Vitamin B2 in Whole Blood - HPLC

Order No.: 37000, for 100 tests
Flavin adenine dinucleotide (FAD), Flavin mononucleotide (FMN), Vitamin B2 (Riboflavin)

Safe results by stabilisation of FAD
Simultaneous determination of FMN
Highly stable calibrators and controls

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

Flavin adenine dinucleotide (FAD)

Flavin mononucleotide (FMN), Vitamin B2 (Riboflavin)

Clinical relevance

Vitamin B2 (riboflavin) belongs to the vitamin B complex, a heterogeneous group of water-soluble vitamins that are all precursors of coenzymes. The coenzymes flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are derived from riboflavin. In their capacity as a prosthetic group in flavoenzymes, they are responsible for the transfer of hydrogen in the respiratory chain. They also play a role in the metabolism of carbohydrates, fatty acids, and amino acids. In addition to its influence on other vitamins such as vitamin B6, riboflavin is essential for maintaining the glutathione reductase activity, thereby having an indirect antioxidative effect.

In Western Europe, a deficiency of riboflavin is observed particularly with vegans, smokers, diabetics and as a result of excessive consumption of alcohol. Typical signs of a vitamin B2 deficiency are poorly healing cracks at the corners of the mouth, inflammation of the oral mucosa and tongue, conjunctivitis and skin inflammation. A severe vitamin B2 deficiency may also promote the development of cataracts.


Product advantages

  • Reliable measurement results due to stabilisation of FAD
  • Simultaneous determination of riboflavin, FMN and FAD
  • Stable calibrators and controls


The present method developed by Chromsystems allows for the fast, simple and specific determination of riboflavin and its active metabolites FAD and FMN in whole blood using an isocratic HPLC system. Due to their native fluorescence, the analytes can be detected directly, without derivatisation. Sample preparation requires only one effective protein precipitation step and subsequent stabilisation of the analytes. A special HPLC column and an optimised mobile phase enable precise and reliable quantification of the analytes.

More Information
Method of Analysis HPLC
Number of Tests 100
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

FAD 3 µg/l
FMN 1 µg/l
Riboflavin 2 µg/l


FAD up to 1000 µg/l
FMN up to 500 µg/l
Riboflavin up to 500 µg/l

Recovery FAD 106 %, FMN 100 %, Rivoflavin 108 %

CV < 3 %


CV < 5.9 %

Analysis Time aproxx. 9 min
Specimen Whole Blood
Pre-analytic Treatment

The patient samples are stable at +2 to +8 °C for up to 1 day, light-protected in a closed vial. Samples not analysed immediately must be stored below -18 °C.

Sample Preparation
  • Pipette the following solutions into an amber reaction vial (light protected) and mix immediately for 30 s (vortex):
    200 μl whole blood (or reconstituted calibrator/control)
    + 200 μl Extraction Buffer
    + 400 μl Precipitation Reagent.

  • Centrifuge for 10 minutes at a minimum of 9000 x g.

  • Pipette 100 μl of Neutralisation Buffer into an amber autosampler vial (light protected), then add 500 μl supernatant and mix well (vortex).

  • Inject 50 μl of this mixture into the HPLC system.

Sample Stability The prepared samples are stable at ambient temperature for 24 hours and at +2 to +8 °C for 7 days, light-protected in a closed vial For longer storage (max. 2 weeks) keep samples below -18 °C.
Injection Volume 50 µl
Flow Rate 1.2 ml/min
Column Temperature ambient (~ 20 °C)

EX 465 nm

EM 525 nm

Gradient isocratic
Additional Info

Every isocratic HPLC system with fluorescence detector is suitable.

Parameter Flavin adenine dinucleotide (FAD), Flavin mononucleotide (FMN), Vitamin B2 (Riboflavin)
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