Overview
EN ISO 17375:2006 (ISO 17375:2006) specifies a validated analytical method for the determination of aflatoxin B1 in animal feeding stuffs. The method uses reverse‑phase high‑performance liquid chromatography (RP‑HPLC) with post‑column derivatization (PCD) (bromination) and fluorescence detection. It is applicable to feed matrices with up to 50% fat and demonstrates a limit of quantification (LOQ) better than 0.5 µg/kg (S/N = 6).
Key topics and technical requirements
- Sampling and scope: Designed for animal feeding stuffs (feed and compound feeds) up to 50% fat content.
- Extraction: Test portion extracted with an acetone/water (85 + 15 v/v) solvent.
- Clean‑up: Immunoaffinity columns (IACs) specific to aflatoxin B1 are used for selective cleanup. IACs must have a capacity ≥ 40 ng and provide ≥ 80% recovery for a 0.25 ng spike.
- Chromatography and detection:
- RP‑HPLC separation followed by post‑column derivatization (PCD) by bromination.
- PCD can be achieved by electrochemical bromine generation or by using pyridinium hydrobromide perbromide (PBPB).
- Final detection by fluorescence.
- Reagents and mobile phases: Specifies HPLC‑grade solvents (acetonitrile, methanol, water), mobile phase recipes (options for PBPB or electrochemical bromination), and reagents such as PBPB, potassium bromide, and nitric acid when applicable.
- Calibration: Detailed procedures for preparing aflatoxin B1 stock and calibration solutions (toluene/acetonitrile stock; Options A & B for working standards) and spectrophotometric concentration verification.
- Quality and safety: Guidance on storage, light protection of aflatoxin standards, and warnings about toxic/flammable solvents and carcinogenic nature of aflatoxins.
- Performance characteristics: The standard includes sections on precision (interlaboratory testing, repeatability, reproducibility) and reporting requirements.
Applications and users
- Routine and regulatory testing of feed ingredients and compound feeds for aflatoxin B1 contamination.
- Used by: feed testing laboratories, contract analytical labs, feed manufacturers, food safety authorities, and researchers monitoring mycotoxin exposure in livestock.
- Supports compliance with feed safety regulations and risk‑based monitoring programs.
Related standards
- ISO 3696 (Water for analytical laboratory use) is referenced for reagent water quality.
- Prepared by ISO/TC 34 and adopted by CEN (EN ISO 17375:2006) for harmonized European application.
Keywords: aflatoxin B1, animal feeding stuffs, EN ISO 17375:2006, ISO 17375:2006, HPLC, post‑column derivatization, immunoaffinity column, feed testing, limit of quantification.