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Novel nanofibrous sorbents for the extraction and determination of resveratrol in wine
Talanta 206 (2020) 120181
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Novel nanofibrous sorbents for the extraction and determination of resveratrol in wine
T
Martina Hákováa, Lucie Chocholoušová Havlíkováa, František Šveca, Petr Solicha, Jakub Erbenb, Jiří Chvojkab, Dalibor Šatínskýa,* a
Charles University, Faculty of Pharmacy, The Department of Analytical Chemistry, Ak. Heyrovského 1203, 500 05, Hradec Králové, Czech Republic The Technical University of Liberec, Faculty of Textile Engineering, Department of Nonwovens and Nanofibrous Materials, Studentská 1402/2, 46001, Liberec 1, Czech Republic
b
ARTICLE INFO
ABSTRACT
Keywords: Nanofiber polymers On-line solid phase extraction Resveratrol Red wine Coating Chromatography
On-line SPE HPLC method using nanofibrous sorbents for the extraction and determination of resveratrol in wine was developed and validated. Different types of nanofibrous and microfibrous polymers were tested and compared with commercial monolithic C18 sorbent. Polyamide and polyacrylonitrile nanofibers and composite materials composed of respective polycaprolactone and poly(vinylidene difluoride) nanofibers at microfibrous scaffold were included among tested materials. Two different polycaprolactone-based materials were prepared and their effect on the extraction properties studied. Alternatively, dopamine-coated polycaprolactone fibers were also used. Poly(vinylidene difluoride) nanofibers/polycaprolactone microfibers composite was found as the most effective sorbent and utilized for the method validation. Resveratrol in red wine was determined using our validated on-line SPE HPLC method.
1. Introduction Polyphenolic compounds present in grapes have significant health benefits [1] since they possess antioxidant activity, which plays a role in preventing and repairing damage caused by oxidative stress. Resveratrol and its effects on human body were widely reported [2–4]. It has anti-inflammatory and antioxidant [3] as well as anticancer effects [5]. It helps to cure neurodegenerative processes, ischema reperfusion, and metabolic diseases [6,7]. These health-related properties led to development of a broad spectrum of analytical methods enabling determination of resveratrol in various products such as wine, berries, and grapes [8,9]. For example, some of these methods used direct injection of wine in absence of extraction procedure [10–12]. However, this simple approach decreased the sensitivity and selectivity of resveratrol determination in wine matrix. Most of the alternative analytical methods included sample pretreatment for matrix removal, and increase in selectivity and sensitivity. Among them, conventional procedures such as solid phase extraction (SPE) [13,14] and liquid-liquid extraction (LLE) [14,15] were favored. These time-consuming methods required multiple steps and often consumed large volume of organic
solvents. Modern approaches such as solid phase microextraction (SPME) were also used [9]. For example, the miniaturized solvent-free SPME method can be used as an alternative to conventional sample pretreatment methods [16]. Yet, SPME is time-consuming and demands high operator skills. Vinas et al. published a paper in 2008 describing the determination of resveratrol in wines, musts, and juices that included SPME followed by HPLC [17]. They compared two techniques, SPME and stir bar extraction. The complete procedure including extraction and desorption required more than 40 min. Aresta et al. tested different types of SPME fibers. Their extraction procedure was 45 min long [9]. Recent trends in sample pretreatment focus on saving solvents, time, and operator skills. The on-line extraction methods meet these requirements. Several alternative extraction sorbents coupled on-line to SPE-HPLC currently exist including molecularly imprinted polymers, different types of polymers, monolithic columns, and nanofibrous materials. Only one paper describing the determination of resveratrol using the on-line SPE HPLC method was published. Its authors used the molecularly imprinted polymers for the extraction of resveratrol from the Polygonum Cuspidatum, a plant used in the traditional Chinese
Abbreviations: PA6, (polyamide 6); PCL, (polycaprolactone); PCL-D, (dopamine-coated polycaprolactone); PVDF, (poly(vinylidene difluoride)); PAN, (polyakrylonitrile); SPE, (solid phase extraction); SPME, (solid phase microextraction); MIP, (molecularly imprinted polymer); nPVDF/μPCL, (poly(vinylidene difluoride) nanofibers on polycaprolactone microfibrous scaffold); PTFE, (poly(tetrafluoroethylene)); UHPLC, Ultra High Performance Liquid Chromatography * Corresponding author. E-mail address: [email protected] (D. Šatínský). https://doi.org/10.1016/j.talanta.2019.120181 Received 15 April 2019; Received in revised form 24 July 2019; Accepted 25 July 2019 Available online 26 July 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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Table 1 Comparison of our method with previously published methods in terms of extraction, detection, and limit of detection. Method
Matrix
Detection
LOD (μg L−1)
Reference
On-line SPE HPLC Direct injection into micro LC SPME Direct injection SPE SPE, LLE LLE SPME, stir bar extraction
wine wine wine, spirit, grape juice wine wine wine wine wine, must, fruit juice
UV FL UV UV UV, FL UV FL, UV FL
2 5 0.5–1.1 60 20 (UV) 3 (FL) 20 20 (UV), 3 (FL) 2; 0.1
Present work [8] [9] [10] [13] [14] [15] [17]
medicine [18]. To our best knowledge, no method describing the online extraction of resveratrol from red wine has been published. Table 1 summarizes the reported methods for the determination of resveratrol in wine using HPLC and classical off-line extraction. In this report, we present a new on-line SPE-UHPLC method using different types of nanofibrous materials as sorbents for the determination of resveratrol in wine. The fibrous materials applied were polyamide 6 (PA6) and polyacrylonitrile (PAN) nanofibers, composite polycaprolactone nanofibers placed on polycaprolactone microfibrous scaffold (nPCL/μPCL), and poly(vinylidene difluoride) nanofibers on polycaprolactone microfibrous scaffold (nPVDF/μPCL). We also introduced a novel sorbent, dopamine-coated nPCL/μPCL (PCL-D). Results obtained with these fibrous sorbents were compared with a commercial C18 monolithic sorbent. Method using the fibers was most effective for sample clean-up and recovery. Finally, our method using nPVDF/μPCL composite material for on-line SPE was validated and applied to real-life samples - wines.
filtered through a 0.22 μm poly(tetrafluorethylene) (PTFE) filter and injected directly into the SPE-UHPLC system. Injection volume was 50 μL. All wines were stored in a freezer to ensure their stability. 2.4. Preparation of nanofibers and microfibers 2.4.1. Polyacrylonitrile PAN solution in N,N-dimethylformamide (7% w/w) was used for the electrospinning process. Nanofibers were prepared using Nanospider™ Production Line NS 1WS500U (Elmarco, Liberec, Czech Republic) under the following processing conditions: wire electrode distance 200 mm, collecting electrode −20 kV, spinning electrode +40 kV. The ambient temperature and relative humidity were set during the process to 23 °C and 50%, respectively. 2.4.2. Polyamide 6 PA6 nanofibers were also prepared using the electrospinning technique. Detailed description of the electrospinning process was published elsewhere [19]. The concentration of PA6 solution was 15% w/w in formic acid/acetic acid. Surface density of the produced nanofibrous sheet was 0.75 g/m2. The ambient temperature and relative humidity were set during the process to 22 °C and 40%, respectively.
2. Materials and methods 2.1. Chemicals and materials All used chemicals are summarized in the supplementary materials (Section S1 - Chemicals, materials).
2.4.3. Polycaprolactone microfiber and nanofiber composite The set-up for the preparation of composite micro and nanomaterial was described previously [19]. The meltblown extruder was loaded with 100 g polymer per hour. The nozzle (105 × 0.4 mm in diameter orifices at 100 mm width) was heated to 250 °C. Stretched fibers were collected by air stream at a distance of 200 mm from nozzle. Polycaprolactone (PCL) solution (16 wt %) was used for electrospinning. Final applied voltages were −14 kV and +35 kV. Electrospun nanofibers formed a stable composite material with melted microfibers. Small discs with a diameter of 5 mm were cut from the nPCL/μPCL layer.
2.2. Instrumentation and software The meltblown system (Laboratory equipment J&M Laboratories, USA) equipped with small needleless electrospinning was used for the preparation of the composite fibers. Detailed description of the process was published elsewhere [19]. A scheme of device is presented in supplementary material (Fig. S1). Production of nanofibers using electrospinning is green-marked while production of microfibers using the meltblown is blue-marked [20]. The nanospider NS1WS500U (Elmarco, Czech Republic) was used for the preparation of the polyacrylonitrile and polyamide 6 nanofibers. A VEGA3 scanning electron microscope (Tescan, Brno, Czech Republic) enabled the structural analysis of our fibers. Evaluation of morphology of all tested nanomaterials is presented in Supplementary material section (Fig. S3). On-line SPE UHPLC was carried out with Nexera X2 UHPLC system (Shimadzu Corporation, Kyoto, Japan), equipped with a CBM-20A communication module, LC30AD solvent delivery systems, a DGU-20 A5R degassing unit, an SIL30AS autosampler, a CTO-20AC column oven with an FCV-12AH high pressure six-port switching valve, as well as detectors SPD-M30A DAD and an RF-10AXL.
2.4.4. Polydopamine-coated polycaprolactone microfiber and nanofiber composite nPCL/μPCL discs described in previous section were placed into a polymerization mixture composed of dopamine (2 g L−1), tris(hydroxymethyl)aminomethane (1.2 g L−1), and water, and held under constant stirring rate of 250 rpm and a temperature of 22 °C. The polymerization was terminated after 4 h, the discs were washed five times with pure water and placed in water in an ultrasonic bath for 30 s. Polydopamine uncoated and coated fibers are shown in the supplementary material (Fig. S2).
2.3. Preparation of standard solutions and samples
2.4.5. Polycaprolactone microfiber and polyvinylidene difluoride nanofiber composite Preparation of nPVDF/μPCL was detailed in our previous work [19]. The extruder was loaded with 100 g polymer per hour. The dyeing nozzle (105 × 0.4 mm in diameter orifices on 100 mm width) was heated to 250 °C. Stretched PCL microfibers were collected by air stream at a distance of 300 mm from the jet. The final concentration of
A standard stock solution at a concentration of 1 g L−1 was prepared by dissolving resveratrol in methanol. The solution was stored in freezer. A working solution with a concentration of 10 mg L−1 for the method development was prepared by diluting the standard stock solution with water on the day of measurement. The wine samples were 2
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PVDF in 2:1 dimethylformamide/acetone used for electrospinning was 16% w/w. The applied voltage on the stringed roller was +35 kV and −15 kV at the opposite electrode.
15 min in an ultrasonic bath at a temperature of 35 °C. Detailed description of the procedures was published elsewhere [19,21]. PCL based sorbents were not stable in organic solvents at higher temperature. Therefore, the column oven temperature for the analysis was set to 20 °C. Furthermore, the stability of polydopamine layer on PCL was tested in 0.01 mol L−1 sodium hydroxide and 0.01 mol L−1 hydrochloric acid. The polydopamine layer was stable in acids and in organic solvents. Coloring of the solution caused by degradation and dissolution of the polydopamine layer was observed during stability test in the sodium hydroxide.
2.5. Preparation of the extraction pre-columns Cylindrical arrangement of fibrous materials was chosen as the most suitable format for the on-line SPE-UHPLC. Materials were weighted and manually packed in 5 × 4.6 mm i. d. column cartridge that was placed in guard precolumn holder. Fully packed pre-column with minimum possible void volume was connected to the on-line SPEUHPLC system and washed with 100% acetonitrile for 15 min at a flow rate increasing from 0.1 to 1 mL min−1 with the aim to optimize the arrangement of the material in the column.
3.2. Optimization of the on-line SPE-UHPLC extraction procedure Equal testing protocols were used with each fibrous polymer. Optimum composition of the washing mobile phase and a flow rate of 1 mL min−1 was used for the washing step. The specific gradient program is presented in Table 2. Duration of the washing step was 1 min. Optimum content of the organic modifier, methanol or acetonitrile, in the washing mobile phase is shown in Fig. 1. The peak area of resveratrol in absence of organic modifier was considered 100% recovery and each change in recovery was related to this value. Resveratrol was well retained on all PCL based sorbents. The highest retention of resveratrol was monitored on composite PVDF nanofibers at PCL microfiber. The highest content of methanol and acetonitrile in the washing mobile phase that did not lead to analyte loss was 30 and 15%, respectively. The effect of the organic modifier on the peak shape was also observed and further tested during recovery experiments. Increase of tailing factor was observed while using stronger washing mobile phase containing a higher percentage of organic modifier. The effect of organic phase vs. Peak tailing is seen in Fig. 2.
2.6. UHPLC column-switching analysis An on-line SPE-UHPLC system was used for the simultaneous preconcentration and determination of resveratrol in red wine. SPE was carried out using our pre-columns filled with fibers or, for comparison, with C18 sorbent. At the beginning of the testing, the elution profile of resveratrol from each sorbent was determined. A different washing mobile phase was used for each polymer at a flow rate of 1.0 mL min−1. The recovery studies and optimization of the extraction process was carried out using a YMC-Triart C18 ExRS (100 × 4.6 mm, S-3μm, Kyoto, Japan) analytical column upon a gradient elution. The mobile phase consisted of the acetonitrile and 0.5% aqueous formic acid. Gradient program is presented in Table 2. The valve switching time was set at 1.0 min since the extraction step also lasted for 1 min. The gradient program started after the valve switched. Analysis of real red wines was carried out using a Kinetex 5 μm PFP 100A (100 × 4.6 mm), core-shell analytical column (Phenomenex, Torrance, CA, USA) and using a gradient of the mobile phase (Table 2). The wine (50 μL) was injected in the extraction pre-column. During this step, the analytical column was equilibrated to the initial conditions of the gradient. The resveratrol was detected at 300 nm. Total run time was 10 min.
3.3. Real-life samples analysis nPVDF/μPCL was used in these experiments because this sorbent well retained the resveratrol and the change in the peak shape at increasing percentage of organic modifier was minimized. Two representative samples of red wines, Merlot and Blue Portugal from the Czech Republic, were used for the method development as models with different matrix background. Two types of reversed phase columns, pentafluorophenyl and C18, were tested for chromatography of resveratrol in red wines. Kinetex PFP 100A (100 × 4.6 mm, 5 μm) coreshell column provided both a better peak shape and the separation from residual matrix interferences compared to the YMC-Triart C18 ExRS (100 × 4.6 mm, S-3μm) analytical column. Therefore, the former was used for the analysis of red wines. Table 2 presents analytical conditions and gradient program. The critical step was the separation of the peak of resveratrol from an unknown interfering peak. Changes in the gradient program and the mobile phase composition did not help to improve the resolution of these two peaks. Higher level of the organic modifier increased tailing of resveratrol peak while the resolution of interference and resveratrol peaks was less than 1.5, which is insufficient. Furthermore, we discovered that the clean-up of the wines was similar with 5 and 30% methanol in the washing mobile phase. Therefore, we used 5% methanol in 0.5% aqueous formic acid as the final washing mobile phase for analysis of the red wines. Comparison of all nanofibrous sorbents was carried out using this method. Fig. 3 compares chromatograms demonstrating the sample clean-up using each sorbent in red wine analysis. The performance of the micro/ nanofiber sorbents was also compared with a commercial C18 silicabased monolith (50 × 4.5 mm). The recovery of resveratrol from this column using 0 and 5% aqueous methanol in the washing step was again considered 100% and the recoveries obtained using each nanofibrous sorbent were related to this value. The recoveries are summarized in Table 3. PAN nanofibers provided the lowest recovery most likely related to the polarity of the polymer thanks to the nitrile groups. PA 6 nanofibers were also not suitable for this application since the
3. Results and discussion 3.1. Stability of the fibrous polymers in solvents Long-term stress stability of all our micro/nanofibrous materials was explored before their use. The long-term stability was tested for 24 h at a temperature of 25 °C while the stress stability was tested for Table 2 Analytical conditions and gradient programs for resveratrol recovery evaluation in model solution and red wine. Stationary phase
Mobile phase
Gradient program Time (min)
Organic (%)
Resveratrol solution YMC-Triart C18 ExRS (100 × 4.6 mm, S-3μm)
0.5% Formic acid: acetonitrile
Red wine Kinetex PFP 100A (100 × 4.6 mm, 5 μm)
0.0 3.0 4.5 5.0 5.1 8.0
30 60 100 100 30 30
0.5% Formic acid: acetonitrile
0.0 1.0 6.0 7.0 7.5 10.0
20 20 50 100 20 20
3
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Fig. 1. Bar charts of the effect of organic modifier in the washing mobile phase on the extraction efficiency of resveratrol.
resveratrol at a concentration of 10 mg L−1 was injected six times to calculate the SST parameters. The peak capacity was 64.56 and the tailing factor of resveratrol peak was 1.7. The repeatability of injection evaluated in terms of the relative standard deviation was 0.12%. Method linearity was tested in the range from 0.1 to 10 mg L−1 using six calibration points. The parameters of the calibration curve including intercept and slope are also summarized in Table 4. The intra-day precision was determined using spiked wine samples at three concentration levels, 0.5, 5, and 10 mg L−1. Wine filtered through the 0.22 μm PTFE filter was used to dilute the standard stock solution of resveratrol to the final concentrations. Each concentration was prepared six times and injected twice. The values of the intra-day precision are summarized again in Table 4. The inter-day reproducibility was tested for three days using the standard solution at a concentration of 10 mg L−1. The RSD value was 1%. Trueness of the method was evaluated by determining the recovery using the standard addition method. Fortified wine samples and not-spiked wine samples were compared to evaluate recovery over three concentration levels. The limit of the detection (LOD) and limit of the quantification (LOQ) were calculated by means of the signal to noise ratio and are shown in Table 4.
recovery was only about 30% in comparison with C18 sorbent. Both PA 6 and PAN nanofibers were used in 2D-cloth format, which can also negatively affect the retention of the analyte due to the poor contact of the sample and the sorbent. Our results confirmed that nPVDF/μPCL composite facilitated a better clean-up than dopamine coated nPCL/ μPCL. However, dopamine coated material – PCL-D, retained more hydrophilic impurities. All three PCL based sorbents provided similar recoveries comparable with that of the C18 sorbent. Better sample clean-up was obtained with all PCL based sorbents compared to the C18 monolith. The difference in clean-up in the red wine analysis between the C18 monolith and the composite nPVDF/μPCL using the 5% aqueous methanol in the washing mobile phase is shown in Fig. 4. 3.4. Validation Validation of the on-line extraction method using the composite nPVDF/μPCL sorbent was carried out to demonstrate the reliability of the method for determination of resveratrol in red wine. The validation parameters are summarized in Table 4. The system suitability test (SST) and validation parameters including intra- and inter-day precision, trueness, and linearity were evaluated. The standard solution of
Fig. 2. Bar charts of the effect of organic modifier in the washing mobile phase on the peak shape of resveratrol. 4
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Table 4 Chromatography system suitability test and validation parameters of the on-line SPE extraction using the nPVDF/μPCL composite fibers combined with HPLC. Parameter
Value
Peak capacity Repeatability of injection (RSD, %) (n=6) Peak symmetry Calibration range (mg L−1) Standard calibration Regression coefficient (r2) Limit of quantification (μg L−1) Limit of detection (μg L−1) Accuracy – spike recovery (%) in winea Intra-day precision (RSD, %)b Inter-day reproducibility (RSD, %) (n = 3)c
64.56 0.12 1.7 0.1–10 y = 350387× - 18941 0.9995 8.1 2.4 98.0; 91.5; 92.8 0.8; 0.5; 2.6 1.0
a
Accuracy was determined as a method recovery using six spiked wine samples at three concentration levels 0.5, 5, 10 mg L−1 b Repetitive determination of six spiked wines at three concentration levels 0.5, 5, 10 mg L−1 c Repetitive determination of standards at one concentration level 10 mg L−1 at three different days.
3.5. Determination of resveratrol in wines Analysis of 41 red wines from different grapes species and countries was carried out using the validated method with nPVDF/μPCL composite as the sorbent for on-line SPE UHPLC. Each wine was filtered through the 0.22 μm PTFE filter and directly injected in the column switching system. Quantities of the resveratrol found in the wines are presented in Table 5. Fig. 3. Comparison of clean-up efficiency and resveratrol retention on different types of sorbents using 5% methanol in 0.5% aqueous formic acid as washing mobile phase for the red wine analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
3.6. Comparison with other methods The major aim of this work was to demonstrate the use nano/microfibrous polymers as sorbents in on-line SPE-UHPLC analysis of red wines. Comparison of our method with those reported in the literature is presented in Table 1. The levels of determined resveratrol were in a good agreement with those found by others [22].
Table 3 Recovery from nanofiber sorbents related to C18 sorbent. Sorbent
C18 PA6 PAN PCL PCL-D PCL/PVDF
Recovery (%) 0% methanol
5% methanol
100.00 37.33 23.32 99.50 99.58 99.41
100.00 32.82 22.64 99.59 99.46 99.37
4. Conclusion Five types of nanofibrous or microfibrous polymers were tested and compared as sorbents for the on-line SPE-UHPLC extraction of resveratrol from red wines. Two different versions of the PCL were among the tested materials and their extraction properties were evaluated. Compared to nPVDF/μPCL and nPCL/μPCL polymers, dopamine coated fibers featured an enhancement in retention of polar impurities present Fig. 4. Comparison of chromatograms demonstrating the clean-up efficiency on nanofibrous composite nPVDF/μPCL (violet line) and C18 monolith (dashed line) using 5% methanol in the washing mobile phase. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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carried out. For effective on-line extraction 45 mg nano/microfibrous sorbent was sufficient and the analysis time including extraction and separation step was mere 10 min.
Table 5 Content of resveratrol in various wines. Wine Czech Republic Svatovavřinecké Svatovavřinecké Frankovka Frankovka Blue portugal Blue portugal Pinot noir Pinot noir André Cabernet moravia Cabernet moravia Donfelder Merlot Australia Shiraz Shiraz Shiraz Shiraz Shiraz Shiraz cabernet Shiraz cabernet Shiraz cabernet Shiraz cabernet sauvignon Spain Cubero Rioja Ribera del dvero Petit verdot Cabernet sauvignon Italy Merlot Primitivo di manduria Shiraz Hungary Blue portugal Blue portugal Slovakia Svatovavřinecké Macedonia Kadarka Africa Pinotage Germany Donfelder Greece Imiglykos Moldavia Cabernet sauvignon France Cabernet sauvignon Argentina Malbec Portugal Tejo
Vintage
Resveratrol content (mg L−1)
2018 2016 2017 2018 2016 2015 2018 2016 2017 2017 2016 2017 2018
0.27 0.59 1.53 2.90 1.19 4.55 1.87 1.03 0.11 0.12 0.58 0.13 1.09
2017 2017 2017 2017 2017 2017 2017 2017 2018
0.18 0.80 1.29 1.91 1.05 2.20 1.86 2.05 0.29
2018 2017 2016 2016 2014
0.16 0.58 0.32 1.29 0.19
2017 2016 2017
0.31 0.29 0.93
2017 2017
0.54 0.76
2018
0.43
2018
0.61
2018
0.15
2018
0.91
2017
0.35
1997
2.15
2017
0.41
2017
0.69
2014
1.05
Acknowledgements Financial support of the GACR project no. 17-08738S and EFSACDN project (No. CZ.02.1.01/0.0/0.0/16_019/0000841) co-funded by ERDF is gratefully acknowledged. M. H. acknowledges the financial support of specific research of Charles University no. SVV 260 412. The result was obtained through the financial support of the Ministry of Education, Youth and Sports of the Czech Republic and the European Union (European Structural and Investment Funds - Operational Program Research, Development and Education) in the frames of the project “Modular platform for autonomous chassis of specialized electric vehicles for freight and equipment transportation”, Reg. No. CZ.02.1.01/0.0/0.0/16_025/0007293. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.120181. References [1] H. Amira-Guebailia, J. Valls, T. Richard, X. Vitrac, J.P. Monti, J.C. Delaunay, J.M. Merillon, Centrifugal partition chromatography followed by HPLC for the isolation of cis-epsilon-viniferin, a resveratrol dimer newly extracted from a red Algerian wine, Food Chem. 113 (2009) 320–324. [2] C.H. Cottart, V. Nivet-Antoine, J.L. Beaudeux, Review of recent data on the metabolism, biological effects, and toxicity of resveratrol in humans, Mol. Nutr. Food Res. 58 (2014) 7–21. [3] A. Csiszar, Anti-inflammatory effects of resveratrol: possible role in prevention of agerelated cardiovascular disease, Ann. N. Y. Acad. Sci. 1215 (2011) 117–122. [4] A. Rauf, M. Imran, M.S. Butt, M. Nadeem, D.G. Peters, M.S. Mubarak, Resveratrol as an anti-cancer agent: a review, Crit. Rev. Food Sci. Nutr. 58 (2018) 1428–1447. [5] M. Ndiaye, R. Kumar, N. Ahmad, Resveratrol in cancer management: where are we and where we go from here? Resveratrol and Health 1215 (2011) 144–149. [6] T. Richard, A.D. Pawlus, M.L. Iglesias, E. Pedrot, P. Waffo-Teguo, J.M. Merillon, J.P. Monti, Neuroprotective properties of resveratrol and derivatives, Resveratrol and Health 1215 (2011) 103–108. [7] B. Juhaz, D.K. Das, A. Kertesz, A. Juhasz, R. Gesztelyi, B. Varga, Reduction of blood cholesterol and ischemic injury in the hypercholesteromic rabbits with modified resveratrol, logevinex, Mol. Cell. Biochem. 348 (2011) 199–203. [8] R. Lopez, P. Dugo, L. Mondello, Determination of trans-resveratrol in wine by microHPLC with fluorescence detection, J. Sep. Sci. 30 (2007) 669–672. [9] A. Aresta, P. Cotugno, F. Massari, C. Zambonin, Determination of trans-resveratrol in wines, spirits, and grape juices using solid-phase micro extraction coupled to liquid chromatography with UV diode-array detection, Food Anal. Method. 11 (2018) 426–431. [10] N. Ratola, J.L. Faria, A. Alves, Analysis and quantification of trans-resveratrol in wines from Alentejo region (Portugal), Food Technol. Biotechnol. 42 (2004) 125–130. [11] M. Lopez, F. Martinez, C. Del Valle, C. Orte, M. Miro, Analysis of phenolic constituents of biological interest in red wines by high-performance liquid chromatography, J. Chromatogr. A 922 (2001) 359–363. [12] L. Vlase, B. Kiss, S.E. Leucuta, S. Gocan, A rapid method for determination of resveratrol in wines by HPLC-MS, J. Liq. Chromatogr. Relat. Technol. 32 (2009) 2105–2121. [13] M.A. Rodriguez-Delgado, G. Gonzalez, J.P. Perez-Trujillo, F.J. Garcia-Montelongo, Transresveratrol in wines from the Canary Islands (Spain). Analysis by high performance liquid chromatography, Food Chem. 76 (2002) 371–375. [14] S. Malovana, F.J.G. Montelongo, J.P. Perez, M.A. Rodriguez-Delgado, Optimisation of sample preparation for the determination of trans-resveratrol and other polyphenolic compounds in wines by high performance liquid chromatography, Anal. Chim. Acta 428 (2001) 245–253. [15] M.A. Rodriguez-Delgado, S. Malovana, J.P. Perez, T. Borges, F.J.G. Montelongo, Separation of phenolic compounds by high-performance liquid chromatography with absorbance and fluorimetric detection, J. Chromatogr. A 912 (2001) 249–257. [16] H. Piri-Moghadam, M.N. Alam, J. Pawliszyn, Review of geometries and coating materials in solid phase microextraction: opportunities, limitations, and future perspectives, Anal. Chim. Acta 984 (2017) 42–65. [17] P. Vinas, N. Campillo, M. Hernandez-Perez, M. Hernandez-Cordoba, A comparison of solid-phase microextraction and stir bar sorptive extraction coupled to liquid chromatography for the rapid analysis of resveratrol isomers in wines, musts and fruit juices, Anal. Chim. Acta 611 (2008) 119–125. [18] X.L. Zhuang, X.C. Dong, S.J. Ma, T. Zhang, Selective on-line extraction of trans-resveratrol and emodin from Polygonum cuspidatum using molecularly imprinted polymer, J.
in the red wines. nPVDF/μPCL exhibited a similar clean-up potential as nPCL/μPCL. However, resveratrol was more retained on the nPVDF/ μPCL and a better peak shape was also observed. All PCL based materials had similar extraction efficiency as commercial C18 sorbent but a better sample clean-up potential. Only 5% of methanol in the washing mobile phase was needed for efficient clean-up of the wines. This reduction in the organic solvent consumption is in line with current trends in the “green” analytical chemistry. The nPVDF/μPCL composite was found the most suitable sorbent for the on-line extraction of the resveratrol from red wine and the complete method validation was
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M. Háková, et al. Chromatogr. Sci. 46 (2008) 739–742. [19] M. Háková, L.C. Havlíková, J. Chvojka, J. Erben, P. Solich, F. Švec, D. Šatínský, A comparison study of nanofiber, microfiber, and new composite nano/microfiber polymers used as sorbents for on-line solid phase extraction in chromatography system, Anal. Chim. Acta 1023 (2018) 44–52. [20] M. Háková, L.C. Havlíková, P. Solich, F. Švec, D. Šatínský, Electrospun nanofiber polymers as extraction phases in analytical chemistry – the advances of the last decade, Trends Anal. Chem. 110 (2019) 81–96.
[21] M. Háková, L.C. Havlíková, J. Chvojka, F. Švec, P. Solich, D. Šatínský, Nanofiber polymers as novel sorbents for on-line solid phase extraction in chromatographic system: a comparison with monolithic reversed phase C18 sorbent, Anal. Chim. Acta 1018 (2018) 26–34. [22] L. Di Donna, D. Taverna, S. Indelicato, A. Napoli, G. Sindona, F. Mazzotti, Rapid assay of resveratrol in red wine by paper spray tandem mass spectrometry and isotope dilution, Food Chem. 229 (2017) 354–357.
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Novel nanofibrous sorbents for the extraction and determination of resveratrol in wine
T
Martina Hákováa, Lucie Chocholoušová Havlíkováa, František Šveca, Petr Solicha, Jakub Erbenb, Jiří Chvojkab, Dalibor Šatínskýa,* a
Charles University, Faculty of Pharmacy, The Department of Analytical Chemistry, Ak. Heyrovského 1203, 500 05, Hradec Králové, Czech Republic The Technical University of Liberec, Faculty of Textile Engineering, Department of Nonwovens and Nanofibrous Materials, Studentská 1402/2, 46001, Liberec 1, Czech Republic
b
ARTICLE INFO
ABSTRACT
Keywords: Nanofiber polymers On-line solid phase extraction Resveratrol Red wine Coating Chromatography
On-line SPE HPLC method using nanofibrous sorbents for the extraction and determination of resveratrol in wine was developed and validated. Different types of nanofibrous and microfibrous polymers were tested and compared with commercial monolithic C18 sorbent. Polyamide and polyacrylonitrile nanofibers and composite materials composed of respective polycaprolactone and poly(vinylidene difluoride) nanofibers at microfibrous scaffold were included among tested materials. Two different polycaprolactone-based materials were prepared and their effect on the extraction properties studied. Alternatively, dopamine-coated polycaprolactone fibers were also used. Poly(vinylidene difluoride) nanofibers/polycaprolactone microfibers composite was found as the most effective sorbent and utilized for the method validation. Resveratrol in red wine was determined using our validated on-line SPE HPLC method.
1. Introduction Polyphenolic compounds present in grapes have significant health benefits [1] since they possess antioxidant activity, which plays a role in preventing and repairing damage caused by oxidative stress. Resveratrol and its effects on human body were widely reported [2–4]. It has anti-inflammatory and antioxidant [3] as well as anticancer effects [5]. It helps to cure neurodegenerative processes, ischema reperfusion, and metabolic diseases [6,7]. These health-related properties led to development of a broad spectrum of analytical methods enabling determination of resveratrol in various products such as wine, berries, and grapes [8,9]. For example, some of these methods used direct injection of wine in absence of extraction procedure [10–12]. However, this simple approach decreased the sensitivity and selectivity of resveratrol determination in wine matrix. Most of the alternative analytical methods included sample pretreatment for matrix removal, and increase in selectivity and sensitivity. Among them, conventional procedures such as solid phase extraction (SPE) [13,14] and liquid-liquid extraction (LLE) [14,15] were favored. These time-consuming methods required multiple steps and often consumed large volume of organic
solvents. Modern approaches such as solid phase microextraction (SPME) were also used [9]. For example, the miniaturized solvent-free SPME method can be used as an alternative to conventional sample pretreatment methods [16]. Yet, SPME is time-consuming and demands high operator skills. Vinas et al. published a paper in 2008 describing the determination of resveratrol in wines, musts, and juices that included SPME followed by HPLC [17]. They compared two techniques, SPME and stir bar extraction. The complete procedure including extraction and desorption required more than 40 min. Aresta et al. tested different types of SPME fibers. Their extraction procedure was 45 min long [9]. Recent trends in sample pretreatment focus on saving solvents, time, and operator skills. The on-line extraction methods meet these requirements. Several alternative extraction sorbents coupled on-line to SPE-HPLC currently exist including molecularly imprinted polymers, different types of polymers, monolithic columns, and nanofibrous materials. Only one paper describing the determination of resveratrol using the on-line SPE HPLC method was published. Its authors used the molecularly imprinted polymers for the extraction of resveratrol from the Polygonum Cuspidatum, a plant used in the traditional Chinese
Abbreviations: PA6, (polyamide 6); PCL, (polycaprolactone); PCL-D, (dopamine-coated polycaprolactone); PVDF, (poly(vinylidene difluoride)); PAN, (polyakrylonitrile); SPE, (solid phase extraction); SPME, (solid phase microextraction); MIP, (molecularly imprinted polymer); nPVDF/μPCL, (poly(vinylidene difluoride) nanofibers on polycaprolactone microfibrous scaffold); PTFE, (poly(tetrafluoroethylene)); UHPLC, Ultra High Performance Liquid Chromatography * Corresponding author. E-mail address: [email protected] (D. Šatínský). https://doi.org/10.1016/j.talanta.2019.120181 Received 15 April 2019; Received in revised form 24 July 2019; Accepted 25 July 2019 Available online 26 July 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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Table 1 Comparison of our method with previously published methods in terms of extraction, detection, and limit of detection. Method
Matrix
Detection
LOD (μg L−1)
Reference
On-line SPE HPLC Direct injection into micro LC SPME Direct injection SPE SPE, LLE LLE SPME, stir bar extraction
wine wine wine, spirit, grape juice wine wine wine wine wine, must, fruit juice
UV FL UV UV UV, FL UV FL, UV FL
2 5 0.5–1.1 60 20 (UV) 3 (FL) 20 20 (UV), 3 (FL) 2; 0.1
Present work [8] [9] [10] [13] [14] [15] [17]
medicine [18]. To our best knowledge, no method describing the online extraction of resveratrol from red wine has been published. Table 1 summarizes the reported methods for the determination of resveratrol in wine using HPLC and classical off-line extraction. In this report, we present a new on-line SPE-UHPLC method using different types of nanofibrous materials as sorbents for the determination of resveratrol in wine. The fibrous materials applied were polyamide 6 (PA6) and polyacrylonitrile (PAN) nanofibers, composite polycaprolactone nanofibers placed on polycaprolactone microfibrous scaffold (nPCL/μPCL), and poly(vinylidene difluoride) nanofibers on polycaprolactone microfibrous scaffold (nPVDF/μPCL). We also introduced a novel sorbent, dopamine-coated nPCL/μPCL (PCL-D). Results obtained with these fibrous sorbents were compared with a commercial C18 monolithic sorbent. Method using the fibers was most effective for sample clean-up and recovery. Finally, our method using nPVDF/μPCL composite material for on-line SPE was validated and applied to real-life samples - wines.
filtered through a 0.22 μm poly(tetrafluorethylene) (PTFE) filter and injected directly into the SPE-UHPLC system. Injection volume was 50 μL. All wines were stored in a freezer to ensure their stability. 2.4. Preparation of nanofibers and microfibers 2.4.1. Polyacrylonitrile PAN solution in N,N-dimethylformamide (7% w/w) was used for the electrospinning process. Nanofibers were prepared using Nanospider™ Production Line NS 1WS500U (Elmarco, Liberec, Czech Republic) under the following processing conditions: wire electrode distance 200 mm, collecting electrode −20 kV, spinning electrode +40 kV. The ambient temperature and relative humidity were set during the process to 23 °C and 50%, respectively. 2.4.2. Polyamide 6 PA6 nanofibers were also prepared using the electrospinning technique. Detailed description of the electrospinning process was published elsewhere [19]. The concentration of PA6 solution was 15% w/w in formic acid/acetic acid. Surface density of the produced nanofibrous sheet was 0.75 g/m2. The ambient temperature and relative humidity were set during the process to 22 °C and 40%, respectively.
2. Materials and methods 2.1. Chemicals and materials All used chemicals are summarized in the supplementary materials (Section S1 - Chemicals, materials).
2.4.3. Polycaprolactone microfiber and nanofiber composite The set-up for the preparation of composite micro and nanomaterial was described previously [19]. The meltblown extruder was loaded with 100 g polymer per hour. The nozzle (105 × 0.4 mm in diameter orifices at 100 mm width) was heated to 250 °C. Stretched fibers were collected by air stream at a distance of 200 mm from nozzle. Polycaprolactone (PCL) solution (16 wt %) was used for electrospinning. Final applied voltages were −14 kV and +35 kV. Electrospun nanofibers formed a stable composite material with melted microfibers. Small discs with a diameter of 5 mm were cut from the nPCL/μPCL layer.
2.2. Instrumentation and software The meltblown system (Laboratory equipment J&M Laboratories, USA) equipped with small needleless electrospinning was used for the preparation of the composite fibers. Detailed description of the process was published elsewhere [19]. A scheme of device is presented in supplementary material (Fig. S1). Production of nanofibers using electrospinning is green-marked while production of microfibers using the meltblown is blue-marked [20]. The nanospider NS1WS500U (Elmarco, Czech Republic) was used for the preparation of the polyacrylonitrile and polyamide 6 nanofibers. A VEGA3 scanning electron microscope (Tescan, Brno, Czech Republic) enabled the structural analysis of our fibers. Evaluation of morphology of all tested nanomaterials is presented in Supplementary material section (Fig. S3). On-line SPE UHPLC was carried out with Nexera X2 UHPLC system (Shimadzu Corporation, Kyoto, Japan), equipped with a CBM-20A communication module, LC30AD solvent delivery systems, a DGU-20 A5R degassing unit, an SIL30AS autosampler, a CTO-20AC column oven with an FCV-12AH high pressure six-port switching valve, as well as detectors SPD-M30A DAD and an RF-10AXL.
2.4.4. Polydopamine-coated polycaprolactone microfiber and nanofiber composite nPCL/μPCL discs described in previous section were placed into a polymerization mixture composed of dopamine (2 g L−1), tris(hydroxymethyl)aminomethane (1.2 g L−1), and water, and held under constant stirring rate of 250 rpm and a temperature of 22 °C. The polymerization was terminated after 4 h, the discs were washed five times with pure water and placed in water in an ultrasonic bath for 30 s. Polydopamine uncoated and coated fibers are shown in the supplementary material (Fig. S2).
2.3. Preparation of standard solutions and samples
2.4.5. Polycaprolactone microfiber and polyvinylidene difluoride nanofiber composite Preparation of nPVDF/μPCL was detailed in our previous work [19]. The extruder was loaded with 100 g polymer per hour. The dyeing nozzle (105 × 0.4 mm in diameter orifices on 100 mm width) was heated to 250 °C. Stretched PCL microfibers were collected by air stream at a distance of 300 mm from the jet. The final concentration of
A standard stock solution at a concentration of 1 g L−1 was prepared by dissolving resveratrol in methanol. The solution was stored in freezer. A working solution with a concentration of 10 mg L−1 for the method development was prepared by diluting the standard stock solution with water on the day of measurement. The wine samples were 2
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PVDF in 2:1 dimethylformamide/acetone used for electrospinning was 16% w/w. The applied voltage on the stringed roller was +35 kV and −15 kV at the opposite electrode.
15 min in an ultrasonic bath at a temperature of 35 °C. Detailed description of the procedures was published elsewhere [19,21]. PCL based sorbents were not stable in organic solvents at higher temperature. Therefore, the column oven temperature for the analysis was set to 20 °C. Furthermore, the stability of polydopamine layer on PCL was tested in 0.01 mol L−1 sodium hydroxide and 0.01 mol L−1 hydrochloric acid. The polydopamine layer was stable in acids and in organic solvents. Coloring of the solution caused by degradation and dissolution of the polydopamine layer was observed during stability test in the sodium hydroxide.
2.5. Preparation of the extraction pre-columns Cylindrical arrangement of fibrous materials was chosen as the most suitable format for the on-line SPE-UHPLC. Materials were weighted and manually packed in 5 × 4.6 mm i. d. column cartridge that was placed in guard precolumn holder. Fully packed pre-column with minimum possible void volume was connected to the on-line SPEUHPLC system and washed with 100% acetonitrile for 15 min at a flow rate increasing from 0.1 to 1 mL min−1 with the aim to optimize the arrangement of the material in the column.
3.2. Optimization of the on-line SPE-UHPLC extraction procedure Equal testing protocols were used with each fibrous polymer. Optimum composition of the washing mobile phase and a flow rate of 1 mL min−1 was used for the washing step. The specific gradient program is presented in Table 2. Duration of the washing step was 1 min. Optimum content of the organic modifier, methanol or acetonitrile, in the washing mobile phase is shown in Fig. 1. The peak area of resveratrol in absence of organic modifier was considered 100% recovery and each change in recovery was related to this value. Resveratrol was well retained on all PCL based sorbents. The highest retention of resveratrol was monitored on composite PVDF nanofibers at PCL microfiber. The highest content of methanol and acetonitrile in the washing mobile phase that did not lead to analyte loss was 30 and 15%, respectively. The effect of the organic modifier on the peak shape was also observed and further tested during recovery experiments. Increase of tailing factor was observed while using stronger washing mobile phase containing a higher percentage of organic modifier. The effect of organic phase vs. Peak tailing is seen in Fig. 2.
2.6. UHPLC column-switching analysis An on-line SPE-UHPLC system was used for the simultaneous preconcentration and determination of resveratrol in red wine. SPE was carried out using our pre-columns filled with fibers or, for comparison, with C18 sorbent. At the beginning of the testing, the elution profile of resveratrol from each sorbent was determined. A different washing mobile phase was used for each polymer at a flow rate of 1.0 mL min−1. The recovery studies and optimization of the extraction process was carried out using a YMC-Triart C18 ExRS (100 × 4.6 mm, S-3μm, Kyoto, Japan) analytical column upon a gradient elution. The mobile phase consisted of the acetonitrile and 0.5% aqueous formic acid. Gradient program is presented in Table 2. The valve switching time was set at 1.0 min since the extraction step also lasted for 1 min. The gradient program started after the valve switched. Analysis of real red wines was carried out using a Kinetex 5 μm PFP 100A (100 × 4.6 mm), core-shell analytical column (Phenomenex, Torrance, CA, USA) and using a gradient of the mobile phase (Table 2). The wine (50 μL) was injected in the extraction pre-column. During this step, the analytical column was equilibrated to the initial conditions of the gradient. The resveratrol was detected at 300 nm. Total run time was 10 min.
3.3. Real-life samples analysis nPVDF/μPCL was used in these experiments because this sorbent well retained the resveratrol and the change in the peak shape at increasing percentage of organic modifier was minimized. Two representative samples of red wines, Merlot and Blue Portugal from the Czech Republic, were used for the method development as models with different matrix background. Two types of reversed phase columns, pentafluorophenyl and C18, were tested for chromatography of resveratrol in red wines. Kinetex PFP 100A (100 × 4.6 mm, 5 μm) coreshell column provided both a better peak shape and the separation from residual matrix interferences compared to the YMC-Triart C18 ExRS (100 × 4.6 mm, S-3μm) analytical column. Therefore, the former was used for the analysis of red wines. Table 2 presents analytical conditions and gradient program. The critical step was the separation of the peak of resveratrol from an unknown interfering peak. Changes in the gradient program and the mobile phase composition did not help to improve the resolution of these two peaks. Higher level of the organic modifier increased tailing of resveratrol peak while the resolution of interference and resveratrol peaks was less than 1.5, which is insufficient. Furthermore, we discovered that the clean-up of the wines was similar with 5 and 30% methanol in the washing mobile phase. Therefore, we used 5% methanol in 0.5% aqueous formic acid as the final washing mobile phase for analysis of the red wines. Comparison of all nanofibrous sorbents was carried out using this method. Fig. 3 compares chromatograms demonstrating the sample clean-up using each sorbent in red wine analysis. The performance of the micro/ nanofiber sorbents was also compared with a commercial C18 silicabased monolith (50 × 4.5 mm). The recovery of resveratrol from this column using 0 and 5% aqueous methanol in the washing step was again considered 100% and the recoveries obtained using each nanofibrous sorbent were related to this value. The recoveries are summarized in Table 3. PAN nanofibers provided the lowest recovery most likely related to the polarity of the polymer thanks to the nitrile groups. PA 6 nanofibers were also not suitable for this application since the
3. Results and discussion 3.1. Stability of the fibrous polymers in solvents Long-term stress stability of all our micro/nanofibrous materials was explored before their use. The long-term stability was tested for 24 h at a temperature of 25 °C while the stress stability was tested for Table 2 Analytical conditions and gradient programs for resveratrol recovery evaluation in model solution and red wine. Stationary phase
Mobile phase
Gradient program Time (min)
Organic (%)
Resveratrol solution YMC-Triart C18 ExRS (100 × 4.6 mm, S-3μm)
0.5% Formic acid: acetonitrile
Red wine Kinetex PFP 100A (100 × 4.6 mm, 5 μm)
0.0 3.0 4.5 5.0 5.1 8.0
30 60 100 100 30 30
0.5% Formic acid: acetonitrile
0.0 1.0 6.0 7.0 7.5 10.0
20 20 50 100 20 20
3
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Fig. 1. Bar charts of the effect of organic modifier in the washing mobile phase on the extraction efficiency of resveratrol.
resveratrol at a concentration of 10 mg L−1 was injected six times to calculate the SST parameters. The peak capacity was 64.56 and the tailing factor of resveratrol peak was 1.7. The repeatability of injection evaluated in terms of the relative standard deviation was 0.12%. Method linearity was tested in the range from 0.1 to 10 mg L−1 using six calibration points. The parameters of the calibration curve including intercept and slope are also summarized in Table 4. The intra-day precision was determined using spiked wine samples at three concentration levels, 0.5, 5, and 10 mg L−1. Wine filtered through the 0.22 μm PTFE filter was used to dilute the standard stock solution of resveratrol to the final concentrations. Each concentration was prepared six times and injected twice. The values of the intra-day precision are summarized again in Table 4. The inter-day reproducibility was tested for three days using the standard solution at a concentration of 10 mg L−1. The RSD value was 1%. Trueness of the method was evaluated by determining the recovery using the standard addition method. Fortified wine samples and not-spiked wine samples were compared to evaluate recovery over three concentration levels. The limit of the detection (LOD) and limit of the quantification (LOQ) were calculated by means of the signal to noise ratio and are shown in Table 4.
recovery was only about 30% in comparison with C18 sorbent. Both PA 6 and PAN nanofibers were used in 2D-cloth format, which can also negatively affect the retention of the analyte due to the poor contact of the sample and the sorbent. Our results confirmed that nPVDF/μPCL composite facilitated a better clean-up than dopamine coated nPCL/ μPCL. However, dopamine coated material – PCL-D, retained more hydrophilic impurities. All three PCL based sorbents provided similar recoveries comparable with that of the C18 sorbent. Better sample clean-up was obtained with all PCL based sorbents compared to the C18 monolith. The difference in clean-up in the red wine analysis between the C18 monolith and the composite nPVDF/μPCL using the 5% aqueous methanol in the washing mobile phase is shown in Fig. 4. 3.4. Validation Validation of the on-line extraction method using the composite nPVDF/μPCL sorbent was carried out to demonstrate the reliability of the method for determination of resveratrol in red wine. The validation parameters are summarized in Table 4. The system suitability test (SST) and validation parameters including intra- and inter-day precision, trueness, and linearity were evaluated. The standard solution of
Fig. 2. Bar charts of the effect of organic modifier in the washing mobile phase on the peak shape of resveratrol. 4
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Table 4 Chromatography system suitability test and validation parameters of the on-line SPE extraction using the nPVDF/μPCL composite fibers combined with HPLC. Parameter
Value
Peak capacity Repeatability of injection (RSD, %) (n=6) Peak symmetry Calibration range (mg L−1) Standard calibration Regression coefficient (r2) Limit of quantification (μg L−1) Limit of detection (μg L−1) Accuracy – spike recovery (%) in winea Intra-day precision (RSD, %)b Inter-day reproducibility (RSD, %) (n = 3)c
64.56 0.12 1.7 0.1–10 y = 350387× - 18941 0.9995 8.1 2.4 98.0; 91.5; 92.8 0.8; 0.5; 2.6 1.0
a
Accuracy was determined as a method recovery using six spiked wine samples at three concentration levels 0.5, 5, 10 mg L−1 b Repetitive determination of six spiked wines at three concentration levels 0.5, 5, 10 mg L−1 c Repetitive determination of standards at one concentration level 10 mg L−1 at three different days.
3.5. Determination of resveratrol in wines Analysis of 41 red wines from different grapes species and countries was carried out using the validated method with nPVDF/μPCL composite as the sorbent for on-line SPE UHPLC. Each wine was filtered through the 0.22 μm PTFE filter and directly injected in the column switching system. Quantities of the resveratrol found in the wines are presented in Table 5. Fig. 3. Comparison of clean-up efficiency and resveratrol retention on different types of sorbents using 5% methanol in 0.5% aqueous formic acid as washing mobile phase for the red wine analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
3.6. Comparison with other methods The major aim of this work was to demonstrate the use nano/microfibrous polymers as sorbents in on-line SPE-UHPLC analysis of red wines. Comparison of our method with those reported in the literature is presented in Table 1. The levels of determined resveratrol were in a good agreement with those found by others [22].
Table 3 Recovery from nanofiber sorbents related to C18 sorbent. Sorbent
C18 PA6 PAN PCL PCL-D PCL/PVDF
Recovery (%) 0% methanol
5% methanol
100.00 37.33 23.32 99.50 99.58 99.41
100.00 32.82 22.64 99.59 99.46 99.37
4. Conclusion Five types of nanofibrous or microfibrous polymers were tested and compared as sorbents for the on-line SPE-UHPLC extraction of resveratrol from red wines. Two different versions of the PCL were among the tested materials and their extraction properties were evaluated. Compared to nPVDF/μPCL and nPCL/μPCL polymers, dopamine coated fibers featured an enhancement in retention of polar impurities present Fig. 4. Comparison of chromatograms demonstrating the clean-up efficiency on nanofibrous composite nPVDF/μPCL (violet line) and C18 monolith (dashed line) using 5% methanol in the washing mobile phase. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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carried out. For effective on-line extraction 45 mg nano/microfibrous sorbent was sufficient and the analysis time including extraction and separation step was mere 10 min.
Table 5 Content of resveratrol in various wines. Wine Czech Republic Svatovavřinecké Svatovavřinecké Frankovka Frankovka Blue portugal Blue portugal Pinot noir Pinot noir André Cabernet moravia Cabernet moravia Donfelder Merlot Australia Shiraz Shiraz Shiraz Shiraz Shiraz Shiraz cabernet Shiraz cabernet Shiraz cabernet Shiraz cabernet sauvignon Spain Cubero Rioja Ribera del dvero Petit verdot Cabernet sauvignon Italy Merlot Primitivo di manduria Shiraz Hungary Blue portugal Blue portugal Slovakia Svatovavřinecké Macedonia Kadarka Africa Pinotage Germany Donfelder Greece Imiglykos Moldavia Cabernet sauvignon France Cabernet sauvignon Argentina Malbec Portugal Tejo
Vintage
Resveratrol content (mg L−1)
2018 2016 2017 2018 2016 2015 2018 2016 2017 2017 2016 2017 2018
0.27 0.59 1.53 2.90 1.19 4.55 1.87 1.03 0.11 0.12 0.58 0.13 1.09
2017 2017 2017 2017 2017 2017 2017 2017 2018
0.18 0.80 1.29 1.91 1.05 2.20 1.86 2.05 0.29
2018 2017 2016 2016 2014
0.16 0.58 0.32 1.29 0.19
2017 2016 2017
0.31 0.29 0.93
2017 2017
0.54 0.76
2018
0.43
2018
0.61
2018
0.15
2018
0.91
2017
0.35
1997
2.15
2017
0.41
2017
0.69
2014
1.05
Acknowledgements Financial support of the GACR project no. 17-08738S and EFSACDN project (No. CZ.02.1.01/0.0/0.0/16_019/0000841) co-funded by ERDF is gratefully acknowledged. M. H. acknowledges the financial support of specific research of Charles University no. SVV 260 412. The result was obtained through the financial support of the Ministry of Education, Youth and Sports of the Czech Republic and the European Union (European Structural and Investment Funds - Operational Program Research, Development and Education) in the frames of the project “Modular platform for autonomous chassis of specialized electric vehicles for freight and equipment transportation”, Reg. No. CZ.02.1.01/0.0/0.0/16_025/0007293. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.120181. References [1] H. Amira-Guebailia, J. Valls, T. Richard, X. Vitrac, J.P. Monti, J.C. Delaunay, J.M. Merillon, Centrifugal partition chromatography followed by HPLC for the isolation of cis-epsilon-viniferin, a resveratrol dimer newly extracted from a red Algerian wine, Food Chem. 113 (2009) 320–324. [2] C.H. Cottart, V. Nivet-Antoine, J.L. Beaudeux, Review of recent data on the metabolism, biological effects, and toxicity of resveratrol in humans, Mol. Nutr. Food Res. 58 (2014) 7–21. [3] A. Csiszar, Anti-inflammatory effects of resveratrol: possible role in prevention of agerelated cardiovascular disease, Ann. N. Y. Acad. Sci. 1215 (2011) 117–122. [4] A. Rauf, M. Imran, M.S. Butt, M. Nadeem, D.G. Peters, M.S. Mubarak, Resveratrol as an anti-cancer agent: a review, Crit. Rev. Food Sci. Nutr. 58 (2018) 1428–1447. [5] M. Ndiaye, R. Kumar, N. Ahmad, Resveratrol in cancer management: where are we and where we go from here? Resveratrol and Health 1215 (2011) 144–149. [6] T. Richard, A.D. Pawlus, M.L. Iglesias, E. Pedrot, P. Waffo-Teguo, J.M. Merillon, J.P. Monti, Neuroprotective properties of resveratrol and derivatives, Resveratrol and Health 1215 (2011) 103–108. [7] B. Juhaz, D.K. Das, A. Kertesz, A. Juhasz, R. Gesztelyi, B. Varga, Reduction of blood cholesterol and ischemic injury in the hypercholesteromic rabbits with modified resveratrol, logevinex, Mol. Cell. Biochem. 348 (2011) 199–203. [8] R. Lopez, P. Dugo, L. Mondello, Determination of trans-resveratrol in wine by microHPLC with fluorescence detection, J. Sep. Sci. 30 (2007) 669–672. [9] A. Aresta, P. Cotugno, F. Massari, C. Zambonin, Determination of trans-resveratrol in wines, spirits, and grape juices using solid-phase micro extraction coupled to liquid chromatography with UV diode-array detection, Food Anal. Method. 11 (2018) 426–431. [10] N. Ratola, J.L. Faria, A. Alves, Analysis and quantification of trans-resveratrol in wines from Alentejo region (Portugal), Food Technol. Biotechnol. 42 (2004) 125–130. [11] M. Lopez, F. Martinez, C. Del Valle, C. Orte, M. Miro, Analysis of phenolic constituents of biological interest in red wines by high-performance liquid chromatography, J. Chromatogr. A 922 (2001) 359–363. [12] L. Vlase, B. Kiss, S.E. Leucuta, S. Gocan, A rapid method for determination of resveratrol in wines by HPLC-MS, J. Liq. Chromatogr. Relat. Technol. 32 (2009) 2105–2121. [13] M.A. Rodriguez-Delgado, G. Gonzalez, J.P. Perez-Trujillo, F.J. Garcia-Montelongo, Transresveratrol in wines from the Canary Islands (Spain). Analysis by high performance liquid chromatography, Food Chem. 76 (2002) 371–375. [14] S. Malovana, F.J.G. Montelongo, J.P. Perez, M.A. Rodriguez-Delgado, Optimisation of sample preparation for the determination of trans-resveratrol and other polyphenolic compounds in wines by high performance liquid chromatography, Anal. Chim. Acta 428 (2001) 245–253. [15] M.A. Rodriguez-Delgado, S. Malovana, J.P. Perez, T. Borges, F.J.G. Montelongo, Separation of phenolic compounds by high-performance liquid chromatography with absorbance and fluorimetric detection, J. Chromatogr. A 912 (2001) 249–257. [16] H. Piri-Moghadam, M.N. Alam, J. Pawliszyn, Review of geometries and coating materials in solid phase microextraction: opportunities, limitations, and future perspectives, Anal. Chim. Acta 984 (2017) 42–65. [17] P. Vinas, N. Campillo, M. Hernandez-Perez, M. Hernandez-Cordoba, A comparison of solid-phase microextraction and stir bar sorptive extraction coupled to liquid chromatography for the rapid analysis of resveratrol isomers in wines, musts and fruit juices, Anal. Chim. Acta 611 (2008) 119–125. [18] X.L. Zhuang, X.C. Dong, S.J. Ma, T. Zhang, Selective on-line extraction of trans-resveratrol and emodin from Polygonum cuspidatum using molecularly imprinted polymer, J.
in the red wines. nPVDF/μPCL exhibited a similar clean-up potential as nPCL/μPCL. However, resveratrol was more retained on the nPVDF/ μPCL and a better peak shape was also observed. All PCL based materials had similar extraction efficiency as commercial C18 sorbent but a better sample clean-up potential. Only 5% of methanol in the washing mobile phase was needed for efficient clean-up of the wines. This reduction in the organic solvent consumption is in line with current trends in the “green” analytical chemistry. The nPVDF/μPCL composite was found the most suitable sorbent for the on-line extraction of the resveratrol from red wine and the complete method validation was
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