Pesticides are used extensively on a global scale to protect crops, ensuring they can be successfully grown, stored, and transported to meet consumer demands. The type of pesticide used varies widely depending on the produce in question, with insecticides, herbicides, rodenticides, and fungicides being the most common. A recent review by the Pesticide Action Network showed that there are more than 17,000 pesticide products currently on the market.
Solvent-based pesticides have traditionally been the pesticide of choice, but in light of growing health concerns, less toxic ionic pesticides are being more widely adopted. For example, glyphosate—an anionic pesticide—is now the most widely used pesticide in the world on GMO-engineered glyphosate-resistant crops. Recently, though, there has been growing public concern that any pesticide contamination in food could be a potential health risk, especially as pesticides can often remain in food at trace levels. This has resulted in increased attention from regulatory agencies and health researchers, who are seeking to better understand and monitor these residues.
To ensure that only minimal levels of pesticides are present in food, accurate quantification is required. Many methods exist for determination of pesticides, but gas chromatography (GC) and liquid chromatography (LC) combined with mass spectrometry (MS) are the standard techniques in regulatory test methods; however, these traditional analytical methods aren’t as effective for determining ionic pesticides, as the compounds are too polar to be retained and separated. In addition, it is difficult to maintain low baselines when analyzing ionic pesticides, making them an analytical headache. These challenges in current analytical approaches have been driving the need for more effective analytical techniques to continue protecting public health.
Taking Charge with IC-MS/MS
Ion chromatography coupled with tandem mass spectrometry (IC-MS/MS) can be used to effectively overcome the challenges faced by existing methods when it comes to anionic pesticide determination (see figure 1). Crucially, the technique is ideal for separating polar compounds and has been used to determine anionic polar pesticides such as glyphosate and glufosinate.
IC-MS/MS has a number of benefits that make it ideally suited for this application. The technique offers high selectivity and sensitivity, as tandem MS detection using selected reaction monitoring (SRM) eliminates sample matrix interference by only scanning for ions of interest. The method also provides low chemical noise, overcoming the baseline issue of GC-MS and LC-MS. With this technique, analytes are also provided in their ionic form, meaning electrospray can be used and the molecular ion retained. Further improvements in pesticide determination are enabled by the electrolytic suppressor, which neutralizes eluent and lowers the background while offering increased sensitivity for conductivity detection and improving the compatibility for MS.
Anionic samples are typically prepared for IC-MS/MS using the quick polar pesticides method (QuPPE) developed by the European Union Reference Laboratory for Pesticide Residues in Fruits and Vegetables (EURL-FV). This acidified methanol-based extraction method has been widely used and accepted for extraction of polar pesticides, according to a 2012 review published in the journal Analytical and Bioanalytical Chemistry, giving excellent results. IC-MS/MS used with QuPPE extraction provides a highly useful and sensitive approach for anionic pesticide determination, ultimately helping analytical scientists to better protect public health.
The Rise of Cationic Pesticides
Cationic quaternary amines, or quats, are a new class of ionic pesticide now gaining popularity. Unlike glyphosate, quats are permanently charged species, regardless of pH. Of these, paraquat, diquat, mepiquat, and chlormequat (see figure 2) are among the most important and commonly used.
Although ionic pesticides are generally less toxic than solvent-based ones, compounds such as paraquat and diquat are still highly toxic. Often, these pesticides are used late in the plant’s life as desiccants to kill the plant before the harvest. By doing this, farmers can bring the crops in earlier, before they are contaminated with mold during the rainy season. While this practice helps to guarantee the food supply, the late addition of these pesticides to the crop can cause problems as they can bind to the plant, creating a higher risk of food supply contamination.
The use of these cationic pesticides, and the risk of contamination, varies globally. Paraquat, for example, is a restricted-use pesticide in the U.S., and neither paraquat nor diquat are approved in the EU, but chlormequat and mepiquat are allowed. Alongside country-by-country restrictions on usage of different pesticides, the permissible quantities of these pesticides vary too. For chlormequat and mepiquat, the EU’s Maximum Residue Levels (MRLs) generally range from 0.01 – 0.05 mg/kg. These differences in approvals and MRLs mean that for products to meet the individual requirements of different countries, it is essential to be able to chromatographically resolve different ionic pesticides from each other to allow separate quantitation.
But to date, cationic polar pesticide analysis has lagged behind analysis of anionic pesticides, even by IC-MS/MS. Most notably, analysis is hindered by poor chromatographic resolution and high costs. While the permanent charge of quats makes them highly effective as pesticides, this feature also makes them highly impractical to derivatize for detection. Second, it also means they adhere, often irreversibly, to glass, metal surfaces, and particles such as clay. This leads to tricky sample preparation, and means chromatographic separation is not reproducible.
IC-MS/MS: A Powerful Quat Pesticide Determination Approach
With recent improvements in column stationary phases, IC-MS/MS can now be used to tackle challenging separations of cationic polar pesticides, including paraquat and diquat, which has been exceptionally difficult due to their similar structures and close m/z values for molecular and fragment ions (a difference of less than 2 a.m.u) (see figure 3).
Thanks to these advances, IC-MS/MS has been used to analyze quat pesticide levels in a range of some of the most widely consumed foods, showing promising results.
Here, we highlight two such studies: one examining cereals, and the other investigating wheat flour, baby food, and tea. In these experiments, the samples were prepared for analysis using QuPPE or adaptations of it. Overall, the IC-MS/MS method provided adequate resolution of the analytes of interest from the rest of the complex food matrix, giving more accurate results.
Cereals are a principal component of many diets, yet the EU’s MRLs are much higher for pesticides in oat cereals. This is primarily because more pesticides are expected to be present in the produce due to higher levels used in cereal crop production. Matrix interference from complex samples makes it challenging to obtain accurate values, too, so this is factored into the MRL. With more analytical labs now using IC-MS/MS, these MRLs could be lowered in line with other produce, as matrix interference is reduced with IC-MS/MS.
The study in question demonstrated that quaternary amine pesticides can be accurately and sensitively determined in oat cereals within 15 minutes using IC-MS/MS. Here, the sample extraction followed QuPPE and was passed through a Thermo Scientific Dionex IonPac CS21-Fast-4μm ion exchange column paired with a triple quadrupole mass detector. For determination of paraquat, diquat, mepiquat, and chlormequat, recoveries of 86% to 118% were obtained, and limits of detection (LODs) <0.1 μg/L or 0.5 μg/kg (see table 1).
Wheat flour is another dietary staple across the globe, and USDA estimates that 131.1 pounds of wheat flour was consumed in the U.S. per capita in 2019. Grain and grain products have particularly complex matrices, making samples challenging to prepare for determination. A simplified version of the QuPPE method has been used for the extraction of anionic polar pesticides, and this approach was also used for extracting cationic polar pesticides from wheat flour in the second study. Using IC-MS/MS here delivered excellent results, with apparent recoveries in QuPPE extracted wheat flour ranging from 97% to 113% (see table 2).
MRLs in the EU for specific prohibited pesticides in baby food were previously set between 3–8 μg/kg; however, the European Food Safety Authority (EFSA) believes this may not be sufficiently protective for infants younger than 16 weeks of age. Yet, to date, there have been no further reductions to MRLs, as suitable analytical methods for detection with improved sensitivity are scarcely used.
IC-MS/MS can be used for effective determination of quaternary pesticides in baby food, though. Following the approach used with wheat flour (in the same study), the simplified QuPPE method can be used to prepare samples and extract pesticides from carrot baby food. The IC-MS/MS method, using the Dionex IonPac CS21-Fast-4μm ion exchange column paired with a triple quadrupole mass detector, worked extremely well, giving apparent recoveries of the pesticides ranging from 96% to 103% (see table 3).
Testing and regulation of beverages have also greatly increased over recent years. One of the most widely consumed beverages—tea—can suffer from pesticide contamination; that is, pesticides that remain in tea leaves can leach into the drink when hot water is added. Determination of these compounds is therefore essential.
To show the versatility of the IC-MS/MS method, tea infusions from both green tea and white tea were prepared as part of the second study and filtered for analysis by IC-MS/MS. The method effectively separated the four common quat pesticides, and corrected apparent recoveries were 94% to 102% for green tea and 92% to 106% for white tea (see table 4).
Paving the Way for Food Safety
Quaternary ionic pesticide use is growing, which is bringing many advantages to food production and distribution. While cationic compounds have typically proved difficult to analyze, advances in ion chromatography column technology are now enabling IC-MS/MS methods that can accurately and sensitively determine them while significantly simplifying analysis.
Eventually, as such IC-MS/MS approaches continue to gain traction for the analysis of quats, the possibility opens for MRLs to be lowered. These lower MRLs will drive improved agricultural practices, alleviating concerns for consumers and regulators and ultimately improving the protection of human health.
Man is product marketing manager specializing in ion chromatography at Thermo Fisher Scientific. Reach her at [email protected].