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

Order No.: 52952/UHPLC, for 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

CE-IVD validated product ready for IVDR within timeframes and transition periods specified by the IVDR-2022 regulation

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

  • 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


This UHPLC assay 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 assays 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.


Content for 1000 analyses
Mobile Phase A, 2 x 1000 ml
Mobile Phase B, 1000 ml
UHPLC Column, 1 pc.
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.

More Information
Method of Analysis UHPLC
Number of Tests 1000
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.
Limit of Quantification PLP 5 μg/l (whole blood) TPP 5 μg/l (whole blood) PLP 1.4 μg/l (plasma)
Linearity Vit. B6 (PLP) 500 μg/l Vit. B1 (TPP) 750 μg/l
Recovery 91–106 %
Intraassay CV ≤ 3.8 %
Interassay CV ≤ 6 %
Analysis Time approx. 3 min
Specimen 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
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

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

Parameter Vitamin B1 (TPP, thiamine pyrophosphate), Vitamin B6 (PLP, pyridoxal 5’-phosphate)
Pressure minimum 450 bar
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