ISO 21446:2019

Overview - ISO 21446:2019 (vitamin K1, normal phase HPLC)

ISO 21446:2019 specifies a validated analytical method for the quantitative determination of trans and total (cis + trans) vitamin K1 (phytonadione) in infant formula, pediatric and adult nutritionals. The method uses normal phase (NP) high-performance liquid chromatography (HPLC) with a post‑column reduction step and fluorescence detection to convert vitamin K to fluorescent derivatives for sensitive measurement. The procedure demonstrates good linearity (~2 μg/L to 80 μg/L trans vitamin K1) and reported limits of quantification (LOQ) of 0.4 μg/L for standards and 0.09 μg/100 g ready‑to‑feed (RTF) for samples (based on 4 g sample diluted to 10 mL).

Key topics and technical requirements

• Analytical principle: Extraction with iso‑octane after protein precipitation and lipid release, separation on a silica (normal phase) column, post‑column mixing with a zinc chloride/sodium acetate/acetic acid ethanolic solution, reduction in a zinc reactor, and fluorescence detection (excitation 245 nm / emission 440 nm).
• Chromatography: Silica analytical column (e.g., 150 mm × 3.0 mm, 3 μm), iso‑octane/isopropanol mobile phase (mobile phase prepared with small % isopropanol for baseline resolution).
• Standards and calibration: Primary phytonadione reference standard, multi‑level working standards prepared from defined stock dilutions; spectrophotometric concentration verification at 248 nm.
• Sensitivity and linearity: Linear range ~2–80 μg/L; LOQ values provided for standards and sample matrix.
• Apparatus and setup: Two isocratic pumps, autosampler (20 μL injection), fluorescence detector, post‑column reactor (zinc), centrifuge, HPLC degasser/tee, and yellow light or shielding to prevent light degradation of vitamin K.
• Sample handling: Procedures for liquid and powder (ready‑to‑feed and reconstituted) samples, extraction and centrifugation steps; emphasis on light protection and class A volumetry.

Applications - who should use ISO 21446:2019

• Quality control and analytical laboratories testing infant formula, pediatric and adult nutritional products for compliance with nutritional labeling and safety requirements.
• Manufacturers of milk‑based, soy‑based, hydrolyzed‑protein or nutritionally complete liquid products performing in‑house vitamin K1 assays.
• Regulatory agencies and standards bodies evaluating nutrient content or comparing methods.
• Method developers and interlaboratory validation teams (the document aligns with AOAC Official Method 2015.09).

Related standards

• ISO 21446:2019 includes an informative comparison with EN 14148 (see Annex C) and is published in collaboration with AOAC INTERNATIONAL. For implementation or cross‑reference, consult national regulatory guidance and the original ISO text.