ISO INTERNATIONAL STANDARD 21446 First edition 2019-01 Infant formula and adult nutritionals Determination of trans and total (cis + trans) vitamin Ki content Normal phase HPLC Formulesinfantiles etproduitsnutritionnelspouradultes Détermination de la teneur en vitamine Ki trans et totale (cis + trans) — Chromatographie liquide a haute performance (CLHP) en phase normale Reference number IS0 21446:2019(E) os1 @ IS0 2019 IS0 21446:2019(E) COPYRIGHTPROTECTEDDOCUMENT @ ISO 2019 All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can be requested from either IsO at the address below or Iso's member body in the country of the requester. ISO copyright office CP 401 : Ch. de Blandonnet 8 CH-1214 Vernier, Geneva Phone: +4122 749 0111 Fax: +41 22 749 09 47 Email: [email protected] Website: www.iso.org Published in Switzerland ii @ IS0 2019 - All rights reserved IS0 21446:2019(E) Contents Page Foreword ..iv 1 Scope .1 2 Normative references 3 Terms and definitions ..1 4 Principle 5 Reagents and materials ..2 6 Standard and solution preparation .2 6.1 Mixture with volume fraction of 10 % isopropanol in iso-octane .2 6.2 Mobilephase .2 6.3 Post-column electrolyte solution .3 6.4 Vitamin K1 (phytonadione) stock standard solution. .3 6.4.1 Vitamin K1 (phytonadione) stock standard solution .3 6.4.2 Stock standard concentration (for non UsP standard) .3 6.5 Vitamin K1 (phytonadione) intermediate I standard solution 3 6.6 Vitamin K1 (phytonadione) intermediate II standard solution .3 6.7 Vitamin K1 (phytonadione) working standard solutions. ..3 7 Apparatus ..4 8 Procedure .4 8.1 Sample preparation .4 8.1.1 Liquid samples 4 8.1.2 Powder samples .5 8.1.3 Dry blended/non-homogenous powder samples .5 8.1.4 Wet blended powder samples. .5 8.1.5 Extraction. .5 8.2 HPLC analysis.. .6 8.2.1 Instrumental operating conditions ..6 8.2.2 Instrument start-up. .6 8.3 HPLC of standards and samples .7 8.4 Instrument shut-down ..8 8.4.1 Short-term shut-down ..8 8.4.2 Long-term shut-down .8 9 .8 Calculations. 10 Precision .10 10.1 General .10 10.2 Repeatability .10 10.3 Reproducibility ..10 Annex A (informative) Example chromatograms ..12 Annex B (informative) Precision data .14 Annex C (informative) Comparison between this document and EN 14148 .18 Bibliography ..20 @ IS0 2019 - All rights reserved iii