Vitamin E

Figure 1. Structures of tocopherols.

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

The group vitamin E includes eight compounds that have similar biological activity as ?-tocopherol. These compounds are ?-, ?-, ?- and ?-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 ?-tocopherol only ) . This definition has been adopted by many European food databases.

Synonyms for vitamin E:

• tocopherol, ephanyl

Summary

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Golden standard

• EN 12822:2000Foodstuffs - Determination of vitamin E by high performance liquid chromatography - Measurement of ?-, ?-, ?- and ?-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 ?-, ?-, ?- and ?-tocopherols.

Principle

Sample treatments usually include saponification and extraction. ?-, ?-, ?- and ?-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 ?- and ?-tocopherols, but ?- and ?-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 ?-, ?-, ?- or ?-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.

 

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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

• 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 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 ?-Tocopheryl acetate (supplemental) in foods and feeds
  • colorimetric
• AOAC 971.30?-Tocopherol and ?-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 989.09?-Tocopheryl acetate in supplemental vitamin E concentrates 
  • gas chromatographic
• AOAC 992.03 Vitamin E activity (all-rac-?-tocopherol) in milk-based infant formula
  • liquid chromatographic

 

 Literature

•  see separate child page below

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