ISO 21446:2019
Infant formula and adult nutritionals — Determination of trans and total (cis + trans) vitamin K1 content — Normal phase HPLC
This document specifies a method for the quantitative determination of trans and total (cis + trans) vitamin K1 in infant, pediatric and adult nutritionals using normal phase (NP) high-performance liquid chromatography (HPLC) with post-column reduction and fluorescence detection. The method demonstrated good linearity over a standard range of ~2 μg/l to 80 μg/l trans vitamin K1, and the limit of quantification (LOQ) was estimated to be 0,4 μg/l for standards and 0,09 μg/100 g ready to feed (RTF) for samples assuming 4 grams of sample are diluted to 10 ml.
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Foreword
1 Scope
2 Normative references
3 Terms and definitions
4 Principle
5 Reagents and materials
6 Standard and solution preparation
6.1 Mixture with volume fraction of 10 % isopropanol in iso-octane
6.2 Mobile phase
6.3 Post-column electrolyte solution
6.4 Vitamin K1 (phytonadione) stock standard solution
6.4.1 Vitamin K1 (phytonadione) stock standard solution
6.4.2 Stock standard concentration (for non USP standard)
6.5 Vitamin K1 (phytonadione) intermediate I standard solution
6.6 Vitamin K1 (phytonadione) intermediate II standard solution
6.7 Vitamin K1 (phytonadione) working standard solutions
7 Apparatus
8 Procedure
8.1 Sample preparation
8.1.1 Liquid samples
8.1.2 Powder samples
8.1.3 Dry blended/non-homogenous powder samples
8.1.4 Wet blended powder samples
8.1.5 Extraction
8.2 HPLC analysis
8.2.1 Instrumental operating conditions
8.2.2 Instrument start-up
8.3 HPLC of standards and samples
8.4 Instrument shut-down
8.4.1 Short-term shut-down
8.4.2 Long-term shut-down
9 Calculations
10 Precision
10.1 General
10.2 Repeatability
10.3 Reproducibility
Annex A (informative) Example chromatograms
Annex B (informative) Precision data
Annex C (informative) Comparison between this document and EN 14148
Bibliography
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Foreword
1 Scope
2 Normative references
3 Terms and definitions
4 Principle
5 Reagents and materials
6 Standard and solution preparation
6.1 Mixture with volume fraction of 10 % isopropanol in iso-octane
6.2 Mobile phase
6.3 Post-column electrolyte solution
6.4 Vitamin K1 (phytonadione) stock standard solution
6.4.1 Vitamin K1 (phytonadione) stock standard solution
6.4.2 Stock standard concentration (for non USP standard)
6.5 Vitamin K1 (phytonadione) intermediate I standard solution
6.6 Vitamin K1 (phytonadione) intermediate II standard solution
6.7 Vitamin K1 (phytonadione) working standard solutions
7 Apparatus
8 Procedure
8.1 Sample preparation
8.1.1 Liquid samples
8.1.2 Powder samples
8.1.3 Dry blended/non-homogenous powder samples
8.1.4 Wet blended powder samples
8.1.5 Extraction
8.2 HPLC analysis
8.2.1 Instrumental operating conditions
8.2.2 Instrument start-up
8.3 HPLC of standards and samples
8.4 Instrument shut-down
8.4.1 Short-term shut-down
8.4.2 Long-term shut-down
9 Calculations
10 Precision
10.1 General
10.2 Repeatability
10.3 Reproducibility
Annex A (informative) Example chromatograms
Annex B (informative) Precision data
Annex C (informative) Comparison between this document and EN 14148
Bibliography