ANNEXES TO TECHNICAL COMMITTEE REPORT

GROUPE D’EXPERTS CHIMISTES POUR L’ÉTUDE DE MÉTHODES D’ANALYSE POUR LES HUILES D’OLIVE ET LES HUILES DE GRIGNONS D’OLIVE

PAYS

ALGÉRIE

EXPERT RETENU

EXPERT NON RETENU

OBSERVATIONS

Ramdane Boussenadji Ministère du Commerce ALGER

C.E. Allemagne

Hans-Jochen Fiebig MRI - Max Rubner Institut MÜNSTER

Espagne

Hipólito Gª Toledo Laboratorio Agroalimentario ATARFE José Ramón García Hierro Laboratorio Arbitral Agroalimentario MADRID

Ne représente pas de frais car il provient de Madrid

Juan Ramón Izquierdo Laboratorio Arbitral Agroalimentario MADRID

Ne représente pas de frais car elle provient de Madrid

Mª Pinar Heredero Laboratorio Central de Aduanas MADRID

Ne représente pas de frais car il provient de Madrid

Eduardo Alonso Laboratorio Central de Aduanas MADRID

Ne représente pas de frais car il provient de Madrid

Ramón Aparicio Instituto de la Grasa SEVILLA Arturo Cert Instituto de la Grasa SEVILLA Wenceslao Moreda Instituto de la Grasa SEVILLA Hermenegildo Cobo Laboratorio del SOIVRE SEVILLA

France

José Linares SOS Cuétara S.A. ANDÚJAR

À ses frais

Carme Gomà Aceites Borges Pont S.A. TÁRREGA

À ses frais

Florence Lacoste Institut des Corps Gras ITERG PESSAC Michel Blanc EXPERAGRO – SAINT-CLOUD Denis Ollivier Laboratoire Interrégional MARSEILLE

Grèce

Efstathia Kremmida-Christopoulou Ministry of Development - ATHENS Vassiliki Panagiotopoulou Ministry of Finance - ATHENS

Italie

Lanfranco Conte Università di UDINE Carlo Mariani Stazione Sperimentale per lo Studio delle Sostanze Grasse - MILANO Pierangela Rovellini Stazione Sperimentale per lo Studio delle Sostanze Grasse - MILANO Luciana Di Giacinto C.R.A. CITTÀ-SANT’ANGELO Fabrizio Apruzzese Direzione Regionale per il Lazio e l’Umbria - ROMA Maurizio Servili Università degli Studi di PERUGIA Raffaele Sacchi Università di Napoli - PORTICI Angelo Faberi Laboratorio Centrale di ROMA

Portugal

Giorgio Cardone CHEMISERVICSE s.a.s. MONOPOLI

À ses frais

Ana Maria Cane UNILEVER ITALIA

À ses frais

Alissa Mattei Carapelli Firenze s.p.a. TAVARNELLE VAL DI PESA

À ses frais

Mª Celeste Gomes (suplente: Fernanda Ramalho) Ajutoridade Segurança Alimentar e Economica - LISBOA Paula Vasconcelos (ou Ana Helena Alegre) Instituto Superior de Agronomia LISBOA

Slovénie

Bojan Butinar ZRS – LABS d.o.o. - IZOLA

ÉGYPTE

Mounir Fahmy Khalil Agricultural Research Center - GIZA

Non retenu Rabiei Zohreh Faculty of New Technologies and Energy Engineering TEHRAN

IRAN

Thamasebi Enferadi Sattar National Institute of Genetic Engineering and Biotechnology TEHRAN

ISRAEL

Zohar Kerem Faculty of Agriculture of the Hebrew University – REHOVOT

Non retenu

JORDANIE

Rafat Abdul-Munem Nimer Ahmad Royal Scientific Society - AMMAN

MAROC

Nadia Maata (ou El-Maâti Benazzouz) Laboratoire Officiel d’Analyses et de Recherches Chimiques CASABLANCA

SYRIE

Zakwan Bido Olive Oil Laboratoires GCSAR IDLEB

TUNISIE

Kamel Ben Ammar Office National de l’Huile - TUNIS

Hamzeh Aliyan Salim Al-Juneidi – Royal Scientif Society AMMAN

Non retenu

Hana’a Al Ghizzawi Jordan Institution for Standards and Metrology Certification AMMAN

Non retenu

Ruba Ayeid AlThawabeyah Jordan Institution for Standards and Technology - AMMAN

Non retenu

INVITÉS AUSTRALIE

Rodney Mayler (Agri. Res. Inst.)

À ses frais

CANADA

Angela Santagati (CFIA)

À ses frais

ÉTATS-UNIS

Richard Cantrill (AOCS)

À ses frais

California Olive Oil Council

À ses frais

Richard E. Mc Donald (FDA)

À ses frais

James V. Falk (US Dept. Agric.)

À ses frais

CODEX

Selma H. Doyran

À ses frais

ISO

un représentant

À ses frais

COMITÉ CONSULTA TIF

un représentant

À ses frais

C.E.

un représentant

À ses frais

TOTAL

33 experts retenus (4 sans frais provenant de Madrid)

5 experts désignés non retenus

15 invités à leurs frais

COMMISSION D’EXPERTS POUR LE CONTRÔLE DES LABORATOIRES PAYS

EXPERT RETENU

EXPERT NON RETENU

Hakim Daoud Ministère du Commerce ALGER

ALGÉRIE

OBSERVATIONS

Ne provient pas d’un laboratoire agréé par le COI

C.E. Espagne

José Ramón García Hierro Laboratorio Arbitral MADRID

France

Michel Blanc EXPERAGRO SAINT-CLOUD

Ne représente pas de frais car il provient de Madrid

Efstathia Kremmida-Christopoulou Participe directement aux analyses de son Minsitry of Development laboratoire en vue de ATHENS l’agrément du COI

Grèce

Italie

Lanfranco Conte Università di Udine UDINE

Portugal

Elvira Esteves Autoridade de Segurança Alimentar e Económica LISBOA Mª Fernanda Ramalho Autoridade de Segurança Alimentar e Económica LISBOA

Participe directement aux analyses de son laboratoire en vue de l’agrément par le COI

COMMISSION RESTREINTE POUR L’ÉTUDE DE LA PRÉSENCE ÉVENTUELLE DE CONTAMINANTS DANS LES HUILES D’OLIVE

PAYS

EXPERT RETENU

EXPERT NON RETENU

OBSERVATIONS

C.E. Espagne

Ne représente pas de frais car elle provient de Madrid

Isabel Hernández Ortíz Laboratorio Arbitral Agroalimentario MADRID Wenceslao Moreda Instituto de la Grasa SEVILLA

France

Florence Lacoste ITERG PESSAC

Grèce

Stella Kontou Pesticide Residues Laboratory Ministry of Finance ATHENS

Italie

Ana Maria Cane UNILEVER – Bestfoods Italia s.r.l. INVERUNO Tiziana Generali Pesticide Residue SEction Istituto Superiore di Sanità ROMA Sabrina Moret Università di Udine UDINE Angelo Faberi Ministero Politiche Alimentari e Forestali ROMA

ISRAEL

Zohar Kerem Faculty of Agriculture REHOVOT

TOTAL

9 experts désignés retenus

Agricole,

GROUPE D’EXPERTS EN ÉVALUATION ORGANOLEPTIQUE DE L’HUILE D’OLIVE PAYS

EXPERT RETENU

EXPERT NON RETENU

Ahmed Rachid Ministère du Commerce ALGER

ALGÉRIE

OBSERVATIONS

Il n’est pas chef d’un des jurys de dégustation agréés par le COI

C.E.

Espagne

Hipólito García Toledo Laboratorio Agroalimentario ATARFE – GRANADA Juan Ramón Izquierdo Laboratorio Arbitral MADRID Wenceslao Moreda Instituto de la Grasa SEVILLA Juan Salas Gómez Laboratorio Agroalimentario CÓRDOBA Mª Ángels Calvo Panel de catadores de aceite de oliva virgen de Cataluña REUS

France

Christian Pinatel AFIDOL/CTO AIX-EN-PROVENCE Florence Lacoste ITERG PESSAC Denis Ollivier Laboratoire Interrégional MARSEILLE

Grèce

Maria Panousopoulou Lazaraki Minsitry of Development ATHENS

Italie

Luciana Di Giacinto

Ne représente pas de frais car il provient de Madrid

Centro Ricerca Olivicoltura CITTÀ SANT’ANGELO (PE) Silvia Tagliabue Stazione Sperimentale Industrie Oli e Grassi MILANO Catarina Forenza Laboratorio Chimico Regionale ROMA Expert en statistique appliquée à l’analyse sensorielle de l’huile d’olive

Andrea Giomo Experto estadístico ABBIATEGRASSO (MI)

Portugal

Paula Vasconcelos Instituto Superior Agronomia LISBOA Laboratoire privé: à ses frais

Ana Carrilho Olivais do Sul, Sociedade Agropecuária Lda MONTE DO TRIGO

Slovénie

Milena Bucar-Miklavcic Science & Research Center of Koper IZOLA

ISRAEL

Fathi Abd El Hadi Israeli Olive Oil Board AFULA

JORDANIE

TUNISIE

Sifi Samira Office National de l’Huile TUNIS

TOTAL

17 experts désignés retenus

Raul Botas Autoridade Segurança Alimentar LISBOA

Il n’est pas chef d’un des jurys de dégustation agréés par le COI

Shimon Lavee Institute of Plant Science REHOVOT

Il n’est pas chef d’un des jurys de dégustation agréés par le COI

Itzhak Tzipori Israeli Southern Research Center GILAT

Il n’est pas chef d’un des jurys de dégustation agréés par le COI

Jamal Mousa Al-Batsh Ministry of Agriculture AMMAN

Il n’est pas chef d’un des jurys de dégustation agréés par le COI

Said Moh’d S. Al Addasi Altoor for Olive Products Co. AMMAN

Il n’est pas chef d’un des jurys de dégustation agréés par le COI

6 experts désignés non retenus

1 expert invité à ses frais

GROUPE D’EXPERTS SUR L’ÉVALUATION ORGANOLEPTIQUE DES OLIVES DE TABLE

PAYS

ALGÉRIE

EXPERT RETENU

EXPERT NON RETENU

OBSERVATIONS

Nourreddine Haridi Direction Qualité et Consommation Ministère du Commerce ALGER

C.E. Espagne

Juan Ramón Izquierdo Laboratorio Arbitral Agroalimentario MADRID Antonio de Mora ASEMESA SEVILLA Luis Rejano Navarro Instituto de la Grasa SEVILLA Antonio Higinio Sánchez Gómez Instituto de la Grasa SEVILLA José Álvarez Calderón SOIVRE Ministerio de Industria, Turismo y Comercio SEVILLA

France

Anne Laurent AFIDOL NYONS

Grèce

Nikos Vallis ROVIES Agricultural Cooperative ROVIES – EVIA Eystathios Panagou National Foundation for Agricultural Research LIKOBRISSI

Ne représente pas de frais car elle provient de Madrid

Italie

Barbara Lanza CRA – OLI Centro di Ricerca per l’Olivicoltura CITTÀ SANT’ANGELO Roberto de Andreis Laboratorio Chimico Merceologico ALBENGA Andrea Giomo ABBIATEGRASSO

Portugal

Cidália Peres INRB/L-INIA OEIRAS

TOTAL

13 experts désignés retenus

Expert en statistique appliquée à l’analyse sensorielle des olives de table

GROUPE D’EXPERTS SUR LES NORMES POUR LES OLIVES DE TABLE

PAYS

ALGÉRIE

EXPERT RETENU

EXPERT NON RETENU

OBSERVATIONS

Nourreddine Haridi Direction Qualité et Consommation Ministère du Commerce ALGER

C.E. Espagne

Alfonso Ruiz López Servicio Control Calidad Ministerio de Medio Ambiente y Medio Rural y Marino MADRID

Ne représente pas de frais car elle provient de Madrid

Magdalena Gálvez Djouma Subdirección General de Frutas y Hortalizas, Aceite de oliva y Vitivinicultura Ministerio de Medio Ambiente y Medio Rural y Marion MADRID

Ne représente pas de frais car elle provient de Madrid

Antonio de Mora ASEMESA SEVILLA

Antonio Garrido Fernández Instituto de la Grasa SEVILLA

Luis Rejano Navarro Instituto de la Grasa SEVILLA

José Álvarez Calderón SOIVRE Ministerio de Industria, Turismo y Comercio SEVILLA

France

Anne Laurent AFIDOL NYONS

Grèce

Nikos Vallis ROVIES Agricultural Cooperative ROVIES – EVIA Eystathios Panagou National Foundation for Agricultural Research LIKOBRISSI Daniellos Georgoudis Panhellenic Association of Table Olive Processors, Packers and Exporters VOLOS

Italie

Barbara Lanza CRA – OLI Centro di Ricerca per l’Olivicoltura CITTÀ SANT’ANGELO Francesco La Croce Ditta Peruzza Nicolò e C. s.c.n. CASTELVETRANO

Portugal

Cidália Peres INRB/L-INIA OEIRAS

MAROC

El-Maâti Benazzouz Laboratoire Officiel d’Analyses Recherches Chimiques CASABLANCA

TOTAL

15 experts désignés retenus

et

de

GROUPE D’EXPERTS POUR L’ÉTUDE DE MÉTHODES D’ANALYSE SUR LES OLIVES À HUILE

PAYS

ALGÉRIE

EXPERT RETENU

EXPERT NON RETENU

OBSERVATIONS

Ahmed Sebai Institut Technique de l’Arboriculture Fruitière SIDI AICH Soraya Idres Qualité et Répression des Fraudes Ministère du Commerce ALGER Mustapha Chabour Ministère de l’Agriculture THÉNIA - BOUMERDÈS

C.E. Espagne

Ne représente pas de frais car il provient de Madrid

Juan Ramón Izquierdo Laboratorio Arbitral Agroalimentario MADRID José Álvarez Calderón SOIVRE Ministerio de Industria, Turismo y Comercio SEVILLA José Alba Mendoza Instituto de la Grasa SEVILLA

France

Christian Pinatel AFIDOL AIX-EN-PROVENCE

Grèce

Efstathia Kremmida-Christopoulou Directorate of Technical Control Consumer Protection Ministry of Development ATHENS

Italie

for

Enzo Perri CRA – Istituto Sperimentale per l’Olivicoltura RENDE Angelo Cichelli Università degli Studi di Pescara PESCARA

Portugal

Paula Vasconcelos (ou Ana Helena Alegre) Instituto Superior de Agronomia LISBOA

ISRAEL

Benjamin Avidan

En attente de recevoir coordonnées et CV

Arnon Dag

En attente de recevoir coordonnées et CV

LIBAN

Georges Hassoun Faculté des Sciences Agronomiques JBEIL

MAROC

El-Maâti Benazzouz Laboratoire Officiel d’Analyses Recherches Chimiques CASABLANCA

et

de

SYRIE

Mohamed Ghassan Ejbara General Commission for Scientific and Agriculture Research IDLEB

TUNISIE

Kamel Ben Ammar Office National de l’Huile TUNIS

TOTAL

17 experts désignés retenus

GROUPE D’EXPERTS SUR LA COMPOSITION NUTRITIONNELLE DES OLIVES DE TABLE

PAYS

ALGÉRIE

EXPERT RETENU

EXPERT NON RETENU

OBSERVATIONS

Nourreddine Haridi Direction Qualité et Consommation Ministère du Commerce ALGER

C.E. Espagne

Alfonso Ruiz López Servicio Control Calidad Ministerio de Medio Ambiente y Medio Rural y Marino MADRID Mª Teresa Marín Tapia Laboratorio Arbitral Agroalimentario MADRID Antonio Garrido Fernández Instituto de la Grasa SEVILLA José Álvarez Calderón SOIVRE Ministerio de Industria, Turismo y Comercio SEVILLA

France

Anne Laurent AFIDOL NYONS

Grèce

Maria Kapsokefalou Agricultural University ATHENS

Italie

Barbara Lanza CRA – OLI Centro di Ricerca per l’Olivicoltura CITTÀ SANT’ANGELO

Portugal

Cidália Peres INRB/L-INIA OEIRAS

Ne représente pas de frais car elle provient de Madrid

Ne représente pas de frais car elle provient de Madrid

EGYPTE

Wael Abas Ahmed Horticulture Research Institute GIZA

MAROC

Hassan Debbarh Fédération des Industries de la Conserve CASABLANCA

TUNISIE

Mokhtar Zarrouk Centre de Biotechnologie de Borj-Cdria (CBBC) HAMMAM-LIF

TOTAL

12 experts désignés retenus

GROUPE D’EXPERTS POUR L’ÉTUDE DE LA COMPOSITION DES HUILES D’OLIVE ET L’IDENTIFICATION VARIÉTALE

EXPERT RETENU

PAYS

ALGÉRIE

EXPERT NON RETENU

OBSERVATIONS

Sonia Keciri Institut Technique de l’Arboriculture Fruitière et de la Vigne SIDI AICH Ou Mustapha Chabour Ministère de l’Agriculture THÉNIA-BOUMERDÈS

C.E. Chypre

Androula Georgiou Agricultural Research Institute NICOSIA

Espagne

Ana Isabel Blanch Laboratorio Arbitral MADRID

France

Christian Pinatel AFIDOL/CTO AIX-EN-PROVENCE ITERG PESSAC Denis Ollivier Laboratoire Interrégional MARSEILLE

Grèce

Efstathia Kremmida-Christopoulou Minsitry of Development ATHENS

Italie

Lanfranco Conte Università di Udine UDINE Maurizio Servili Università degli Studi PERUGIA

Ne représente pas de frais car elle provient de Madrid

Portugal

Paula Vasconcelos (ou Ana Helena Alegre) Instituto Superior Agronomia LISBOA

ISRAEL

Shimon Lavee (ou Zohar Kerem) Faculty of Agriculture REHOVOT

MAROC

Abderraouf El Antari Institut National de la Recherche Agronomique MARRAKECH

SYRIE

Mustafa Moalem Ministry of Agriculture and Agrarian Reform IDLEB

TUNISIE

Sifi Samira Office National de l’Huile TUNIS

TOTAL

13 experts désignés retenus

GROUPE D’EXPERTS JURY INTERNATIONAL PRIX À LA QUALITÉ DU CONSEIL OLEÍCOLE INTERNATIONAL “MARIO SOLINAS”

PAYS

EXPERT RETENU

EXPERT NON RETENU

Koli Sami Ministère du Commerce ALGER

ALGÉRIE

OBSERVATIONS

Il n’est pas chef d’un des jurys de dégustation agréés par le COI

C.E.

Espagne

Hipólito García Toledo Laboratorio Agroalimentario ATARFE – GRANADA Juan Ramón Izquierdo Laboratorio Arbitral MADRID

Ne représente pas de frais car il provient de Madrid

Juan Salas Gómez Laboratorio Agroalimentario CÓRDOBA

France

Denis Ollivier Laboratoire Interrégional MARSEILLE

Grèce

Maria Panousopoulou Lazaraki Minsitry of Development ATHENS

Italie

Luciana Di Giacinto Centro Ricerca Olivicoltura CITTÀ SANT’ANGELO (PE) Silvia Tagliabue Stazione Sperimentale Industrie Oli e Grassi MILANO

Andrea Giomo Experto estadístico ABBIATEGRASSO (MI)

Portugal

José Gouveia Instituto Superior Agronomia LISBOA

Expert en statistique appliquée à l’analyse sensorielle de l’huile d’olive

ISRAEL

TUNISIE

Sifi Samira Office National de l’Huile TUNIS

TOTAL

10 experts désignés retenus

Raul Botas Autoridade Segurança Alimentar LISBOA

Il n’est pas chef d’un des jurys de dégustation agréés par le COI

Fathi Abd El Hadi Israeli Olive Oil Board AFULA

Son jury n’a pas l’ancienneté requise en tant que jury agréé par le COI (*)

3 experts désignés non retenus

(*) Le Jury international du Prix à la qualité du Conseil Oléicole International “Mario Solinas” est formé par 9 membres + l’expert en statistique appliquée à l’analyse sensorielle des huiles d’olive. Les membres de ce Jury doivent être chefs de jury ayant été reconnu par le COI au moins pendant 5 années consécutives, raison pour laquelle la composition du jury est variable.

GROUPE D’EXPERTS POUR L’ÉTUDE DES NORMES DOC/IGP

PAYS

ALGÉRIE

EXPERT RETENU

Seddi Nacéra Ministère du Commerce ALGER

C.E. Portugal

David Gouveia Gabinete de Planeamento e Politicas LISBOA

Espagne

Carmen Serrano Balazote Ministerio de Medio Ambiente y Medio Rural y Marino MADRID

ISRAEL

Arnon Dag (Shimon Lavee) Faculty of Agriculture REHOVOT

TOTAL

4 experts désignés retenus

EXPERT NON RETENU

OBSERVATIONS

INTERNATIONAL OLIVE COUNCIL

Príncipe de Vergara, 154 – 28002 Madrid – España Telef.: +34 915 903 638 Fax: +34 915 631 263 - e-mail: [email protected] - http://www.internationaloliveoil.org/

- Draft -

DECISION No DEC-…/97-V/2009

TRADE STANDARD APPLYING TO OLIVE OILS AND OLIVE-POMACE OILS

THE COUNCIL OF MEMBERS OF THE INTERNATIONAL OLIVE COUNCIL,

Having regard to Decision No DEC-22/96-V/08 of 21 November 2008 whereby the Council adopted the trade standard applying to olive oils and olivepomace oils referenced COI/T.15/NC No 3/Rev. 3 pursuant to the International Agreement on Olive Oil and Table Olives, 2005, in particular articles 21 and 25 thereof, Having regard to the recommendation framed by the Technical Committee to adopt the duly justified methods for the Determination of the content of waxes, fatty acid methyl and ethyl esters by capillary gas chromatography referenced COI/T.20/Doc. No 28 and the Determination of biophenols in olive oils by HPLC referenced COI/T.20/Doc. No 29, Whereas it is therefore necessary to create sections 4.9, 11.24 and 11.25 in the trade standard,

-2-

DECIDES

The trade standard applying to olive oils and olive-pomace oils referenced COI/T.15/NC No 3/Rev. 4 of November 2009 shall replace and rescind the trade standard applying to olive oils and olive-pomace oils referenced COI/T.15/NC No 3/Rev. 3 of November 2008. The Members shall take whatever measures are appropriate, in the manner required by their legislation, to apply the standard adopted and shall notify the Executive Secretariat of any such measures as soon as they are taken. Non-Member States involved in international trade in olive oils and olivepomace oils shall be invited to take into consideration the standard adopted and to adapt their regulations to the provisions thereof.

Madrid (Spain), … November 2009

__________

COI/T.15/NC No 3/Rev. 4 November 2009

INTERNATIONAL OLIVE

ENGLISH Original: FRENCH

COUNCIL

Príncipe de Vergara, 154 – 28002 Madrid – España Telef.: +34 915 903 638 Fax: +34 915 631 263 - e-mail: [email protected] - http://www.internationaloliveoil.org/

- Draft -

TRADE STANDARD APPLYING TO OLIVE OILS AND OLIVE-POMACE OILS

__________

INTERNATIONAL OLIVE

COI/T.15/NC No 3/Rev. 4 November 2009 ENGLISH Original: FRENCH

COUNCIL

Príncipe de Vergara, 154 – 28002 Madrid – España Telef.: +34 915 903 638 Fax: +34 915 631 263 – e-mail: [email protected] - http://www.internationaloliveoil.org/

TRADE STANDARD APPLYING TO OLIVE OILS AND OLIVE-POMACE OILS

1.

SCOPE

This standard applies to olive oils and olive-pomace oils that are the object of international trade or of concessional or food aid transactions.

2.

DESIGNATIONS AND DEFINITIONS

2.1. Olive oil is the oil obtained solely from the fruit of the olive tree (Olea europaea L.), to the exclusion of oils obtained using solvents or re-esterification processes and of any mixture with oils of other kinds. It is marketed in accordance with the following designations and definitions: 2.1.1. Virgin olive oils are the oils obtained from the fruit of the olive tree solely by mechanical or other physical means under conditions, particularly thermal conditions, that do not lead to alterations in the oil, and which have not undergone any treatment other than washing, decantation, centrifugation and filtration. 2.1.1.1. Virgin olive oils fit for consumption as they are include: (i) Extra virgin olive oil: virgin olive oil which has a free acidity, expressed as oleic acid, of not more than 0.8 grams per 100 grams, and the other characteristics of which correspond to those fixed for this category in this standard.

COI/T.15/NC No 3/Rev. 4 page 2

(ii) Virgin olive oil: virgin olive oil which has a free acidity, expressed as oleic acid, of not more than 2 grams per 100 grams and the other characteristics of which correspond to those fixed for this category in this standard. (iii) Ordinary virgin olive oil: virgin olive oil which has a free acidity, expressed as oleic acid, of not more than 3.3 grams per 100 grams and the other characteristics of which correspond to those fixed for this category in this standard.1/ 2.1.1.2. Virgin olive oil not fit for consumption as it is, designated lampante virgin olive oil, is virgin olive oil which has a free acidity, expressed as oleic acid, of more than 3.3 grams per 100 grams and/or the organoleptic characteristics and other characteristics of which correspond to those fixed for this category in this standard. It is intended for refining or for technical use. 2.1.2. Refined olive oil is the olive oil obtained from virgin olive oils by refining methods which do not lead to alterations in the initial glyceridic structure. It has a free acidity, expressed as oleic acid, of not more than 0.3 grams per 100 grams and its other characteristics correspond to those fixed for this category in this standard. 2/ 2.1.3. Olive oil is the oil consisting of a blend of refined olive oil and virgin olive oils fit for consumption as they are. It has a free acidity, expressed as oleic acid, of not more than 1 gram per 100 grams and its other characteristics correspond to those fixed for this category in this standard.3/ 2.2. Olive-pomace oil is the oil obtained by treating olive pomace with solvents or other physical treatments, to the exclusion of oils obtained by re-esterification processes and of any mixture with oils of other kinds. It is marketed in accordance with the following designations and definitions: 2.2.1. Crude olive-pomace oil is olive-pomace oil whose characteristics correspond to those fixed for this category in this standard. It is intended for refining for use for human consumption, or it is intended for technical use. ____________

1/

This designation may only be sold direct to the consumer if permitted in the country of retail sale. If not permitted, the designation of this product shall comply with the legal provisions of the country concerned.

2/

This designation may only be sold direct to the consumer if permitted in the country of retail sale.

3/

The country of retail sale may require a more specific designation.

COI/T.15/NC No 3/Rev. 4 page 3

2.2.2. Refined olive-pomace oil is the oil obtained from crude olive-pomace oil by refining methods which do not lead to alterations in the initial glyceridic structure. It has a free acidity, expressed as oleic acid, of not more than 0.3 grams per 100 grams and its other characteristics correspond to those fixed for this category in this standard.1/ 2.2.3. Olive-pomace oil is the oil comprising the blend of refined olive-pomace oil and virgin olive oils fit for consumption as they are. It has a free acidity of not more than 1 gram per 100 grams and its other characteristics correspond to those fixed for this category in this standard.2/ In no case shall this blend be called "olive oil".

3.

PURITY CRITERIA

The identity characteristics comprising the purity criteria shall be applicable to olive oils and olive-pomace oils. The limits established for each criterion include the precision values of the attendant recommended method. 3.1.

Fatty acid composition as determined by gas chromatography (% m/m methyl esters): -

Myristic acid Palmitic acid Palmitoleic acid Heptadecanoic acid Heptadecenoic acid Stearic acid Oleic acid Linoleic acid Linolenic acid Arachidic acid Gadoleic acid (eicosenoic) Behenic acid Lignoceric acid

< 0.05 7.5 - 20.0 0.3 - 3.5 < 0.3 < 0.3 0.5 - 5.0 55.0 - 83.0 3.5 - 21.0 < 1.0 < 0.6 < 0.4 < 0.2* < 0.2

__________ 1/

This product may only be sold direct to the consumer if permitted in the country of retail sale.

2/

The country of retail sale may require a more specific designation.

*

Limit raised to < 0.3 for olive-pomace oils.

COI/T.15/NC No 3/Rev. 4 page 4

3.2.

Trans fatty acid content (% trans fatty acids) C18:1 T

-

Edible virgin olive oils Lampante virgin olive oil Refined olive oil Olive oil Crude olive-pomace oil Refined olive-pomace oil Olive-pomace oil

3.3.

Sterol and triterpene dialcohol composition

3.3.1.

Desmethylsterol composition (% total sterols)

- Cholesterol - Brassicasterol - Campesterol - Stigmasterol - Delta-7-stigmastenol - Beta-sitosterol + delta-5-avenasterol + delta-5-23-stigmastadienol + clerosterol + sitostanol + delta 5-24-stigmastadienol

__________ * Limit raised to < 0.2 for olive-pomace oils.

% _______

C18:2 T + C18:3 T % _______

< 0.05 < 0.10 < 0.20 < 0.20 < 0.20 < 0.40 < 0.40

< 0.05 < 0.10 < 0.30 < 0.30 < 0.10 < 0.35 < 0.35

< 0.5 < 0.1 * < 4.0 < campesterol in edible oils < 0.5

> 93.0

COI/T.15/NC No 3/Rev. 4 page 5

3.3.2. -

3.3.3. 3.4.

Total sterol content

Virgin olive oils Refined olive oil Olive oil Crude olive-pomace oil Refined olive-pomace oil Olive-pomace oil

Erythrodiol and uvaol content

> 1000 > 2500 > 1800 > 1600

(% total sterols)

Edible virgin olive oils Lampante virgin olive oil Refined olive oil Olive oil Crude olive-pomace oil Refined olive-pomace oil Olive-pomace oil Wax content

-

(mg/kg)

3502/ > 350 > 350

__________ 1/

When the oil has a wax content between 300 mg/kg and 350 mg/kg it is considered a lampante virgin olive oil if the total aliphatic alcohol content is < 350 mg/kg or the erythrodiol + uvaol content is < 3.5%.

2/

When the oil has a wax content between 300 mg/kg and 350 mg/kg it is considered a crude olive-pomace oil if the total aliphatic alcohol content is > 350 mg/kg and the erythrodiol + uvaol content is > 3.5%.

COI/T.15/NC No 3/Rev. 4 page 6

3.5. Maximum difference between the actual and theoretical ECN 42 triacylglycerol content -

3.6.

Edible virgin olive oils Lampante virgin olive oil Refined olive oil Olive oil Crude olive-pomace oil Refined olive-pomace oil Olive-pomace oil

Stigmastadiene content -

3.7.

0.2 0.3 0.3 0.3 0.6 0.5 0.5

(mg/kg) < 0.10 < 0.50

Edible virgin olive oils Lampante virgin olive oil

Content of 2-glyceryl monopalmitate -

Edible virgin olive oils and olive oil: C:16:0 < 14.0%; 2P < 0.9% C:16:0 > 14.0%, 2P < 1.0%

-

Non-edible virgin olive oils and refined olive oils: C:16:0 < 14.0%; 2P < 0.9% C:16:0 > 14.0%, 2P < 1.1%

-

Olive-pomace oils

< 1.2%

-

Crude and refined olive-pomace oils

< 1.4%

3.8.

Unsaponifiable matter -

Olive oils Olive-pomace oils

(g/kg) < 15 < 30

COI/T.15/NC No 3/Rev. 4 page 7

4. QUALITY CRITERIA The limits established for each criterion and designation include the precision values of the attendant recommended method

Extra virgin olive oil

Virgin olive oil

Ordinary virgin olive oil

Lampante virgin olive oil *

Refined olive oil

Olive oil

acceptable

Crude olivepomace oil

Refined olivepomace oil

Olivepomace oil

good

acceptable

good

- colour

light yellow

light, yellow to green

light, yellow to brownish yellow

light, yellow to green

- aspect at 20oC for 24 hours

limpid

limpid

limpid

limpid

4.1 Organoleptic characteristics - odour and taste - odour and taste (on a continuous scale): . median of defect . median of the fruity attribute

*

Me = 0 Me > 0

0 < Me < 3.5 Me > 0

3.5 6.0

4.2. Free acidity % m/m expressed in oleic acid

< 0.8

< 2.0

< 3.3

> 3.3

< 0.3

< 1.0

no limit

< 0.3

< 1.0

4.3. Peroxide value in milleq. Peroxide oxygen per kg/oil

< 20

< 20

< 20

no limit

150 mg/kg or ratio FAEEs/FAMEs >1.5 mg/kg

This determination is solely for application by commercial partners on an optional basis.

** Commercial partners in the country of retail sale may require compliance with these limits when the oil is made available to the end consumer. *** After passage of the sample through activated alumina, absorbency at 270 nm shall be equal to or less than 0.11.

< 1.5

> 120oC

COI/T.15/NC No 3/Rev. 4 page 9

5.

FOOD ADDITIVES

5.1.

Virgin olive oils and crude olive-pomace oil: none permitted.

5.2. Refined olive oil, olive oil, refined olive-pomace oil and olive-pomace oil: alpha-tocopherol permitted to restore natural tocopherol lost in the refining process. Maximum level: 200 mg/kg of total alpha-tocopherol in the final product.

6.

CONTAMINANTS

6.1.

Heavy metals

The products covered by this standard shall comply with maximum limits being established by the Codex Alimentarius Commission but in the meantime the following limits will apply:

Maximum permissible concentration Lead (Pb) Arsenic (As)

6.2.

0.1 mg/kg 0.1 mg/kg

Pesticide residues

The products covered by this standard shall comply with those maximum residue limits established by the Codex Alimentarius Commission for these commodities.

6.3.

Halogenated solvents -

Maximum content of each halogenated solvent Maximum content of the sum of all halogenated solvents

0.1 mg/kg 0.2 mg/kg

COI/T.15/NC No 3/Rev. 4 page 10

7.

HYGIENE

7.1. It is recommended that the products intended for human consumption covered by the provisions of this standard be prepared and handled in accordance with the appropriate sections of the Recommended International Code of Practice – General Principles of Food Hygiene (CAC/RP 1-1969, Rev. 3 – 1997), and other relevant Codex texts such as Codes of Hygienic Practice and Codes of Practice. 7.2. The products intended for human consumption should comply with any microbiological criteria established in accordance with the Principles for the Establishment and Application of Microbiological Criteria (CAC/GL 21-1997).

8.

PACKING

Olive oils and olive-pomace oils intended for international trade shall be packed in containers complying with the General Principles of Food Hygiene recommended by the Codex Alimentarius Commission (CAC/RCP 1 - 1969, Rev. 3 1997), and other relevant texts such as Codes of Hygienic Practice and Codes of Practice. The containers used may be: 8.1. tanks, containers, vats, which permit the transportation in bulk of olive oils and olive-pomace oils; 8.2. metal drums, in good condition, hermetically-sealed, which should be internally covered with a suitable varnish; 8.3. metal tins and cans, lithographed, new, hermetically-sealed, which should be internally covered with a suitable varnish; 8.4. demi-johns, glass bottles or bottles made of suitable macromolecular material.

9.

CONTAINER FILLING TOLERANCE

The volume occupied by the contents shall under no circumstances be less than 90% of the capacity of the container, except in the case of tin containers with a capacity of, or less than, 1 litre in which the volume occupied shall under no circumstances be less than 80% of the capacity of the container; this capacity is equal to the volume of distilled water at 20oC which the container can hold when full.

COI/T.15/NC No 3/Rev. 4 page 11

10.

LABELLING

In addition to sections 2, 3, 7 and 8 of the Codex General Standard for the Labelling of Pre-packaged Foods (CODEX STAN 1-1985, Rev.1 - 1991) and the guidelines applying to food not intended for direct sale to consumers, the specific provisions providing the following information shall be applied: 10.1.

On containers intended for direct sale to consumers

10.1.1.

Name of the product

The labelling on each container shall indicate the specific designation of the product contained, complying in every way with the relevant provisions of this standard. 10.1.1.1. Designations of olive oils: -

Extra virgin olive oil Virgin olive oil Ordinary virgin olive oil1/ Refined olive oil1/ Olive oil2/

10.1.1.2. Designations of olive-pomace oils: - Refined olive-pomace oil1/ - Olive-pomace oil2/ 10.1.2.

Net contents

The net contents shall be declared by volume in the metric system ("Système International" units). 10.1.3.

Name and address

The name and address of the manufacturer, packer, distributor, importer, exporter or seller shall be declared.

_________________ 1/

2/

This product may only be sold direct to the consumer if permitted in the country of retail sale. The country of retail sale may require a more specific designation.

COI/T.15/NC No 3/Rev. 4 page 12

10.1.4.

Country of origin

The name of the country of origin shall be declared. When the product undergoes substantial processing in a second country, the country in which such processing is carried out shall be considered as the country of origin for labelling purposes. 10.1.5.

Indications of source and appellations of origin

10.1.5.1. Indications of source The labels of virgin olive oils may indicate their source (country, region or locality) when they have been empowered to do so by their country of origin and when such virgin olive oils have been produced, packed and originate exclusively in the country, region or locality mentioned. 10.1.5.2. Appellations of origin The labels of extra virgin olive oils may indicate their appellation of origin (country, region or locality) when they have been awarded such an appellation, in accordance with the terms provided under the regulations of their country of origin and when such extra virgin olive oil has been produced, packed and originates exclusively in the country, region or locality mentioned. 10.1.6.

Lot identification

Each container shall be embossed or otherwise permanently marked in code or in clear to identify the producing factory and the lot. 10.1.7.

Date marking and storage conditions

10.1.7.1. Date of minimum durability In the case of pre-packaged products intended for the end consumer, the date of minimum durability (preceded by the words "best before end") shall be declared by the month and year in uncoded numerical sequence. The month may be indicated by letters in those countries where such use will not confuse the consumer; if the shelf life of the product is valid to December, the expression "end (stated year)" may be used as an alternative. 10.1.7.2. Storage instructions Any special conditions for storage shall be declared on the label if the validity of the date of minimum durability depends thereon.

COI/T.15/NC No 3/Rev. 4 page 13

10.2.

On forwarding packs of oils intended for human consumption

In addition to the details noted under section 10.1., the following inscription shall appear: - number and type of containers held in pack. 10.3. On containers allowing the transportation in bulk of olive oils and olive-pomace oils The labelling on each container shall include: 10.3.1.

Name of the product

The name shall indicate the specific designation of the product contained, complying in every way with the provisions of this standard. 10.3.2.

Net contents

The net contents shall be declared by weight or volume in the metric system ("Système International" units). 10.3.3.

Name and address The name and address of the manufacturer, distributor or exporter shall be

declared. 10.3.4.

Country of origin The name of the exporting country shall be declared.

11.

METHODS OF ANALYSIS AND SAMPLING

The methods of analysis and sampling given below are international referee methods. The latest version of these methods should be used. 11.1.

Sampling According to ISO 5555, "Animal and vegetable fats and oils - Sampling".

COI/T.15/NC No 3/Rev. 4 page 14

11.2.

Preparation of the test sample

According to ISO 661, "Animal and vegetable fats and oils - Preparation of the test sample".

11.3.

Determination of the fatty acid composition

According to COI/T.20/Doc. No 24, "Preparation of the fatty acid methyl esters from olive oil and olive-pomace oil", and ISO 5508, "Analysis by gas chromatography of methyl esters of fatty acids" or AOCS Ch 2–91.

11.4.

Determination of the trans fatty acid content

According to COI/T.20/Doc. No 17/Rev. 1, "Determination of trans unsaturated fatty acids by capillary column gas chromatography”, or ISO 15304 or AOCS Ce 1f–96.

11.5.

Determination of the sterol composition and total sterol content

According to COI/T.20/Doc. No 10/Rev. 1, "Determination of the composition and content of sterols by capillary-column gas chromatography", or AOCS Ch 6-91.

11.6.

Determination of the content of erythrodiol + uvaol

According to IUPAC no. 2.431, "Determination of the erythrodiol content". Capillary columns are recommended.

11.7.

Determination of the wax content

According to COI/T.20/Doc. No 18/Rev. 2 “Determination of wax content by capillary-column gas chromatography” or AOCS Ch 8-02.

11.8.

Determination of the aliphatic alcohol content

According to COI/T.20/Doc. No 26 “Determination of aliphatic alcohols content by capillary gas chromatography”.

COI/T.15/NC No 3/Rev. 4 page 15

11.9. Determination of the difference between the actual and theoretical ECN 42 triacylglycerol content According to COI/T.20/Doc. No 20/Rev. 2, "Determination of the difference between actual and theoretical content of triacylglycerols with ECN 42", or AOCS 5b-89. 11.10.

Determination of the stigmastadiene content

According to COI/T.20/Doc. No 11/Rev. 2, "Determination of stigmastadienes in vegetable oils", or COI/T.20/Doc. no. 16/Rev. 1, "Determination of sterenes in refined vegetable oils", or ISO 15788-1 or AOCS Cd 26-96.

11.11.

Determination of the content of 2-glyceryl monopalmitate

According to COI/T.20/Doc. No 23, "Determination of the percentage of 2glyceryl monopalmitate ". 11.12.

Determination of the unsaponifiable matter

According to ISO 3596, “Determination of the unsaponifiable matter – Method using diethyl ether extraction”, or AOCS Ca 6b-53 or ISO 18609. The results should be expressed in g/unsaponifiable matter per kg/oil. 11.13.

Determination of the organoleptic characteristics

According to COI/T.20/Doc. No 15/Rev. 2, "Organoleptic assessment of virgin olive oil". 11.14.

Determination of the free acidity

According to ISO 660, "Determination of acid value and acidity", or AOCS Cd 3d-63.

11.15.

Determination of the peroxide value

According to ISO 3960, “Determination of the peroxide value”, or AOCS Cd 8b-90.

COI/T.15/NC No 3/Rev. 4 page 16

11.16.

Determination of the absorbency in ultra-violet

According to COI/T.20/Doc. No 19/Rev. 2, "Spectrophotometric investigation in the ultraviolet", or ISO 3656 or AOCS Ch 5-91. 11.17.

Determination of the moisture and volatile matter According to ISO 662, "Determination of the moisture and volatile matter".

11.18.

Determination of the insoluble impurities in light petroleum According to ISO 663, "Determination of the insoluble impurities".

11.19.

Determination of the flash point According to the FOSFA International method.

11.20.

Detection of trace metals

According to ISO 8294, "Determination of copper, iron and nickel by direct graphite furnace atomic absorption spectrometry". 11.21.

Determination of the alpha-tocopherol

According to ISO 9936, “Determination of tocopherols and tocotrienols contents – Method using high-performance liquid chromatography”. 11.22.

Determination of traces of heavy metals

-

Determination of lead: according to ISO 12193 or AOCS Ca 18c-91 or AOAC 994.02.

-

Determination of arsenic: according to AOAC 952.13 or AOAC 942.17 or AOAC 985.16.

11.23.

Detection of traces of halogenated solvents

According to COI/T.20/Doc. No 8/Corr. 1 “Determination tetrachloroethylene in olive oils by gas-liquid chromatography”.

of

COI/T.15/NC No 3/Rev. 4 page 17

11.24.

Determination of the content of waxes and alkyl esters

According to COI/T.20/Doc. No 28 “Determination of the content of waxes, fatty acid methyl esters and fatty acid ethyl esters by capillary gas chromatography“.

11.25.

Determination of biophenols

According to COI/T.20/Doc. No 29 “Determination of biophenols in olive oils by HPLC“.

__________

INTERNATIONAL OLIVE COUNCIL

COI/T.20/Doc No 29 May 2009 ENGLISH Original: ITALIAN

Príncipe de Vergara, 154 – 28002 Madrid – España Telef.: +34 915 903 638 Fax: +34 915 631 263 - e-mail: [email protected] - http://www.internationaloliveoil.org/

DETERMINATION OF BIOPHENOLS IN OLIVE OILS BY HPLC

1

PURPOSE This method describes a procedure for the extraction and HPLC quantification of the biophenolic minor polar (BMP) compounds in olive oils, such as the natural and oxidised derivatives of oleuropein and ligstroside, lignans, flavonoids and phenolic acids. The range of measurement is from 30 mg/kg to 800 mg/kg. WARNING: This method may require the use of dangerous apparatus and chemicals or the performance of dangerous operations. It does not specify all the safety issues connected with its use. Users are therefore responsible for taking all appropriate safety measures beforehand and for observing any legal requirements.

2

PRINCIPLE The method is based on direct extraction of the biophenolic minor polar compounds from olive oil by means of a methanol solution and subsequent quantification by HPLC with the aid of a UV detector at 280 nm. Syringic acid is used as the internal standard. The content of the natural and oxidised oleuropein and ligstroside derivatives, lignans, flavonoids and phenolic acids is expressed in mg/kg of tyrosol.

3

EQUIPMENT

3.1

High-performance ternary gradient liquid chromatograph (HPLC), equipped with C18 reverse-phase column (4.6 mm x 25 cm), Spherisorb ODS-2 5µm, 100 A°, with spectrophotometric UV detector at 280 nm and integrator. Room temperature. Spectral recording for identification purposes is facilitated by using a photodiode detector with a spectral range from 200 nm to 400 nm.

3.2

Flasks, 10 ml and 100 ml, Class A.

3.3

Electronic pipette, 100 µl, 1000 µl and 5000 µl.

3.4

Test tubes, with screw cap, 10 ml.

3.5

Agitator for test tubes1

3.6

Ultrasonic extraction bath.

3.7

Syringe filters ∅13 mm, PVDF type 0.45 µm.

1

Vortex type.

COI/T. 20/Doc. No 29 Page 2

3.8

Centrifuge capable of working at a speed of 5000 rev/min.

3.9

Balance, accurate to ± 0.001 g.

3.10

Plastic syringes, 5 ml.

3.11

Usual laboratory glassware.

4

REAGENTS Reagents should be pure HPLC chromatography grade.

4.1

Orthophosphoric acid, 85% (V/V).

4.2

Methanol, chromatography grade.

4.3

Acetonitrile, chromatography grade.

4.4

Water, chromatography grade.

4.5

Ternary linear elution gradient: water 0.2 % H3PO4 (V/V) (A), methanol (B), acetonitrile (C). Elution solvents should be de-gassed. Gradient elution should be performed as follows: Gradient elution Time min

Flow ml/min

A %

B %

C %

0

1.00

96

2

2

40

1.00

50

25

25

45

1.00

40

30

30

60

1.00

0

50

50

70

1.00

0

50

50

72

1.00

96

2

2

82

1.00

96

2

2

4.6

2- (4 - hydroxyphenyl) ethanol (tyrosol) ≥ 98 %.

4.7

3,5 dimethoxy 4- hydroxy benzoic acid (syringic acid) ≥ 97 % .

4.8

Extraction solution: methanol/water 80/20 (V/V).

4.9

Solution of external calibration standards (tyrosol and syringic acid). Accurately weigh 0.030 g of tyrosol (4.6) and 0.015 g of syringic acid (4.7) into a 10 ml volumetric flask (3.2). Make up to volume with the solution of methanol/water 80/20 (V/V) (4.8). Transfer 100 µl (3.3) of the solution to a 10 ml volumetric flask. Make up to volume with the solution of methanol/water 80/20 (V/V) (4.8). The concentrations of the external calibration solution are as follows: tyrosol 0.030 mg/ml, syringic acid 0.015 mg/ml. The solution is stable if kept for three months in the refrigerator at + 4°C.

COI/T. 20/Doc. No 29 Page 3

4.10

Preparation of the internal standard solution (syringic acid). Weigh accurately 0.015 g (4.7) of syringic acid into a 10 ml volumetric flask and make up to volume with the solution of methanol/water 80/20 (V/V) (4.8). Transfer 1 ml (3.3) of the solution to a 100 ml volumetric flask (3.2). Make up to volume with the solution of methanol/water 80/20 (V/V) (4.8). The final concentration is 0.015 mg/ml. The solution is stable if kept for three months in the refrigerator at + 4°C.

5

PROCEDURE

5.1

Sample preparation

In a 10 ml screw-cap test tube (3.4) accurately weigh 2.0 g of olive oil. Transfer 1 ml of the internal standard solution (4.10) to the previously weighed sample. Seal with the screw cap and shake (3.5) for exactly 30 sec. Add 5 ml (3.3) of the methanol/water 80/20 (V/V) extraction solution (4.8). Shake (3.5) for exactly 1 min. Extract in the ultrasonic bath (3.6) for 15 min at room temperature. Centrifuge at 5000 rev/min for 25 min (3.8). Take an aliquot of the supernatant phase and filter through a 5 ml plastic syringe (3.10), with a 0.45 µm PVDF filter (3.7). 5.2

HPLC analysis Switch on the UV spectrophotometer at least 1 hour before analysis. The chromatography column should be conditioned for at least 15 min with the elution solvent (initial composition) (water 0.2 % H3PO4 (V/V) /methanol/acetonitrile 96/2/2 (V/V/V)) (gradient elution). A preliminary empty gradient chromatographic run should always be done (to make sure there are no interfering co-elution peaks) by injecting 20 µl of methanol/water 80/20 (V/V) (4.8) into the HPLC system. Inject 20 µl of the external calibration standard solution (4.9) and record the chromatogram at 280 nm. Calculate the values of the response factors RF for 1 µg of tyrosol and 1 µg of syringic acid (6.1). Calculate the ratio of the response factor of syringic acid to tyrosol, called RRFsyr/tyr. Note down the values (6.2). Inject 20 µl of the final sample solution into the HPLC system and record the chromatogram at 280 nm. Perform two independent determinations on the same sample and check that the results lie inside the precision values of the method. Figure 1 shows a typical chromatogram of the biophenols in an extra virgin olive oil characterised by individual component. The sum of the areas of the individual peaks should be taken into account to calculate the total content. At the end of the day flush methanol/acetonitrile 1/1 (V/V) through the chromatographic column at a rate of 1.0 ml/min for at least 15 min and store the column in methanol/acetonitrile 1/1 (V/V).

COI/T. 20/Doc. No 29 Page 4

Figure 1 HPLC chromatogram recorded at 280 nm for biophenols profile present in an extra virgin olive oil

6

EXPRESSION OF RESULTS

6.1

Calculation of the response factors of the external calibration standards (RF) RF1µg (syringic acid) = Area syringic acid/ µg syringic acid injected RF1µg (tyrosol) = Area tyrosol/ µg tyrosol injected

6.2

Calculation of the ratio between the two response factors (RRF) RRFsyr/tyr = RF1 µg (syringic acid)/ RF1 µg (tyrosol) The value of RRFsyr/tyr should be constant and should lie inside the range 5.1 ± 0.4. It enables the final result to be expressed as tyrosol, using syringic acid as the internal standard.

COI/T. 20/Doc. No 29 Page 5

6.3

Calculation of the biophenol content of virgin olive oil Biophenol content (natural and oxidised oleuropein and ligstroside derivatives, lignans, flavonoids and phenolic acids), expressed in mg/kg, is calculated by measuring the sum of the areas of the related chromatographic peaks (identification in Table 1) according to the following formula, the result is expressed without decimal place.

(mg/kg) =

(ΣA) × 1000 × RRFsyr/tyr × (W syr. acid)  (A syr. acid) × (W)

where:

(ΣA)

is the sum of the peak areas of the biophenols (hydroxytyrosol, tyrosol, natu ral and oxidised oleuropein and ligstroside derivatives, lignans, flavonoids and phenolic acids) recorded at 280 nm;

A syr. acid

is the area of the syringic acid internal standard recorded at 280 nm;

1000

is the factor used to express the result in mg/kg;

W

is the weight of the oil used, in grams;

RRFsyr/tyr

is the multiplication coefficient for expressing the final results as tyrosol;

W syr. acid

is the weight, in mg, of the syringic acid used as internal standard in 1 ml of solution added to the sample.

COI/T. 20/Doc. No 29 Page 6

Table 1 Identification of biophenols peaks. Maximum absorbance (max UV abs) values and relative retention times (RRT)*

Peak No

Biophenols

RRT*

Max. UV abs. nm

1

Hydroxytyrosol

0.62

230-280

2

Tyrosol

0.80

230-275

3

Vanillic acid

0.96

260

4

Caffeic acid

0.99

325

5

Syringic acid (internal standard)

1.00

280

6

Vanillin

1.10

310

7

Para-coumaric acid

1.12

310

8

Hydroxytyrosyl acetate

1.20

232-285

9

Ferulic acid

1.26

325

10

Ortho-coumaric acid

1.31

325

11;11a

Decarboxymethyl oleuropein aglycone, oxidised dialdehyde form

-

235-280

12

Decarboxymethyl oleuropein aglycone, dialdehyde form

1.45

235-280

13

Oleuropein

1.48

230-280

14

Oleuropein aglycone, dialdehyde form

1.52

235-280

15

Tyrosyl acetate

1.54

230-280

16;16a

Decarboxymethyl ligstroside aglycone, oxidised dialdehyde form

1.63

235-275

17

Decarboxymethyl ligstroside aglycone, dialdehyde form

1.65

235-275

18

Pinoresinol, 1 acetoxy-pinoresinol

1.69

232-280

19

Cinnamic acid

1.73

270

20

Ligstroside aglycone, dialdehyde form

1.74

235-275

21;21a;21b

Oleuropein aglycone, oxidised aldehyde and hydroxylic form

-

235-280

22

Luteolin

1.79

255-350

23

Oleuropein aglycone, aldehyde and hydroxylic form

1.87

235-280

24;24a;24b

Ligstroside aglycone, oxidised aldehyde and hydroxylic form

-

235-275

25

Apigenin

1.98

230-270-340

26

Methyl-luteolin

-

255-350

27

Ligstroside aglycone, aldehyde and hydroxylic form

2.03

235-275

(*) The relative retention time is calculated with respect to the retention time of syringic acid. Identification is performed by HPLC-MS.

7

TEST REPORT The test report should specify the following information: (a) The reference of this method. (b) The test results, expressed in mg/kg of oil (no decimal places). (c) The RRF value used for calculations. (d) Any departure from this method, made by agreement between the parties concerned or for any other reason. (e) The identification details of the laboratory, the date on which the test was performed and the signature of the test supervisor.

COI/T. 20/Doc. No 29 Page 7

PRECISION VALUES 1.

Analysis of the collaborative test results

The precision values of the method are given in the attached table. Seventeen laboratories holding IOC recognition at the time took part in the collaborative test arranged by the Executive Secretariat in 2008. The laboratories were from eight different countries. Sample A – Extra virgin olive oil (Italy) Sample B – Extra virgin olive oil (Spain) Sample C – Extra virgin olive oil (Tunisia) Sample D – Extra virgin olive oil (Slovenia) Sample E – Extra virgin olive oil (Greece) Sample R – Extra virgin olive oil (Italy) The results of the collaborative test organised by the IOC Executive Seecretariat were statistically processed according to the rules laid down in the international standards ISO 5725. Accuracy (trueness and precision) of measurement methods and results. Outliers were examined by applying the Cochran and Grubbs tests to the laboratory results for all the determinations (replicates a and b). The table lists: N

number of participating laboratories.

Outliers

number of laboratories with outlying values.

Mean

mean of the accepted results.

r

value below which the absolute difference between two single independent test results obtained with the same method on identical test material in the same laboratory by the same operator using the same equipment within short intervals of time may be expected to lie with a probability of 95%.

Sr

repeatability standard deviation.

RSDr

(%) repeatability coefficient of variation (Sr x 100 / mean).

R

value below which the absolute difference between two single test results obtained with the same method on identical test material in different laboratories with different operators using different equipment may be expected to lie with a probability of 95%.

SR

reproducibility standard deviation.

RSDR HoR

(%) reproducibility coefficient of variation (SR x 100/mean). is the HORRAT value for reproducibility, [RSDR actual/ RSDR theor] = 2 (1-0.5logC) and C is the concentration of the compound expressed to the power 10. (Horwitz equation).

COI/T. 20/Doc. No 29 Page 8

Precision values for total biophenol content, (mg/1000 g) SAMPLE A

Mean N Outliers non-outlier tests number R Sr RSDr(%) R SR RSDR(%) HOR

2.

SAMPLE SAMPLE SAMPLE SAMPLE SAMPLE B C D E R

mg/kg

mg/kg

mg/kg

mg/kg

mg/kg

mg/kg

694 17 3 14 28 29 10.4

573 17 3 14 28 36 12.7

153 17 1 16 32 18 6.4

343 17 2 15 30 24 8.7

297 17 2 15 30 22 7.7

301 17 2 15 30 17 6.2

2

2

4

3

3

2

101 36,0

84 29,9

60 21,3

63 22,4

77 27,5

32 11,5

5

5

14

7

9

4

0.9

0.8

1.9

1.0

1.4

0.6

References

ISO 5725-1:1994

Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions.

ISO 5725-2:1994

Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of the repeatability and reproducibility of a standard measurement method.

ISO 5725:5:1998

Accuracy (trueness and precision) of measurement methods and results Part 5: Alternative methods for the determination of the precision of a standard measurement method.

ISO 5725:6:1994

Accuracy (trueness and precision) of measurement methods and results Part 6: Use in practice of accuracy values

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INTERNATIONAL OLIVE COUNCIL Príncipe de Vergara, 154 – 28002 Madrid – España Telef.: +34 915 903 638 Fax: +34 915 631 263 - e-mail: [email protected] - http://www.internationaloliveoil.org/

- Draft -

DECISION NO DEC-…/97-V/2009 IDENTIFICATION AND QUANTIFICATION OF THE PHENOLICS IN OLIVE OIL

THE COUNCIL OF MEMBERS OF THE INTERNATIONAL OLIVE COUNCIL,

Having regard to the recommendation made by the Technical Committee at its 1st meeting, held on the occasion of the 94th session of the Council of Members, Having regard to the recommendation made by the Techncial Committee on the occasion of the 97th session of the Council of Members to adopt and include the method for the determination of the biophenols in olive oils by HPLC in the IOC trade standard, Whereas it would be expedient to have another method to facilitate the determination of the phenolics present in the unsaponifiable fraction of olive oils until the chemists define a position on the method displaying a better reliability as well as on acceptable repeatability and reproducibility values; Whereas the chemists have examined the application of the method under consideration, DES DECIDES

The provisional recommendation of the following method shall be recommended to the Members: .

Method of A. Cert, Colorimetric determination of O-Diphenols in olive oils.

This Decision shall replace and rescind Resolution No RES-4/94-V/2006 of 26 November 2006.

Madrid (Spain), … November 2009

COLORIMETRIC METHOD FOR THE DETERMINATION OF O-DIPHENOLIC COMPOUND IN OLIVE OILS REVISED METHOD: December 2007 By Arturo Cert. Ana Romero and R. Cert Instituto de la Grasa, (CSIC), Sevilla, Spain

1.- Material and apparatus 1.1.- Equipment for Solid Phase Extracción (SPE) 1.2.- Diol-bonded phase cartridges of 6 mL (0,5g) (Waters or similar) 1,3.- Syringe filter of cellulose acetate (0.45 -µm of pore size, (Alltech, Scharlab or similar) 1.4.- Rotary evaporator under vacuum 1.5.- UV-vis Spectrophotometer at λ=370 nm, provided with quartz absorption cuvettes of 10.0 mm path length.

2.- Solvents and reactives 2.1.- n-Hexane AR 2.2.- Mixture n-hexane/ethyl acetate 90 :10 (v/v) 2.3.- Methanol for UV-spectrometry. 2.4.- Distilled or deionized water. 2.5.- Sodium molibdate dihydrate, AR (Merck or similar). 2.6.- Pyrocatechol (catechol) (≥99%) (Aldrich or similar). 2.7.- Solution methanol/water 1:1 (v/v). 2.8.- Solution A. Methanol/water 1:1 acidified with hydrochloric acid prepared by addition of 0,5 ml of 6N HCl to 100 ml of solution methanol/water 1:1 (2.7). 2.9.- Solution of 5% sodium molibdate dihydrate in methanol/water 1:1 (2.7).

3.- Calibration line A calibration line (concentration, expressed in millimol/mL, vs absorbance) is obtained by measurement of solutions of pyrocatechol in solution A (2.8). Solutions of pyrocatechol are prepared in the range 0.02 – 0.07 mg/mL. These solutions must be freshly prepared and protected from light exposure. With the aid of a pipette, 2 mL of the phenolic solution are taken and poured into a glass tube. Then, 0,5 mL of sodium molybdate solution (2.9) are added, and the mixture is shacked. The absorbance at λ=370 nm is measured. The absorbance of the phenolic solution is amended with the absorbance of a blank obtained by mixing 2 mL of the phenolic solution with 0.5 mL of methanol/water 1:1 (2.7).

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Solutions of catechol, hydroxytyrosol, and hydroxytyrosyl acetate gave the following equation for an absorbance range of 0.2 – 0.8: Concentration of o-diphenols (mmol/mL) = (-0.170 + 8.236 * ABS) * 10-4

being ABS = absorbance phenolic solution – absorbance of the blank

4.- Isolation of the phenolic extract A sample of olive oil (6±0.001 g) is dissolved in 6 mL of hexane (2.1). A diol-bonded phase 6-mL cartridge (1.2) is placed in a vacuum elution apparatus, and conditioned by the consecutive passing of 9 ml of methanol (2.3) and 9 ml of hexane (2.1). Then the vacuum is released to prevent drying of the column. The oil solution is applied to the column and passed through the cartridge. The sample container is washed with two 4-ml portions of hexane that are run out of the cartridge. The sample container is washed again with 3 ml of the mixture hexane/ethyl acetate (90:10, v/v) (2.2) that are run out of the cartridge and discarded. Then, a 25-mL conical flask is placed and the column is eluted with 15 ml of methanol (2.3). The solvent is evaporated in a rotary evaporator at room temperature under vacuum until dryness. The residue is dissolved in 5 mL of acidified solution A (2.8).

5.- Spectrophotometric determination With the aid of a pipette provided with a syringe filter of cellulose acetate , 2 mL of the phenolic extract are taken and poured into a glass tube. Then, 0,5 mL of sodium molybdate solution (2.9) are added, and the mixture is shacked. The absorbance at λ=370 nm is measured. The absorbance of the phenolic solution is amended with the absorbance of a blank obtained by mixing 2 mL of the phenolic extract with 0.5 mL of methanol/water 1:1 (2.7). If absorbance of phenolic solution is higher than 0.8, a new phenolic extract must be obtained and solved in 10 mL of solution A (2.8) instead of 5 mL. An alternative is the extraction from 3 g of oil. If absorbance of phenolic solution is lower than 0.2, the phenolic extracts of two or more cartridges must be combined and solved in 5 mL of solution A (2.8). Using a single cartridge, the minimum concentration determined is 0.15 mmol/kg of oil.

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5.- Calculations The concentration of o-diphenols in oils, expressed in millimol/kg, is calculated by:

[C] in oil (mmol/kg) = (Cextr * 2.5 * Vextr * 1000) / 2 * Woil

Being: Cextr (mmol/mL) = concentration in the extract calculated by application of the absorbance to the calibration line 2.5 = volume (mL) of the reaction mixture Vextr = total volume (mL) of extract solution (usually 5 mL) 2 = Volume (mL) of extract solution taken for the reaction Woil = weight (g) of oil sample. The results must be expressed in millimol of o-diphenols / Kg of oil.

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