Overview
ISO 9936:2016 defines a validated HPLC method for determining the contents of free α-, β-, γ- and δ‑tocopherols and tocotrienols (collectively tocols) in animal and vegetable fats and oils. The standard specifies sample preparation, chromatography conditions, calibration and result expression for accurate quantification by high‑performance liquid chromatography (HPLC). Products that contain tocopherol/tocotrienol esters require preliminary saponification. Milk and milk products (and fats derived from them) are excluded. Annex B describes a cold saponification procedure for information.
Key topics and technical requirements
- Analytes covered: free α, β, γ, δ tocopherols and tocotrienols (tocols).
- Scope exclusions: milk and milk products.
- Sample prep: homogenization (see ISO 661) and protection from light; saponification required for esterified tocols (cold saponification method given in Annex B as informative).
- Chromatography: HPLC separation with recommended detectors and columns:
- Detector: fluorescence recommended (excitation 295 nm, emission 330 nm); UV at 292 nm is an alternative (less sensitive).
- Columns: diol column (250 × 4 mm) or silica column (250 × 4.6 mm), ~5 μm packing; diol column preferred when β‑tocotrienol is expected because it avoids co‑elution with γ‑tocopherol.
- Mobile phase examples: 3.85% tetrahydrofuran (THF) in n‑heptane; alternate solvent mixes (e.g., 5% t‑butyl methyl ether + 95% n‑heptane) for improved separation of specific peaks.
- Calibration and quantification: prepare stock calibration solutions (e.g., ~10 mg standard in 50 ml n‑heptane), verify concentration by UV spectrometry (absorbance 0.2–0.8) using published division factors for each tocopherol/tocotrienol, and prepare mixed working standards daily.
- Quality metrics: method includes precision data (repeatability, reproducibility) and interlaboratory test results (Annex C) to support method robustness.
Practical applications and users
ISO 9936:2016 is used for:
- Quality control and labeling verification of edible oils, fats and fat‑containing food ingredients.
- Regulatory and compliance testing where tocopherol/tocotrienol content (vitamin E activity and antioxidant levels) is relevant.
- R&D and formulation work in food, feed and nutraceutical industries monitoring natural antioxidant profiles.
Typical users: analytical laboratories, oil processors, food manufacturers, regulatory agencies and research institutions.
Related standards
- ISO 661 - Preparation of test sample (referenced normative).
- ISO 5555 - Recommended sampling for fats and oils (sampling guidance mentioned).
- ISO/TC 34 - Technical committee for food products, responsible for development.
Keywords: ISO 9936:2016, tocopherol, tocotrienol, tocols, HPLC, animal and vegetable fats and oils, saponification, fluorescence detector, chromatography, calibration.