Vitamin B1 (Thiamine Pyrophosphate) HPLC Assay kit:
For Research Use Only
Size: 100 tests
Incubation Time: 20 minutes
Sample Type: EDTA Whole Blood
Sample Size: 50 µL
Reference Values (Normal): 35 – 99 ng/ml
It is suggested that each laboratory establish its own normal ranges
The Eagle Biosciences Vitamin B1 (Thiamine Pyrophosphate) HPLC Assay Kit is intended for the quantitative determination of vitamin B1 (thiamine pyrophosphate or thiamine diphosphate) in EDTA-blood. The Vitamin B1 (Thiamine Pyrophosphate) HPLC Assay kit is for research use only and not to be used in diagnostic procedures.
For the determination of vitamin B1 a precipitation step to remove high molecular substances is performed first. After centrifugation the supernatant is mixed with a derivatization solution and incubated for 10 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 EDTA-blood calibrator; the concentration is calculated via integration of the peak heights respectively areas.
- Pipette into 1.5 ml reaction tubes: 50 µl sample, CAL or CTRL + 150 µl DIL +50 µl PREC
- Mix well. Leave the tubes for 10 minutes at 2-8°C and centrifuge afterwards at 10.000g for 10 minutes.
- Mix 150 µl supernatant + 50 µl REAC + 50 µl DERIVAT
- Incubate for 10 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.
- Inject 50 µl of the supernatant for chromatography into the HPLC-system. The supernatant is stable in the dark for 3 days at 2-8°C.
Vitamin B1 is a water soluble vitamin. It consists out of a pyrimidine and a thiazol ring, which are synthesized independently and linked afterwards. Vitamin B1 is de novo produced by many plants and microorganisms. Humans and animals have to take it up by nutrition. They are only able to do the phosphorylation reaction of the thiamin molecule.
Thiamine is found free and as mono-, di- and triphosphate. The different forms can be transferred into each other. The metabolic active form is thiamin diphosphate in the body and thiamin triphosphate in the brain. Thiamin diphosphate (TDP) works as thermostable coenzyme in many enzymes of the carbohydrate and amino acid metabolism. In most reactions TDP catalyzes the cleavage of C-C bonds. In the nervous system vitamin B1 is needed for the stimulation of nerve cells.
The classical disease of patients with a lack of vitamin B1 is Beriberi, which is known from regions where people eating predominantly white rice. The symptoms are paralysis, muscle dystrophia and heart failure. Other diseases are the Wernicke encephalopathy, the Korsakow syndrome and some forms of the Landry`s paralysis. In industrialized countries alcoholism seems to be the most important reason for a lack of vitamin B1. In alcoholics the thiamin resorption is disturbed. Due to a damage of the liver the storage capacity of vitamin B1 is reduced.
The Eagle Biosciences Vitamin B1 (Thiamine Diphosphate) HPLC Assay Kit 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 (IC2201rp) and the controls (IC2201ko). Both can be supplied by The 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 method
The measurement of serum and plasma samples is possible with the Vitamin B1 (Thiamine Pyrophosphate) HPLC Assay Kit but not recommended, because the concentration are most below the detection limit. Lipemic samples should not be measured.