38038 HPLC Kit Vitamin D3 Vitamin D2 Serum Plasma

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

Order no.: 38038
100 Tests
  • 25-OH-vitamin D2
  • 25-OH-vitamin D3


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

Description

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).

Chromatogram

Technical Data

Method of Analysis

HPLC

Number of Tests

100

Parameter

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

Analysis Time

10 min

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 %

Column temperature

approx. 25 °C

Flow rate

0.7 ml/min

Specimen

Serum/Plasma

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.
  • Cool down 10 min to +4 °C.
  • Centrifuge 5 min at 13 000 rpm.
  • 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.

Injection Volume

25 µl (10-50 µl)

Injection interval

12 min

Additional Info

Isocratic HPLC system with UV detector.

Wavelength

265 nm

Autosampler rinsing solution

Water:acetonitrile = 50:50

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


Order no.: 38031

Internal Standard


Order no.: 38004

Precipitation Reagent


Order no.: 38005

Wash Buffer 1


Order no.: 38006

Wash Buffer 2


Order no.: 38007

Elution Buffer


Order no.: 38009

Sample Clean Up Columns


Order no.: 38008

Reaction vials


Order no.: 33005

Calibrators

Controls

Controls available separately

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