ISO 23942:2022

Determination of hydroxytyrosol and tyrosol content in extra virgin olive oils — Reverse phase high performance liquid chromatography (RP-HPLC) method

This document specifies a method for the quantitative determination of hydroxytyrosol and tyrosol content in extra virgin olive oils using reverse phase high performance liquid chromatography (RP-HPLC) with spectrophotometric detection. The method is also applicable to all other olive oils of a different commercial category.

Détermination de la teneur en hydroxytyrosol et tyrosol dans les huiles d'olive vierges extra — Méthode par chromatographie liquide à haute performance en phase inverse (CLHP-RP)

Le présent document spécifie une méthode de détermination quantitative de la teneur en hydroxytyrosol et tyrosol dans les huiles d’olive vierges extra par chromatographie liquide à haute performance en phase inverse (CLHP-RP) couplée à la détection spectrophotométrique. La méthode est également applicable à toutes les autres huiles d’olive de catégorie commerciale différente.

General Information

• Status: Published
• Publication Date: 01-Nov-2022
• 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: 02-Nov-2022
• Due Date: 01-Mar-2023
• Completion Date: 02-Nov-2022

Relations

• Revises: ISO/TS 23942:2020 - Determination of hydroxytyrosol and tyrosol content in extra virgin olive oils — Reverse phase high performance liquid chromatography (RP-HPLC)
• Effective Date: 06-Jun-2022

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 23942
First edition
2022-11
Determination of hydroxytyrosol
and tyrosol content in extra virgin
olive oils — Reverse phase high
performance liquid chromatography
(RP-HPLC) method
Détermination de la teneur en hydroxytyrosol et tyrosol dans les
huiles d'olive vierges extra — Méthode par chromatographie liquide à
haute performance en phase inverse (CLHP-RP)
Reference number
ISO 23942:2022(E)
© ISO 2022

---------------------- Page: 1 ----------------------
ISO 23942:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022
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
ii
  © ISO 2022 – All rights reserved

---------------------- Page: 2 ----------------------
ISO 23942:2022(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 1
6 Apparatus . 2
7 Sampling . 3
8 Procedure .3
8.1 Sample preparation . 3
8.2 HPLC analysis . 3
8.2.1 General . 3
8.2.2 HPLC conditions . 3
8.2.3 Peak identification . 4
9 Expression of results . 4
10 Precision . 5
10.1 Validation study . 5
10.2 Repeatability, r. 6
10.3 Reproducibility, R . 6
11 Test report . 6
Annex A (informative) Chromatogram 280 nm . 7
Annex B (normative) Limit of detection (LOD) and limit of quantification (LOQ) .8
Annex C (informative) Validation studies . 9
Bibliography .13
iii
© ISO 2022 – All rights reserved

---------------------- Page: 3 ----------------------
ISO 23942:2022(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, Subcommittee SC 11,
Animal and vegetable fats and oils.
This first edition cancels and replaces ISO/TS 23942:2020, which has been technically revised.
The main changes are as follows:
— conversion of a Technical Specification to an International Standard;
— additional validation studies have been added to Annex C.
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 2022 – All rights reserved

---------------------- Page: 4 ----------------------
ISO 23942:2022(E)
Introduction
Biophenolic compounds of a secoiridoid nature, and typical of extra virgin olive oil (Olea europaea L.),
are derived from oleuropein and ligstroside, and are correlated to different beneficial health effects for
human beings other than particular sensorial characteristics. The biophenolic compounds contain, in
an esterified form, two aromatic alcohols, namely hydroxytyrosol and tyrosol. The method given in this
document is based on an extraction of the biophenolic fraction with a methanol/water solution and a
[1][2]
subsequent hydrolysis reaction to produce free tyrosol and hydroxytyrosol.
v
© ISO 2022 – All rights reserved

---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 23942:2022(E)
Determination of hydroxytyrosol and tyrosol content in
extra virgin olive oils — Reverse phase high performance
liquid chromatography (RP-HPLC) method
1 Scope
This document specifies a method for the quantitative determination of hydroxytyrosol and tyrosol
content in extra virgin olive oils using reverse phase high performance liquid chromatography (RP-
HPLC) with spectrophotometric detection.
The method is also applicable to all other olive oils of a different commercial category.
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/
3.1
hydroxytyrosol and tyrosol
aromatic alcohols present in extra virgin olive oil typical of Olea europaea L. species as free or bound
form
4 Principle
Hydroxytyrosol and tyrosol, present in free and esterified forms, are extracted from the oil with
a methanol/water solution and then submitted to hydrolysis reaction with a 10 % of sulphuric acid
ethanolic solution. The components are identified by means of HPLC and a spectrophotometric detector
at 280 nm. The amount of free aromatic alcohols is calculated with the use of an external standard.
5 Reagents
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade.
5.1 Ortho-phosphoric acid, a volume fraction of 85 %.
5.2 Methanol chromatographic grade.
5.3 Acetonitrile chromatographic grade.
5.4 Water chromatographic grade.
1
© ISO 2022 – All rights reserved

---------------------- Page: 6 ----------------------
ISO 23942:2022(E)
5.5 Ethanol, a volume fraction of 96 %.
5.6 Sulphuric acid, a volume fraction of 96 %.
5.7 Methanol/water solution, 80/20 v/v.
5.8 Reference sample: hydroxytyrosol or 2-(3,4-Dihydroxyphenyl)ethanol, e.g. Extrasynthese,
1)
(Cedex, France) .
1)
5.9 Reference sample: tyrosol, e.g. Sigma Aldrich (Germany) .
5.10 Standard calibration solution of hydroxytyrosol and tyrosol.
Prepare the external standard calibration solution of hydroxytyrosol and tyrosol as follows.
Weigh accurately, to the nearest 0,1 mg, about 25 mg of hydroxytyrosol (5.8) and tyrosol (5.9) in a
graduated 50 ml flask (6.2) and make to volume with a solution of methanol/water 80/20 v/v (5.7).
Transfer 1 ml of this solution in another 10 ml flask and fill to volume with the same solution of
methanol/water 80/20 v/v (5.7). The final concentration is 50 mg/l of each external standard. Inject
20 μl of this solution in the HPLC system. The solution is stable for at least six months at –20 °C.
5.11 Hydrolysis solution, consisting of ethanol/water/sulphuric acid 50/40/10 v/v/v.
6 Apparatus
The usual laboratory glassware and the following shall be used.
6.1 Analytical balance suitable for weighing to an accuracy of within ± 0,1 mg.
6.2 10 ml and 50 ml calibrated flasks, class A.
6.3 1 000 μl and 5 000 μl electronic pipette or manual pipette.
6.4 10 ml test tube, with a screw cap.
6.5 Mixer, type vortex.
6.6 Ultrasonic extraction bath.
6.7 PVDF (polyvinyl difluoride) syringe filters, 0,45 μm, 13 mm.
6.8 Centrifuge, able to operate at 5 000 r/min.
6.9 5 ml plastic syringe.
6.10 Thermostatic bath.
6.11 Analytical system, comprising a HPLC ternary pump with a degassing system equipped
with an HPLC column, RP 18 reverse phase. The following column has proven to be adapted for the
1) Extrasynthese (Cedex, France) and Sigma Aldrich (Germany) are examples of a companies that make suitable
products available commercially. This information is given for the convenience of users of this document and does
not constitute an endorsement by ISO of these products.
2
  © ISO 2022 – All rights reserved

---------------------- Page: 7 ----------------------
ISO 23942:2022(E)
2)
determination (internal diameter 4,6 mm, length 25 cm, size 5 μm, 100 Å, type Spherisorb ODS2 ) with
a UV spectrophotometric detector at 280 nm and integration system. A photodiode array detector (PDA)
for spectra recording can be used to facilitate peak identification, by matching the hydroxytyrosol and
tyrosol spectra in the sample extract with the spectra of the external standard.
A system for analysis data and integration is needed.
7 Sampling
It is important that an intact oil sample is delivered to the laboratory, which has not been damaged
or modified during transport or storage. A representative sample is considered for the purpose of the
[3]
analysis. A recommended sampling method is given in ISO 5555 .
8 Procedure
8.1 Sample preparation
Weigh, with an analytical balance (6.1), 2 g of oil well-homogenized in a 10 ml conical test tube (6.4).
Add, using a pipette (6.3), 5 ml of the methanol/water solution 80/20 v/v (5.7). Mix the solution with
the help of a mixer for test tube type vortex (6.5) for 1 min and continue the extraction for 15 min
in an ultrasonic bath (6.6) at room temperature. Centrifuge (6.8) at 5 000 r/min for 25 min. Filter
an aliquot through a PVDF membrane syringe filter (6.7). Transfer 1 ml, using a pipette (6.3), of the
filtered solution into another 10 ml test tube (6.4) and completely dry on a thermostatic bath (6.10) at a
maximum temperature of 40 °C under nitrogen stream. Add 1 ml of hydrolysis solution (5.11) and mix,
followed by reaction at 40 °C for 1 h. Leave the solution at room temperature overnight. Then filter the
solution using a PVDF membrane syringe filter (6.7).
8.2 HPLC analysis
8.2.1 General
Inject 20 µl of the sample into the HPLC system (6.11). The first sample injected as part of a series of
analysis shall be a blank of a methanol/water solution 80/20 v/v (5.7). There shall be no interfering
signals present during the chromatographic run at the same retention time of hydroxytyrosol and
tyrosol.
8.2.2 HPLC conditions
The operating conditions given in Table 1 have proven to be adapted for the determination.
2) The Sphe
...

NORME ISO
INTERNATIONALE 23942
Première édition
2022-11
Détermination de la teneur en
hydroxytyrosol et tyrosol dans les
huiles d'olive vierges extra — Méthode
par chromatographie liquide à haute
performance en phase inverse
(CLHP-RP)
Determination of hydroxytyrosol and tyrosol content in extra virgin
olive oils — Reverse phase high performance liquid chromatography
(RP-HPLC) method
Numéro de référence
ISO 23942:2022(F)
© ISO 2022

---------------------- Page: 1 ----------------------
ISO 23942:2022(F)
DOCUMENT PROTÉGÉ PAR COPYRIGHT
© ISO 2022
Tous droits réservés. Sauf prescription différente ou nécessité dans le contexte de sa mise en œuvre, aucune partie de cette
publication ne peut être reproduite ni utilisée sous quelque forme que ce soit et par aucun procédé, électronique ou mécanique,
y compris la photocopie, ou la diffusion sur l’internet ou sur un intranet, sans autorisation écrite préalable. Une autorisation peut
être demandée à l’ISO à l’adresse ci-après ou au comité membre de l’ISO dans le pays du demandeur.
ISO copyright office
Case postale 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Genève
Tél.: [phone]
E-mail: [e-mail]
Web: www.iso.org
Publié en Suisse
ii
  © ISO 2022 – Tous droits réservés

---------------------- Page: 2 ----------------------
ISO 23942:2022(F)
Sommaire Page
Avant-propos .iv
Introduction .v
1 Domaine d’application . 1
2 Références normatives .1
3 Termes et définitions . 1
4 Principe. 1
5 Réactifs . 1
6 Appareillage . 2
7 Échantillonnage .3
8 Mode opératoire . 3
8.1 Préparation de l’échantillon . 3
8.2 Analyse CLHP . 3
8.2.1 Généralités . 3
8.2.2 Conditions de CLHP . . 3
8.2.3 Identification des pics . 4
9 Expression des résultats . 5
10 Fidélité . 6
10.1 Étude de validation . 6
10.2 Répétabilité, r . 6
10.3 Reproductibilité, R . 6
11 Rapport d’essai .6
Annexe A (informative) Chromatogramme 280 nm . 7
Annexe B (normative) Limite de détection (LOD) et de quantification (LOQ) .8
Annexe C (informative) Études de validation . 9
Bibliographie .13
iii
© ISO 2022 – Tous droits réservés

---------------------- Page: 3 ----------------------
ISO 23942:2022(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,
sous-comité SC 11, Corps gras d’origines animale et végétale.
Cette première édition annule et remplace l’ISO/TS 23942:2020, qui a fait l’objet d’une révision
technique.
Les principales modifications sont les suivantes:
— conversion d’une Spécification technique en une Norme internationale;
— ajout d’études de validation supplémentaires à l’Annexe C.
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/fr/members.html.
iv
  © ISO 2022 – Tous droits réservés

---------------------- Page: 4 ----------------------
ISO 23942:2022(F)
Introduction
Les composés biophénoliques de nature sécoiridoïde et spécifiques de l’huile d’olive vierge extra (Olea
europaea L.), sont issus de l’oleuropéine et du ligstroside et, outre leurs caractéristiques sensorielles
particulières, sont associés à différents effets bénéfiques pour la santé humaine. Les composés
biophénoliques contiennent deux alcools aromatiques sous forme estérifiée, à savoir l’hydroxytyrosol
et le tyrosol. La méthode décrite dans le présent document repose sur une extraction de la fraction
biophénolique avec une solution méthanol/eau et sur une réaction d’hydrolyse ultérieure pour produire
[1][2]
du tyrosol libre et de l’hydroxytyrosol libre .
v
© ISO 2022 – Tous droits réservés

---------------------- Page: 5 ----------------------
NORME INTERNATIONALE ISO 23942:2022(F)
Détermination de la teneur en hydroxytyrosol et tyrosol
dans les huiles d'olive vierges extra — Méthode par
chromatographie liquide à haute performance en phase
inverse (CLHP-RP)
1 Domaine d’application
Le présent document spécifie une méthode de détermination quantitative de la teneur en hydroxytyrosol
et tyrosol dans les huiles d’olive vierges extra par chromatographie liquide à haute performance en
phase inverse (CLHP-RP) couplée à la détection spectrophotométrique.
La méthode est également applicable à toutes les autres huiles d’olive de catégorie commerciale
différente.
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 https:// www .electropedia .org/
3.1
hydroxytyrosol et tyrosol
alcools aromatiques présents sous forme libre ou liée dans l’huile d’olive vierge extra, spécifiques de
l’espèce Olea europaea L.
4 Principe
L’hydroxytyrosol et le tyrosol, présents sous forme libre et sous forme estérifiée, sont extraits de l’huile
avec une solution méthanol/eau puis soumis à une réaction d’hydrolyse avec une solution éthanolique
à 10 % d’acide sulfurique. Les composés sont identifiés par CLHP et par détection spectrophotométrique
à 280 nm. La quantité d’alcools aromatiques libres est calculée par étalonnage externe.
5 Réactifs
Pendant l’analyse, sauf mention contraire, utiliser uniquement des réactifs de qualité analytique
reconnue.
5.1 Acide orthophosphorique, d’une fraction volumique de 85 %.
5.2 Méthanol de qualité chromatographique.
1
© ISO 2022 – Tous droits réservés

---------------------- Page: 6 ----------------------
ISO 23942:2022(F)
5.3 Acétonitrile de qualité chromatographique.
5.4 Eau de qualité chromatographique.
5.5 Éthanol, d’une fraction volumique de 96 %.
5.6 Acide sulfurique, d’une fraction volumique de 96 %.
5.7 Solution méthanol/eau, 80/20 V/V.
5.8 Échantillon de référence: hydroxytyrosol ou 2-(3,4-dihydroxyphényl)éthanol, par exemple
1))
Extrasynthese, (Cedex, France) .
1
5.9 Échantillon de référence: tyrosol, par exemple Sigma Aldrich (Allemagne) .
5.10 Solution d’étalonnage externe d’hydroxytyrosol et de tyrosol.
Préparer la solution d’étalonnage externe d’hydroxytyrosol et de tyrosol comme suit.
Peser exactement, à 0,1 mg près, environ 25 mg d’hydroxytyrosol (5.8) et de tyrosol (5.9) dans une
fiole jaugée de 50 ml (6.2) et compléter au volume avec une solution méthanol/eau 80/20 V/V (5.7).
Transvaser 1 ml de cette solution dans une autre fiole de 10 ml et compléter au volume avec la même
solution méthanol/eau 80/20 V/V (5.7). La concentration finale sera de 50 mg/l pour chaque étalon
externe. Injecter 20 μl de cette solution dans le système CLHP. La solution est stable pendant au moins
six mois à –20 °C.
5.11 Solution d’hydrolyse, constituée d’éthanol/eau/acide sulfurique 50/40/10 V/V/V.
6 Appareillage
La verrerie de laboratoire habituelle et les éléments suivants doivent être utilisés.
6.1 Balance analytique précise à ± 0,1 mg.
6.2 Fioles jaugées de 10 ml et 50 ml, de classe A.
6.3 Pipette électronique de 1 000 μl et 5 000 μl ou pipette manuelle.
6.4 Tube à essai de 10 ml, avec bouchon à vis.
6.5 Mélangeur, de type Vortex.
6.6 Bain d’extraction à ultrasons.
6.7 Filtres seringues en PVDF (polyfluorure de vinylidène), 0,45 μm, 13 mm.
6.8 Centrifugeuse pouvant tourner à 5 000 tr/min.
1) Extrasynthese (Cedex, France) et Sigma Aldrich (Allemagne) sont des exemples de sociétés fabriquant
des produits appropriés disponibles dans le commerce. Cette information est donnée par souci de commodité à
l’intention des utilisateurs du présent document et ne saurait constituer un engagement de l’ISO à l’égard de ces
produits.
2
  © ISO 2022 – Tous droits réservés

---------------------- Page: 7 ----------------------
ISO 23942:2022(F)
6.9 Seringue en plastique de 5 ml.
6.10 Bain thermostatique.
6.11 Système analytique, comprenant une pompe ternaire de CLHP avec système de dégazage
équipée d’une colonne de CLHP, en phase inverse RP 18. La colonne suivante s’est avérée adaptée à la
détermination (diamètre interne 4,6 mm, longueur 25 cm, granulométrie 5 μm, 100 Å, type Spherisorb
2))
ODS2 ) avec détecteur spectrophotométrique UV à 280 nm et système d’intégration. Un détecteur
à barrettes de photodiodes (PDA) pour l’enregistrement des spectres peut être utilisé pour faciliter
l’identification des pics, en faisant correspondre les spectres d’hydroxytyrosol et de tyrosol dans
l’échantillon avec les spectres de l’étalon externe.
Un système d’analyse et d’intégration des données est nécessaire.
7 Échantillonnage
Il est important de fournir au laboratoire un échantillon d’huile intact, non endommagé ou non modifié
pendant le transport ou le stockage. Un échantillon représentatif doit être utilisé pour l’analyse. Une
[3]
méthode d’échantillonnage recommandée est indiquée dans l’ISO 5555 .
8 Mode opératoire
8.1 Préparation de l’échantillon
À l’aide d’une balance analytique (6.1), peser 2 g d’huile bien homogénéisée dans un tube à essai
conique de 10 ml (6.4). Ajouter, à l’aide d’une pipette (6.3), 5 ml de solution méthanol/eau 80/20 V/V
(5.7). Mélanger la solution à l’aide d’un mélangeur pour tube à essai de type Vortex (6.5) pendant 1 min
et poursuivre l’extraction pendant 15 min dans un bain à ultrasons (6.6) à température ambiante.
Centrifuger (6.8) à 5 000 tr/min pendant 25 min. Filtrer une portion aliquote à travers un filtre
seringue à membrane en PVDF (6.7). Transférer, à l’aide d’une pipette (6.3), 1 ml de la solution filtrée
dans un autre tube à essai de 10 ml (6.4) et sécher totalement dans un bain thermostatique (6.10) à
une température maximale de 40 °C sous un courant d’azote. Ajouter 1 ml de la solution d’hydrolyse
(5.11) et mélanger, puis laisser réagir à 40° pendant 1 h. Laisser la solution toute une nuit à température
ambiante. Filtrer ensuite la solution à l’aide d’un filtre seringue à membrane en PVDF (6.7).
8.2 Analyse CLHP
8.2.1 Généralités
Injecter 20 µl de l’échantillon dans le système de CLHP (6.11). Le premier échantillon injecté dans une
série d’analyse doit être un blanc de solution méthanol/eau 80/20 V/V (5.7). Aucun signal interférent ne
doit être présent pendant l’analyse chromatographique au même temps de rétention de l’hydroxytyrosol
et du tyrosol.
8.2.2 Conditions de CLHP
Les conditions de fonctionnement indiquées dans le Tableau 1 se sont avérées adaptées à la
détermination.

2) La colonne Spherisorb ODS2 est un exemple de colonne chromatographique appropriée et disponible da
...

© ISO 2022 – All rights reserved
ISO/DISPRF 23942:2022(E)
Date: 2022-06-0209-07
ISO/TC 34/SC 11
Secretariat: BSI
Determination of hydroxytyrosol and tyrosol content in extra
virgin olive oils — Reverse phase high performance liquid
chromatography (RP-HPLC) method
Détermination de la teneur en hydroxytyrosol et tyrosol dans les huiles d'olive vierges extra —
Méthode par chromatographie liquide à haute performance en phase inverse (CLHP-PIRP)

---------------------- Page: 1 ----------------------
ISO/PRF 23942:2022(E)
© ISO 2022
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-1214 Vernier, Geneva
Phone: + 41 22 749 01 11
Email: [e-mail]
Website: www.iso.org
Published in Switzerland.
ii © ISO 2022 – All rights reserved

---------------------- Page: 2 ----------------------
ISO/PRF 23942:2022(E)
Contents Page
Foreword . iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 1
6 Apparatus . 2
7 Sampling . 3
8 Procedure . 3
8.1 Sample preparation . 3
8.2 HPLC analysis . 3
8.2.1 General . 3
8.2.2 HPLC conditions . 3
8.2.3 Peak identification . 4
9 Expression of results . 4
10 Precision . 5
10.1 Validation study . 5
10.2 Repeatability, r . 5
10.3 Reproducibility, R . 6
11 Test report . 6
Annex A (informative) Chromatogram 280 nm . 7
Annex B (normative) Limit of detection (LOD) and limit of quantification (LOQ) . 8
Annex C (informative) Validation studies . 9
Bibliography . 13

© ISO 2022 – All rights reserved iii

---------------------- Page: 3 ----------------------
ISO/PRF 23942:2022(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, Subcommittee SC 11,
Animal and vegetable fats and oils.
This first edition cancels and replaces ISO/TS 23942:2020, which has been technically revised.
The main changes are as follows:
— conversion of a Technical Specification to an International Standard;
— additional validation studies have been added to Annex C.
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 2022 – All rights reserved

---------------------- Page: 4 ----------------------
ISO/PRF 23942:2022(E)
Introduction
Biophenolic compounds of a secoiridoid nature, and typical of extra virgin olive oil (Olea europaea L.), are
derived from oleuropein and ligstroside, and are correlated to different beneficial health effects for
human beings other than particular sensorial characteristics. The biophenolic compounds contain, in an
esterified form, two aromatic alcohols, namely hydroxytyrosol and tyrosol. The method given in this
document is based on an extraction of the biophenolic fraction with a methanol/water solution and a
[ [6][7] ]
subsequent hydrolysis reaction to produce free tyrosol and hydroxytyrosol . .
© ISO 2022 – All rights reserved v

---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO/PRF 23942:2022(E)

Determination of hydroxytyrosol and tyrosol content in extra
virgin olive oils — Reverse phase high performance liquid
chromatography (RP-HPLC) method
1 Scope
This document specifies a method for the quantitative determination of hydroxytyrosol and tyrosol
content in extra virgin olive oils using reverse phase high performance liquid chromatography (RP-HPLC)
with spectrophotometric detection.
The method is also applicable to all other olive oils of a different commercial category.
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/
3.1
hydroxytyrosol and tyrosol
aromatic alcohols present in extra virgin olive oil typical of Olea europaea L. species as free or bound form
4 Principle
Hydroxytyrosol and tyrosol, present in free and esterified forms, are extracted from the oil with a
methanol/water solution and then submitted to hydrolysis reaction with a 10 % of sulphuric acid
ethanolic solution. The components are identified by means of HPLC and a spectrophotometric detector
at 280 nm. The amount of free aromatic alcohols is calculated with the use of an external standard.
5 Reagents
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade.
5.1 Ortho-phosphoric acid, a volume fraction of 85 %.
5.2 Methanol chromatographic grade.
5.3 Acetonitrile chromatographic grade.
5.4 Water chromatographic grade.
© ISO 2022 – All rights reserved
1

---------------------- Page: 6 ----------------------
ISO/PRF 23942:2022(E)
5.5 Ethanol, a volume fraction of 96 %.
5.6 Sulphuric acid, a volume fraction of 96 %.
5.7 Methanol/water solution, 80/20 v/v.
5.8 Reference sample: hydroxytyrosol or 2-(3,4-Dihydroxyphenyl)ethanol, e.g. Extrasynthese,
1
(Cedex, France) .
1)
5.9 Reference sample: tyrosol, e.g. Sigma Aldrich (Germany) .
5.10 Standard calibration solution of hydroxytyrosol and tyrosol.
Prepare the external standard calibration solution of hydroxytyrosol and tyrosol as follows.
Weigh accurately, to the nearest 0,1 mg, about 25 mg of hydroxytyrosol (5.8) and tyrosol (5.9) in a
graduated 50 ml flask (6.2) and make to volume with a solution of methanol/water 80/20 v/v (5.7).
Transfer 1 ml of this solution in another 10 ml flask and fill to volume with the same solution of
methanol/water 80/20 v/v (5.7). The final concentration is 50 mg/l of each external standard. Inject
20 μl of this solution in the HPLC system. The solution is stable for at least six months at –20 °C.
5.11 Hydrolysis solution, consisting of ethanol/water/sulphuric acid 50/40/10 v/v/v.
6 Apparatus
The usual laboratory glassware and the following shall be used.
6.1 Analytical balance suitable for weighing to an accuracy of within ± 0,1 mg.
6.2 10 ml and 50 ml calibrated flasks, class A.
6.3 1 000 μl and 5 000 μl electronic pipette or manual pipette.
6.4 10 ml test tube, with a screw cap.
6.5 Mixer, type vortex.
6.6 Ultrasonic extraction bath.
6.7 PVDF (polyvinyl difluoride) syringe filters, 0,45 μm, 13 mm.
6.8 Centrifuge, able to operate at 5 000 r/min.
6.9 5 ml plastic syringe.
6.10 Thermostatic bath.
6.11 Analytical system, comprising a HPLC ternary pump with a degassing system equipped with an
HPLC column, RP 18 reverse phase. The following column has proven to be adapted for the determination

1
Extrasynthese (Cedex, France) and Sigma Aldrich (Germany) are examples of a companies that make suitable
products available commercially. This information is given for the convenience of users of this document and does
not constitute an endorsement by ISO of these products.

© ISO 2022 – All rights reserved
2

---------------------- Page: 7 ----------------------
ISO/PRF 23942:2022(E)
2
(internal diameter 4,6 mm, length 25 cm, size 5 μm, 100 Å, type Spherisorb ODS2 ) with a UV
spectrophotometric detector at 280 nm and integration system. A photodiode array detector (PDA) for
spectra recording can be used to facilitate peak identification, by matching the hydroxytyrosol and
tyrosol spectra in the sample extract with the spectra of the external standard.
A system for analysis data and integration is needed.
7 Sampling
It is important that an intact oil sample is delivered to the laboratory, which has not been damaged or
modified during transport or storage. A representative sample is considered for the purpose of the
analysis. A recommended sampling method is given in ISO 5555.
8 Procedure
8.1 Sample preparation
Weigh, with an analytical balance (6.1), 2 g of oil well-homogenized in a 10 ml conical test tube (6.4). Add,
using a pipette (6.3), 5 ml of the methanol/water solution 80/20 v/v (5.7). Mix the solution with the help
of a mixer for test tube type vortex (6.5) for 1 min and continue the extraction for 15 min in an ultrasonic
bath (6.6) at room temperature. Centrifuge (6.8) at 4500 rcf5 000 r/min for 25 min. Filter an aliquot
through a PVDF membrane syringe filter (6.7). Transfer 1 ml, using a pipette (6.3), of the filtered solution
into another 10 ml test tube (6.4) and completely dry on a thermostatic bath (6.10) at a maximum
temperature of 40 °C under nitrogen stream. Add 1 ml of hydrolysis solution (5.11) and mix, followed by
reaction at 40 °C for 1 h. Leave the solution at room temperature overnight. Then filter the solution using
a PVDF membrane syringe filter (6.7).
8.2 HPLC analysis
8.2.1 General
Inject 20 µl of the sample into the HPLC system (6.11). The first sample injected as part of a series of
analysis shall be a blank of a methanol/water solution 80/20 v/v (5.7). There shall be no interfering
signals present during the chromatographic run at the same retention time of hydroxytyrosol and tyrosol.
8.2.2 HPLC conditions
The operating conditions given in Table 1 have proven to be adapted for the determination.
Table 1 — Operating conditions
Time Flow A B C
min ml/min % % %
0 1,00 96 2 2
40 1,00 50 25 25
45 1,00 40 30 30

2
The Spherisorb ODS2 column is an example of suitable chromatographic column that is commercially available.
Alternative columns may be used, such as columns suitable for aan Ultra-HPLC (UHPLC) system, if the operating
conditions in Table 1 isare adjusted and the resulting precision data parameters are equal or better than those
reported in Tables C.3, C.4, and C.5. This information is given for the convenience of users of this document and
does not constitute an endor
...

INTERNATIONAL ISO
STANDARD 23942
First edition
Determination of hydroxytyrosol
and tyrosol content in extra virgin
olive oils — Reverse phase high
performance liquid chromatography
(RP-HPLC) method
Détermination de la teneur en hydroxytyrosol et tyrosol dans les
huiles d'olive vierges extra — Méthode par chromatographie liquide à
haute performance en phase inverse (CLHP-RP)
PROOF/ÉPREUVE
Reference number
ISO 23942:2022(E)
© ISO 2022

---------------------- Page: 1 ----------------------
ISO 23942:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022
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
ii
PROOF/ÉPREUVE © ISO 2022 – All rights reserved

---------------------- Page: 2 ----------------------
ISO 23942:2022(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 1
6 Apparatus . 2
7 Sampling . 3
8 Procedure .3
8.1 Sample preparation . 3
8.2 HPLC analysis . 3
8.2.1 General . 3
8.2.2 HPLC conditions . 3
8.2.3 Peak identification . 4
9 Expression of results . 4
10 Precision . 5
10.1 Validation study . 5
10.2 Repeatability, r. 6
10.3 Reproducibility, R . 6
11 Test report . 6
Annex A (informative) Chromatogram 280 nm . 7
Annex B (normative) Limit of detection (LOD) and limit of quantification (LOQ) .8
Annex C (informative) Validation studies . 9
Bibliography .13
iii
© ISO 2022 – All rights reserved PROOF/ÉPREUVE

---------------------- Page: 3 ----------------------
ISO 23942:2022(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, Subcommittee SC 11,
Animal and vegetable fats and oils.
This first edition cancels and replaces ISO/TS 23942:2020, which has been technically revised.
The main changes are as follows:
— conversion of a Technical Specification to an International Standard;
— additional validation studies have been added to Annex C.
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
PROOF/ÉPREUVE © ISO 2022 – All rights reserved

---------------------- Page: 4 ----------------------
ISO 23942:2022(E)
Introduction
Biophenolic compounds of a secoiridoid nature, and typical of extra virgin olive oil (Olea europaea L.),
are derived from oleuropein and ligstroside, and are correlated to different beneficial health effects for
human beings other than particular sensorial characteristics. The biophenolic compounds contain, in
an esterified form, two aromatic alcohols, namely hydroxytyrosol and tyrosol. The method given in this
document is based on an extraction of the biophenolic fraction with a methanol/water solution and a
[6][7]
subsequent hydrolysis reaction to produce free tyrosol and hydroxytyrosol.
v
© ISO 2022 – All rights reserved PROOF/ÉPREUVE

---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 23942:2022(E)
Determination of hydroxytyrosol and tyrosol content in
extra virgin olive oils — Reverse phase high performance
liquid chromatography (RP-HPLC) method
1 Scope
This document specifies a method for the quantitative determination of hydroxytyrosol and tyrosol
content in extra virgin olive oils using reverse phase high performance liquid chromatography
(RP-HPLC) with spectrophotometric detection.
The method is also applicable to all other olive oils of a different commercial category.
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/o bp
— IEC Electropedia: available at https:// www.e lectropedia. org/
3.1
hydroxytyrosol and tyrosol
aromatic alcohols present in extra virgin olive oil typical of Olea europaea L. species as free or bound
form
4 Principle
Hydroxytyrosol and tyrosol, present in free and esterified forms, are extracted from the oil with
a methanol/water solution and then submitted to hydrolysis reaction with a 10 % of sulphuric acid
ethanolic solution. The components are identified by means of HPLC and a spectrophotometric detector
at 280 nm. The amount of free aromatic alcohols is calculated with the use of an external standard.
5 Reagents
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade.
5.1 Ortho-phosphoric acid, a volume fraction of 85 %.
5.2 Methanol chromatographic grade.
5.3 Acetonitrile chromatographic grade.
5.4 Water chromatographic grade.
1
© ISO 2022 – All rights reserved PROOF/ÉPREUVE

---------------------- Page: 6 ----------------------
ISO 23942:2022(E)
5.5 Ethanol, a volume fraction of 96 %.
5.6 Sulphuric acid, a volume fraction of 96 %.
5.7 Methanol/water solution, 80/20 v/v.
5.8 Reference sample: hydroxytyrosol or 2-(3,4-Dihydroxyphenyl)ethanol, e.g. Extrasynthese,
1)
(Cedex, France) .
1)
5.9 Reference sample: tyrosol, e.g. Sigma Aldrich (Germany) .
5.10 Standard calibration solution of hydroxytyrosol and tyrosol.
Prepare the external standard calibration solution of hydroxytyrosol and tyrosol as follows.
Weigh accurately, to the nearest 0,1 mg, about 25 mg of hydroxytyrosol (5.8) and tyrosol (5.9) in a
graduated 50 ml flask (6.2) and make to volume with a solution of methanol/water 80/20 v/v (5.7).
Transfer 1 ml of this solution in another 10 ml flask and fill to volume with the same solution of
methanol/water 80/20 v/v (5.7). The final concentration is 50 mg/l of each external standard. Inject
20 μl of this solution in the HPLC system. The solution is stable for at least six months at –20 °C.
5.11 Hydrolysis solution, consisting of ethanol/water/sulphuric acid 50/40/10 v/v/v.
6 Apparatus
The usual laboratory glassware and the following shall be used.
6.1 Analytical balance suitable for weighing to an accuracy of within ± 0,1 mg.
6.2 10 ml and 50 ml calibrated flasks, class A.
6.3 1 000 μl and 5 000 μl electronic pipette or manual pipette.
6.4 10 ml test tube, with a screw cap.
6.5 Mixer, type vortex.
6.6 Ultrasonic extraction bath.
6.7 PVDF (polyvinyl difluoride) syringe filters, 0,45 μm, 13 mm.
6.8 Centrifuge, able to operate at 5 000 r/min.
6.9 5 ml plastic syringe.
6.10 Thermostatic bath.
6.11 Analytical system, comprising a HPLC ternary pump with a degassing system equipped
with an HPLC column, RP 18 reverse phase. The following column has proven to be adapted for the
1) Extrasynthese (Cedex, France) and Sigma Aldrich (Germany) are examples of a companies that make suitable
products available commercially. This information is given for the convenience of users of this document and does
not constitute an endorsement by ISO of these products.
2
PROOF/ÉPREUVE © ISO 2022 – All rights reserved

---------------------- Page: 7 ----------------------
ISO 23942:2022(E)
2)
determination (internal diameter 4,6 mm, length 25 cm, size 5 μm, 100 Å, type Spherisorb ODS2 ) with
a UV spectrophotometric detector at 280 nm and integration system. A photodiode array detector (PDA)
for spectra recording can be used to facilitate peak identification, by matching the hydroxytyrosol and
tyrosol spectra in the sample extract with the spectra of the external standard.
A system for analysis data and integration is needed.
7 Sampling
It is important that an intact oil sample is delivered to the laboratory, which has not been damaged
or modified during transport or storage. A representative sample is considered for the purpose of the
analysis. A recommended sampling method is given in ISO 5555.
8 Procedure
8.1 Sample preparation
Weigh, with an analytical balance (6.1), 2 g of oil well-homogenized in a 10 ml conical test tube (6.4).
Add, using a pipette (6.3), 5 ml of the methanol/water solution 80/20 v/v (5.7). Mix the solution with
the help of a mixer for test tube type vortex (6.5) for 1 min and continue the extraction for 15 min
in an ultrasonic bath (6.6) at room temperature. Centrifuge (6.8) at 5 000 r/min for 25 min. Filter
an aliquot through a PVDF membrane syringe filter (6.7). Transfer 1 ml, using a pipette (6.3), of the
filtered solution into another 10 ml test tube (6.4) and completely dry on a thermostatic bath (6.10) at a
maximum temperature of 40 °C under nitrogen stream. Add 1 ml of hydrolysis solution (5.11) and mix,
followed by reaction at 40 °C for 1 h. Leave the solution at room temperature overnight. Then filter the
solution using a PVDF membrane syringe filter (6.7).
8.2 HPLC analysis
8.2.1 General
Inject 20 µl of the sample into the HPLC system (6.11). The first sample injected as part of a series of
analysis shall be a blank of a methanol/water solution 80/20 v/v (5.7). There shall be no interfering
signals present during the chromatographic run at the same retention time of hydroxytyrosol and
tyrosol.
8.2.2 HPLC conditions
The operating conditions given in Table 1 have proven to be adapted for the determination.
2) The Spherisorb ODS2
...