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

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

Order no.: 52052/RUO
100 Tests
  • Vitamin B1
  • Vitamin B6

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

Description

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.

Chromatogram

Technical Data

Method of Analysis

HPLC

Number of Tests

100

Parameter

Vitamin B1, Vitamin B6

Analysis Time

7 - 9 min

Gradient

Gradient profile: specific binary gradients for whole blood and plasma

Column temperature

ambient (~ 25 °C)

Flow rate

1.5 - 2.3 ml/min

Specimen

Whole Blood and Whole Blood/Plasma

Sample Preparation

  • In a light protected vial mix 200 μl whole blood or plasma with 100 μl Internal Standard and 300 μl Precipitation Reagent, mix for at least 30 sec (vortex).

  • Centrifuge for 5 min with at least 9000 x g.

  • In a new light protected vial mix 250 μl Neutralisation Reagent and 100 μl Derivatisation Mix with 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 9000 x g.

  • Transfer supernatant into a light protected autosampler vial, inject 25–50 μl into the HPLC system.

Sample Stability

The research samples are stable at +2 to +8°C for 1day. For longer storage (up to 1week) deep freeze the research samples below -18°C (maximum 2 weeks).

Injection Volume

25-50 µl

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.

Wavelength

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


Order no.: 52001/RUO

Mobile Phase B


Order no.: 52022/RUO

Internal Standard


Order no.: 52044/RUO

Precipitation Reagent


Order no.: 52005/RUO

Neutralisation Reagent


Order no.: 52006/RUO

Derivatisation Reagent 1


Order no.: 52007/RUO

Derivatisation Reagent 2


Order no.: 52008/RUO

Reaction vials


Order no.: 33005/RUO

Calibrators

Calibrators available separately

Controls

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