Overview
ISO 6867:2000 specifies a validated analytical method for the determination of vitamin E (DL-α‑tocopherol) in animal feeding stuffs and pet foods using high-performance liquid chromatography (HPLC). The standard describes sample preparation, saponification, extraction, chromatographic separation, detection and calculation of results, and includes precision data from interlaboratory tests.
Key topics and technical requirements
- Analyte and scope: Quantification of DL-α‑tocopherol in animal feeds and pet foods.
- Sample preparation: Grind and homogenize samples (see ISO 6498 for test-sample preparation), then saponify a test portion with ethanolic potassium hydroxide and sodium ascorbate to protect the vitamin.
- Extraction and cleanup: Extract the unsaponifiable fraction into light petroleum (boiling range 40–60 °C), dry over anhydrous sodium sulfate, evaporate under vacuum (≤40 °C) and reconstitute residue in hexane.
- Chromatography (normal-phase guidance):
- Column: silica, 250 mm × 4.6 mm, 5–10 µm; ≥5 000 theoretical plates recommended.
- Mobile phase: hexane : 1,4‑dioxan (970 : 30 v/v) (filter before use).
- Flow rate: 1.5 ml/min.
- Detector: fluorescence (excitation ~295 nm; emission ~330 nm). Reverse‑phase may be used if equivalent separation is achieved.
- Calibration and standards: Use DL-α‑tocopherol standard (≥96% purity) with single‑point or multi‑point calibration (2–10 µg/ml range suggested). The standard purity should be checked spectrophotometrically (UV λmax ≈ 292 nm).
- Result expression: Convert extract concentration (µg/ml) to International Units per kg (IU/kg) using the formula in Clause 9; include a correction factor of 1.1 for DL‑α‑tocopherol acetate where applicable.
- Performance: Annex A reports interlaboratory precision (examples: mean contents 23–141 IU/kg with repeatability and reproducibility limits provided).
Applications and who uses it
- Feed testing laboratories performing routine vitamin analysis for quality control.
- Pet food and animal feed manufacturers for verification of label claims and batch release.
- Regulatory agencies and accreditation bodies assessing compliance with nutritional standards.
- Animal nutritionists and researchers evaluating feed formulations and stability of vitamin E in matrices.
- Practical benefits include reliable quantification of vitamin E in complex feed matrices, guidance for method validation and reproducibility across labs.
Related standards
- ISO 6498 - Preparation of test samples for animal feeding stuffs.
- ISO 3696 - Water for analytical laboratory use.
- ISO 5725 - Precision (used for interlaboratory study design and statistics).
Keywords: ISO 6867:2000, vitamin E determination, DL-α-tocopherol, animal feeding stuffs, pet foods, HPLC, saponification, fluorescence detection, feed analysis, calibration.