Vitamin E

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Tocopherols.jpg
Figure 1. Structures of tocopherols.

α-tocopherol: C29H50O2, Mw = 430,71 g/mol.

The group vitamin E includes eight compounds that have similar biological activity as α-tocopherol. These compounds are α-, β-, γ- and δ-tocopherols and the corresponding tocotrienols. The measurement of vitamin E is carried out by determination of tocopherols, and vitamin E activity can be calculated taking the activity of each tocopherol into account.

Note:

USDA Food Composition Database for Standard Reference, Release 25 defines vitamin E as α-tocopherol only ) . This definition has been adopted by many European food databases.

Synonyms for vitamin E:

  • tocopherol, ephanyl

Summary



Golden standard

  • EN 12822:2000Foodstuffs - Determination of vitamin E by high performance liquid chromatography - Measurement of α-, β-, γ- and δ-tocopherols.
    • draft prEN 12822:2012

Method indicator

  • Name
  • Code

Scope

The European Standard EN 12822 describes a method for the determination of vitamin E in foodstuffs by high performance liquid chromatography (HPLC). Vitamin E content is determined by measurement of α-, β-, γ- and δ-tocopherols.

Principle

Sample treatments usually include saponification and extraction. α-, β-, γ- and δ-tocopherols are determined by HPLC using fluorometric detection. Also photometric detection (UV-range) can be used but fluorometric detection is preferred.

Key steps

General

  • The sample treatments described below are meant for other samples than oil and fat samples (low water content) containing unesterified tocopherols. 
  • For oil and fat samples with unesterified tocopherols, the following pretreatments are used:
    • n-hexane is added to dissolve the test portion.
    • Solution is diluted prior to chromatography.  
    • Only normal phase chromatography should be used for determination of tocopherols in these oil and fat samples.

Saponification

  • The sample is homogenised prior to saponification.
  • Ethanol or methanol, water, an antioxidant and an aqueous potassium hydroxide are added.
  • Saponification includes heat treatment.
    • 80-100 °C, 15-40 min

Extraction

  • The ratio of alcohol to water should be 1:1 before extraction to avoid emulsions.
  • Extraction is carried out using suitable solvent.
    • Recovery should be checked to exclude the possibility of losses.
  • The extract is washed with water.
  • Solvent is evaporated and sample is redissolved using e.g. mobile phase.

HPLC

  • Liquid chromatographic system consisting of a pump, an injector, a fluorescence detector (excitation wavelength 295 nm and emission wavelength 330 nm) and data evaluation system.
    • Also UV detector (292 nm) may be used.
  • Normal phase column: particle size 5 µm (silica phase), diameter e.g. 4,0-4,6 mm, length 100-250 mm. 
  • Mobile phase: e.g. 3 % 1,4-dioxane in n-hexane.
  • Also other chromatographic systems can be used if equivalent results are guaranteed.
    • Reverse phase columns can be used to quantify α- and δ-tocopherols, but β- and γ-tocopherols cannot be separated.
  • The performance criteria is the baseline separation of the analytes from interferences.

Identification and detection

  • Tocopherols are detected fluorometrically (excitation wavelength 295 nm and emission wavelength 330 nm) or using UV detector (292 nm).
  • Identification of tocopherols 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

  • External standard method is used for quantification.
    • Peak areas or peak heights of the samples are compared to the corresponding values for the standard substances.
  • The linearity of the calibration must be checked.
  • The mass fraction of α-, β-, γ- or δ-tocopherol is calculated and result is reported as mg/100 g of the sample. 
  • For calculation of vitamin E content, biological activities must be taken into account.


 



Remarks

  • The purity of standard substances can vary, thus it is necessary to check the concentration of calibration solution spectrometrically. 
  • Unnecessary exposure to light must be avoided during analysis.
  • Alternative HPLC conditions are described in the Standard.
  • Oldet methods
    • colorimetric
      • gives a total tocopherol content
      • forming complexes unstable
    • GLC

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 E:

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

DLA - Dienstleistung Lebensmittel Analytik GbR: 32/2012 Food Supplement I: Vitamins A, D3, E, K1 and beta-Carotin

DGF - Deutsche Gesellschaft für Fettwissenschaft

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

Other methods available

  • AACC 86-06.01 Analysis of Vitamins A and E by High-Performance Liquid Chromatography
  • AOAC 948.26 α-Tocopheryl acetate (supplemental) in foods and feeds
    • colorimetric
  • AOAC 971.30α-Tocopherol and α-tocopheryl acetate in foods and feeds
    • colorimetric
  • AOAC 975.43Identification of RRR- or all-rac-alpha-tocopherol in drugs and food or feed supplements
    • polarimetric
  • AOAC 988.14 Tocopherol isomers in mixed tocopherols concentrate (FCC, USP, AOAC method)
    • gas chromatographic
  • AOAC 989.09α-Tocopheryl acetate in supplemental vitamin E concentrates 
    • gas chromatographic
  • AOAC 992.03 Vitamin E activity (all-rac-α-tocopherol) in milk-based infant formula
    • liquid chromatographic

 

 Literature

  •  see separate child page below

EuroFIR assistance to this method/guidelines