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

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

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

Vitamin B1 (TPP, thiamine pyrophosphate)

Vitamin B6 (PLP, pyridoxal 5’-phosphate)

Clinical relevance

Vitamin B1 (thiamine) plays an important role in deriving energy from carbohydrates and fats. The active form is thiamine pyrophosphate (TPP), a coenzyme for some enzymatic reactions in carbohydrate metabolism and for pyruvate decarboxylase. TPP is also significantly involved in the oxidative degradation of glucose. The determination of the TPP concentration is more significant than measurements of total thiamine, as by this method only the physiologically active form is taken into account.

Vitamin B6 comprises pyridoxine, pyridoxamine and pyridoxal, as well as their phosphate derivatives. It is ingested with food and transferred via several enzymatic conversions into its active form, pyridoxal-5’-phosphate (PLP). PLP is involved in nearly one hundred different enzymatic reactions, among other things, as co-factor in amino acid metabolism, and in the formation of haemoglobin or neurotransmitters in the brain.

 

Product advantages

  • Specific internal standard for each parameter
  • No post-column derivatisation required
  • One sample preparation and one run for both parameters

 

This Chromsystems kit facilitates the combined analysis of vitamin B1 in whole blood and vitamin B6 in whole blood or plasma. The sample preparation can be processed from different matrices, so that both whole blood samples and plasma samples can easily be measured in the same run. Vitamin molecules are already derivatised during sample preparation, which renders the common post-column derivatisation unnecessary. The separation takes place with a binary gradient. The internal standard for each of the two parameters and special quality controls ensure precise and reliable quantification.

This method is also available with simplified sample preparation in pre-mixed tubes.

There is also a method using UHPLC as well as automated sample preparation for HPLC and UHPLC methods available for laboratories with a higher sample throughput.

More Information
Number of Tests 100
Limit of quantification

TPP 2.0 μg/l (whole blood)
PLP 4.5 μg/l (whole blood), 0.5 μg/l (plasma)

Linearity

PLP 500 μg/l
TPP 750 μg/l

Recovery ≤ 102 %
Intraassay

CV ≤ 2.3 %

Interassay

CV ≤ 5.2 %

Analysis Time 7–9 min
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 30 sec (vortex).
  • Centrifuge for 5 min at 14000 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 at 14000 x g.
  • Transfer supernatant into a light protected autosampler vial, inject 25-50 μl into the HPLC system.
Sample Stability The prepared sample is stable at room temperatur for 1 day, at +2 to +8 °C for 7 days. For longer storage (up to 2 weeks) keep samples below -18 °C.
Specimen Whole Blood/Plasma
Pre-analytic Treatment

The patient samples are stable at +2 to +8 °C for 1 week, light-protected in closed vial. For longer storage (up to 2 weeks) keep 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 binary
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.
Method of Analysis HPLC
Parameter Vitamin B1 (TPP, thiamine pyrophosphate), Vitamin B6 (PLP, pyridoxal 5’-phosphate)
Additional Info Every common HPLC system with binary pump and programmable fluorescence detector is suitable.
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