Overview
ISO 14565:2000 specifies a validated analytical method for determining total vitamin A (retinol) in animal feeding stuffs and pet foods using high-performance liquid chromatography (HPLC). The method quantifies all-trans-retinol and cis‑isomers and expresses results in International Units per kilogram (IU/kg). It is designed for routine laboratory use where accurate vitamin A content measurement is required for quality control, compliance and research.
Key topics and technical requirements
- Scope & analyte: Total vitamin A (all‑trans‑retinol and cis‑isomers) in animal feeds and pet foods; results reported in IU/kg (1 IU vitamin A = 0.300 µg all‑trans‑retinol).
- Principle: Sample saponification with ethanolic KOH, extraction into light petroleum, evaporation, dissolution in 2‑propanol and quantification by reverse‑phase HPLC producing a single retinol peak.
- HPLC conditions & equipment highlights:
- Column: C18, 250 mm × 4.6 mm, 5–10 µm particles, ≥4 000 theoretical plates, k' ≈ 0.6 for all‑trans‑retinol.
- Mobile phase: methanol/water (770:30 by volume).
- Flow rate: 1.0 ml/min; injection volume: 10 µl.
- Detector: UV at 325 nm with integrator/data system.
- Sample handling: Protect retinol from light and air (use amber glassware), work quickly and complete assays within one working day; store extracts under inert gas at 4 °C if delayed.
- Calibration & standards: Hydrolysis of all‑trans‑retinyl acetate for calibration or use certified all‑trans‑retinol standards; spectrophotometric standardization at 325 nm using defined correction equations.
- Expression of results: Calculate IU/kg from retinol concentration in extract and sample mass.
- Precision: Interlaboratory precision data are provided (annex A) showing repeatability and reproducibility ranges across feed types.
Applications and users
- Feed and pet food quality control laboratories verifying label claims and nutritional composition.
- Manufacturers performing in‑house QC on premixes, concentrates and finished feeds.
- Regulatory agencies and testing bodies assessing compliance with vitamin A specifications.
- Research laboratories studying vitamin stability, bioavailability or formulation effects in feeds.
Related standards
- ISO 3696:1987 - Water for analytical laboratory use (reagent water quality).
- ISO 6498 - Preparation of test samples for animal feeding stuffs.
- Annex A of ISO 14565 contains interlaboratory precision data for typical feed matrices.
Keywords: ISO 14565:2000, vitamin A determination, retinol, HPLC, animal feeding stuffs, feed testing, pet food analysis, saponification, IU/kg, C18 column.