Vitamin B6 in Serum/Plasma - HPLC

Order no.: 31000/S/RUO
100 Tests
  • Vitamin B6
  • Suitable for plasma and serum
  • Fast and easy sample preparation
  • Low cost per test

Description

The present reagent kit allows the rapid, easy and specific determination of vitamin B6 (pyridoxal-5'-phosphate, PLP) using a simple isocratic HPLC system. Instead of the usual, technically demanding post-column derivatisation, simple precolumn derivatisation is used. Plasma and serum research specimens (after addition of EDTA or heparin as anticoagulant) are suitable for this kit. The sample clean-up procedure includes an effective protein precipitation and extraction step with optimised conditions. Subsequent derivatisation (water bath, 60 °C) produces a fluorescent PLP-derivative. The chromatographic determination is run on an isocratic HPLC system with fluorescence detection. By this sample clean up procedure highly stable extracts are achieved, allowing the analysis of large batches.

 

Chromatogram

Technical Data

Method of Analysis

HPLC

Number of Tests

100

Parameter

Vitamin B6

Analysis Time

< 8 min

Column temperature

ambient (~ 25°C)

Flow rate

1.0–1.2 ml/min

Specimen

Plasma/Serum

Sample Preparation

  • To 200 μl of plasma/serum (calibrator, control, research specimen) add 300 μl Precipitation Reagent in a light protected vial, vortex-mix for at least 30 s.
  • Incubate for 10 min at +4°C, then centrifuge for 5 min at 16000 x g.
  • Transfer 250 μl supernatant into new light protected vial.
  • Add 250 μl Neutralisation Reagent, mix briefly (a precipitation will form, do not centrifuge!).
  • Add 100 μl Derivatisation Reagent (contains cyanide), mix briefly.
  • Incubate for 20 min at 60°C (water bath).
  • Cool research sample down in cold water, then incubate for 10 min at +4°C; centrifuge for 2 min at 16000 g.
  • Transfer supernatant into light protected autosampler vial, inject 25–50 μl into the HPLC system.

Sample Stability

Keep research samples cool and light protected for transport. They are stable up to to 7 days at +4°C. For longer storage keep frozen below -18°C.

Injection Volume

25–50 µl

Wavelength

EX 320 nm, EM 415 nm

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.: 31001/RUO

Mobile Phase, 10 x 1000 ml


Order no.: 31002/RUO

Precipitation Reagent


Order no.: 31004/RUO

Neutralisation Reagent


Order no.: 31005/RUO

Derivatisation Reagent


Order no.: 31006/RUO

Reaction vials


Order no.: 33005/RUO

Calibrators

Calibrators available separately

Controls

Controls available separately