Microdialysis as a New Technique for Extracting Phenolic Compounds

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Microdialysis as a New Technique for Extracting Phenolic Compounds from Extra Virgin Olive Oil Gianfranco Bazzu,†,‡ Maria Giovanna Molinu,†,§ Antonio Dore,*,§ and Pier Andrea Serra*,‡,§ ‡

Department of Clinical and Experimental Medicine, Section of Pharmacology, University of Sassari, Viale San Pietro 43/b, 07100 Sassari, Italy § Institute of Sciences of Food Production (ISPA), National Research Council (CNR), Traversa La Crucca 3, Regione Baldinca, 07040 Li Punti, Sassari, Italy ABSTRACT: The amount and composition of the phenolic components play a major role in determining the quality of olive oil. The traditional liquid−liquid extraction (LLE) method requires a time-consuming sample preparation to obtain the “phenolic profile” of extra virgin olive oil (EVOO). This study aimed to develop a microdialysis extraction (MDE) as an alternative to the LLE method to evaluate the phenolic components of EVOO. To this purpose, a microdialysis device and dialysis procedure were developed. “Dynamic-oil” microdialysis was performed using an extracting solution (80:20 methanol/water) flow rate of 2 μL min−1 and a constant EVOO stream of 4 μL min−1. The results indicated a strong positive correlation between MDE and the LLE method, providing a very similar phenolic profile obtained with traditional LLE. In conclusion, the MDE approach, easier and quicker in comparison to LLE, provided a reliable procedure to determine the phenolic components used as a marker of the quality and traceability of EVOO. KEYWORDS: extra virgin olive oil, liquid−liquid extraction, microdialysis extraction, polyphenol content, HPLC

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INTRODUCTION In the last few decades, the beneficial effect for human health of the consumption of extra virgin olive oil (EVOO) has been thoroughly studied.1−3 The preventive and protective action of EVOO is attributed to its high level of monounsaturated fatty acids and the presence in the minor fraction of “phenolic compounds”. These include molecules from different chemical classes, phenolic acids, flavones, lignans, phenyl ethyl alcohols, and secoiridoids, contributing to the nutraceutical properties of the oil as well as the flavor, taste, and aroma. Moreover, because of their strong antioxidant properties, they affect the EVOO storability, preventing the processes of oxidative degradation.4 For these reasons, the phenolic components play a major role in determining the quality and, as a consequence, the market price of olive oil. The amount and composition of the phenolic fraction depends upon several factors: cultivar, degree of maturation of the drupes, possible infestations, pedoclimatic conditions, and production methods.5 As a result, the phenolic components are suitable to be used as a marker of the process and traceability and to prevent commercial fraud. Many efforts are currently being addressed in the development of analytical methods for the qualitative and quantitative characterization of phenols in olive oil. Antioxidant capacity and Folin−Ciocalteau colorimetric assay are widely used to evaluate the total phenolic content but do not give any qualitative information on single compounds. On the contrary, instrumental methods based on gas chromatography or high-performance liquid chromatography (HPLC) are sensitive and specific and, when coupled with a mass detector, allow for identification of components unknown or not commercially available as a standard.6 Nowadays, they are the most important analytical methods for the “phenolic profile” determination in olive oil, although they need long and tedious sample preparation. Extraction and © XXXX American Chemical Society

cleanup of phenols from olive oil is usually carried out by partitioning the analytes between two phases: both liquid in liquid−liquid extraction (LLE) and liquid and solid for solidphase extraction (SPE).7,8 The main drawbacks of LLE and SPE are the use of a large or a relatively large amount of solvents and the need of well-skilled personnel. Moreover, they are difficult to automate and/or miniaturized and are timeconsuming. Typically, LLE includes EVOO solubilization in hexane, extraction with methanol/water solution, concentration of the extract, and redissolution in the mobile phase for HPLC analysis. Dependent upon small- or large-scale analysis performed, from 2 to 60 g of olive oil and from 7 to 80 mL of solvents are required.9 When large numbers of samples have to be processed, costs and time of the analyses increase considerably. Therefore, a faster and simpler technique for rapid isolation of phenolic compounds from EVOO is strongly recommended: microdialysis can fit the purpose. Developed in 1972 by Delgado and colleagues10 and popularized by Ungerstedt and co-workers,11 microdialysis is one of the most widely used techniques for in vivo or in vitro sampling of the chemical substances in extracellular fluids of animal tissues or cultured cells. On the basis of a very simple principle, mimicking the function of an artificial capillary blood vessel, microdialysis allows for recovery of low-molecularweight substances topically from a tissue and/or contribution of them to it.12 Different analytical techniques can be coupled with microdialysis to analyze the collected sample, such as spectrophotometric determination,13 microsensors,14 or HPLC, Received: Revised: Accepted: Published: A

December 22, 2016 February 8, 2017 February 12, 2017 February 13, 2017 DOI: 10.1021/acs.jafc.6b05725 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry using electrochemical,15,16 ultraviolet (UV), and fluorescence17 detectors. Recently, microdialysis has been successfully applied to plant studies as a rapid method to evaluate antioxidant capacity.18 In this study, the potential of microdialysis as technique for the fast extraction of the polyphenolic fraction from EVOO was investigated and the comparison to the traditional LLE technique was discussed.

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MATERIALS AND METHODS

Chemicals. Folin−Ciocalteu reagent, sodium carbonate, hydroxytyrosol, tyrosol, oleuropein, vanillic acid, p-coumaric acid, o-coumaric acid, ferulic acid, pinoresinol, luteolin, apigenin, methanol (MeOH, HPLC gradient grade), and acetonitrile (HPLC gradient grade) were purchased from Sigma-Aldrich S.r.l. (Milano, Italy). Gallic acid was obtained from Carlo Erba Reagenti S.p.A. (Rodano, Milano, Italy). Ultrapure water was prepared using a Milli-Q system (Millipore Corporation, Billerica, MA, U.S.A.). Stock standard solutions for polyphenol chromatographic analyses were prepared by dissolving 20−25 mg of powder in 25 mL of methanol/water (80:20). Working phenol solutions were obtained with subsequent dilutions with methanol/water (80:20). All of the mixtures were stored in the dark at −20 °C until use. EVOO. The experiments were carried out on four EVOOs with different polyphenolic contents. EVOOs were provided by local producers and were from Sardinian olive varieties (Olea europaea L.) collected from October to December 2014. LLE of Phenolic Compounds from EVOO. Extraction of phenolic compounds was performed according to the official method.19 Briefly, EVOO (4 g) was weighed in a 50 mL screw-cap test tube, with the addition of 5 mL of 80:20 (v/v) methanol/water, and sonicated in an ultrasonic bath (VWR International, Milano, Italy). After 15 min, the mixture was centrifuged at 6000g for 25 min, the methanol layer was transferred to a calibrated flask, and the olive oil was extracted again. The organic phases were collected and brought to a volume of 10 mL. Finally, the solution was filtered through a 0.45 μm polyvinylidene fluoride (PVDF) filter and subjected to the Folin− Ciocalteu test and HPLC analysis. Total Phenolic Content Determination. The total phenolic content was determined using the Folin−Ciocalteu assay according to Singleton and co-workers.20 Briefly, the methanolic extract (1 mL) was mixed with 1 mL of Folin−Ciocalteu reagent and 10 mL of 7.5% sodium carbonate and brought to a final solution volume of 25 mL with water. The reaction mixture was incubated for 120 min in the dark at room temperature, and then the absorbance was measured at 750 nm [Agilent 8453 ultraviolet−visible (UV−vis) spectrophotometer, Agilent Technologies, Palo Alto, CA, U.S.A.]. Results were expressed as milligrams of gallic acid equivalent (GAE) per 1 kg of oil by means of a calibration curve of gallic acid (10−40 mg L−1; R2 = 0.996). Samples were analyzed in triplicate. Concentric Microdialysis Probe Construction and “StaticOil” Dialytical Extraction of Phenolic Compounds from EVOO. The concentric microdialysis probe has been previously developed and described by our research group.18 The probe has a concentric design and was made using a section of plastic-coated silica tubing (diameter of 0.15 mm; Scientific Glass Engineering, Milton Keynes, U.K.) placed in the center of a semi-permeable polyacrylonitrile dialysis fiber (molecular cutoff weight of 12 kD; Filtral 16 Hospal Industrie, France). The section of silica tubing served as the inlet, and the outlet was also made with a section of plastic-coated silica tubing, positioned in the center of the polythene tubing. Quick-drying epoxy glue was used to seal the tip of the dialysis fibers. The final diameter of the probe was 0.22 mm with an active length of 40 mm. A first series of experiments was performed to attain the best performing microdialysis setup on EVOO phenolic compound extraction, related to the recovery of the probe, sample volume, and analytical system requirement. The experimental setup was assembled as illustrated in Figure 1A, and measurements were performed as follows: the microdialysis probe was placed in a plastic [polyethylene terephthalate (PET)] vial containing 0.5 mL of olive oil. Continuous dialysis at a

Figure 1. Three-dimensional (3D) drawings and graphic representations of the microdialysis devices developed and used in this study. The main differences between the (A) “static-oil” system and (B) “dynamic-oil” system are the geometry of the microdialysis probe (concentric and linear, respectively) and the oil stream (present only in the latter device).

fixed flow rate of 2 μL min−1 was performed using an 80:20 (v/v) methanol/water extracting solution by means of a micro-infusion pump (CMA/100, Solna, Sweden) connected to the microdialysis inlet (1) through a length of polyethylene tubing. The first sample was collected from the microdialysis probe outlet (2) after 30 min of stabilization, and then five dialysates were collected every 20 min. The collected samples were analyzed by HPLC to determine the phenolic compounds of EVOO. Linear Microdialysis Probe Construction and “Dynamic-Oil” Dialytical Extraction of Phenolic Compounds from EVOO. A second series of experiments was carried out using a dual asymmetricflow perfusion and a new linear microdialysis device. The liner microdialysis probe was made as described above, with some modifications (Figure 1B). In brief, both sides of a 40 mm semipermeable polyacrylonitrile dialysis fiber (12 kD cutoff weight) were connected to a section of plastic-coated silica tubing serving as both the microdialysis inlet (1) and outlet (2). The microdialysis probe was placed in the center of a 60 μL glass capillary (75 mm length and 1.4 mm outer diameter), and the device was sealed by quick-drying epoxy glue. A micro-infusion pump (CMA/100, Solna, Sweden) was connected to the microdialysis probe inlet through a length of polyethylene tubing. Continuous dialysis (2 μL min−1 flow rate) was performed using an 80:20 (v/v) methanol/water extracting solution. A second micro-infusion pump was connected to the glass capillary inlet (3) through a length of polyethylene tubing, constantly pumped the EVOO at 4 μL min−1 flow rate. The first microdialysis sample was B

DOI: 10.1021/acs.jafc.6b05725 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Polyphenol Concentration Detected in LLE and MDE Extracts of EVOOsa oil 1 polyphenol 1, hydroxytyrosol 2, tyrosol 3, caffeic acid 4, vanilline 5, p-coumaric acid 6, ferulic acid 7, DAFOA 8, DAFLA 9, luteolin 10, AFOA 11, apigenin 12, peak I 13, peak II TPc

LLE 0.58 1.39 0.82 0.55 0.21 0.35 7.03 69.75 0.97 7.97 0.55 4.81 0.99

oil 2 MDE

a a a a a a a a a a a a a

0.56 1.21 0.81 0.43 0.19 0.32 6.49 61.41 0.73 7.89 0.41 3.60 1.16 220.25

a b a b a a a b b a b b a

LLE 1.66 4.27 0.30 0.32 0.14 0.16 30.36 92.04 1.79 15.99 1.01 4.66 1.15

oil 3 MDE

a a a a a a a a a a a a a

1.71 3.90 0.38 0.31 0.16 0.40 26.78 80.47 1.59 14.33 0.92 4.97 1.23 468.95

a b a a a b b b a a a a b

LLE 0.31 3.23 0.75 0.28 0.08 0.17 2.97 79.45 0.96 3.70 0.41 3.90 4.16

oil 4 MDE

a a a a a a a a a a a a a

0.32 3.19 0.39 0.24 0.09 0.18 2.74 68.74 0.55 1.99 0.21 3.33 4.10 164.11

a a b b a a a b b b b a a

LLE

MDE

0.73 a 1.11 a ND ND ND ND 7.30 a 14.42 a 0.93 a 12.18 a 0.78 a 3.01 a 17.73 a

0.83 b 1.47 b 0.05 ND ND ND 5.82 b 10.39 b 0.52 b 10.54 b 0.52 b 2.02 b 12.73 b 95.05

a

Each result represents a mean of three replicates. Values are expressed as milligrams per liter. Values within a row followed by the same letter are not significantly different (p < 0.05) according to Student’s t test. The average relative standard deviation (RSD, %) was 9.6%. The number of each polyphenol is the peak shown in the HPLC chromatogram of Figure 2. bND = not detectable. cThe total phenolic content (TP) was determined using the Folin−Ciocalteu assay and expressed as milligrams per kilogram of GAE. collected after 30 min of stabilization, and then five dialysates were collected every 20 min and analyzed by HPLC. Polyphenolic Profile of EVOO. An Agilent 1100 LC system (Agilent Technologies, Palo Alto, CA, U.S.A.) equipped with binary pump, degaser, column thermostat, autosampler, and diode array detector was used for the polyphenol analyses. Chromatographic separation was carried out according to the literature.19 Briefly, the column was a Luna C18 (250 × 4.6 mm, 5 μm) from Phenomenex (Torrance, CA, U.S.A.) with a security guard cartridge (4 × 2 mm). The flow rate was 1 mL min−1, and the column temperature was set to 30 °C. The injection volume was 20 μL, and the detection wavelengths were set to 280 and 320 nm. Elution was carried out with a ternary mobile phase of solvent A (water and 0.1% trifluoracetic acid), solvent B (methanol), and solvent C (acetonitrile). The initial percentage eluent composition was 96:2:2 (A/B/C) that was changed according to the following gradient program: 50:25:25 from 0 to 40 min, 40:30:30 from 40 to 45 min, 0:50:50 from 45 to 60 min, and 0:50:50 for 10 min. The system was allowed to come back to the initial solvent composition in 12 min. Identification and peak assignment of polyphenols were based on a comparison of their retention times to those of standards. The concentrations of hydroxityrosol, tyrosol, caffeic acid, vanilline, p-coumaric acid, ferulic acid, luteolin, and apigenin were calculated according to the external standard method curve (five concentrations in duplicate between 1 and 5 mg L−1; R2 = 0.999) and expressed as milligrams per liter. Tyrosol derivatives, dialdehydic form of ligstroside aglycone (DAFLA) and aldehydic form of ligstroside aglycone (AFLA), and hydroxytyrosol derivatives, dialdehydic form of oleuropein aglycone (DAFOA) and aldehydic form of oleuropein aglycone (AFOA), were identified according to the literature19 and quantified according to Tasioula-Margari and Tsabolatidou21 as follows: The tyrosol calibration curve corrected by the molecular weight ratio DAFLA/tyrosol (304/138) or AFLA/ tyrosol (362/138) was used for DAFLA and AFLA, and the oleuropein glycoside calibration curve corrected by the molecular weight ratio hydroxytyrosol/oleuropein glycoside (154/540), DAFOA/oleuropein glycoside (304/138), or AFOA/oleuropein glycoside (362/138) was used for hydroxytyrosol, DAFOA, and AFOA. Statistical Analysis. The concentrations of polyphenols in samples from microdialysis extraction (MDE) or LLE samples were plotted (or tabulated) as the mean ± standard error of the mean (SEM) or standard deviation (SD) and expressed as milligrams per liter or percent changes in comparison to a reference sample (100%). The effect of increasing concentrations of MeOH was studied in a range comprised between 40 and 80% (MeOH %) using MDE, and

the changes of the area of eight HPLC peaks (tyrosol, hydroxytyrosol, DAFOA, DAFLA, luteolin, AFOA, apigenin, and AFLA) were recorded and computed as milligrams per liter by referring to a standard mixture. These results were plotted as concentrations versus MeOH %, and linear regressions and Pearson’s correlation coefficients were calculated. For the validation of MDE versus LLE, the mean, median, and interquartile range (IQR) of the HPLC peak areas were used. The IQR contains 50% of the values included between the 25th and 75th percentiles. The significance of the differences of the means at a 5% level was determined using Student’s t test with StatGraphics (version XV, Manugistics, Rockville, MD, U.S.A.) or Prism (version 5.02, GraphPad, La Jolla, CA, U.S.A.) applications.

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RESULTS AND DISCUSSION “Static-Oil” and “Dynamic-Oil” Dialytical Extraction of Phenolic Compounds from EVOO. EVOOs with polyphenolic contents ranging from 95 to 469 mg kg−1 of GAE (Table 1) were selected for the trials. Concentric and linear microdialysis probe construction and dialysis procedures were developed to maximize the MDE performance. In a first series of experiments, dialysis parameters, such as extracting solution flow rate, sample rate, and oil volume, were studied using the setup illustrated in Figure 1A (“static-oil” dialysis). Different flow rates of the extracting solution, ranging from 1 up to 5 μL min−1, were applied, resulting in a sample volume of 40 μL in a period of time (sample rate) comprised between 8 and 40 min. The flow rate of 2 μL min−1 (sample rate 20 min) was selected as a good compromise between the polyphenol recovery (data not shown) and the sample volume required for HPLC analysis (20 μL). A total of six concentric microdialysis probes were inserted into a PET vial containing 0.5 mL of olive oil (Figure 1A), with 100% success in terms of the functioning of the microfluidic circuit. As showed in Figure 2 (filled circles), the polyphenol content in the “static-oil” microdialysis samples decreased as a function of time. In the first sample, the polyphenol content was the highest and very close to LLE, then it decreased in a linear manner, dropping to 83% after 60 min (p < 0.05 versus sample 1) and 65% after 100 min in comparison to the first sample (100%). Similar changes were previously observed by microdialysis studies on antioxidant capacity in the parenchyma of Opuntia ficus-indica.18 In that C

DOI: 10.1021/acs.jafc.6b05725 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 2. Percent changes of polyphenols in dialysis samples obtained by “static-oil” and “dynamic-oil” microdialysis. The polyphenol content in the “static-oil” microdialysis samples decreased linearly as a function of time, dropping to 83% after 60 min (p < 0.05 versus sample 1) and 65% after 100 min. Continuous dialysis in a small closed vial led to a progressive clearance of polyphenols and to a change in color, fading to white. The above-described phenomena are not present in “dynamic-oil” microdialysis.

study, we demonstrated an exponential decay of the concentration of the analyzed compounds as a function of time as a result of the plant physiology. In particular, the insertion of the probe into cladodes of Opuntia formed a tight channel filled with the fluid of the broken cells very similar to the “static-oil” microdialysis described in the Materials and Methods. As illustrated in Figure 2, continuous dialysis in a small closed vial led to a progressive clearance of polyphenols and to a change in the color of EVOO, fading to white. Although this loss of color indicates an exhaustive extraction of polyphenols from oil, this procedure was not able to provide a series of microdialysis samples (replicates) having the same amount of polyphenols extracted from EVOO. For this reason “static-oil” microdialysis was considered a not reliable approach for repeated measurements. To avoid this time-related decay of the phenolic content of the samples, in a second series of experiments, a new “dynamicoil” microdialysis device was developed. This setup (Figure 1B) takes advantage of asymmetric-flow microdialysis used for in vivo monitoring of brain neurochemicals.16,22 In particular, the combination of the most performing microdialysis flow rate with a constant oil stream (4 μL min−1) allowed for the performance of the dialysis of fresh EVOO for each collected sample. As shown in Figure 2 (unfilled circles), no significant differences were observed in polyphenol extraction all along the sampling in comparison to the first sample. Similarly, no change in color of the collected oil after each sample was perceived, which resulted in a constant over time. The use of a dual perfusion rate was chosen to mimic the animal models in which, even if performed in a small portion of tissue, baseline samples do not significantly differ from each other under fixed conditions. During in vivo microdialysis, this is due to the regional blood flow, responsible for maintaining compound supply and removal of waste products, and the absence of physiological barriers, such as cellular walls in plants.

Figure 3. (A) Effect of increasing concentrations of MeOH (in a range comprised between 40 and 80%) on selected polyphenol content in MDE samples. The linear changes of the area of eight peaks (tyrosol, hydroxytyrosol, DAFOA, DAFLA, luteolin, AFOA, apigenin, and AFLA) were recorded and computed as milligrams per liter by referring to a standard mixture. (B) Linear regression was calculated, and the resulting slopes were compared. (∗) p < 0.05 versus tyrosol, hydroxytyrosol, luteolin, apigenin, and AFLA slopes. (#) p < 0.05 versus DAFOA and DAFLA slopes.

The main feature of “dynamic-oil” microdialysis in comparison to “static-oil” microdialysis was that polyphenol extraction was carried out from fresh EVOO flowing inside the capillary and represents a single extraction procedure for each sample. This arrangement allowed for the performance of repeated measurements using a small amount of oil (80 μL) for each sample, while the fixed experimental conditions permitted the collection of samples containing the same amount of polyphenols representative of the polyphenols present in the original EVOO. This approach, coupled with HPLC analysis, was able to discriminate differences in phenols content among the four EVOOs studied. The results discussed in the following paragraphs have been obtained using “dynamic-oil” microdialysis with an extracting solution flow rate of 2 μL min−1 and a constant oil stream of 4 μL min−1. Effect of the Perfusion Solvent Composition on Phenolic Compound Extraction from EVOO. MDE D

DOI: 10.1021/acs.jafc.6b05725 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 4. HPLC elution profiles at 280 nm of the phenolic extracts from LLE and MDE of oil 1. Peaks correspond to (1) hydroxytyrosol, (2) tyrosol, (3) vanilline, (4) caffeic acid, (5) p-coumaric acid, (6) ferulic acid, (7) DAFOA and its oxidized form, (8) DAFLA and its isomeric form, (9) luteolin, (10) AFLA, (11) apigenin, and (12 and 13) unknown peaks.

efficiency was improved by increasing MeOH percentage in the perfusion solvent. In fact, the concentrations of all studied compounds (tyrosol, hydroxytyrosol, DAFOA, DAFLA, luteolin, AFOA, apigenin, and AFLA) increased linearly (R2 > 0.99) with a slope comprised between 0.218 ± 0.006 mg L−1 (MeOH %)−1 for AFLA and 1.08 ± 0.011 mg L−1 (MeOH %)−1 for AFOA (Figure 3). On the basis of the MeOH effect on the MDE efficiency, polyphenols can be divided in three groups: (i) high sensitivity to MeOH changes (AFOA), (ii) intermediate sensitivity to MeOH changes (DAFOA and DAFLA), and (iii) low sensitivity to MeOH changes (tyrosol, hydroxytyrosol, luteolin, apigenin, and AFLA). Groups i and ii showed a higher sensitivity to MeOH changes in comparison to group iii (p < 0.05), while the computed slope of AFOA was significantly higher than the slopes of DAFOA and DAFLA (p < 0.05). The analysis of the correlation between the MeOH % and the recovery percentage in the MDE samples showed Pearson’s coefficient r > 0.99 for all compounds (p < 0.05). On the basis of these results, the MeOH/H2O ratio (v/v) was fixed to 80:20 in the extracting solution used for MDE. To assure that this concentration of MeOH did not negatively affect the microdialysis membrane performance, a bundle of microdialysis hollow fibers was immersed in 80:20 MeOH/H2O for 48 h and then used for microdialysis probe construction. No differences were observed in MDE performances (data not shown). Microdialysis versus LLE of Polyphenols from EVOO. Identification of polyphenols was made by the means of standards (peaks 1−6, 9, and 11 in Figure 4) or, when not commercially available, by comparison to data reported in the literature (peaks 7, 8, and 10).19 All EVOOs showed a similar qualitative polyphenolic composition. The main components were secoiridoids: DAFOA co-eluted with an oxidized form19,21,23,24 (peak 7), DAFLA co-eluted with pinoresinol19,21,24 (peak 8), and finally AFOA19,23 (peak 10). Peaks 12 and 13 were not identified but could be the AFLA compound, as reported by Kotsiou and co-workers,23 TasioulaMargari and Tsabolatidou,21 and Daskalasi and colleagues.24 Minor components were hydroxytyrosol, tyrosol, caffeic acid, vanilline, p-coumaric acid, ferulic acid, luteolin, and apigenin. As expected, quantitative differences (Table 1) in the single phenols were detected among the oils because of the different

Figure 5. (A) Plotting of all raw LLE values (n = 156) versus the corresponding MDE values. (B and C) Descriptive statistics of LLE and MDE data distributions. Red arrows, means; blue arrows, medians. IQR = interquartile range.

origin (olive variety, pedoclimatic conditions, processing methods, and storage conditions).25 MDE and LLE yielded the same polyphenolic profile. The most striking evidence was the perfect overlapping of MDE and LLE chromatograms for each oil analyzed (Figure 4). Although differences were found between MDE and LLE for single compounds, overall, the polyphenol content in both extracts is comparable, suggesting that the two analytical techniques are equivalent from either a qualitative or quantitative point of view. The final microdialysis setting that we used (“dynamic-oil” dialytical extraction with 80:20 MeOH/H2O) seems to have performed the same results of LLE on 4 g of olive oil. Plotting all raw LLE values (n = 156) versus the corresponding MDE values (Figure 5A) showed a strong positive correlation between the two extracting techniques (Pearson’s coefficient r = 0.9971; R2 = 0.994; p < 0.0001). The linear fit with 95% confidence interval resulted in a slope of 0.875 ± 0.005 (R2 = 0.994), indicating a 12.5% E

DOI: 10.1021/acs.jafc.6b05725 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

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difference between MDE and LLE extracting performances. Data distribution shown in panels B and C of Figure 5 confirmed the slight overestimation (around 13%) of LLE in comparison to MDE (LLE mean = 69.27 ± 9.28, and MDE mean = 60.16 ± 8.14) and indicates an overlapping distribution between LLE and MDE confirmed by the medians (LLE median = 22.32, and MDE median = 20.13) and the IQRs. In conclusion, the “dynamic-oil” microdialysis setup provided a reliable procedure and statistically consistent data. It also allowed for highlighting differences in polyphenol composition between the analyzed oils. This novel approach in polyphenol extraction could be a rapid, reliable, and useful tool to compare the phenolic content from different EVOOs and to monitor how it changes during the aging of oil. For these reasons, it is our opinion that the microdialysis approach could, in the future, play a role in determining the quality and storability of EVOO. Moreover, MDE is easier, operator-independent, quicker, and cheaper in comparison to LLE. It provided small volume samples ready for HPLC analysis, suggesting the possibility of automation and miniaturization of the whole extraction/ analytical process. No sample manipulation is required in microdialysis, minimizing human error and sample degradation. On the other hand, the probe and microdialysis device construction is a time-consuming and user-dependent activity. Differences at this stage of the process could affect the reliability of polyphenol extraction. To our knowledge, it is the first time microdialysis has been used for polyphenol extraction from food matrices.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Gianfranco Bazzu: 0000-0002-1018-8196 Author Contributions †

Gianfranco Bazzu and Maria Giovanna Molinu contributed equally to this work. Funding

The research was supported by Regione Sardegna P.O.R. SARDEGNA F.S.E. 2007−2013, Obiettivo Competitività Regionale e Occupazione, Asse IV Capitale Umano, Linea di Attività l.3.1. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED EVOO, extra virgin olive oil; DAFOA, dialdehydic form of oleuropein aglycone; AFOA, aldehydic form of oleuropein aglycone; AFLA, aldehydic form of ligstroside aglycone; DAFLA, dialdehydic form of ligstroside aglycone; LLE, liquid−liquid extraction; SPE, solid-phase extraction; MDE, microdialysis extraction; HPLC, high-performance liquid chromatography; PVDF, polyvinylidene fluoride; GAE, gallic acid equivalent; IQR, interquartile range; SEM, standard error of the mean

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REFERENCES

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DOI: 10.1021/acs.jafc.6b05725 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acs.jafc.6b05725 J. Agric. Food Chem. XXXX, XXX, XXX−XXX