ISO 20639:2015

Overview

ISO 20639:2015 specifies a laboratory method for the quantitative determination of pantothenic acid (vitamin B5), excluding bound forms, in infant formula and adult nutritionals (powdered products) using ultra high performance liquid chromatography and tandem mass spectrometry (UHPLC‑MS/MS). The standard defines sample preparation, extraction (0.4 mol/L ammonium acetate buffer, pH 3.8), chromatographic and MS/MS conditions, calibration with an isotopically labelled internal standard, and calculation procedures for reporting results on a mg/100 g basis.

Key Topics and Requirements

• Scope and principle: Extraction of free pantothenic acid followed by UHPLC‑MS/MS quantitation. Bound forms are not determined by this method.
• Reagents and standards: Use of analytical‑grade solvents; calcium d‑pantothenate as reference standard and an isotopically labelled calcium pantothenate as internal standard (IS).
• Sample preparation: Procedures for dry blended powders, wet blended powders and liquids; enzymatic treatment (α‑amylase) recommended if starch is present to reduce viscosity.
• Extraction and cleanup: 25 mL of 0.4 mol/L ammonium acetate (pH 3.8) added to test portion, stirring and filtration before analysis.
• UHPLC conditions: Examples provided (injection 2 µL, column 30 °C, flow 0.45 mL/min, formic acid in water / acetonitrile gradient). Direct LC flow to MS only during early elution to limit source fouling.
• MS/MS conditions: Positive ESI, indicative instrument settings given (e.g., capillary 2.2 kV, collision energy ~14 V). Monitored transitions: m/z 220.2 → 90.1 (PA) and m/z 224.2 → 94.1 (IS).
• Calibration and calculation: Five‑point calibration curve using peak area ratio (PA/IS), recommended 1/x weighted regression; formula provided to compute pantothenic acid mass fraction (mg/100 g).
• Precision and validation: Interlaboratory precision data and validation references are included (Annex B).

Applications

• Routine quality control and nutritional analysis of infant formulas and adult nutritional powders.
• Compliance testing for product labeling and regulatory requirements related to vitamin B5 content.
• Method adoption in analytical laboratories for accurate, traceable determination of free pantothenic acid.
• Research and product development where precise quantitation of free pantothenic acid is required.

Who Would Use This Standard

• Food testing laboratories and contract analytical services
• Infant formula and nutritional product manufacturers (QC and R&D)
• Regulatory agencies and conformity assessment bodies
• Accreditation bodies and laboratories seeking validated, harmonized methods

Related Standards

• The method is published in collaboration with AOAC and is equivalent to AOAC Official Method 2012.16. ISO/TC 34 (Food products) oversaw development. Validation references include interlaboratory study procedures such as ISO 5725‑2.

Keywords: ISO 20639:2015, pantothenic acid, infant formula, adult nutritionals, UHPLC‑MS/MS, ultra high performance liquid chromatography, tandem mass spectrometry, vitamin B5 determination.