Overview
ISO/TS 15495:2010 (IDF/RM 230) provides guidance for the quantitative determination of melamine and cyanuric acid in milk, powdered milk products and infant formulae using electrospray ionization liquid chromatography tandem mass spectrometry (LC‑MS/MS). The Technical Specification outlines the analytical principle, sample preparation, LC and MS requirements, validation expectations and reporting details. Annexes A and B give example LC‑MS/MS methods (HPLC/UPLC with triple quadrupole or ion‑trap instruments).
Key topics and technical requirements
- Analytical principle: Extraction of analytes from homogenized or reconstituted samples, protein precipitation, centrifugation and analysis by LC‑MS/MS using electrospray ionization (ESI) and selected reaction monitoring (SRM).
- Chromatography: Hydrophilic interaction liquid chromatography (HILIC) is recommended to separate melamine and cyanuric acid. Minimum retention time must be at least twice the column void volume; retention time ratios to internal standards must match calibration within ±2.5%.
- Mass spectrometry: Triple‑quadrupole instruments preferred. For each analyte, monitor at least two SRM transitions (quantifier and qualifier); internal standards require at least one transition (two recommended). Qualifier/quantifier ion ratio tolerances are specified for confirmation.
- Quantification: Isotope dilution using stable isotope internal standards for both melamine and cyanuric acid.
- Sample preparation: Spike isotope‑labelled standards at the start; use ≥5 mL extraction solvent per gram of sample, with final extraction solvent ≥70% organic; extract for ≥5 minutes, then centrifuge and filter.
- Performance & validation:
- Limit of quantification (LoQ) should be at least 10× lower than any maximum limit (ML).
- Typical trueness/recovery: 95–105% (acceptance 80–110%).
- Repeatability CV typically 3–6% (limit