ISO 20636:2018

Overview

ISO 20636:2018 - Infant formula and adult nutritionals: Determination of vitamin D by liquid chromatography‑mass spectrometry (LC‑MS) specifies a validated analytical method for the quantitative determination of vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) in powdered and liquid infant formulas and adult nutritional products. The method covers sample types including powders, ready‑to‑feed liquids and liquid concentrates, with application ranges from 0.15 µg/100 g (LOQ) to 59 µg/100 g for vitamin D2 and 0.25 µg/100 g to 65 µg/100 g for vitamin D3.

Key topics and technical requirements

• Analytical principle: alkaline saponification → extraction into isooctane → PTAD derivatisation → re‑extraction into acetonitrile → reversed‑phase LC separation → detection by mass spectrometry (MRM).
• Derivatisation reagent: 4‑phenyl‑1,2,4‑triazoline‑3,5‑dione (PTAD) to form easily ionisable vitamin D adducts.
• Internal standards: use of stable isotope labelled d6‑vitamin D2 and d6‑vitamin D3 to correct for extraction, derivatisation and ionisation variability.
• Calibration and quantitation: daily calibration standards prepared with non‑labelled working solutions and labelled internal standards; quantitative ranges and limits of quantification defined in the standard.
• Reagents and instrument requirements: LC‑MS (LC coupled to MS/MS), LC‑MS grade solvents (acetonitrile, methanol, formic acid), isooctane, KOH saponification, PTAD reagent.
• Quality and safety: method includes precision data (repeatability, reproducibility); warns about hazardous materials and the need for laboratory safety practices.
• Sample preparation: procedures for powder, slurry and liquid samples are specified to ensure consistent extraction.

Practical applications

• Routine quality control and specification testing of fortified infant formulas and adult nutritionals.
• Regulatory compliance testing by food safety authorities and third‑party laboratories.
• Product development and stability studies in infant nutrition and medical nutrition formulations.
• Research on vitamin D fortification levels and bioanalytical method validation.

Who should use this standard

• Food testing laboratories and LC‑MS analytical teams
• Infant formula and clinical nutrition manufacturers (QC/R&D)
• Regulatory agencies and inspection bodies
• Accreditation and proficiency testing organizations

Related standards

• ISO 20636:2018 is equivalent to AOAC Official Method 2016.05 (as noted in the introduction).
• Annex D of ISO 20636 provides a comparison with EN 12821, useful for laboratories comparing methods.

Keywords: ISO 20636:2018, vitamin D determination, LC‑MS, infant formula testing, PTAD derivatisation, d6 internal standards, MRM, vitamin D2, vitamin D3.