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Figure 1. Biotin, C10H16N2O3S, Mw = 224,31 g/mol

Biotin occurs in foods as the free vitamin or bound to protein.

Synonyms for biotin:

  • vitamin B7, co-enzyme R, vitamin H


Golden standard

EN 15607:2009 Foodstuffs - Determination of d-biotin by HPLC

Method indicator

  • Name
  • Code


The European Standard EN 15607 specifies a method for the determination of the mass fraction of d-biotin in foodstuffs by high performance liquid chromatography (HPLC). This method can also be used for estimation of d-biocytin, but it has to be noted that the method has not been validated for the determination of d-biocytin.


Pretreatments include enzymatic treatment followed by extraction. Quantification is done by HPLC with post-column binding reaction. Post-column reaction of d-biotin with avidin protein that is bound to a fluorescent marker, provides a specific method.

Key steps


  • The sample should be homogenised prior to extraction.
  • Gluthatione solution, EDTA solution, citrate buffer solution and papain solution are added.
    • Use of taka-diastase may be needed if the sample contains high amounts of starch.
  • The sample solution is incubated overnight (37 °C).
  • The solution is filtered.


  • Liquid chromatographic system consisting of a pump, an injector, a fluorescence detector (excitation wavelength 490 nm and emission wavelength 520 nm) and a data evaluation system.
  • Reverse phase column: particle size 5 µm, diameter 4,0 mm, length 250 mm
  • Mobile phase: phosphate buffer (pH 6):methanol (8:2, v:v)
  • Also other chromatographic systems can be used if equivalent results are guaranteed.
  • The performance criteria is the baseline separation of d-biotin from interferences.
  • The system should include a post-column reactor derivatisation unit with a suitable reagent delivery system.  
    • d-biotin is derivatised with fluorescent avidin-fluorescein 5-isothiocyanate.

Identification and detection

  • D-biotin is detected fluorometrically (excitation wavelength 490 nm, emission wavelength 520 nm)
  • Identification of d-biotin 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 the standard solution to the sample test solution.

Quantification and calculations

  • Quantification is done by external calibration.
  • Peak areas or heights are integrated and second degree calibration curve is used.
  • The mass fraction of d-biotin in µg/100 g of the sample is calculated.  



  • Other methods for the determination of d-biotin:
    • microbiological method using Lactobacillus plantarum
    • radio-assays using specific binding protein
  • The method described in the standard EN 15607 can also be used for estimation of d-biocytin.

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 biotin:

PTA - Proficiency Testing Australia: Report No. 651, Food Proficiency Testing Program, Round 31 - Vitamins

DLA - Dienstleistung Lebensmittel Analytik GbR: 33/2012 Food Supplement II: Biotin, Vitamin C, Niacin and Pantothenic Acid

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

Other methods available


  •  see separate child page below

EuroFIR assistance to this method/guidelines