Infant formula and adult nutritionals -- Determination of free and total choline and free and total carnitine -- Liquid chromatography tandem mass spectrometry (HPLC-MS/MS)

This document specifies a method for the determination of total or free choline and carnitine in infant formula and adult nutritionals by liquid chromatography and tandem mass spectrometry (HPLC-MS/MS).

Formules infantiles et produits nutritionnels pour adultes -- Détermination de la teneur en choline totale et la teneur en carnitine par chromatographie en phase liquide et spectrométrie de masse en tandem (CL-SM/SM)

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Publication Date
05-Oct-2020
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ISO 21468:2020 - Infant formula and adult nutritionals -- Determination of free and total choline and free and total carnitine -- Liquid chromatography tandem mass spectrometry (HPLC-MS/MS)
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INTERNATIONAL ISO
STANDARD 21468
First edition
2020-10
Infant formula and adult
nutritionals — Determination of free
and total choline and free and total
carnitine — Liquid chromatography
tandem mass spectrometry (HPLC-
MS/MS)
Formules infantiles et produits nutritionnels pour adultes —
Détermination de la teneur en choline totale et la teneur en carnitine
par chromatographie en phase liquide et spectrométrie de masse en
tandem (CL-SM/SM)
Reference number
ISO 21468:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 21468:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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.
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Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 21468:2020(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 1

5 Reagents and materials ................................................................................................................................................................................. 1

6 Preparation of solutions ............................................................................................................................................................................... 2

6.1 Mobile phase A ....................................................................................................................................................................................... 2

6.2 Mobile phase B ........................................................................................................................................................................................ 2

6.3 HPLC injector wash ............................................................................................................................................................................. 2

6.4 Preparation of choline and carnitine stock solutions ............................................................................................ 3

6.5 Preparation of working standard solutions ................................................................................................................... 3

7 Apparatus ..................................................................................................................................................................................................................... 4

8 Procedure..................................................................................................................................................................................................................... 5

8.1 Sample preparation ............................................................................................................................................................................ 5

8.1.1 Samples needing reconstitution ......................................................................................................................... 5

8.1.2 Analysis of free carnitine and choline ........................................................................................................... 5

8.1.3 Analysis of total carnitine and choline .......................................................................................................... 5

8.2 Instrument parameters ................................................................................................................................................................... 6

8.2.1 HPLC parameters ............................................................................................................................................................. 6

8.2.2 LC-MS parameters .......................................................................................................................................................... 7

9 Calculations................................................................................................................................................................................................................ 8

10 Precision ....................................................................................................................................................................................................................... 9

10.1 General ........................................................................................................................................................................................................... 9

10.2 Repeatability ............................................................................................................................................................................................. 9

10.3 Reproducibility ....................................................................................................................................................................................... 9

11 Test report ................................................................................................................................................................................................................11

Annex A (informative) Example chromatograms .................................................................................................................................12

Annex B (informative) Precision data ..............................................................................................................................................................13

Bibliography .............................................................................................................................................................................................................................16

© ISO 2020 – All rights reserved iii
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ISO 21468:2020(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 2015.10: Determination

of Free and Total Choline and Free and Total Carnitine in Infant Formula and Adult/Pediatric Nutritional

Formula by Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS).

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 2020 – All rights reserved
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INTERNATIONAL STANDARD ISO 21468:2020(E)
Infant formula and adult nutritionals — Determination of
free and total choline and free and total carnitine — Liquid
chromatography tandem mass spectrometry (HPLC-MS/MS)
1 Scope

This document specifies a method for the determination of total or free choline and carnitine in infant

formula and adult nutritionals by liquid chromatography and tandem mass spectrometry (HPLC-MS/MS).

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 .electropedia .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

Samples are extracted in water for free carnitine and choline. For total carnitine and choline, samples

are digested with nitric acid and microwave-assisted heating. Free and total extracts are both diluted

with water, mixed with acetonitrile, and analysed using liquid chromatography (LC) with tandem mass

spectrometric (MS/MS) detection.
5 Reagents and materials

During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and high

purity MS grade water or water of equivalent purity.

Non-specific binding can occur with these analytes when using glassware, so plasticware should be used

at all times for standard/sample preparation. All laboratory plasticware should be single use whenever

possible. Positive displacement pipets are also mandatory for pipetting to avoid contamination and for

accuracy with organic solvents.
© ISO 2020 – All rights reserved 1
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ISO 21468:2020(E)
5.1 Water, purified, MS grade or equivalent purity.
5.2 Acetonitrile, MS grade.
5.3 Ammonium formate, MS grade or equivalent.
5.4 Formic acid, MS grade or equivalent.
5.5 Nitric acid, a mass fraction of 70 %, ACS grade or equivalent.

WARNING — All preparation steps with nitric acid should be performed within a fume hood. The

necessary personal protective equipment should be used when handling.
5.6 Isopropanol, MS grade or equivalent.
5.7 Desiccant.
5.8 L-carnitine, inner salt, primary reference standard. Store in a desiccator.
5.9 Choline bitartrate, primary reference standard.
5.10 L-carnitine-d HCL, primary reference standard.
5.11 Choline-1,1,2,2-d chloride, primary reference standard.
6 Preparation of solutions

CAUTION — All mobile phase bottles should be rinsed thoroughly prior to use with purified

water and isopropanol.
6.1 Mobile phase A

A mixture of 1 part per volume of 5 mmol/l ammonium formate in water and 1 part per volume of

acetonitrile, with 0,2 % formic acid.

Weigh 0,32 g ± 0,01g of ammonium formate into a 1 l bottle (7.16) containing 500 ml of purified water.

Add a stir bar and then mix on a stir plate until dissolved. Add 500 ml of acetonitrile and 2,00 μl of

formic acid to the mobile phase container. Mix on a stir plate until thoroughly mixed, typically for about

2 min. The solution is stable for two weeks when stored at room temperature.
6.2 Mobile phase B

A mixture of 1 part per volume of 30 mmol/l of ammonium formate in water and 1 part per volume of

acetonitrile, with 0,2 % formic acid.

Weigh 1,89 g ± 0,01g of ammonium formate into a 1 l mobile phase bottle containing 500 ml of purified

water. Add a stir bar and then mix on a stir plate until dissolved. Add 500 ml of acetonitrile and 2,00 μl

of formic acid to the mobile phase container. Mix on a stir plate until thoroughly mixed, typically for

about 2 min. The solution is stable for two weeks when stored at room temperature.

6.3 HPLC injector wash
Mobile phase B (see 6.2) or as recommended by supplier.
2 © ISO 2020 – All rights reserved
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ISO 21468:2020(E)
6.4 Preparation of choline and carnitine stock solutions

6.4.1 Stock and working standards are stable for six months when stored in a refrigerator set to

maintain 2 °C to 8 °C. Protect standard solutions from actinic light. Alternate weights or volumes may be

used to scale these preparations.
6.4.2 Choline stock solution, mass concentration, ρ ≈ 25 mg/ml.

Weigh 0,520 0 g ± 0,01 g of choline bitartrate into a 20 ml polypropylene container. Dissolve with

10,0 ml of purified water. Correct the final concentration of this solution for purity, moisture, and form

to represent free choline ion concentration in solution. The molecular weight (MW) of choline ion is

104,17, The MW of choline bitartrate is 253,25.
6.4.3 Carnitine stock solution, ρ ≈ 25 mg/ml.

Weigh 0,250 0 g ± 0,01 g of carnitine into a 20 ml polypropylene vial. Dissolve with 10,0 ml of

purified water.
Correct for moisture content and purity to represent carnitine concentration.
6.4.4 L-carnitine-d stock solution, IS 1, ρ ≈ 2,00 mg/ml.

Weigh 0,025 g ± 0,001 g of L-carnitine-d HCl into a 20 ml polypropylene vial. Dissolve with 10,0 ml of

purified water.
6.4.5 Choline-1,1,2,2-d stock solution, IS 2, ρ ≈ 2,00 mg/ml.

Weigh 0,031 00 g ± 0,001 g of choline-1,1,2,2-d chloride into a 20 ml polypropylene container. Dissolve

with 10,0 ml of purified water.
6.5 Preparation of working standard solutions

6.5.1 Prepare the working standard solutions according to Table 1 in polypropylene vials.

Table 1 — Preparation of working standard solutions
Source
Source Purified Final Prepared Extracted
concen-
Standard Source
volume water volume concentration concentration
tration
solution solution ID
mg/ml ml ml ml μg/ml ng/ml
stock
STD 6 solutions 25,0 2,00 each 6,00 10,0 5 000 5 000
(6.4.2, 6.4.3)
STD 5 stock
solutions 25,0 1,60 each 6,80 10,0 4 000 4 000
(6.4.2, 6.4.3)
STD 4 stock
solutions 25,0 0,800 each 8,40 10,0 2 000 2 000
(6.4.2, 6.4.3)
STD 3 STD6 5,00 1,00 9,00 10,0 500 500
STD 2 STD3 0,500 0,400 9,60 10,0 20 20
STD 1 STD3 0,500 0,200 9,80 10,0 10 10
NOTE Alternate masses or volumes may be used to scale these preparations.
© ISO 2020 – All rights reserved 3
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ISO 21468:2020(E)

6.5.2 Mixture of L-carnitine-d and choline-1,1,2,2-d internal standard working solution (IWS),

3 4
ρ ≈ 200 μg/ml.

Transfer 10 ml of IS 1 solution (6.4.4) and 10 ml of IS 2 solution (6.4.5) into a 100 ml tube (7.13). Add

80 ml of purified water to bring to a final volume of 100 ml.
7 Apparatus
7.1 HPLC system, Prominence (Shimadzu, Kyoto, Japan) or equivalent.

7.2 MS/MS system, API 4000 with Electrospray Ionization (ESI) (ABSciex, Framingham, MA) or

equivalent.
7.3 Mass spectrometry software, Analyst (ABSciex) or equivalent.

7.4 Analytical column, Zorbax 300-SCX, 3,0 mm × 50 mm, 5 µm (Agilent, Santa Clara, CA) or

equivalent.

7.5 Microwave, a commercial microwave designed for laboratory use, with a closed-vessel system and

controlled temperature ramping capability. Use manufacturer-recommended vessels, i.e. MARS6 or

equivalent.
1) 1) 1)

7.6 Microwave turntable, liner, and cap., MARSXpress , 55 ml PFA Teflon® , 40 position (CEM) or

equivalent.
7.7 Vortex mixer.
7.8 Analytical balance, precision to 0,001 g.
7.9 Horizontal shaker.
7.10 Magnetic stir plate.
7.11 Positive displacement pipets.
7.12 Repeater positive displacement pipet.
7.13 Polypropylene tubes, assorted sizes
7.14 Digestion vessels for microwave digestion.
®1)
7.15 Graduated polypropylene tube, e.g. Digitube or equivalent.
7.16 Glass containers, 1 l to2 l bottle.
7.17 Syringe filters, 0,45 µm PTFE (polytetrafluorethene).
7.18 Disposable syringes, 3 ml.

1) This is an example of a suitable product available commercially. This information is given for the convenience of

users of this document and does not constitute and endorsement by ISO of the product named. Equivalent products

may be used if they can be shown to lead to the same results.
4 © ISO 2020 – All rights reserved
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ISO 21468:2020(E)
7.19 Graduated cylinders, assorted sizes.
7.20 Magnetic stir bars.
7.21 Autosampler vials and caps, 1,5 ml silanized crimp top.
7.22 Microcentrifuge tubes, 1,5 ml polypropylene.
7.23 Bottle top dispenser, 5 ml acid resistant.
7.24 Desiccator, glass.
8 Procedure
8.1 Sample preparation
8.1.1 Samples needing reconstitution

Weigh 10,00 g ± 0,500 g of sample in a suitable disposable cup or beaker. Add purified water to bring the

total mass (including the powder mass) to 100,00 g ± 1,00 g. Add a stir bar and stir as fast as possible

without causing the sample to splatter. Stir for at least 10 min, but no longer than 30 min.

Masses may be adjusted as needed to accommodate different powder types and levels of analytes.

8.1.2 Analysis of free carnitine and choline

Weigh 0,1 g to 1,0 g of ready-to-feed (RTF) or reconstituted sample into a tared 50 ml tube (7.15)

depending on the concentration of each analyte (to fall within calibration curve). A reagent blank, a

reagent blank + internal standard (ISTD), and working standards shall be included for each analysis

and treated the same as samples through the analysis.

Add 50 μl of each working solution to separate 50 ml tubes (7.15). The final nominal concentrations for

these after they have gone through the sample preparation (diluted 1 000 times) are listed in 6.4 and

6.5 to be used for construction of the calibration curve.

Add 50 μl of mixed internal standard working solution (6.5.2) to each sample, working standards

solutions and reagent blank + ISTD.

Dilute to 25 ml with purified water, cap, and thoroughly mix the sample on a horizontal shaker. This

sample extract is stable for three days when stored refrigerated and protected from light.

If further dilution of samples is required, dilute the samples to appropriate concentrations using the

reagent blank + ISTD using polypropylene vessels to do the dilutions.

Transfer a 0,5 ml aliquot of extract into a microcentrifuge tube, along with 0,5 ml of acetonitrile. Mix well.

Filter samples t
...

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