Overview
ISO 12080-2:2009 (IDF 142-2:2009) specifies a laboratory method for the determination of vitamin A in dried skimmed milk using high-performance liquid chromatography (HPLC). The method applies to dried skimmed milk samples containing at least 10 IU vitamin A per gram and provides a validated HPLC procedure-covering saponification, extraction, chromatographic separation and quantification by UV or fluorescence detection.
Key Topics and Requirements
- Scope and applicability: Designed specifically for dried skimmed milk with ≥10 IU/g vitamin A.
- Principle: Sample saponification and solvent extraction followed by HPLC separation; quantification using an UV detector (typical 325 nm) or fluorescence detector (excitation 325 nm, emission 450 nm).
- Reagents and solvents: Includes 95% ethanol, methanol (HPLC grade), potassium hydroxide (KOH), sodium ascorbate, butylated hydroxytoluene (BHT) as antioxidant, and light petroleum (boiling range 40–60 °C or 60–80 °C).
- Chromatographic conditions: Example column 250 × 4.6 mm, 10 μm C8 or C18 packing; typical flow 2 ml/min, injection 20 μl, and mobile phase ~ methanol:water 90:10 (v/v) (adjustment may be needed by equipment/column).
- Sample preparation: Dissolve dried milk, saponify (reflux ~30 min), extract into light petroleum, evaporate, reconstitute in methanol and inject.
- Calculation and units: Results expressed as micrograms retinol per gram (µg/g) or International Units (IU/g) using the reference standard and peak-area ratios.
- Precision: Repeatability - difference >14% of mean in not more than 5% of cases; Reproducibility - difference >42% in not more than 5% of cases.
- Safety: Warns about hazardous materials and standard laboratory practices; work in subdued light and use antioxidants to prevent degradation.
Applications and Users
- Who uses it: Dairy quality-control laboratories, analytical service labs, milk powder manufacturers, regulatory agencies, and research institutions monitoring vitamin fortification and nutritional labelling.
- Practical uses:
- Verification of vitamin A fortification levels in dried skimmed milk and infant-milk formulas (where applicable).
- Quality assurance during production and shelf-life studies (stability of vitamin A).
- Regulatory compliance testing and product specification verification.
- Method comparison and validation against colorimetric methods (see related standards).
Related Standards
- ISO 12080-1 / IDF 142-1: Colorimetric method for vitamin A in dried skimmed milk.
- ISO 707 / IDF 50: Recommended sampling of milk powder.
- ISO/IDF joint publications and ISO 5725 series for precision evaluation inform method validation and interlaboratory studies.
Keywords: ISO 12080-2:2009, dried skimmed milk, vitamin A determination, HPLC method, high-performance liquid chromatography, IDF 142-2, saponification, milk powder analysis, vitamin A assay, dairy laboratory.