Determination of Phenols, Flavones, and Lignans in Olive Oil
J. Agric. Food Chem., Vol. 49, No. 5, 2001 2191
(6) Tsimidou, M.; Papadopoulos, G.; Boskou, D. Determi-
nation of phenolic compounds in virgin olive oil by
reverse-phase HPLC with emphasis on UV detection.
Food Chem. 1992, 44, 53-60.
(7) Montedoro, G.; Servili, M.; Baldioli, M.; Miniati, E.
Simple and hydrolyzable phenolic compounds in virgin
olive oil. I. Their extraction, separation, and semiquan-
titative evaluation by HPLC. J . Agric. Food Chem. 1992,
40, 1571-1576.
good repeatability of the complete method. The recover-
ies relative to the standard solutions were excellent
(Table 1).
The HPLC method was compared with the colorimet-
ric quantification of o-diphenols by reaction with mo-
lybdate. For this, the colorimetric method was applied
to several hydroxytyrosol solutions, and the following
calibration line was obtained:
(8) Brenes, M.; Garc´ıa, A.; Garc´ıa, P.; R´ıos, J . J .; Garrido,
A. Phenolic compounds in spanish olive oils. J . Agric.
Food Chem. 1999, 47, 3535-3540.
(9) Espartero, J . L.; Mateos, R.; Trujillo, M.; Cert, A.;
Alcudia, F. Structural characterization of new polyphe-
nols in olive oil. In Proceedings of the 27th Biennial
Meeting of the Royal Spanish Chemistry Society; La
Laguna, Spain, J uly 1999; p 165.
(10) Brenes, M.; Hidalgo, F. J .; Garcı´a, A.; R´ıos, J . J .; Garc´ıa,
P.; Zamora, R.; Garrido, A. Pinoresinol and 1-acetoxy-
pinoresinol, two new phenolic compounds identified in
olive oil. J . Am. Oil Chem. Soc. 2000, 77, 715-720.
(11) Owen, R. W.; Mier, W.; Giacosa, A.; Hull, W. E.;
Spiegelhalder, B.; Bartsch, H. Identification of lignans
as major components in the phenolic fraction of olive
oil. Clin. Chem. 2000, 46, 976-988.
(12) Va´zquez-Roncero, A.; J aner, C.; J aner, M. L. Phenolic
components in olive fruits. III. Polyphenols in olive oil.
Grasas Aceites 1976, 27, 185-191.
(13) Cortesi, N.; Azzolini, M.; Rovellini, P. Determination of
minor polar components in virgin olive oils. Riv. Ital.
Sostanze Grasse 1995, 72, 333-338.
(14) Gutie´rrez, F.; Albi, M. A.; Palma, R.; R´ıos, J . J .; Ol´ıas,
J . M. Bitter taste of virgin olive oil: Correlation of
sensory evaluation and instrumental HPLC analysis. J .
Food Sci. 1989, 54, 68-70.
(15) Mannino, S.; Cosio, M. S.; Bertuccioli, M. High perfor-
mance liquid chromatography of phenolic compounds in
virgin olive oils using amperometric detection. Ital. J .
Food Sci. 1993, 4, 363-370.
C (mmol/mL) ) 1.36 × 10-3 × absorbance + 1.2 ×
10-5
The method was then applied to standard solutions
of phenolic compounds possessing different functional
groups. As expected, vanillin, tyrosol, and R-tocopherol
did not react with molybdate, whereas the molar
responses of hydroxytyrosol, hydroxytyrosyl acetate, the
aldehydic form 1, and luteolin were similar. Application
of the colorimetric method to methanol extracts obtained
by SPE on diol phase yielded results similar to those
obtained with the HPLC method, when only compounds
having o-diphenol structure (hydroxytyrosol, hydroxy-
tyrosol derivatives, and luteolin) were considered. The
results for Picual olive oil were 0.52 and 0.52, for
Manzanilla olive oil, 1.01 and 1.04, and for Arbequina
olive oil, 0.16 and 0.17 mmol/kg, respectively.
For comparing SPE on diol-phase and liquid-liquid
extraction, phenolic extracts were obtained from a
Picual virgin olive oil using both analytical methods,
each one in duplicate. The four extracts were analyzed
by HPLC using the described method. The concentra-
tions of each phenolic compound obtained by both
extraction methods did not show differences at 95% of
statistical significance level. In summary, the SPE on
diol phase followed by HPLC with UV detection is a
simple and precise analytical method for determining
phenolic compounds, lignans, flavones, and cinnamic
acid in olive oils.
(16) Pirisi, F. M.; Cabras, P.; Cao, C. F.; Migliorini, M.;
Muggelli, M. Phenolic compounds in virgin olive oil. 2.
Reappraisal of the extraction, HPLC separation, and
quantification procedures. J . Agric. Food Chem. 2000,
48, 1191-1196.
(17) Andreoni N.; Fiorentini, R. Determination of phenolic
compounds in olive oil. Riv. Ital. Sostanze Grasse 1995,
72, 163-164.
(18) Servili, M.; Baldioli, M.; Selvaggini, R.; Miniati, E.;
Macchioni, A.; Montedoro, G. High-performance liquid
chromatography evaluation of phenols in olive fruit,
virgin olive oil, vegetation waters, and pomace and 1D-
and 2D-nuclear magnetic resonance characterization. J .
Am. Oil Chem. Soc. 1999, 76, 873-882.
(19) Evangelisti, F.; Zunin, P.; Tiscornia, E.; Petacchi, R.;
Drava, G.; Lanteri, S. Stability to oxidation of virgin
olive oils as related to olive conditions: Study of polar
compounds by chemometric methods. J . Am. Oil Chem.
Soc. 1997, 74, 1017-1023.
ACKNOWLEDGMENT
We are indebted to Rosario Gonza´lez Cordones and
Manuel Rodr´ıguez Aguilar for technical assistance.
LITERATURE CITED
(1) Tsimidou, M. Polyphenols and quality of virgin olive oil
in retrospect. Ital. J . Food Sci. 1998, 10, 99-116.
(2) Angerosa, F.; d’Alessandro, N.; Corona, F.; Mellerio, G.
Characterization of phenolic and secoiridoid aglycons
present in virgin olive oil by gas chromatography-
chemical ionization mass spectrometry. J . Chromatogr.
A 1996, 736, 195-203.
(20) Rovellini, P.; Cortesi, N.; Fedeli, E. Ana´lisis of flavonoids
from Olea europea by HPLC-UV and HPLC-electro-
spray-MS. Riv. Ital. Sostanze Grasse 1997, 74, 273-279.
(21) Baraldi, P. G.; Simoni, D.; Manfredini, S.; Menziani, E.
Preparation of 3,4-dihydroxy-1-benzeneethanol: A re-
investigation. Liebigs Ann. Chem. 1983, 684-686.
(22) LaLonde, R. T.; Wong, Ch.; Tsai, A. Polyglucosidic
metabolites of Oleaceae. The chain sequence of oleoside
aglucon, tyrosol, and glucose units in three metabolites
from Fraxinus americana. J . Am. Chem. Soc. 1976, 98,
3007-3012.
(23) Capasso, R.; Evidente, A.; Visca, C.; Gianfreda, L.;
Maremonti, M.; Greco, G. Production of glucose and
bioactive aglycons by chemical and enzymatic hydrolysis
of purified oleuropein from Olea europea. Appl. Biochem.
Biotechnol. 1996, 61, 365-377.
(3) Cortesi, N.; Azzolini, M.; Rovellini, P.; Fedeli, E. Minor
polar components of virgin olive oil. A hypothetical
structure by LC-MS. Riv. Ital. Sostanze Grasse 1995,
72, 241-251.
(4) Pirisi, F. M.; Angioni, A.; Cabras, P.; Garau, V. L.;
Sanjust di Teulada, M. T.; Karin dos Santos, M.;
Bandino, G. Phenolic compounds in virgin olive oils. I.
Low-wavelength quantitative determination of complex
phenols by high-performance liquid chromatography
under isocratic elution. J . Chromatogr. A 1997, 768,
207-213.
(5) Montedoro, G. M.; Servili, M.; Baldioli, M.; Selvaggini,
R.; Miniati, E.; Macchioni, A. Simple and hydrolyzable
compounds in virgin olive oil. 3. Spectroscopic charac-
terization of the secoiridoid derivatives. J . Agric. Food
Chem. 1993, 41, 2228-2234.