Advances in MS Methods for the Detection of Pesticides in Food

Pesticides can be very effective at protecting food crops; however, there are food safety concerns over the levels of pesticide residues found in foods for human consumption. This article discusses the advantages and limitations of mass spectrometry (MS) applied to the detection of pesticide residues in food.

The world's population continues to grow, placing pressure on food producers to increase crop yields. The use of pesticides, fungicides, and herbicides to prevent crop spoilage therefore also continues to grow; however, there are concerns that the compounds and their residues can enter the food supply chain. Strict regulations and controls are in place to ensure that pesticides are used at safe levels because they can potentially have a negative effect on human health.

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Control of pesticide use is a worldwide problem. Different pesticides are used in different regions depending on the environment where the produce is grown. For example, in warmer climates such as Latin America, insects cause more crop destruction and the use of insecticides may be more common here than in other regions. It is therefore difficult to restrict the types of pesticides that can be used and this can cause complications when importing food from one country to another. Europe has the lowest maximum residue levels (MRLs) of pesticides authorized in food — MRLs in European Food Regulations (Regulation in EC No 396/2005) are typically between 0.01–10 mg/kg depending on the pesticide or combination of pesticides used. Countries outside of Europe tend to adhere to the same control measures because Europe is one of the biggest importers of food, especially fruits and vegetables. Fruits, vegetables, cereals, and grains are the foods that are the main focus of testing.

Professor Fernandez-Alba, head of the European Reference Laboratory for Pesticide Residues in Fruits and Vegetables from the University of Almeria (Almeria, Spain), estimates that between 300 and 500 different pesticides are used on food crops, requiring laboratories to analyze thousands of samples each year. He commented: "One of the most important tasks is to facilitate the communication between laboratories to cover the large scope of pesticides and to agree on an adequate limit of quantification."

Pesticide Detection Using Mass Spectrometry

Mass spectrometry (MS) is currently used in all pesticide testing laboratories and is considered a fundamental technique. An important advantage of MS over other methods is that it allows the detection of hundreds of compounds in a single run or single analysis. The most widely used MS techniques include liquid chromatography tandem mass spectrometry (LC–MS–MS) and gas chromatography tandem mass spectrometry (GC–MS–MS). Some new polar pesticides such as triadimenol, dimethomorph, and difenoconazole are only amenable to detection by LC techniques and therefore LC–MS–MS is becoming the more dominant method.¹

Mass spectrometry techniques have advanced over the years with developments in instrumentation improving analysis capability, selectivity, and detection limits. Several different analysis systems that can enhance tandem MS capabilities such as the triple quadrupole (QqQ), ion-trap (IT), quadrupole time-of-flight (QTOF), and quadrupole-orbitrap. Quadrupole MS systems have high ion-filtering capabilities that allow reliable detection and quantification of up to 400 residues in complex food matrices in a single run.² Triple quadrupole instruments are particularly popular because they have a multiple reaction monitoring (MRM) mode. MRM is a double mass filtering approach, which allows greater sensitivity, selectivity, and accurate quantification of many compounds in one analytical run.

In addition to advances in MS techniques improvements have also been made in the methods used to extract pesticides from food. For example, QuEChERS (quick, easy, cheap, effective, rugged, and safe) is straightforward to use, relatively cheap, and can separate lots of pesticides in a single extraction.³ Another advantage of newer methods is that they consume less organic solvent compared with more traditional methods.⁴

Case Study

Professor Fernandez-Alba and his team have developed a robust method that uses a micro liquid chromatography (micro LC) system coupled with a triple quadrupole system for the detection of pesticides.

LC Method Parameters: Column: 0.5 × 50 mm, 2.7-μm 90 Å HALO C18 (AB Sciex); mobile phases: acetonitrile and water (0.1% formic acid); injection volume: 3 μL; flow: 30 μL/min; total run time: 14 min

QqQ–MS Method Parameters: Source: ESI (+) with microFlow electrode (AB Sciex); pesticide identification: 2 transitions.

Source Parameters: Nebulizer and collision gas: nitrogen; gas temperature: 300 °C; curtain gas (CUR): 20; ion spray voltage (IS): 5000 V.

Micro LC uses smaller volumes of flow than traditional LC, that is 30 μL/min compared with 400 μL/min. This means that 10 times less solvent is required and the sensitivity of the technique is increased 20–40 times (see Figure 1).⁵ In full-scan mode the method increased sensitivity by 100-fold. The full-scan mode enabled more comprehensive compound identification using full-scan MS–MS fingerprints and compound library searching for enhanced screening workflows and simultaneous quantitation. Advances in MS methods allow contaminants to be rapidly detected at very low levels, with detection limits in the region of 1 μg/kg.⁶

Figure 1: Extracted ion chromatograms (XIC) for the multiple reaction monitoring (MRM)1:404→372 and MRM2:404→344 transitions of Azoxystrobin corresponding to a spiked orange matrix at a concentration of 10 μg/kg comparing a conventional LC at 400 μL/min flow rate (blue peak) with a microLC at 30 μL/min (red peak).

Limitations of MS Methods

One of the main difficulties faced when interpreting MS results are matrix effects; natural components in the sample matrix can affect the quantification of pesticides in food samples, with the same pesticide giving different results in different matrices (see Figure 2). Different approaches therefore have to be applied to quantify pesticides in one food matrix compared with another, increasing the time and cost.

Figure 2: Evaluation of matrix effects in pesticide multiresidue analysis by mapping natural components using liquid chromatography high-resolution mass spectrometry (LC–HRMS).

An effective approach to reduce matrix effects is to dilute extracts before LC injection (for example, 0.1 g sample/mL) as shown in Figure 3. Alternative instrumental methods can also help overcome the effect of food matrices — the use of micro LC allows the sample to be diluted 30 times, which can dilute away the matrix and reduce its impact on the measurements. Lower injection volumes and greater dilution results in less matrix being injected into the system, increasing the ability of the system to handle long runs of complex samples. Matrix interference makes it important for laboratories to use instrumentation and techniques that are very sensitive, and to adhere to methods that use dilution of samples. Matrix effects cannot be completely overcome, but the impact of matrix interference can be reduced and the speed of analysis can be increased.⁷

Figure 3: Different dilution factors applied for quantification of acetamiprid in different matrices including leek, orange, and tomato.

Future Directions

In the future, it is likely that more pesticide detection laboratories will adopt microflow or low-flow devices for chromatography coupled with high-resolution MS. Higher sensitivity MS techniques allow the detection of targeted compounds as well as the evaluation of unknown or non-target compounds. This will enable food safety scientists to analyze both compounds known to them (targeted compounds) and monitor other potential hazards (non-targeted compounds) present in a sample in the same analysis. Pesticide compound libraries that include hundreds of full scan MS–MS spectra for commonly tested residues and metabolites are also useful for laboratories to make comparisons and give confidence in results.

Professor Fernandez-Alba explained, "We are using MS instruments that are so much better than they were 10 years ago. We can correctly identify hundreds of pesticides in a single analysis with much higher sensitivity, meaning we can rapidly detect very low concentrations of pesticides in food."

Summary

Testing for pesticide residues in foods can be challenging because of the broad range of pesticides used, the use of multiple pesticides on a single crop, and the interference of matrix interfaces. However, advances in methods for quantifying the levels of pesticides in foods allow results to be determined more simply, quickly, and accurately. These improvements make it easier for regulatory laboratories to assess pesticide residue levels in foods, meaning that they can respond more quickly if samples are over the MRL. In the long term it is important to make sure that MRLs are adhered to globally and are well controlled to help to limit the negative effects of pesticides on human health and the environment.

References

1. A.R. Fernandez-Alba and J.F. García-Reyes, Trends in Analytical Chemistry 27(11), 973–990 (2008).

2. S. Grimalt, J.V. Sancho, O.J. Pozo, and F. Hernández, Journal of Mass Spectrometry 45(4), 421–36 (2010).

3. S.J. Lehotay, K.J. Son, H. Kwon, U. Koesukwiwat, W. Fu, K. Mastovska, E. Hoh, and N. Leepipatpiboon, Journal of Chromatography A. 1217(16), 2548–2560 (2011).

4. http://www.thenfl.com/blog/why-change-your-pesticides-screens

5. http://www.eurl-pesticides.eu/userfiles/file//EPRW2014.pdf

6. S. Lacorte and A.R. Fernandez-Alba, Mass Spectrometry Reviews 25, 866–880 (2006).

7. C. Ferrer, A. Lozano, A. Aguera, A. Jimenez Giron, and A.R. Fernandez-Alba, Journal of Chromatography A 1218, 7634–7639 (2011).

Ashley Sage is currently the senior manager of the European Market Development team at AB Sciex for the food and environmental business unit. After gaining his PhD in analytical chemistry in separation, mass spectrometry, and spectroscopic science, Ashley moved into the commercial world in 1996 when he joined Micromass as an applications scientist. In this role, he developed mass spectrometry solutions on both quadrupole, triple quad, and time-of-flight instruments for food, pharmaceutical, and life science applications. After seven years, Ashley moved into the business and marketing side of the scientific instrumentation industry and became the global product manager for the time-of-flight MS platform and continued to develop the use of high resolution MS for both life science and applied applications.

E-mail: ashley.sage@absceix.com

Website: www.absciex.com

This article is from The Column. The full issue can be found here:http://images2.advanstar.com/PixelMags/lctc/digitaledition/November07-2014-uk.html