Overview - ISO 14939:2001 (Carbadox determination by HPLC)
ISO 14939:2001 specifies a laboratory method for the determination of carbadox in animal feeding stuffs and premixtures using high-performance liquid chromatography (HPLC). The standard covers sample preparation, extraction, cleanup, chromatographic conditions and detection. It applies to:
- animal feeding stuffs with carbadox mass fractions from 0.5 mg/kg (LOQ) up to 100 mg/kg, and
- premixtures with carbadox up to 10 %.
The limit of detection (LOD) is 0.1 mg/kg. Carbadox is identified by reverse‑phase HPLC with UV detection at 365 nm (or optionally by post‑column derivatization with NaOH, detection at 420 nm).
Key topics and technical requirements
- Scope and ranges: LOQ 0.5 mg/kg (feeds), LOD 0.1 mg/kg; premixtures up to 10% carbadox.
- Analytical principle: Extraction with a 1:1 acetonitrile:methanol solvent; feeds are pre‑wetted with water; extracts from feeds are cleaned on a short aluminium oxide column; final analysis by reverse‑phase HPLC with UV detection.
- Mobile phase & reagents: Mobile phase prepared from a sodium acetate solution mixed with acetonitrile; detailed reagent and standard preparation steps (stock and working solutions) are specified.
- Chromatography & detection: Requirements for guard and analytical columns (C18 bonded phases), flow rates, injection volumes and capacity factor (K’) ≥ 1.0 for carbadox retention. UV detection at 365 nm is primary; diode array detector or post‑column derivatization (NaOH, 420 nm) are provided for confirmation.
- Interferences & confirmation: Potential interferences include dimetridazole, nitrofurazone and sulfadimidine sodium; the standard describes co‑chromatography and detector/derivatization options for confirmation.
- Safety & handling: Carbadox and several reagents are light‑sensitive and toxic; operations should be done in reduced light and under appropriate laboratory controls (fume hood, PPE).
- Quality / performance: Clauses on precision, repeatability, reproducibility and interlaboratory testing are included.
Applications and users
ISO 14939:2001 is intended for:
- Feed testing laboratories performing quantitative residue analysis.
- Feed and premixture manufacturers for quality control of medicated feeds.
- Regulatory and inspection bodies monitoring compliance with feed additive regulations.
- Veterinary diagnostic labs assessing carbadox levels in piglet growth‑promoting feeds.
Keywords: ISO 14939:2001, carbadox, HPLC method, animal feeding stuffs, premixtures, limit of detection, reverse-phase HPLC, feed testing, analytical standard.
Related standards
- ISO 6498:1998 - Preparation of test samples (referenced normative document).
- ISO 6497 (referenced for recommended sampling methods).