Vitamin B1 in Whole Blood and Vitamin B6 in Whole Blood/Plasma - HPLC

Order No.: 52052/RUO, for 100 tests
Parameters:
Vitamin B1 (TPP, thiamine pyrophosphate), Vitamin B6 (PLP, pyridoxal 5’-phosphate)

Each analyte safeguarded by a specific internal standard
No post-column derivatisation required
One sample preparation and one run for both analytes

Vitamin B1 (TPP, thiamine pyrophosphate)

Vitamin B6 (PLP, pyridoxal 5’-phosphate)

This Chromsystems reagent kit allows for the HPLC determination of thiamine pyrophosphate and pyridoxal-5’-phosphate, analogues of Vitamin B1 and Vitamin B6, in one run, with one sample preparation and without complex post-column derivatisation. Sample preparation is restricted to a single precipitation step and subsequent derivatisation in a reaction vial. The derivatised research sample is analysed using fluorescence detection with a wavelength switching, and the chromatographic separation is performed with 2 mobile phases in less than 9 minutes. The use of a tailor-made internal standard for each parameter ensures precise and accurate results.

More Information
Method of Analysis HPLC
Number of Tests 100
Analysis Time 7 - 9 min
Specimen Whole Blood and Whole Blood/Plasma
Sample Preparation
  • Mix 200 μl reserach sample/calibrator/control with 100 μl Internal Standard and 300 μl Precipitation Reagent in an amber vial (light protection) and shake 30 s (vortex).
  • Centrifuge for 5 min with at least 14000 x g.
  • Pipette 250 μl Neutralisation Reagent and 100 μl derivatisation mix into a new vial. Add 250 μl supernatant obtained above, mix briefly.
  • Incubate for 25 min at 60 °C (waterbath).
  • Cool sample for 10 min at +2 to +8 °C, then centrifuge for 2 min with at least 14000 x g.
  • Transfer supernatant into a light protected autosampler vial, inject 25–50 μl into the HPLC system.
Sample Stability Light protected the prepared research samples are stable up to 1 day at room temperature and up to 7 days at +2 to +8°C. For longer storage (maximum 2 weeks) deep freeze the prepared samples below -18°C.
Injection Volume 25-50 µl
Flow rate 1.5 - 2.3 ml/min
Column temperature ambient (~ 25 °C)
Wavelength

Start EX 320 nm, EM 415 nm
after approx. 3.8 min switch to EX 367 nm, EM 435 nm

Gradient

Gradient profile: specific binary gradients for whole blood and plasma

Additional Info

For the combined analysis of vitamins B1 and B6 in whole blood or plasma every common HPLC system with binary pump and programmable fluorescence detector is suitable.

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.
Parameter Vitamin B1 (TPP, thiamine pyrophosphate), Vitamin B6 (PLP, pyridoxal 5’-phosphate)