Infant formula and adult nutritionals -- Simultaneous determination of total vitamins B1, B2, B3 and B6 -- Enzymatic digestion and LC-MS/MS

This document specifies a method for the simultaneous quantitative determination of four water-soluble vitamins in infant formula and related nutritional products, including relevant forms of vitamins B1, B2, B3 and B6 by enzymatic digestion and UHPLC-MS/MS. This document is not intended to be used on products where vitamins have not been added.

Préparations pour nourrissons et produits nutritionnels pour adultes -- Détermination simultanée de la teneur en vitamines B1, B2, B3 et B6 -- Digestion enzymatique et CL-SM/SM

Le présent document spécifie une méthode de détermination quantitative simultanée de la teneur en quatre vitamines hydrosolubles dans les préparations pour nourrissons et les produits nutritionnels associés, notamment les formes pertinentes de vitamines B1, B2, B3 et B6 par digestion enzymatique et CLUHP-SM/SM. Le présent document n'est pas destiné ŕ ętre utilisé pour des produits dans lesquels aucune vitamine n'a été ajoutée.

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Publication Date
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INTERNATIONAL ISO
STANDARD 21470
First edition
2020-11
Infant formula and adult
nutritionals — Simultaneous
determination of total vitamins B , B ,
1 2
B and B — Enzymatic digestion and
3 6
LC-MS/MS
Formules infantiles et produits nutritionnels pour adultes —
Détermination simultanée de la teneur en vitamines B , B , B et B
1 2 3 6
— Digestion enzymatique et CL-SM/SM
Reference number
ISO 21470:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 21470:2020(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.
ISO copyright office
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CH-1214 Vernier, Geneva
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 21470:2020(E)
Contents Page

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

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

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

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

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

5 Reagents and materials ................................................................................................................................................................................. 2

6 Standard and solution preparation .................................................................................................................................................. 3

6.1 Mobile phases and prepared solutions .............................................................................................................................. 3

6.2 Stable isotope labelled compounds, individual, internal standard stock solutions .................... 4

6.3 Stock standard solutions of native compounds .......................................................................................................... 5

6.4 Working standard solution preparation ........................................................................................................................... 6

6.5 Summary of standard and solution preparation ...................................................................................................... 7

7 Apparatus ..................................................................................................................................................................................................................... 7

8 Procedure..................................................................................................................................................................................................................... 9

8.1 Sample preparation ............................................................................................................................................................................ 9

8.1.1 Powdered products ....................................................................................................................................................... 9

8.1.2 Reconstituted powders and liquid products ........................................................................................... 9

8.2 Enzymatic digestion ........................................................................................................................................................................... 9

8.3 UHPLC-MS/MS analysis ................................................................................................................................................................... 9

8.3.1 UHPLC conditions ........................................................................................................................................................... 9

8.3.2 MS tune conditions .....................................................................................................................................................10

8.3.3 Mass transitions ............................................................................................................................................................10

8.3.4 LC-MS/MS equilibration .........................................................................................................................................11

8.4 Quality control ......................................................................................................................................................................................11

8.4.1 General...................................................................................................................................................................................11

8.4.2 Calibration curve ..........................................................................................................................................................11

9 Calculations.............................................................................................................................................................................................................11

10 Precision data .......................................................................................................................................................................................................13

10.1 General ........................................................................................................................................................................................................13

10.2 Repeatability ..........................................................................................................................................................................................13

10.3 Reproducibility ....................................................................................................................................................................................13

11 Test report ................................................................................................................................................................................................................15

Annex A (informative) Precision data ..............................................................................................................................................................16

Annex B (informative) Comparison between this document and EN 14122............................................................25

Annex C (informative) Comparison between this document and EN 14152 ............................................................27

Annex D (informative) Comparison between this document and EN 14164 ...........................................................29

Bibliography .............................................................................................................................................................................................................................31

© ISO 2020 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 21470:2020(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 2015.14: Simultaneous

Determination of Total Vitamins B , B , B , and B in Infant Formula and Related Nutritionals by Enzymatic

1 2 3 6
Digestion and 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
---------------------- Page: 4 ----------------------
INTERNATIONAL STANDARD ISO 21470:2020(E)
Infant formula and adult nutritionals — Simultaneous
determination of total vitamins B , B , B and B —
1 2 3 6
Enzymatic digestion and LC-MS/MS
1 Scope

This document specifies a method for the simultaneous quantitative determination of four water-

soluble vitamins in infant formula and related nutritional products, including relevant forms of vitamins

B , B , B and B by enzymatic digestion and UHPLC-MS/MS. This document is not intended to be used

1 2 3 6
on products where vitamins have not been added.
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/o bp
— 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

Samples are prepared by enzymatic digestion with papain and α-amylase to hydrolyse protein and

complex carbohydrate and acid phosphatase to free phosphorylated vitamin forms. Stable-isotope

labelled internal standards are incorporated into the sample preparation to correct for variability in

both the sample preparation and instrument response. A series of six mixed working standard solutions

spanning two orders of magnitude in vitamin concentration are used to generate calibration curves

based on the peak response ratio of the analyte to its stable-isotope labelled internal standard.

Prepared samples and working standard solutions are injected onto ultra-high pressure liquid

chromatograph (UPLC) interfaced to a triple-quadrupole mass spectrometer (MS/MS) for analysis. The

MS/MS is configured to monitor precursor-fragment ion pairs for each analyte and internal standard.

This reaction forms the basis for method selectivity. Analytes are quantified by least squares regression

using the response ratio of the analyte to its internal standard.
© ISO 2020 – All rights reserved 1
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ISO 21470:2020(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 Niacinamide (nicotinamide) (MW = 122,12), primary reference standard, e.g. USP Reference

Standard, catalogue #1462006 . Follow the manufacturer’s storage and handling directions.

5.2 Niacin (nicotinic acid) (MW = 123,11), primary reference standard, e.g. USP Reference Standard,

catalogue # 1461003 . Follow the manufacturer’s storage and handling directions.

5.3 Pyridoxine hydrochloride (MW = 205,64), primary reference standard, e.g. USP Reference

Standard, catalogue # 1587001 . Follow the manufacturer’s storage and handling directions.

5.4 Riboflavin (MW = 376,36), primary reference standard, e.g. USP Reference Standard, catalogue #

1603006 . Follow the manufacturer’s storage and handling directions.

5.5 Thiamine hydrochloride (MW = 337,27), primary reference standard, e.g. USP Reference Standard,

catalogue #1656002 . Follow the manufacturer’s storage and handling directions. Measure the moisture

content of the powder prior to use or use the supplier certificate of analysis (COA) moisture value.

5.6 Pyridoxamine dihydrochloride, Fluka Analytical Standard, catalogue #P9380 .
5.7 Pyridoxal hydrochloride, Sigma, catalogue #P9130 .
2 1)
5.8 H -Niacinamide, CDN Isotopes, catalogue #D-3457 .
2 1)
5.9 H -Nicotinic acid, CDN Isotopes, catalogue #D-4368 .
13 13

5.10 C-Pyridoxine: pyridoxine:HCl (4,5-bis( hydroxymethyl)- C ), Cambridge Isotope

4 4
Laboratory, catalogue #CLM-7563 .
2 1)
5.11 H -Pyridoxal, IsoSciences, catalogue #7098 .
2 1)
5.12 H -Pyridoxamine, IsoSciences, catalogue #7099 .
13 1)
5.13 C -Thiamine chloride, IsoSciences, catalogue #9209 .
13 15 1)
5.14 C , N-Riboflavin, IsoSciences, catalogue #7072 .
4 2

5.15 Acid phosphatase, type II from potato, 0,5 U/mg to 3,0 U/mg, Sigma, catalogue #P3752 .

5.16 Papain from Carica papaya, ≥ 3 U/mg, Sigma, catalogue #76220 .
5.17 α–amylase from aspergillus oryzae, 150 U/mg, Sigma, catalogue #A9857 .

5.18 Hydrochloric acid concentrated (substance concentration c = 12 mol/l), ACS grade, or equivalent.

5.19 Ammonium formate, for mass spectrometry (purity ≥ 99,0 %), Fluka 70221 or equivalent .

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 an endorsement by ISO of the product named. Equivalent products

may be used if they can be shown to lead to the same results.
2 © ISO 2020 – All rights reserved
---------------------- Page: 6 ----------------------
ISO 21470:2020(E)
5.20 Glacial acetic acid, Sigma ACS reagent grade, or equivalent .
5.21 Formic acid, Sigma ACS reagent grade, or equivalent .
5.22 Laboratory water, 18,0 MΩ, < 10 µg/kg TOC, or equivalent.
5.23 Methanol, Fisher LC-MS/MS Optima grade or EMD Omni-Solve LC-MS grade .

5.24 Ethylenediaminetetracetic acid, disodium salt dihydrate (EDTA), ACS grade (99 % to 101 %),

or equivalent.
5.25 Potassium phosphate dibasic, ACS grade (purity > 98 %), or equivalent.
5.26 meta-Phosphoric acid, ACS grade (33,5 % to 36,5 %), or equivalent.
5.27 Buffer solutions for pH meter calibration, pH = 4,0, 7,0 and 10,0.
5.28 Phosphoric acid, 85 g/100 g, ACS grade, or equivalent.
5.29 Potassium hydroxide, 40 g/100 g, ACS grade, or equivalent.
6 Standard and solution preparation
6.1 Mobile phases and prepared solutions

6.1.1 Mobile phase A, substance concentration c = 0,020 mol/l ammonium formate in water.

Using a graduated cylinder, transfer 500 ml laboratory water to a mobile phase reservoir. Add 0,631 g of

ammonium formate (5.19) and mix well. Expiration is three days.
6.1.2 Mobile phase B, methanol.
6.1.3 HCl solution, c = 0,12 mol/l.

Add approximately 300 ml of water to a 500 ml graduated cylinder. Add 5,0 ml ± 0,1 ml of concentrated

HCl solution (5.18) and swirl to mix. Bring to 500 ml with laboratory water and mix well.

6.1.4 Acetic acid solution, 1,0 ml/100 ml.

Add approximately 30 ml of water to a 500 ml graduated cylinder. Add 5,0 ml ± 0,1 ml of glacial acetic

acid (5.20) and swirl to mix. Bring to 500 ml with laboratory water and mix well.

6.1.5 Weak needle wash, 10 ml/100 ml methanol in water, expiration three months. Alternatively,

use week needle wash as recommended by the supplier.
6.1.6 Strong needle wash, methanol or as recommended by the supplier.
6.1.7 Ammonium formate solution, c = 0,050 mol/l.

Using a graduated cylinder, transfer 1 400 ml of laboratory water to an appropriate reservoir. Add

4,41 g of ammonium formate (5.19) and mix well. One 400 ml is adequate for 6 working standards and

32 samples. Scale as needed. Expiration is three days.
© ISO 2020 – All rights reserved 3
---------------------- Page: 7 ----------------------
ISO 21470:2020(E)
6.1.8 Mixed enzyme solution.

Using a graduated cylinder, transfer 200 ml of ammonium formate buffer (6.1.7) to an appropriate

reservoir. Add 200 mg ± 10 mg of acid phosphatase (5.15), 80 mg ± 5 mg of α-amylase (5.17) and

400 mg ± 10 mg of papain (5.16). Mix for 10 min with a magnetic stir plate and stir bar. Check pH and

adjust to 4,25 ± 0,25 with formic acid (5.21, approximately 100 μl). 200 ml is adequate for 6 working

standards and 32 samples. Scale as needed. Prepare fresh daily.

6.2 Stable isotope labelled compounds, individual, internal standard stock solutions

6.2.1 Internal standard stock solutions have an expiration of six months. However, the following

guidelines can be used to troubleshoot internal standards and, when documented as part of routine

system suitability checks, extend the expiration dates indefinitely.

Based on US FDA bioanalytical method validation guidelines, which state that the lowest-level

[9]

calibration shall be five times the analyte response of the blank, the channel of the non-labelled

analyte of interest shall be monitored to ensure the stable isotope-labelled internal standard does not

contribute more than 20 % of the area count of the lowest-level calibration standard. No response should

be generated in any other channels being monitored in the method, as this is a sign of contamination, in

which case fresh solution should be prepared or fresh lot of material should be ordered.

The area count of the internal standard should be at least three times the area count of the analyte in

the lowest-level calibration standard and the lowest level matrix-based QC sample.

6.2.2 H -Niacinamide stock solution, mass concentration ρ ≈ 560 µg/ml.

Weigh 14,0 mg ± 0,1 mg into a tared weighing vessel. Quantitatively transfer to a 25 ml volumetric

flask with laboratory water and fill to the mark with laboratory water. Mix well and transfer to a 50 ml

amber bottle and store refrigerated (2 °C to 8 °C). For expiration, see 6.2.1.
6.2.3 H -Nicotinic acid stock solution, ρ ≈ 500 µg/ml.

Weigh 12,5 mg ± 0,1 mg into a tared weighing vessel. Quantitatively transfer to a 25 ml volumetric

flask with laboratory water and fill to the mark with laboratory water. Mix well and transfer to a 50 ml

amber bottle and store refrigerated (2 °C to 8 °C). For expiration, see 6.2.1.
6.2.4 C -Pyridoxine stock solution, ρ ≈ 70 µg/ml.

Weigh 7,0 mg ± 0,1 mg into a tared weighing vessel. Quantitatively transfer to a 100 ml volumetric flask

with laboratory water and fill to the mark with laboratory water. Mix well and transfer to a 100 ml

amber bottle and store refrigerated (2 °C to 8 °C). For expiration, see 6.2.1.
6.2.5 H -Pyridoxal stock solution, ρ ≈ 40 µg/ml.

Weigh 4,0 mg ± 0,1 mg into a tared weighing vessel. Quantitatively transfer to a 100 ml volumetric flask

with laboratory water and fill to the mark with laboratory water. Mix well and transfer to a 100 ml

amber bottle and store refrigerated (2 °C to 8 °C). For expiration, see 6.2.1.
6.2.6 H -Pyridoxamine stock solution, ρ ≈ 40 µg/ml.

Weigh 4,0 mg ± 0,1 mg into a tared weighing vessel. Quantitatively transfer to a 100 ml volumetric flask

with laboratory water and fill to the mark with laboratory water. Mix well and transfer to a 100 ml

amber bottle and store refrigerated (2 °C to 8 °C). For expiration, see 6.2.1.
4 © ISO 2020 – All rights reserved
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ISO 21470:2020(E)
6.2.7 C -Thiamine chloride stock solution, ρ ≈ 100 µg/ml.

Weigh 5,0 mg ± 0,1 mg of C -thiamine into a tared weighing vessel. Quantitatively transfer to a 50 ml

volumetric flask with HCl solution (6.1.2) and fill to the mark with HCl solution (6.1.2). Mix well and

transfer to a 100 ml amber bottle and store refrigerated (2 °C to 8 °C). For expiration, see 6.2.1.

13 15
6.2.8 C , N-Riboflavin stock solution, ρ ≈ 73 µg/ml.
4 2
13 15

Weigh 7,3 mg ± 0,1 mg of C , N -riboflavin into a tared weighing vessel. Quantitatively transfer to

4 2

a 100 ml volumetric flask with acetic acid solution (6.1.3) and fill to the mark with acetic acid solution

(6.1.3). Mix well and transfer to a 100 ml amber bottle and store refrigerated (2 °C to 8 °C). For

expiration, see 6.2.1.
6.2.9 Internal standard stock mixture (ISSM).

Combine 2 500 μl of ammonium formate solution (6.1.7) with 250 μl of H -niacinamide stock solution

2 13

(6.2.2), 250 μl of H -nicotinic acid stock solution (6.2.3), 250 μl of C -pyridoxine stock solution (6.2.4),

4 4
2 2

200 μl of H -pyridoxal stock solution (6.2.5), 50 μl of H -pyridoxamine stock solution (6.2.6), 250 μl

3 3
13 13 15

of C -thiamine stock solution (6.2.7) and 250 μl of C , N -riboflavin stock solution (6.2.8). Volume

4 4 2

provides sufficient ISSM for 6 working standards and 32 samples. Scale as needed. Prepare fresh daily.

6.2.10 Phosphate buffer solution, pH = 5,0 (0,010 mol/l potassium phosphate dibasic, 1 g/100 g EDTA,

2 g/100 g metaphosphoric acid.

Weigh 20,0 g ± 0,2 g of EDTA into a tared weighing vessel and quantitatively transfer to a 2 000 ml

beaker containing approximately 1 800 ml laboratory water and add a magnetic stir bar.

Weigh 34,8 g ± 0,1 g of potassium phosphate dibasic into a tared weighing vessel and quantitatively

transfer to the 2 000 ml beaker already containing approximately 1 800 ml laboratory water and

EDTA. Mix by stirring on a magnetic stir plate until both the EDTA and potassium phosphate dibasic is

completely dissolved.

Weigh 40,0 g ± 0,2 g of metaphosphoric acid into a tared weighing vessel and quantitatively transfer

to the 2 000 ml beaker containing approximately 1 800 ml laboratory water, EDTA, and potassium

phosphate dibasic. Mix by stirring on a magnetic stir plate until the metaphosphoric acid is completely

dissolved.

Adjust the pH of the solution to pH = 5,00 ± 0,02 using 40 g/100 g potassium hydroxide or 85 g/100 g

phosphoric acid. Quantitatively transfer the solution to a 2 000 ml volumetric flask and dilute to volume

with laboratory water. Expiration: 48 hours.
6.3 Stock standard solutions of native compounds
6.3.1 Vitamin standard stock mixture (VSSM).

Accurately weigh the indicated amounts for the following standards using separate weighing funnels

or other appropriate weighing vessels and quantitatively transfer to a 100 ml volumetric flask using

phosphate buffer (pH = 5).
a) Niacinamide (5.1): 70,5 mg ± 0,5 mg.
b) Thiamine hydrochloride (5.5): 10,5 mg ± 0,2 mg.

Determine the moisture of the thiamine hydrochloride reference standard (5.5) as directed on

the container immediately prior to weighing or use moisture content from the supplier COA. The

per cent moisture determined for the reference standard is used to calculate the concentration of

thiamine in the VSSM.
c) Riboflavin (5.4): 7,0 mg ± 0,2 mg.
© ISO 2020 – All rights reserved 5
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ISO 21470:2020(E)
d) Pyridoxine hydrochloride (5.3): 10,8 mg ± 0,2 mg.

Fill to volume with phosphate buffer (pH = 5) solution. Heat and slowly stir until the standards have

completely dissolved (riboflavin dissolves more slowly) and the solution is clear. Do not heat the

solution for more than 40 min and do not exceed 90 °C. Store refrigerated (2 °C to 8 °C). Expiration:

three months.
6.3.2 Nicotinic acid stock solution, ρ = 550 mg/ml.

Accurately weigh 13,7 mg ± 0,1 mg niacin primary reference standard (5.2). Quantitatively transfer the

nicotinic acid to a 25 ml volumetric flask. Add laboratory water to a total volume of about 20 ml and swirl

until completely dissolved. Bring to volume with laboratory water. Mix well. Expiration: three months.

6.3.3 Pyridoxal stock solution, ρ = 140 mg/ml.

Accurately weigh 17,0 mg ± 0,5 mg pyridoxal dihydrochloride standard (5.7). Quantitatively transfer

to a 100 ml volumetric flask. Add laboratory water to a total volume of about 70 ml and swirl until

completely dissolved. Bring to volume with laboratory water. Mix well. Expiration: three months.

6.3.4 Pyridoxamine stock solution, ρ = 160 mg/ml.

Accurately weigh 23,0 mg ± 0,5 mg pyridoxamine hydrochloride standard (5.6). Quantitatively transfer

to a 100 ml volumetric flask. Add laboratory water to a total volume of about 70 ml and swirl until

completely dissolved. Bring to volume with laboratory water. Mix well. Expiration: three months.

6.3.5 Mixed working standard (MWS).

Combine 500 μl VSSM (6.3.1), 25 μl pyridoxamine stock (6.3.4), 25 μl pyridoxal stock (6.3.3), and 65 μl

nicotinic acid stock solutions (6.3.2) in a 10 ml volumetric flask containing approximately 5 ml of

ammonium formate solution (6.1.7). Bring to volume with ammonium formate solution (6.1.7) and mix

well. Prepare fresh daily.
6.4 Working standard solution preparation
6.4.1 Working solution (WS) 1.

Add 20 μl of MWS (6.3.5) and 980 μl of ammonium formate (6.1.7) to a 50 ml centrifuge tube. Add

100 μl of ISSM (6.2.9), and vortex to mix. Prepare fresh daily.
6.4.2 Working solution (WS) 2.

Add 50 μl of MWS (6.3.5) and 950 μl of ammonium formate (6.1.7) to a 50 ml centrifuge tube. Add

100 μl of ISSM (6.2.9), and vortex to mix. Prepare fresh daily.
6.4.3 Working solution (WS) 3.

Add 100 μl of MWS (6.3.5) and 900 μl of ammonium formate (6.1.7) to a 50 ml centrifuge tube. Add

100 μl of ISSM (6.2.9), and vortex to mix. Prepare fresh daily.
6.4.4 Working solution (WS) 4.

Add 200 μl of MWS (6.3.5) and 800 μl of ammonium formate (6.1.7) to a 50 ml centrifuge tube. Add

100 μl of ISSM (6.2.9), and vortex to mix. Prepare fresh daily.
6 © ISO 2020 – All rights reserved
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ISO 21470:2020(E)
6.4.5 Working solution (WS) 5.

Add 500 μl of MWS (6.3.5) and 500 μl of ammonium formate (6.1.7) to a 50 ml centrifuge tube. Add

100 μl of ISSM (6.2.9), and vortex to mix. Prepare fresh daily.
6.4.6 Working solution (WS) 6.

Add 1 000 μl of MWS (6.3.5). Add 100 μl of ISSM (6.2.9), and vortex to mix. Prepare fresh daily.

6.5 Summary of standard and solution preparation
See Table 1.
Table 1 — Summary of standard and solution preparation
Compound Mass Purity Mois- Volume Aliquot Volume Aliquot Aliquot Final
ture cor- stock stock of MWS of MWS of ISSM volume
rection solution (6.3.5) (6.2.9)
mg ml µl ml µl µl ml
Niacinamide
70,5 ± 0,5 0,999 1,000 100 500 10 see 6.4 100 30
(5.1)
Thiamine HCl
a b
10,5 ± 0,2 0997 0961 100 500 10 see 6.4 100 30
(5.5)
Riboflavin
7,0 ± 0,2 0,986 1,000 100 500 10 see 6.4 100 30
(5.4)
Pyridoxine
10,8 ± 0,2 0,999 1,000 100 500 10 see 6.4 100 30
(5.3)
Pyridoxal (5.7) 17,0 ± 0,5 0,990 1,000 100 25 10 see 6.4 100 30
Pyridoxamine
23,0 ± 0,5 0,980 1,000 100 25 10 see 6.4 100 30
(5.6)
Niacin
(nicotinic 13,7 ± 0,1 0,998 1,000 25 65 10 see 6.4 100 30
acid) (5.2)
Purity of the standard as defined by the manufacturer.

Moisture correction (1 – moisture content, from measurement or from the COA provided by the manufacturer).

7 Apparatus
7.1 Waters® Acquity BEH C18 column or equivalent, 2,1 mm x 100 mm, 1,7 μm.
7.2 UHPLC system, Waters Acquity Classic , or equivalent.

7.3 Tandem quadrupole mass spectrometer with ESI probe, Waters Xevo TQ-S , or equivalent.

7.4 Analytical balances.

A balance capable of accurately weighing 5,00 mg (for standards), a six-place balance, an analytical

five-place balance for sa
...

NORME ISO
INTERNATIONALE 21470
Première édition
2020-11
Préparations pour nourrissons et
produits nutritionnels pour adultes —
Détermination simultanée de la
teneur en vitamines B , B , B et B —
1 2 3 6
Digestion enzymatique et CL-SM/SM
Infant formula and adult nutritionals — Simultaneous
determination of total vitamins B , B , B and B — Enzymatic
1 2 3 6
digestion and LC-MS/MS
Numéro de référence
ISO 21470:2020(F)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 21470:2020(F)
DOCUMENT PROTÉGÉ PAR COPYRIGHT
© ISO 2020

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Publié en Suisse
ii © ISO 2020 – Tous droits réservés
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ISO 21470:2020(F)
Sommaire Page

Avant-propos ..............................................................................................................................................................................................................................iv

1 Domaine d’application ................................................................................................................................................................................... 1

2 Références normatives ................................................................................................................................................................................... 1

3 Termes et définitions ....................................................................................................................................................................................... 1

4 Principe .......................................................................................................................................................................................................................... 1

5 Réactifs et matériaux ....................................................................................................................................................................................... 2

6 Préparation des étalons et des solutions .................................................................................................................................... 3

6.1 Phases mobiles et solutions préparées .............................................................................................................................. 3

6.2 Composés marqués aux isotopes stables et solutions mères individuelles d’étalons

internes .......................................................................................................................................................................................................... 4

6.3 Solutions mères d’étalons de composés natifs ............................................................................................................ 6

6.4 Préparation de la solution d’étalons de travail ........................................................................................................... 7

6.5 Résumé de la préparation des étalons et des solutions ....................................................................................... 8

7 Appareillage .............................................................................................................................................................................................................. 8

8 Mode opératoire.................................................................................................................................................................................................... 9

8.1 Préparation de l’échantillon ........................................................................................................................................................ 9

8.1.1 Produits en poudre ........................................................................................................................................................ 9

8.1.2 Poudres reconstituées et produits liquides ...........................................................................................10

8.2 Digestion enzymatique .................................................................................................................................................................10

8.3 Analyse CLHP-SM/SM ....................................................................................................................................................................10

8.3.1 Conditions de CLUHP ................................................................................................................................................10

8.3.2 Conditions de réglage du SM ..............................................................................................................................11

8.3.3 Transitions des masses ...........................................................................................................................................11

8.3.4 Équilibrage du CL-SM/SM .....................................................................................................................................12

8.4 Contrôle qualité ...................................................................................................................................................................................12

8.4.1 Généralités .........................................................................................................................................................................12

8.4.2 Courbe d’étalonnage ..................................................................................................................................................12

9 Calculs ...........................................................................................................................................................................................................................13

10 Données de fidélité .........................................................................................................................................................................................14

10.1 Généralités ...............................................................................................................................................................................................14

10.2 Répétabilité .............................................................................................................................................................................................14

10.3 Reproductibilité ..................................................................................................................................................................................14

11 Rapport d’essai ....................................................................................................................................................................................................17

Annexe A (informative) Données de fidélité ..............................................................................................................................................19

Annexe B (informative) Comparaison entre le présent document et l’EN 14122 ...............................................28

Annexe C (informative) Comparaison entre le présent document et l’EN 14152 ...............................................31

Annexe D (informative) Comparaison entre le présent document et l’EN 14164...............................................33

Bibliographie ...........................................................................................................................................................................................................................35

© ISO 2020 – Tous droits réservés iii
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ISO 21470:2020(F)
Avant-propos

L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes

nationaux de normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est

en général confiée aux comités techniques de l'ISO. Chaque comité membre intéressé par une étude

a le droit de faire partie du comité technique créé à cet effet. Les organisations internationales,

gouvernementales et non gouvernementales, en liaison avec l'ISO participent également aux travaux.

L'ISO collabore étroitement avec la Commission électrotechnique internationale (IEC) en ce qui

concerne la normalisation électrotechnique.

Les procédures utilisées pour élaborer le présent document et celles destinées à sa mise à jour sont

décrites dans les Directives ISO/IEC, Partie 1. Il convient, en particulier, de prendre note des différents

critères d'approbation requis pour les différents types de documents ISO. Le présent document a été

rédigé conformément aux règles de rédaction données dans les Directives ISO/IEC, Partie 2 (voir www

.iso .org/ directives).

L'attention est attirée sur le fait que certains des éléments du présent document peuvent faire l'objet de

droits de propriété intellectuelle ou de droits analogues. L'ISO ne saurait être tenue pour responsable

de ne pas avoir identifié de tels droits de propriété et averti de leur existence. Les détails concernant

les références aux droits de propriété intellectuelle ou autres droits analogues identifiés lors de

l'élaboration du document sont indiqués dans l'Introduction et/ou dans la liste des déclarations de

brevets reçues par l'ISO (voir www .iso .org/ brevets).

Les appellations commerciales éventuellement mentionnées dans le présent document sont données

pour information, par souci de commodité, à l’intention des utilisateurs et ne sauraient constituer un

engagement.

Pour une explication de la nature volontaire des normes, la signification des termes et expressions

spécifiques de l'ISO liés à l'évaluation de la conformité, ou pour toute information au sujet de l'adhésion

de l'ISO aux principes de l’Organisation mondiale du commerce (OMC) concernant les obstacles

techniques au commerce (OTC), voir www .iso .org/ avant -propos.

Le présent document a été élaboré par le comité technique ISO/TC 34, Produits alimentaires, en

collaboration avec l’AOAC INTERNATIONAL. Il est publié par l’ISO, et séparément par l’AOAC

INTERNATIONAL. La méthode décrite dans le présent document est l’équivalent de la méthode officielle

de l’AOAC 2015.14: Simultaneous Determination of Total Vitamins B , B , B , and B in Infant Formula and

1 2 3 6
Related Nutritionals by Enzymatic Digestion and LC-MS/MS.

Il convient que l'utilisateur adresse tout retour d'information ou toute question concernant le présent

document à l'organisme national de normalisation de son pays. Une liste exhaustive desdits organismes

se trouve à l'adresse www .iso .org/ members .html.
iv © ISO 2020 – Tous droits réservés
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NORME INTERNATIONALE ISO 21470:2020(F)
Préparations pour nourrissons et produits nutritionnels
pour adultes — Détermination simultanée de la teneur
en vitamines B , B , B et B — Digestion enzymatique et
1 2 3 6
CL-SM/SM
1 Domaine d’application

Le présent document spécifie une méthode de détermination quantitative simultanée de la teneur en

quatre vitamines hydrosolubles dans les préparations pour nourrissons et les produits nutritionnels

associés, notamment les formes pertinentes de vitamines B , B , B et B par digestion enzymatique

1 2 3 6

et CLUHP-SM/SM. Le présent document n’est pas destiné à être utilisé pour des produits dans lesquels

aucune vitamine n’a été ajoutée.
2 Références normatives
Le présent document ne contient aucune référence normative.
3 Termes et définitions

Pour les besoins du présent document, les termes et définitions suivants s’appliquent.

L’ISO et l’IEC tiennent à jour des bases de données terminologiques destinées à être utilisées en

normalisation, consultables aux adresses suivantes:

— ISO Online browsing platform: disponible à l’adresse https:// www .iso .org/ obp

— IEC Electropedia: disponible à l’adresse http:// www .electropedia .org/
3.1
produit nutritionnel pour adultes

aliment spécialement formulé, complet sur le plan nutritionnel, consommé sous forme liquide, qui peut

constituer la seule source d’alimentation, constitué de n’importe quelle combinaison de lait, soja, riz,

lactosérum, protéine hydrolysée, amidon et acides aminés, avec et sans protéine intacte

3.2
préparation pour nourrissons

substitut du lait maternel spécialement fabriqué pour satisfaire à lui seul les besoins nutritionnels

des nourrissons pendant les premiers mois de leur vie, jusqu’à l’introduction d’une alimentation

complémentaire appropriée
[SOURCE: Codex Standard 72-1981]
4 Principe

Des échantillons sont préparés par digestion enzymatique avec de la papaïne et de l’α-amylase pour

hydrolyser les protéines, les glucides complexes et la phosphatase acide en des formes de vitamines

phosphorylées libres. Des étalons internes marqués aux isotopes stables sont incorporés dans la

préparation des échantillons pour corriger la variabilité de préparation des échantillons et de réponse

de l’instrument. Une série de six solutions de travail du mélange d’étalons couvrant deux ordres de

grandeur en termes de concentration en vitamines est utilisée pour générer des courbes d'étalonnage

d’après le facteur de réponse maximale de l’analyte à son étalon interne marqué aux isotopes stables.

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ISO 21470:2020(F)

Les échantillons préparés et les solutions d’étalons de travail sont injectés dans un chromatographe

liquide à ultra-haute pression (CLUHP) couplé à un spectromètre de masse triple quadripôle (SM/SM)

en vue d’être analysés. Le SM/SM est configuré pour suivre pour chaque analyte et étalon interne des

paires d’ions précurseur-fragment. Cette réaction constitue la base de la sélectivité de la méthode. Les

analytes sont quantifiés à l’aide de la méthode des moindres carrés en utilisant le facteur de réponse de

l’analyte à son étalon interne.
5 Réactifs et matériaux

Pendant l’analyse, sauf indication contraire, utiliser uniquement des réactifs de qualité analytique

reconnue, et de l'eau distillée ou déminéralisée ou de l'eau d'une pureté équivalente.

5.1 Niacinamide (nicotinamide) (MM = 122,12), étalon de référence primaire, par exemple étalon

de référence USP, référence 1462006 . Respecter les instructions de conservation et de manipulation

données par le fabricant.

5.2 Niacine (acide nicotinique) (MM = 123,11), étalon de référence primaire, par exemple étalon

de référence USP, référence 1461003 . Respecter les instructions de conservation et de manipulation

données par le fabricant.

5.3 Chlorhydrate de pyridoxine (MM = 205,64), étalon de référence primaire, par exemple étalon

de référence USP, référence 1587001 . Respecter les instructions de conservation et de manipulation

données par le fabricant.

5.4 Riboflavine (MM = 376,36), étalon de référence primaire, par exemple étalon de référence USP,

référence 1603006 . Respecter les instructions de conservation et de manipulation données par le

fabricant.

5.5 Chlorhydrate de thiamine (MM = 337,27), étalon de référence primaire, par exemple étalon

de référence USP, référence 1656002 . Respecter les instructions de conservation et de manipulation

données par le fabricant. Mesurer la teneur en humidité de la poudre avant emploi ou utiliser la valeur

d’humidité indiquée dans le certificat d’analyse du fournisseur.
5.6 Dichlorhydrate de pyridoxamine, Fluka Analytical Standard, référence P9380 .
5.7 Chlorhydrate de pyridoxal, Sigma, référence P9130 .
2 1)
5.8 H -niacinamide, CDN Isotopes, référence D-3457 .
2 1)
5.9 H -acide nicotinique, CDN Isotopes, référence D-4368 .
13 13

5.10 C -pyridoxine: pyridoxine: HCl (4,5-bis(hydroxyméthyl)- C ), Cambridge Isotope

4 4
Laboratory, référence CLM-7563 .
2 1)
5.11 H -pyridoxal, IsoSciences, référence 7098 .
2 1)
5.12 H -pyridoxamine, IsoSciences, référence 7099 .
13 1)
5.13 C -chlorure de thiamine, IsoSciences, référence 9209 .

1) Exemple de produit approprié disponible sur le marché. Cette information est donnée à l'intention des

utilisateurs du présent document et ne signifie nullement que l'ISO approuve ou recommande l'emploi exclusif du

produit ainsi désigné. Des produits équivalents peuvent être utilisés s'il est démontré qu'ils aboutissent aux mêmes

résultats.
2 © ISO 2020 – Tous droits réservés
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ISO 21470:2020(F)
13 15 1)
5.14 C , N-riboflavine, IsoSciences, référence 7072 .
4 2

5.15 Phosphatase acide, type II de pomme de terre, 0,5 U/mg à 3,0 U/mg, Sigma, référence P3752 .

5.16 Papaïne de Carica papaya, ≥ 3 U/mg, Sigma, référence 76220 .
5.17 α-amylase d’Aspergillus oryzae, 150 U/mg, Sigma, référence A9857 .

5.18 Acide chlorhydrique concentré (concentration c = 12 mol/l), qualité ACS, ou équivalent.

5.19 Formiate d’ammonium, pour la spectrométrie de masse (pureté ≥ 99,0 %), Fluka 70221 ou

équivalent .
5.20 Acide acétique glacial, Sigma, qualité réactif ACS, ou équivalent .
5.21 Acide formique, Sigma, qualité réactif ACS, ou équivalent .
5.22 Eau de laboratoire, 18,0 MΩ, < 10 µg/kg COT, ou équivalente.

5.23 Méthanol, Fisher, qualité LC-MS/MS Optima, ou EMD, qualité Omni-Solve LC-MS .

5.24 Acide éthylènediaminetétracétique, sel disodique dihydraté (EDTA), qualité ACS (99 % à

101 %), ou équivalent.

5.25 Phosphate de potassium dibasique, qualité ACS (pureté > 98 %), ou équivalent.

5.26 Acide métaphosphorique, qualité ACS (33,5 % à 36,5 %), ou équivalent.
5.27 Solutions tampons pour l’étalonnage du pH-mètre, pH = 4,0, 7,0 et 10,0.
5.28 Acide phosphorique, 85 g/100 g, qualité ACS, ou équivalent.
5.29 Hydroxyde de potassium, 40 g/100 g, qualité ACS, ou équivalent.
6 Préparation des étalons et des solutions
6.1 Phases mobiles et solutions préparées

6.1.1 Phase mobile A, concentration c = 0,020 mol/l de formiate d’ammonium dans l’eau.

À l’aide d’une éprouvette graduée, transférer 500 ml d’eau de laboratoire dans un réservoir de phase

mobile. Ajouter 0,631 g de formiate d’ammonium (5.19) et bien mélanger. La durée de conservation est

de trois jours.
6.1.2 Phase mobile B, méthanol.
6.1.3 Solution de HCl, c = 0,12 mol/l.

Ajouter environ 300 ml d’eau dans une éprouvette graduée de 500 ml. Ajouter 5,0 ml ± 0,1 ml de solution

de HCl concentrée (5.18) et remuer. Compléter à 500 ml avec de l’eau de laboratoire et bien mélanger.

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ISO 21470:2020(F)
6.1.4 Solution d’acide acétique, 1,0 ml/100 ml.

Ajouter environ 30 ml d’eau dans une éprouvette graduée de 500 ml. Ajouter 5,0 ml ± 0,1 ml d’acide

acétique glacial (5.20) et remuer. Compléter à 500 ml avec de l’eau de laboratoire et bien mélanger.

6.1.5 Liquide doux de rinçage de l’aiguille, 10 ml/100 ml de méthanol dans l’eau, d’une durée de

conservation de trois mois. Il est également possible d’utiliser le liquide doux de rinçage de l’aiguille

recommandé par le fournisseur.

6.1.6 Liquide fort de rinçage de l’aiguille, méthanol ou tel que recommandé par le fournisseur.

6.1.7 Solution de formiate d’ammonium, c = 0,050 mol/l.

À l’aide d’une éprouvette graduée, transférer 1 400 ml d’eau de laboratoire dans un réservoir approprié.

Ajouter 4,41 g de formiate d’ammonium (5.19) et bien mélanger. Un volume de 400 ml convient pour

6 solutions d’étalons de travail et 32 échantillons. Adapter si nécessaire. La durée de conservation est

de trois jours.
6.1.8 Solution du mélange d’enzymes

À l’aide d’une éprouvette graduée, transférer 200 ml de solution tampon formiate d’ammonium (6.1.7)

dans un réservoir approprié. Ajouter 200 mg ± 10 mg de phosphatase acide (5.15), 80 mg ± 5 mg

d’α-amylase (5.17) et 400 mg ± 10 mg de papaïne (5.16). Mélanger pendant 10 min avec une plaque

d’agitation magnétique et un barreau aimanté. Contrôler le pH et ajuster à 4,25 ± 0,25 avec de l’acide

formique (5.21, environ 100 μl). Un volume de 200 ml convient pour 6 solutions d’étalons de travail et

32 échantillons. Adapter si nécessaire. À préparer chaque jour.

6.2 Composés marqués aux isotopes stables et solutions mères individuelles d’étalons

internes

6.2.1 La durée de conservation des solutions mères d’étalons internes est de six mois. Toutefois, les

lignes directrices suivantes peuvent être utilisées pour résoudre les problèmes liés aux normes internes

et, lorsqu’elles sont documentées dans le cadre des contrôles de routine d’aptitude du système, pour

prolonger indéfiniment les dates limites d'utilisation.

Conformément aux lignes directrices de validation de la méthode bioanalytique de l’USFDA, qui

indiquent que le point le plus bas de la gamme d’étalonnage doit correspondre à cinq fois la réponse

[9]

de l’analyte du blanc , le canal de l’analyte d’intérêt non marqué doit être contrôlé pour s’assurer que

l'étalon interne marqué aux isotopes stables ne contribue pas à plus de 20 % de l’aire de l’étalon de

plus bas niveau. Il convient qu’aucune réponse ne soit générée dans les autres canaux surveillés dans

le cadre de la méthode. Dans le cas contraire, cela traduit une contamination, auquel cas il convient de

préparer une nouvelle solution ou de commander un nouveau lot de matériau.

Il convient que l’aire de l'étalon interne représente au moins trois fois l’aire de l’analyte de plus bas

niveau dans la gamme d’étalonnage et dans l'échantillon contrôle qualité de plus bas niveau.

6.2.2 Solution mère de H -niacinamide, concentration massique ρ ≈ 560 µg/ml.

Peser 14,0 mg ± 0,1 mg dans un récipient de pesée taré. Transférer quantitativement dans une fiole

jaugée de 25 ml contenant de l’eau de laboratoire et compléter jusqu’au trait avec de l’eau de laboratoire.

Bien mélanger et transférer dans un flacon ambré de 50 ml et conserver au réfrigérateur (entre 2 °C et

8 °C). Pour connaître la durée de conservation, voir 6.2.1.
6.2.3 Solution mère de H -acide nicotinique, ρ ≈ 500 µg/ml.

Peser 12,5 mg ± 0,1 mg dans un récipient de pesée taré. Transférer quantitativement dans une fiole

jaugée de 25 ml contenant de l’eau de laboratoire et compléter jusqu’au trait avec de l’eau de laboratoire.

4 © ISO 2020 – Tous droits réservés
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ISO 21470:2020(F)

Bien mélanger et transférer dans un flacon ambré de 50 ml et conserver au réfrigérateur (entre 2 °C et

8 °C). Pour connaître la durée de conservation, voir 6.2.1.
6.2.4 Solution mère de C -pyridoxine, ρ ≈ 70 µg/ml.

Peser 7,0 mg ± 0,1 mg dans un récipient de pesée taré. Transférer quantitativement dans une fiole jaugée

de 100 ml contenant de l’eau de laboratoire et compléter jusqu’au trait avec de l’eau de laboratoire. Bien

mélanger et transférer dans un flacon ambré de 100 ml et conserver au réfrigérateur (entre 2 °C et

8 °C). Pour connaître la durée de conservation, voir 6.2.1.
6.2.5 Solution mère de H -pyridoxal, ρ ≈ 40 µg/ml.

Peser 4,0 mg ± 0,1 mg dans un récipient de pesée taré. Transférer quantitativement dans une fiole jaugée

de 100 ml contenant de l’eau de laboratoire et compléter jusqu’au trait avec de l’eau de laboratoire. Bien

mélanger et transférer dans un flacon ambré de 100 ml et conserver au réfrigérateur (entre 2 °C et

8 °C). Pour connaître la durée de conservation, voir 6.2.1.
6.2.6 Solution mère de H -pyridoxamine, ρ ≈ 40 µg/ml.

Peser 4,0 mg ± 0,1 mg dans un récipient de pesée taré. Transférer quantitativement dans une fiole jaugée

de 100 ml contenant de l’eau de laboratoire et compléter jusqu’au trait avec de l’eau de laboratoire. Bien

mélanger et transférer dans un flacon ambré de 100 ml et conserver au réfrigérateur (entre 2 °C et

8 °C). Pour connaître la durée de conservation, voir 6.2.1.
6.2.7 Solution mère de C -chlorure de thiamine, ρ ≈ 100 µg/ml.

Peser 5,0 mg ± 0,1 mg de C -thiamine dans un récipient de pesée taré. Transférer quantitativement

dans une fiole jaugée de 50 ml contenant une solution de HCl (6.1.2) et compléter jusqu’au trait avec la

solution de HCl (6.1.2). Bien mélanger et transférer dans un flacon ambré de 100 ml et conserver au

réfrigérateur (entre 2 °C et 8 °C). Pour connaître la durée de conservation, voir 6.2.1.

13 15
6.2.8 Solution mère de C , N-riboflavine, ρ ≈ 73 µg/ml.
4 2
13 15

Peser 7,3 mg ± 0,1 mg de C , N -riboflavine dans un récipient de pesée taré. Transférer

4 2

quantitativement dans une fiole jaugée de 100 ml contenant une solution d’acide acétique (6.1.3) et

compléter jusqu’au trait avec la solution d’acide acétique (6.1.3). Bien mélanger et transférer dans un

flacon ambré de 100 ml et conserver au réfrigérateur (entre 2 °C et 8 °C). Pour connaître la durée de

conservation, voir 6.2.1.
6.2.9 Solution mère d’étalons internes (ISSM)

Mélanger 2 500 μl de solution de formiate d’ammonium (6.1.7) avec 250 μl de solution mère de H -

niacinamide (6.2.2), 250 μl de solution mère de H -acide nicotinique (6.2.3), 250 μl de solution mère de

13 2 2

C -pyridoxine (6.2.4), 200 μl de solution mère de H -pyridoxal (6.2.5), 50 μl de solution mère de H -

4 3 3

pyridoxamine (6.2.6), 250 μl de solution mère de C -thiamine (6.2.7) et 250 μl de solution mère de

13 15

C , N -riboflavine (6.2.8). Un volume suffisant d’ISSM convient pour 6 solutions d’étalons de travail

4 2
et 32 échantillons. Adapter si nécessaire. À préparer chaque jour.

6.2.10 Solution tampon phosphate, pH = 5,0 (0,010 mol/l de phosphate de potassium dibasique,

1 g/100 g d’EDTA, 2 g/100 g d’acide métaphosphorique).

Peser 20,0 g ± 0,2 g d’EDTA dans un récipient de pesée taré, transférer quantitativement dans un bécher

de 2 000 ml contenant environ 1 800 ml d’eau de laboratoire et ajouter un barreau aimanté.

Peser 34,8 g ± 0,1 g de phosphate de potassium dibasique dans un récipient de pesée taré et transférer

quantitativement dans le bécher de 2 000 ml contenant déjà environ 1 800 ml d’eau de laboratoire

et d’EDTA. Mélanger en agitant sur une plaque d'agitation magnétique jusqu’à ce que l’EDTA et le

phosphate de potassium dibasique soient complètement dissous.
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ISO 21470:2020(F)

Peser 40,0 g ± 0,2 g d’acide métaphosphorique dans un récipient de pesée taré et transférer

quantitativement dans le bécher de 2 000 ml contenant environ 1 800 ml d’eau de laboratoire, d’EDTA

et de phosphate de potassium dibasique. Mélanger en agitant sur une plaque d'agitation magnétique

jusqu’à ce que l’acide métaphosphorique soit complètement dissous.

Ajuster le pH de la solution à pH = 5,00 ± 0,02 en utilisant 40 g/100 g d’hydroxyde de potassium ou

85 g/100 g d’acide phosphorique. Transférer quantitativement la solution dans une fiole jaugée de

2 000 ml et diluer au volume avec de l’eau de laboratoire. Durée de conservation: 48 h.

6.3 Solutions mères d’étalons de composés natifs
6.3.1 Solution mère d’étalons de vitamines (VSSM)

Peser exactement les quantités indiquées pour les étalons suivants en utilisant des entonnoirs de pesée

séparés ou d’autres récipients de pesée appropriés et transférer quantitativement dans une fiole jaugée

de 100 ml en utilisant le tampon phosphate (pH = 5).
a) Niacinamide (5.1): 70,5 mg ± 0,5 mg.
b) Chlorhydrate de thiamine (5.5): 10,5 mg ± 0,2 mg.

Déterminer la teneur en humidité de l’étalon de référence chlorhydrate de thiamine (5.5) comme

indiqué sur le conditionnement juste avant de peser, ou utiliser la teneur en humidité notée dans le

certificat d’analyse du fournisseur. Le pourcentage d’humidité déterminé pour l’étalon de référence

sert à calculer la concentration en thiamine de la VSSM.
c) Riboflavine (5.4): 7,0 mg ± 0,2 mg.
d) Chlorhydrate de pyridoxine (5.3): 10,8 mg ± 0,2 mg.

Compléter au volume avec la solution tampon phosphate (pH = 5). Chauffer et agiter lentement jusqu’à ce

que les étalons soient complètement dissous (la riboflavine se dissout plus lentement) et que la solution

soit claire. Ne pas chauffer la solution pendant plus de 40 min et ne pas dépasser 90 °C. Conserver au

réfrigérateur (entre 2 °C et 8 °C). Durée de conservation: trois mois.
6.3.2 Solution mère d’acide nicotinique, ρ = 550 mg/ml.

Peser exactement 13,7 mg ± 0,1 mg d'étalon primaire de référence niacine (5.2). Transférer

quantitativement l’acide nicotinique dans une fiole jaugée de 25 ml. Ajouter de l’eau de laboratoire

jusqu’à atteindre un volume total d’environ 20 ml et remuer jusqu’à dissolution complète. Compléter au

volume avec de l’eau de laboratoire. Bien mélanger. Durée de conservation: trois mois.

6.3.3 Solution mère de pyridoxal, ρ = 140 mg/ml.

Peser exactement 17,0 mg ± 0,5 mg d'étalon dichlorhydrate de pyridoxal (5.7). Transférer

quantitativement dans une fiole jaugée de 100 ml. Ajouter de l’eau de laboratoire jusqu’à atteindre un

volume total d’environ 70 ml et remuer jusqu’à dissolution complète. Compléter au volume avec de l’eau

de laboratoire. Bien mélanger. Durée de conservation: trois mois.
6.3.4 Solution mère de pyridoxamine, ρ = 160 mg/ml.

Peser exactement 23,0 mg ± 0,5 mg d'étalon chlorhydrate de pyridoxamine (5.6). Transfér

...

DRAFT INTERNATIONAL STANDARD
ISO/DIS 21470
ISO/TC 34 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2019-12-25 2020-03-18
Infant formula and adult nutritionals — Simultaneous
determination of total vitamins B1, B2, B3 and B6 —
Enzymatic digestion and LC-MS/MS

Formules infantiles et produits nutritionnels pour adultes — Détermination simultanée de la teneur en

vitamines B1, B2, B3 et B6 - Digestion enzymatique et CL-SM/SM
ICS: 67.050
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
This document is circulated as received from the committee secretariat.
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 21470:2019(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. ISO 2019
---------------------- Page: 1 ----------------------
ISO/DIS 21470:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
---------------------- Page: 2 ----------------------
ISO/DIS 21470:2019(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 .................................................................................................................................................. 4

7 Apparatus ..................................................................................................................................................................................................................... 8

8 Procedure..................................................................................................................................................................................................................... 9

8.1 Sample preparation ............................................................................................................................................................................ 9

8.1.1 Powdered products. ...................................................................................................................................................... 9

8.1.2 Reconstituted powders and liquid products. .......................................................................................... 9

8.2 Enzymatic digestion ........................................................................................................................................................................... 9

8.3 UPLC-MS/MS analysis ....................................................................................................................................................................10

8.3.1 UPLC conditions ............................................................................................................................................................10

8.3.2 MS tune conditions .....................................................................................................................................................10

8.3.3 Mass transitions ............................................................................................................................................................10

8.3.4 LC-MS/MS equilibration .........................................................................................................................................11

8.4 Quality control ......................................................................................................................................................................................11

8.4.1 General...................................................................................................................................................................................11

8.4.2 Calibration curve ..........................................................................................................................................................11

9 Calculations.............................................................................................................................................................................................................11

10 Precision data .......................................................................................................................................................................................................13

10.1 General ........................................................................................................................................................................................................13

10.2 Repeatability ..........................................................................................................................................................................................13

10.3 Reproducibility ....................................................................................................................................................................................13

11 Test report ................................................................................................................................................................................................................15

Annex A (informative) Precision data ..............................................................................................................................................................16

Bibliography .............................................................................................................................................................................................................................24

© ISO 2019 – All rights reserved iii
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ISO/DIS 21470:2019(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 2015.14: Simultaneous

Determination of Total Vitamins B , B , B , and B in Infant Formula and Related Nutritionals by Enzymatic

1 2 3 6
Digestion and 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 2019 – All rights reserved
---------------------- Page: 4 ----------------------
DRAFT INTERNATIONAL STANDARD ISO/DIS 21470:2019(E)
Infant formula and adult nutritionals — Simultaneous
determination of total vitamins B1, B2, B3 and B6 —
Enzymatic digestion and LC-MS/MS
1 Scope

This document specifies a method for the simultaneous quantitative determination of four water-

soluble vitamins in infant formula and related nutritional products, including relevant forms of vitamins

B , B , B and B by enzymatic digestion and UHPLC-MS/MS.
1 2 3 6
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 prepared by enzymatic digestion with papain and α-amylase to hydrolyze protein and

complex carbohydrate and acid phosphatase to free phosphorylated vitamin forms. Stable-isotope

labelled internal standards are incorporated into the sample preparation to correct for variability in

both the sample preparation and instrument response. A series of six mixed working standard solutions

spanning two orders of magnitude in vitamin concentration are used to generate calibration curves

based on the peak response ratio of the analyte to its stable-isotope labelled internal standard.

Prepared samples and working standard solutions are injected onto ultra-high pressure liquid

chromatograph (UPLC) interfaced to a triple-quadrupole mass spectrometer (MS/MS) for analysis. The

MS/MS is configured to monitor parent-daughter (precursor-fragment) ion pairs for each analyte and

internal standard. This reaction forms the basis for method selectivity. Analytes are quantified by least

squares regression using the response ratio of the analyte to its internal standard.

© ISO 2019 – All rights reserved 1
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ISO/DIS 21470:2019(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 Nicotinamide, primary reference standard, for example USP Reference Standard, Catalog #

1462006 .
Store as indicated on label.

5.2 Niacin (nicotinic acid), primary reference standard, for example USP Reference Standard,

Catalog # 1461003 .
Stored as indicated on label.

5.3 Pyridoxine hydrochloride, primary reference standard, for example USP Reference Standard,

Catalog # 1587001 .

Store in desiccator protected from white light. Dry according to manufacturer’s instructions prior to use.

5.4 Riboflavin, primary reference standard, for example USP Reference Standard,

Catalog # 1603006 .

Store in desiccator protected from white light. Dry according to manufacturer’s instructions prior to use.

5.5 Thiamine hydrochloride, primary reference standard, for example USP Reference Standard,

Catalog # 1656002 .

Store in desiccator protected from white light. Measure the moisture content of the powder prior to use.

5.6 Pyridoxamine dihydrochloride
Fluka Analytical Standard, catalog# P9380.
5.7 Pyridoxal hydrochloride
Sigma, catalog# P9130.
5.8 H -Nicotinamide
CDN Isotopes, Catalog # D-3457 .
5.9 H -Nicotinic acid
CDN Isotopes; Catalog # D-4368 .
13 13
5.10 C -Pyridoxine: pyridoxine:HCl (4,5-bis(h ydroxymethyl)- C )
4 4
Cambridge Isotope Laboratory;Catalog # CLM-7563 .
5.11 H -Pyridoxal.
IsoSciences; Catalog # 7098 .

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 an endorsement by ISO of the product named. Equivalent products

may be used if they can be shown to lead to the same results.
2 © ISO 2019 – All rights reserved
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ISO/DIS 21470:2019(E)
5.12 H -Pyridoxamine.
IsoSciences; Catalog # 7099 .
5.13 C -Thiamine chloride.
IsoSciences; Catalog # 9209 .
13 15
5.14 C , N-Riboflavin.
4 2
IsoSciences, Catalog# 7072 .
5.15 Acid phosphatase, type II from potato, 0,5 U/mg to 3,0 U/mg.
Sigma, catalog# P3752 .
5.16 Papain from Carica papaya, ≥ 3 U/mg.
Sigma, catalog# 76220 .
5.17 α–amylase from aspergillus oryzae, 150 U/mg.
Sigma, catalog# A9857 .
5.18 Hydrochloric acid concentrated (substance concentration c = 12 mol/l).
ACS grade, or equivalent.
5.19 Ammonium formate, for mass spectrometry (purity ≥ 99,0%).
Fluka 70221 or equivalent .
5.20 Glacial acetic acid.
Sigma ACS Reagent Grade, or equivalent .
5.21 Formic acid.
Sigma ACS Reagent Grade, or equivalent .
5.22 Laboratory water.
18,0 MΩ, < 10 ppb TOC, or equivalent.
5.23 Methanol.
Fisher LC-MS/MS Optima grade or EMD Omni-Solve LC-MS grade .
5.24 Ethylenediaminetetracetic acid, disodium salt dihydrate (EDTA).
ACS grade (99 % to 101 %), or equivalent.
5.25 Potassium phosphate dibasic.
ACS grade (purity > 98 %), or equivalent.
© ISO 2019 – All rights reserved 3
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ISO/DIS 21470:2019(E)
5.26 meta-Phosphoric acid.
ACS grade (33,5 % to 36,5 %), or equivalent.
5.27 Buffer solutions for pH meter calibration, pH = 4,0, 7,0, and 10,0.
5.28 Phosphoric acid, 85 %.
ACS grade, or equivalent.
5.29 Potassium hydroxide, 40 %.
ACS grade, or equivalent.
6 Standard and solution preparation

6.1 Mobile phase A, substance concentration c = 0,020 mol/l ammonium formate in water.

Using a graduated cylinder, transfer 500 ml laboratory water to a mobile phase reservoir. Add 0,631 g of

ammonium formate (5.19) and mix well. Expiration is 3 days.
6.2 HCl solution, c = 0,12 mol/l.

Add approximately 300 ml of water to a 500 ml graduated cylinder. Add 5,0 ml + 0,1 ml of concentrated

HCl solution (5.18) and swirl to mix. Bring to 500 ml with laboratory water and mix well.

6.3 Acetic acid solution, 1,0 %.

Add approximately 30 ml of water to a 500 ml graduated cylinder. Add 5,0 ml + 0,1 ml of glacial acetic

acid (5.20) and swirl to mix. Bring to 500 ml with laboratory water and mix well.

6.4 Mobile phase B, methanol.
6.5 Weak needle wash, 10 % methanol in water, expiration 3 months.
6.6 Strong needle wash, methanol.
6.7 Ammonium formate solution, c = 0,050 mol/l.

Using a graduated cylinder, transfer 1 400 ml of laboratory water to an appropriate reservoir. Add

4,41 g of ammonium formate (5.19) and mix well. 1 400 ml is adequate for 6 working standards and 32

samples. Scale as needed. Expiration is 3 days.
6.8 Mixed enzyme solution

Using a graduated cylinder, transfer 200 ml of ammonium formate buffer (6.7) to an appropriate

reservoir. Add 200 mg ± 10 mg of acid phosphatase (5.15), 80 mg ± 5 mg of α-amylase (5.17) and 400 mg

± 10 mg of papain (5.16). Mix for 10 min with a magnetic stir plate and stir bar. Check pH and adjust

to 4,25 + 0,25 with formic acid (5.21, ~ 100 μl). 200 ml is adequate for 6 working standards and 32

samples. Scale as needed. Prepare fresh daily.
4 © ISO 2019 – All rights reserved
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ISO/DIS 21470:2019(E)

6.9 Stable isotope labelled compounds, individual, internal standard stock solutions

6.9.1 General

Internal standard stock solutions have an expiration of 6 months. However, the following guidelines can

be used to troubleshoot internal standard and, when documented as part of routine system suitability

checks, extend the expiration dates indefinitely.

Based on U.S. FDA bioanalytical method validation guidelines that the lowest-level calibration shall be 5

[9]

times the analyte response of the blank, see, the channel of the non-labelled analyte of interest shall

be monitored to ensure the stable isotope-labelled internal standard does not contribute more than

20 % of the area count of the lowest-level calibration standard. No response should be generated in any

other channels being monitored in the method, as this is a sign of contamination, in which case fresh

solution should be prepared or fresh lot of material should be ordered.

The area count of the internal standard should be at least 3 times the area count of the analyte in the

lowest-level calibration standard and the lowest level matrix-based QC sample.
6.9.2 H -Nicotinamide stock solution, mass concentration ρ ≈ 560 µg/ml.

Weigh 14,0 mg + 0,1 mg into a tarred weighing vessel. Quantitatively transfer to a 25 ml volumetric

flask with laboratory water and fill to the mark with laboratory water. Mix well and transfer to a 50 ml

amber bottle and store refrigerated (2 ˚C to 8 ˚C). Expiration, see 6.9.1.
6.9.3 H -Nicotinic acid stock solution, ρ ≈ 500 µg/ml.

Weigh 12,5 mg + 0,1 mg into a tarred weighing vessel. Quantitatively transfer to a 25 ml volumetric

flask with laboratory water and fill to the mark with laboratory water. Mix well and transfer to a 50 ml

amber bottle and store refrigerated (2 ˚C to 8 ˚C). Expiration, see 6.9.1.
6.9.4 C -Pyridoxine stock solution, ρ ≈ 70 µg/ml.

Weigh 7,0 mg + 0,1 mg into a tarred weighing vessel. Quantitatively transfer to a 100 ml volumetric

flask with laboratory water and fill to the mark with laboratory water. Mix well and transfer to a 100 ml

amber bottle and store refrigerated (2 ˚C to 8 ˚C). Expiration, ee 6.9.1.
6.9.5 H -Pyridoxal stock solution, ρ ≈ 40 µg/ml.

Weigh 4,0 mg + 0,1 mg into a tarred weighing vessel. Quantitatively transfer to a 100 ml volumetric

flask with laboratory water and fill to the mark with laboratory water. Mix well and transfer to a 100 ml

amber bottle and store refrigerated (2 ˚C to 8 ˚C). Expiration, ee 6.9.1.
6.9.6 H -Pyridoxamine stock solution, ρ ≈ 40 µg/ml.

Weigh 4,0 mg + 0,1 mg into a tarred weighing vessel. Quantitatively transfer to a 100 ml volumetric

flask with laboratory water and fill to the mark with laboratory water. Mix well and transfer to a 100 ml

amber bottle and store refrigerated (2 ˚C to 8 ˚C). Expiration, ee 6.9.1.
6.9.7 C -Thiamine chloride stock solution, ρ ≈ 100 µg/ml.

Weigh 5,0 mg + 0,1 mg of C -thiamine into a tarred weighing vessel. Quantitatively transfer to a

50 ml volumetric flask with HCl solution (6.2) and fill to the mark with HCl solution (6.2). Mix well and

transfer to a 100 ml amber bottle and store refrigerated (2 ˚C to 8 ˚C). Expiration, ee 6.9.1.

© ISO 2019 – All rights reserved 5
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ISO/DIS 21470:2019(E)
13 15
6.9.8 C , N-Riboflavin stock solution, ρ ≈ 73 µg/ml.
4 2
13 15

Weigh 7,3 mg + 0,1 mg of C , N -riboflavin into a tarred weighing vessel. Quantitatively transfer to a

4 2

100 ml volumetric flask with acetic acid solution (6.3) and fill to the mark with acetic acid solution (6.3).

Mix well and transfer to a 100 ml amber bottle and store refrigerated (2 ˚C to 8 ˚C). Expiration, ee 6.9.1.

6.10 Internal standard stock mixture (ISSM).

Combine 2 500 μl of ammonium formate solution (6.7) with 250 μl of H -nicotinamide stock solution

2 13

(6.9.2), 250 μl of H -nicotinic acid stock solution (6.9.3), 250 μl of C -pyridoxine stock solution (6.9.4),

4 4
2 2

200 μl of H -pyridoxal stock solution (6.9.5), 50 μl of H -pyridoxamine stock solution (6.9.6), 250 μl

3 3
13 13 15

of C -thiamine stock solution (6.9.7) and 250 μl of C , N -riboflavin stock solution (6.9.8). Volume

4 4 2

provides sufficient ISSM for 6 working standards and 32 samples. Scale as needed. Prepare fresh daily.

6.11 Phosphate buffer solution, pH = 5,0 (0,010 mol/l potassium phosphate dibasic, 1 % EDTA, 2 %

metaphosphoric acid.

Weigh 20,0 g ± 0,2 g of EDTA into a tarred weighing vessel and quantitatively transfer to a 2 000 ml

beaker containing approximately 1 800 ml laboratory water and add a magnetic stir bar.

Weigh 34,8 g ± 0,1 g of potassium phosphate dibasic into a tarred weighing vessel and quantitatively

transfer to the 2 000 ml beaker already containing approximately 1 800 ml laboratory water and

EDTA. Mix by stirring on a magnetic stir plate until both the EDTA and potassium phosphate dibasic is

completely dissolved.

Weigh 40,0 g ± 0,2 g of metaphosphoric acid into a tarred weighing vessel and quantitatively transfer

to the 2 000 ml beaker containing approximately 1 800 ml laboratory water, EDTA, and potassium

phosphate dibasic. Mix by stirring on a magnetic stir plate until the metaphosphoric acid is completely

dissolved.

Adjust the pH of the solution to pH = 5,00 ± 0,02 using 40 % potassium hydroxide or 85 % phosphoric

acid. Quantitatively transfer the solution to a 2 000 ml volumetric flask and dilute to volume with

laboratory water. Expiration: 48 hours.
6.12 Stock standard solutions of native compounds
6.12.1 Vitamin standard stock mixture (VSSM)

Accurately weigh the indicated amounts for the following standards using separate weighing funnels

or other appropriate weighing vessel and quantitatively transfer to a 100 ml volumetric flask using

phosphate buffer (pH = 5):
a) niacinamide: 70,5 mg ± 0,5 mg;
b) thiamine hydrochloride: 10,5 mg ± 0,2 mg ;

Determine the moisture of the thiamine hydrochloride reference standard (5.5) as directed on

the container immediately prior to weighing. The percent moisture determined for the reference

standard is used to calculate the concentration of thiamine in the VSSM.
c) riboflavin: 7,0 mg ± 0,2 mg;

Dry an appropriate amount of the riboflavin reference standard (5.4) at 105°C ± 1 °C for 2 h (±

10 min) prior to weighing.
d) pyridoxine hydrochloride: 10,8 mg ± 0,2 mg;

Dry an appropriate amount of the pyridoxine hydrochloride reference standard (5.3) over

indicating absorbent in vacuo for 4 h prior to weighing.
6 © ISO 2019 – All rights reserved
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ISO/DIS 21470:2019(E)

Fill to volume with phosphate buffer (pH = 5) solution. Heat and slowly stir until the standards have

completely dissolved (riboflavin dissolves more slowly) and the solution is clear. Do not heat the solution

for more than 40 min and do not exceed 90 °C. Store refrigerated (2 ˚C - 8 ˚C). Expiration: 3 months.

6.12.2 Nicotinic acid stock solution, ρ = 550 mg/ml.

Accurately weigh 13,7 mg ± 0,1 mg niacin primary reference standard (5.2). Quantitatively transfer the

nicotinic acid to a 25 ml volumetric flask. Add lab water to a total volume of about 20 ml and swirl until

completely dissolved. Bring to volume with lab water. Mix well. Expiration: 3 months.

6.12.3 Pyridoxal stock solution, ρ = 140 mg/ml.

Accurately weigh 17,0 mg ± 0,5 mg pyridoxal dihydrochloride standard (5.7). Quantitatively transfer

to a 100 ml volumetric flask. Add lab water to a total volume of about 70 ml and swirl until completely

dissolved. Bring to volume with lab water. Mix well. Expiration: 3 months.
6.12.4 Pyridoxamine stock solution, ρ = 160 mg/ml.

Accurately weigh 23,0 mg ± 0,5 mg pyridoxamine hydrochloride standard (5.6). Quantitatively transfer

to a 100 ml volumetric flask. Add lab water to a total volume of about 70 ml and swirl until completely

dissolved. Bring to volume with lab water. Mix well. Expiration: 3 months.
6.13 Mixed working standard (MWS)

Combine 500 μl VSSM (6.12.1), 25 μl pyridoxamine stock (6.12.4), 25 μl pyridoxal stock (6.12.3), and

65 μl nicotinic acid stock solutions (6.12.2) in a 10 ml volumetric flask containing approximately 5 ml

of ammonium formate solution (6.7). Bring to volume with ammonium formate solution (6.7) and mix

well. Prepare fresh daily.
6.14 Working standard solution preparation
6.14.1 Working solution (WS) 1.

Add 20 μl of MWS (6.13) and 980 μl of ammonium formate (6.7) to a 50 ml centrifuge tube. Add 100 μl

of ISSM (6.10), and vortex to mix. Prepare fresh daily.
6.14.2 Working solution (WS) 2.

Add 50 μl of MWS (6.13) and 950 μl of ammonium formate (6.7) to a 50 ml centrifuge tube. Add 100 μl

of ISSM (6.10), and vortex to mix. Prepare fresh daily.
6.14.3 Working solution (WS) 3.

Add 100 μl of MWS (6.13) and 900 μl of ammonium formate (6.7) to a 50 ml centrifuge tube. Add 100 μl

of ISSM (6.10), and vortex to mix. Prepare fresh daily.
6.14.4 Working solution (WS) 4.

Add 200 μl of MWS (6.13) and 800 μl of ammonium formate (6.7) to a 50 ml centrifuge tube. Add 100 μl

of ISSM (6.10), and vortex to mix. Prepare fresh daily.
6.14.5 Working solution (WS) 5.

Add 500 μl of MWS (6.13) and 500 μl of ammonium formate (6.7) to a 50 ml centrifuge tube. Add 100 μl

of ISSM (6.10), and vortex to mix. Prepare fresh daily.
© ISO 2019 – All rights reserved 7
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ISO/DIS 21470:2019(E)
6.14.6 Working solution (WS) 6.

Add 1 000 μl of MWS (6.13). Add 100 μl of ISSM (6.10), and vortex to mix. Prepare fresh daily.

7 Apparatus
7.1 Waters® Acquity BEH C18 column or equivalent, 2,1 mm x 100 mm, 1,7 μm.
7.2 UPLC system, Waters Acquity Classic , or equivalent.

7.3 Tandem quadrupole mass spectrometer with ESI probe, Waters Xevo TQ-S , or equivalent.

7.4 Analytical balances.

7.4.1 A balance capable of accurately weighing 5,00 mg (for standards), 6-place balance.

7.4.2 An analytical five-place balance for samples, and

7.4.3 A top loading two place balance capable of weighing to several hundred grams.

7.5 Water purifier, Millipore Milli-Q Water Purification System , or equivalent.

7.6 Water bath shaker, capable of maintaining 37 °C, Lab-Line Orbit , or equivalent.

7.7 Bottle-top dispenser, capable of dispensing volumes of approximately 24 ml.
7.8 pH meter, capable of measuring pH = 4,0 to pH = 5,0.
7.9 Vortex mixer.
7.10 Multi-position magnetic stir plate.

7.11 Room light shields, A.L.P. Protect-A-Lamp , UV cutoff at 460 nm, or equivalent.

7.12 Graduated cylinders, various sizes, including 10 ml, 100 ml, 500 ml and 1 000 ml.

7.13 Beakers, various sizes, including 100 ml, 200 ml, 400 ml, 600 ml, 1 000 ml and 2 000 ml.

7.14 Volumetric flasks, various sizes, including 10 ml, 25 ml, 50 ml, 100 ml, 250 ml and 2 000 ml.

7.15 Mobile phase bottles, glass, various sizes, including 250 ml, 500 ml, 1 000 ml and 2 000 ml.

7.16 Disposable plastic Pasteur pipettes.

7.17 Amber bottles, volume capacity of 50 ml and 100 m (for stock standard storage).

7.18 Weighing vessels, various, including disposable weighing boats and glass weighing funnels.

2) 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 an endorsement by ISO of the product named. Equivalent products

may be used if they can be shown to lead to the same results.
8 © ISO 2019 –
...

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