A P P L I C AT I O N N O T E

Liquid Chromatography/
Mass Spectrometry
Authors:
Robert Seward
Catherine Stacey
PerkinElmer, Inc.
Waltham, MA

PerkinElmer is the exclusive global distributor

of the TIBCO™ Spotfire® platform for certain
scientific and clinical R&D applications.

Markers for Spanish Olive

Oil Cultivars – Statistical Introduction
Analysis of Polar Compounds For high value olive oil,
the country of origin and
from LC/MS Results type of olive cultivar has
a dramatic effect on the
price. Falsifying the provenance of such oils is an attractive proposition to
fraudulent suppliers.

Polar components such as phenolic acids and lignans are present in olive oils.1
Secoiridoids; phenolics which include an elenolic acid moiety, are unique to
the Olearaceae family. These compounds are known to have beneficial health
effects due to their antioxidant properties and are associated with the
organoleptic properties. Measuring the levels of these polar components in
oils from different cultivars and geographic origins is of interest for nutritional
and authenticity reasons.

In this study a statistical analysis of the polar chemical components of olive

oils was used to determine potential markers for olive cultivars. These markers
could be used to confirm the provenance of unknown samples.
 Method
Spanish Olive Oil Samples
A number of authentic branded Spanish Extra Virgin Olive oils An example separation of components for one sample is shown,
(EVOOs) were analyzed. Each of the oils were cold pressed from with the same gradient used for both ionization modes (Figure 1
either a single cultivar olive or from a known blend of cultivars and Figure 2). Many compounds were selectively detected in either
grown in a distinct geographic region in Spain. The single cultivars positive or negative modes, although some compounds were
were Manzanilla Cacereña, Arbequina, Picual, Arbequina, Empeltre detected in both modes. In negative mode (Figure 1), long chain
and Cornicabra and the blends were comprised of various mixtures fatty acids and a number of phenolics are detected. In positive
of Hojiblanco, Picudo and Picual cultivars. mode (Figure 2) a number of phenolics, terpenes and fatty acids
are observed as [M + H]+ or [M + Na]+ ions.
LC/MS Analysis of Polar Components in Olive Oils
Polar Compound Extraction For components of interest, the accurate mass and isotope
Liquid-liquid extraction was performed to enrich the polar patterns of the molecular ions in the original datasets were used
components of the oils for improved sensitivity. Extraction also to obtain elemental formulas, which were correlated to known
reduced the high levels of hydrophobic triacylglycerides and compounds in olive oil. The names and elemental formulas of
diacylglycerides, which would be retained on reversed-phase all the compounds detected in both modes are summarized in a
columns during the HPLC separation of the polar components. table (see Table 1).
Olive oil samples were extracted three times with N,N- Statistical Analysis
dimethylformamide (DMF). Extracts were pooled and washed twice All of the LC/MS datasets were processed with a proprietary
with hexane to remove hydrophobic components. Residual hexane algorithm to extract the intensities for significant LC peaks. The
was removed from the DMF extracts by centrifugal evaporation. results were compiled into a table, with a row for each dataset,
The DMF extracts were diluted 1:20 with water and 2 µL of each and columns of intensities for each significant peak. Each column
extract were injected for LC/MS analysis. was labeled with a text summary of the rounded m/z and time
values for that peak. The table was imported into TIBCO Spotfire®
LC/MS Method
for statistical analysis using an S-Plus Principal Component Analysis
Extracts were analyzed in triplicate with a single chromatographic
(PCA) function and for graphical display of result in color-coded
method, with separate analyzes for the detection of components
scatter plots and bar charts.
either with positive or negative mode electrospray ionization.
Methods for extracting the significant features from each LC/MS
Components were separated by reversed-phase gradients on a
dataset and for the PCA of these features with Spotfire have been
Brownlee SPP C18 column with a Flexar™ FX-10 UHPLC pump,
described in previous applications notes.
using water/methanol eluents at 0.4 mL/min, with a gradient from
10% methanol to 50% methanol over 10 mins, then detected
with an AxION® 2 TOF MS fitted with an Ultraspray™ 2 ion source.

Figure 1. Example separation showing EVOO polar components detected in negative ion mode, with peaks labeled with the nominal mass of each compound.
2
Negative Mode Statistical Results
The PCA of negative mode features for all the samples gave The components which most strongly contribute to the cultivar
component scores and loadings which were displayed in scatter differences are shown in the corresponding Loadings Plot (Figure 3
plots. A Scores Plot of PC 2 v. 1 (Figure 3 left panel) shows a clear right panel). Significant differentiators of the groups include the
cultivar grouping, with Arbequina and Empeltre samples grouped levels of oleic acid, together with the phenolics hydroxytyrosyl
in the left side of the plot and are distinguished from Cornicabra, acyclodihydroelenolate (HT-ACDE) and elenolic acid.
Picual and blends on the right side.

Figure 2. Example separation showing EVOO polar components detected in positive ion mode, with peaks labeled with the nominal mass of each compound.

Figure 3. PCA Scores Plots and Loadings Plots from the negative mode results for all detected compounds. Grouping of samples by cultivar (left panel) most strongly correlates to
the levels of the compounds HT-ACDE and elenolic acid as shown in the Loadings Plot (right panel). 3
 All cultivars had similar levels of fatty acids, except for one Empeltre Oleuropein levels are reduced during the ripening of the olives. A
cultivar which had higher levels. Normally free fatty acid levels in recent report1 analyzed the levels of oleocanthal and oleacein in
EVOOS are low, but high levels are related to the breakdown of Greek and Californian oils and found levels varied by both cultivar
TAGs in the olive oil. The high levels in this one sample suggest and processing temperature.
that the oil may be degraded. These results suggest that levels of Positive Mode Statistical Results
free fatty acids are not reliable markers for cultivar or origin. The PCA of positive mode phenolic compounds levels produced
The levels of phenolics vary widely between cultivars and between a Scores Plot of PC 2 v. 1 (Figure 6, left panel), which showed
different brands within each cultivar. Some brands have extremely cultivar grouping. The Loadings Plot showed that the compounds
high levels of elenoic acid. Because of a chemical transformation of ligstroside aglycone (p-HPEA-EA or oleocanthal), oleuropein
elenolic acid in acidic eluents, a broad peak was observed with the aglycone (DHPEA-EA), acetoxypinoresinol and pinoresinol were
chromatography method used. Thus, the reported peak height and most correlated to the cultivars.
area were not as reproducible between samples as for other The lignans pinoresinol and acetoxypinoresinol showed widely
compounds, leading to a wide scatter of sample points in the initial varying levels between cultivars. All Manzanilla Cacereña, some
Scores Plot. Cornicabra and Picual cultivars and blended samples have high
A new PCA of the negative mode results excluded the elenolic acid levels of both lignans; Empeltre and Arbequina brands had no
and fatty acids component intensities which had shown large detectable acetoxypinoresinol and moderate levels of pinoresinol.
variability in the first analysis. The new Scores Plots (Figure 4 left These lignans have been detected previously2,3 in Spanish olive oils.
panel) now show groupings due only to the variation in levels of Levels of acetoxy-pinoresinol were reported to be abundant in
the phenolic compounds. Arbequina, Empeltre and Picual samples Arbequina and Hojiblanco oils, but low in Picual and Hojiblanco
are each closely grouped, although Cornicabra and Manzanilla oils; with pinoresinol abundant in Picual and Cornicabra cultivars.
Cacereña samples were scattered.
Our findings differ, but a large variation in levels of these lignans
The compounds in the Loadings Plot (Figure 4 right panel) which between oils from different brands was predictable. Pinoresinols
were most related to the cultivar grouping were oleuropein are major components of olive seeds, so levels may be related to
aglycone, oleocein, HT-ACDE and ligstroside aglycone the different olive crushing conditions rather than cultivar.
decarboxymethyl (p-HPEA-EDA or oleocanthal).
Free fatty acids are also detected in positive mode as [M + H]+ ions.
Two of these compounds, oleuropein and oleocanthal, contribute Oleic acid has high levels in one Empeltre cultivar, similar to the
to the bitterness of the oils. Levels are lowest in the Arbequina and findings from negative ion results. 2-oleoylglycerol, resulting from
Empeltre cultivars, which have low bitterness levels, as shown in a oxidative degradation of TAGs, was also detected in positive mode
bar chart (Figure 5). and has highest levels in the same Empeltre cultivar sample. Other
Variability of oleuropein levels for brands within each cultivar may compounds proposed to be oxidized forms of oleic acid are also
be due to different ripeness levels of the olives used for the oils. higher in all Empeltre samples.

Figure 4. PCA Scores Plots and Loadings Plots of phenolics detected in negative mode. Scores Plot (left panel) shows grouping correlated to levels of oleuropein aglycone,
4 oleocein, HT-ACDE and ligstroside aglycone (p-HPEA-EDA).
Figure 5. Levels of the bitter compounds oleuropein (top) and oleocanthal (bottom) for each sample, ordered and color coded by cultivar. Levels of both are lowest in the
Arbequina (red) and Empeltre (green) cultivars.

Figure 6. PCA Scores and Loadings Plots of the positive mode results. Scores Plot (left panel) shows grouping into cultivars dominated by levels of acetoxypinoresinol, pinoresinol,
ligstroside aglycone HPEA-EA and oleuropein aglycone DHPEA-EA.

5
Table 1. Accurate masses of the compounds. Conclusion
Molecule [M-H]- [M+H]+ [M+Na]+ Oils from a number of olive cultivars could be partly differentiated
Tyrosol (p-HPEA) 137.0608 139.0754 161.0578 by the levels of polar component features detected in either
Hydroxytyrosol (3,4-DHPEA) 153.0557 155.0703 177.0528 positive or negative mode analysis. The most significant
Hydroxytyrosol aetate (3,4-DHPEA-AC) 195.0663 197.0808 219.0633 differentiators determined from PCA are certain lignans and
Hydroxylated decarboxymethyl
199.0612 201.0757 223.0582 secoiridoids, including pinoresinol, acetoxypinoresinol, oleuropein
elenolic acid
Hydroxylated decarboxyl elenolic acid 213.0768 215.0914 237.0739 and oleocanthal.
Desoxy elenolic acid 225.0768 227.0914 249.0739 Methods combining these results, from separate analytical
Myristic acid 227.2017 229.2162 251.1987 methods, for more of a complete statistical evaluation of the oils,
Elenolic acid (EA) 241.0718 243.0863 265.0688 will be covered in a separate application note.
Palmitoleic acid (16:1) 253.2173 255.2319 277.2143
Elenolic acid methyl ester 255.0874 257.1020 279.0845 References
Palmitic acid (16:0) 255.2330 257.2475 279.2300
1. Evangelia Karkoula, Angeliki Skantzari, Eleni Melliou and
Hydroxylated elenolic acid 257.0667 259.0812 281.0637
Prokopios Magiatis. Quantitative Measurement of Major
Apigenin 269.0455 271.0601 293.0426
Secoiridoid Derivatives in Olive Oil Using qNMR. Proof of the
Linolenic acid (18:3) 277.2173 279.2319 301.2143
Artificial Formation of Aldehydic Oleuropein and Ligstroside
Linoleic acid (18:2) 279.2330 281.2475 303.2300
Aglycon Isomers. J. Agric. Food Chem., 62, 3 (2014), 600–607.
Oleic acid (18:1) 281.2486 283.2632 305.2456
Steric acid (18:0) 283.2643 285.2788 307.2613 2. M. Brenes, F. J. Hdalgo, A. Garcia, J.J.Rios, P. Garcia, R. Zamora
Luteolin 285.0405 287.0550 309.0375 and A. Garrido. Pinoresinol and acetoxypinoresinol, two new
Tyrosol hexoside 299.1136 301.1282 323.1107 phenolic compounds indentified in olive oil. JAOCS, 77, 7
Ligstroside aglycone decarboxymethyl (2000), 715-720.
303.1238 305.1384 327.1208
(p-HPEA-EDA, Oleocanthal)
Hydroxytyrosol 4-O-glucoside 315.1085 317.1231 339.1056 3. Carrasco-Pancorbo, Alegria. Rapid Quantification of the
Oleuropein aglycone decarboxymethyl Phenolic Fraction of Spanish Virgin Olive Oils by Capillary
319.1187 321.1333 343.1158
(3,4-DHPEA-EDA, Oleacein) Electrophoresis with UV Detection. J Agric Food Chem,
Lactone (ester with hydroxytyrosol) 321.1344 323.1489 345.1314
54 (2006), 7984-7991.
10-OH-Oleuropein aglycone
335.1136 337.1282 359.1107
decarboxymethyl
Pinoresinol 357.1344 359.1489 381.1314
Ligstroside aglycone dehydro 359.1136 361.1282 383.1107
Ligstroside aglycone (p-HPEA-EA) 361.1293 363.1438 385.1263
(+)-1-Hydroxypinoresinol 373.1293 375.1438 397.1263
Oleuropein aglycone dehydro 375.1085 377.1231 399.1056
Oleuropein aglycone (3,4-DHPEA-EA) 377.1242 379.1387 401.1212
Hydroxytyrosol acyclodihydroelenolate
381.1555 383.1700 405.1525
(HT-ACDE)
Oleoside / Secologanoside 389.1089 391.1235 413.1060
Methyl oleuropein aglycone 391.1398 393.1544 415.1369
Hydroxy-oleuropein aglycone 393.1191 395.1337 417.1162
Oleoside 11-methylester / oleoside
403.1246 405.1391 427.1216
7-methyl ester / 8-epikingiside
(+)-1-Acetoxypinoresinol 415.1398 417.1544 439.1369
Oleoside dimethylester 417.1402 419.1548 441.1373
Ligstroside aglycone + MeOH + FA 439.1610 441.1755 463.1580
Oleanolic acid 455.3531 457.3676 479.3501
Maslinic acid 471.3480 473.3625 495.3450

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