ISO 20631:2024

Overview

ISO 20631:2024 specifies a validated analytical method for determining total folate in infant formula and adult nutritionals. The method combines trienzyme extraction (protease, α‑amylase and rat plasma conjugase) to release folate monoglutamates with ultra high performance liquid chromatography tandem mass spectrometry (UHPLC‑MS/MS) for selective quantitation. The standard is intended for laboratories performing folate analysis for labeling, quality control, formulation and regulatory compliance.

Key topics and technical requirements

• Scope: Total folate in powdered and ready‑to‑feed infant formulas and adult nutritional products.
• Extraction principle: Trienzyme digestion in buffered conditions (deconjugation of polyglutamate folates to monoglutamates), followed by purification.
• Cleanup: Weak anion exchange (WAX) solid‑phase extraction (SPE) to concentrate and purify folate analytes prior to analysis.
• Instrumental analysis: UHPLC‑MS/MS with specific MS/MS transitions for folic acid, 5‑methyl‑tetrahydrofolate (5‑CH3‑THF) and 5‑formyl‑tetrahydrofolate (5‑CHO‑THF).
• Internal standards: Isotopically labelled 13C internal standards for folic acid, 5‑CH3‑THF and 5‑CHO‑THF are required for accurate quantitation.
• Reagents and handling: LC‑MS grade reagents; folate standards and extracts are light‑sensitive and must be handled under UV‑filtered or yellow light and stored appropriately (e.g., −20 °C).
• Calculations & reporting: Calibration curves, peak area ratios to internal standards, and reporting of folate as folic acid equivalents (µg/100 g). The standard includes precision (repeatability and reproducibility) performance data.
• Sample preparation: Procedures for homogenizing products and reconstituting powders to ready‑to‑feed form are specified.

Applications and users

ISO 20631:2024 is targeted at:

• Food testing and analytical laboratories performing folate analysis by UHPLC‑MS/MS.
• Infant formula and adult nutritional product manufacturers for quality control, formulation verification and stability studies.
• Regulatory agencies and compliance laboratories for nutritional labeling verification and surveillance.
• Research groups studying folate bioavailability and fortification effects in complex food matrices.

Practical uses include verifying declared folate content, optimizing fortification processes, monitoring production consistency, and supporting product registration or market authorization.

Related standards

• The method in ISO 20631:2024 is equivalent to AOAC Official Method 2011.06 (Total Folate in Infant Formula and Adult Nutritionals by Trienzyme Extraction and LC‑MS/MS).
• Developed under ISO/TC 34 (Food products) guidance for analytical method standardization.

Keywords: ISO 20631:2024, trienzyme extraction, UHPLC‑MS/MS, total folate, infant formula, adult nutritionals, folic acid, 5‑CH3‑THF, WAX SPE, isotopically labelled internal standards, folate analysis, food testing.