ISO 4214:2022 PDF

Overview

ISO 4214:2022 - Milk and milk products - Determination of amino acids in infant and adult/paediatric nutritional formulas and dairy products - specifies a validated laboratory method for the quantitative determination of total amino acids in milk-derived and related matrices. The method uses 6‑aminoquinolyl‑N‑hydroxysuccinimidyl carbamate (AQC/AccQ·Tag) derivatization followed by ultra‑high‑performance liquid chromatography (UHPLC) separation and ultraviolet (UV) detection. It covers a single analysis for 17 amino acids (see Key Topics) and was validated in a wide range of matrices including infant formulas, adult nutritional powders, milk powders, whey, sodium caseinate, cereals and pet food.

Key Topics and Technical Requirements

• Analytes covered: alanine, arginine, aspartic acid (with asparagine), cystine (cysteine + cystine as S‑2‑carboxyethylthiocysteine), glutamic acid (with glutamine), glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine and valine.
  • Tryptophan is explicitly excluded from this method.
• Analytical principle:
  • Protein hydrolysis in 6 mol/L HCl for 24 h at 110 °C in the presence of phenol, 3,3′‑dithiodipropionic acid (DDP) and norvaline (internal standard).
  • Conversion of cysteine/cystine to a stable derivative (XCys) using DDP.
  • AQC (AccQ·Tag) derivatization of liberated amino acids.
  • Separation by reversed‑phase UHPLC and detection by UV at 260 nm (fluorescence allowed if equivalence demonstrated).
• Reagents and standards: AccQ·Tag Ultra kit or equivalent, cystine and mixed amino acid calibration standards, norvaline internal standard, specified HCl/NaOH concentrations and ultra‑pure water.
• Workflow elements: sample preparation, hydrolysis, neutralization/dilution, derivatization, UHPLC separation, peak identification/integration, calibration curve construction and calculation of results.
• Validation and performance: precision data (repeatability and reproducibility) and chromatogram examples are provided (Annex A and B).

Practical Applications and Users

• Intended for food testing laboratories, dairy and infant formula manufacturers, contract testing labs, regulatory and food control authorities, and research groups requiring reliable amino acid profiles in:
  • Infant and toddler formulas (milk‑ and soy‑based, hydrolysed and elemental products)
  • Adult/paediatric nutritional powders and ready‑to‑use formulas
  • Milk, whey and milk powders, sodium caseinate
  • Cereals, breakfast cereals and selected pet foods
• Benefits include a single, standardized UHPLC‑based assay for multiple amino acids, traceable calibration, and improved comparability of amino acid composition data for quality control, labeling compliance and nutritional assessment.

Related Standards and Publication

• ISO 4214:2022 was developed jointly by ISO and the International Dairy Federation (IDF) and is also published separately by AOAC INTERNATIONAL. For laboratory accreditation and conformity, refer to national standards bodies and relevant accreditation guidance.