Overview
EN ISO 13903:2005 - "Animal feeding stuffs - Determination of amino acids content" (endorsed by CEN) specifies a laboratory method for measuring both free (synthetic and natural) and total (peptide‑bound + free) amino acids in animal feed. The procedure uses an amino acid analyser or HPLC with ion‑exchange chromatography and post‑column ninhydrin derivatization for photometric detection. It covers key amino acids routinely monitored in feed formulation and quality control.
Key topics and technical requirements
- Analytes covered: sum of cystine + cysteine, methionine, lysine, threonine, alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, phenylalanine, proline, serine, tyrosine, valine.
- Exclusions: method is not valid for tryptophan or hydroxy‑analogues of amino acids.
- Forms not differentiated: salts of amino acids and D/L stereoisomers are not distinguished.
- Principle (brief):
- Free amino acids extracted with dilute HCl; macromolecules precipitated with sulfosalicylic acid.
- Total amino acids: oxidation (performic acid/phenol) when required (e.g., cyst(e)ine, methionine), then acid hydrolysis (6 mol/L HCl, 23 h), neutralization to pH 2.20.
- Separation by ion‑exchange chromatography; detection by ninhydrin at 570 nm (440 nm for proline).
- Instrumentation & reagents: amino acid analyser or HPLC with ion‑exchange column, stable pumps, ninhydrin reagent, internal standard (e.g., norleucine), performic acid oxidation reagents and controlled hydrolysis.
- Limits of quantification (equipment dependent; typical achievable levels):
- Total lysine ≈ 0.3 g/kg; total methionine ≈ 0.25 g/kg; total cystine+cysteine ≈ 0.35 g/kg; total threonine ≈ 0.2 g/kg.
- Free amino acids: free lysine ≈ 0.035 g/kg; free methionine ≈ 0.035 g/kg; free threonine ≈ 0.03 g/kg.
- NOTE: lower detection limits may be achievable but must be validated by the user.
Applications
- Quality control of compound feeds, premixtures and raw materials to verify amino acid composition.
- Feed formulation and nutrition - ensuring required levels of limiting amino acids (lysine, methionine, threonine, cystine) for livestock performance.
- Regulatory compliance and labeling - verifying declared amino acid contents.
- Contract and research laboratories - routine analysis, interlaboratory studies, method development and validation.
Who uses this standard
- Feed manufacturers and nutritionists, analytical and contract testing laboratories, regulatory agencies, research institutes and suppliers of feed additives and premixes.
Related standards
- ISO 13903:2005 (original international text) - adopted as EN ISO 13903:2005 by CEN.
- The standard is based on Commission Directive 98/64/EC (as noted in the ISO foreword).
Keywords: EN ISO 13903:2005, amino acids determination, animal feeding stuffs, HPLC, amino acid analyser, free and total amino acids, feed quality control, lysine methionine threonine cystine.