Overview
ISO 13903:2005 - Animal feeding stuffs - Determination of amino acids content - specifies laboratory procedures for measuring free (synthetic and natural) and total (peptide‑bound plus free) amino acids in animal feed using an amino acid analyser or HPLC with ion‑exchange separation and post‑column ninhydrin detection. The method covers 17 amino acids (including lysine, methionine, threonine, cystine+cysteine, alanine, arginine, glutamic and aspartic acids, leucine, isoleucine, valine, phenylalanine, tyrosine, proline, serine, histidine and glycine). It does not determine tryptophan or hydroxy‑analogues, nor distinguish salts or D/L stereoisomers.
Key Topics
- Scope: Determination of specified free and total amino acids in feeding stuffs (ISO 13903:2005).
- Principle:
- Free amino acids: extraction with dilute HCl, protein precipitation (5‑sulfosalicylic acid), pH adjustment and separation by ion‑exchange chromatography; detection with ninhydrin (photometric at 570 nm).
- Total amino acids: selective oxidation (performic acid/phenol) for cyst(e)ine and methionine when required, acid hydrolysis (6 mol/L HCl, typically 23 h), pH adjustment, ion‑exchange separation and ninhydrin detection (440 nm for proline).
- Sample preparation: grinding to 0.5 mm; drying or freeze‑drying for high‑moisture samples; defatting if high fat content.
- Instrumentation: amino acid analyser or HPLC with ion‑exchange column, stable pumping, post‑column ninhydrin derivatization and photometric detector.
- Reagents & calibration: use of citrate buffer (pH 2.20), internal standard (e.g., norleucine), cysteic acid and methionine sulfone standards for oxidized analyses.
- Performance: Limits of quantification depend on chromatographic equipment; typical achievable levels include 0.3 g/kg total lysine, 0.25 g/kg total methionine, 0.35 g/kg total cystine+cysteine, and low‑level free amino acid quantification down to ~0.03–0.035 g/kg for some analytes.
- Limitations: not valid for tryptophan or hydroxy analogues; no discrimination between salts or D/L forms.
Applications
- Quality control and formulation verification in feed manufacturing (e.g., ensuring essential amino acid content).
- Regulatory compliance testing and official control laboratories analyzing animal feeding stuffs.
- Research and nutritional studies assessing amino acid composition of feed ingredients and premixtures.
- Verification of synthetic amino acid supplementation and free amino acid levels in premixes.
Who Uses This Standard
- Feed manufacturers and QA/QC laboratories
- Regulatory and official control labs
- Agricultural research institutions and nutritionists
- Contract testing laboratories offering amino acid analysis by HPLC or amino acid analyzers
Related
- Prepared by ISO/TC 34 (Food products), Subcommittee SC 10 (Animal feeding stuffs). ISO 13903:2005 is based on Commission Directive 98/64/EC and complements other feed and food analytical standards on nutrient and contaminant determination.