ISO 21446:2019

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
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: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
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
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
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
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso
.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 wrapped
tightly in foil. All standard solutions shall be prepared using Class A volumetric glassware.
6.1 Mixture with volume fraction of 10 % isopropanol in iso-octane
Add about 70 ml of iso-octane (5.4) to a 100 ml volumetric flask. Add 10 ml isopropanol (5.5) to the
volumetric flask and dilute to volume with iso-octane. Expiration is 6 months.
6.2 Mobile phase
Add about 900 ml iso-octane (5.4) to a 1 000 ml volumetric flask. Add 3 ml to 4 ml 10 % isopropanol
(6.1) to the volumetric flask and dilute to volume with iso-octane. Expiration is 6 months if stored in
tightly stoppered container.
NOTE The isopropanol concentration in the mobile phase can be adjusted slightly until baseline resolution
of cis and trans vitamin K from other peaks present in some samples is achieved, see Figures A.2 and A.3.
2 © ISO 2019 – All rights reserved

6.3 Post-column electrolyte solution
Transfer 0,5 g ± 0,05 g zinc chloride (5.12) and 0,20 g ± 0,02 g sodium acetate anhydrous (5.10) to a
1 000 ml volumetric flask with ethanol (5.9). Add 150 μl ± 15 μl glacial acetic acid (5.1) and dilute to
volume with ethanol. Mix solution for about 30 min or until solution is clear and all salts are dissolved.
Expiration is 6 months.
6.4 Vitamin K (phytonadione) stock standard solution
6.4.1 Vitamin K (phytonadione) stock standard solution
Accurately weigh to 0,000 01 g about 0,055 00 g vitamin K (phytonadione) (5.7) into a 250 ml
volumetric flask. Dissolve standard and dilute to volume with iso-octane (5.4). Store in a refrigerator in
a tightly stoppered container protected from light. Expiration is 6 months.
6.4.2 Stock standard concentration (for non USP standard)
To determine the stock standard concentration, evaporate 0,5 ml of vitamin K stock standard under
a stream of nitrogen and redissolve the residue in 10,0 ml of hexane. Measure the absorbance of this
solution in a 1 cm cell against a reference of hexane at wavelength of 248 nm with a spectrophotometer.
Calculate the vitamin K stock standard concentration, ρ , in milligrams per litre, using Formula (1):
1 ss
A ××10 000 20
ρ = (1)
ss
where
A is the absorbance of the solution at 248 nm;
1%
is the A value of vitamin K in hexane at 248 nm;
1cm
1%
10 000
is the conversion of A to milligrams per litre;
1cm
20 is the dilution of the stock standard solution.
6.5 Vitamin K (phytonadione) intermediate I standard solution
Dilute 1,0 ml vitamin K stock standard (6.4.1) to 100 ml with iso-octane. Prepare from stock standard
solution each time new working standards are made.
6.6 Vitamin K (phytonadione) intermediate II standard solution
Dilute 10,0 ml vitamin K intermediate I standard (6.5) to 50 ml with iso-octane. Prepare each time
new working standards are made.
6.7 Vitamin K (phytonadione) working standard solutions
Dilute 1,0 ml, 3,0 ml, 6,0 ml, 10,0 ml and 20,0 ml intermediate II standard (6.6) to 100 ml with iso-
octane. Store at 2 °C to 8 °C in a refrigerator in tightly closed containers protected from light. Expiration
is 3 months.
Transfer working standards to autosampler vials with Pasteur pipets or equivalent glass. Do not pour
the standards from the volumetric flasks into vials.
7 Apparatus
7.1 HPLC system.
Two isocratic pumps; autosampler capable of injecting 20 μl; fluorescence detector; instrument
degasser (optional), high-pressure mixing tee or T-junction, and post-column reactor column about
20 mm × 4 mm stainless steel e.g. old HPLC column with packing removed or equivalent. The system
should be configured as shown in Figure 1.
7.2 Analytical column, silica 150 mm × 3,0 mm, 3 μm, 60 Å, or equivalent.
7.3 Analytical balance, capable of weighing to the nearest 0,000 01 g.
7.4 Beakers, glass, assorted sizes.
7.5 Centrifuge.
7.6 Centrifuge tubes and caps, 50 ml glass tubes with caps lined with polytetrafluoroethylene (PTFE).
7.7 Cylinders, graduated, glass, assorted sizes.
7.8 Gas regulator, compatible with helium or nitrogen.
7.9 Gas sparge, tubing and filtering assembly.
7.10 Magnetic stirrer and stir bar, with rack to hold centrifuge tubes.
7.11 Pipet, disposable glass, Pasteur.
7.12 Pipet, mechanical, variable volume, 0,5 ml to 5 ml and 10 μl to 100 μl.
7.13 Pipet, repeating 5 ml and 25 ml or equivalent.
7.14 Spectrophotometer, capable of measuring absorbance at 248 nm.
7.15 Volumetric flasks, glass, Class A, assorted sizes.
7.16 Volumetric pipets, glass, Class A, assorted sizes.
7.17 Vortex mixers.
7.18 Yellow lights or yellow shields, with cutoff of at least 440 nm.
8 Procedure
8.1 Sample preparation
8.1.1 Liquid samples
For ready-to-feed liquids, mix samples well or homogenize to ensure homogeneity and accurately weigh
to 0,001 g, up to 4 g of sample into 50 ml centrifuge tubes. To liquids with sample masses less than 4 g,
4 © ISO 2019 – All rights reserved

add enough water (5.8) to the tubes so that the sample mass plus the amount of water added (g or ml)
equal about 4 and mix well.
8.1.2 Powder samples
If the powder sample homogeneity is unknown, assume that it is non-homogenous and proceed as for
dry-blended/non-homogenous powder samples.
8.1.3 Dry blended/non-homogenous powder samples
For dry blended/non-homogenous powder samples, accurately weigh approximately 25,0 g of powder
and add 200 g of water (5.8). Record all masses. Mechanically stir or mix by hand until a homogeneous
suspension is obtained. A homogenizer can be used when necessary. Accurately weigh to 0,001 g up to
4 g of homogeneous suspension into 50 ml centrifuge tubes. If less than 4 g of homogeneous suspension
are weighed, add enough water to the tubes so that the sample mass plus the amount of water added (g
or ml) equal about 4 and mix well.
8.1.4 Wet blended powder samples
For wet blended homogenous powder samples, accurately weigh to 0,000 1 g up to 0,5 g of powder into
50 ml centrifuge tubes. Add 4 ml of water (5.8) and mix well.
8.1.5 Extraction
Add 25 ml ± 2,0 ml methanol (5.6) to each sample just prior to vortexing or stirring. Methanol should
not be added to more than two samples consecutively without vortexing or stirring. Cap each centrifuge
tube. Vortex each sample for at least 30 s at a rate that causes a vortex within the tube and then allow
samples to sit undisturbed for at least 10 min, but no more than 40 min, or add a magnetic stir bar
(7.10) to each centrifuge tube, cap tubes and place onto a magnetic stir plate, and stir samples for at
least 10 min, but not more than 40 min, at a spin rate that causes a vortex. Begin timing after vortex
forms in the tubes.
Quantitatively add 10 ml ± 0,05 ml iso-octane (5.4) to each sample with a volumetric pipet and cap
tubes. Iso-octane can be added to all samples before vortexing or stirring any of the samples. Vortex
each sample for at least 45 s or stir each sample for at least 45 s at a rate that causes a vortex to form
within the tubes. Begin timing after vortex forms in the tubes. Add 5 ml ± 0,5 ml laboratory water (5.8)
to each sample and cap tubes. Vortex or shake each sample for at least 20 s or stir each sample for at
least 20 s at a spin rate that causes a vortex to form within the sample. Begin timing after vortex forms
in the tubes.
Centrifuge the samples until a clean separation of the isooctane and aqueous–methanol layers results.
The iso-octane layer should be a clear layer at the top of the centrifuge tube, and the aqueous–methanol
layer should be a cloudy layer below the iso-octane layer. In some samples, there may be a small emulsion
layer between the iso-octane and aqueous-methanol layers. A good separation of solvent layers can
usually be achieved by centrifuging samples for approximately 10 min at 800 relative centrifugal force.
Do not add ethanol to samples to remove emulsions. Ethanol will change the final dilution volume and
affect results.
Remove samples from the centrifuge and inspect each sample to verify that the iso-octane and
aqueous–methanol layers are separated. With a glass pipet, carefully rinse down the upper walls of the
centrifuge tube with a portion of the iso-octane layer. If the layers become mixed together, centrifuge
the sample again. Pipette a portion of the clear iso-octane layer into a labelled autosampler vial and cap
the vial.
8.2 HPLC analysis
8.2.1 Instrumental operating conditions
HPLC analytical column mobile phase flow rate: 0,4 ml/min;
Post-column flow rate: 0,4 ml/min;
Injection volume: 20 μl;
Run time: 20 min;
Fluorescence excitation and emission: 245 nm and 440 nm, respectively.
8.2.2 Instrument start-up
The system should be configured as shown in Figure 1.
Figure 1 — Vitamin K system configuration
If necessary, remove used zinc and repack the post-column reactor column with fresh zinc (5.11).
The zinc reactor column should be repacked whenever the S/N in the lowest standard is too low to
accurately integrate the vitamin K peak, linearity requirements (r ≥ 0,999) cannot be met unless the
highest standard is excluded from the curve, peak responses from injections of the same standard drop
6 © ISO 2019 – All rights reserved

by more than 7 % and the drop cannot be attributed to other system components, or the system back
pressure through the zinc reactor increases significantly and vitamin K peak widths begin to increase.
To repack the zinc reactor column, remove the hex nuts and retainers from both ends of the column
and force the used zinc out of the column with a thin wire or similar apparatus. Flush the zinc reactor
column with ethanol to remove residual zinc. Replace the hex nut and retainer on one end of the zinc
reactor column. Carefully transfer a small amount of fresh zinc powder to the reactor column with a
...