1st International Symposium
on Recent Developments in Pesticide Analysis

Abstract: The analysis of pesticide residues is undoubtedly one of the most challenging analytical tasks that a routine laboratory can get involved in. What makes pesticide residue analysis complicated? There are three major factors: a large number of potential analytes, a large number of potential sample matrix types, and low concentrations of pesticide residues that need to be detected, identified, and accurately quantitated. Modern analytical instruments provide sensitivity, selectivity, and speed, enabling large analytical scope (in terms of both analytes and matrices) and very low limits of quantitation. The increasing analytical scope and analyte detectability, however, bring additional challenges that routine laboratories need to deal with. On top of that, there are many other factors that contribute to the complexity of pesticide residue analysis, such as regulatory issues, lab accreditation requirements, global pesticide uses and global trade (sourcing of raw materials and distribution of products), or client/stakeholder demands, especially when it comes to turn-around-times. This presentation will discuss these challenges and ways to handle them in a routine pesticide residue testing environment.

Abstract: A myriad of toxic substances are released into our environment daily, either deliberately or accidentally produced. Aquatic systems throughout the world are increasingly under a wide array of anthropogenic stressors. The concentration of persistent organic pollutants (POPs) was assessed in wild pelagic and benthopelagic fishes in order to evaluate the relative ecological risk of these chemical compounds. The presence of twenty-seven heavy metals, selected congeners of polychlorinated biphenyls and organochlorine pesticides, was investigated in muscle, liver, and renal tissues collected from 46 wild fishes distributed among Thunnus thynnus, Pagellus bogaraveo, and Dentex dentex from the Mediterranean Sea. The mean concentration of each element was calculated and compared to the provisional tolerable daily or weekly intake (PTDI, PTWI) or to the established tolerable upper intake when possible. The maximum safe consumption (MSC) for adult intakes was achieved. Hg, Fe, Co, Ni, Zn, As, Se, Sr, Mo, Cd, Sn, Ba were variably present at higher level within muscular samples of the three different fish species. The maximum safe consumption calculated for mercury lead to a limited recommended weekly intake for all the tested fish species. In addition, Accelerated Solvent Extraction (ASE) was successfully optimized to extract and clean up in a single step the POPs compounds in combination with gas-chromatography and mass-spectrometry with triple quadrupole (QqQ).

Abstract: Thermo Fisher Scientific offers an incredibly wide range of LC-MS solutions for the analysis of pesticides in food and environmental samples. These range from specific targeted analysis using triple quadrupole systems, to targeted and non-targeted screening, confirmation and quantification using cutting edge Orbitrap technologies.

This presentation will focus on the use of LC-MS and IC-MS systems in multi-residue analysis of pesticides, and associated software solutions for such data analysis. Additionally, we will discuss on-line sample preparation techniques for improving productivity in high throughput laboratories.

Abstract: Some polar ionic pesticides and their associated metabolites are not “amenable” to common multi residue methods, such as QuEChERS. These compounds need alternative extraction conditions for improved recovery and/or alternative separation conditions for improved retention and/or peak shape. Currently the polar pesticides tend to be treated as a series of selective single residue methods adding significant cost to the analysis and therefore, are often excluded from pesticide monitoring programs. At Fera we have coupled the QuPPe generic extraction conditions with determination by suppressed ion chromatography tandem quadrupole mass spectrometry. This presentation will describe our experiences and challenges

faced in implementing this method, presenting validation data in a cereals matrix.

Abstract: Current lifestyle has caused that the society is looking for food alternatives in order to obtain a good nutritional diet in fast way. The large amount of bioactive substan-ces in plants has contributed to the appearance of nutraceutical products.

In general, various kinds of microbial contaminants could be found in medicinal plants. Such botanicals are widely regarded as valuable agricultural crops and pesticides may be used against mold and insects that may cause plant damage.

Nutraceutical products and dried herb materials typically represent very complex matrices for pesticide residue analysis. This is a greater challenge during the development of analytical methods, and sensitive instrumentation is required for detecting low concentration of pesticide levels1. The main objective of this study was the development of analytical methods based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction procedure for analysis of more than 170 pesticides by gas chromatography- tandem mass spectrometry (GC/MS-MS). New analytical methods were developed for the determination of pesticides in soya- based1 and ginkgo biloba-based2 nutraceuticals. Different extraction solvents and clean-up approaches were tested, obtaining the most efficient conditions with a mixture of sorbents (PSA, C18, GBC and Zr-Sep+). GC/MS-MS was performed with a triple quadrupole benchtop detector with electron ionization. Satisfactory validation parameters were obtained. Matrix-matched standard solutions were used to eliminate the matrix effect. The applicability of the method was proved by analyses of different types of nutraceuticals3. It is well known that pesticides could be degraded in the environment. These degradation products can be even more hazardous as the original compound.The transformation products of pesticides were identified by LC-Orbitrap-MS. Although these compounds were not included in the original method, they were monitored as post-target compounds.

Abstract: Analysis within the food safety is diverse due to the variety of matrices as well as in terms of numerous contaminants, from pesticides and mycotoxins, to veterinary drugs and processing contaminants. Depending on compound characteristics and the requirements of the analysis, different techniques may be used. Current mass spectrometry (GC-MS, LC-MS and high resolution accurate mass) instruments typically form the basis of multi-analyte methods, whereby hundreds of compounds can be screened and/or quantified at the same time. Needless to say a lot of data is generated, thus clear and fast data processing is required to integrate, identify and quantify the data accurately, and then to report the result in the minimum time possible all with minimum effort. Having one software solution for multiple techniques also reduces the training of use of different software for different techniques. In principal the approach by GC-MS is no different from LC-MS(/MS). Minor differences depend on requirements of the analysis and in some cases legislation. However, screening analysis will have different requirements to quantitative analysis. In triple quadrupole analysis there is a requirement to identification criteria by using a second SRM transition, whereas, in HRAM identification using a fragment ion, isotope ratio or matching a spectrum with a library might be preferred. In this presentation the Thermo Scientific™ TraceFinder™ software structure, workflows and options will be explained with examples obtained using different analytical techniques.

Abstract: Mediterranean rivers are characterized by important fluctuations in the flow rates and heavy contamination pressures from extensive urban, industrial and agricultural active-ties. This translates in contamination levels most often higher than in other European basins. For instance, the Llobregat River basin (Catalonia, NE Spain) suffers from ex-treme and frequent flow fluctuations (1–100 m3 s-1) and receives the effluent dischar-ges of more than 55 WWTPs, and at some points, especially in drought periods, the effluents may represent almost 100% of the total flow of the river. Thus, it is not strange to find considerably high levels of organic contaminants along the river and increasing concentrations when moving downstream due to the also increasing number of WWTPs and population pressure. Besides conventional contaminants such as pesticides, Hg, organochlorine compounds, polycyclic aromatic hydrocarbons (PAHs), alkylphenols and polybrominated diphenyl ethers (PBDEs), emerging contaminants such as pharma-ceuticals, some industrial compounds and polar pesticides represent an overgrowing portion of trace organic contaminants of concern in Iberian rivers such as those consi-dered in the present work: Ebro, Jucar, Guadalquivir, Ter and Llobregat.

This presentation summarizes the findings of several studies and monitoring programs conducted with the aim of getting a better insight about the sources and fate of polar pesticides in the aquatic environment in Spain. The occurrence of these contaminants is discussed in relationship to their removal in WWTPs, influence of hydrological condi-tions in the receiving river waters, sediments and dilution factors. The removal of some of them (e.g., triazines, organophosphates, phenylureas, acidic herbicides, chloroacet-anilides) with advanced water treatments and their toxicity is also presented. In addi-tion, the risk posed by these emerging contaminants in both surface and effluent waste waters was assessed towards different aquatic organisms (algae, daphnids and fish) by determining hazard indexes. Whereas in concentration terms pollution loads are domi-na-ted by industrial compounds and pharmaceuticals, risk is mainly attributable to pesti-cides. These risk based indexes are further correlated with some biological descriptors characterizing both the structure and functioning of the receiving aquatic ecosystems such as biofilms or the macroinvertebrate community.

The study completes with an analysis of the strategies under discussion within the European Countries in order to reduce the environmental risk posed by the occurrence of the most critical emerging contaminants in surface waters. In particular the recent European directive 2013/39/UE as regards priority substances in the field of water policy and the Swiss Micropoll Strategy are presented and their main issues discussed in terms of relevant compounds to monitor regularly, priority actions to reduce their occurrence, and best techniques to adopt in upgrading existing wastewater treatment plants

Abstract: Pesticides used in agriculture can be transported during rain events to surface waters, where they can have adverse effect on aquatic organism even at low concentration levels. Thus, the regular monitoring of pesticides in surface water must be undertaken. In Europe, pesticides with high application rate, mobility, persistence and toxicity are typically included in monitoring campaigns along with the regulated priority substances as defined in the European Water Framework Directive (WFD) in order to assess the water quality. As new pesticides are constantly released on the market and the pesticide use pattern can vary strongly on a regional scale, the adjustment of monitoring program to river basin specific pesticides is challenging and relevant pesticides can be easily overseen. The goal of this study was to perform a complete analytical pesticide screening, assess the associated risk for the aquatic environment and compare this with the risk obtained when only the subset of pesticides as required by the WFD is analyzed.

Five rivers impacted by agricultural activities in the Swiss plateau were investigated. Biweekly composite water samples were analyzed over 6 months by a screening method using solid phase extraction (SPE) liquid chromatography coupled to high resolution tandem mass spectrometry (LC-HRMS/MS, Thermo Scientific™ Q Exactive™MS). The acquired data were processed using a target and suspect screening approach. Using this method 86% of all registered synthetic organic pesticides in Switzerland (310) could be detected in low concentrations (0.5-10 ng/L).

The results of the complete screening showed that on average 30-40 pesticides were detected per sample. The sum of individual risk quotients following the concept of concentration addition for plants and invertebrates exceeded the critical value by factors of 4-6 and 2-3, respectively. It was found that only 50% of the detected substances would have been measured if a subset of only 30-40 pesticides had been investigated. Also 30-40% of the total risk, especially the risk caused by insecticides would have been missed by such a reduced monitoring program, because relevant substances such as neonicotinoids were not included in the subset of analytes. The findings clearly show that only a complete pesticide screening can reliably represent the water quality of a river.

Abstract: Shoot-and-Dilute GC-MS/MS uses split injection paired with a very sensitive triple-quadrupole mass spectrometer to alleviate matrix-related issues occurring at the GC inlet and column. There are well-known problems associated with splitless injection of dirty samples (e.g. QuEChERS extracts), most notably compound degradation and the loss of relatively active and involatile pesticides. These issues lead to inaccurate quantification, and in some cases, completely missing the pesticide of interest. GC inlet problems can occur very quickly with real samples, sometimes with a single splitless injection of a particularly dirty sample. This leads to time-consuming inlet and column maintenance to restore instrument performance.

An easy way to mitigate the above problems is to use split injection when possible.

If LOD and LOQ requirements are achievable using split injections, increased flow through the inlet minimizes residence time inside the inlet liner, which decreases sensitive pesticide degradation. In addition, GC oven start temperature can be higher, thus reducing overall run time as well as the time needed to re-equilibrate the GC oven.

Shoot-and-Dilute GC-MS/MS was tested using a screening method for multi-class pesticides in celery and orange QuEChERS extracts and compared with a splitless injection method.

Abstract: Prince Edward Island produces over 30 percent of Canada’s total potato production annually. Correspondingly, pesticide use in PEI is substantial, with as many as 20 different pesticides applied to potato crops each season. Several of these pesticides are classified as “probable human carcinogens” by the U.S. Environmental Protection Agency. Pesticides can leach into the highly permeable, fractured sandstone aquifer, contaminating the sole source of drinking water for PEI residents. Both the degree of exposure to pesticides and the quantitative characterization of pesticide disposition in the body remain unknown.

The primary objective of this study is to characterize exposure to 27 pesticides in groundwater, air, soil and exposed individuals in PEI. The role of eccrine sweat glands in ridding the body of bioaccumulated pesticides will also be investigated through identification and quantitation of pesticides and key metabolites in sweat and compared with concentrations in blood and urine. All samples will be analyzed using ultra high-pressure liquid chromatography (UHPLC) and gas chromatography (GC) coupled with mass spectrometry (MS). The Thermo Scientific™ Accela™ UHPLC System coupled with the Thermo Scientific™ Exactive™ Plus Orbitrap MS will be employed for analysis of 12 polar and highly water-soluble analytes, including diquat, glyphosate and metabolite AMPA; preliminary trials using an ion-pairing reagent have resulted in the successful retention of diquat. The Thermo Scientific Focus GC and ISQ single quadrupole MS will be used to analyze 15 pesticides and metabolites, including chlorothalonil, phorate and atrazine. This study will provide critical exposure data through analyses of air, soil and groundwater, potentially major sources of pesticide exposure, and human sweat as a possible excretion route. Ultimately, pesticide excretion in sweat may be a vital means of eliminating deleterious pesticides that may otherwise accumulate in the body over time and may lead to the development of cancers.

Abstract: The Thermo Scientific™ Q Exactive™ LC-MS has been evaluated for detection, identification and quantitation of pesticide residues included in the European Union Monitoring Program. Pesticides were analyzed in four matrices representing wide range of difficulty (tomato, pepper, orange and green tea).

The first objective was to compare and then select the optimum resolution in full scan analysis. Resolutions of 17,500, 35,000 and 70,000 FWHM (m/z 200) were studied. The lowest resolution was found to be insufficient because of numerous false negative results (with 5 ppm mass tolerance). A resolution of 35,000 assured good results in tomato, pepper and orange, however for analysis of green tea only 70,000 could be recommended. It was also observed that an increase of resolution improved chromato-graphic peak shape and peak area reproducibility. The response for all of the investi-gated pesticides were linear up to 500 µg/kg, and no saturation effects were observed within the range (10-500 µg/kg).

Furthermore a second part was focused in the simultaneous application of MS and MS/MS operating in Data Dependent mode. In this mode data were acquired in full scan, but if an ion from a predefined inclusion list was detected, the Q Exactive system switched to MS/MS and carried out one scan. Pre-studied fragment ions and ratio abundance were selected for identification. Full scan data was acquired at a resolution of 70,000, whereas MS/MS data were acquired using 17,500. Atf 10 µg/kg, 97% of pesticides in tomato extract were detected confirmed by at least one fragment ion, whereas in green tea (the most difficult of analyzed matrices) 86%. Finally, Data Dependent mode was applied for analysis of 100 real samples of fruits and vegetables. Identification by MS/MS fragments helped to discard all false positive results obtained in full scan. Obtained results showed that, despite very high resolution applied in full scan mode (70,000), MS/MS identification was necessary. Quantification was very close to the obtained by the application of LC-QqQ-MS.

Abstract: Based on the proven Thermo Scientific™Q Exactive™ platform, the Q Exactive Focus MS provides durable and reliable performance for routine laboratories doing automated screening, profiling and quantification analysis. Full scan confirmation mode and parallel reaction monitoring (PRM) provide reproducible quantitation results and targeted screening capabilities. Variable data-independent analysis (vDIA) provides complete qualitative coverage for unknown screening without compromising proven quantitative attributes.

The Q Exactive Focus system provides best in class data quality at a value-price. It is ideal for routine labs performing food safety residue analysis, environmental analysis, forensic toxicology, sports doping, clinical research, metabolomics, and routine pharmaceutical analyses.

Abstract: Pesticide residue analysis is a challenging task because of the high number (>800) of substances that needs to be analyzed. Of these, some 100 are often present while the others are found only at (very) low frequencies. This calls for two analytical approaches: a quantitative method with all associated analytical quality control (AQC), and a screening method to cover as many as possible additional pesticides with minimal efforts in terms of data handling and AQC-burden, respectively.

With the recent improvements in high resolution MS it is possible to combine the two approaches in one measurement. This presentation will focus on application of full scan analysis using chromatography coupled to Q Orbitrap mass spectrometry. The different non-target acquisition options of the instrument will be explained.

For LC, the combination of full scan and vDIA (variable Data Independent Acquisition) was found to provide optimal sensitivity selectivity and identification. Both quantitative and qualitative performance were evaluated according to the EU guidelines for validation and AQC (SANCO/12571/2013) and it is demonstrated that the new technology is fit-for-purpose and can be applied as a more efficient and effective alternative to LC-triple quadrupole MS/MS.

For pesticides not amenable to LC-MS, it would be highly desirable to follow a similar approach in GC analysis. However, unlike LC, there has been a gap in the capabilities of full scan MS for GC in terms of mass resolving power, sensitivity and/or quantitative performance. The coupling of Orbitrap MS to GC may fill this gap. The first results of the use of a GC-Orbitrap system will be presented and are promising. It is foreseen that within the near future, full scan high resolution MS could replace triple quadrupole MS/MS as main stream detector in chromatographic residue analysis.

Abstract: This presentation will discuss the results and learning experience from a joint collaboration between the Fera Science Ltd., UK and Thermo Fisher Scientific. The sensitivity, robustness, speed, and ease of use of the Thermo Scientific™ TSQ™ 8000 GC-MS/MS and TSQ™ 8000 Evo GC-MS/MS systems were evaluated for the routine multi-residue analysis of pesticides in food. Extracts have been analysed for the detection, identification, and quantification of more than 100 pesticides at concentrations as low as 1 ng/g in a diverse range of sample matrices. Mean recoveries for the majority of pesticides spiked at 1 ng/g in baby foods were 80–100% with associated %RSDs below 20, which is in compliance with SANCO/12571/2013. To obtain satisfactory linearity and precision requires careful optimisation of injector parameters, including temperature, injection volume, choice of liner, retention gap, etc., especially for the direct-injection of QuEChERS acetonitrile extracts. Information will be provided on maintenance procedures to obtain consistent performance during the analysis of large numbers of extracts over the period of more than one year. The increase in productivity and screening options possible due to the ultra fast acquisition rates of the TSQ 8000 Evo GC-MS/MS systems will also be highlighted.

Abstract: Among recent official methods introduced by the U.S. Environmental Protection Agency (U.S. EPA) EPA Method 1699 can be found [1]. This method is used for the determi-nation of organochlorine, organophosphorus, triazine and pyrethroid pesticides in environmental samples by high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS) using isotope dilution and internal standard quantitation techniques. This EPA method is generally applied to aqueous, solid, tissue and biosolids matrices. The aim of our study was to extend the scope of applicable matrices for this method to include food samples. Furthermore the compatibility of this method with QuEChERS extracts has been investigated. Tea and Rucola salad samples were prepared via the QuEChERS method. Extracts were analyzed according to EPA method 1699 on a Thermo Scientific™ DFS™ High Resolution GC-MS coupled to two Thermo Scientific™ TRACE™ Ultra GC supported by an extra-wide Thermo Scientific™ TriPlus™ autosampler. Standards according to the EPA method were self prepared and a high resolution MS multi window selected ion monitoring (SIM) method was set up including the usage of suitable reference masses (FC43).

A mass spectrometer resolution of 10,000 (10 % valley) was employed for ultimate selectivity. Extracts and standards were injected in splitless mode via a temperature programmable PTV injector on a 30 m DB17ms column (0.25mm ID, 0.25 um film thickness). The instrument sensitivity using this EPA method was proven with standard measurements and resulted in the low pg/ul (ppb) range. For a number of components even sub ppb sensitivity can be achieved, which includes pestizides with strong tendency to fragment like Dieldrin, Aldrin, Endosulfans, Endosulfansulfate.

The measurements of QuEChERS extracts from different food samples showed very good selectivity for most of the targeted pesticides with sensitivity well in the range requested by food regulations.

Abstract: Pesticides represent a diverse group of chemicals that have been used to kill or

control undesired insects, weeds, rodents, fungi, bacteria or other organisms.

The use of pesticides and other chemicals in the production of food has increased considerably over the past 35 years, polluted the environment, threatens beneficial insects (e.g., bees) and wildlife species (e.g., birds) and may pose a risk to human health and the environment.

Consistent sampling methods coupled with sensitive, selective and reliable analytical methods are required to monitor pesticides in environmental matrices.

A new analytical workflow for the quantitative determination of 198 pesticides and qualitative screening of 603 targeted pesticides is presented. Field grab samples were analysed by direct aqueous injection, liquid chromatography separation and high reso-lution Orbitrap mass spectrometer (LC-Orbitrap MS) under full mass scan and data independent data acquisition modes to achieve good data quality and operation efficiency. Polar organic chemical integrative samples collected from surface water and waste water treatment plant effluents were ultrasonically extracted and analysed by using the same LC-Orbitrap MS. Target pesticides were identified by LC retention time (RT), isotopic patterns, EU SANCO/12571/2013 criteria and library search; and quantitated by area counts obtained from extracted ion chromatograms using a mass extraction window of 5 ppm. The same data were also used in the screening of non-targeted pesticides and depending on the occurrence, can be included in the targeted lists for future monitoring needs.