Overview
EN ISO 14939:2001 - "Animal feeding stuffs - Determination of carbadox content - Method using high-performance liquid chromatography (HPLC)" is a CEN-adopted International Standard that specifies a validated HPLC procedure to quantify carbadox in premixtures and animal feeding stuffs. The method covers feeds with carbadox mass fractions from 0.5 mg/kg (limit of quantification) to 100 mg/kg, premixtures up to 10%, and has a lower limit of detection of 0.1 mg/kg. Carbadox is a quinoxaline chemotherapeuticum used as a growth-promoting feed additive for piglets.
Key topics and technical requirements
- Analytical technique: Reverse-phase HPLC with UV detection at 365 nm; post-column derivatization with NaOH and detection at 420 nm is an alternative for confirmation.
- Sample types & ranges: Applicable to premixtures (up to 10% carbadox) and animal feeding stuffs (0.5–100 mg/kg; LOD 0.1 mg/kg).
- Extraction and cleanup: Carbadox is extracted using an acetonitrile:methanol (1:1) solvent. Feeding stuffs are prewetted with water and extracts are purified on a short aluminium oxide column; premixtures are directly diluted.
- Mobile phase & reagents: Mobile phase based on sodium acetate solution and acetonitrile; specific reagent preparations and standard solutions (stock and working) are specified.
- Apparatus requirements: HPLC pump (0.5–1.5 ml/min), injection loops (20–100 µl), UV detector (365 nm) or diode-array detector for confirmation, specified guard and analytical C18 columns.
- Interferences & confirmation: Potential interferences from dimetridazole, nitrofurazone, and sulfadimidine are noted. Confirmation can be performed by co‑chromatography, diode-array spectra or post-column derivatization.
- Quality & safety: Method includes requirements for standard materials, light-sensitive handling (carbadox and some standards), and preparation/storage of stock solutions. The standard contains annexes with a flowchart and interlaboratory precision data.
Applications and users
- Who uses it: Analytical laboratories, feed manufacturers, regulatory agencies, and quality-control teams involved in feed safety and compliance testing.
- Practical uses: Routine quantification of carbadox residues in premixtures and finished feeds, regulatory monitoring, supplier quality control, and investigation of suspected contamination or non‑compliance.
- Value: Provides a standardized, comparable HPLC method with defined limits of detection/quantification and documented precision for feed safety programs.
Related standards
- ISO 6498:1998 - referenced for preparation of test samples (sampling and sample prep guidance).
- Adopted by CEN (EN ISO 14939:2001) for harmonized use across European national standards bodies.
Keywords: EN ISO 14939:2001, carbadox, animal feeding stuffs, HPLC, high-performance liquid chromatography, feed analysis, premixtures, limit of detection, feed additive.