Vitamin B2 - Riboflavin

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B2 riboflaviini.JPG
Figure 1. Riboflavin, C17H20N4O6, Mw= 376,36 g/mol

Vitamin B2 occurs in foods as the free riboflavin, riboflavin-5'-phosphate (FMN) or flavin adenine dinucleotide (FAD).

Synonyms for vitamin B2:

  • riboflavin, lactoflavin, ovoflavin


Golden standard

EN 14152:2003 Foodstuffs - Determination of vitamin B2 by HPLC

  • corrigendum EN 14152:2003/AC (2005)
  • draft pfEN 14152:2012 under discussion (March 2012)

Method indicator

  • Name
  • Code


The European Standard EN 14152:2003 specifies a method for the determination of vitamin B2 in foods by high performance liquid chromatography (HPLC). The determination of B2 is carried out by measurement of riboflavin.

This method has been tested in two interlaboratory studies:

  • the first set of tested samples: milk powder and pig's liver 
    • vitamin B2 concentration: 1,45-10,68 mg/100g
  • the second set of tested samples: tube feeding solution, baby food, powdered milk, meal with fruits, yeast, cereal and chocolate powder 
    • vitamin B2 concentration: 0,21-87,1 mg/100 g


Sample pretreatments include acid hydrolysis and enzymatic dephosphorylation. Riboflavin in a sample solution is determined by HPLC using fluorometric detection.

Key steps


  • Sample is homogenised before extraction.
  • Extraction phase includes acid hydrolysis using dilute hydrochloric acid or sulfuric acid. Solution is heated during this phase.
    • heat treatment: autoclaving in 121 °C for 30 min or heating in 100 °C for 60 min
  • The pH of the sample solution should not be higher than pH=2,0.

Enzyme treatment

  • Riboflavin-5'-phosphate is added to the extract to check the enzyme activity. All riboflavin-5'-phosphate should be converted to riboflavin.
  • The dephosphorylation can depend on the sample matrix and on the enzyme used. Taka diastase is used currently.
  • The pH of the extract is adjusted to 4,0 when using taka diastase
    • Optimal pH, incubation time and incubation temperature should be checked for each enzyme used.
  • Enzymes may contain riboflavin and this should be considered while calculating the results.


  • Filtering and/or centrifugation may be necessary before HPLC analysis.
  • Liquid chromatographic system consisting a pump, an injector, a fluorescence detector (excitation wavelength e.g. 468 nm, emission wavelength e.g. 520 nm) and a data evaluation system.
  • Reversed phase column: particle size 3-10 µm, diameter 4,0-4,6 mm, length 100-250 mm.
  • Mobile phase: methanol and phosphate buffer containing tetraethlyammoniumchloride and sodium heptanesulfonate.
  • Also other chromatographic systems can be used if equivalent results are guaranteed.
  • The performance criteria is the baseline separation of the riboflavin from interferences.

Identification and detection

  • Riboflavin is detected fluorometrically (excitation wavelength e.g. 468 nm, emission wavelength e.g. 520 nm).
  • Identification of riboflavin is done by the comparison of the retention time obtained with the standard test solution to that of the sample test solution.
    • Identification can also be done by adding a small amount of the appropriate standard solution to the sample test solution.

Quantification and calculations

  • Appropriate volumes of the standard solution and the sample test solution are injected. Quantification is done by external standard method by comparing the integrated peak areas or peak heights to the corresponding values of the standard substance.
  • Linearity of the calibration should be checked.
  • The mass fraction of vitamin B2 in mg/100 g of the sample is calculated. The result of vitamin B2 is reported as riboflavin in mg/100 g.


  • Vitamin B2 is very sensitive to light, and exposure to light should be minimised for example by using brown glassware.
    • Vitamin B2 is also sensitive to alkaline conditions.
  • Prolonged heating should be avoided because of the losses caused.
  • The purity of riboflavin standard substance can vary, and therefore the concentration of the stock solution should be checked spectrophotometrically.
  • The EN standard describes alternative HPLC conditions.
  • Old data
    • HPLC
    • Fluorometry
    • Microbiological
    • These three methods give similar results (van den Berg, 1996)
  • Latest review
    • Blake CJ. 2007. Analytical procedures for water-soluble vitamins in foods and dietary supplements:a review. Analytical Bioanalytical Chemistry. 389:63-76
    • Chen P, Wolf WR. 2007. LC/UV/MS-MRM for the simultaneous determination of water-soluble vitamins in multi-vitamin dietary supplements. Anal Bioanal Chem387(7):2441-8 

Criteria for analytical performance and analytical quality control

Certified Reference Materials/Standard Reference Material

Proficiency testing schemes 

Here are listed some completed, on-going and/or upcoming proficiency testing schemes concerning vitamin B2.

DRRR - Deutsches Referenzbüro für Lebensmittel-Ringversuche und Referenzmaterialien

API - American Proficiency Institute


PTA - Proficiency Testing Australia: Report No. 651 Foor Proficiency Testing Program (Round 31) - Vitamins

Livsmedelsverket, National Food Agency of Sweden - Report 22 (2008) Vitamins in Foods - Round V-6

 Some upcoming proficiency testing schemes can be found in the EPTIS database.

Other methods available

  • AACC 86-70.01 Riboflavin---Fluorometric Method
  • AACC 86-72.01 Riboflavin---Microbiological Method
  • AOAC 940.33 Riboflavin (Vitamin B2) in Vitamin Preparations
    • Microbiological
  • AOAC 970.65 Riboflavin (Vitamin B2) in Foods and Vitamin Preparations
    • Fluorometric
  • AOAC 981.15 Riboflavin in Foods and Vitamin Preparations
    • Automated, fluorometric
  • AOAC 985.31 Riboflavin in Ready-To-Feed Milk-Based Infant Formula
    • Fluorometric


  •  see separate child page below

EuroFIR assistance to this method/guidelines