Vitamin B6 Whole Blood HPLC Assay


The Vitamin B6 Whole Blood HPLC Assay is intended for the quantitative determination of vitamin B6 in EDTA-blood. The Vitamin B6 HPLC Assay kit is for research use only and not to be used in diagnostic procedures.

Vitamin B6 Whole Blood HPLC Assay

The Vitamin B6 Whole Blood HPLC Assay is For Research Use Only

Size: 100 tests
Sensitivity: 2.5 U/mL
Dynamic Range: 10 – 270 U/mL
Incubation Time: 30 minutes
Sample Type: EDTA Whole Blood
Sample Size: 100 µL
Reference Values (Normal):
EDTA blood:    8.6 – 27.2 ng/ml
G.J. den Ottolander, Diagnostisch Kompas1997, S.441
It is suggested that each laboratory establish its own normal ranges

Assay Background

The vitamins pyridoxin, pyridoxal and pyridoxamin and the appropriate phosphate products are summarized as vitamin B6. All forms can be transformed into the active form pyridoxal-5- phosphate. Vitamin B6 functions as a coenzyme and is essential for more than 50 reactions in the protein, carbohydrate and fatty acid metabolism thereby synthesizing, transforming or degrading amino acids. In protein metabolism vitamin B6 supports the resorption of amino acids and their transport into the cells. Furthermore vitamin B6 contributes to the synthesis of neurotransmitters and amine products (histamine).

Due to the fact that vitamin B6 contributes to a variety of different reactions lack of vitamin B6 results in various clinical pictures as muscle dystrophia, skin diseases, or disturbances of the nervous system. High risk groups for reduced vitamin B6 concentrations in serum are lactating women, women taking oral contraceptives with high amount of estrogen and chronic drinkers.  The Eagle Biosciences Vitamin B6 HPLC Assay makes it possible to determine the vitamin in an easy, fast and precise method. The kit includes all reagents in ready to use form for preparation and separation of the samples with exception of the columns (IC2100rp) and the controls (IC2101ko). Both can be supplied by Eagle Biosciences. Beside the complete test kits it is possible to order all components separately. Please request our single component price list.

Related Products

Vitamin B6 HPLC Assay Kit Control Set
Vitamin B6 HPLC Assay Kit Column
Vitamin B6 Microtiter Plate Assay

Additional Information

Assay Principle

For the determination of vitamin B6 a precipitation step to remove high molecular substances is performed first. After centrifugation the supernatant is mixed with a derivatisation solution and incubated for 20 min at 60°C. The fluorescent probe is then cooled (2-8°C), centrifuged and injected into the HPLC system. The isocratic separation via HPLC at 30°C lasts 10 minutes. The chromatograms are recorded by a fluorescence detector. The quantification is performed with the delivered plasma calibrator; the concentration is calculated via integration of the peak heights respectively areas.

Assay Procedure

  1. Pipette into 1.5 ml reaction tubes:  100 µl sample, CAL or CTRL + 150 µl PREC
  2. Mix well. Leave the tubes for 10 minutes at 2-8°C and centrifuge afterwards at 10.000g for 2 minutes.
  3. Mix 100 µl supernatant + 250 µl DERIVAT
  4. Incubate for 20 minutes at 60°C on a shaker or in a water bath; cool to 2-8°C and centrifuge at 10.000g for 5 minutes.
  5. Inject 20 µl of the supernatant for chromatography into the HPLC-system.  The supernatant is stable in the dark for 5 days at 2-8°C.

Limitations of the Assay

For the measurement of serum and plasma samples, use the Vitamin B6 HPLC Assay Kit from Eagle Biosciences VB631-H100.

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