Overview
ISO 21468:2020 specifies a validated analytical method for the determination of free and total choline and free and total carnitine in infant formula and adult nutritional products using liquid chromatography with tandem mass spectrometric detection (HPLC‑MS/MS or LC‑MS/MS). The method covers sample reconstitution, extraction for free analytes, microwave‑assisted nitric acid digestion for total analytes, chromatographic separation, MS/MS quantification and calculation/reporting of results.
Key topics and technical requirements
- Scope: Quantification of free and total choline and carnitine in ready‑to‑feed and powdered infant/adult nutritionals.
- Analytical principle: Aqueous extraction for free forms; nitric acid digestion with microwave heating for total forms; dilution, protein precipitation (acetonitrile), and LC‑MS/MS analysis.
- Reagents and solvents: MS‑grade water, acetonitrile, ammonium formate and formic acid; nitric acid (70 %) for digestion.
- Mobile phases: Two ammonium formate/acetonitrile mixtures with 0.2 % formic acid (Mobile phase A and B) are specified for HPLC operation.
- Standards and internal standards: Primary reference standards (choline bitartrate, L‑carnitine) and isotopically labelled internal standards (L‑carnitine‑d HCl; choline‑1,1,2,2‑d) are used for accurate quantitation. Stock/working solutions stability and preparation guidance are provided.
- Apparatus: Typical instrumentation listed includes an HPLC system, ESI‑MS/MS, an ion‑exchange/SCX column (example: Zorbax 300‑SCX), microwave digestion system, and single‑use plasticware to avoid non‑specific binding. Positive‑displacement pipets are mandatory for solvent accuracy.
- Quality and performance: The standard includes procedures for calibration, blanks, internal standard recovery, precision (repeatability and reproducibility) and required reporting elements. Example chromatograms and precision data are supplied in informative annexes.
Practical applications
- Routine compliance testing of infant formula and nutritional products for nutritional labelling and safety.
- Quality control during product development and manufacturing to verify choline and carnitine content.
- Regulatory and conformity assessment testing by government laboratories and notified bodies.
- Method transfer and proficiency testing for analytical laboratories implementing LC‑MS/MS assays for vitamins and nutrient markers.
Who should use this standard
- Food and infant formula manufacturers and their QC/analytical teams.
- Contract testing and commercial analytical laboratories with LC‑MS/MS capability.
- Regulatory agencies, research institutions and standardization committees focused on infant/adult nutrition analysis.
Related standards
- AOAC Official Method 2015.10 - equivalent method (LC/MS/MS) referenced in the ISO foreword.
- Codex Standard 72‑1981 - definition of infant formula referenced for scope.
Keywords: ISO 21468:2020, infant formula, adult nutritionals, choline, carnitine, HPLC‑MS/MS, LC‑MS/MS, analytical method, microwave digestion, internal standards, quality control.
Open preview (PDF)Infant formula and adult nutritionals — Determination of free and total choline and free and total carnitine — Liquid chromatography tandem mass spectrometry (HPLC-MS/MS)
