52952-UHPLC kit vitamin B1 vitamin B6 whole blood plasma

High Throughput UHPLC, combined Vitamin B1/B6 Analysis - UHPLC

Order no.: 52952/UHPLC
1000 Tests
  • Vitamin B1 (TPP, thiamine pyrophosphate)
  • Vitamin B6 (PLP, pyridoxal 5’-phosphate)

UHPLC separation in less than 3 min
Each parameter safeguarded by a specific internal standard


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

  • High throughput due to separation in less than 3 minutes
  • Reliable and reproducible results with a specific internal standard for each parameter
  • Easy sample preparation with Pre-mixed Tubes


The new UHPLC kit facilitates the combined analysis of vitamin B1 in whole blood and vitamin B6 in whole blood or plasma. Due to faster analysis times in less than 3 minutes, this method fulfils the requirements of clinical laboratories with higher sample throughput. The easy and efficient sample preparation consists of a single precipitation step and a final derivatisation in a reaction vial. The internal standard for each of the two parameters and special quality controls ensure precise and reliable quantification.

Chromsystems also offers two more kits that feature determination using HPLC: a standard version as well as one with pre-mixed tubes.

An automated method using HPLC is also available for laboratories with higher sample throughput.


Kit content

Mobile Phase A, 2 x 1000 ml
Mobile Phase B, 1000 ml
Whole Blood Calibration Standard (lyoph.), 5 x 5 x 1 ml
Internal Standard, 10 x 10 ml
Precipitation Reagent, 10 x 30 ml
Pre-mixed Neutralisation Tubes, 10 x 100 pcs.
Derivatisation Reagent 1 (lyoph.), 10 x 2 x 0.3 ml
Derivatisation Reagent 2 , 10 x 15 ml
Reaction vials, amber coloured (light protection), 10 x 100 pcs.
UHPLC column, 1 pc.


Technical Data

Method of Analysis


Number of Tests



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

Analysis Time

approx. 3 min

Limit of quantification

PLP 5 μg/l (whole blood) TPP 5 μg/l (whole blood) PLP 1.4 μg/l (plasma)


Vit. B6 (PLP) 500 μg/l Vit. B1 (TPP) 750 μg/l


91–106 %


CV ≤ 3.8 %


CV ≤ 6 %


Whole Blood/Plasma

Pre-analytic Treatment

The patient samples are stable at +2 to +8 °C for 1 week, light-protected in a closed vial. For longer storage (up to two weeks) keep samples below -18 °C.

Sample Preparation

  • Mix 200 μl whole blood/plasma with 100 μl Internal Standard and 300 μl Precipitation Reagent in an amber vial (light protection).
  • Shake 30 s (vortex).
  • Centrifuge 5 min at 14000 x g.
  • Add 100 μl Derivatisation Mix in a labelled Pre-mixed Neutralisation Tube and add 250 μl of the supernatant from step 1-3, mix briefly.
    Do not centrifuge the precipitate!
  • Incubate 25 min at 60° C (water bath).
  • Incubate for 10 min at +2 to +8 °C and centrifuge for 2 min at 14000 x g.
  • Transfer the supernatant into a light protected autosampler vial, inject 2.5–10 μl into the UHPLC system.

Sample Stability

The prepared samples are stable at room temperature for 1 day, at +2 to +8 °C for 7 days. For longer storage (up to 2 weeks) keep samples below -18 °C

Additional Info

Any major UHPLC system with binary pump and programmable fluorescence detector is suitable.

Injection Volume

2.5–10 μl

Flow rate

0.7 ml/min



Column temperature

ambient (~ 25°C)


Start EX 320 nm, EM 415 nm,

after approx. 1.6 min switch to EX 367 nm, EM 435 nm


minimum 450 bar

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


Calibrators available separately