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 analysed by liquid chromatography–tandem
mass spectrometry (LC–MS–MS). The cleanup procedure was simple and required less than 1 min for each sample. Satisfactory
recoveries ranging from 81.6% to 112.2% with relative standard deviations less than 10.8% achieved. The linear dynamic range
was 2–400 ng/mL with a correlation coefficient greater than 0.9940. The limit of detection (LOD) and limit of quantification
(LOQ) 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.
Approximately, 26 billion litres of wine were produced worldwide and about 24 billion litres were consumed, according to the
International Organization of Vine and Wine, in 2010 (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 labourintensive, 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-fibre 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 clean up 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 sulphate and primary secondary amine (PSA)
sorbent, residual water is adsorbed onto the anhydrous magnesium sulphate, 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 fullscan chromatograms after cleanup
with four traditional dSPE approaches containing different amounts of PSA sorbent and the rapid mini-cartridge filtration
approach. The rapid minicartridge approach produced a slightly cleaner extract than the dSPE approach containing the same
amount of magnesium sulphate and PSA sorbent. However, the cleanup procedure with push-through minicartridge 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 analysed 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 analysed 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).
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