Determination of Pesticides in Red Wine by QuEChERS Extraction, Novel Cleanup with Rapid Mini-cartridge Filtration, and LC–MS-MS Detection

Oct 01, 2012

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.

Figure 1: Classes, structures, LogP, and pKa values of the eight pesticides selected in this study.
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 pK a 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.

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).