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A validated method for the determination of selected phenolics in olive oil using high-performance liquid chromatography with coulometric electrochemical detection and a fused-core column
Food Chemistry 138 (2013) 1663–1669
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Analytical Methods
A validated method for the determination of selected phenolics in olive oil using high-performance liquid chromatography with coulometric electrochemical detection and a fused-core column Banu Bayram a,b, Beraat Ozcelik b, Gerhard Schultheiss c, Jan Frank d, Gerald Rimbach a,⇑ a
Institute of Human Nutrition and Food Science, Christian-Albrechts-University, Hermann-Rodewald-Strasse 6, 24098 Kiel, Germany Department of Food Engineering, Istanbul Technical University, 34469 Maslak, Istanbul, Turkey Christian-Albrechts-University, Olshausen Strasse 40, 24098 Kiel, Germany d Institute of Biological Chemistry and Nutrition, University of Hohenheim, Garbenstrasse 30, 70599 Stuttgart, Germany b c
a r t i c l e
i n f o
Article history: Received 16 August 2012 Received in revised form 23 October 2012 Accepted 17 November 2012 Available online 5 December 2012 Keywords: Fused core column Olive oil Phenolics Electrochemical detector HPLC
a b s t r a c t A liquid chromatographic method with a coulometric electrochemical detector (ECD) and a fused-core column was developed for the quantification of the olive oil phenolics tyrosol, hydroxytyrosol, oleuropein, pinoresinol, and caffeic, ferulic, vanillic, and p-coumaric acid. The method was validated according to guidelines of the U.S. Food and Drug Administration. The selectivity, linearity, lower limit of quantification (LOQ), lower limit of detection (LOD), precision, accuracy, recovery, as well as the stabilities of the phenolic standards and quality control samples were determined. The separation of the eight phenolic compounds was achieved within 16 min and the total analysis time (35 min) was ca. 3-fold shorter than that of conventional HPLC methods. The LOQ range was 0.3–15.3 ng/mL, which is at least 5-fold lower than those of other methods. Recovery was between 75% and 101%. Overall the method has the advantages of being sensitive, selective, fast and provides simultaneous qualitative and quantitative analysis of phenolics. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Olive oil, the primary source of fat in the Mediterranean diet, has been suggested to have potential antioxidant (Turner et al., 2010), antiinflammatory (Zhang, Cao, & Zhong, 2009), cardioprotective (Bayram et al., 2012), anticancer (Gill et al., 2005), antidiabetic (Rigacci et al., 2010) and neuroprotective activity (Schaffer et al., 2007). Olive oil is characterized by a high content of oleic acid and is an important source of hydrophilic phenolic compounds that contribute to stability, sensory, technological, and nutritional properties of olive oil (Servili et al., 2004). A wide range of phenolic compounds, belonging to many different classes including phenolic acids and alcohols, flavonoids, hydroxy-isochromans, secoiridoids, and lignans, have been identified in virgin olive oil (Carrasco-Pancorbo et al., 2005; Servili et al., 2004) with tyrosol and hydroxytyrosol, being the quantitatively major phenolic compounds. Phenolic acids, such as vanillic acid, caffeic acid, p-coumaric acid, and ferulic acid, constitute another important group of olive oil phenolics. The lignan pinoresinol is a newly recognized abundant compound in olive oil (Brenes, Garcia, Garcia, & Garrido, 2000). ⇑ Corresponding author. Tel.: +49 431 880 2583; fax: +49 431 880 2628. E-mail address: [email protected] (G. Rimbach). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.11.122
The identification and quantification of phenolic compounds in olive oil is of great interest due to their nutritional and technological importance. Various methods have been used for the characterization of phenolic compounds in olive oil, such as high-performance liquid chromatography (HPLC) coupled with ultraviolet detection (Tsimidou, Papadopoulos, & Boskou, 1992), diode array detection (Pirisi et al., 1997) coulometric electrochemical detection (Brenes et al., 2000), mass spectrometry (MS) (Suárez, Macià, Romero, & Motilva, 2008), gas chromatography coupled to MS (García-Villalba et al., 2011), capillary electrophoresis (CE) (Bonoli, Bendini, Cerretani, Lercker, & Gallina-Toschi, 2004), nuclear magnetic resonance spectroscopy (Christophoridou, Dais, Tseng, & Spraul, 2005) and infrared spectroscopy (Montedoro et al., 1993). Among these techniques, LC–MS is the predominent method used for the quantification of phenolics in olive oil. Coulometric electrochemical detector (ECD) provides high sensitivity and specificity for substances that are either oxidized or reduced at the applied potential. It provides an analytical tool for resolving and accurately detecting trace amounts of electroactive compounds, including phenolics, in a wide range of samples. Thus, due to its high selectivity, sensitivity, low detection limits and enhanced resolving power (Achilli, Cellerino, & Hamache, 1993; Svendsen, 1993) coulometric electrochemical detection has become increasingly popular for the analysis of plant bioactives
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in foods and beverages. Furthermore HPLC coupled to ECD has been applied in the analysis of phenolic compounds in fruits (Aaby, Skrede, & Wrolstad, 2005), vegetables (Xu, Yu, & Chen, 2006) and pharmaceutical products (Peng, Yuan, Liu, & Ye, 2005). In addition to sensitivity, run times are an important aspect of chromatographic methods. The total analysis times of published methods for olive oil phenolics are often longer than 40 min (Bonoli et al., 2004; Tovar, Motilva, & Romero, 2001). In the literature CE methods with run times under 15 min are available but CE has the disadvantage of a relatively low sensitivity (Ballus, Meinhart, Bruns, & Godoy, 2011; Bonoli et al., 2004). Recent developments in column technology, such as the newly developed stationary phases with fused-core particles, offer increased chromatographic efficiency and resolution, shorter analysis times, and increased sensitivity at lower operating pressures (McCalley, 2010). To the best of our knowledge there is currently no method available in the literature for the analysis of olive oil phenolics combining the sensitivity of HPLC–ECD with the superior separation and speed of a fused-core column. Therefore, we aimed to develop a rapid and sensitive fused-core column HPLC–ECD method for the analysis of selected phenolic compounds (oleuropein, tyrosol, hydroxytyrosol, pinoresinol, ferulic acid, caffeic acid, vanillic acid and p-coumaric acid) in olive oil and to validate it according to the FDA guidelines for bioanalytical method validation (FDA, 2001). 2. Materials and methods 2.1. Oil samples Six olive oils from six different countries and produced from different olive varieties were directly obtained from the producers and analysed for their content of phenolics. All oil samples were kept at 4 °C under a nitrogen atmosphere until analysis. Polyphenol-free olive oil was obtained from Sigma Aldrich (Steinheim, Germany). 2.2. Chemicals HPLC grade methanol was obtained from J.T. Baker (Deventer, The Netherlands) and acetonitrile from Sigma Aldrich (Steinheim, Germany). Standards of tyrosol (CAS No. 501-94-0), vanillic acid (CAS No. 121-34-6), caffeic acid (CAS No. 331-39-5), p-coumaric acid (CAS No. 501-98-4), and ferulic acid (CAS No. 1135-24-6) were purchased from Sigma Aldrich (Schnelldorf, Germany). Oleuropein (CAS No. 32619-42-4) and hydroxytyrosol (CAS No. 10597-60-1) were supplied by Extrasynthese (Genay Cedex, France) and pinoresinol by Separation Research (Turku, Finland). Reference standards of all analyzed compounds were HPLC grade with purities higher than 98% except for oleuropein, which was >90%. HPLC grade 60% perchloric acid was obtained from Fisher Scientific (Leicestershire, UK).
to 1.5 g of a phenolic-free olive oil to yield oils with low and high concentrations of phenolics. The highest amounts of phenolic compounds added to the olive oil were set based on literature and were (all in mg/kg): oleuropein, 12; tyrosol, 35; hydroxytyrosol, 50; pinoresinol, 90; ferulic acid, 15; caffeic acid, 15; vanillic acid, 5.3; and p-coumaric acid, 15. The phenolic mixture was diluted 20-fold to obtain the control oils with low concentrations of phenolics. 2.5. Extraction of phenolic compounds from olive oil The phenolic fraction of the olive oils was obtained by solidphase extraction (SPE) using diol-bonded SPE cartridges (Varian, Bond Elut Diol 500 mg/3 mL) according to a protocol modified from Mateos et al. (2001). Briefly, an SPE cartridge was placed in a vacuum elution apparatus (Phenomenex, Aschaffenburg, Germany) and conditioned by the consecutive passing of 6 mL of methanol and 6 mL of hexane. A 1.5 g oil sample was mixed with 1.5 mL of hexane, and applied to the column. The solvent was pulled through, leaving the sample on the solid phase. The sample container was washed with two 3 mL portions of hexane, and the sample container was washed again with 4 mL of hexane/ethyl acetate (90:10, v/v). The column was eluted with 10 mL of methanol and the solvent evaporated in a centrifugal evaporator (Jouan RC, Saint Herblain, France) at 35 °C. The phenolic residue was dissolved in 1 mL of methanol. Extracts were filtered through a 0.2 lm PTFE membrane filter (Pall Corporation, Michigan, USA) and directly injected into the HPLC system or stored at 20 °C. 2.6. HPLC analysis of olive oil phenolics HPLC analyses of olive oil phenolics were carried out on a Jasco system (Jasco GmbH Deutschland, Gross-Umstadt, Germany) consisting of a pump (PU-2085) and autosampler (XLC-3059AS), while detection was carried out on a 4-channel ESA 5600A CoulArray detector with integrated column oven (ESA Inc., Chelmsford, MA, USA). For analyte detection, increasing potentials of +250, +400, +500 and +750 mV were applied. Separation of phenolics was achieved on a Kinetex C18 column (100 4.6 mm, 2.6 lm, Phenomenex, Aschaffenburg, Germany) using gradient elution with water (pH 3.1, solvent A), acetonitrile (solvent B), and methanol (solvent C) each containing 60 mmol/L LiClO4. The solvent gradient changed according to the following conditions: from 96% A – 3% B – 1% C to 85% A – 10% B – 5% C in 4 min; 82.5% A – 11.5% B – 6% C in 2 min; 80% A – 12% B – 8% C in 2 min; 65% A – 24% B – 11% C in 7 min; 85% B – 15% C in 6 min; 85% B – 15% C for 5 min; 96% A – 3% B – 1% C to 85% A in 4 min; the final conditions were maintained for 5 min. The pH of the aqueous eluent was adjusted to 3.1 with perchloric acid, and the eluent was vacuum-filtered through a 0.2 lm hydrophilic polypropylene membrane filter (Pall Corporation, Michigan, USA). The column temperature was maintained at 40 °C, the flow rate set at 1.25 mL/min, and the injection volume was 10 lL. The analytes were quantified against authentic compounds as external standards using the sum of peak heights. 2.7. Method validation
2.3. Preparation of stock and working standard solutions Stock solutions of olive oil phenolic standards were prepared in methanol at a concentration of 2 mg/mL and stored at 20 °C in amber-coloured bottles. The working standard solutions were prepared by diluting stock solution with H2O/methanol (50:50, v/v).
The analytical method for the determination of olive oil phenolics was validated according to the requirements of FDA guidelines for bioanalytical method validation (FDA, 2001). The method was validated in terms of linearity, selectivity, LOQ, LOD, accuracy, precision, recovery, stock solution stability, short-term stability, freeze-and-thaw stability, and bench-top stability.
2.4. Preparation of quality control samples The quality control samples were prepared by diluting the stock solutions with methanol and adding 75 lL of the phenolic mixture
2.7.1. Selectivity For evaluation of selectivity, olive oil free of phenolic compounds (Sigma Aldrich, Steinheim, Germany) was used as a blank
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Table 1 Linear ranges, regression coefficients, slope, intercept and lower limits of quantification (LOQ) and detection (LOD) of the phenolic compounds. The linear range of the detector response was assessed by serial dilution of a mixture of the studied compounds dissolved in H2O:MeOH (50:50, v/v). Calibrations curves were obtained by plotting the sum of peak heights against the analyte concentrations (n = 3).
Fig. 1. Representative chromatogram of olive oil phenolics injected with a standard solution or extracted from spiked olive oil and resolved by gradient elution on a Kinetex™ C18 fused-core column and detected by 4-channel coulometric electrochemical detection. The retention times of the analytes (in minutes) are given in brackets: 1, hydroxytyrosol (2.7); 2, tyrosol (4.1); 3, caffeic acid (5.2); 4, vanillic acid (5.4); 5, ferulic acid (7.4); 6, p-coumaric acid (8.4); 7, oleuropein (13.2); and 8, pinoresinol (15.3).
Compound
Linear range (ng/ mL – lg/mL)
Regression coefficient
LOD (ng/mL)
LOQ (ng/mL)
Tyrosol Hydroxytyrosol Pinoresinol Oleuropein Caffeic acid Ferulic acid Vanillic acid p-Coumaric acid
0.3–16.7 15.3–62.5 2.0–33.3 3.4–222.0 5.4–22.2 3.4–27.8 0.6–40.0 1.4–22.2
0.9993 0.9998 0.9991 0.9998 0.9999 0.9996 0.9999 0.9969
0.07 0.06 0.50 0.11 0.30 1.70 0.15 0.03
0.30 15.3 2.00 3.40 5.40 3.40 0.60 1.40
2.7.3. Precision and accuracy The precision and accuracy of the method were determined on 3 consecutive days by five replicate analyses of quality control samples with known amounts of olive oil phenolics at two concentrations (low and high). The olive oil was extracted and the calculated analyte concentrations of spiked oil samples were compared with the known concentrations within the same day (intra-day) and from day-to-day (inter-day). The precision is given as the intra- and inter-day coefficients of variation (% CV). The accuracy was expressed as the absolute error percentage and calculated as follows: Accuracy (%) = [Mean of measured concentration Known added concentration]/Known added concentration 100. 2.7.4. Recovery The recoveries of the phenolics were calculated by analyzing five replicate spiked olive oil samples for each concentration (low and high) and comparing the detector responses with those of calibration standards with identical concentrations. The recovery (in percent) was calculated as follows: Recovery (%) = [Measured concentration/Expected concentration] 100.
Fig. 2. Representative chromatogram of olive oil phenolics extracted from a representative olive oil sample and resolved by gradient elution on a Kinetex™ C18 fused-core column and detected by 4-channel coulometric electrochemical detection. The retention times of the analytes (in minutes) are given in brackets: 1, hydroxytyrosol (2.7); 2, tyrosol (4.1); 3, caffeic acid (5.2); 4, vanillic acid (5.4); 5, ferulic acid (7.4); 6, p-coumaric acid (8.4); 7, oleuropein (13.2); and 8, pinoresinol (15.3).
and extracted and analyzed to check for the presence of interfering peaks.
2.7.2. Linearity, LOD and LOQ The linear range of the detector response was determined by serial dilution of a mixture of the studied compounds. Calibration curves were obtained by plotting the sum of peak heights of the respective analyte versus its concentration for eight different concentrations. Standard solutions corresponding to each point in the calibration curve were injected in triplicate and regression parameters were calculated. The LOQ was defined as the lowest concentration that could be determined with a deviation from the actual concentration and a coefficient of variation of precision of less than 20%, respectively. The LOD was the lowest concentration of the analyte of interest that could be reliably discerned from the baseline (signal-to-noise ratio 3).
2.7.5. Stock solution stability The stability of stock solutions was determined at two concentration levels (low and high). The aliquots of stock solutions were stored at room temperature (RT), 4 °C and 20 °C for 24 h. The initial detector response of solutions was compared to that after storage and results were expressed as percent degredation. 2.7.6. Short- and long-term stability Five aliquots of spiked oil samples were prepared at two concentrations (low and high). The aliquots were analysed and the remaining samples stored at RT, 4 °C, 20 °C, 80 °C for 24 h. For the long term stability the remaining samples were stored at 20 °C and 80 °C for 6 months. The initial values were compared to those obtained after storage and results were expressed as percent degredation. 2.7.7. Freeze-and-thaw stability Three aliquots of spiked oil samples were prepared at two concentrations (low and high). The aliquots were analysed and the remaining samples frozen at 80 °C for 24 h, thawed at room temperature and frozen again for 12-24 h at 80 °C. The freezeand-thaw-cycle was repeated two additional times and the stability determined by comparing initial values to those obtained after repeated freezing–thawing and results were expressed as percent degredation.
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Table 2 Intra-day and inter-day precision, accuracy, and recovery of phenolic compounds added at low or high concentrations to phenolic-free olive oil and determined in five replicate analyses (n = 5).
Tyrosol Intra-day Inter-day Hydroxytyrosol Intra-day Inter-day Oleuropein Intra-day Inter-day Pinoresinol Intra-day Inter-day Caffeic acid Intra-day Inter-day Ferulic acid Intra-day Inter-day Vanillic acid Intra-day Inter-day p-Coumaric acid Intra-day Inter-day
Nominal concentration (mg/kg)
Calculated concentration (mg/kg)
1.75 35.00 1.75 35.00
1.64 29.90 1.68 30.30
93.8 85.5 95.8 86.5
6.33 14.65 4.26 13.56
2.56 6.93 3.77 6.01
2.50 50.00 2.50 50.00
1.92 38.63 1.99 39.90
76.6 77.3 79.8 79.8
23.44 22.73 20.23 20.24
3.64 6.69 3.78 6.21
0.60 12.00 0.60 12.00
0.61 10.45 0.56 9.79
100.9 87.1 94.1 81.6
0.86 12.86 5.89 18.41
3.27 3.24 5.75 10.88
4.75 95.00 4.75 95.00
4.12 84.52 4.30 89.60
86.8 89.0 90.6 94.3
13.21 11.03 9.40 5.68
3.57 7.43 4.24 6.51
0.75 15.00 0.75 15.00
0.56 13.39 0.57 13.23
74.5 83.9 76.4 88.2
25.54 16.13 23.52 11.79
9.00 5.44 7.68 5.79
0.75 15.00 0.75 15.00
0.67 13.94 0.67 13.76
89.9 92.9 89.8 91.8
10.11 7.06 10.23 8.25
3.85 4.74 4.46 5.55
0.27 5.30 0.27 5.30
0.26 4.46 0.26 4.46
96.0 84.2 95.5 84.1
4.03 15.81 4.50 15.94
6.24 4.53 5.49 5.78
0.75 15.00 0.75 15.00
0.72 14.88 0.69 10.80
96.3 99.2 91.6 95.0
3.68 0.76 8.42 5.04
3.63 4.56 4.44 5.37
2.7.8. Bench-top stability Three aliquots of spiked oil samples were prepared at two concentrations (low and high) and the stability of samples in the autosampler was determined by injection of spiked oil samples at 6 h intervals (at 6, 12, and 18 h). The samples were injected from the same HPLC vial and the screw-caps were replaced after each injection to minimize solvent loss. The results were expressed as percent degradation.
3. Results and discussion 3.1. Optimization of the chromatographic method In order to optimize the analysis time, selectivity, and sensitivity of our method for all analytes, chromatographic conditions including mobile phase composition and gradient, flow rate, column temperature, column type, salt type and concentration, as well as working potentials applied on the CoulArray detector were varied. To achieve optimum electron transfer in coulometric electrochemical detection, a mobile phase with an electrolyte concentration between 50 and 100 mmol/L is recommended. Among the salts and buffer systems tested, problems with solubility and pH
Recovery [%]
Accuracy [%]
Precision [CV%]
occurred with sodium acetate/acetic acid, sodium dihydrogen phosphate/phosphoric acid, disodium hydrogen phosphate/phosphoric acid. Lithium perchlorate (LiCIO4), added at a concentration of 60 mmol/L to each eluent, and adjustment of the pH to 3.1 with perchloric acid resulted in better peak shapes. High salt concentrations may be problematic for the HPLC system and salt crystals were frequently observed in our system, which therefore had to be regularly washed with different solvents. This may be considered as a drawback of the developed method, albeit one that will be difficult to circumvent in electrochemical detection. Furthermore, different potentials and potential intervals were studied and it was observed that limiting the number of channels from originally eight to four improved the sensitivity of our method (data not shown). Therefore, only four channels were used for all subsequent experiments. Trials were carried out with different volumes of acetonitrile and methanol in the mobile phase to improve the resolution of the analytes. The use of methanol instead of acetonitrile as organic solvent resulted in loss of resolution for oleuropein and pinoresinol. Isocratic elution did not resolve all eight studied phenolics. The best peak shape and optimum resolution were obtained with the given mobile phase composition, which uses methanol as organic modifier in low percentages together with acetonitrile. The resolution of caffeic and vanillic acid was particularly improved
B. Bayram et al. / Food Chemistry 138 (2013) 1663–1669 Table 3 Stock solution stability of the phenolic standards at low or high concentrations after 24 h storage at RT, 4 °C, or 20 °C (n = 5). Stock solution stability [Degradation %] Concentration (lg/mL)
RT
4 °C
20 °C
Tyrosol 4.170 0.260 0.004
6.8 2.5 1.7
3.5 1.6 0.9
5.5 0.5 0.8
Hydroxytyrosol 15.63 0.976 0.030
0.9 0.6 1.3
6.7 2.0 0.2
18.3 0.3 1.5
Oleuropein 55.66 3.472 0.054
0.6 0.7 1.2
5.0 2.4 0.5
4.0 0.1 0.4
Pinoresinol 8.330 0.520 0.008
0.6 3.0 1.0
4.0 1.8 0.6
2.4 0.1 1.2
Caffeic acid 5.560 0.347 0.005
23.7 2.6 1.8
8.1 8.8 1.5
21.0 3.8 2.0
Ferulic acid 6.940 0.434 0.007
8.1 3.3 1.9
12.4 2.2 1.3
3.6 0.1 1.0
Vanillic acid 10.00 0.625 0.009
1.4 0.1 0.6
0.6 2.0 0.1
1.6 0.1 1.2
p-Coumaric acid 5.560 0.347 0.005
9.9 1.5 1.7
0.3 2.1 1.2
2.0 0.3 0.2
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3.3. Linearity, lower limit of detection (LOD), and lower limit of quantification (LOQ) The linearity of the detector response for the eight phenolics was evaluated by triplicate injection of standard solutions corresponding to each point on the standard curve on 2 different days. The detector responses for all compounds were linear from concentrations in the low ng/mL range and up to 17–222 lg/mL and regression coefficients (R2) for all compounds were higher than 0.99 (Table 1). The LOQ and LOD of the phenolics ranged from 0.3 ng/mL to 15.3 ng/mL and 0.03 ng/mL to 1.7 ng/mL, respectively (Table 1) and were lower than those reported using GC/MS (García-Villalba et al., 2011), capillary electrophoresis (Ballus et al., 2011), UPLC– MS (Suárez et al., 2008) and capillary electrophoresis-electrospray ˇ alvo, Robledo, & Martínez, 2009) as ionization-MS (Nevado, Pen shown in the Supplementary Table 1. Overall, our data indicate that coulometric electrochemical detection is a highly sensitive method for the quantification of olive oil phenolics. 3.4. Precision, accuracy and recovery
with the mobile phase composition. The developed gradient elution allowed the separation of the eight olive oil phenolics in less than 16 min (Fig. 1). The total chromatographic run time, including equilibration, was 35 min, which is shorter than that of HPLC methods for olive oil phenolics reported in the literature (Bonoli et al., 2004; Franconi et al., 2006; Tovar et al., 2001). Although the separation of compounds with very different polarities often requires gradient elution, it is not frequently used with electrochemical detection, because it results in significant baseline shifts (Paterson, Cowie, & Jackson, 1996). Due to such fluctuations in the chromatogram that affect the sensitivity of the method, it was not possible to reduce the analysis time further by using faster gradients. Experiments were also performed with different types of conventional (Gemini and Luna C18) and fused-core (Kinetex PFP and Kinetex C18) columns from Phenomenex. The best chromatogram with good peak symmetry and reproducibility was obtained using a Kinetex C18 column. The impact of column temperature on analyte separation and peak symmetry was studied at 25, 30, 35, and 40 °C. Higher temperatures resulted in slightly better peak shapes and faster elution (data not shown). Therefore, 40 °C was selected as the column temperature for all further experiments. 3.2. Selectivity Following the extraction of the polyphenol-free olive oil, no interfering peaks were detected. Good separation of all eight phenolics was achieved within 16 min; for individual retention times (RT) see Fig. 1.
Precision, accuracy, and recovery were determined by adding low and high concentrations of the standard compounds to phenolic-free olive oil, which was then extracted and the phenolics quantified in five replicate analyses (Table 2). Intra- and inter-day repeatability studies were carried out by analyzing five spiked olive oil samples on 3 consecutive days. Intra-day precision was in the range of 2.6–9.0% and inter-day precision was 3.8–10.9% (Table 2). The precision was thus within the limits established by the FDA (620% for LOQ and 615% for other concentration levels). Satisfactory accuracy values were obtained ranging from 25.5% to 0.8%, although for some of the phenolics, the FDA limits were exceeded (620% at concentrations near the LOQ and 615% at higher concentrations). The recoveries of phenolic compounds from spiked olive oil samples ranged from 75% to 101% (Table 2). 3.5. Stability The stability of phenolic compounds prior to analysis is an important issue, which is too often neglected during method development. Therefore in the present study, we determined the stabilities of olive oil phenolics in stock solutions (low and high concentrations) and spiked olive oil samples (at low and high concentrations). Stock solution aliquots were stored at RT, 4 °C, or 20 °C for 24 h and the detector response after storage was compared with the initial response. Results are given as percent degradation (Table 3). The highest degradation in stock solutions was observed at low concentrations of caffeic acid at RT (24%) and 20 °C (21%) and at low concentrations of hydroxytyrosol after storage at 20 °C (18%), the degradation of all other phenolics was less than 12% (Table 3). For the short-term stability, five aliquots of spiked oil samples were prepared at low and high concentrations and analysed after storage at RT, 4 °C, 20 °C, 80 °C for 24 h and detector responses compared with initial responses. Under the conditions investigated, the stability of phenolics in stock solution was comparable with that in oils, with the highest degradation of 15% observed for high concentrations of vanillic acid at 80 °C. No clear differences in the stabilities of the phenolics in oil were observed between 24 h-storage at RT, 4 °C, or 20 °C (Table 4). For the long-term stability five aliquots of spiked oil samples were prepared at low and high concentrations and analysed after storage at 20 °C and 80 °C for 6 months. A significant degradation was observed for the low concentrations of pinoresinol (72%), hydroxytyrosol (64.4%), vanillic acid (51.7%) and high
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Table 4 Short-term, long-term, freeze-and-thaw, and post-preparative stabilities (expressed as percent degradation) of phenolics extracted from spiked (at low and high concentrations) olive oil. For short-term stability the samples were stored at RT, 4, 20 and 80 °C for 24 h (n = 5) and for long-term stability the samples were stored at 20 and 80 °C for 6 months. The samples were left in the autosampler at RT for up to 18 h for post-preparative stability. Short-term stability [Degradation %]
Long-term stability [Degradation %]
Nominal Concentration [mg/kg]
80 °C
20 °C
Tyrosol 1.75 35
+4 °C
3.2 11.5
3.7 10.2
4.1 9.1
Hydroxytyrosol 2.5 50
2.3 12.5
3.1 11.0
Oleuropein 0.6 12
3.3 11.7
Pinoresinol 4.75 95
RT
Freeze-and-thaw stability [Degradation %]
Post-preparative stability [Degradation %]
80 °C
20 °C
6.1 7.5
11.7 4.1
11.5 17.5
10.1 4.0
0.0 0.3
0.2 0.7
0.6 0.7
1.0 9.0
4.4 5.5
13.6 12.9
64.4 7.6
10.3 4.2
2.0 0.4
3.9 0.3
0.1 1.4
5.5 11.1
9.7 9.2
9.0 6.2
29.4 1.4
66.4 23.3
1.6 8.0
5.6 0.1
11.1 0.2
1.3 1.4
6.4 14.5
6.8 13.9
2.7 12.1
6.6 9.6
16.6 14.5
72.0 4.0
9.0 1.5
1.6 0.2
1.9 0.0
0.9 1.0
Caffeic acid 0.75 15
5.8 13.7
5.5 12.1
5.1 10.1
6.8 6.2
4.9 8.5
9.2 43.0
8.1 2.3
5.9 0.5
18.2 1.2
5.3 1.2
Ferulic acid 0.75 15
4.8 13.2
5.2 11.8
6.0 7.0
7.6 7.2
3.6 0.1
8.1 44.1
9.3 2.5
1.1 0.6
2.8 0.6
0.1 1.3
Vanillic acid 0.27 5.3
5.1 15.0
4.1 14.0
5.6 12.4
7.4 9.6
6.9 12.1
51.7 22.8
11.3 0.1
0.8 0.0
7.0 0.3
0.5 0.3
p-Coumaric acid 0.75 15
4.5 11.6
4.9 10.3
4.9 9.1
7.6 6.3
20.8 21.1
8.9 0.2
1.0 0.6
2.3 0.5
0.7 1.8
0.10 1.9
concentrations of ferulic acid (44.1%) and caffeic acid (43%) at 20 °C. It may be stated that for the long-term stability of olive oil phenolics, storage at 80 °C is better as less degradation of phenolics (0.1–29.4%) was observed (Table 4). The stabilities of phenolics in spiked oil samples were analysed in triplicate after three freeze-and-thaw-cycles. Percent degradation was higher at low concentrations and ranged from 0.1% to 11.3%. Thus, the studied olive oil phenolics remained relatively stable during three freeze-and-thaw cycles (Table 4). The stability of extracted samples during the time in the autosampler was investigated using three aliquots of spiked oil samples prepared at low and high concentrations. Extracted samples were injected three times at 6 h intervals from the same HPLC vial. The investigated phenolics were stable for 18 h in the autosampler (Table 4). The highest degradation was observed after 12 h for caffeic acid (18.2%) and oleuropein (11.2%) at low concentrations.
6h
12 h
18 h
3.6. Application of the method for the quantification of phenolics in olive oil samples The developed and validated method was used to analyse the content of tyrosol, hydroxytyrosol, oleuropein, pinoresinol, and caffeic, ferulic, vanillic, and p-coumaric acids in olive oils produced from olives of different varieties and harvest years and collected from six different countries (Table 5). A representative chromatogram of phenolics extracted from olive oil is shown in Fig. 2. The qualitative and quantitative composition of olive oil phenolics in these oils varies significantly. Tyrosol, hydroxytyrosol, and pinoresinol were the most abundant phenolic compounds in our oil samples and phenolic acids were present only in trace amounts (Table 5). These results are in accordance with those reported in the literature for the phenolic content of olive oils (Andjelkovic et al., 2008; Baccouri et al., 2007).
Table 5 Concentrations (mg/kg; mean ± SD) of phenolic compounds in extra virgin olive oil samples quantified by HPLC–ECD and separated with a fused-core column. Olive oil sample Country of origin Olive variety Harvest year
1 Italy Coratina 2008
2 Spain Picual 2008
3 France Brun 2008
4 Turkey Saurani 2008
5 Greece Kalamata 2007
6 USA Manzanillo & Mission 2007
Tyrosol Hydroxytyrosol Oleuropein Pinoresinol Caffeic acid Vanillic acid Ferulic acid p-Coumaric acid
20.8 ± 0.21 18.9 ± 0.14 1.33 ± 0.03 3.56 ± 0.07 0.56 ± 0.02 0.34 ± 0.001 0.09 ± 0.001 0.13 ± 0.02
48.0 ± 0.07 12.9 ± 0.00 0.69 ± 0.007 2.08 ± 0.00 0.43 ± 0.006 0.02 ± 0.0013 0.09 ± 0.0001 0.31 ± 0.0007
53.4 ± 0.49 11.3 ± 0.14 0.50 ± 0.05 2.37 ± 0.02 1.28 ± 0.73 0.46 ± 0.17 0.18 ± 0.07 1.09 ± 0.23
33.1 ± 0.21 14.6 ± 0.14 2.38 ± 0.04 2.37 ± 0.03 0.01 ± 0.0009 0.02 ± 0.002 0.03 ± 0.0001 0.09 ± 0.002
9.99 ± 0.16 2.89 ± 0.04 0.27 ± 0.009 1.69 ± 0.02 0.02 ± 0.001 0.07 ± 0.003 0.01 ± 0.0007 0.05 ± 0.003
28.3 ± 0.49 21.6 ± 0.49 0.20 ± 0.01 0.75 ± 0.01 0.11 ± 0.09 0.08 ± 0.005 0.10 ± 0.008 0.59 ± 0.006
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4. Conclusion In this study a HPLC method with coulometric electrochemical detection was developed for the rapid quantification of eight selected phenolic compounds in olive oil. The method provides good resolution of all analytes and, compared to most published methods, superior limits of quantification and detection, respectively. In fact, the LOD of all analytes were at least 5-fold lower than those reported for other HPLC detectors and were comparable with the most sensitive methods reported in the literature. The use of a fused-core column with 2.6 lm particles allowed the analysis of all eight phenolic compounds within 16 min and a total analysis time of only 35 min, which is a nearly 3-fold reduction in the analysis time over published HPLC methods, while operating at a back pressure that can be handled by most conventional HPLC systems. Thus, our method has the advantage of being compatible with standard HPLC pumps and the use of an electrochemical detector, which is less expensive in acquisition and maintenance than a mass detector, further improves its wide applicability and usefulness. The developed method was validated and successfully applied to olive oil samples. The validated method is thus highly sensitive, selective, and fast, and provides simultaneous qualitative and quantitative analysis of tyrosol, hydroxytyrosol, oleuropein, pinoresinol, and caffeic, ferulic, vanillic, and p-coumaric acid in oil samples. Our method may be an alternative to classical HPLCUV/DAD/MS methods when samples with low olive oil phenolic content are under investigation. It needs to be established whether our method can also be applied for the analysis of olive oil phenolics in biological samples such as blood and urine. Acknowledgements B.B. is supported by TUBITAK (The Scientific and Technological Research Council of Turkey). The authors thank Dr. Chi Vinh Duong (Phenomenex, Aschaffenburg, Germany) for his valuable advice and assistance, Dr. Nicholas Santiago (ESA Inc., USA) for technical support, and Dr. Esra Capanoglu Güven (Istanbul Technical University, Turkey) for her kind assistance. References Aaby, K., Skrede, G., & Wrolstad, R. E. (2005). Phenolic composition and antioxidant activities in flesh and achenes of strawberries (Fragaria ananassa). Journal of Agricultural Food Chemistry, 53, 4032–4040. Achilli, G., Cellerino, G. P., & Hamache, P. H. (1993). Identification and determination of phenolic constituents in natural beverages and plant extracts by means of a coulometric electrode array system. Journal of Chromatography, 632, 111–117. Andjelkovic, M., Van Camp, J., Pedra, M., Renders, K., Socaciu, C., & Verhé, R. (2008). Correlations of the phenolic compounds and the phenolic content in some Spanish and French olive oils. Journal of Agricultural Food Chemistry, 56, 5181–5187. Baccouri, O., Cerretani, L., Bendini, A., Caboni, M. F., Zarrouk, M., Pirrone, L., et al. (2007). Preliminary chemical characterization of Tunisian monovarietal virgin olive oils and comparison with Sicilian ones. European Journal of Lipid Science and Technology, 109, 1208–1217. Ballus, C. A., Meinhart, A. D., Bruns, R. E., & Godoy, H. T. (2011). Use of multivariate statistical techniques to optimize the simultaneous separation of 13 phenolic compounds from extra-virgin olive oil by capillary electrophoresis. Talanta, 83, 1181–1187. Bayram, B., Ozcelik, B., Grimm, S., Roeder, T., Schrader, C., Ernst, I. M. A., et al. (2012). A diet rich in olive oil phenolics reduces oxidative stress in the heart of SAMP8 mice by induction of Nrf2-dependent gene expression. Rejuvenation Research, 15, 71–81. Bonoli, M., Bendini, A., Cerretani, L., Lercker, G., & Gallina-Toschi, T. (2004). Qualitative and semiquantitative analysis of phenolic compounds in extra virgin olive oils as a function of the ripening degree of olive fruits by different analytical techniques. Journal of Agricultural Food Chemistry, 52, 7026–7032. Brenes, M., Garcia, A., Garcia, P., & Garrido, A. (2000). Rapid and complete extraction of phenols from olive oil and determination by means of a coulometric electrode array system. Journal of Agricultural Food Chemistry, 48, 5178–5183. Carrasco-Pancorbo, A., Cerretani, L., Bendini, A., Segura-Carretero, A., Gallina-Toschi, T., & Fernandez-Gutiérrez, A. (2005). Analytical determination of polyphenols in olive oils. Journal of Separation Science, 28, 837–858.
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Christophoridou, S., Dais, P., Tseng, L. H., & Spraul, M. (2005). Separation and identification of phenolic compounds in olive oil by coupling high-performance liquid chromatography with postcolumn solid-phase extraction to nuclear magnetic resonance spectroscopy (LC-SPE-NMR). Journal of Agricultural Food Chemistry, 53, 4667–4679. FDA (2001). Guidance for Industry: Bioanalytical Method Validation. US Department of Health and Human Services, FDA, Center for Drug Evaluation and Research, Rockville, USA. Available from:. Franconi, F., Coinu, R., Carta, S., Urgeghe, P. P., Ieri, F., Mulinacci, N., et al. (2006). Antioxidant effect of two virgin olive oils depends on the concentration and composition of minor polar compounds. Journal of Agricultural Food Chemistry, 54, 3121–3125. García-Villalba, R., Pacchiarotta, T., Carrasco-Pancorbo, A., Segura-Carretero, A., Fernández-Gutiérrez, A., Deelder, A. M., et al. (2011). Gas chromatographyatmospheric pressure chemical ionization-time of flight mass spectrometry for profiling of phenolic compounds in extra virgin olive oil. Journal of Chromatography A, 1218, 959–971. Gill, C. I. R., Boyd, A., McDermott, E., McCann, M., Servili, M., Selvaggini, R., et al. (2005). Potential anti-cancer effects of virgin olive oil phenols on colorectal carcinogenesis models in vitro. International Journal of Cancer, 117, 1–7. Mateos, R., Espartero, J. L., Trujillo, M., Rios, J. J., Leon-Camacho, M., Alcudia, F., et al. (2001). Determination of phenols, flavones and lignans in virgin olive oils by solid phase extraction and high performance liquid chromatography with diode array ultraviolet detection. Journal of Agricultural Food Chemistry, 49, 2185–2192. McCalley, D. V. A. (2010). Instrumental considerations for the effective operation of short, highly efficient fused-core columns. Investigation of performance at high flow rates and elevated temperatures. 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Journal of Pharmaceutical and Biomedical Analysis, 39, 431–437. Pirisi, F. M., Angioni, A., Cabras, P., Garau, V. L., di Teulada, M. T. S., dos Santos, M. K., et al. (1997). Phenolic compounds in virgin olive oils I. Low-wavelength quantitative determination of complex phenols by high-performance liquid chromatography under isocratic elution. Journal of Chromatography A, 768, 207–213. Rigacci, S., Guidotti, V., Bucciantina, M., Parri, M., Nediani, C., Cerbai, E., et al. (2010). Oleuropein aglycon prevents cytotoxic amyloid aggregation of human amylin. Journal of Nutritional Biochemistry, 21, 725–726. Schaffer, S., Podstawa, M., Visioli, F., Bogani, P., Muller, W. E., & Eckert, G. P. (2007). Hydroxytyrosol-rich olive mill wastewater extract protects brain cells in vitro and ex vivo. Journal of Agricultural Food Chemistry, 55, 5043– 5049. Servili, M., Selvaggini, R., Esposto, S., Taticchi, A., Montedoro, G., & Morozzi, G. (2004). 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Determination of phenolic compounds in virgin olive oil by reversed-phase HPLC with emphasis on UV detection. Food Chemistry, 44, 53–60. Turner, R., Etiene, N., Garcia-Alonso, M., de Pascual-Teresa, S., Minihane, A. M., Weinberg, P. D., et al. (2010). Antioxidant and anti-atherogenic activities of olive oil phenolics. International Journal of Vitaminology and Nutrition Research, 75, 61–70. Xu, X., Yu, L., & Chen, G. (2006). Determination of flavonoids in Portulaca oleracea L. by capillary electrophoresis with electrochemical detection. Journal of Pharmaceutical and Biomedical Analysis, 41, 493–499. Zhang, X., Cao, J., & Zhong, L. (2009). Hydroxytyrosol inhibits pro-inflammatory cytokines, iNOS, and COX-2 expression in human monocytic cells. NaunynSchmiedebergs Archives of Pharmacology, 379, 581–586.
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Analytical Methods
A validated method for the determination of selected phenolics in olive oil using high-performance liquid chromatography with coulometric electrochemical detection and a fused-core column Banu Bayram a,b, Beraat Ozcelik b, Gerhard Schultheiss c, Jan Frank d, Gerald Rimbach a,⇑ a
Institute of Human Nutrition and Food Science, Christian-Albrechts-University, Hermann-Rodewald-Strasse 6, 24098 Kiel, Germany Department of Food Engineering, Istanbul Technical University, 34469 Maslak, Istanbul, Turkey Christian-Albrechts-University, Olshausen Strasse 40, 24098 Kiel, Germany d Institute of Biological Chemistry and Nutrition, University of Hohenheim, Garbenstrasse 30, 70599 Stuttgart, Germany b c
a r t i c l e
i n f o
Article history: Received 16 August 2012 Received in revised form 23 October 2012 Accepted 17 November 2012 Available online 5 December 2012 Keywords: Fused core column Olive oil Phenolics Electrochemical detector HPLC
a b s t r a c t A liquid chromatographic method with a coulometric electrochemical detector (ECD) and a fused-core column was developed for the quantification of the olive oil phenolics tyrosol, hydroxytyrosol, oleuropein, pinoresinol, and caffeic, ferulic, vanillic, and p-coumaric acid. The method was validated according to guidelines of the U.S. Food and Drug Administration. The selectivity, linearity, lower limit of quantification (LOQ), lower limit of detection (LOD), precision, accuracy, recovery, as well as the stabilities of the phenolic standards and quality control samples were determined. The separation of the eight phenolic compounds was achieved within 16 min and the total analysis time (35 min) was ca. 3-fold shorter than that of conventional HPLC methods. The LOQ range was 0.3–15.3 ng/mL, which is at least 5-fold lower than those of other methods. Recovery was between 75% and 101%. Overall the method has the advantages of being sensitive, selective, fast and provides simultaneous qualitative and quantitative analysis of phenolics. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Olive oil, the primary source of fat in the Mediterranean diet, has been suggested to have potential antioxidant (Turner et al., 2010), antiinflammatory (Zhang, Cao, & Zhong, 2009), cardioprotective (Bayram et al., 2012), anticancer (Gill et al., 2005), antidiabetic (Rigacci et al., 2010) and neuroprotective activity (Schaffer et al., 2007). Olive oil is characterized by a high content of oleic acid and is an important source of hydrophilic phenolic compounds that contribute to stability, sensory, technological, and nutritional properties of olive oil (Servili et al., 2004). A wide range of phenolic compounds, belonging to many different classes including phenolic acids and alcohols, flavonoids, hydroxy-isochromans, secoiridoids, and lignans, have been identified in virgin olive oil (Carrasco-Pancorbo et al., 2005; Servili et al., 2004) with tyrosol and hydroxytyrosol, being the quantitatively major phenolic compounds. Phenolic acids, such as vanillic acid, caffeic acid, p-coumaric acid, and ferulic acid, constitute another important group of olive oil phenolics. The lignan pinoresinol is a newly recognized abundant compound in olive oil (Brenes, Garcia, Garcia, & Garrido, 2000). ⇑ Corresponding author. Tel.: +49 431 880 2583; fax: +49 431 880 2628. E-mail address: [email protected] (G. Rimbach). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.11.122
The identification and quantification of phenolic compounds in olive oil is of great interest due to their nutritional and technological importance. Various methods have been used for the characterization of phenolic compounds in olive oil, such as high-performance liquid chromatography (HPLC) coupled with ultraviolet detection (Tsimidou, Papadopoulos, & Boskou, 1992), diode array detection (Pirisi et al., 1997) coulometric electrochemical detection (Brenes et al., 2000), mass spectrometry (MS) (Suárez, Macià, Romero, & Motilva, 2008), gas chromatography coupled to MS (García-Villalba et al., 2011), capillary electrophoresis (CE) (Bonoli, Bendini, Cerretani, Lercker, & Gallina-Toschi, 2004), nuclear magnetic resonance spectroscopy (Christophoridou, Dais, Tseng, & Spraul, 2005) and infrared spectroscopy (Montedoro et al., 1993). Among these techniques, LC–MS is the predominent method used for the quantification of phenolics in olive oil. Coulometric electrochemical detector (ECD) provides high sensitivity and specificity for substances that are either oxidized or reduced at the applied potential. It provides an analytical tool for resolving and accurately detecting trace amounts of electroactive compounds, including phenolics, in a wide range of samples. Thus, due to its high selectivity, sensitivity, low detection limits and enhanced resolving power (Achilli, Cellerino, & Hamache, 1993; Svendsen, 1993) coulometric electrochemical detection has become increasingly popular for the analysis of plant bioactives
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in foods and beverages. Furthermore HPLC coupled to ECD has been applied in the analysis of phenolic compounds in fruits (Aaby, Skrede, & Wrolstad, 2005), vegetables (Xu, Yu, & Chen, 2006) and pharmaceutical products (Peng, Yuan, Liu, & Ye, 2005). In addition to sensitivity, run times are an important aspect of chromatographic methods. The total analysis times of published methods for olive oil phenolics are often longer than 40 min (Bonoli et al., 2004; Tovar, Motilva, & Romero, 2001). In the literature CE methods with run times under 15 min are available but CE has the disadvantage of a relatively low sensitivity (Ballus, Meinhart, Bruns, & Godoy, 2011; Bonoli et al., 2004). Recent developments in column technology, such as the newly developed stationary phases with fused-core particles, offer increased chromatographic efficiency and resolution, shorter analysis times, and increased sensitivity at lower operating pressures (McCalley, 2010). To the best of our knowledge there is currently no method available in the literature for the analysis of olive oil phenolics combining the sensitivity of HPLC–ECD with the superior separation and speed of a fused-core column. Therefore, we aimed to develop a rapid and sensitive fused-core column HPLC–ECD method for the analysis of selected phenolic compounds (oleuropein, tyrosol, hydroxytyrosol, pinoresinol, ferulic acid, caffeic acid, vanillic acid and p-coumaric acid) in olive oil and to validate it according to the FDA guidelines for bioanalytical method validation (FDA, 2001). 2. Materials and methods 2.1. Oil samples Six olive oils from six different countries and produced from different olive varieties were directly obtained from the producers and analysed for their content of phenolics. All oil samples were kept at 4 °C under a nitrogen atmosphere until analysis. Polyphenol-free olive oil was obtained from Sigma Aldrich (Steinheim, Germany). 2.2. Chemicals HPLC grade methanol was obtained from J.T. Baker (Deventer, The Netherlands) and acetonitrile from Sigma Aldrich (Steinheim, Germany). Standards of tyrosol (CAS No. 501-94-0), vanillic acid (CAS No. 121-34-6), caffeic acid (CAS No. 331-39-5), p-coumaric acid (CAS No. 501-98-4), and ferulic acid (CAS No. 1135-24-6) were purchased from Sigma Aldrich (Schnelldorf, Germany). Oleuropein (CAS No. 32619-42-4) and hydroxytyrosol (CAS No. 10597-60-1) were supplied by Extrasynthese (Genay Cedex, France) and pinoresinol by Separation Research (Turku, Finland). Reference standards of all analyzed compounds were HPLC grade with purities higher than 98% except for oleuropein, which was >90%. HPLC grade 60% perchloric acid was obtained from Fisher Scientific (Leicestershire, UK).
to 1.5 g of a phenolic-free olive oil to yield oils with low and high concentrations of phenolics. The highest amounts of phenolic compounds added to the olive oil were set based on literature and were (all in mg/kg): oleuropein, 12; tyrosol, 35; hydroxytyrosol, 50; pinoresinol, 90; ferulic acid, 15; caffeic acid, 15; vanillic acid, 5.3; and p-coumaric acid, 15. The phenolic mixture was diluted 20-fold to obtain the control oils with low concentrations of phenolics. 2.5. Extraction of phenolic compounds from olive oil The phenolic fraction of the olive oils was obtained by solidphase extraction (SPE) using diol-bonded SPE cartridges (Varian, Bond Elut Diol 500 mg/3 mL) according to a protocol modified from Mateos et al. (2001). Briefly, an SPE cartridge was placed in a vacuum elution apparatus (Phenomenex, Aschaffenburg, Germany) and conditioned by the consecutive passing of 6 mL of methanol and 6 mL of hexane. A 1.5 g oil sample was mixed with 1.5 mL of hexane, and applied to the column. The solvent was pulled through, leaving the sample on the solid phase. The sample container was washed with two 3 mL portions of hexane, and the sample container was washed again with 4 mL of hexane/ethyl acetate (90:10, v/v). The column was eluted with 10 mL of methanol and the solvent evaporated in a centrifugal evaporator (Jouan RC, Saint Herblain, France) at 35 °C. The phenolic residue was dissolved in 1 mL of methanol. Extracts were filtered through a 0.2 lm PTFE membrane filter (Pall Corporation, Michigan, USA) and directly injected into the HPLC system or stored at 20 °C. 2.6. HPLC analysis of olive oil phenolics HPLC analyses of olive oil phenolics were carried out on a Jasco system (Jasco GmbH Deutschland, Gross-Umstadt, Germany) consisting of a pump (PU-2085) and autosampler (XLC-3059AS), while detection was carried out on a 4-channel ESA 5600A CoulArray detector with integrated column oven (ESA Inc., Chelmsford, MA, USA). For analyte detection, increasing potentials of +250, +400, +500 and +750 mV were applied. Separation of phenolics was achieved on a Kinetex C18 column (100 4.6 mm, 2.6 lm, Phenomenex, Aschaffenburg, Germany) using gradient elution with water (pH 3.1, solvent A), acetonitrile (solvent B), and methanol (solvent C) each containing 60 mmol/L LiClO4. The solvent gradient changed according to the following conditions: from 96% A – 3% B – 1% C to 85% A – 10% B – 5% C in 4 min; 82.5% A – 11.5% B – 6% C in 2 min; 80% A – 12% B – 8% C in 2 min; 65% A – 24% B – 11% C in 7 min; 85% B – 15% C in 6 min; 85% B – 15% C for 5 min; 96% A – 3% B – 1% C to 85% A in 4 min; the final conditions were maintained for 5 min. The pH of the aqueous eluent was adjusted to 3.1 with perchloric acid, and the eluent was vacuum-filtered through a 0.2 lm hydrophilic polypropylene membrane filter (Pall Corporation, Michigan, USA). The column temperature was maintained at 40 °C, the flow rate set at 1.25 mL/min, and the injection volume was 10 lL. The analytes were quantified against authentic compounds as external standards using the sum of peak heights. 2.7. Method validation
2.3. Preparation of stock and working standard solutions Stock solutions of olive oil phenolic standards were prepared in methanol at a concentration of 2 mg/mL and stored at 20 °C in amber-coloured bottles. The working standard solutions were prepared by diluting stock solution with H2O/methanol (50:50, v/v).
The analytical method for the determination of olive oil phenolics was validated according to the requirements of FDA guidelines for bioanalytical method validation (FDA, 2001). The method was validated in terms of linearity, selectivity, LOQ, LOD, accuracy, precision, recovery, stock solution stability, short-term stability, freeze-and-thaw stability, and bench-top stability.
2.4. Preparation of quality control samples The quality control samples were prepared by diluting the stock solutions with methanol and adding 75 lL of the phenolic mixture
2.7.1. Selectivity For evaluation of selectivity, olive oil free of phenolic compounds (Sigma Aldrich, Steinheim, Germany) was used as a blank
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Table 1 Linear ranges, regression coefficients, slope, intercept and lower limits of quantification (LOQ) and detection (LOD) of the phenolic compounds. The linear range of the detector response was assessed by serial dilution of a mixture of the studied compounds dissolved in H2O:MeOH (50:50, v/v). Calibrations curves were obtained by plotting the sum of peak heights against the analyte concentrations (n = 3).
Fig. 1. Representative chromatogram of olive oil phenolics injected with a standard solution or extracted from spiked olive oil and resolved by gradient elution on a Kinetex™ C18 fused-core column and detected by 4-channel coulometric electrochemical detection. The retention times of the analytes (in minutes) are given in brackets: 1, hydroxytyrosol (2.7); 2, tyrosol (4.1); 3, caffeic acid (5.2); 4, vanillic acid (5.4); 5, ferulic acid (7.4); 6, p-coumaric acid (8.4); 7, oleuropein (13.2); and 8, pinoresinol (15.3).
Compound
Linear range (ng/ mL – lg/mL)
Regression coefficient
LOD (ng/mL)
LOQ (ng/mL)
Tyrosol Hydroxytyrosol Pinoresinol Oleuropein Caffeic acid Ferulic acid Vanillic acid p-Coumaric acid
0.3–16.7 15.3–62.5 2.0–33.3 3.4–222.0 5.4–22.2 3.4–27.8 0.6–40.0 1.4–22.2
0.9993 0.9998 0.9991 0.9998 0.9999 0.9996 0.9999 0.9969
0.07 0.06 0.50 0.11 0.30 1.70 0.15 0.03
0.30 15.3 2.00 3.40 5.40 3.40 0.60 1.40
2.7.3. Precision and accuracy The precision and accuracy of the method were determined on 3 consecutive days by five replicate analyses of quality control samples with known amounts of olive oil phenolics at two concentrations (low and high). The olive oil was extracted and the calculated analyte concentrations of spiked oil samples were compared with the known concentrations within the same day (intra-day) and from day-to-day (inter-day). The precision is given as the intra- and inter-day coefficients of variation (% CV). The accuracy was expressed as the absolute error percentage and calculated as follows: Accuracy (%) = [Mean of measured concentration Known added concentration]/Known added concentration 100. 2.7.4. Recovery The recoveries of the phenolics were calculated by analyzing five replicate spiked olive oil samples for each concentration (low and high) and comparing the detector responses with those of calibration standards with identical concentrations. The recovery (in percent) was calculated as follows: Recovery (%) = [Measured concentration/Expected concentration] 100.
Fig. 2. Representative chromatogram of olive oil phenolics extracted from a representative olive oil sample and resolved by gradient elution on a Kinetex™ C18 fused-core column and detected by 4-channel coulometric electrochemical detection. The retention times of the analytes (in minutes) are given in brackets: 1, hydroxytyrosol (2.7); 2, tyrosol (4.1); 3, caffeic acid (5.2); 4, vanillic acid (5.4); 5, ferulic acid (7.4); 6, p-coumaric acid (8.4); 7, oleuropein (13.2); and 8, pinoresinol (15.3).
and extracted and analyzed to check for the presence of interfering peaks.
2.7.2. Linearity, LOD and LOQ The linear range of the detector response was determined by serial dilution of a mixture of the studied compounds. Calibration curves were obtained by plotting the sum of peak heights of the respective analyte versus its concentration for eight different concentrations. Standard solutions corresponding to each point in the calibration curve were injected in triplicate and regression parameters were calculated. The LOQ was defined as the lowest concentration that could be determined with a deviation from the actual concentration and a coefficient of variation of precision of less than 20%, respectively. The LOD was the lowest concentration of the analyte of interest that could be reliably discerned from the baseline (signal-to-noise ratio 3).
2.7.5. Stock solution stability The stability of stock solutions was determined at two concentration levels (low and high). The aliquots of stock solutions were stored at room temperature (RT), 4 °C and 20 °C for 24 h. The initial detector response of solutions was compared to that after storage and results were expressed as percent degredation. 2.7.6. Short- and long-term stability Five aliquots of spiked oil samples were prepared at two concentrations (low and high). The aliquots were analysed and the remaining samples stored at RT, 4 °C, 20 °C, 80 °C for 24 h. For the long term stability the remaining samples were stored at 20 °C and 80 °C for 6 months. The initial values were compared to those obtained after storage and results were expressed as percent degredation. 2.7.7. Freeze-and-thaw stability Three aliquots of spiked oil samples were prepared at two concentrations (low and high). The aliquots were analysed and the remaining samples frozen at 80 °C for 24 h, thawed at room temperature and frozen again for 12-24 h at 80 °C. The freezeand-thaw-cycle was repeated two additional times and the stability determined by comparing initial values to those obtained after repeated freezing–thawing and results were expressed as percent degredation.
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Table 2 Intra-day and inter-day precision, accuracy, and recovery of phenolic compounds added at low or high concentrations to phenolic-free olive oil and determined in five replicate analyses (n = 5).
Tyrosol Intra-day Inter-day Hydroxytyrosol Intra-day Inter-day Oleuropein Intra-day Inter-day Pinoresinol Intra-day Inter-day Caffeic acid Intra-day Inter-day Ferulic acid Intra-day Inter-day Vanillic acid Intra-day Inter-day p-Coumaric acid Intra-day Inter-day
Nominal concentration (mg/kg)
Calculated concentration (mg/kg)
1.75 35.00 1.75 35.00
1.64 29.90 1.68 30.30
93.8 85.5 95.8 86.5
6.33 14.65 4.26 13.56
2.56 6.93 3.77 6.01
2.50 50.00 2.50 50.00
1.92 38.63 1.99 39.90
76.6 77.3 79.8 79.8
23.44 22.73 20.23 20.24
3.64 6.69 3.78 6.21
0.60 12.00 0.60 12.00
0.61 10.45 0.56 9.79
100.9 87.1 94.1 81.6
0.86 12.86 5.89 18.41
3.27 3.24 5.75 10.88
4.75 95.00 4.75 95.00
4.12 84.52 4.30 89.60
86.8 89.0 90.6 94.3
13.21 11.03 9.40 5.68
3.57 7.43 4.24 6.51
0.75 15.00 0.75 15.00
0.56 13.39 0.57 13.23
74.5 83.9 76.4 88.2
25.54 16.13 23.52 11.79
9.00 5.44 7.68 5.79
0.75 15.00 0.75 15.00
0.67 13.94 0.67 13.76
89.9 92.9 89.8 91.8
10.11 7.06 10.23 8.25
3.85 4.74 4.46 5.55
0.27 5.30 0.27 5.30
0.26 4.46 0.26 4.46
96.0 84.2 95.5 84.1
4.03 15.81 4.50 15.94
6.24 4.53 5.49 5.78
0.75 15.00 0.75 15.00
0.72 14.88 0.69 10.80
96.3 99.2 91.6 95.0
3.68 0.76 8.42 5.04
3.63 4.56 4.44 5.37
2.7.8. Bench-top stability Three aliquots of spiked oil samples were prepared at two concentrations (low and high) and the stability of samples in the autosampler was determined by injection of spiked oil samples at 6 h intervals (at 6, 12, and 18 h). The samples were injected from the same HPLC vial and the screw-caps were replaced after each injection to minimize solvent loss. The results were expressed as percent degradation.
3. Results and discussion 3.1. Optimization of the chromatographic method In order to optimize the analysis time, selectivity, and sensitivity of our method for all analytes, chromatographic conditions including mobile phase composition and gradient, flow rate, column temperature, column type, salt type and concentration, as well as working potentials applied on the CoulArray detector were varied. To achieve optimum electron transfer in coulometric electrochemical detection, a mobile phase with an electrolyte concentration between 50 and 100 mmol/L is recommended. Among the salts and buffer systems tested, problems with solubility and pH
Recovery [%]
Accuracy [%]
Precision [CV%]
occurred with sodium acetate/acetic acid, sodium dihydrogen phosphate/phosphoric acid, disodium hydrogen phosphate/phosphoric acid. Lithium perchlorate (LiCIO4), added at a concentration of 60 mmol/L to each eluent, and adjustment of the pH to 3.1 with perchloric acid resulted in better peak shapes. High salt concentrations may be problematic for the HPLC system and salt crystals were frequently observed in our system, which therefore had to be regularly washed with different solvents. This may be considered as a drawback of the developed method, albeit one that will be difficult to circumvent in electrochemical detection. Furthermore, different potentials and potential intervals were studied and it was observed that limiting the number of channels from originally eight to four improved the sensitivity of our method (data not shown). Therefore, only four channels were used for all subsequent experiments. Trials were carried out with different volumes of acetonitrile and methanol in the mobile phase to improve the resolution of the analytes. The use of methanol instead of acetonitrile as organic solvent resulted in loss of resolution for oleuropein and pinoresinol. Isocratic elution did not resolve all eight studied phenolics. The best peak shape and optimum resolution were obtained with the given mobile phase composition, which uses methanol as organic modifier in low percentages together with acetonitrile. The resolution of caffeic and vanillic acid was particularly improved
B. Bayram et al. / Food Chemistry 138 (2013) 1663–1669 Table 3 Stock solution stability of the phenolic standards at low or high concentrations after 24 h storage at RT, 4 °C, or 20 °C (n = 5). Stock solution stability [Degradation %] Concentration (lg/mL)
RT
4 °C
20 °C
Tyrosol 4.170 0.260 0.004
6.8 2.5 1.7
3.5 1.6 0.9
5.5 0.5 0.8
Hydroxytyrosol 15.63 0.976 0.030
0.9 0.6 1.3
6.7 2.0 0.2
18.3 0.3 1.5
Oleuropein 55.66 3.472 0.054
0.6 0.7 1.2
5.0 2.4 0.5
4.0 0.1 0.4
Pinoresinol 8.330 0.520 0.008
0.6 3.0 1.0
4.0 1.8 0.6
2.4 0.1 1.2
Caffeic acid 5.560 0.347 0.005
23.7 2.6 1.8
8.1 8.8 1.5
21.0 3.8 2.0
Ferulic acid 6.940 0.434 0.007
8.1 3.3 1.9
12.4 2.2 1.3
3.6 0.1 1.0
Vanillic acid 10.00 0.625 0.009
1.4 0.1 0.6
0.6 2.0 0.1
1.6 0.1 1.2
p-Coumaric acid 5.560 0.347 0.005
9.9 1.5 1.7
0.3 2.1 1.2
2.0 0.3 0.2
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3.3. Linearity, lower limit of detection (LOD), and lower limit of quantification (LOQ) The linearity of the detector response for the eight phenolics was evaluated by triplicate injection of standard solutions corresponding to each point on the standard curve on 2 different days. The detector responses for all compounds were linear from concentrations in the low ng/mL range and up to 17–222 lg/mL and regression coefficients (R2) for all compounds were higher than 0.99 (Table 1). The LOQ and LOD of the phenolics ranged from 0.3 ng/mL to 15.3 ng/mL and 0.03 ng/mL to 1.7 ng/mL, respectively (Table 1) and were lower than those reported using GC/MS (García-Villalba et al., 2011), capillary electrophoresis (Ballus et al., 2011), UPLC– MS (Suárez et al., 2008) and capillary electrophoresis-electrospray ˇ alvo, Robledo, & Martínez, 2009) as ionization-MS (Nevado, Pen shown in the Supplementary Table 1. Overall, our data indicate that coulometric electrochemical detection is a highly sensitive method for the quantification of olive oil phenolics. 3.4. Precision, accuracy and recovery
with the mobile phase composition. The developed gradient elution allowed the separation of the eight olive oil phenolics in less than 16 min (Fig. 1). The total chromatographic run time, including equilibration, was 35 min, which is shorter than that of HPLC methods for olive oil phenolics reported in the literature (Bonoli et al., 2004; Franconi et al., 2006; Tovar et al., 2001). Although the separation of compounds with very different polarities often requires gradient elution, it is not frequently used with electrochemical detection, because it results in significant baseline shifts (Paterson, Cowie, & Jackson, 1996). Due to such fluctuations in the chromatogram that affect the sensitivity of the method, it was not possible to reduce the analysis time further by using faster gradients. Experiments were also performed with different types of conventional (Gemini and Luna C18) and fused-core (Kinetex PFP and Kinetex C18) columns from Phenomenex. The best chromatogram with good peak symmetry and reproducibility was obtained using a Kinetex C18 column. The impact of column temperature on analyte separation and peak symmetry was studied at 25, 30, 35, and 40 °C. Higher temperatures resulted in slightly better peak shapes and faster elution (data not shown). Therefore, 40 °C was selected as the column temperature for all further experiments. 3.2. Selectivity Following the extraction of the polyphenol-free olive oil, no interfering peaks were detected. Good separation of all eight phenolics was achieved within 16 min; for individual retention times (RT) see Fig. 1.
Precision, accuracy, and recovery were determined by adding low and high concentrations of the standard compounds to phenolic-free olive oil, which was then extracted and the phenolics quantified in five replicate analyses (Table 2). Intra- and inter-day repeatability studies were carried out by analyzing five spiked olive oil samples on 3 consecutive days. Intra-day precision was in the range of 2.6–9.0% and inter-day precision was 3.8–10.9% (Table 2). The precision was thus within the limits established by the FDA (620% for LOQ and 615% for other concentration levels). Satisfactory accuracy values were obtained ranging from 25.5% to 0.8%, although for some of the phenolics, the FDA limits were exceeded (620% at concentrations near the LOQ and 615% at higher concentrations). The recoveries of phenolic compounds from spiked olive oil samples ranged from 75% to 101% (Table 2). 3.5. Stability The stability of phenolic compounds prior to analysis is an important issue, which is too often neglected during method development. Therefore in the present study, we determined the stabilities of olive oil phenolics in stock solutions (low and high concentrations) and spiked olive oil samples (at low and high concentrations). Stock solution aliquots were stored at RT, 4 °C, or 20 °C for 24 h and the detector response after storage was compared with the initial response. Results are given as percent degradation (Table 3). The highest degradation in stock solutions was observed at low concentrations of caffeic acid at RT (24%) and 20 °C (21%) and at low concentrations of hydroxytyrosol after storage at 20 °C (18%), the degradation of all other phenolics was less than 12% (Table 3). For the short-term stability, five aliquots of spiked oil samples were prepared at low and high concentrations and analysed after storage at RT, 4 °C, 20 °C, 80 °C for 24 h and detector responses compared with initial responses. Under the conditions investigated, the stability of phenolics in stock solution was comparable with that in oils, with the highest degradation of 15% observed for high concentrations of vanillic acid at 80 °C. No clear differences in the stabilities of the phenolics in oil were observed between 24 h-storage at RT, 4 °C, or 20 °C (Table 4). For the long-term stability five aliquots of spiked oil samples were prepared at low and high concentrations and analysed after storage at 20 °C and 80 °C for 6 months. A significant degradation was observed for the low concentrations of pinoresinol (72%), hydroxytyrosol (64.4%), vanillic acid (51.7%) and high
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Table 4 Short-term, long-term, freeze-and-thaw, and post-preparative stabilities (expressed as percent degradation) of phenolics extracted from spiked (at low and high concentrations) olive oil. For short-term stability the samples were stored at RT, 4, 20 and 80 °C for 24 h (n = 5) and for long-term stability the samples were stored at 20 and 80 °C for 6 months. The samples were left in the autosampler at RT for up to 18 h for post-preparative stability. Short-term stability [Degradation %]
Long-term stability [Degradation %]
Nominal Concentration [mg/kg]
80 °C
20 °C
Tyrosol 1.75 35
+4 °C
3.2 11.5
3.7 10.2
4.1 9.1
Hydroxytyrosol 2.5 50
2.3 12.5
3.1 11.0
Oleuropein 0.6 12
3.3 11.7
Pinoresinol 4.75 95
RT
Freeze-and-thaw stability [Degradation %]
Post-preparative stability [Degradation %]
80 °C
20 °C
6.1 7.5
11.7 4.1
11.5 17.5
10.1 4.0
0.0 0.3
0.2 0.7
0.6 0.7
1.0 9.0
4.4 5.5
13.6 12.9
64.4 7.6
10.3 4.2
2.0 0.4
3.9 0.3
0.1 1.4
5.5 11.1
9.7 9.2
9.0 6.2
29.4 1.4
66.4 23.3
1.6 8.0
5.6 0.1
11.1 0.2
1.3 1.4
6.4 14.5
6.8 13.9
2.7 12.1
6.6 9.6
16.6 14.5
72.0 4.0
9.0 1.5
1.6 0.2
1.9 0.0
0.9 1.0
Caffeic acid 0.75 15
5.8 13.7
5.5 12.1
5.1 10.1
6.8 6.2
4.9 8.5
9.2 43.0
8.1 2.3
5.9 0.5
18.2 1.2
5.3 1.2
Ferulic acid 0.75 15
4.8 13.2
5.2 11.8
6.0 7.0
7.6 7.2
3.6 0.1
8.1 44.1
9.3 2.5
1.1 0.6
2.8 0.6
0.1 1.3
Vanillic acid 0.27 5.3
5.1 15.0
4.1 14.0
5.6 12.4
7.4 9.6
6.9 12.1
51.7 22.8
11.3 0.1
0.8 0.0
7.0 0.3
0.5 0.3
p-Coumaric acid 0.75 15
4.5 11.6
4.9 10.3
4.9 9.1
7.6 6.3
20.8 21.1
8.9 0.2
1.0 0.6
2.3 0.5
0.7 1.8
0.10 1.9
concentrations of ferulic acid (44.1%) and caffeic acid (43%) at 20 °C. It may be stated that for the long-term stability of olive oil phenolics, storage at 80 °C is better as less degradation of phenolics (0.1–29.4%) was observed (Table 4). The stabilities of phenolics in spiked oil samples were analysed in triplicate after three freeze-and-thaw-cycles. Percent degradation was higher at low concentrations and ranged from 0.1% to 11.3%. Thus, the studied olive oil phenolics remained relatively stable during three freeze-and-thaw cycles (Table 4). The stability of extracted samples during the time in the autosampler was investigated using three aliquots of spiked oil samples prepared at low and high concentrations. Extracted samples were injected three times at 6 h intervals from the same HPLC vial. The investigated phenolics were stable for 18 h in the autosampler (Table 4). The highest degradation was observed after 12 h for caffeic acid (18.2%) and oleuropein (11.2%) at low concentrations.
6h
12 h
18 h
3.6. Application of the method for the quantification of phenolics in olive oil samples The developed and validated method was used to analyse the content of tyrosol, hydroxytyrosol, oleuropein, pinoresinol, and caffeic, ferulic, vanillic, and p-coumaric acids in olive oils produced from olives of different varieties and harvest years and collected from six different countries (Table 5). A representative chromatogram of phenolics extracted from olive oil is shown in Fig. 2. The qualitative and quantitative composition of olive oil phenolics in these oils varies significantly. Tyrosol, hydroxytyrosol, and pinoresinol were the most abundant phenolic compounds in our oil samples and phenolic acids were present only in trace amounts (Table 5). These results are in accordance with those reported in the literature for the phenolic content of olive oils (Andjelkovic et al., 2008; Baccouri et al., 2007).
Table 5 Concentrations (mg/kg; mean ± SD) of phenolic compounds in extra virgin olive oil samples quantified by HPLC–ECD and separated with a fused-core column. Olive oil sample Country of origin Olive variety Harvest year
1 Italy Coratina 2008
2 Spain Picual 2008
3 France Brun 2008
4 Turkey Saurani 2008
5 Greece Kalamata 2007
6 USA Manzanillo & Mission 2007
Tyrosol Hydroxytyrosol Oleuropein Pinoresinol Caffeic acid Vanillic acid Ferulic acid p-Coumaric acid
20.8 ± 0.21 18.9 ± 0.14 1.33 ± 0.03 3.56 ± 0.07 0.56 ± 0.02 0.34 ± 0.001 0.09 ± 0.001 0.13 ± 0.02
48.0 ± 0.07 12.9 ± 0.00 0.69 ± 0.007 2.08 ± 0.00 0.43 ± 0.006 0.02 ± 0.0013 0.09 ± 0.0001 0.31 ± 0.0007
53.4 ± 0.49 11.3 ± 0.14 0.50 ± 0.05 2.37 ± 0.02 1.28 ± 0.73 0.46 ± 0.17 0.18 ± 0.07 1.09 ± 0.23
33.1 ± 0.21 14.6 ± 0.14 2.38 ± 0.04 2.37 ± 0.03 0.01 ± 0.0009 0.02 ± 0.002 0.03 ± 0.0001 0.09 ± 0.002
9.99 ± 0.16 2.89 ± 0.04 0.27 ± 0.009 1.69 ± 0.02 0.02 ± 0.001 0.07 ± 0.003 0.01 ± 0.0007 0.05 ± 0.003
28.3 ± 0.49 21.6 ± 0.49 0.20 ± 0.01 0.75 ± 0.01 0.11 ± 0.09 0.08 ± 0.005 0.10 ± 0.008 0.59 ± 0.006
B. Bayram et al. / Food Chemistry 138 (2013) 1663–1669
4. Conclusion In this study a HPLC method with coulometric electrochemical detection was developed for the rapid quantification of eight selected phenolic compounds in olive oil. The method provides good resolution of all analytes and, compared to most published methods, superior limits of quantification and detection, respectively. In fact, the LOD of all analytes were at least 5-fold lower than those reported for other HPLC detectors and were comparable with the most sensitive methods reported in the literature. The use of a fused-core column with 2.6 lm particles allowed the analysis of all eight phenolic compounds within 16 min and a total analysis time of only 35 min, which is a nearly 3-fold reduction in the analysis time over published HPLC methods, while operating at a back pressure that can be handled by most conventional HPLC systems. Thus, our method has the advantage of being compatible with standard HPLC pumps and the use of an electrochemical detector, which is less expensive in acquisition and maintenance than a mass detector, further improves its wide applicability and usefulness. The developed method was validated and successfully applied to olive oil samples. The validated method is thus highly sensitive, selective, and fast, and provides simultaneous qualitative and quantitative analysis of tyrosol, hydroxytyrosol, oleuropein, pinoresinol, and caffeic, ferulic, vanillic, and p-coumaric acid in oil samples. Our method may be an alternative to classical HPLCUV/DAD/MS methods when samples with low olive oil phenolic content are under investigation. It needs to be established whether our method can also be applied for the analysis of olive oil phenolics in biological samples such as blood and urine. Acknowledgements B.B. is supported by TUBITAK (The Scientific and Technological Research Council of Turkey). The authors thank Dr. Chi Vinh Duong (Phenomenex, Aschaffenburg, Germany) for his valuable advice and assistance, Dr. Nicholas Santiago (ESA Inc., USA) for technical support, and Dr. Esra Capanoglu Güven (Istanbul Technical University, Turkey) for her kind assistance. References Aaby, K., Skrede, G., & Wrolstad, R. E. (2005). Phenolic composition and antioxidant activities in flesh and achenes of strawberries (Fragaria ananassa). Journal of Agricultural Food Chemistry, 53, 4032–4040. Achilli, G., Cellerino, G. P., & Hamache, P. H. (1993). Identification and determination of phenolic constituents in natural beverages and plant extracts by means of a coulometric electrode array system. Journal of Chromatography, 632, 111–117. Andjelkovic, M., Van Camp, J., Pedra, M., Renders, K., Socaciu, C., & Verhé, R. (2008). Correlations of the phenolic compounds and the phenolic content in some Spanish and French olive oils. Journal of Agricultural Food Chemistry, 56, 5181–5187. Baccouri, O., Cerretani, L., Bendini, A., Caboni, M. F., Zarrouk, M., Pirrone, L., et al. (2007). Preliminary chemical characterization of Tunisian monovarietal virgin olive oils and comparison with Sicilian ones. European Journal of Lipid Science and Technology, 109, 1208–1217. Ballus, C. A., Meinhart, A. D., Bruns, R. E., & Godoy, H. T. (2011). Use of multivariate statistical techniques to optimize the simultaneous separation of 13 phenolic compounds from extra-virgin olive oil by capillary electrophoresis. Talanta, 83, 1181–1187. Bayram, B., Ozcelik, B., Grimm, S., Roeder, T., Schrader, C., Ernst, I. M. A., et al. (2012). A diet rich in olive oil phenolics reduces oxidative stress in the heart of SAMP8 mice by induction of Nrf2-dependent gene expression. Rejuvenation Research, 15, 71–81. Bonoli, M., Bendini, A., Cerretani, L., Lercker, G., & Gallina-Toschi, T. (2004). Qualitative and semiquantitative analysis of phenolic compounds in extra virgin olive oils as a function of the ripening degree of olive fruits by different analytical techniques. Journal of Agricultural Food Chemistry, 52, 7026–7032. Brenes, M., Garcia, A., Garcia, P., & Garrido, A. (2000). Rapid and complete extraction of phenols from olive oil and determination by means of a coulometric electrode array system. Journal of Agricultural Food Chemistry, 48, 5178–5183. Carrasco-Pancorbo, A., Cerretani, L., Bendini, A., Segura-Carretero, A., Gallina-Toschi, T., & Fernandez-Gutiérrez, A. (2005). Analytical determination of polyphenols in olive oils. Journal of Separation Science, 28, 837–858.
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