Overview
ISO 14892:2002 (IDF 177:2002) specifies a validated analytical method for the determination of vitamin D in dried skimmed milk using high-performance liquid chromatography (HPLC). The procedure is intended for test samples containing at least 10 µg vitamin D per 100 g (equal to 400 IU/100 g) and combines saponification, solvent extraction, semi‑preparative clean-up and reverse‑phase HPLC with UV detection.
Key topics and technical requirements
- Scope & detection limits: Method applicable when the sample contains ≥ 10 µg/100 g vitamin D (≥ 400 IU/100 g).
- Analytes: Distinguishes between vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). The method uses one as an internal standard for the other; reliability requires that the sample contains only the target vitamin (no native signal of the internal standard). Verification is done by running the procedure without the internal standard.
- Sample preparation: Test portions are saponified and solvent‑extracted, with antioxidants (e.g., BHT) used to protect analytes.
- Clean-up: Semi‑preparative normal‑phase HPLC (n‑hexane + n‑pentanol mobile phase) is used to separate vitamin D from matrix interferences; fractions are collected prior to analysis.
- Quantification: Reverse‑phase HPLC (C18/ODS column) with UV detection at ~265 nm determines vitamin D content; acetonitrile/propan‑2‑ol/water (90:7:3) is the analytical mobile phase.
- Apparatus & parameters: Typical columns are 250 mm × 4.6 mm, 5 µm; clean-up flow ~3 ml/min (injection 600 µl), analytical flow ~2 ml/min (injection 200 µl). Detector sensitivity and system suitability tests are specified.
- Standards & reagents: Stock and working standard preparations for cholecalciferol and ergocalciferol (storage at about −18 °C for up to 1 month), ISO 3696 water, solvents (HPLC grade), KOH solutions, BHT antioxidant, etc.
- Precision & validation: Method selection is based on interlaboratory studies; precision descriptors (repeatability, reproducibility) and reporting requirements are included in the standard.
Applications and users
- Quality control in the dairy industry: Routine testing of fortified and naturally occurring vitamin D in dried skimmed milk.
- Regulatory compliance & labeling: Supporting nutritional claims, fortification verification and compliance with food regulatory limits.
- Contract and food testing laboratories: Accredited labs performing HPLC-based vitamin analysis.
- Research and product development: Food scientists assessing stability, fortification efficiency or processing effects on vitamin D.
Related standards & references
- Normative reference: ISO 3696 (Water for analytical laboratory use).
- The method was developed jointly by ISO, IDF and AOAC and aligns with established pharmacopoeial principles for vitamin D analysis.
Keywords: ISO 14892:2002, dried skimmed milk, vitamin D determination, HPLC, vitamin D3, vitamin D2, cholecalciferol, ergocalciferol, dairy quality control, food testing.