Biotin

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

Summary

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Golden standard

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

Method indicator

• Name
• Code

Scope

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.

Principle

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

Extraction

• 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.
  

HPLC

• 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.  

 

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Remarks

• 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

• Greenfield and Southgate discuss the criteria for analytical performance and quality of analytical data in their book.
  • Greenfield H, Southgate DAT. 2003. Food composition data - production, management and use. Elsevier Applied Science, London, UK.

•  link to precision data

Certified Reference Materials/Standard Reference Material

• link to detailed data

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.

 Literature

•  see separate child page below

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