ISO 20638:2015

Overview

ISO 20638:2015 defines a validated analytical method for the quantitative determination of 5′-mononucleotides in infant formula (powders, ready-to-feed liquids, and liquid concentrates) using liquid chromatography. The standard specifies sample preparation, solid-phase extraction (SPE), chromatographic separation on a C18 stationary phase, UV detection with a photodiode array detector, and quantitation using an internal standard (thymidine 5′-monophosphate, TMP). The method is equivalent to AOAC Official Method 2011.20 and was developed by ISO/TC 34 (Food products).

Key topics and requirements

• Analytes covered: uridine (UMP), inosine (IMP), adenosine (AMP), guanosine (GMP), cytidine (CMP) 5′-mononucleotides.
• Principle: extraction into a high-salt/EDTA solution to limit matrix interactions, cleanup by strong-anion exchange SPE, then HPLC separation with gradient elution and UV detection.
• Chromatography: C18 column (example: Gemini C18, 4.6 × 250 mm, 5 µm), mobile phases of phosphate buffer and methanol, controlled gradient and column oven at 40 °C; monitoring wavelengths specified per nucleotide.
• Quantitation: internal standard technique using TMP; calibration via ratio of peak areas (nucleotide/TMP) with linear regression forced through zero.
• Reagents & apparatus: lists analytical-grade reagents (KH2PO4, KBr, NaCl, EDTA, methanol, etc.), SPE cartridges (strong-anion exchange), HPLC system with diode-array detector, syringe filters, and centrifugation devices.
• Documentation: includes procedures for standard preparation, extraction, SPE conditioning, elution, filtration, chromatographic conditions, calculations for concentrations and purity, and example chromatograms and precision data.

Applications and users

ISO 20638:2015 is practical for:

• Infant formula manufacturers for product formulation verification and batch quality control.
• Independent testing laboratories performing compositional analysis and regulatory compliance testing.
• Regulatory agencies monitoring nucleotide labeling and safety in infant nutrition products.
• Research groups studying nucleotide supplementation or stability in milk-based formulas.

Benefits include reproducible, traceable quantitation of nucleotide additives and native nucleotides for labeling accuracy, product development, and safety assessment.

Related standards

• AOAC Official Method 2011.20 (equivalent analytical method)
• Codex Standard 72-1981 (definition of infant formula referenced in ISO 20638:2015)

Keywords: ISO 20638:2015, infant formula, nucleotides, 5′-mononucleotides, liquid chromatography, HPLC, solid-phase extraction, SPE, photodiode array detector, TMP internal standard.