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.

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

• Status: Published
• Publication Date: 06-Jan-2019
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: ISO/TC 34 - Food products
• Drafting Committee: ISO/TC 34/WG 14 - Vitamins, carotenoids and other nutrients
• Current Stage: 6060 - International Standard published
• Due Date: 09-Aug-2018
• Completion Date: 07-Jan-2019

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 21446:2019(E)
ISO 2019
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ISO 21446:2019(E)
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© ISO 2019

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ii © ISO 2019 – All rights reserved
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ISO 21446:2019(E)
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

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ISO 21446:2019(E)
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
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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.
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ISO 21446:2019(E)
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.

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ISO 21446:2019(E)
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
248
ρ = (1)
419
where
A is the absorbance of the solution at 248 nm;
248
419
is the A value of vitamin K in hexane at 248 nm;
1cm
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.
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ISO 21446:2019(E)
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,

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ISO 21446:2019(E)

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.
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ISO 21446:2019(E)
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

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ISO 21446:2019(E)

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

spatula, and press down on the zinc in the column with an old HPLC piston or similar apparatus to pack

it tightly. Continue adding zinc and pressing it down until the level of zinc is even with the top of the

column. After the reactor column is full, replace the second retainer and hex nut. The more tightly zinc

is packed into the reactor column, the more symmetrical the vitamin K peaks will be.

When using a helium or nitrogen, sparge the mobile phase and post-column electrolyte solutions by

bubbling helium or nitrogen through them at a flow rate just fast enough to cause small ripples on the

surface of the mobile phase and post-column solutions. To maximize the life of the zinc reactor column,

sparge the mobile phase and post-column electrolyte solution for at least 30 min before connecting

the zinc reactor column if mobile phase and post-column solutions are flowing, or do not pump mobile

phase and post-column electrolyte solutions through the zinc reactor column until at least 30 mins

after sparging begins. Once the mobile phase and post-column electrolyte solutions have been sparged,

allow the column and post-column reactor to equilibrate with mobile phase flowing at 0,4 ml/min and

post-column electrolyte solution flowing at 0,4 ml/min for at least 30 min prior to the first injection

if the zinc reactor has been used for previous analyses, or for several hours if the zinc post-column

reactor has been freshly packed. Once the mobile phase and post-column solutions have been sparged,

reduce the helium or nitrogen flow rate so that only a small stream of helium or nitrogen bubbles are

visible in the mobile phase and post-column solutions and there is minimal disturbance to the surface

of these solutions. Bubble helium or nitrogen very slowly through the mobile phase and post-column

electrolyte solutions continuously throughout the entire run. Once the run has started, do not adjust

the helium or nitrogen flow rate.

When using an instrument degasser, allow the column and post-column reactor to equilibrate with

mobile phase flowing at 0,4 ml/min and post-column electrolyte solution flowing at 0,4 ml/min for at

least 30 min prior to the first injection.

Allow the fluorescence detector lamp to warm up 30 min prior to the first injection.

When the mobile phase and post-column electrolyte solution are continuously sparged with helium or

nitrogen or flow through an instrument degasser throughout a run, it is not necessary to pack the post-

column reactor with zinc at the beginning of every run. It should be possible to analyse hundreds of

extracts before the zinc reactor column shall be repacked.
8.3 HPLC of standards and samples

Inject the most concentrated standard (approximately 80 μg/l) and observe the response on the

fluorescence detector. If necessary, adjust the detector gain and sensitivity settings so that the standard

response is within the range of the detector. Once the detector settings have been determined, inject

the most concentrated standard 3 to 4 times and note the peak areas. If the system is equilibrated, the

RSD of the standard peak areas should be ≤ 2 %, and the peak areas should not steadily increase or

decrease by more than 4 % from the first injection to the third or fourth injection. If the RSD is > 2 %,

locate the source of the imprecision and correct it before beginning the sample analysis. If peak areas

steadily increase or decrease by more than 4 %, the system is not equilibrated and shall be allowed to

equilibrate longer. Once the system has reached equilibrium and the injection precision is ≤ 2 % RSD,

inject a set of standards, sample extracts, and another set of standards. Every set of sample extracts

shall be bracketed by standards.
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ISO 21446:2019(E)
8.4 Instrument shut-down
8.4.1 Short-term shut-down

After analysing a set of sample extracts, simultaneously turn off the flow on the mobile phase and post-

column electrolyte solution pumps. Remove the helium or nitrogen sparge lines from the mobile phase

and post-column electrolyte solutions and turn off the helium or nitrogen. Turn off the fluorescence

detector lamp.
8.4.2 Long-term shut-down

Remove the helium or nitrogen sparge lines from the mobile phase and post-column electrolyte solution

and turn off the helium or nitrogen. Turn off the fluorescence detector lamp. Remove and cap the zinc

reactor column. Flush the post-column pump and fluorescence detector cell with reagent alcohol to

remove residual zinc chloride, sodium acetate, and acetic acid.
9 Calculations

The vitamin K concentrations of samples analysed on the HPLC system are determined by comparison

of peak areas from samples of known weight with t
...