Vitamin K1 HPLC Assay kit:
For Research Use Only
Size: 100 tests
Incubation Time: 10 minutes
Sample Type: Plasma, Serum
Sample Size: 1 mL
Reference Values (Normal): 0.5 – 5 ng/mL
It is suggested that each laboratory establish its own normal ranges
The Eagle Biosciences Vitamin K1 HPLC Assay Kit is intended for the quantitative determination of vitamin K1 in plasma and serum. The Vitamin K1 HPLC Assay is for research use only and not to be used in diagnostic procedures.
For the determination of vitamin K1 an internal standard is added first. After a solid phase extraction on SPE-cartridges, the samples are precipitated. The supernatant is then extracted with an organic solvent and evaporated. After that the samples are re-suspended and injected into the HPLC system. A post-column reduction reactor reduces vitamin K and enables the measurement of vitamin K with a fluorescence detector. The isocratic separation at 30°C using a “reverse phase” column lasts 20 minutes. The chromatograms are recorded by a fluorescence detector. The quantification is performed by the delivered plasma calibrator; the concentration is calculated by the internal standard method via integration of the peak areas resp. heights.
- The cartridge is rinsed with 3 ml methanol and 3 ml deionized water.
- Add 1ml sample, CAL or CTRL + 10 µl IS
- Pipette the mixture on the SPE cartridge. Let it soak through by vacuum and collect the break-through in a glass vial.
- Add 2 ml PREC , vortex for 1 min. and centrifuge for 5 min. at 10.000 x g.
- Pipette the supernatant in a fresh glass vial and add 4 ml of EXTR.
- Vortex for 2 min and centrifuge for 5 min at 3.500 x g.
- Transfer the upper layer in a new glass vial and evaporate to dryness. The dried sample is stable for 8 days at 4-8°C.
- Connect the post-column reduction reactor in the HPLC-system, as described above and wait for equilibration (30-45 min).
- Check the performance of the reactor by the injection of 100 µl of ISOP and determine the signal to noise ratio, which should be greater than 25.
- Re-suspend the dried sample in 150 µl ELU and inject 100 µl in the HPLC-system.
Vitamin K was first discovered by Dam in 1929. He fed chicken with ether extracted food and recognized deadly bleeding. Chicken supplemented with the ether extract normalized the symptoms. The hypothetic coagulation factor was called vitamin K. Vitamin K1 (phylloquinone) is synthesized by green plants and taken up by nutrition, whereas vitamin K2 (menaquinone) is produced by bacteria in the gut.
Vitamin K works as cofactor of an oxygen dependent carboxylase, which carboxylates specific glutamic acid residues in g-position. Because of that modification, blood clotting factors can be activated by cleavage. In bone metabolism the g-carboxylation enables osteocalcin to bind to hydroxyapatite of the bone matrix. Vitamin K can be stored only for a short time, whereby deficiency occur after a view days. A lack of vitamin K mainly manifests in clotting disorders. Beside this, disorders in bone metabolism caused by a deficient modification of osteocalcin have been reported.
The Eagle Biosciences Vitamin K1 HPLC Assay kit makes it possible to determine the vitamin in an easy, fast and precise method. The kit includes all reagents for the preparation and separation of the samples with exception of the columns (IC2400rp) and the controls (IC2400ko). Both can be supplied by Eagle Biosciences. Beside the complete test kit it is possible to order all components separately. Please request our single component price list.
Limitations of the Assay:
Strong hemolytic and lipemic samples should not be measured with the Vitamin K1 HPLC Assay kit.