HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN"L 90/52
EN
Official Journal of the European Union
28.3.2013
COMMISSION IMPLEMENTING REGULATION (EU) No 299/2013
of 26 March 2013
amending Regulation (EEC) No 2568/91 on the characteristics of olive oil and olive-residue oil and
on the relevant methods of analysis
THE EUROPEAN COMMISSION,
(5) In
order
to
continue
the
process
of
harmonisation
with
the international standards laid down by the IOC certain
methods of analysis laid down in Regulation (EEC) No
Having regard to the Treaty on the Functioning of the European
2568/91 should be updated. Consequently, the method
Union,
of analysis provided for in Annex XVIII to that Regu
lation should be replaced by a more efficient method. It
is also appropriate to remedy some inconsistencies and
Having regard to Council Regulation (EC) No 1234/2007 of
imperfections of the methods of analysis provided for in
22 October 2007 establishing a common organisation of agri
Annex IX thereto.
cultural markets and on specific provisions for certain agri

cultural products (Single CMO Regulation) ( 1 ), and in particular
Article 113, paragraph 1, point (a), and Article 121, first para
(6) A
transitional
period
is
needed
for
Member
States
in
graph, point (a), in conjunction with Article 4 thereof,
order to apply the new rules laid down by this Regu
lation.
Whereas:
(7) The
Commission
has
developed
an
information
system
(1) Commission
Regulation
(EEC)
No
2568/91
of
11
July
that allows managing documents and procedures elec
1991 on the characteristics of olive oil and olive-
tronically in its own internal working procedures and
residue oil and on the relevant methods of analysis (
in its relations with the authorities involved in the

2 )
defines the chemical and organoleptic characteristics of
common agricultural policy. It is considered that the
olive and olive-residue oil and stipulates methods of
notification obligations provided for in Regulation
assessing these characteristics. Those methods should be
(EEC) No 2568/91 can be fulfilled via that system in
updated on the basis of the opinion of chemical experts
accordance with Commission Regulation (EC) No
and in line with the work carried out within the Inter
792/2009 of 31 August 2009 laying down detailed
national Olive Council (hereinafter ‘IOC’).
rules for the Member States’ notification to the
Commission of information and documents in imple
mentation of the common organisation of the markets,
(2) Pursuant
to
Article
113(3)
of
Regulation
(EC)
No
the direct payments’ regime, the promotion of agri
1234/2007, Member States are to check whether olive
cultural products and the regimes applicable to the
oils and olive-residue oils conform to the marketing
outermost regions and the smaller Aegean islands ( 3 ).
standards laid down in Regulation (EEC) No 2568/91
and are to apply penalties as appropriate. Articles 2
and 2a of Regulation (EEC) No 2568/91 provide
(8) Regulation
(EEC)
No
2568/91
should
therefore
be
detailed rules for those conformity checks. Those rules
amended accordingly.
should ensure that olive oil for which a quality standard
has been laid down effectively complies with that
standard. The rules should be further detailed, including
(9) The
Management
Committee
for
the
Common
Organi
a risk analysis. For the purpose of those conformity
sation of Agricultural Markets has not delivered an
checks, the term ‘marketed olive oil’ should be defined.
opinion within the time limit set by its chairman,
(3) Experience
has
shown
certain
risks
of
fraud
impeding
the HAS ADOPTED THIS REGULATION:
full effect of the consumer protection offered by Regu
lation (EEC) No 2568/91. Holders of olive oil should
therefore keep entry and withdrawal for each category
Article 1
of oils in a register. In order to avoid excessive adminis
Regulation (EEC) No 2568/91 is amended as follows:
trative burdens without undermining the objectives of
the olive oil register, the gathering of information
should be limited until the stage of bottling of olive oil.
(1) Article 2a is replaced by the following:
(4) In
order
to
ensure
the
follow-up
and
evaluate
the
‘Article 2a
measures of Regulation (EEC) No 2568/91, Member
States should notify the Commission not only of the
1. For
the
purpose
of
this
Article,
“olive
oil
marketed”
national implementing measures, but also report results
means total quantity of olive oil and olive pomace oil of a
of the conformity checks.
relevant Member State that is consumed in that Member
State or exported from that Member State.
( 1 ) OJ L 299, 16.11.2007, p. 1.
( 2 ) OJ L 248, 5.9.1991, p. 1.
( 3 ) OJ L 228, 1.9.2009, p. 3.

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28.3.2013 Official
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European
Union L
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2. Member
States
shall
ensure
that
conformity
checks
are
without prejudice to any other penalties, apply effective,
carried out selectively, based on a risk analysis, and with
proportionate and dissuasive penalties to be determined in
appropriate frequency, so as to ensure that the olive oil
the light of the seriousness of the irregularity detected.
marketed is consistent with the category declared.
Where checks reveal significant irregularities, Member States
3. The
criteria
to
assess
the
risk
may
include:
shall increase the frequency of checks in relation to
marketing stage, oil category, origin, or other criteria.’;
(a) the category of oil, the period of production, the price
of oils in relation to other vegetable oils, the blending
and packing operations, the storage facilities and
(3) the following Article 7a is inserted:
conditions, the country of origin, the country of desti
nation, the means of transport or the volume of the lot;
‘Article 7a
Natural or legal persons and groups of persons who hold
(b) the position of the operators in the marketing chain, the
olive oil and olive pomace oil from the extraction at the
volume and/or value marketed by them, the range of oil
mill up to the bottling stage included, for whatever profes
categories they market, the type of business carried out
sional or commercial purposes, shall be required to keep
such as milling, storage, refining, blending, packaging or
entry and withdrawal registers for each category of such
retail sale;
oils.
(c) findings made during previous checks including the
number and type of defects found, the usual quality
Member State shall ensure that the obligation laid down in
of oils marketed, the performance of technical
the first paragraph is duly complied with.’;
equipment used;
(4) Article 8 is replaced by the following:
(d) the reliability of operators’ quality assurance systems or
self-checking systems related to the conformity to
‘Article 8
marketing standards;
1. Member
States
shall
notify
the
Commission
of
the
(e) the place where the check is carried out, in particular if
measures implementing this Regulation. They shall inform
it is the first point of entry into the Union, the last
the Commission of any subsequent amendments.
point of exit from the Union or the place where the
oils are produced, packaged, loaded or sold to the final
2. No
later
than
31
May
of
each
year,
Member
States
consumer;
shall transmit to the Commission a report on the imple
mentation of this Regulation during the previous calendar
(f) any other information that might indicate a risk of non-
year. The report shall contain at least the results of the
compliance.
conformity checks carried out on olive oils as per the
templates set out in Annex XXI.
4. Member
States
shall
lay
down
in
advance:
3. The
notifications
referred
to
in
this
Regulation
shall
be
(a) the criteria for assessing the risk of non-conformity of
made in accordance with Commission Regulation (EC) No
lots;
792/2009 (*).
(b) on the basis of a risk analysis for each risk category, the
minimum number of operators or lots and/or quantities
___________
which will be subject to a conformity check.
(*) OJ L 228, 1.9.2009, p. 3.’;
At least one conformity check per thousand tonnes of olive
oil marketed in the Member State shall be carried out per
(5) Annex IX is replaced by the text set out in Annex I to this
year.
Regulation;
5. Member
States
shall
verify
compliance
by:
(6) Annex XVIII is replaced by the text set out in Annex II to
this Regulation;
(a) carrying out, in any order, the analyses set out in Annex
I; or
(7) Annex XXI, the text of which is set out in Annex III to this
Regulation, is added.
(b) following the order set out in Annex Ib on the decision
tree until one of the decisions appearing in the decision
tree is reached.’;
Article 2
This Regulation shall enter into force on the seventh day
(2) Article 3 is replaced by the following:
following that of its publication in the Official Journal of the
European Union.
‘Article 3
Where it is found that an oil does not correspond to its
It shall apply from 1 January 2014. However, Article 8(2) shall
category description, the Member State concerned shall,
apply from 1 January 2015.

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L 90/54
EN
Official Journal of the European Union
28.3.2013
This Regulation shall be binding in its entirety and directly applicable in all Member States.
Done at Brussels, 26 March 2013.
For the Commission
The President
José Manuel BARROSO

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EN
28.3.2013 Official
Journal
of
the
European
Union L
90/55
ANNEX I
‘ANNEX IX
SPECTROPHOTOMETRIC INVESTIGATION IN THE ULTRAVIOLET
FOREWORD
Spectrophotometric examination in the ultraviolet can provide information on the quality of a fat, its state of preservation
and changes brought about in it by technological processes.
The absorption at the wavelengths specified in the method is due to the presence of conjugated diene and triene systems.
These absorptions are expressed as specific extinctions E 1 % 1 cm (the extinction of 1 % solution of the fat in the specified
solvent, in a thickness of 1 cm) conventionally indicated by K (also referred to as ‘extinction coefficient’).
1. SCOPE
The method describes the procedure for performing a spectrophotometric examination of olive oil (as described in
the Appendix) in the ultraviolet.
2. PRINCIPLE
OF
THE
METHOD
The fat in question is dissolved in the required solvent and the extinction of the solution is then determined at the
specified wavelengths with reference to pure solvent. Specific extinctions are calculated from the spectropho
tometer readings. The specific absorbance at 232 nm and 268 nm in iso-octane or 232 nm and 270 nm in
cyclohexane for a concentration of 1 g per 100 ml in a 10 mm cell is calculated.
3. EQUIPMENT
3.1. A spectrophotometer for measuring extinction in the ultraviolet between 220 and 360 nm, with the possibility of
reading individual nanometric units. Before use it is recommended that the wavelength and absorbance scales of
the spectrometer be checked as follows.
3.1.1. Wavelength scale: This may be checked using a reference material consisting of an optical glass filter containing
holmium oxide which has distinct absorption bands. The reference material is designed for the verification and
calibration of the wavelength scales of visible and ultraviolet spectrophotometers having nominal spectral band
widths of 5 nm or less. The holmium glass filter is measured in the absorbance mode against an air blank, over the
wavelength range of 640 to 240 nm. For each spectral bandwidth (0,10 – 0,25 – 0,50 – 1,00 – 1,50 – 2,00 and
3,00), a baseline correction is performed with an empty cell holder. The wavelengths of the spectral bandwidth are
listed in the certificate of the reference material in ISO 3656.
3.1.2. Absorbance scale: This may be checked using a reference material consisting of 4 solutions of potassium dichromate
in perchloric acid sealed in four UV quartz cells to measure the linearity and photometric accuracy reference in the
UV. The potassium dichromate filled cells (40 mg/ml, 60 mg/ml, 80 mg/ml and 100 mg/ml) are measured against
a perchloric acid blank. The net absorbance values are listed in the certificate of the reference material in ISO
3656.
3.2. Rectangular quartz cells, with covers, having an optical length of 1 cm. When filled with water or other suitable
solvent the cells should not show differences between them of more than 0,01 extinction units.
3.3. 25
ml
graduated
flasks.
3.4. Analytical
balance,
capable
of
being
read
to
the
nearest
0,0001
g.
4. REAGENTS
Use only reagents of recognized analytical grade, unless otherwise stated.
Solvent: Iso-octane (2,2,4-trimethylpentane) for the measurement at 232 nm and 268 nm or cyclohexane for the
measurement at 232 nm and 270 nm, having an absorbance less than 0,12 at 232 nm and less than 0,05 at
250 nm against distilled water, measured in a 10 mm cell.
5. PROCEDURE
5.1. The
sample
in
question
must
be
perfectly
homogeneous
and
without
suspected
impurities.
Oils
which
are
liquid
at
ambient temperature are to be filtered through paper at a temperature of approximately 30 °C, hard fats are to be
homogenized and filtered at a temperature of not more than 10 °C above the melting point.

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L 90/56
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Official Journal of the European Union
28.3.2013
5.2. Weigh accurately approximately 0,25 g (to the nearest 1 mg) of the sample so prepared into a 25 ml graduated
flask, make up to the mark with the solvent specified and homogenize. The resulting solution must be perfectly
clear. If opalescence or turbidity is present filter quickly through paper.
5.3. Fill a quartz cell with the solution obtained and measure the extinctions at an appropriate wavelength between 232
and 276 nm, using the solvent used as a reference.
The extinction values recorded must lie within the range 0,1 to 0,8. If not the measurements must be repeated
using more concentrated or more dilute solutions as appropriate.
NOTE: It may not be necessary to measure the absorbance over the full wavelength range.
6. EXPRESSION
OF
THE
RESULTS
6.1. Record the specific extinctions (extinction coefficients) at the various wavelengths calculated as follows:
A E
K

λ

!
λ
where:
c s
Κλ = specific extinction at wavelength λ,
Ελ = extinction measured at wavelength λ;
c =
concentration
of
the
solution
in
g/100
ml;
s =
thickness
of
the
quartz
cells
in
cm.
The results are to be expressed to two decimal places.
6.2. Variation of the specific extinction (ΔΚ)
Spectrophotometric analysis of olive oil in accordance with the official method in the Union legislation involves
also the determination of the variation of the absolute value of the specific extinction (ΔΚ), which is given by:
A K
ΔK
m – 4
jK
!
m – K m 4
2 j
where Km is the specific extinction at wavelength m, the wavelength for maximum absorption depends on the
solvent used: 270 for cyclohexane and 268 for iso-octane.

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28.3.2013 Off
Appendix
OLIVE OIL CHARACTERISTICS
Difference:
Organoleptic
Fatty acid methyl esters
Peroxide
Stigmas
ECN42
K 270 (*)
Organoleptic
'
evaluation
Category
(FAMES) and fatty acid
Acidity
index
Waxes
tadiene (HPLC) and K
K 270 Delta-K
evaluation

Fruity
EN
232 (*)
ethyl esters (FAEEs)
(%) (*)
mEq
mg/kg (**)
2 glyceril monopalmitate (%)
mg/kg (
ECN42
or K
(*) (5 )
Median defect
1 )
median

02/kg (*)
(theoretical
268 (5 )'
(Md) (*)
calculation)
(Mf) (*)
1. Extra
virgin
olive
oil
Σ FAME + FAEE
≤ 0,8
≤ 20
≤ 250
≤ 0,9 if total palmitic acid % ≤ 14 % ≤ 0,10
≤ 0,2
≤ 2,50 ≤ 0,22 ≤ 0,01 Md
=
0 Mf
>
0
≤75 mg/kg or
75 mg/kg <Σ FAME +
≤ 1,0 if total palmitic acid % > 14 %
FAEE ≤150 mg/kg and
(FAEE/FAME) ≤1,5
2. Virgin
olive
oil
—
≤ 2,0
≤ 20
≤ 250
≤ 0,9 if total palmitic acid % ≤ 14 % ≤ 0,10
≤ 0,2
≤ 2,60 ≤ 0,25 ≤ 0,01 Md
≤ 3,5 Mf
>
0
≤ 1,0 if total palmitic acid % > 14 %
icial
3. Lampante
olive
oil
— >
2,0
—
≤ 300 (3 ) ≤ 0,9 if total palmitic acid % ≤ 14 % ≤ 0,50
≤ 0,3
—
—
— Md
>
3,5
(2 )
—
Jour
nal
≤ 1,1 if total palmitic acid % > 14 %
of
4. Refined
olive
oil
—
≤ 0,3
≤ 5
≤ 350
≤ 0,9 if total palmitic acid % ≤ 14 %
—
≤ 0,3
—
≤ 1,10 ≤ 0,16
—
—
the
≤ 1,1 if total palmitic acid % > 14 %
European
5. Olive oil composed of
—
≤ 1,0
≤ 15
≤ 350
≤ 0,9 if total palmitic acid % ≤ 14 %
—
≤ 0,3
—
≤ 0,90 ≤ 0,15
—
—
refined and virgin olive
oils
≤ 1,0 if total palmitic acid % > 14 %
Union
6. Crude
olive-pomace
oil
—
—
— >
350
(4 )
≤ 1,4
—
≤ 0,6
—
—
—
—
—
7. Refined
olive-pomace
oil
—
≤ 0,3
≤ 5 >
350
≤ 1,4
—
≤ 0,5
—
≤ 2,00 ≤ 0,20
—
—
8. Olive-pomace
oil
≤ 1,0
≤ 15 >
350
≤ 1,2
—
≤ 0,5
—
≤ 1,70 ≤ 0,18
—
—
(1 ) Total isomers which could (or could not) be separated by capillary column.
(2 ) Or where the median defect is less than or equal to 3,5 and the fruity median is equal to 0.
(3 ) Oils with a wax content of between 300 mg/kg and 350 mg/kg are considered to be lampante olive oil if the total aliphatic alcohol content is less than or equal to 350 mg/kg or if the erythrodiol and uvaol content is less than or equal
to 3,5 %.
(4 ) Oils with a wax content of between 300 mg/kg and 350 mg/kg are considered to be crude olive-pomace oil if the total aliphatic alcohol content is above 350 mg/kg and if the erythrodiol and uvaol content is greater than 3,5 %.
(5 ) K 270 if solvant is cyclohexane, K 268 if solvant is iso-octane.’
L
90/57

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L 90/58
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Official Journal of the European Union
28.3.2013
ANNEX II
‘ANNEX XVIII
DETERMINATION OF THE DIFFERENCE BETWEEN ACTUAL AND THEORETICAL CONTENT OF
TRIACYLGLYCEROLS WITH ECN 42
1. SCOPE
Determination of the absolute difference between the experimental values of triacylglycerols (TAGs) with
equivalent carbon number 42 (ECN42 HPLC ) obtained by determination in the oil by high performance liquid
chromatography and the theoretical value of TAGs with an equivalent carbon number of 42 (ECN 42 theoretical )
calculated from the fatty acid composition.
2. FIELD
OF
APPLICATION
The standard is applicable to olive oils. The method is applicable to the detection of the presence of small
amounts of seed oils (rich in linoleic acid) in every class of olive oils.
3. PRINCIPLE
The content of triacylglycerols with ECN 42 determined by HPLC analysis and the theoretical content of
triacylglycerols with ECN 42 (calculated on the basis of GLC determination of fatty acid composition) correspond
within a certain limit for genuine olive oils. A difference larger than the values adopted for each type of oil
points out that the oil contains seed oils.
4. METHOD
The method for the calculation of the theoretical content of triacylglycerols with ECN 42 and of the difference
with respect to the HPLC data is essentially made by the coordination of analytical data obtained by means of
other methods. It is possible to distinguish three phases: determination of fatty acid composition by capillary gas
chromatography, calculation of theoretical composition of triacylglycerols with ECN 42, HPLC determination of
ECN 42 triacylglycerols.
4.1. Apparatus
4.1.1. Round-bottomed
flasks,
250
and
500
ml.
4.1.2. Beakers
100
ml.
4.1.3. Glass chromatographic column, 21 mm internal diameter, 450 mm length, with cock and normalised cone
(female) at the top.
4.1.4. Separating funnels, 250 ml, with normalised cone (male) at the bottom, suitable for connection to the top of the
column.
4.1.5. Glass
rod,
600
mm
length.
4.1.6. Glass
funnel,
80
mm
diameter.
4.1.7. Volumetric flasks, 50 ml.
4.1.8. Volumetric flasks, 20 ml.
4.1.9. Rotary
evaporator.
4.1.10. High performance liquid chromatograph, allowing thermostatic control of column temperature.
4.1.11. Injection units for 10 μl delivery.
4.1.12. Detector: differential refractometer. The full scale sensitivity should be at least 10 –4 units of refractive index.
4.1.13. Column: stainless steel tube 250 mm length x 4,5 mm internal diameter packed with 5 μm diameter particles of
silica with 22 to 23 % carbon in the form of octadecylsilane.
4.1.14. Data processing software.
4.1.15. Vials, of about 2 ml volumes, with Teflon-layered septa and screw caps.
4.2. Reagents
The reagents should be of analytical purity. Elution solvents should be de-gassed, and may be recycled several
times without effect on the separations.

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European
Union L
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4.2.1. Petroleum
ether
40–
60
°C
chromatographic
grade
or
hexane.
4.2.2. Ethyl
ether,
peroxide-free,
freshly
distilled.
4.2.3. Elution solvent for purifying the oil by column chromatography mixture petroleum ether/ethyl ether 87/13 (v/v).
4.2.4. Silica
gel,
70-230
mesh,
type
Merck
7734,
with
water
content
standardised
at
5
%
(w/w/).
4.2.5. Glass
wool.
4.2.6. Acetone
for
HPLC.
4.2.7. Acetonitrile
or
propionitrile
for
HPLC.
4.2.8. HPLC elution solvent: acetonitrile + acetone (proportions to be adjusted to obtain the desired separation; begin
with 50:50 mixture) or propionitrile.
4.2.9. Solubilisation
solvent:
acetone.
4.2.10. Reference triglycerides: commercial triglycerides (tripalmitin, triolein, etc.) may be used and the retention times
then plotted in accordance with the equivalent carbon number, or alternatively reference chromatograms
obtained from soya oil, mixture 30:70 soya oil — olive oil and pure olive oil (see notes 1 and 2 and figures
1 to 4).
4.2.11. Solid phase extraction column with silica phase 1 g, 6 ml.
4.3. Sample
preparation
As a number of interfering substances can give rise to false positive results, the sample must always be purified
according to IUPAC method 2.507, used for the determination of polar compounds in frying fats.
4.3.1. Chromatographic
column
preparation
Fill the column (4.1.3) with about 30 ml of elution solvent (4.2.3), then introduce inside the column some glass
wool (4.2.5) pushing it to the bottom of the column by means of the glass rod (4.1.5).
In a 100 ml beaker, suspend 25 g of silica gel (4.2.4) in 80 ml of elution mixture (4.2.3), then transfer it to the
column by means of a glass funnel (4.1.6).
To ensure the complete transfer of the silica gel to the column, wash the beaker with the elution mixture and
transfer the washing portions to the column too.
Open the cock and let the solvent elute from the column until its level is about 1 cm over the silica gel.
4.3.2. Column
chromatography
Weigh with the accuracy of 0,001 g, 2,5 ± 0,1 g of oil, previously filtered, homogenised and anhydrified, if
necessary, in a 50 ml volumetric flask (4.1.7).
Dissolve it in about 20 ml of elution solvent (4.2.3). If necessary, slightly heat it to make the dissolution easily.
Cool at room temperature and adjust the volume with elution solvent.
By means of a volumetric pipette, introduce 20 ml of solution inside the column prepared according to 4.3.1,
open the cock and let the solvent elute to the silica gel layer level.
Then elute with 150 ml of elution solvent (4.2.3), adjusting the solvent rate at about 2 ml/min (150 ml will take
about 60-70 minutes to pass through the column).
The eluate is recovered in a 250 ml round-bottomed flask (4.1.1) previously tared in an oven and exactly
weighed. Eliminate the solvent at reduced pressure in a rotary evaporator (4.1.9) and weigh the residue that
will be used to prepare the solution for HPLC analysis and for methyl ester preparation.
The sample recovery from the column must be 90 % at least for the extra virgin, virgin, ordinary, refined and
olive oil categories, and a minimum of 80 % for lampante and olive-pomace oils.

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4.3.3. SPE
purification
Silica SPE column is activated by passing 6 ml of hexane (4.2.3) under vacuum, avoiding dryness.
Weigh to an accuracy of 0,001 g, 0,12 g in a 2 ml vial (4.1.15) and dissolve with 0,5 ml of hexane (4.2.3).
Load the SPE column with the solution and elute with 10 ml of hexane-diethyl ether (87:13 v/v) (4.2.3) under
vacuum.
The collected fraction is evaporated to dryness in a rotary evaporator (4.1.9) under reduced pressure at room
temperature. The residue is dissolved in 2 ml of acetone (4.2.6) for triacylglycerol (TAG) analysis.
4.4. HPLC
analysis
4.4.1. Preparation
of
the
samples
for
chromatographic
analysis
A 5 % solution of the sample to be analysed is prepared by weighing 0,5 ± 0,001 g of the sample into a 10 ml
graduated flask and making up to 10 ml with the solubilisation solvent (4.2.9).
4.4.2. Procedure
Set up the chromatographic system. Pump elution solvent (4.2.8) at a rate of 1,5 ml/min to purge the entire
system. Wait until a stable base line is obtained.
Inject 10 μl of the sample prepared as in point 4.3.
4.4.3. Calculation
and
expression
of
results
Use the area normalisation method, i.e. assume that the sum of the areas of the peaks corresponding to TAGs
from ECN 42 up to ECN 52 is equal to 100 %.
Calculate the relative percentage of each triglyceride using the formula:
% triglyceride = area of peak × 100/sum of peak areas.
The results should be given to at least two decimal places.
See notes 1 to 4.
4.5. Calculation
of
triacylglycerols
composition
(moles
%)
from
fatty
acid
composition
data
(area
%)
4.5.1. Determination
of
fatty
acid
composition
Fatty acid composition is determined by ISO 5508 by means of a capillary column. The methyl esters are
prepared according to COI/T.20/Doc. No 24.
4.5.2. Fatty
acids
for
calculation
Glycerides are grouped by their Equivalent Carbon Number (ECN), taking into account the following equival
encies between ECN and fatty acids. Only fatty acids with 16 and 18 carbon atoms were taken into consider
ation, because only these are important for olive oil. The fatty acids should be normalised to 100 %.
Fatty acid (FA) Abbreviation
Molecular weight
(MW)
ECN
Palmitic acid
P 256,4 16
Palmitoleic acid Po 254,4 14
Stearic acid
S 284,5 18
Oleic acid
O 282,5 16
Linoleic acid
L 280,4 14
Linolenic acid Ln 278,4 12
4.5.3. Conversion of area % into moles for all fatty acids (1)
area % P
area % S
area % Po
moles P
moles S
moles Po
MW P
MW S
MW Po
area % O
area % L
area % Ln
moles O
moles L
moles Ln
MW O
MW L
MW Ln

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EN
28.3.2013 Official
Journal
of
the
European
Union L
90/61
4.5.4. Normalisation
of
fatty
acid
moles
to
100
%
(2)
moles P
moles % P 1,2,3
100
moles P S Po O L Ln
moles S
moles % S 1,2,3
100
moles P S Po O L Ln
moles Po
moles % Po 1,2,3
100
moles P S Po O L Ln
moles O
moles % O 1,2,3
100
moles P S Po O L Ln
moles L
moles % L 1,2,3
100
moles P S Po O L Ln
moles Ln
moles % Ln 1,2,3
100
moles P S Po O L Ln
The result gives the percentage of each fatty acid in moles % in the overall (1, 2, 3–) position of the TAGs.
Then the sum of the saturated fatty acids P and S (SFA) and the unsaturated fatty acids Po, O, L and Ln (UFA) are
calculated (3):
moles % SFA = moles % P + moles % S
moles % UFA = 100 – moles % SFA
4.5.5. Calculation of the fatty acid composition in 2- and 1, 3- positions of TAGs
The fatty acids are distributed to three pools as follows: one for 2- position and two identical for 1- and 3-
positions, with different coefficients for the saturated (P and S) and unsaturated acids (Po, O, L and Ln).
4.5.5.1. Saturated fatty acids in 2-position [P(2) and S(2)] (4):
moles % P(2) = moles % P (1,2,3) * 0,06
moles % S(2) = moles % S (1,2,3) * 0,06
4.5.5.2. Unsaturated fatty acids in 2-position [Po(2), O(2), L(2) and Ln(2)] (5):
moles % Po
moles % Po2
1,2,3
moles % UFA 100 – moles % P2 – moles % S2
moles % O
moles % O2
1,2,3
moles % UFA 100 – moles % P2 – moles % S2
moles % L
moles % L2
1,2,3
moles % UFA 100 – moles % P2 – moles % S2
moles % Ln
moles % Ln2
1,2,3
moles % UFA 100 – moles % P2 – moles % S2
4.5.5.3. Fatty acids in 1,3-positions [P(1,3), S(1,3), Po(1,3), O(1,3), L(1,3) and Ln(1,3)] (6):
moles % P
moles % P1,3
1,2,3 – moles % P2
2
moles % P1,2,3
moles % S
moles % S1,3
1,2,3 – moles % S2
2
moles % S1,2,3
moles % Po
moles % Po1,3
1,2,3 – moles % Po2
2
moles % Po1,2,3

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L 90/62
EN
Official Journal of the European Union
28.3.2013
moles % O
moles % O1,3
1,2,3 – moles % O2
2
moles % O1,2,3
moles % L
moles % L1,3
1,2,3 – moles % L2
2
moles % L1,2,3
moles % Ln
moles % Ln1,3
1,2,3 – moles % Ln2
2
moles % Ln1,2,3
4.5.6. Calculation
of
triacylglycerols
4.5.6.1. TAGs with one fatty acid (AAA, here LLL, PoPoPo) (7)
moles % A
moles % AAA
1,3 moles % A2 moles % A1,3
10 000
4.5.6.2. TAGs with two fatty acids (AAB, here PoPoL, PoLL) (8)
moles % A
moles % AAB
1,3 moles % A2 moles % B1,3 2
10 000
moles % A
moles % ABA
1,3 moles % B2 moles % A1,3
10 000
4.5.6.3. TAGs with three different fatty acids (ABC, here OLLn, PLLn, PoOLn, PPoLn) (9)
moles % A
moles % ABC
1,3 moles % B2 moles % C1,3 2
10 000
moles % B
moles % BCA
1,3 moles % C2 moles % A1,3 2
10 000
moles % C
moles % CAB
1,3 moles % A2 moles % B1,3 2
10 000
4.5.6.4. Triacylglycerols with ECN42
The triacylglycerols with ECN42 are calculated according to equations 7, 8 and 9 and are then given in order of
expected elution in HPLC (normally only three peaks).
LLL
PoLL and the positional isomer LPoL
OLLn and the positional isomers OLnL and LnOL
PoPoL and the positional isomer PoLPo
PoOLn and the positional isomers OPoLn and OLnPo
PLLn and the positional isomers LLnP and LnPL
PoPoPo
SLnLn and the positional isomer LnSLn
PPoLn and the positional isomers PLnPo and PoPLn
The triacylglycerols with ECN42 are given by the sum of the nine triacylglycerols including their positional
isomers. The results should be given to at least two decimal places.
5. EVALUATION
OF
THE
RESULTS
The calculated theoretical content and the content determined by the HPLC analysis are compared. If the
difference in the absolute value of the HPLC data minus the theoretical data is greater than the values stated
for the appropriate oil category in the standard, the sample contains seed oil.
Results are given to two decimal figures.

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EN
28.3.2013 Official
Journal
of
the
European
Union L
90/63
6.
EXAMPLE (THE NUMBERS REFER TO THE SECTIONS IN THE TEXT OF THE METHOD)
— 4.5.1. Calculation
of
moles
%
fatty
acids
from
GLC
data
(normalised
area
%)
The following data are obtained for the fatty acid composition by GLC:
FA
P
S Po
O
L Ln
MW 256,4 284,5 254,4 282,5 280,4 278,4
Area % 10,0 3,0 1,0 75,0 10,0 1,0
— 4.5.3 Conversion of area % into moles for all fatty acids (see formula (1))
10
moles P = 256,4 0,03900 moles P
3
moles S = 284,5 0,01054 moles S
1
moles Po = 254,4 0,00393 moles Po
75
moles O = 282,5 0,26549 moles O
10
moles L = 280,4 0,03566 moles L
1
moles Ln = 278,4 0,00359 moles Ln
Total =
0,35821
moles
TAGs
— 4.5.4 Normalisation
of
fatty
acid
moles
to
100
%
(see
formula
(2))
0,03900 moles P
moles % P(1,2,3) =
100
0,35821 moles 10,887 %
0,01054 moles S
moles % S(1,2,3) =
100
0,35821 moles 2,942 %
0,00393 moles Po
moles % Po(1,2,3) =
100
0,35821 moles 1,097 %
0,26549 moles O
moles % O(1,2,3) =
100
0,35821 moles 74,116 %
0,03566 moles L
moles % L(1,2,3) =
100
0,35821 moles 9,955 %
0,00359 moles Ln
moles % Ln(1,2,3) =
100
0,35821 moles 1,002 %
Total moles %
= 100 %
Sum of the saturated and unsaturated fatty acids in the 1,2,3-position of TAGs (see formula (3)):
moles % SFA = 10,887 % + 2,942 % = 13,829 %
moles % UFA = 100,000 % – 13,829 % = 86,171 %

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L 90/64
EN
Official Journal of the European Union
28.3.2013
— 4.5.5 Calculation of the fatty acid composition in 2- and 1,3-positions of the TAGs
— 4.5.5.1 Saturated fatty acids in 2-position [P(2) and S(2)] (see formula (4))
moles % P(2) = 10,887 % * 0,06 = 0,653 moles %
moles % S(2) = 2,942 % * 0,06 = 0,177 moles %
— 4.5.5.2 Unsaturated fatty acids in 2-position [Po(1,3), O(1,3), L(1,3) and Ln(1,3)] (see formula (5))
1,097 %
moles % Po2 86,171 % 100 – 0,653 – 0,177 1,262 moles %
74,116 %
moles % O2 86,171 % 100 – 0,653 – 0,177 85,296 moles %
9,955 %
moles % L2 86,171 % 100 – 0,653 – 0,177 11,457 moles %
1,002 %
moles % Ln2 86,171 % 100 – 0,653 – 0,177 1,153 moles %
— 4.5.5.3 Fatty acids in 1,3-positions [P(1,3), S(1,3), Po(1,3), O(1,3), L(1,3) and Ln(1,3)] (see formula (6))
10,887 – 0,653
moles % P1,3
2
10,887 16,004 moles %
2,942 – 0,177
moles % S1,3
2 2,942 4,325 moles %
1,097 – 1,262
moles % Po1,3
2 1,097 1,015 moles %
74,116 – 85,296
moles % O1,3
2
74,116 68,526 moles %
9,955 – 11,457
moles % L1,3
2
9,955 9,204 moles %
1,002 – 1,153
moles % Ln1,3
2 1,002 0,927 moles %
— 4.5.6. Calculation
of
triacylglycerols
From the calculated fatty acid composition in sn-2- and sn-1,3-positions:
FA in 1,3-pos 2-pos
P 16,004
% 0,653
%
S 4,325
% 0,177
%
Po 1,015
% 1,262
%
O 68,526
% 85,296
%
L 9,204
% 11,457
%
Ln 0,927
% 1,153
%
Sum 100,0
% 100,0
%
the following triacylglycerols are calculated:
LLL
PoPoPo
PoLL with 1 positional isomer
SLnLn with 1 positional isomer

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EN
28.3.2013 Official
Journal
of
the
European
Union L
90/65
PoPoL with 1 positional isomer
PPoLn with 2 positional isomers
OLLn with 2 positional isomers
PLLn with 2 positional isomers
PoOLn with 2 positional isomers
— 4.5.6.1. TAGs with one fatty acid (LLL, PoPoPo) (see formula (7))
9,204 %
mol % LLL
11,457 % 9,204 % , = 0,09706 mol LLL
10 000
1,015 %
mol % PoPoPo
1,262 % 1,015 %
10 000
0,00013 mol PoPoPo
— 4.5.6.2 TAGs with two fatty acids (PoLL, SLnLn, PoPoL) (see formula (8))
1,015 %
mol % PoLL LLPo
11,457 % 9,204 % 2
10 000
0,02141
9,204 %
mol % LPoL
1,262 % 9,204 %
10 000
0,01069
0,03210 mol PoLL
4,325 %
mol % SLnLn LnLnS
1,153 % 0,927 % 2
10 000
0,00092
0,927 %
mol % LnSLn
0,177 % 0,927 %
10 000
0,00002
0,00094 mol SLnLn
1,015 %
mol % PoPoL LPoPo
1,262 % 9,204 % 2
10 000
0,00236
1,015 %
mol % PoLPo
11,457 % 1,015 %
10 000
0,00118
0,00354 mol PoPoL
— 4.5.6.3 TAGs with three different fatty acids (PoPLn, OLLn, PLLn, PoOLn) See formula (9)
16,004 %
mol % PPoLn
1,262 % 0,927 % 2
10 000
0,00374
0,927 %
mol % LnPPo
0,653 % 1,015 % 2
10 000
0,00012
1,015 %
mol % PoLnP
1,153 % 16,004 % 2
10 000
0,00375
0,00761 mol PPoLn
68,526 %
mol % OLLn
11,457 % 0,927 % 2
10 000
0,14556
0,927 %
mol % LnOL
85,296 % 9,204 % 2
10 000
0,14555
9,204 %
mol % LLnO
1,153 % 68,526 % 2
10 000
0,14544
0,43655 mol OLLn

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L 90/66
EN
Official Journal of the European Union
28.3.2013
16,004 %
mol % PLLn
11,457 % 0,927 % 2
10 000
0,03399
0,927 %
mol % LnPL
0,653 % 9,204 % 2
10 000
0,00111
9,204 %
mol % LLnP
1,153 % 16,004 % 2
10 000
0,03397
0,06907 mol PLLn
1,015 %
mol % PoOLn
85,296 % 0,927 % 2
10 000
0,01605
0,927 %
mol % LnPoO
1,262 % 68,526 % 2
10 000
0,01603
68,526 %
mol % OLnPo
1,153 % 1,015 % 2
10 000
0,01604
0,04812 mol PoOLn
ECN42 = 0,69512 mol TAGs
Note 1: The elution order can be determined by calculating the equivalent carbon numbers, often defined by the
relation ECN = CN – 2n, where CN is the carbon number and n is the number of double bonds; it can be
calculated more precisely by taking into account the origin of the double bond. If n o , n l and n ln are the numbers
of double bonds attributed to oleic, linoleic and linolenic acids respectively, the equivalent carbon number can be
calculated by means of the relation of the formula:
EN = CN – d o n o – d l n l – d ln n ln
where the coefficient d o , d l and d ln can be calculated by means of the reference triglycerides. Under the
conditions specified in this method, the relation obtained will be close to:
ECN = CN – (2,60 n o ) – (2,35 n l ) – (2,17 n ln )
Note 2: With several reference triglycerides, it is also possible to calculate the resolution with respect to triolein:
α = RT 1 /RT triolein
by use of the reduced retention time RT 1 = RT – RT solvent
The graph of log α against f (number of double bonds) enables the retention values to be determined for all the
triglycerides of fatty acids contained in the reference triglycerides — see Figure 1.
Note 3: The efficiency of the column should permit clear separation of the peak of trilinolein from the peaks of
the triglycerides with an adjacent RT. The elution is carried out up to ECN 52 peak.
Note 4: A correct measure of the areas of all peaks of interest for the present determination is ensured if the
second peak corresponding to ECN 50 is 50 % of full scale of the recorder.

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EN
28.3.2013 Official
Journal
of
the
European
Union L
90/67
Figure 1
Graph of log α against f (number of double bonds)
Number of double bonds
La: lauric acid; My: myristic acid; P: palmitic acid; S: stearic acid; O: oleic acid; L: linoleic acid; Ln: linolenic acid

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L 90/68
EN
Official Journal of the European Union
28.3.2013
Figure 2
Low linoleic olive oil
(a)
With solvent: Acetone/Acetonitrile.
PROFILE a: Main components of chromatographic peaks: ECN42: (1) LLL + PoLL; (2) OLLn + PoOLn; (3) PLLn; ECN44:
(4) OLL + PoOL; (5) OOLn + PLL; (6) POLn + PPoPo; (7) OOL + PoOO; ECN46: (8) OOL + LnPP; (9) PoOO; (10) SLL +
PLO; (11) PoOP + SPoL + SOLn + SPoPo; (12) PLP; ECN48: (13) OOO + PoPP; (14 + 15) SOL + POO; (16) POP; ECN50:
(17) SOO; (18) POS + SLS.
(b)
With solvent: Propionitrile
PROFILE b: Main components of chromatographic peaks: ECN42: (1) LLL; (2) OLLn + PoLL; (3) PLLn; ECN44: (4) OLL;
(5) OOLn + PoOL; (6) PLL + PoPoO; (7) POLn + PPoPo + PPoL; ECN46: (8) OOL + LnPP; (9) PoOO; (10) SLL + PLO; (11)
PoOP + SPoL + SOLn + SPoPo; (12) PLP; ECN48: (13) OOO + PoPP; (14) SOL; (15) POO; (16) POP; ECN50: (17) SOO;
(18) POS + SLS

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EN
28.3.2013 Official
Journal
of
the
European
Union L
90/69
Figure 3
High linoleic olive oil
(a)
With solvent: Acetone/Acetonitrile (50:50).
Profile a: Main components of chromatographic peaks: ECN42: (1’) LLL + PoLL; (2’) OLLn + PoOLn; (3’) PLLn; ECN44:
(4’) OLL + PoOL; (5’) OOLn + PLL; (6’) POLn + PPoPo; ECN46: (7’) OOL + PoOO; (8’) PLO + SLL + PoOP; (9’) PLP +
PoPP; ECN48: (10’) OOO; (11’) POO + SLL + PPoO; (12’) POP + PLS; ECN50: (13’) SOO; (14’) POS + SLS
(b)
With solvent: Propionitrile.
Profile b: Main components of chromatographic peaks: ECN42: (1) LLL; (2 + 2’) OLLn + PoLL; (3) PLLn; ECN44: (4) OLL;
(5) OOLn + PoOL; (6) PLL + PoPoO; (7) POLn + PPoPo + PPoL; ECN46: (8) OOL + LnPP; (9) PoOO; (10) SLL + PLO; (11)
PoOP + SPoL + SOLn + SPoPo; ECN48: (12) PLP; (13) OOO + PoPP; (14) SOL; (15) POO; (16) POP; ECN50: (17) SOO;
(18) POS + SLS; ECN52: (19) AOO.’

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L 90/70
ANNEX III
‘ANNEX XXI
Results of conformity checks carried out on olive oils referred to in Article 8(2)
EN
Labelling Chemical
parameters Organoleptic
characteristics
(4 ) Final
conclusion

Parameters
If so, please
Sample Category
Country
Place of
Legal
Designation of
Storage
Erroneous
Fruity
of origin inspection (
out of limit indicate which C/NC (3 ) Median
1 )
name
origin
conditions
information
Legibility C/NC
(3 )
Y/N
one(s) (
defect
Median C/NC (3 )
Required
action
Sanction
2 )
Off
icial
Jour
nal
of
the
European
(1 ) Internal market (mill, bottlers, retail stage), export, import.
(2 ) Each characteristic of olive oil set out in Annex I shall have a code.
(3 ) Conform/not conform.
Union
(4 ) Not required for olive oil and pomace-oil.’
28.3.2013

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Document Outline

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