Determination of Neonicotinoid Insecticides and their Metabolites, and Strobilurin Fungicides in Atmospheric Particles using Liquid Chromatography-Positive Electrospray Ionization-Tandem Mass Spectrometry

Special Article - Pesticides

Austin Environ Sci. 2021; 6(1): 1052.

Determination of Neonicotinoid Insecticides and their Metabolites, and Strobilurin Fungicides in Atmospheric Particles using Liquid Chromatography-Positive Electrospray Ionization-Tandem Mass Spectrometry

Behdarvandan A and Raina-Fulton R*

Department of Chemistry and Biochemistry, University of Regina, Canada

*Corresponding author: Renata Raina-Fulton, Department of Chemistry and Biochemistry, University of Regina, 3737 Wascana Parkway, Regina, Saskatchewan S4S 0A2, Canada

Received: January 18, 2021; Accepted: February 09, 2021; Published: February 16, 2021


A liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous determination of metabolites of neonicotinoid insecticides with neonicotinoid insecticides and strobilurin fungicides in the particle phase fraction of atmospheric samples. Pressurized solvent extraction was used to extract the target analytes from particles collected on glass fiber filters, followed by a C18 SPE cleanup step. Recoveries of 85.9 to 108.3% and relative standard deviation <13% were obtained for all analytes. The method detection limit for metabolites of neonicotinoid insecticides were 0.5-3 ng/ mL (air concentrations of 0.44-2.66 pg/m³). Matrix effects were compensated for using standard addition calibration for quantitation. This paper provides the first detection of desmethyl-thiamethoxam which coincided with higher concentrations of thiamethoxam in atmospheric particles. Other neonicotinoid insecticides and strobilurin fungicides detected in the particle phase in the atmosphere included acetamiprid, clothianidin, imidacloprid, azoxystrobin, kresoxim-methyl, pyraclostrobin, and trifloxystrobin. This research highlights the potential of not only neonicotinoid insecticides and strobilurin fungicides moving in the atmosphere in the particle phase, but also a metabolite of thiamethoxam (desmethyl-thiamethoxam) in a region with orchards and vineyards where foliar applications with air blast sprayers dominate.

Keywords: Metabolites; Neonicotinoid insecticides; Strobilurin fungicides; Liquid chromatography-tandem mass spectrometry; Desmethyl-thiamethoxam; Atmospheric particles


dSPE: Dispersive Solid Phase Extraction; GC-MS: Gas Chromatograph-Mass Spectrometry; LC-ESI+-MS/MS: Liquid Chromatography-Positive Electrospray-Tandem Mass Spectrometry; MDL: Method Detection Limit; QuEChERS: Quick, Easy, Cheap, Effective Rugged, and Safe; SPE: Solid Phase Extraction; US-EPA: United States Environmental Protection Agency.


Neonicotinoid insecticides have been identified as a concern to the health of pollinators and aquatic insects [1-3]. Health Canada published three pollinator re-evaluations in April 2019 and subsequently cancelled the uses of three neonicotinoid insecticides (thiamethoxam, clothianidinm and imidacloprid) in Canada for foliar applications on crops including pome fruit and stone fruit, while stopping spraying on some other crops such as berries and fruiting vegetables, before and after bloom [4]. Further decisions and re-evaluations of these neonicotinoid insecticides in agriculture in Canada are still pending due to COVID-19 restriction delays and expected by 2022. In January 2020, the United States Environmental Protection Agency (US-EPA) also published their proposed interim decision for several neonicotinoids (thiamethoxam, clothiandin, imidacloprid, acetamiprid, and dinotefuran) which allows for continued usage in agricultural regions with label changes to reduce spray drift and runoff [5-9]. There were no specific changes to requirements for air blast sprayers which are commonly used in orchards and vineyards. Thiacloprid was canceled voluntarily by the registrant such that its registration review was cancelled in 2014. In 2018, the European Union also extended the ban on the use of neonicotinoid insecticides to all field crops as a result of growing evidence that these insecticides cause harm to pollinators such as honey bees [10]. Fungicides have also been identified as a potential concern to bee health and our previous study identified that some fungicides can be transported in the atmosphere in both the gas and particle phase [11-14].

A liquid chromatography-positive ion electrospray-tandem mass spectrometry method was developed for the simultaneous analysis of neonicotinoid insecticides and strobilurin fungicides in particles in the atmosphere at trace levels and provided the first detection of these active ingredients in particles in the atmosphere in an agricultural region with orchards and vineyards where appliations are typically using air blast sprayer [13]. The Okanagan Valley in Canada is in close proximity to the Okanogan County of Washington State in the United States with both regions having similar crops (orchards and vineyards dominant) and foliar spray applications of neonicotinoid insecticides and strobilurin fungicides. We aimed to assess whether with regulations and best management practices that existed in 2016 and 2018 if there were neonicotinoid insecticides and strobilurin fungicides as well as metabolites of neonicotinid insecticides present in particles in the atmosphere in this region of the United States.

Metabolites of neonicotinoid insecticides have been identified from plant and animal studies but measurements in environmental media have been very limited due to analytical method development changes [15-18]. Consequently, we screened for viable target metabolites of neonicotinoid insecticides from plant extract studies, water quality studies, or measurements in biological fluids, that could be analyzed with selective methods using Liquid Chromatography- Positive Electrospray Ionization Tandem Mass Spectrometry (LCESI+- MS/MS) to assess the feasibility for analysis of an environmental sample matrix at trace level [19]. Based on this review we included desmethyl-acetamiprid, desmethyl-Thiamethoxam (dm-THM), imidcloprid-urea, imidacloprid-olefin, and 6-chloronicotinic acid (a common metabolite of acetamiprid, clothianidin, dinotefuran, imidaclorpid, nitenpyram, thiacloprid, and thamethoxam) into the method development for a new method that could provide simultaneous analysis of metabolites of neonicotinoid insecticides with neonicotinoid insecticides and strobilurin fungicides and improve on the recovery of selected target analytes such as nitenpyram, a less commonly studied neonicotinoid insecticide with prior methods with recoveries as low as 60% in some sample matrices [13,20]. Metabolites of neonicotinoid insecticides are also more polar than their parent active ingredients and more prone to losses in sample preparation often requiring selective methods [19].

The neonicotinoid insecticides and strobilurin fungicides were identified in our studies as candidate active ingredients in formulations used in foliar applications in orchards and vineyards for transport in the atmosphere in the particle phase due to their low volatility. Metabolites were also of interest due to the large variation in concentrations of neonicotinoid insecticides observed in particles in the atmosphere in an adjacent agricultural region (Okanagan Valley in Canada) such that it was proposed that breakdown of the active ingredients may be occurring before or during atmospheric transport [13,19]. This study represents the first analysis of atmospheric particles for metabolites of neonicotinoid insecticides worldwide and was of interest to determine if metabolites of neonicotinoid insecticides were present in the atmosphere on particles in a region with expected foliar spray applications such that atmospheric transport could be a new pathway of transport in the environment. The Okanogan County and some surrounding counties of Washington state has both historical usage and usage of both neonicotinoid insecticides and strobilurin fungicides [21,22]. The target list of analytes was also expanded to include one chemical alternative to neonicotinoid insecticides, sulfoxaflor, which is a more recently registered sulfoximine insecticide and is currently registered for use in the United States [21]. Sulfoxaflor has four stereoisomers (2S,3S-sulfoxaflor, 2R,3Ssulfoxaflor, 2S,3R-sulfoxafor, and 2R, 3R-sufoxafor) and has not been included in other methods for neonicotinoid insecticides and in the analyses of food matrices has been analyzed in a selective method for only sufoxaflor or sufoxaflor and its two metabolites using either normal phase, reversed phase or chiral LC separations [23-27]. In addition, we also added picoxystrobin to the list of target analytes in the method as it is not commonly included in analyses of strobilurin fungicides as it can be more challenging to analyze with LC-MS/MS as compared to GC-MS methods which can be used for the analysis of strobilurin fungicides, but GC is not typically used for analysis of neonicotinoid insecticides [28-36].

Sample preparation methods for neonicotinoid insecticides and strobilurin fungicides have largely been developed for other matrices including honey, pollen, fruits and vegetables or soil often involving modified QuECHERS methods but these methods are not typically used for analysis of air samples [36]. Sampling for pesticides and other semi-volatiles in the atmosphere uses materials such as quartz fiber filters for the collection of particles with the pesticides most commonly extracted from these materials into an organic solvent with pressurized solvent-extraction followed by clean-up with solid phase extraction (SPE) or dispersive solid phase extraction (dSPE) methods. Materials used for air sampling have been shown not to contributed significantly to the matrix issues observed [13,14]. A uniquely aspect to atmospheric sample analyses is that the composition of the matrix can vary significantly over time (collection period during the agricultural season) and is often unknown due to the variety of other semi-volatile organics that can also be collected with high-volume air sampling with the concentration of these semi-volatile organics expecting to change with varying contributions from agricultural, industrial, and residential emission sources, and other combustion sources such as forest fires which have increased in occurrence in the study region. Consequently, calibration methods used for quantitation typically include the use of deuterated internal standards (internal standard calibration) or standard addition calibration rather than matrix matched calibration standards.

The goal of this study was to determine if metabolites of neonicotinoid insecticides, neonicotinoid insecticides, and strobilurin fungicides were present in particles in the atmosphere at Omak, WA. The sampling location was selected to be at Omak in Washington State as this county (Okanogan County) has a higher proportion of apple orchards and vineyards than the Okanagan Valley in Canada, which has more variation in stone and pome fruit trees. There is known usage of neonicotinoid insecticides and strobilurin fungicides regionally (Okanogan county and surrounding counties) in 2016 and 2018 [21,22].

Materials and Methods

Chemical and general details

Ethyl acetate, acetonitrile, and methanol were of pesticide grade and supplied by Fisher Scientific. Deionized water with resistivity <18MO cm was obtained from Nanopure Diamond system (Barnstead International, Dubuque, IA, USA). Formic acid with concentration >88.0% was obtained from VWR Scientific (West Chester, PA, USA). Solid Imidacloprid-d4 (IMI-d4), Clothianidin-d3 (CLO-d3), and Thiamethoxam-d3 (THM-d3) were obtained from C/D/N Isotopes Inc. (Pointe Claire, QC, Canada). Solids or stock solutions at 100μg/mL in acetonitrile or methanol of strobilurin fungicides (Azoxystrobin (AZOXY), Dimoxystrobin (DIMOXY), Fluoxastrobin (FLUOXA), Kresoxim-Methyl (KRES), Picoxystrobin (PICOXY), Pyraclostrobin (PYRA), and Trifloxystrobin (TRIFLOXY)) and neonicotinoid insecticides (Acetamiprid (ACE), Clothianidin (CLO), Dinotefuran (DIN), Imidacloprid (IMI), Nitenpyram (NIT), Sulfoxaflor (SULF), Thiamethoxam (THM), and Thiacloprid (THC)) and metabolites of neonicotinoid insecticides (6-Chloronicotinic Acid (CINA), Imidacloprid-olefin (IMI-olefin), Imidacloprid-urea (IMI-urea), Desmethyl Thiamethoxam (dm-THM), and Desmethyl Acetamiprid (dm-ACE) were purchased from Chem Service Inc. (West Chester, PA, USA).

Pesticide standards

Thiamethoxam-d3 (N-methyl-d3) (THM-d3) was used as a surrogate to evaluate recoveries in samples (SRMs 295.1→132.0 (cone voltage 20, collision energy 15); 295.1→184.0 (20, 22);. CLO-d3 (SRMs 253.1→172.1 (17, 15); 253.1→132.0 (15, 12)); was used to determine the final volume of an extract of the dried fraction F1 from the SPE cleanup step after the addition of internal standards and was approximately 1.0 mL. IMI-d4 (SRMs 260.1→179.0 (20, 14); 260.1→213.1 (20, 14)); was used as an internal standard for calibration purposes. Matrix effects for assessed for all samples collected in 2016 and selected samples in 2018 due to the expected potential influence of forest fires. The internal standard was not used in the determination of slopes from the solvent-based and standard addition calibration curves in the % ME calculation shown in Supplementary material, but was used in the standard addition calibration curves used for quantitation.

Individual stock solutions of pesticides in methanol were prepared by dissolving solids of individual pesticides (~1 mg) in 1 mL of methanol and stored at -4°C. Calibration standards were prepared from a stock solution containing a mixture of the standards at 1.000μg/mL in methanol. Internal Standard (IS), IMI-d4 at 75ng/ mL was used in calibration standards and all samples. Preparation of samples and calibration standards were carried out on the analysis day. The calibration range for solvent-based calibration curve was MDL -30ng/mL, but could be extended to 100ng/mL if required. The lowest prepared calibration standard was generally selected to be 0.5ng/mL. Standard addition calibration was completed with the dilution factor of the sample of ½ with standard concentrations added also to 30 ng/mL with IMI-d4 added as the internal standard at 75ng/mL.

Sample collection and preparation of particle extracts

Polyurethane Foam (PUF) air sampler (TE-1000BL, Tisch Environmental) was used to collect air samples at the Confederated Tribes of the Colville Reservation operated air monitoring site located at Omak within the Okanogan County. The PUF sampler motor operated at a flow rate of ~225 L/min with an air volume of approximately 350 to 370 m³ per day with most samples collected continuously over a 2-week sampling period. Air samples were collected from during 2016 from March 12 to August 30, and from May 23 to September 05, 2018. For the purposes of matrix evaluation, 3 samples from 2018 with higher atmospheric particle concentrations and a sample collected at the end of the agricultural season were used to assess potential additional matrix effects from wildfire sources to the atmosphere.

In the top portion of the sampling module a quartz microfiber filter (10.2cm diameter, Tisch Environmental) is inserted between two teflon rings. Filters are weighed in a glove bag under nitrogen atmosphere to ±0.00002g before and after sampling. The lower portion of the sampling module contains a glass cartridge with polyurethane foam (PUF, 12.7cm length X 7.3cm diameter) for gas phase concentrations of pesticides, which were not analyzed as part of this study. The Polyurethane Foam (PUF) was purchased from Tisch Environmental and certified to be flame retardant free and was pre-cleaned using pressurized solvent extraction with ethyl acetate as the extraction solvent using the same extraction procedures as sample extraction. The PUFs were air dried in the dark prior to use. The sampling modules are shipped to the sampling site or the materials are exchanged during instrument calibration visits. The sampling module is equipped with a PM2.5 cyclone designed such that particles <2.5μm are collected on filters. Particle concentrations (PM2.5) reported herein were determined by gravimetric analysis of filters obtained from the high-volume air sampler and ranged from 1.4 to 23.8 μg/m³ during 2016 and 10.2 to 75.7 μg/m³ during 2018 sampling.

Extraction and cleanup

The quartz filters were extracted according to Raina-Fulton method with modifications described herein briefly [13]. The filters were transferred to 30 mL extraction cells and extracted using an ASE100 pressurized solvent extraction system (Dionex, Sunnyvale, CA, USA) with ethyl acetate as the extraction solvent. The extraction procedure held the cell at 100°C and pressure of 1500 psi during the 30 min static mode followed by a 60% flush with ethyl acetate of the volume of the cell. Three static stages were used to ensure complete extraction of the target analytes from the particles collected on the quartz filter. A second extraction showed no detectable levels of target analytes. At the end of the extraction, the extraction cell is purged with nitrogen for 600s. The total extraction volume is approximately 70 mL. To this extract 1 mL of 2-propanol added as a keeper for the drying step.

This extract from pressurized solvent extraction s concentrated to <2mL, transferred to 15 mL vials, and dried again to ~1mL at 0.5mL/ hr with a slight vacuum <1kPa. All extracts are stored at -4°C until sample cleanup. Sample cleanup of the extract was completed using C18 SPE (6mL, 1000mg, Canadian Life Science, Peterborough, ON, Canada) and the cleanup was modified from the prior method13 to improve recoveries of metabolites of neonicotinoids. C18 SPE cartridges were conditioned with 6 mL of methanol and 6 mL of water. The sample extract (500μL) and 3μL of 1μg/mL THM-d3 were loaded onto the preconditioned SPE tube. This was followed by loading of 450μL of water, which was eluted into the F0 fraction and contained no target pesticides or metabolites. The pesticides of interest (neonicotinoid insecticides, metabolites of neonicotinoid insecticides, and strobilurin fungicides) were eluted into a fraction, F1, with 5mL of 100% methanol. A volume of 50μL 2-propanol was added to the F1 extract prior to drying. The eluted extracts from SPE were concentrated to ~1mL at 0.5mL/h with a slight vacuum <1kPa with an additional 50μL of 2-propanol added when the volume was reduced to 2.5mL and 1.5mL. The final volume of this fraction was measured by adding 20μL of 10μg/mL clothianidin-d3 (volume check standard) and ranged from 0.5-1.1 mL. Extracts were then generally diluted with methanol at a dilution factor of 1/2 prior to LC-MS/MS analysis with internal standard, imidacloprid-d4, added at 75ng/mL. A second fraction eluted from the SPE cartridge shown no detectable levels of target analytes or THM-d3. The amount of pesticides measured in the extract was divided by the total volume of air sampled during each sampling period to determine concentrations of pesticides (pg/m3) in the atmosphere in the particle phase.

LC-MS/MS analysis

A Waters LC system consisting of a 1525μ binary pump and column heater was utilized to conduct LC analysis. A LEAP Technologies autosampler (Carrboro, NC, USA) was used for 10μL injections at 100μL/s. In order to minimize the sample carry-over, two pre- and post-cleans with ethyl acetate followed by methanol were carried out. A guard column (4×2.0 mm, C18 Gemini) was connected to phenyl-hexyl column, 50×2.00 mm i.d., 2.5μm (Phenomenex, Torrance, CA, USA). It was then placed in a column heater at 30°C. Initial mobile phase was 40 v% acetonitrile containing 0.1 v% formic acid and 60 v% water with 3 v% methanol, 2 v% 2-propanol, 0.05 v% formic acid at a flow rate of 0.15mL/min. The following linear gradual change in mobile phase gradient of acetonitrile with 0.1 v% formic acid was applied: 0.0 min, 15 v%;1.5 min, 30 v%; 3 min, 33 v%; 7 min, 40 v%; 10 min, 50 v%; 12 min, 55 v%; 15 min, 70 v%, held for 3 min. The elution of all analytes was completed in less than 18.0 min. A pre-injection of 10μL of 2-propanol with a 20 min elution time at initial mobile phase conditions was also used to minimize carry-over issues as some samples exhibited high matrix interferences and this was found to improve retention time stability (±0.1 min) and column performance over time.

The Waters LC system was attached to a Quattro Premier (Milford, MA, USA) triple quadrupole with electrospray ionization in positive ion mode (ESI+). The temperature of the source was set to 120°C, desolvation temperature of 300°C, desolvation gas at 750 L/h, and cone gas at 150 L/h. The optimized settings for ESI were: extractor voltage of 4 V, capillary voltage of 3.50 kV, and RF lens of 0.1 V. The collision gas used for SRM was argon (UHP) at 0.15mL/ min or 9.3×10-4 mbar. Infusion experiments were performed for the new target analytes to determine the SRM conditions in ESI+ with a syringe pump flow rate of 50μL/min. Quantitative and confirmation SRMs used for all target analytes and the retention times of the target analytes with the optimized gradient program (Table 1).