Overview
ISO 18329:2004 (IDF 193:2004) defines a standardized analytical method for the quantitative determination of furosine (ε-furoylmethyl-lysine) in milk and milk products. The method uses ion-pair reverse-phase high-performance liquid chromatography (IP-RP HPLC) with UV detection at 280 nm to evaluate the early stage of the Maillard reaction - particularly useful for assessing effects of heat treatment on raw and processed milk and on cheese.
Key topics and technical requirements
- Analyte and units: Furosine content expressed as milligrams per 100 g of protein.
- Principle: Warm acid hydrolysis converts ε-lactulosyl-lysine into furosine; furosine is quantified by comparison with a certified furosine standard.
- Sample types: Raw or heat‑treated milk and cheese.
- Hydrolysis: Nitrogen-flushed vials; heat 23 h at 110 °C; filter hydrolysate; filtered hydrolysate may be stored at −20 °C.
- Protein determination: Nitrogen by Kjeldahl (ISO 8968-1) and conversion factor 6.38.
- Solid-phase extraction (SPE): C18 cartridge (≈500 mg) pre-wet with methanol then water; load 0.5 ml hydrolysate; elute with HCl solution III (3.0 mol/L). Purified hydrolysate stable ~1 week at −20 °C.
- HPLC conditions:
- Column: Dedicated furosine RP column (4.6 × 250 mm, 5 µm) or equivalent.
- Detector: UV at 280 nm; sensitivity such that 10 pmol injection S/N > 10; min AUFS 0.010.
- Elution solvents: Dilute acetic acid (solvent A) and KCl in dilute acetic acid (solvent B).
- Gradient (example): 0–12.5 min 100% A; 19.5 min to 50% A/50% B; hold to 22.0 min; return to 100% A by 24.0 min; total run 32 min.
- Flow rate: 1.2 ml/min; column temp: 30–35 °C; injection volume: 20–50 µl.
- Quantification: Single-point comparison to furosine standard solution (approx. 1 nmol/ml); furosine peak should elute ~20–24 min and be baseline-resolved.
- Performance: Standard includes interlaboratory precision data (repeatability and reproducibility).
Applications and users
ISO 18329 is used by:
- Dairy quality-control and analytical laboratories monitoring heat treatment and Maillard reaction progression.
- Cheese and milk processors for process control, shelf‑life assessment, and product quality evaluation.
- Regulatory bodies and research labs studying thermal damage, protein modification, and food chemistry.
Practical uses include verifying pasteurization/sterilization impacts, comparing processing conditions, and supporting product specifications that reference furosine as a marker of protein glycation.
Related standards
- ISO 8968-1 / IDF 20-1 - Milk - Determination of nitrogen (Kjeldahl) (used to convert nitrogen to protein).
- ISO 707 - Recommended sampling for milk and milk products (sampling guidance).
Keywords: ISO 18329, furosine, milk analysis, HPLC, ion-pair reverse-phase, Maillard reaction, heat treatment, cheese testing, dairy quality control.
Open preview (PDF)Milk and milk products — Determination of furosine content — Ion-pair reverse-phase high-performance liquid chromatography method
