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Sample preparation in this study involved QuEChERS extraction followed by a filter-based cleanup method that is easier and faster than dispersive solid-phase extraction.
In this study a novel, simple, rapid, and effective method was successfully developed for the determination of pesticide residues in red wine samples. Sample preparation involved extraction of pesticide residues into acetonitrile by QuEChERS (quick, easy, cheap, effective, rugged, and safe) and cleanup with a rapid push-through mini-cartridge filter instead of dispersive solid-phase extraction (dSPE). The red wine extract was cleaned up by passing it through a mini-cartridge containing primary secondary amine sorbent that retains organic acids, sugars, and polyphenolic pigments. The cleaned extract was analyzed by liquid chromatography–tandem mass spectrometry (LC–MS-MS). The cleanup procedure was simple and required less than 1 min per sample. Satisfactory recoveries ranging from 81.6% to 112.2% with relative standard deviations less than 10.8% were achieved. The linear dynamic range was 2–400 ng/mL with a correlation coefficient greater than 0.9940. The limit of detection and limit of quantification were in the range of 0.01–0.40 and 0.05–1.33 ng/mL, respectively. Six commercially available red wine samples were tested in this study, three of which were found to be positive for the presence of pesticides.
According to the International Organization of Vine and Wine, in 2010 about 26 billion liters of wine were produced worldwide, of which about 24 billion liters were consumed (1). Wine, especially red wine, is a rich source of polyphenols such as resveratrol, catechin, and epicatechin. These polyphenolic compounds are antioxidants that protect cells from oxidative damage caused by free radicals. Research on antioxidants found in red wine has shown that they may inhibit the development of certain cancers such as prostate cancer (2). In addition, consumption of red wines has been believed to have heart-healthy benefits (2). The application of pesticides such as fungicides and insecticides to improve grape yields is a common practice in vineyards. However, the applied pesticides may permeate through plant tissues and remain in the harvested grapes and subsequent processed products, such as grape juice and wine. Because pesticide residues are a potential source of toxic substances that are harmful to human beings, it is important to test for the levels of pesticide residues in grapes, juice, and wine. Although the European Union (EU) has set maximum residue levels (MRLs) for pesticide residues in wine grapes of 0.01–10 mg/kg (3,4), it has not yet established MRLs for wine. A study of 40 bottles of wine bought within the EU revealed that 34 of the 40 bottles contained at least one pesticide. The average number of pesticides per bottle was more than four, while the highest number of pesticides found in a single bottle was 10 (5).
The analysis of pesticide residues in red wine is challenging because of the complexity of the matrix, which contains alcohol, organic acids, sugars, phenols, and pigments (such as anthocyanins). Traditional red wine sample preparation methods include liquid–liquid extraction (LLE) with different organic solvents (6,7) and solid-phase extraction (SPE) with reversed-phase C18 and polymeric sorbents (8–10). However, LLE is labor intensive, consumes large amounts of organic solvents, and sometimes forms emulsions, making it difficult to separate the organic and aqueous phases. In contrast, SPE uses less solvent without emulsion formation, but demands more effort for method development. Other methods such as solid-phase microextraction (SPME) (11,12), hollow-fiber liquid-phase microextraction (13), and stir-bar sorptive extraction (SBSE) (14) use little or no organic solvent but are less reproducible. Typical instrumental detections systems include gas chromatography (GC), GC coupled to mass spectrometry (GC–MS), and liquid chromatography coupled to tandem mass spectrometry (LC–MS-MS) (6–14).
QuEChERS (quick, easy, cheap, effective, rugged, and safe) is a promising sample preparation method that was first reported in 2003 by Anastassiades, Lehotay, and colleagues for the determination of pesticide residues in vegetables and fruits (15). Since then QuEChERS has been widely used for the analysis of pesticides and other compounds of concern in various food, oil, and beverage matrices (16–18). The QuEChERS procedure involves extraction of pesticides from a sample with high water content into acetonitrile with the addition of salts to separate the phases and partition the pesticides into the organic layer. This is followed by dispersive solid-phase extraction (dSPE) to clean up various matrix coextractives and is achieved by mixing an aliquot of sample extract with sorbents prepacked in a centrifuge tube.
The aim of this study is to develop a method using QuEChERS extraction, but an easier and faster cleanup method compared to dSPE to cleanup red wine coextractives. This novel sample cleanup method is based on a filter-and-clean concept: The red wine extract is pushed through a mini-cartridge containing anhydrous magnesium sulfate and primary secondary amine (PSA) sorbent, residual water is adsorbed onto the anhydrous magnesium sulfate, and red wine coextractives are retained by the PSA sorbent. The purified extract is collected into an autosampler vial and injected into an LC–MS-MS system for analysis without the need for further filtration with a syringe filter. This cleanup procedure is simple and takes less than 1 min per sample. Red wine extracts were assessed for cleanliness based on visual appearance and full-scan chromatograms after cleanup with four traditional dSPE approaches containing different amounts of PSA sorbent and the rapid mini-cartridge filtration approach. The rapid mini-cartridge approach produced a slightly cleaner extract than the dSPE approach containing the same amount of magnesium sulfate and PSA sorbent. However, the cleanup procedure with push-through mini-cartridge filtration was found to be much faster than dSPE. Eight pesticides belonging to insecticide, fungicide, and parasiticide classes were selected for analysis in this study. Polarities of the eight selected pesticides were very different, with the logarithms of the octanol water partition coefficient (LogP) ranging from -0.779 to 5.004. The classes, structures, LogP, and pKa values are listed in Figure 1. Among the eight pesticides analyzed in this study, cyprodinil was most often detected on grapes, with chlorpyrifos, diazinone, and methamidophos also frequently found on grapes (19). The recoveries of planar pesticides included in this study (carbendazim, thiabendazole, pyrimethanil, and cyprodinil) are often adversely affected by graphitized carbon black (GCB), a sorbent that is widely used in dSPE to clean up pigmented samples. In this study, PSA sorbent was used instead of GCB for cleanup of red wine samples and the recoveries of these planar pesticides are reported.
Figure 1: Classes, structures, LogP, and pKa values of the eight pesticides selected in this study.
Finally, six commercially available red wine samples were analyzed using this simple, rapid, and effective sample preparation method. Carbendazim was detected in three red wine samples, although the detected concentrations (parts per billion) are much lower than the European or Japanese regulated levels (parts per million) in grapes (20,21).
Standards and Reagents
HPLC-grade acetonitrile and LC–MS-grade methanol were purchased from Spectrum. Methamidophos, carbendazim, pyrimethanil, diazinone, and chlorpyrifos (all 100 ppm) were purchased from Chem Service. Thiabendazole (1000 ppm), cyprodinil (100 ppm), and pyrazophos were purchased from Ultra Scientific. Triphenyl phosphate (TPP, 5000 ppm) was purchased from Cerilliant and was used as the internal standard (IS) in this study.
A 100 ppm thiabendazole solution was made by mixing 200 µL of the 1000 ppm stock solution with 1.8 mL acetonitrile. A 2 ppm pesticide working standard solution was made by adding 80 µL each of the eight 100 ppm standards with 3.36 mL of acetonitrile. A 5 ppm IS solution was made by diluting 10 µL of the 5000 ppm TPP stock solution with 10 mL of acetonitrile.
The six red wine samples tested in this study were provided by coworkers. Portions (10 mL) of the red wine samples were added into 50-mL polypropylene centrifuge tubes (UCT). To prepare fortified samples, red wine samples were spiked with appropriate amounts of the 2 ppm pesticide working standard solution, vortexed for 30 s and allowed to equilibrate for 15 min. A 10-mL volume of acetonitrile was added to each sample and then shaken for 1 min. Salts (4000 mg of anhydrous magnesium sulfate and 2000 mg of sodium chloride) packed in a Mylar pouch (UCT) were added, and the samples were shaken vigorously for 1 min and then centrifuged at 5000 rpm for 5 min. The upper layer red wine extract was then ready for cleanup.
Two cleanup methods, traditional dSPE and rapid push-through mini-cartridge filtration, were compared for cleanup efficiency of the red wine extract. Four 2-mL dSPE tubes containing 110 mg MgSO4 and 25 mg PSA (A); 110 mg MgSO4 and 50 mg PSA (B); 110 mg MgSO4 and 100 mg PSA (C); and 110 mg MgSO4 and 180 mg PSA (D) were tested to compare the cleanup efficiency against the rapid push-through mini-cartridge containing 110 mg MgSO4 and 180 mg PSA (UCT, ECPURMPSMC). For dSPE cleanup, 1 mL of the red wine extract was transferred into the 2-mL dSPE tube, shaken for 30 s, then centrifuged at 10,000 rpm for 5 min. A 0.5-mL volume of the cleaned extract was transferred into a 2-mL autosampler vial and 10 µL of the 5 ppm TPP (IS) solution was added. For rapid push-through mini-cartridge cleanup, 1 mL of the red wine extract was loaded using a nonsterile latex-free syringe with a Luer-lock tip (VWR), the loaded syringe was attached to the mini-cartridge and the extract was pushed through in a slow, drop-wise fashion. The first 0.5-mL portion of the cleaned extract was collected in a 2-mL autosampler vial and 10 µL of the 5 ppm TPP (IS) solution was added. For both cleanup methods, about half the 1-mL portion of the red wine extract was adsorbed onto the sorbents in the dSPE tube or mini-cartridge.
An Agilent 6890 GC system coupled with a model 5975C single-quadrupole mass-selective detector (MSD, Agilent) was used in this study for the acquisition of full-scan chromatograms of extracts that were prepared using the different cleanup methods. The GC system was equipped with a 30 m × 0.25 mm, 0.25-µm df Rtx-5MS capillary column integrated with a 10-m guard column (Restek). A splitless liner with dimensions of 4 mm × 6.5 mm × 78.5 mm (i.d. × o.d. × L) packed with deactivated glass wool (UCT) was used to introduce the extract onto the GC column. Splitless injections (1 µL) at 250 °C were made with a 50-mL/min split vent at 1 min. Ultrahigh-purity helium at a constant flow rate of 1.2 mL/min was used as the carrier gas. The oven temperature was initially held at 40 °C for 1 min; ramped at 10 °C/min to 300 °C and held for 3 min. The total run time was 30 min with data acquisition beginning at 4 min. The detector interface, ion source, and quadrupole temperatures were set at 280 °C, 250 °C, and 150 °C, respectively. Chromatograms of the red wine extracts were obtained in full-scan mode with a scanning range of 35–700 amu.
An Accela 1250 LC system coupled to a TSQ Vantage triple-quadrupole MS system was supplied by Thermo Fisher Scientific. A PAL autosampler (CTC Analytics) was equipped for automated sample injections. Xcalibur (version 2.1) software (Thermo Fisher Scientific) was used for data acquisition and processing. The separation of the eight target pesticides was performed on a 100 mm × 2.1 mm, 3-µm dp Sepax HP-C18 column with a 20 mm × 2.1 mm, 3-µm dp Restek C18 guard column. The column temperature was maintained at room temperature (~20 °C). The injection volume was 10 µL at 15 °C. Mobile-phase A was 0.1% formic acid in Milli-Q water (EMD Millipore), and mobile-phase B was 0.1% formic acid in methanol. A flow rate of 200 µL/min was used. The gradient program was as follows: 5% B for 1 min, 5–50% B over 2 min, 50–95% B over 5 min, 95% B for 6 min, 95–5% B in 0.2 min, and 5% B for 2 min.
Tandem MS was operated with heated electrospray ionization (HESI) in positive mode, and the conditions were as follows: spray voltage: 3000 V; sheath gas: nitrogen at 40 psi; auxiliary gas: nitrogen at 10 psi; ion transfer capillary temperature: 350 °C; collision gas: argon at 1.5 mTorr; Q1 peak width: 0.2 Da FWHM (full width half maximum); Q3 peak width: 0.7 Da FWHM. Optimization of the MS-MS transitions (collision energies and S-Lens RF values) was performed individually for each pesticide by infusing 1-µg/mL standard in acetonitrile at 10 µL/min with 50:50 (v/v) mobile phases A and B at a flow rate of 200 µL/min. The two most intense and characteristic precursor–product ion transitions were chosen for selected reaction monitoring (SRM). Acquisition was divided into three segments (0–5 min, 5.01–11 min, and 11.01–16 min) based on the retention times of the target analytes. The retention times, precursor and product ions, collision energies, S-Lens RF values, and dwell times are listed in Table I.
Table I: Retention times, SRM transitions, and dwell times for target analytes and internal standard (IS)
Results and Discussion
Evaluation of Cleanup Efficiency
Red wine extracts that underwent cleanup with the rapid mini-cartridge filtration, and with four traditional dSPE tubes containing 110 mg anhydrous MgSO4 and different amounts of PSA were compared as outlined in the "Cleanup" section. Red wine extracts without any cleanup, with dSPE cleanup A, B, C, and D, and with rapid mini-cartridge filtration are illustrated in Figure 2. The red color in the extracts decreases as the amounts of the PSA sorbent increases in the dSPE tubes. The samples analyzed with dSPE (D) and the rapid mini-cartridge containing the same amounts of magnesium sulfate and PSA yielded a similar colorless appearance. Large amounts of PSA (180 mg) contributed to the efficient removal of various matrix coextractives such as organic acids, sugars, phenols, and pigments in the red wine. Figure 3 shows the full-scan chromatograms of four extracts that underwent cleanup with traditional dSPE (Figures 3a–3d) and one with mini-cartridge filtration (Figure 3e). The chromatogram of rapid mini-cartridge filtration was slightly cleaner than that with dSPE (D). In addition, the rapid mini-cartridge filtration approach based on the filter-and-clean concept was simpler and faster than the dSPE approach, and was therefore selected for the cleanup of the red wine samples in this study.
Figure 2: Photograph of red wine extracts without any cleanup, cleaned up with dSPE A, B, C, and D, and cleaned up with rapid mini-cartridge filtration.
Matrix Matched Calibration, LOD, and LOQ
Calibration curves were obtained by analyzing matrix matched standards, which were prepared by spiking appropriate amounts of the 2 ppm pesticide working solution into blank red wine extracts after cleanup with rapid mini-cartridge filtration. Six matrix matched calibration standards at concentrations of 2, 10, 40, 100, 200, and 400 ng/mL were analyzed. The linear dynamic ranges, regression equations, and correlation coefficients (R2 ) are listed in Table II.
Table II: Matrix matched calibration, LODs, and LOQs
The limit of detection (LOD) and limit of quantification (LOQ) are the concentrations that give signal-to-noise ratios (S/N) of 3 and 10, respectively. In this study they were estimated according to the S/N values of the lowest matrix matched calibration level of 2 ng/mL. The calculated LOD ranged from 0.01 to 0.40 ng/mL and the LOQ ranged from 0.05 to 1.33 ng/mL (see Table II). The minimum reporting limit (MRL) in this study was set at the lowest calibration level of 2 ng/mL.
Figure 3: Full-scan chromatograms of red wine extracts cleaned up with (a) dSPE A, (b) dSPE B, (c) dSPE C, (d) dSPE D, and (e) rapid mini-cartridge filtration.
A chromatogram of red wine sample 1 fortified with 10 ng/mL of the target pesticides is shown in Figure 4. All the peaks, except for methamidophos, were sharp and offered reliable quantification. The satisfactory separation of the eight pesticides is also evident in the chromatogram. This allowed the data acquisition to be divided into three segments which ensured the optimal performance of the MS system, including dwell time (scanning speed) for each analyte.
Figure 4: Chromatogram of red wine sample 1 fortified with pesticides (10 ng/mL).
Accuracy and Precision Data
Red wine samples fortified with 10, 50, and 100 ng/mL of the target pesticides were extracted with QuEChERS and cleaned up using the rapid mini-cartridge filtration procedure. The recovery and relative standard deviation (RSD) data are listed in Table III. Recoveries of 81.6–112.2%, with an overall recovery of 97.0%, were achieved with this simple, rapid, and easy-to-use procedure. RSDs of four replicates for each of the three spiking levels were less than 10.8%, which indicated that this method is suitable for the determination of pesticide residues in red wine samples.
Table III: Accuracy and precision data
Application to Red Wine Samples
Six red wine samples were tested using the newly developed and validated method. The results of the red wine samples tested are listed in Table IV. Several of the pesticides were detected at low concentrations less than the method MRL. Carbendazim was the only pesticide detected above the MRL. The detected carbendazim concentrations were at 10.2, 8.7 and 2.3 ng/mL in samples 4, 5, and 6, respectively. However, the detected concentrations are much lower than the European (0.5 mg/kg) or Japanese (3 mg/kg) regulated levels in grapes.
Table IV: Pesticide residues detected in six red wine samples. The minimum reporting limit (MRL) of the method is 2 ng/mL.
A simple, fast, novel, and effective cleanup method for red wine samples was successfully developed. Pesticide residues in red wine samples were extracted using the nonbuffered QuEChERS procedure. Cleanup was carried out by passing 1 mL of the red wine extract through a mini-cartridge containing magnesium sulfate and PSA. The magnesium sulfate adsorbed residual water remaining in the acetonitrile extract, and the PSA sorbent retained matrix coextractives, including organic acids, sugars, phenols, and pigments. The cleanup method based on a filter-and-clean concept took less than 1 min per sample, thereby providing higher throughput than the traditional dSPE procedure. Cleaned extract was injected directly into an LC–MS-MS system for analysis. The analytical run required only 16 min and the target pesticides were chromatographically well resolved.
Good sensitivity and selectivity were achieved for the clean extracts obtained using the rapid mini-cartridge filter and LC–MS-MS detection. Good linearity, low LODs and LOQs, and satisfactory accuracy and precision data were obtained, indicating that this method was suitable for the analysis of pesticide residues in red wine samples. Six commercially available red wine samples were tested with the newly developed and validated method. Carbendazim was present in three red wine samples, although the detected concentrations were far below the European and Japanese regulated levels in grapes.
Thomas August and Lisa Snyder are acknowledged for the arrangement of the UCT products needed for this study. Catherine Messinger and Evelyn Scanlon are thanked for providing red wine samples. Dr. Brian Kinsella is thanked for proofreading the manuscript and providing valuable discussions and suggestions.
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Xiaoyan Wang and Michael J. Telepchak are with UCT in Bristol, Pennsylvania. Direct correspondence to: firstname.lastname@example.org.