ISO 5132:2024

Animal and vegetable fats and oils — High-performance liquid chromatography (HPLC) analysis of phenolic antioxidants

This document specifies a method for the analysis of phenolic antioxidants by high-performance liquid chromatography (HPLC). It is applicable to quantifying the following synthetic phenolic compounds added to animal and vegetable fats, oils and shortenings as antioxidants, at concentrations normally added to oils: — propyl gallate (PG); — octyl gallate (OG); — dodecyl gallate (also called “lauryl gallate (LG)”); — 2,4,5-trihydroxybutyrophenone (THBP); — tert-butylhydroquinone (TBHQ); — nordihydroguaiaretic acid (NDGA); — 2- and 3-tert-butyl-4-hydroxyanisole (BHA); — 2,6-di-tert-butyl-4-(hydroxymethyl)phenol (BHT Alcohol or Ionox-100); — 2,6-di-tert-butyl-4-hydroxytoluene (BHT). A method for determining the absence of an antioxidant, or the maximum trace amount, within the limits of the analysis, is given in Annex B. The issue of canolol, a naturally occurring substance in rapeseed, interfering with the analysis is addressed in Annex C.

Titre manque

General Information

• Status: Published
• Publication Date: 22-Oct-2024
• ICS: 67.200.10 - Animal and vegetable fats and oils
• Technical Committee: ISO/TC 34/SC 11 - Animal and vegetable fats and oils
• Drafting Committee: ISO/TC 34/SC 11 - Animal and vegetable fats and oils
• Current Stage: 6060 - International Standard published
• Start Date: 23-Oct-2024
• Due Date: 23-Oct-2024
• Completion Date: 23-Oct-2024

Standards Content (Sample)

International
Standard
ISO 5132
First edition
Animal and vegetable fats and
oils — High-performance liquid
chromatography (HPLC) analysis of
phenolic antioxidants
PROOF/ÉPREUVE
Reference number
© ISO 2024
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: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
PROOF/ÉPREUVE
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 2
6 Apparatus . 3
7 Procedure . 3
7.1 Extraction of liquid oils, animal fats and shortenings .3
7.2 Chromatography .4
7.2.1 HPLC column .4
7.2.2 HPLC conditions .4
8 Calculations . 5
9 Validation . . 5
Annex A (informative) Validation study . 6
Annex B (informative) Estimation of the absence of an antioxidant . 9
Annex C (informative) Resolution of canolol from TBHQ in rapeseed (canola) oil .12
Bibliography . 14
PROOF/ÉPREUVE
iii
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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, Subcommittee SC 11, Animal
and vegetable fats and oils.
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.
PROOF/ÉPREUVE
iv
Introduction
This document describes the analysis of synthetic phenolic antioxidants intentionally added to oils and fats,
or inadvertently added during manufacturing processes. Information is also included for estimating the
absence of an antioxidant from oils and fats within the limitations of the method. Interference from natural
compounds present in rapeseed (canola) oil are also addressed.
[1]
This document represents AOCS Official Method Ce 6a-2021 .
PROOF/ÉPREUVE
v
International Standard ISO 5132:2024(en)
Animal and vegetable fats and oils — High-performance
liquid chromatography (HPLC) analysis of phenolic
antioxidants
1 Scope
This document specifies a method for the analysis of phenolic antioxidants by high-performance liquid
chromatography (HPLC).
It is applicable to quantifying the following synthetic phenolic compounds added to animal and vegetable
fats, oils and shortenings as antioxidants, at concentrations normally added to oils:
— propyl gallate (PG);
— octyl gallate (OG);
— dodecyl gallate (also called “lauryl gallate (LG)”);
— 2,4,5-trihydroxybutyrophenone (THBP);
— tert-butylhydroquinone (TBHQ);
— nordihydroguaiaretic acid (NDGA);
— 2- and 3-tert-butyl-4-hydroxyanisole (BHA);
— 2,6-di-tert-butyl-4-(hydroxymethyl)phenol (BHT Alcohol or Ionox-100);
— 2,6-di-tert-butyl-4-hydroxytoluene (BHT).
A method for determining the absence of an antioxidant, or the maximum trace amount, within the limits of
the analysis, is given in Annex B.
The issue of canolol, a naturally occurring substance in rapeseed, interfering with the analysis is addressed
in Annex C.
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 terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
4 Principle
The sample is diluted in hexane- The antioxidants are extracted into acetonitrile, diluted with isopropanol
and analysed by reversed-phase gradient HPLC with ultraviolet (UV) detection at 280 nm.
PROOF/ÉPREUVE
5 Reagents
5.1 Acetonitrile, HPLC grade.
5.2 2-Propanol, analytical grade.
5.3 Hexane, isohexane or similar volatile non-polar solvent, analytical grade.
5.4 High purity water.
5.5 Acetic acid, > 99,8 %, HPLC Grade, for acidifying mobile phases.
5.6 HPLC mobile phases:
a) Eluent A: Water containing 5 % acetic acid. Add 900 ml high purity water to a 1 l volumetric flask, add
50 ml acetic acid and bring to 1 litre with high purity water.
b) Eluent B: Acetonitrile containing 5 % acetic acid. Add 50 ml acetic acid to a 1 l volumetric flask, add
acetonitrile with mixing to bring to 1 litre.
5.7 Antioxidant standards: PG, OG, LG, THBP, TBHQ, NDGA, BHA (mixture of 2- and 3-isomers), Ionox-100
and/or BHT. In practice, only standards for the antioxidants of interest should be prepared.
5.8 Standard solutions. Refrigerate all antioxidant solutions out of direct light. Prepare all solutions with
2-propanol + acetonitrile (1:1).
a) Stock standard (~1 g/l): Accurately weigh and transfer about 50 mg of each antioxidant into a 50 ml
volumetric flask, dissolve, dilute to volume and mix.
b) Working standard, target 80 mg/l: Pipet 4 ml stock solution into a 50 ml volumetric flask, dilute to
volume and mix. Other standard concentrations can be prepared if desired.
c) Calculate the exact standard concentrations as shown by Formula (1):
C = m × V × 0,4 (1)
AS
where
C is the concentration of the working standard in mg/l;
m is the mass of the antioxidant standard, in mg, added to make 50 ml stock standard;
AS
V is the volume of stock standard solution used to make the working standard;
0,4 is (1 000 mg/g)/(50 ml stock standard volume × 50 ml working standard volume).
5.9 Extraction solvents. Saturate hexane and acetonitrile by mixing and shaking together for 2 min
and separate. Unless otherwise specified, use these saturated solvents for the extraction described in 7.1.
Significant amounts of hexane will dissolve in the acetonitrile; therefore, allow for extra hexane. Determine
how much acetonitrile and hexane are needed for all samples (each sample will require at least 20 ml hexane
and 150 ml acetonitrile).
PROOF/ÉPREUVE
6 Apparatus
6.1 Gradient HPLC system, consisting of a gradient pumping system, sample injection system, column
heater, UV or photodiode array detector and data analysis system. The system shall be capable of pumping
at a pressure compatible with an acceptable flow rate for the selected column.
6.2 C18 Reversed-phase HPLC column, available from a wide variety of manufacturers. Most
C18 columns will be capable of the needed separation. A guard column is highly recommended to protect the
analytical column. Narrow diameter columns consume less solvent and also have a higher response. Smaller
particle size improves resolution, but also increases back pressure.
6.3 Borosilicate beakers, 50 ml and 150 ml.
6.4 Separatory funnels, 125 ml and 250 ml.
6.5 Volumetric flasks, 50 ml.
6.6 Class A volumetric pipet, 4 ml.
6.7 Graduated glass cylinders, with ground-glass stoppers, 10 ml.
6.8 Graduated cylinders, 50 ml and 1 litre.
6.9 Solvent evaporation system: either a multi-vessel nitrogen blow-down evaporator such as TurboVap
1)
(Biotage, Uppsala, Sweden) or a rotary evaporator.
6.10 Appropriate evaporation vessels: either vessels for the nitrogen evaporation system (e.g. TurboVap
vials, 250 ml) or round-bottomed flasks, 250 ml, for the rotary evaporator.
6.11 Analytical balance, capable of weighing to the nearest 0,001 g.
7 Procedure
7.1 Extraction of liquid oils, animal fats and shortenings
7.1.1 Weigh, to the nearest 0,01 g, approximately 4 g of oil into a 50 ml beaker. Quantitatively transfer to
a 125 ml separatory funnel using about 20 ml hexane saturated with acetonitrile and rinse the beaker with
saturated hexane. Close and shake the separatory funnel to completely dissolve (for solid fats) and mix.
7.1.2 Extract the oil-hexane mixture with 50 ml of acetonitrile saturated with hexane. If an emulsion
forms, break by holding the 125 ml separatory funnel under hot tap water for 5 s to 10 s. Collect the extract
(bottom phase) in a 250 ml separatory funnel and repeat the extraction with 50 ml acetonitrile saturated
with hexane twice. Drain the combined extracts into a 250 ml round-bottomed flask or TurboVap vial (see
7.1.3). The draining shall be carried out slowly to prevent the inclusion of hexane-oil droplets.
The 150 ml acetonitrile extract may be stored overnight under refrigeration and protected from li
...

ISO/DIS PRF 5132:2024(E)
Date: 2024-07-17
ISO/TC 34/SC 11
Secretariat: BSI
Date: 2024-08-28
Animal and vegetable fats and oils — High-performance liquid
chromatography (HPLC) analysis of phenolic antioxidants
PROOF
ISO/PRF 5132:2024(en)
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
E-mail: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
ISO/PRF 5132:2024(en)
Contents
Foreword . iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 2
6 Apparatus . 3
7 Procedure . 3
7.1 Extraction of liquid oils, animal fats and shortenings . 3
7.2 Chromatography . 4
8 Calculations . 5
9 Validation . 5
Annex A (informative) Validation study . 6
Annex B (informative) Estimation of the absence of an antioxidant . 9
Annex C (informative) Resolution of canolol from TBHQ in rapeseed (canola) oil . 12
Bibliography . 14

iii
ISO/PRF 5132:2024(en)
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent rights
in respect thereof. As of the date of publication of this document, ISO had not received notice of (a) patent(s)
which may be required to implement this document. However, implementers are cautioned that this may not
represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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, Subcommittee SC 11, Animal
and vegetable fats and oils.
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/PRF 5132:2024(en)
Introduction
This document describes the analysis of synthetic phenolic antioxidants intentionally added to oils and fats,
or inadvertently added during manufacturing processes. Information is also included for estimating the
absence of an antioxidant from oils and fats within the limitations of the method. Interference from natural
compounds present in rapeseed (canola) oil are also addressed.
[1] [1]
This document represents AOCS Official Method Ce 6a-2021 .The AOCS revised the original AOCS Ce 6-86
“Antioxidants, Liquid Chromatographic Method” and this document is an adoption of the revised AOCS Ce 6a-
[2]
2021 “HPLC Analysis of Phenolic Antioxidants” method.

v
Animal and vegetable fats and oils — High-performance liquid
chromatography (HPLC) analysis of phenolic antioxidants
1 Scope
This document specifies a method for the analysis of phenolic antioxidants by high-performance liquid
chromatography (HPLC).
It is applicable to quantifying the following synthetic phenolic compounds added to animal and vegetable fats,
oils and shortenings as antioxidants, at concentrations normally added to oils:
— propyl gallate (PG);
— octyl gallate (OG);
— dodecyl gallate (also called “lauryl gallate (LG)”);
— 2,4,5-trihydroxybutyrophenone (THBP);
— tert-butylhydroquinone (TBHQ);
— nordihydroguaiaretic acid (NDGA);
— 2- and 3-tert-butyl-4-hydroxyanisole (BHA);
— 2,6-di-tert-butyl-4-(hydroxymethyl)phenol (BHT Alcohol or Ionox-100);
— 2,6-di-tert-butyl-4-hydroxytoluene (BHT).
A method for determining the absence of an antioxidant, or the maximum trace amount, within the limits of
the analysis, is given in Annex B.
The issue of canolol, a naturally occurring substance in rapeseed, interfering with the analysis is addressed in
Annex C.
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 terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
4 Principle
The sample is diluted in hexane- The antioxidants are extracted into acetonitrile, diluted with isopropanol and
analysed by reversed-phase gradient HPLC with ultra-violetultraviolet (UV) detection at 280 nm.
ISO/PRF 5132:2024(en)
5 Reagents
5.1 Acetonitrile, HPLC grade.
5.2 2-Propanol, analytical grade.
5.3 Hexane, isohexane or similar volatile non-polar solvent, analytical grade.
5.4 High purity water.
5.5 Acetic acid, > 99,8 %, HPLC Grade, for acidifying mobile phases.
5.6 HPLC mobile phases:
a) Eluent A: Water containing 5 % acetic acid. Add 900 ml high purity water to a 1 -l a volumetric flask, add
50 ml acetic acid and bring to 1 litre with high purity water.
b) Eluent B: Acetonitrile containing 5 % acetic acid. Add 50 ml acetic acid to a 1 -l a volumetric flask, , add
acetonitrile with mixing to bring to 1 litre.
5.7 Antioxidant standards: PG, OG, LG, THBP, TBHQ, NDGA, BHA (mixture of 2- and 3-isomers), Ionox-100
and/or BHT. In practice, only standards for the antioxidants of interest should be prepared.
5.8 Standard solutions. Refrigerate all antioxidant solutions out of direct light. Prepare all solutions with
2-propanol + acetonitrile (1:1).
a) Stock standard (~1 g/l): Accurately weigh and transfer about 50 mg of each antioxidant into a 50 ml
volumetric flask, dissolve, dilute to volume and mix.
b) Working standard, target 80 mg/l: Pipet 4 ml stock solution into a 50 ml volumetric flask, dilute to volume
and mix. Other standard concentrations can be prepared if desired.
c) Calculate the exact standard concentrations as shown by Formula (1):
C = mAS × V × 0,4 (1)
where
C is the concentration of the working standard concentration in mg/l;
m is the mass of the antioxidant standard, in mg, added to make 50 ml stock standard;
AS
V is the volume of stock standard solution used to make the working standard;
0,4
is (1 000 mg/g)/(50 ml stock standard volume x× 50 ml working standard volume).
5.9 Extraction solvents. Saturate hexane and acetonitrile by mixing and shaking together for 2 min and
separate. Unless otherwise specified, use these saturated solvents for the extraction described in 7.1.
Significant amounts of hexane will dissolve in the acetonitrile; therefore, allow for extra hexane. Determine
ISO/PRF 5132:2024(en)
how much acetonitrile and hexane are needed for all samples (each sample will require at least 20 ml hexane
and 150 ml acetonitrile).
6 Apparatus
6.1 Gradient HPLC system, consisting of a gradient pumping system, sample injection system, column
heater, UV or photodiode array detector and data analysis system. The system shall be capable of pumping at
a pressure compatible with an acceptable flow rate for the selected column.
6.2 C18 Reversed-phase HPLC column, available from a wide variety of manufacturers. Most C18 columns
will be capable of the needed separation. A guard column is highly recommended to protect the analytical
column. Narrow diameter columns consume less solvent and also have a higher response. Smaller particle size
improves resolution, but also increases back pressure.
6.3 Borosilicate beakers, 50 ml and 150 ml.
6.4 Separatory funnels, 125 ml and 250 ml.
6.5 Volumetric flasks, 50 ml.
6.6 Class A volumetric pipet, 4 ml.
6.7 Graduated glass cylinders, with ground-glass stoppers, 10 ml.
6.8 Graduated cylinders, 50 ml and 1 litre.
6.9 Solvent evaporation system: either a multi-vessel nitrogen blow-down evaporator such as TurboVap
1)
(Biotage, Uppsala, Sweden) or a rotary evaporator.
6.10 Appropriate evaporation vessels: either vessels for the nitrogen evaporation system (e.g. TurboVap
vials, 250 ml) or round-bottomed flasks, 250 ml, for the rotary evaporator.
6.11 Analytical balance, capable of weighing to the nearest 0,001 g.
7 Procedure
7.1 Extraction of liquid oils, animal fats and shortenings
7.1.1 Weigh, to the nearest 0,01g01 g, approximately 4g4 g of oil into a 50 ml beaker. Quantitatively transfer
to a 125 ml separatory funnel using about 20 ml hexane saturated with acetonitrile and rinse the beaker with
saturated hexane. Close and shake the separatory funnel to completely dissolve (for solid fats) and mix.
7.1.2 Extract the oil-hexane mixture with 50 ml of acetonitrile saturated with hexane. If an emulsion forms,
break by holding the 125 ml separatory funnel under hot tap water for 5 s to 10 s. Collect the extract (bottom
phase) in a 250 ml separatory funnel and repeat the extraction with 50 ml acetonitrile saturated with hexane
twice. Drain the combined extracts into a 250 ml round-bottomed flask or TurboVap vial (see 7.1.3). The
draining shall be carried out slowly to prevent the inclusion of hexane-oil droplets.
The 150 ml acetonitrile extract may be stored overnight under refrigeration and protected from light if
necessary.
1)
TurboVap (Biotage, Uppsala, Sweden) 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 this product.
ISO/PRF 5132:2024(en)
7.1.3 For evaporation, use one of the following two options:
a) Option 1: Nitrogen evaporation. Collect the samples in appropriate evaporation flasks. To an additional
evaporation flask, add 10,0 ml of the working standard and approximately 140 ml acetonitrile. If there are
many samples, prepare one flask containing the working standard to run with each evaporation batch.
Place the samples plus one flask containing the working standard in the evaporator set to 40 °C and start
the nitrogen flow. Evaporate until there is less than 4 ml volume remaining, but not to dryness, to obtain
concentrated acetonitrile extracts. Correct the antioxidant results for the recovery of the working
standard (see Clause 8).
b) Option 2: Rotary Evaporation: Collect the samples in 250 ml round-bo
...