Overview
ISO 9231:2008 (IDF 139:2008) specifies a validated analytical method for the determination of benzoic acid and sorbic acid in milk and milk products. The method is applicable to milk, dried milk, yogurt and other fermented milks, cheese and processed cheese, and is suitable for measuring both preservatives at levels above 5 mg/kg. The procedure combines Carrez precipitation for removal of fats and proteins with high‑performance liquid chromatography (HPLC) on a reversed‑phase C18 column and UV detection.
Key topics and technical requirements
- Principle: Remove fats and proteins by Carrez precipitation, dilute with methanol, clarify and analyse by reversed‑phase HPLC.
- Sample preparation:
- Yogurt/fermented milks: homogenize and weigh 20 g into 100 mL volumetric flask.
- Other milk products: weigh 3 g, disperse in ~10 mL water and transfer to 100 mL volumetric flask.
- Precipitation and clarification: Add 25 mL 0.1 M NaOH, heat (ultrasonic bath or 70 °C water bath) for 15 min, adjust pH to 8 ± 1 with sulfuric acid, add potassium hexacyanoferrate(II) and zinc acetate, dilute with methanol and filter through 0.45 µm.
- HPLC conditions: Reversed‑phase C18 column (250 × 4 mm), mobile phase = 10% methanol / 90% phosphate buffer (pH 6.7), flow ≈ 1.2 mL/min, UV detection at 227 nm (benzoic acid) and 250 nm (sorbic acid). Typical retention times: benzoic ~5.5 min, sorbic ~7 min.
- Calibration & quantification: Linear calibration; single‑point calibration acceptable if linearity is verified. Dilute samples if peaks are off scale. Check UV signal ratios to detect co‑eluting interferences (e.g., hippuric acid in milk).
- Reagents & apparatus: Methanol, phosphate buffer, Carrez reagents (potassium hexacyanoferrate(II), zinc acetate), HPLC system with dual‑wavelength or diode‑array detector, 1 cm flow cell.
- Performance: The standard includes precision information (interlaboratory testing, repeatability and reproducibility) and reporting requirements.
Applications and users
ISO 9231 is intended for:
- Dairy industry QA/QC labs verifying preservative levels in raw and processed dairy products.
- Food safety and regulatory laboratories enforcing legal limits for benzoic and sorbic acids.
- Contract analytical laboratories offering preservative testing services.
- Research groups studying preservative occurrence and migration in dairy matrices.
Keywords: ISO 9231, HPLC method, benzoic acid analysis, sorbic acid analysis, milk and milk products, preservative testing, Carrez precipitation, dairy testing, food safety.
Related standards
- ISO 707 | IDF 50 (sampling of milk and milk products) - recommended sampling guidance.
- ISO 1042 - specification for one‑mark volumetric flasks used in the procedure.
This internationally recognized ISO/IDF method provides a reproducible HPLC‑based approach for preservative analysis in diverse dairy matrices, supporting compliance, quality control and food safety monitoring.