25-OH-Vitamin D3/D2 in Serum/Plasma - HPLC

Order No.: 38038, for 100 tests
Parameters:
25-OH-Vitamin D2, 25-OH-Vitamin D3


Vitamins D3 and D2 with one analysis
Improved separation
Quality controls and calibrator available

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
  • Reliable separation
  • Reliable quantification with a stable internal standard

 

This Chromsystems kit allows the simultaneous and reliable chromatographic determination of 25-OH-Vitamin D3 and 25-OH-Vitamin D2 on an isocratic HPLC system with UV detection. By means of efficient protein precipitation and selective solid phase extraction interfering components are removed. A non-physiological derivative of vitamin D is used as a stable internal standard to produce reliable quantification. This way, reproducible and reliable results can be produced at a low expenditure of cost and time.

Alternatively, for high sample throughput Chromsystems offers an online method with selective online solid phase extraction (order no. 38900/1000) as well as methods for analysis using mass spectrometry (order no. 62000).

More Information
Number of Tests 100
Limit of quantification

1.1 µg/l - 1.4 µg/l

Linearity up to at least 250 µg/l
Recovery 25-OH-Vitamin D3: 86 %; 25-OH-Vitamin D2: 87 %
Intraassay

25-OH-Vitamin D3: CV 3.0 %
25-OH-Vitamin D2: CV 1.8 %

Interassay

25-OH-Vitamin D3: CV 3.3 %
25-OH-Vitamin D2: CV 4.6 %

Analysis Time 10 min
Sample Preparation
  • Mix 500 µl serum/plasma with 50 µl Internal Standard in a light-protected reaction vial.
  • Add 500 µl Precipitation Reagent, vortex-mix for 20 s.
  • Incubate 10 min to +4 °C.
  • Centrifuge 5 min at 15 000 x g.
  • Apply complete supernatant to the extraction column and draw through by centrifugation or suction, discard effluent.
  • Draw 2 x 1 ml Wash Buffer 1 through the column by centrifugation or suction, discard effluents.
  • Draw 75 µl Wash Buffer 2 through column by centrifugation or suction, discard effluent.
  • Elute 25-OH-Vitamin D3 and D2 with 200 µl Elution Buffer and dilute with 20 µl dist. water.
  • Inject 25 µl eluate into the HPLC system.
Sample Stability At ambient temperature up to 3 days, at +2 to +8 °C up to 1 week. It is recommended to keep samples cool for transport. Freeze for longer storage (up to 1 year) at -20 °C.
Specimen Serum/Plasma
Injection Volume 25 µl (10-50 µl)
Flow rate 0.7 ml/min
Column temperature approx. 25 °C
Wavelength 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.
Method of Analysis HPLC
Parameter 25-OH-Vitamin D2, 25-OH-Vitamin D3
Complete Specification
Linearity: up to at least 250 µg/l
Limit of quantification: D3: 1.4 µg/l
D2: 1.1 µg/l
Recovery: D3: 86 %
D2: 87 %
Intra-assay: CV 0.8-3.0 %
Inter-assay: CV 1.9-4.6 %
Injection cycle time: 12 min

HPLC parameters

Isocratic HPLC system with UV detector.

Injection volume: 25 µl (10-50 µl)
Flow rate: 0.7 ml/min
Wavelength: 265 nm
Column temperature: aproxx. 25 °C
Autosampler rinsing solution: water:acetonitrile = 50:50
Additional Info Isocratic HPLC system with UV detector.
Injection interval 12 min
Autosampler rinsing solution Water:acetonitrile = 50:50
  1. 38031 HPLC mobile phase vitamin D3 vitamin D2 serum plasma
    Mobile Phase
    Order no.: 38031
    25-OH-Vitamin D3/D2 in Serum/Plasma - HPLC
    Details
  2. 38031 HPLC mobile phase vitamin D3 vitamin D2 serum plasma
    Mobile Phase, 10 x 1000 ml
    Order no.: 38032
    25-OH-Vitamin D3/D2 in Serum/Plasma - HPLC
    Details
  3. 38033 HPLC serum calibration standard vitamin D3 vitamin D2
    Vitamin D3/D2 Serum Calibration Standard
    Order no.: 38033
    25-OH-Vitamin D3/D2 in Serum/Plasma - HPLC
    Details
  4. 38004 HPLC internal standard vitamin D3 vitamin D2 serum plasma
    Internal Standard
    Order no.: 38004
    25-OH-Vitamin D3/D2 in Serum/Plasma - HPLC
    Details
  5. 38005 HPLC precipitation reagent vitamin D3 vitamin D2 serum plasma
    Precipitation Reagent
    Order no.: 38005
    25-OH-Vitamin D3/D2 in Serum/Plasma - HPLC
    Details
  6. 38006 HPLC wash buffer vitamin D3 vitamin D2 serum plasma
    Wash Buffer 1
    Order no.: 38006
    25-OH-Vitamin D3/D2 in Serum/Plasma - HPLC
    Details
  7. 38007 HPLC wash buffer vitamin D3 vitamin D2 serum plasma
    Wash Buffer 2
    Order no.: 38007
    25-OH-Vitamin D3/D2 in Serum/Plasma - HPLC
    Details
  8. 38009 HPLC elution buffer vitamin D3 vitamin D2 serum plasma
    Elution Buffer
    Order no.: 38009
    25-OH-Vitamin D3/D2 in Serum/Plasma - HPLC
    Details
  9. 38008 HPLC sample clean up columns vitamin D3 vitamin D2 serum plasma
    Sample Clean Up Columns
    Order no.: 38008
    25-OH-Vitamin D3/D2 in Serum/Plasma - HPLC
    Details
  10. Reaction Vials, 1.5 ml
    Reaction Vials, 1.5 ml
    Order no.: 33005
    Amber colour, light protected
    Details
  1. 38033 HPLC serum calibration standard vitamin D3 vitamin D2
    Vitamin D3/D2 Serum Calibration Standard
    Order no.: 38033
    25-OH-Vitamin D3/D2 in Serum/Plasma - HPLC
    Details
  2. 62028 3PLUS1 Calibrator Vitamin D
    3PLUS1® Multilevel Serum Calibrator Set 25-OH-Vitamin D3/D2
    Order no.: 62028

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

    Details
  1. 0028 0029 0030 HPLC 25-OH-vitamin D3 25-OH-vitamin D2 serum controls
    25-OH-Vitamin D3/D2 Serum Controls
    Order no.: 0028/0029/0030
    Details
  1. PEEK-encased Prefilter, 2 µm
    Details
  2. 38130 HPLC column vitamin D3 vitamin D2 serum plasm
    HPLC column
    Order no.: 38130
    25-OH-Vitamin D3/D2 in Serum/Plasma - HPLC
    Details
  3. PEEK Prefilter Housing
    PEEK Prefilter Housing
    Order no.: 15010
    Details
  4. Precolumn Cartridge Holder 4/10
    Details
  5. Precolumn cartridge 4/10
    Precolumn cartridge 4/10
    Order no.: 18038
    25-OH-Vitamin D3/D2 in Serum/Plasma - HPLC
    Details
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