Overview
EN ISO 13904:2016 - "Animal feeding stuffs - Determination of tryptophan content" is a CEN-endorsed ISO method for measuring total and free tryptophan in animal feeds. The standard covers complete and complementary feeds, supplements, raw materials, ingredients and concentrates, and it also addresses free tryptophan in commercial pure substances and premixtures containing more than 2 % tryptophan. The method does not distinguish between D‑ and L‑forms of tryptophan.
Key topics and technical requirements
- Scope: Determination of total and free tryptophan in feeding stuffs; free tryptophan in pure products/premixtures >2 %.
- Principle:
- Total tryptophan: alkaline hydrolysis with saturated barium hydroxide and heating to 110 °C for 20 h; internal standard added after hydrolysis.
- Free tryptophan: mild acidic extraction with internal standard (in some pure products the internal standard can be added post‑extraction).
- Quantification by reversed‑phase C18 HPLC with fluorescence detection.
- Standards and reagents:
- Tryptophan reference substance and control (purity ≥ 99 %).
- α‑methyltryptophan as internal standard (purity ≥ 99 %).
- Key reagents: barium hydroxide octahydrate, sodium hydroxide, orthophosphoric and hydrochloric acids, HPLC‑grade methanol.
- Calibration and controls: Preparation of concentrated standard and internal standard solutions; calibration solutions for feeding stuffs and pure products; routine control of standard purity (noted in the standard).
- Performance: The document includes sections on precision, repeatability, reproducibility and results from interlaboratory tests, plus informative annexes on observations and sample preparation.
Applications
- Quality control and routine analysis in feed laboratories to verify nutritional composition and label claims.
- Raw‑material testing for feed manufacturers and premix producers.
- Regulatory compliance testing by governmental and certification bodies monitoring feed safety and nutrient content.
- Research laboratories studying amino acid content or processing effects on tryptophan availability.
Who should use this standard
- Feed testing laboratories and contract analytical services
- Feed manufacturers, premix formulators and ingredient suppliers
- Regulatory agencies and standardization organizations
- Academic and industry researchers working on animal nutrition and feed composition
Related standards
- Supersedes EN ISO 13904:2005; developed by ISO/TC 34 (Food products) in collaboration with CEN/TC 327 (Animal feeding stuffs - Methods of sampling and analysis).
Keywords: EN ISO 13904:2016, tryptophan determination, animal feeding stuffs, HPLC fluorescence, alkaline hydrolysis, α‑methyltryptophan, feed analysis, total and free tryptophan.