ISO 21446:2019
(Main)Infant formula and adult nutritionals — Determination of trans and total (cis + trans) vitamin K1 content — Normal phase HPLC
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.
Formules infantiles et produits nutritionnels pour adultes — Détermination de la teneur en vitamine K1 trans et totale (cis + trans) — Chromatographie liquide à haute performance (CLHP) en phase normale
General Information
Standards Content (Sample)
INTERNATIONAL ISO
STANDARD 21446
First edition
2019-01
Infant formula and adult
nutritionals — Determination of trans
and total (cis + trans) vitamin K
content — Normal phase HPLC
Formules infantiles et produits nutritionnels pour adultes —
Détermination de la teneur en vitamine K trans et totale (cis +
trans) — Chromatographie liquide à haute performance (CLHP) en
phase normale
Reference number
©
ISO 2019
© ISO 2019
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ii © ISO 2019 – All rights reserved
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
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 Mobile phase . 2
6.3 Post-column electrolyte solution . 3
6.4 Vitamin K (phytonadione) stock standard solution . 3
6.4.1 Vitamin K (phytonadione) stock standard solution . 3
6.4.2 Stock standard concentration (for non USP standard) . 3
6.5 Vitamin K (phytonadione) intermediate I standard solution . 3
6.6 Vitamin K (phytonadione) intermediate II standard solution . 3
6.7 Vitamin K (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 Calculations. 8
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
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
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ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
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.org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 34, Food products in collaboration with
AOAC INTERNATIONAL. It is being published by ISO and separately by AOAC INTERNATIONAL. The
method described in this document is equivalent to the AOAC Official Method 2015.09: Trans vitamin K
in Infant, Pediatric, and Adult Nutritionals, HPLC with Fluorescence Detection.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO 2019 – All rights reserved
INTERNATIONAL STANDARD ISO 21446:2019(E)
Infant formula and adult nutritionals — Determination of
trans and total (cis + trans) vitamin K content — Normal
phase HPLC
1 Scope
This document specifies a method for the quantitative determination of trans and total (cis + trans)
vitamin K 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 K , 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.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:/ /www. iso. org/obp
— IEC Electropedia: available at http:/ /www.e lectropedia. org/
3.1
adult nutritional
nutritionally complete, specially formulated food, consumed in liquid form, which may constitute the
sole source of nourishment, made from any combination of milk, soy, rice, whey, hydrolysed protein,
starch and amino acids, with and without intact protein
3.2
infant formula
breast-milk substitute specially manufactured to satisfy, by itself, the nutritional requirements of
infants during the first months of life up to the introduction of appropriate complementary feeding
[SOURCE: Codex Standard 72-1981]
4 Principle
Vitamin K is extracted from products with iso-octane after precipitation of proteins and release of
lipids with methanol. Prepared samples are injected onto a silica HPLC column where cis and trans
vitamin K are separated with an iso-octane–isopropanol mobile phase. The column eluent is mixed
with a dilute ethanolic solution of zinc chloride, sodium acetate, and acetic acid, and cis and trans
vitamin K are reduced to fluorescent derivatives in a zinc reactor column. The resulting fluorescent
compounds are then detected by fluorescence at an excitation wavelength of 245 nm and an emission
wavelength of 440 nm.
5 Reagents and materials
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and
distilled or demineralized water or water of equivalent purity.
5.1 Acetic acid, glacial, > 99 %, American Chemical Society (ACS).
5.2 Helium or nitrogen, zero grade or equivalent helium or nitrogen.
5.3 Hexane, HPLC grade.
5.4 Iso-octane (2,2,4-trimethylpentane), HPLC grade.
5.5 Isopropanol (isopropyl alcohol), HPLC grade.
5.6 Methanol, HPLC grade.
5.7 Phytonadione/phylloquinone (vitamin K ), primary reference standard. Store per label
instructions.
5.8 Laboratory or distilled water, with conductivity of 0,067 μS/cm (15 Mohm/cm).
5.9 Ethanol, 95 %, ACS.
5.10 Sodium acetate, anhydrous, ACS.
5.11 Zinc, < 150 μm, 99,995 % or equivalent.
5.12 Zinc chloride, > 97 %, ACS.
6 Standard and solution preparation
CAUTION — Since vitamin K is light-sensitive, all standards shall be prepared, handled, and
stored in the dark or under yellow shielded lighting unless otherwise stated. If the standards
are transported through or into an area without yellow shielded lighting, they shall be w
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