Overview
EN ISO 9936:2016 (ISO 9936:2016) specifies an HPLC method for the determination of free tocopherol and tocotrienol contents (collectively tocols) in animal and vegetable fats and oils. The method covers identification and quantification of α-, β-, γ- and δ-tocopherols and tocotrienols by high-performance liquid chromatography (HPLC). Milk and milk products (and fats derived from them) are explicitly excluded. For samples containing tocopherol/tocotrienol esters a preliminary saponification step is required; a cold saponification procedure is provided in Annex B for information.
Key topics and technical requirements
- Analyte scope: Free α-, β-, γ-, δ-tocopherols and tocotrienols (tocol content reported in mg/kg).
- Sample preparation: Dissolution of test portion in n‑heptane; saponification required for esterified tocols.
- Chromatography:
- Preferred detectors: fluorescence (excitation 295 nm, emission 330 nm). UV detection at 292 nm is permitted but not recommended.
- Column options: 250 mm × 4 mm diol column (≈5 µm) or 250 mm × 4.6 mm silica column (≈5 µm).
- Mobile phase examples: 3.85% (v/v) tetrahydrofuran (THF) in n‑heptane is described; alternative solvent mixtures (e.g., t‑butyl methyl ether + n‑heptane) can improve separation of γ‑tocopherol and β‑tocotrienol.
- Calibration and standards:
- Use HPLC‑grade reagents and defined tocopherol/tocotrienol standards.
- If pure standards are unavailable, reference oils (e.g., wheat germ, soya, palm oil) can aid peak identification.
- Purity of commercial standards can vary; the standard advises verifying calibration solution concentration (e.g., by UV spectrometry).
- Data and reporting:
- Calculation of individual tocol contents using calibration factors.
- Precision, repeatability and reproducibility are addressed, with interlaboratory test results provided (Annex C).
- Supporting material: Annex A (example chromatograms), Annex B (saponification procedure), Annex C (interlaboratory results).
Applications and typical users
- Food and oil laboratories performing quality control and compositional analysis of edible oils, vegetable oils, animal fats, and oilseed products.
- Oilseed processors and edible oil manufacturers monitoring natural vitamin E (tocol) content for nutrition labeling, product development and shelf‑life studies.
- Research and regulatory laboratories involved in nutritional analysis, authenticity testing or compliance with food safety standards.
- Suppliers of standards, HPLC column manufacturers and instrument vendors referencing method requirements for method validation and instrument configuration.
Related standards
This standard is essential for accurate, reproducible tocopherol and tocotrienol analysis by HPLC in fats and oils and supports consistent vitamin E quantification across laboratories.