/D2 in Serum/Plasma - LC-MS/MS

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

  • 25-OH-vitamin D2
  • 25-OH-vitamin D3

Order No.: 62000, 62000/1000
200 / 1000 Tests

Isotopically labelled internal standard
Multilevel calibrators
Online sample preparation
Upgrade available for the determination of 3-epi-25-OH-Vitamin D3/D2

* Required Fields


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

  • Isotopically labelled internal standard
  • Online sample preparation
  • 6PLUS1® Multilevel Calibrator Set
  • Upgrade available for the determination of epimeric forms


This Chromsystems kit allows the specific determination of 25-OH-Vitamin D3 and 25-OH-Vitamin D2 in serum and plasma with LC-MS/MS. The manual sample preparation is limited to simple and effective protein precipitation. After this, the analytes are concentrated using an online trap column and interfering matrix components are separated. The trap column is connected by a simple switching system to the HPLC column, which provides further purification of the analytes. This method is characterised by the use of atmospheric pressure chemical ionisation (APCI). Isotopically labelled internal standards assure a high level of precision and robustness of the method and minimise matrix effects ("ion suppression"). The 3PLUS1® and 6PLUS1® multilevel calibrators are optionally available and guarantee a high level of precision of the results.

An upgrade for the determination of the epimeric forms of 25-OH-Vitamin D3 and 25-OH-Vitamin D2 is also available for this kit (see accessories), as well as a variant for automated sample preparation in 96 well filter plates (order no. 620000/1000/F).


Kit content 62000                                 

Mobile Phase A, 1000 ml                     
Mobile Phase B, 1000 ml                      
Precipitation Reagent, 5 ml                 
Internal Standard, 40 ml                        
Rinsing Solution, 1000 ml                    
Reaction vials, 2 x 100 pcs.        


Kit content 62000/1000    

Mobile Phase A, 5 x 1000 ml                                                                   
Mobile Phase B, 5 x 1000 ml                                                                  
Precipitation Reagent, 5 x 5 ml                                                                
Internal Standard, 5 x 40 ml                                                                    
Rinsing Solution, 2 x 1000 ml                                                        
Reaction vials, 10 x 100 pcs.        
Analytical column, 1 pc.           
Trap column, 2 pcs.                   
3PLUS1® 25-OH-Vitamin D3/D2 Multilevel Serum  Calibrator Set (lyoph.), 4 x 1 ml            
MassCheck® 25-OH-Vitamin D3/D2 Serum Control, Level I (lyoph.), 5 x 1 ml
MassCheck® 25-OH-Vitamin D3/D2 Serum Control, Level II (lyoph.), 5 x 1 ml 


Technical Data

Method of Analysis



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

Analysis Time

5 min

Limit of quantification

25-OH-Vitamin D2: 2.2 µg/l
25-OH-Vitamin D3: 3.0 µg/l


up to 250 μg/l


CV < 5 %


CV < 6 %


The trap column and the analytical column are linked to the mass spectrometer through a 2-position 6-port valve.



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 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 1 week at +2 to +8 °C. For longer storage periods keep samples frozen below -18 °C.

Injection Volume

10 to 50 μl


Pump A: 0.02–1.00 ml/min
Pump B: 0.60–1.00 ml/min

Additional Info

HPLC parameters

Pump A delivers 100 % Mobile Phase A for loading the trap column.
Pump B delivers 100 % Mobile Phase A for separating analytes on the analytical column.


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.

Kit Components

Components available separately

Mobile Phase A

Order no.: 62001

Mobile Phase B

Order no.: 62002

Precipitation Reagent

Order no.: 62003

Internal Standard

Order no.: 62004

Rinsing Solution

Order no.: 62009

Reaction vials

Order no.: 3006



Controls available separately