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

Order No.: 38900/1000, for 1000 tests
Parameters:
25-OH-Vitamin D2, 25-OH-Vitamin D3

Small sample volume: 100 µl
Online sample preparation
Reliable separation

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 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).

 

Content

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.

More Information
Number of Tests 1000
Limit of quantification 5 µg/l
Linearity 5 - 250 µg/l
Recovery 25-OH-Vitamin D3: 99 %; 25-OH-Vitamin D2: 93 %
Intraassay

25-OH-Vitamin D3: CV ≤ 3.2 %
25-OH-Vitamin D2: CV ≤ 4.1 %

Interassay

25-OH-Vitamin D3: CV ≤ 5.6 %
25-OH-Vitamin D2: CV ≤ 5.7 %

Analysis Time 10 min
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.
Specimen Serum/Plasma
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)
Wavelength 265 nm
Information

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

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 250 µg/l
Limit of detection: 5 µg/l



Intra-assay: 25-OH-Vitamin D3: CV 3.2 %

25-OH-Vitamin D2: CV 4.1 %



Inter-assay: 25-OH-Vitamin D3: CV 5.6 %

25-OH-Vitamin D2: CV 5.7 %



Recovery: 25-OH-Vitamin D3: 99 %

25-OH-Vitamin D2: 93 %



Run time: 10 min

HPLC Parameters:

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

Injection volume: 75 µl
Flow rates: Mobile Phase A (trap column): 1.0 ml/min

Mobile Phase B (analytical column): 0.7 ml/min
Wavelength: 265 nm
Column temperature: ambient (~25 °C)
  1. 38901 HPLC mobile phase A vitamin D3 vitamin D2 serum plasma
    Mobile Phase A
    Order no.: 38901
    25-OH-Vitamin D3/D2 in Serum/Plasma (Online Method) - HPLC
    Details
  2. 38902 HPLC mobile phase B vitamin D3 vitamin D2 serum plasma
    Mobile Phase B
    Order no.: 38902
    25-OH-Vitamin D3/D2 in Serum/Plasma (Online Method) - HPLC
    Details
  3. 38904 HPLC internal standard vitamin D3 vitamin D2 serum plasma
    Internal Standard
    Order no.: 38904
    25-OH-Vitamin D3/D2 in Serum/Plasma (Online Method) - HPLC
    Details
  4. 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
  5. 38910 HPLC column vitamin D3 vitamin D2 serum plasma
    HPLC Column 25-OH-Vitamin D3/D2 in Serum/Plasma (Online Method)
    Order no.: 38910
    25-OH-Vitamin D3/D2 in Serum/Plasma (Online Method) - HPLC
    Details
  6. 38920 HPLC trap column vitamin D3 vitamin D2 serum plasma
    Trap column
    Order no.: 38920
    25-OH-Vitamin D3/D2 in Serum/Plasma (Online Method) - HPLC
    Details
  7. PEEK Prefilter Housing
    PEEK Prefilter Housing
    Order no.: 15010
    Details
  8. Reaction Vials, transparent, 1.5 ml
    Reaction Vials, transparent, 1.5 ml
    Order no.: 33006
    Transparent
    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
  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. PEEK Prefilter Housing
    PEEK Prefilter Housing
    Order no.: 15010
    Details
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