Determination of hydroxytyrosol in plasma by HPLC

Article abstract of DOI:10.1021/ac000121h

Hydroxytyrosol (2-(3,4-dihydroxyphenyl)ethanol), a phenolic compound present in extravirgin olive oil, has been reported to contribute to the prevention of cardiovascular disease. The present study describes an accurate and reproducible reversed-phase HPLC method to measure hydroxytyrosol in plasma. This compound was extracted from acidified plasma by solid-phase extraction using an Oasis HLB copolymer. The plasma sample was rinsed with water and methanol in water (5:95; v/v). Hydroxytyrosol was eluted with methanol, which was subsequently evaporated under a nitrogen stream. Analysis by HPLC with diode array-UV detection was carried out using a C₁₈ column and a gradient elution with acidified water and methanol/acetonitrile (50:50; v/v). The method was validated by the analyses of plasma samples spiked with pure hydroxytyrosol, obtaining a linear correlation (0.9986) and precision with a coefficient of variation ranging from 0.79 to 6.66%. The recovery was ?100%, and the limit of detection was 37 ng/mL. The oral administration of hydroxytyrosol to rats and its subsequent detection in plasma showed that the method is suitable for pharmacokinetic studies.

Full text of DOI:10.1021/ac000121h

Anal. Chem. 2000, 72, 4458-4461
Technical Notes
Determination of Hydroxytyrosol in Plasma by
HPLC
Valentina Ruiz-Gutie´ rrez,† M. Emı´lia Juan,‡ Arturo Cert,† and Joana M. Planas*,‡
Instituto de la Grasa y sus derivados, Consejo Superior de Investigaciones Cient´ıficas, E-41012 Sevilla, Spain, and
Departament de Fisiologia-Divisio´ IV, Facultat de Farma`cia, Universitat de Barcelona, E-08028 Barcelona, Spain
Hydroxytyrosol (2 -(3 ,4 -dihydroxyphenyl)ethanol), a phe-
nolic compound present in extravirgin olive oil, has been
reported to contribute to the prevention of cardiovascular
disease. The present study describes an accurate and
reproducible reversed-phase HP LC method to measure
hydroxytyrosol in plasma. This compound was extracted
from acidified plasma by solid-phase extraction using an
Figure 1. Chemical structure of hydroxytyrosol.
Oasis HLB copolymer. The plasma sample was rinsed
with water and methanol in water (5 :9 5 ; v/ v). Hydroxy-
tyrosol was eluted with methanol, which was subsequently
evaporated under a nitrogen stream. Analysis by HP LC
with diode array-UV detection was carried out using a C1 8
column and a gradient elution with acidified water and
methanol/ acetonitrile (5 0 :5 0 ; v/ v). The method was
validated by the analyses of plasma samples spiked with
pure hydroxytyrosol, obtaining a linear correlation (0.9986)
and precision with a coefficient of variation ranging from
0 .7 9 to 6 .6 6 %. The recovery was ∼1 0 0 %, and the limit
of detection was 3 7 ng/ mL. The oral administration of
hydroxytyrosol to rats and its subsequent detection in
plasma showed that the method is suitable for pharma-
cokinetic studies.
also be due to the presence of antioxidants in the nonsaponifiable
fraction8-11 such as hydroxytyrosol (2-(3,4-dihydroxyphenyl)-
ethanol) (Figure 1) and its secoiridoid derivatives.12
Experiments in vitro have shown that hydroxytyrosol has many
effects that could be beneficial in vivo, including antioxidant
activity,13 protection of lipoproteins against oxidative and free-
radical damage,10,14-16 and inhibition of platelet aggregation and
eicosanoid formation.17
Notwithstanding the effects described in vitro, the mechanisms
of hydroxytyrosol absorption, bioavailability, and tissue distribution
in humans are still undefined. This lack of information is due not
only to the difficulty of developing sensitive methods for measur-
ing hydroxytyrosol in plasma and body fluids but also to the
absence of a commercially available pure standard.
Here we develop a simple, accurate, and sensitive method for
identifying and quantifying hydroxytyrosol in human plasma. We
present a rapid procedure for sample preparation, avoiding
successive plasma manipulations by means of an SPE cartridge,
followed by HPLC analysis with photodiode array detection. The
The healthy effects of the Mediterranean diet have been
attributed, at least in part, to the intake of fat with a high content
of monounsaturated fatty acids and a lower consumption of
saturated fatty acids.1,2 Olive oil is the main source of fat containing
the higher monounsaturated/ saturated ratio in the Mediterranean
diet,3,4 and it is believed to contribute to the prevention of
cardiovascular disease3,5 and cancer.6,7 Its beneficial effects may
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G. J. Lipid Res. 1 9 9 2 , 33, 627-633.
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Atherosclerosis 1 9 9 6 , 12, 15-33.
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247, 60-64.
(11) De la Puerta, R.; Ru´ız-Gutierrez, V.; Hoult, J. R. S. Biochem. Pharmacol.
* Corresponding author: (tel) +34.93.402.45.05; (fax) +34.93.403.59.01;
(e-mail) jplanas@farmacia.far.ub.es.
† Consejo Superior de Investigaciones Cient´ıficas.
1 9 9 9 , 445-449.
‡ Universitat de Barcelona.
(12) Montedoro, G. F.; Servilli, M.; Baldioli, M.; Selvaggini, R.; Miniati, E.;
Macchioni, A. J. Agric. Food Chem. 1 9 9 3 , 41, 2228-2234.
(13) Chimi, H.; Cillard, J.; Cillard, P.; Rahmani, M. J. Am. Oil Chem. Soc. 1 9 9 1 ,
68 (5), 307-312.
(14) Grignaffini, P.; Roma, P.; Galli, C.; Catapano, A. L. Lancet 1 9 9 4 , 343, 1296-
1297.
(1) Mensink, R. P.; Katan, M. B. Arterioscler. Thromb. 1 9 9 2 , 12, 911-919.
(2) Nestle, M. Am. J. Clin. Nutr. 1 9 9 5 , 61, 1313S-1320S.
(3) Willett, W. C.; Sacks, S.; Trichopoulou, A.; Drescher, G.; Ferro-Luzzi, A.;
Helsing, E.; Trichopoulos, D. Am. J. Clin. Nutr. 1 9 9 5 , 61, 1402S-1406S.
(4) Serra-Majem, Ll.; Ribas, L.; Ramon, J. M. Br. J. Nutr. 1 9 9 9 , 81 (2), S105-
S112.
(15) Visioli, F.; Bellomo, G.; Montedoro, G. F.; Galli, C. Atherosclerosis 1 9 9 5 ,
(5) Katan, M. B.; Zock, P. L.; Mensink, R. P. Am. J. Clin. Nutr. 1 9 9 5 , 61,
117, 25-32.
1368S-1373S.
(16) Manna, C.; Galleti, P.; Cucciolla, V.; Moltedo, O.; Leone, A.; Zappia, V. J.
Nutr. 1 9 9 7 , 127, 286-292.
(17) Petroni, A.; Blasevich, M.; Salami, M.; Papini, N.; Montedoro, G. F.; Galli,
(6) Trichopoulou, A. Cancer Causes Control 1 9 9 5 , 6, 475-476.
(7) Lipworth, L.; Martinez, M. E.; Angell, J.; Hsien, C. C.; Trichopoulos, D.
Prev. Med. 1 9 9 7 , 26, 181-190.
C. Thromb. Res. 1 9 9 5 , 78 (2), 151-160.
4458 Analytical Chemistry, Vol. 72, No. 18, September 15, 2000
10.1021/ac000121h CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/03/2000

performance of the method was assessed by oral administration
of hydroxytyrosol to Wistar rats and its measurement in plasma.
°C. The mobile phase included solvent A, consisting of 3% acetic
acid in 97% deionized water, and solvent B, containing equal
volumes of acetonitrile and methanol. The flow rate was 1 mL/
min. Separation was performed using the gradient elution pro-
gram: 0 min, 95% A and 5% B; 25.0 min, 70% A and 30% B; 27.5
min, 60% A and 40% B; 30.0 min, 30% A and 70% B; 40 min, 0% A
and 100% B; 45 min, 95% A and 5% B. There was a 5-min delay
prior to the injection of the next sample to ensure reequilibration
of the column.
The chromatograms were obtained according to the retention
time with detection at 280 nm, at which the absorbance of
hydroxytyrosol presents a maximum. Peak identification of this
compound was carried out by comparison of the retention time
and its UV spectra (from 200 to 400 nm) with those of a standard.
Method Validation. The solid-phase extraction of hydroxy-
tyrosol from human plasma followed by HPLC analysis was
validated according to The United States Pharmacopoeia.20
Samples. A pool of human plasma samples obtained from fasted
healthy individuals by venipuncture (Blood Bank, Hospital Cl´ınic
i Provincial de Barcelona, Spain) was prepared. Aliquots of the
pooled plasma were stored at -20 °C until the analyses were
performed, and the samples were prepared as indicated in
preparation of plasma samples.
EXPERIMENTAL SECTION
Instrument. HPLC analyses were performed on a 1100
Hewlett-Packard (HP) (Palo Alto, CA) gradient liquid chromato-
graph equipped with a Rheodyne injection valve with a 20-µL
fixed loop, a Lichrospher 100 RP-18 reversed-phase column
(250 mm × 4 mm, 5 µm) from Merck (Darmstadt, Germany),
preceded by a precolumn of the same material as the stationary
phase, and a HP 1100 diode array UV-visible detector. An HP
Chemstation software system controlled all the equipment and
carried out the data processing.
Chemicals and Reagents. A chemically pure standard of
hydroxytyrosol was synthesized by LiAlH4 reduction of 3,4-
dihydroxy-1-benzeneacetic acid as described by Baraldi et al.18 The
internal standard, catechol, was obtained from Merck (Darmstadt,
Germany). Acetonitrile, methanol, and acetic acid, all HPLC grade,
were purchased from Romil Ltd. (Cambridge, England). All other
chemicals were analytical grade and were used without further
purification. Water used in all experiments was passed through a
Milli-Q (Millipore, Milan, Italy) water purification system (18 mΩ).
A stock solution of catechol (1.02 mg/ mL) in water was used as
internal standard.
Linearity. A stock solution of hydroxytyrosol was obtained by
dissolving the pure standard in water; the solution was stored at
4 °C and used within 4 weeks. Spiked plasma samples containing
increasing concentrations of hydroxytyrosol: 0.5, 0.75, 1, 2.5, 5,
7.5, and 10 µg/ mL and a fixed concentration of catechol as internal
standard (IS) (10 µg/ mL) were analyzed according to the
procedure described above. Linearity was checked by performing
linear regression analysis of the peak area ratio of hydroxytyrosol/
catechol against hydroxytyrosol concentration.
P reparation of P lasma Samples. Blood samples were col-
lected in tubes containing EDTA-K3 and centrifuged at 2700 rpm
(Beckman, model TJ-6 centrifuge, rotor TH-4 with buckets) for
15 min, and the plasma was immediately separated from cells. A
standard solution (10 µg/ mL) of catechol and 10 µL of phosphoric
acid (85% v/ v) were added to 1-mL aliquots of plasma and mixed
in a vortex for 1 min.
Solid-P hase Extraction (SP E) of P lasma Samples. Solid-
phase purification of hydroxytyrosol from plasma samples was
carried out on an Oasis HLB extraction cartridge (30 mg, 1 mL;
WAT 094226) purchased from Waters (Milford, MA). The
cartridge was placed in a device fitted with a small vacuum pump
and a waste receiver. The pressure was carefully monitored during
the critical cartridge conditioning, equilibration, and sample
loading steps, thus preventing changes in the separation efficiency
due to variations in the flow rate. The cartridge was conditioned
at a flow rate of 4-5 mL/ min, and the analyte was loaded, washed,
and eluted at 1.5-2 mL/ min. Prior to use, the cartridge was
conditioned with 1 mL of methanol followed by equilibration with
1 mL of water. A 1-mL aliquot of spiked acidified plasma was slowly
loaded into the cartridge, followed by 0.5 mL of water, and then
1 mL of 5% (v/ v) methanol in water. The vacuum was released to
replace the waste receiver with a conical glass tube containing
10 µL of 1% ascorbic acid solution. The SPE cartridge was eluted
under vacuum with 2 mL of methanol. The conical tube containing
the eluted fraction was capped with Parafilm and stored at 4 °C
until HPLC analysis. Just before the HPLC analysis, the eluted
fraction was evaporated under a nitrogen stream to a final volume
of 0.2 mL.
Precision. The precision of the analytical method was deter-
mined by assaying a sufficient number of plasma samples (n )
4-6) at seven different concentrations of hydroxytyrosol ranging
from 0.5 to 10 µg/ mL and was expressed as relative standard
deviation (coefficient of variation). Peak areas were considered
for the calculation of the concentration and establish the precision.
Accuracy. One milliliter of plasma and 1 mL of water as control
were spiked with hydroxytyrosol at concentrations of 1 and 10
µg/ mL together with 10 µg/ mL of catechol as internal standard.
The samples were purified by SPE and analyzed by HPLC as
described. Accuracy was calculated by comparing the values for
the plasma samples and control; results are expressed as percent-
ages of analyte recovered.
Sensitivity. The limit of detection (LD) and the limit of
quantification (LQ) were calculated by measuring the analytical
background response, running six blanks of plasma using the
maximum sensitivity allowed by the system. The signal-to-noise
ratio was used to determine the LD, and it was estimated as the
concentration of hydroxytyrosol in plasma samples that generated
a peak with an area at least 3 times higher than the baseline noise.
LQ was considered to be 10 times the standard deviation of the
six blank samples analyzed using the maximum sensitivity allowed
by the system. The LQ was subsequently validated by the analysis
of six plasma samples known to be near the LQ.
HP LC P rocedure. A 20-µL aliquot of the evaporated sample
was injected into a Lichrospher 100 RP-18 (Merck) reversed-phase
column (250 mm × 4 mm) with a 5-µm particle operating at 30
(19) Bai, C.; Yan, X.; Takenaka, M.; Sekiya, K.; Nagata, T. J. Agric. Food Chem.
1 9 9 8 , 46, 3998-4001.
(20) U.S. Pharmacopoeia, USP XXIII 1 9 9 5 , 1225, 1982-1984.
(18) Baraldi, P. G.; Simoni, D.; Manfredini, S.; Menziani, E. Liebigs Ann. Chem.
1 9 8 3 , 684-686.
Analytical Chemistry, Vol. 72, No. 18, September 15, 2000 4459

Animal Studies. Male Wistar rats (200-250 g) were housed
three per cage, in a temperature-controlled room, with a light-
dark cycle of 12 h and free access to water and a commercial rat
chow. Animal treatment was in full accordance with the European
Community Guidelines for the care and management of laboratory
animals. Hydroxytyrosol dissolved in water was administered
orally to overnight-fasted rats at a dose of 20 mg/ kg, except for
the control group, which was given only water. Animals were
anesthetized with ether and blood was collected by cardiac
puncture and transferred to a tube containing EDTA-K3 as
anticoagulant, at 5 and 10 min after the administration. Im-
mediately after obtaining the plasma by centrifugation, the analyte
was purified using the SPE procedure described above.
RESULTS AND DISCUSSION
Optimization of Solid-P hase Extraction. Methanol was the
solvent of choice since hydroxytyrosol is usually obtained by
extraction with this solvent from extravirgin olive oil.21,22 Further-
more, it proved to be an efficient solvent, breaking the hydro-
phobic bond, eluting hydroxytyrosol, and rendering excellent
recoveries.
Sample acidification was taken into account in order to disrupt
hydroxytyrosol-protein binding and enhance recovery. Therefore,
in initial attempts, extractions were carried out by mixing the
plasma sample with 10 and 20 µL/ mL phosphoric acid and without
acidification. The addition of 10 µL/ mL phosphoric acid was
selected because it yielded the best recovery.
The optimal amount of water and the percentage of methanol
in water that was sufficient to wash the sample without eluting
hydroxytyrosol were carefully evaluated due to the high polarity
of this compound. An Oasis cartridge was loaded with a known
amount of hydroxytyrosol dissolved in water, and the washout
fractions were monitored by injecting them in the HPLC. The
optimal volume of water for the first rinsing step was studied over
a range from 0.5 to 2 mL and found to be 0.5 mL. For the second
washing step, different amounts of methanol in water were
assessed: 2.5, 5, and 7.5%. A concentration of 5% proved to be
the optimal solution to eliminate interferences of plasma without
eluting the analyte. No hydroxytyrosol was detected in the optimal
washout fractions, and it was completely recovered when 2 mL
of 100% methanol was loaded into the cartridge. One milliliter of
methanol was sufficient to elute the analyte; however, an excess
of 1 mL is advised.
The concentration of the eluted fraction under a nitrogen
stream to a final volume of 0.2 mL enhanced the sensitivity of the
analytical method allowing the quantification of very low amounts
of this compound. However, volumes lower than 0.2 mL were
prone to erratic recoveries. During concentration, methanol was
evaporated; thus, the 0.2 mL corresponded to an aqueous solution
of hydroxytyrosol that, when injected into the HPLC, gave a
chromatogram (Figure 2) with excellent peak shapes.
Figure 2. HPLC chromatogram at 280 nm of hydroxytyrosol in
human plasma after isolation. Peak A, hydroxytyrosol; peak B,
catechol (internal standard).
The samples were extracted as described in the Experimental
Section, and the recovery was ∼98%.
Stability of Hydroxytyrosol. In the course of prior experi-
ments (data not shown), a decrease in hydroxytyrosol stability
was observed. Although this lack of stability was not noticed in
the SPE extraction from plasma, it was thought advisable to add
an antioxidant. The addition of 10 µL of 1% ascorbic acid and
storage of the final methanolic extract in a tube capped with
Parafilm at 4 °C, followed by evaporation under nitrogen stream
just before the HPLC analysis, stabilized the hydroxytyrosol for
at least one week. These conditions allowed a large number of
samples to be extracted and stored for later HPLC analysis.
Method Validation. The analytical performance parameters
assessed for the overall assay were linearity, precision, accuracy,
sensitivity, and selectivity.
Linearity. The response of hydroxytyrosol was checked in the
range of application of the analytical method by linear regression
analysis by the least-squares method of peak area ratio of
hydroxytyrosol/ catechol against different hydroxytyrosol concen-
trations. The response was linear in the range of concentrations
evaluated from 0.5 to 10 µg/ mL, giving an equation of y )
0.062x + 0.001 (n ) 31) and a regression coefficient of 0.9986.
Precision. The precision expressed as coefficient of variation
(CV) ranged from 0.79 to 6.66%, at seven different concentrations
ranging from 0.5 to 10 µg/ mL, indicating that the analytical
method is repeatable. The values obtained were as follows (CV,
%): 6.66 (0.50 µg/ mL), 3.63 (0.75 µg/ mL), 1.60 (1.00 µg/ mL),
3.01 (2.50 µg/ mL), 3.57 (5.00 µg/ mL), 0.79 (7.50 µg/ mL), and
1.90 (10.00 µg/ mL). The results were lower than those acceptable
according to the CV established by Horwitz23 for intralaboratory
analysis.
Selection of the Internal Standard. Catechol, an alcohol with
structural similarity to hydroxytyrosol, which eluted at 9.05 min
and was not present in plasma samples, was used as IS. To confirm
that catechol was retained by the Oasis cartridge, 1 mL of plasma
and 1 mL of water as control were spiked with 10 µL/ mL catechol.
Accuracy. The accuracy was determined for the overall assay
by measuring the percentage of recovery after the addition of
known amounts of standard to a control and to a pool of human
(21) Va´zquez-Roncero, A.; Janer del Valle, C.; Janer del Valle, M. L. Grasas Aceites
1 9 7 6 , 26, 14-18.
(22) Gutfinger, T. J. Am. Oil Chem. Soc. 1 9 8 1 , 58, 96-968.
(23) Horwitz, W. Anal. Chem. 1 9 8 2 , 54, 67A-76A.
4460 Analytical Chemistry, Vol. 72, No. 18, September 15, 2000

plasma, at two different concentrations. The mean recoveries at
1 and 10 µg/ mL were 98.7 and 99.8%, respectively. The values
indicate that hydroxytyrosol is quantitatively extracted using this
method.
Sensitivity. The limit of detection was 37 ng/ mL on the basis
of a signal-to-noise ratio of 3. The limit of quantification was 96
ng/ mL.
Selectivity. Hydroxytyrosol was quantified at 280 nm, its
maximum absorbance to improve selectivity. Hydroxytyrosol was
well resolved and free from interference peaks (Figure 2). The
identity of the chromatographic peak was confirmed not only by
its retention time but also by its spectrum (Figure 3).
Verification of the Method. Hydroxytyrosol was orally
administered to rats at a dose of 20 mg/ kg, to assess the validity
of the method. Blood taken by cardiac puncture was analyzed as
described in the Experimental Section, resulting in a hydroxy-
tyrosol plasmatic concentration of 1.22 µg/ mL at 5 min after
administration, and 1.91 µg/ mL at 10 min. The results showed
that the method developed was reliable, reproducible, and easily
applied to biological samples.
In contrast to the large number of methods published to
determine hydroxytyrosol in olive oil,24-27 there are few studies
that analyze this compound in biological samples. The first
procedure to determine hydroxytyrosol in rat plasma samples was
reported by Bai et al.19 This method included organic-phase
extraction and derivatization prior to the analysis by gas chroma-
tography. Solid-phase extraction of hydroxytyrosol with an Oasis
cartridge was suitable for the determination of this compound
since it avoided analyte losses and provided maximal sensitivity
Figure 3. UV spectra of standard hydroxytyrosol (dotted line),
hydroxytyrosol in human plasma after isolation (straight line), and
hydroxytyrosol in rat plasma after isolation (dashed line), all obtained
by diode array detection.
with minimal handling.
In conclusion, a simple, precise, sensitive, reproducible, and
reliable method has been developed to study the levels of
hydroxytyrosol in plasma. The handling of samples is easy and
can be performed automatically. The oral administration of
hydroxytyrosol to experimental animals and its detection in plasma
has provided evidence of its absorption.
ACKNOWLEDGMENT
M.E.J. was a recipient of Beca de Recerca i Doce`ncia from
the Universitat de Barcelona. We thank Ms. Raquel Mateos for
excellent technical assistance. This work was supported by Grants
PB96-1255 from the DGES and OLI96-2126 from the CICYT,
Ministerio de Educacio´n y Cultura, Spain.
(24) Montedoro, G. F.; Servilli, M.; Baldioli, M.; Miniati, E. J. Agric. Food Chem.
1 9 9 2 , 40, 1571-1576.
(25) Tsimidou, M.; Papadopoulos, G.; Boskou, D. Food Chem. 1 9 9 2 , 44, 53-
60.
(26) Akasbi, M.; Shoeman D. W.; Csallany, S. J. Am. Oil Chem. Soc. 1 9 9 3 , 70,
Received for review February 3, 2000. Accepted June 14,
2000.
367-370.
(27) Angerosa, F.; D’Alessandro, N.; Konstantinou, P.; Di Giacinto, L. J. Agric.
Food Chem. 1 9 9 5 , 43, 1802-1806.
AC000121H
Analytical Chemistry, Vol. 72, No. 18, September 15, 2000 4461