Determination of vitamin B6 by HPLC

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Golden standard

EN 14164:2008 Foodstuffs - Determination of vitamin B6 by HPLC

  • draft pfEN 14164:2012 under discussion (March 2012)


Method indicator

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The European Standard EN 14164:2008 specifies a method for the determination of vitamin B6 in foodstuffs by high performance liquid chromatography (HPLC). Vitamin B6 is the mass fraction of the sum of pyridoxine, pyridoxal and pyridoxamine including their phosphorylated derivatives determined as pyridoxine. This standard EN 14164:2008 does not take glycosylated forms into account, but these forms of pyridoxine can be determined with the method given in EN 14663:2005.


Sample pretreatments include acid hydrolysis and enzymatic dephosphorylation. Pyridoxamine is transformed to pyridoxal by reaction with glyoxylic acid, and pyridoxal is reduced to pyridoxine. The reduction is carried out in alkaline medium by adding sodium borohydride. The pyridoxine is then quantified by HPLC using fluorometric detection.

Key steps


  • The sample must be homogenised before extraction.
  • Acid hydrolysis is carried out by adding hydrochloric acid and heating in boiling water bath for 30 min.
  • Also sample treatment without acid hydrolysis phase is described.

Enzyme treatment and transformation

  • The pH is adjusted to 4,5 before addition of glyoxylic acid solution, ferrous sulfate solution and phosphatase solution.
    • Reaction with glyoxylic acid catalysed by Fe2+ transforms pyridoxamine to pyridoxal
  • Sodium borohydride solution is added to the sample solution after incubation.
    • Pyridoxal is transformed to pyridoxine by reaction with sodium borohydride.
  • The sample solution is filtered.


  • Liquid chromatographic system consisting of a pump, an inejctor, a fluorescence detector (excitation wavelength 290 nm, emission wavelength 395 nm) and a data evaluation system.
  • Reversed phase column: particle size 5 µm, diameter 4,0 mm, length 250 mm.
  • Other particle sizes and column dimensions may be used if equivalent results can be guaranteed.
  • Mobile phase: acetonitrile in water with sodium octanesulfonate or sodium heptanesulfonate, and potassium dihydrogen phosphate.
  • Also other chromatographic systems can be used if equivalent results are guaranteed.
  • The performance criteria is the baseline separation of the analytes from interferences.

Identification and detection

  • Pyridoxine is detected fluorometrically (excitation wavelength 290 nm, emission wavelength 395 nm).
  • Identification of pyridoxine 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 B6 in mg/100 g of the sample is calculated. The result of vitamin B6 is reported as pyridoxine in mg/100 g.


  • Use of taka diastase instead of acid phosphatase does not obtain complete hydrolysis of phosphorylated forms of vitamin B6, thus acid phosphatase must be used.
  • Vitamin B6 is sensitive to light, heat and high pH.
  • The EN standard describes also pretreatments without acid hydrolysis.
  • There is an alternative to the conversion phase, although this is not described in the EN 14164:2008.
    • All the vitamers may be quantified separately and these are calculated as pyridoxine and summed to represent the total vitamin B6 concentration.
  • Latest review:
    • Blake, C.J.,(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

EuroFIR assistance to this method/guidelines