Methods of Analysis—Determination of Pesticides in Filtered Water and Suspended Sediment using Liquid Chromatography- and Gas Chromatography-Tandem Mass Spectrometry
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Abstract
The widespread application of pesticides in agricultural and urban areas leads to their presence in surface waters. Presence of these biologically active chemicals in environmental waters potentially has adverse effects on nontarget organisms. To better understand the environmental fate of these contaminants, a robust method to capture chemicals with wide-ranging physicochemical properties has been developed. The method was developed by the U.S. Geological Survey’s Organic Chemistry Research Laboratory to monitor pesticides, pesticide degradates, and other agrochemicals in environmental surface waters throughout the country. The analysis involves a multiresidue method to determine 183 pesticides and pesticide degradates in filtered water samples and 178 pesticides and pesticide degradates in paired suspended sediment samples. After the filtration of whole water, contaminants are individually measured in the filtered water and the collected suspended sediment. Filtered water is extracted via solid-phase extraction, whereas suspended sediment is extracted using an ultrasonication, solid-liquid extraction. Samples are analyzed by liquid chromatography-tandem mass spectrometry using an electrospray ionization source in positive and negative modes and analyzed by gas chromatography-tandem mass spectrometry using an advanced electron ionization source in positive mode. Instrument parameters were optimized for the highest sensitivity, and at least two transitions (quantifier and qualifier) were monitored for each analyte.
Recoveries in test filtered water (n=9; 183 analytes) from the American River, California, and suspended sediment (n=9; 178 analytes) samples fortified at 15 nanograms per liter (ng/L) ranged from 70.1 to 121.0 and 71.1 to 117.0 percent in water and suspended sediment filter samples, respectively. Method detection limits of pesticides and pesticide degradates ranged from 0.5 to 10.6 ng/L in water and 0.7 to 11.8 ng/L in suspended sediment filters. Reporting limits were 1.1–21.1 ng/L and 1.5–23.7 ng/L in water and filter samples, respectively. The developed method is applied to surface-water samples for the analysis of pesticides, pesticide degradates, and other agrochemicals.
Introduction
Pesticide applications can increase crop production, improve crop quality, and reduce proliferation of pest-borne diseases. The United States (U.S.) applies over 450 million kilograms (kg) of pesticides annually, representing 20–25 percent of the world market (Atwood and Paisley-Jones, 2017). Widespread use of pesticides has caused concerns about potential adverse effects because pesticides and their degradates are easily transported into and throughout the environment. Hundreds of synthetic pesticides of wide-ranging physicochemical properties are registered for use in the U.S. with multiple pesticides often being detected in individual surface-water samples (Gilliom and others, 2006; Orlando and others, 2014). The pesticide classes, compounds, and concentrations detected in environmental waters are affected by multiple factors including intensity of use, geographic location, seasonal dependence; and by soil, climate, and hydrologic characteristics (Gilliom and others, 2006). Sensitive (low nanograms per liter [ng/L]) analytical methods that target a diverse array of agrochemicals could improve understanding of the fate and transport of these chemicals in the environment.
The U.S. Geological Survey (USGS) Organic Chemistry Research Laboratory (OCRL) in Sacramento, California, has analyzed pesticides in environmental media since the 1990s. Early methods collected pesticides on octylsilane (C8) solid-phase extraction (SPE) cartridges (Domagalski and Kuivila, 1993; Crepeau and others, 2000). In 2008, SPE was transitioned to hydrophilic-lipophilic balance (HLB) cartridges for the analysis of 62 pesticides and degradates in water (Hladik and others, 2008). Pyrethroids, synthetic organophosphate insecticides, were reported to preferentially adsorb to suspended sediments (Hladik and Kuivila, 2009), highlighting the importance of individually measuring the dissolved and suspended phases of surface-water samples. With the introduction of new pesticides and the acquisition of new instrumentation, the method has been adapted through the years to include additional analytes and improve sensitivity (Hladik and Calhoun, 2012; Orlando and others, 2013, 2014; Sanders and others, 2018). The most recent report included analysis of 154 pesticides and pesticide degradates in surface water and suspended sediment from the Sacramento–San Joaquin Delta (De Parsia and others, 2019). Methods described in De Parsia and others (2019) have been further updated and validated in this report, with compounds shifted from gas chromatography-tandem mass spectrometry (GC-MS/MS) to liquid chromatography-tandem mass spectrometry (LC-MS/MS) and new analytes added. At the time of this study, water samples are analyzed for 183 analytes and suspended sediments are analyzed for 178 compounds using LC-MS/MS and GC-MS/MS. Support for this report was provided by the California Water Science Center.
Purpose and Scope
The purpose of this report is to describe an analytical procedure for the extraction and quantification of pesticides and pesticide degradates in surface-water samples using LC-MS/MS and GC-MS/MS. Pesticide concentrations are determined individually in filtered water and suspended sediment (project dependent) from a single whole-water sample. The method is an expansion of previous methods (Hladik and others, 2008; Hladik and Calhoun, 2012; Orlando and others, 2013, 2014; Sanders and others, 2018; De Parsia and others, 2019) and increases the total number of target analytes to 183 in water and 178 in suspended sediment. New analytes include pesticide degradates and pesticides that have recently been registered or increasing in application (California Department of Pesticide Regulation, 2023; U.S. Environmental Protection Agency, 2023b). Whole-water samples were collected in 1-liter (L) amber-glass bottles and filtered through a pre-weighed 0.7-micrometer (µm) glass-fiber filter. Filtered water was extracted using SPE and combined with bottle washes that captured chemicals adsorbed to the glass. Suspended sediment samples on filters were air-dried and extracted via sonication. The analytical procedure described will contribute to a better understanding of the occurrence, fate, and transport of pesticides in the environment.
Compound recoveries, analytical precision, method detection limits (MDLs), and reporting limits (RLs) were determined for each analyte from spiked surface water collected from the American River, California. Water from the American River was used in place of laboratory reagent water to better represent environmental media. The American River has low suspended sediment and low dissolved organic carbon (Hladik and Calhoun, 2012). Zero target analytes (pesticides and pesticide degradates) were detected in blank samples during the development of this method. The MDLs were calculated in filtered surface water and suspended sediment samples following the U.S. Environmental Protection Agency (EPA) procedure (U.S. Environmental Protection Agency, 2016). Additional samples were monitored for background concentrations of analytes. Surrogate compounds and internal standards were added to each sample before sample preparation and analysis, respectively, to monitor recoveries, issues with matrix interferences and effects, or method performance. The method is applicable to pesticide analyses of filtered surface water and suspended sediment samples.
Methods of Study
Applications of pesticides in urban and agricultural settings are constantly changing due to changes in regulations, changes in pest pressures, and new chemicals being introduced to the market. As a result, analytical methods must be updated and validated to monitor the presence and fate of these current-use pesticides in surface waters.
Approach
Previous target lists were examined for compounds that could be removed from or added to analysis during updates to the analytical method. Criteria that were monitored when deciding on removing a chemical included (1) current applications of the pesticide, (2) persistence, and (3) toxicity (U.S. Environmental Protection Agency, 2022; California Department of Pesticide Regulation, 2023; Food and Agriculture Organization of the United Nations, 2023). Chemicals that have been registered for use in the U.S. or have seen increased use were added to the method after determining their suitability and applications (California Department of Pesticide Regulation, 2023; U.S. Environmental Protection Agency, 2023b). Analytical standards for pesticides and pesticide degradates were acquired from the EPA National Pesticide Standard Repository (Fort Meade, Maryland; U.S. Environmental Protection Agency, 2023a). If standards were unavailable, they were purchased from Sigma-Aldrich (St. Louis, Missouri), HPC Standards (Atlanta, Georgia), or Accustandard (New Haven, Connecticut). Mass-labeled surrogate and internal standards were purchased from Cambridge Isotope Laboratories (Tewksbury, Massachusetts), Sigma-Aldrich, or HPC.
Standards were individually optimized on LC-MS/MS or GC-MS/MS instrumentation, generating precursor and product ion(s) for each analyte. If necessary, chemicals were optimized in positive and negative mode by LC-MS/MS to choose the ionization mode that provided the best sensitivity. Instrument conditions were further optimized to produce the highest sensitivity for most analytes. The method was validated through different tests including analyte recovery, precision, and MDL.
Previous Studies
The reported method has been adapted from previous studies done by the OCRL (Hladik and others, 2008; Hladik and Kuivila, 2009; Hladik and Calhoun, 2012; Orlando and others, 2013, 2014; Sanders and others, 2018; De Parsia and others, 2019). Extractions with HLB SPE and analyses using GC-MS/MS and LC-MS/MS have provided sufficient sensitivity (low ng/L) and selectivity for the detection and quantification of pesticides and pesticide degradates in filtered water and suspended sediment samples.
Analytical Method
An analytical method is presented for the analysis of 183 pesticides and pesticide degradates in filtered surface waters as well as 178 pesticides and pesticide degradates in paired suspended sediments. Pesticides are analyzed in samples are analyzed by LC-MS/MS and GC-MS/MS. The USGS method number is O-4442-23 and method code is GLC02.
Method Number, Schedule, and Code
The analytical method and validation described in this report were developed by the USGS OCRL. The method was approved as USGS method O-4442-23. Pesticides and pesticide degradates are extracted from filtered water using SPE and analyzed using LC-MS/MS in electrospray ionization (ESI) positive and negative mode and using GC-MS/MS. Suspended sediment is extracted via sonication and analyzed using LC-MS/MS in ESI positive and negative mode and using GC-MS/MS.
Scope and Application
Method O-4442-23 is applied for the determination of 183 agrochemicals in whole-water samples, with individual measurements in filtered water (183 analytes) and suspended sediment (178 analytes). Due to low recoveries (less than 70 percent) in suspended sediment, 5 analytes (bentazon, imazalil, penoxsulam, tebuconazole t-butylhydroxy, and thiamethoxam degradate (NOA-407475) were removed from suspended sediment analyses with the remaining analytes analyzed in both matrices. The method is applicable to surface waters, but the collection and analyses of suspended sediment samples are project dependent. Surface waters with low suspended sediment concentrations may not contain enough suspended sediment in 1 L of water for analysis. Analyses are completed using LC-MS/MS in the ESI positive (n=138) and negative (n=12) modes and using GC-MS/MS (n=33) with advanced electron ionization (AEI). The laboratory method code for this method is GLC02 (U.S. Geological Survey, 2023a). Analytical parameters, including compound names, retention times, quantifier transitions, and qualifier transitions, are reported in tables 1–3 for each analysis.
Table 1.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) retention times, instrument parameters, and transitions for target compounds in electrospray ionization positive (ESI[+]) mode.[CE represents the collision energy reported in electronvolts (eV) for the fragmentation of precursor to the product for each transition monitored. Abbreviations: min, minute; V, volt; m/z, mass-to-charge ratio]
Table 2.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) retention times, instrument parameters, and transitions for target compounds in electrospray ionization negative (ESI[−]) mode.[CE represents the collision energy reported in electronvolts (eV) for the fragmentation of precursor to the product for each transition monitored. Abbreviations: min, minute; V, volt; m/z, mass-to-charge ratio]
Table 3.
Gas chromatography-tandem mass spectrometry (GC-MS/MS) retention times, instrument parameters, and transitions for target compounds.[CE represents the collision energy reported in electronvolts (eV) for the fragmentation of precursor to the product for each transition monitored. Abbreviations: min, minute; m/z, mass-to-charge ratio]
Summary of Method
Water samples are collected in the field following methods described in the USGS National Field Manual (U.S. Geological Survey, variously dated), and the samples are typically collected using 1-L pre-cleaned, baked amber-glass bottles. Samples are shipped overnight or transported on ice to the USGS OCRL for processing and analysis. Samples are processed within 7 days of receipt, and final extracts may be stored up to 30 days before analysis.
Samples for pesticide analyses were filtered through pre-weighed, 0.7-μm glass-fiber filters (Whatman Grade GF/F; Piscataway, New Jersey) to remove suspended materials. After filtration, the filter paper containing the suspended sediments was dried in the dark at room temperature overnight and then stored in a freezer at −20 degrees Celsius (°C) until extraction. Before extraction, the filtered water and dried suspended sediment fractions were each spiked with 50 microliters (µL) of a 1 nanogram per microliter (ng/µL) recovery surrogate solution containing atrazine-13C3, fipronil-13C4,15N2, imidacloprid-d4, metolachlor-13C6, cis-permethrin-13C6, p,p′-DDE-13C12, tebuconazole-13C3, and trifluralin-d14.
Water was loaded under vacuum onto an Oasis HLB (Waters, Milford, Mass.; 6 milliliters [mL], 500 milligrams [mg]) cartridge that had been conditioned before use with one column volume of dichloromethane followed by one column volume of acetone and two column volumes of deionized water. The water samples were pulled through the SPE cartridge under vacuum at a flow rate of approximately 10 milliliters per minute (mL/min). After extraction, the SPE cartridge was dried under vacuum. Analytes were eluted with 10 mL of 1:1 volume per volume (v:v) acetone:dichloromethane. The eluent was evaporated to less than 0.5 mL under a gentle stream of dry nitrogen (Organomation N-Evap; Berlin, Mass.) and solvent-exchanged into acetonitrile. The final sample volume was 0.2 mL.
The suspended sediment fraction on the filter paper was extracted twice with 50 mL of dichloromethane via sonication (Fisherbrand 11211; Waltham, Mass.) for 10 minutes. The extract was filtered through sodium sulfate and evaporated under nitrogen using a Turbovap II (Biotage; Uppsala, Sweden) to 0.5 mL. The solvent was exchanged into acetonitrile and further evaporated to less than 0.2 mL using a gentle stream of dry nitrogen. The final sample volume was 0.2 mL.
An internal standard solution containing 2.5 ng/µL of acenaphthene-d10, bifenthrin-d5, clothianidin-d3, myclobutanil-d4, and oxyfluorfen-d5 was then added (20 µL) to the extracts of both fractions. Sample extracts were stored in a freezer at −20 °C until analysis, up to 30 days.
Safety Considerations
Appropriate personal protective equipment must always be worn during sample handling and processing, including safety glasses/goggles, nitrile gloves, and laboratory coats. Steps that use organic solvents must be completed in a well-vented fume hood. Proper precautions should be taken when handling heated zones (that is, injector, oven, and MS sources) of the LC-MS/MS and GC-MS/MS instrumentation, which can be upwards of 320 °C. Zones should be given time to cool before instrument maintenance procedures. Laboratory personnel should receive hazardous materials safety training and understand the hazards associated with solvents, target compounds, and reagents related to this method. Liquid waste produced during sample preparation and analysis must be collected in appropriate containers (glass bottles or plastic carboys) for proper disposal.
Interferences
Interferences that cause positive and negative analytical biases potentially lead to inaccurate identification or quantitation of target analytes. Matrix interferences, including additional environmental contaminants, natural organic matter, and salts, may occupy active sites on the SPE stationary phase or coelute with target analytes, causing lower recoveries or matrix effects in instrumental analysis. To address and recognize potential interferences, quality assurance/quality control (QA/QC) samples are necessary, including instrument blanks, laboratory blanks, field blanks, field replicates, matrix spikes, and continuous calibration verification (CCV) samples. Additional QA/QC protocols, such as surrogate and internal standards, will help correct for or interpret these interferences through monitoring recovery and instrument response. Furthermore, because many of these target analytes are used in household pest controls, it is important for field and laboratory personnel to limit contamination concerns related to repellent-treated clothing and contamination from applications of pesticides at home or near the laboratory before sample handling.
Equipment and Supplies
The following equipment and supplies are used for water and suspended sediment method development, sample collection, and sample preparation. If appropriate, equivalent equipment and supplies may be used. All laboratory materials must be properly cleaned before sample preparation to avoid possible contamination. Glassware is washed with Liquinox solution, rinsed with hot tap water, and then rinsed with organic-free water. After washing, glassware is baked at 450 °C in a muffle furnace. Glass-fiber filters, glass wool, and sodium sulfate are all baked at 450 °C in a muffle furnace. Materials that cannot be baked, including stainless-steel spatulas and tweezers, are solvent-rinsed with acetone.
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• Analytical balances—An analytical balance capable of weighing to the nearest 0.1 mg is used for weighing filter papers pre- and post-filtering (Sartorius Quintix). An analytical balance capable of weighing to the nearest 0.002 mg is used for weighing and preparing neat standards (Sartorius Cubis II). Analytical balances are internally calibrated before use and externally calibrated yearly. The accuracy is checked as necessary.
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• Concentrator tubes—Samples are eluted into, transferred into, and evaporated under nitrogen in 15 mL and 200 mL glass concentrator tubes (DWK Life Sciences Snap Cap Centrifuge Tube and Biotage Evaporation Tube).
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• Diaphragm pumps—Diaphragm pumps are used to pump water through filters or to pull water through SPE cartridges (Masterflex L/S with polytetrafluoroethylene [PTFE], Diaphragm Pumphead and a Gast Diaphragm Vacuum/Pressure Pump).
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• Electronic pipettes—A 10–100 µL electronic pipette is used to fortify samples with surrogate standard, internal standard, and matrix spike. Additional capacities of electronic pipettes or manual pipettes (0.1–2 µL, 0.5–10 µL, 20–300 µL, 100–1,000 µL, 500–5,000 µL, and 1–10 mL) may be necessary for standard preparation. Appropriate disposable polypropylene tips are used with the pipettes. Electronic and manual pipettes are calibrated yearly, and accuracy is checked as necessary.
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• Erlenmeyer flasks—Suspended sediment filter papers are extracted in a 250-mL glass Erlenmeyer flask.
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• Filter holder—An aluminum filter holder (Geotech, 142 millimeter [mm]) is used for filtering water samples.
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• GC-MS/MS Column—An Agilent Technologies DB-5MS analytical column (30 meter [m]×0.25 mm×0.25 μm) is used for gas chromatography (GC) separation. The column contains a 10-m integrated guard column made from deactivated fused silica tubing at the front of the analytical column.
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• Glass bottles—Amber-glass bottles (1 L) are used to collect grab samples in the field.
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• Glass-fiber filter—A Whatman Grade GF/F glass-fiber filter (142 mm, 0.7 µm) is used to filter water samples and collect suspended sediment.
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• Glass funnels are used in filtering and drying suspended sediment extracts.
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• Glass wool is used to plug glass funnels to which sodium sulfate is added, and suspended sediment samples are poured through following extraction.
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• Graduated cylinders—Water samples are filtered into 1-L graduated glass cylinders. Additional sizes may be necessary in preparation of mobile phases and organic solvent mixtures.
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• LC-MS/MS column—An Agilent Technologies Zorbax Eclipse XDB-C18 column (2.1 mm×150 mm, 3.5 µm) preceded by a Zorbax Eclipse XDB-C8 guard cartridge (2.1 mm×12.5 mm, 5 µm) is used for liquid chromatography (LC) separation.
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• Muffle furnace—Glassware is baked at 450 °C for a minimum of 4 hours after washing to remove organic contaminants before use (Thermo Scientific Lindberg Blue M).
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• Nitrogen generators produce nitrogen (up to 99.9 percent) for evaporators and LC-MS/MS instrumentation (Claind Nigen LCMS 40-1, FDGSi Maestro 64-1).
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• Nitrogen evaporators are used to concentrate samples (Organomation N-Evap, Biotage Horizon XcelVap, and Biotage TurboVap).
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• Oasis HLB SPE cartridges—Water samples are extracted with 500 mg, 6 mL Waters Oasis HLB SPE cartridges.
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• Pasteur pipettes—Glass Pasteur pipettes are used for the transfer of samples.
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• Solvent dispensers—Solvent bottles are equipped with 1–10 mL solvent dispensers (BrandTech Dispensette S) for volumetric additions of solvent to samples.
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• A sonicator is used to extract suspended sediment filter samples (Fisherbrand 11211).
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• The SPE tube adapters allow SPE cartridges to be eluted in series if one clogs during sample loading.
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• The SPE tubing is used to load 1-L samples under vacuum onto SPE cartridges. Tubing is made from PTFE and contains an adapter to fit in the cartridge and a weighted end to sit at the bottom of the sample bottle.
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• Vacuum manifold—A vacuum manifold is used for SPE of water samples (Supelco Visiprep 12). The manifold includes a vial rack to hold concentrator tubes.
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• Vial inserts—Final sample extracts are placed in 250-µL vial inserts.
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• Vials and caps—Amber-glass screw top vials (2 mL) and screw caps are used for final sample extracts. Extracts are placed in a 250 µL vial insert and housed in the screw top vials. Vials must fit in instrument autosampler trays.
Instrumentation
Surface water and suspended sediment sample extracts are analyzed by LC-MS/MS and GC-MS/MS. Instrumentation are described below, including columns for chromatography.
Liquid Chromatography-Tandem Mass Spectrometry
The LC-MS/MS analysis is completed on an Agilent Technologies (Santa Clara, Calif.) 1260 infinity bio-inert high-performance liquid chromatograph coupled to a 6430 triple quadrupole mass spectrometer. The instrumentation is equipped with a 1260 bio-inert high-performance autosampler, a bio-inert quaternary pump, and a 1290 thermostatted column compartment. An Agilent Technologies Zorbax Eclipse XDB-C18 column (2.1 mm×150 mm, 3.5 µm) preceded by a Zorbax Eclipse XDB-C8 guard cartridge (2.1 mm×12.5 mm, 5 µm) is used for separation. Analyses are completed in positive and negative ion modes following an ESI source.
Gas Chromatography-Tandem Mass Spectrometry
The GC-MS/MS analysis is completed on a Trace 1310 gas chromatograph coupled to a TSQ 9000 triple quadrupole mass spectrometer (Thermo Scientific, Waltham, Mass.). The instrument is equipped with a TriPlus RSH autosampler, a programmable temperature vaporizing (PTV) inlet, and an AEI source. An Agilent Technologies DB-5MS analytical column (30 m×0.25 mm×0.25 μm) with a 10-m integrated guard column is used for GC separation.
Analytical Standards and Reagents
Analytical standards and reagents used for method validation, sample preparation, and analyte quantification are described below. Equivalent standards and reagents may be used, but should be validated to ensure no contamination.
Neat Standards, Standard Solutions, and Reagents
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• Analyte protectants—Analyte protectants of ethylglycerol, gulonolactone, and sorbitol were purchased from Sigma-Aldrich. Analyte protectants are used to limit matrix effects in GC-MS/MS analysis.
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• Analytical standards—Neat analytical standards of pesticides and pesticide degradates were obtained from the EPA National Pesticide Standard Repository (U.S. Environmental Protection Agency, 2023a). Standards for compounds that were not available at the pesticide repository were purchased from Sigma-Aldrich, HPC Standards, or Accustandard.
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• Internal standards—Internal standard compounds of acenaphthene-d10, bifenthrin-d5, clothianidin-d3, myclobutanil-d4, and oxyfluorfen-d5 were purchased from Sigma-Aldrich.
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• Sodium sulfate—Certified American Chemical Society (ACS) grade, granular, 10–60 mesh purchased from Fisher Scientific.
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• Surrogate standards—Surrogate standard compounds of atrazine-13C3, fipronil-13C4,15N2, imidacloprid-d4, metolachlor-13C6, cis-permethrin-13C6, p,pʹ-DDE-13C12, and trifluralin-d14 were purchased from Cambridge Isotope Laboratories. The compound tebuconazole-13C3 was purchased from Sigma-Aldrich.
Solvents and Gases
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• Acetone—Optima grade (Fisher Chemical, A929-4).
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• Acetonitrile—OmniSolv liquid chromatography-mass spectrometry (LC-MS) grade (MilliporeSigma, AX0156).
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• Compressed gases—Argon (99.999 percent), helium (99.999 percent), and nitrogen (99.999 percent) were purchased from local suppliers. Argon and nitrogen are used as collision gases for the GC-MS/MS and LC-MS/MS, respectively. Helium is used as the GC-MS/MS carrier gas. Nitrogen generators are used to produce nitrogen for the LC-MS/MS ESI source and for evaporation during sample preparation.
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• Dichloromethane—GC Resolv grade (Fisher Chemical, D154-4).
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• Ethyl acetate—Optima grade (Fisher Chemical, E196-4).
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• Formic acid—Optima LC-MS grade (Fisher Chemical, A117-50).
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• Isopropanol—Certified ACS grade (Fisher Chemical, A416-4).
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• Methanol—Optima grade (Fisher Chemical, A454-4).
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• Organic-free water—Generated from purification of house deionized water using a PURELAB flex 2 (ELGA LabWater, Woodridge, Illinois) system. The PURELAB flex 2 delivers ultrapure type I (18.2 megaohm-centimeters [MΩ∙cm]) water.
Standards Preparation
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1. Primary standard solutions—Individual stock solutions of neat analytical standards are prepared at 1 milligram per milliliter (mg/mL) by accurately weighing, using a calibrated microbalance, 2–5 mg of the neat standard into a 7-mL amber-glass vial. Using appropriate electronic pipettes, add 1 mL of acetone per milligram of the weighed standard. If analyte is not dissolvable in acetone, an appropriate solvent is used, or the standard is prepared at a lower concentration.
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2. LC-MS/MS ESI(+) stock solution—A 5-ng/µL stock solution is prepared by adding 125 µL of 1-mg/mL primary standard solutions (138 unlabeled compounds, table 1) to a 25-mL volumetric flask and bringing to volume with acetonitrile. Appropriate volumes are added for primary standard solutions that are less than 1 mg/mL.
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3. LC-MS/MS ESI(−) stock solution—A 5-ng/µL stock solution is prepared by adding 125 µL of 1-mg/mL primary standard solutions (12 unlabeled compounds, table 2) to a 25-mL volumetric flask and bringing to volume with acetonitrile. Appropriate volumes are added for primary standard solutions that are less than 1 mg/mL.
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4. GC-MS/MS stock solution—A 10-ng/µL stock solution is prepared by adding 250 µL of 1-mg/mL primary standard solutions (33 unlabeled compounds, table 3) to a 25-mL volumetric flask and bringing to volume with acetonitrile. Appropriate volumes are added for primary standard solutions that are less than 1 mg/mL.
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5. Intermediate stock solutions—Intermediate stock solutions at concentrations of 2.5 ng/µL for the analytes and surrogates and 0.25 ng/µL for the internal standards are prepared for the LC-MS/MS ESI(+), LC-MS/MS ESI(−), and GC-MS/MS stock solutions. Add 2.5 mL of 5-ng/µL stock or 1.25 mL of 10-ng/µL stock to a 5-mL volumetric flask. Dependent upon analysis, the appropriate surrogates and internal standards are added to each solution. Surrogate volumes are 250 µL for 50-ng/µL solutions, 125 µL for 100-ng/µL solutions, and 12.5 µL for 1-mg/mL solutions. Add 0.5 mL of a 2.5-ng/µL solution of the appropriate internal standards and bring to volume with acetonitrile. The GC-MS/MS intermediate solution also contains 0.5 mL of the analyte protectant stock.
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6. Analyte protectant stock solution—An analyte protectant stock solution is prepared by adding 2.5-g (in grams) ethylglycerol, 0.25-g gulonolactone, and 0.25-g sorbitol to a 25-mL volumetric flask. Add 8.75 mL of organic-free water and bring to volume with acetonitrile. Final concentrations are 100-mg/mL ethylglycerol and 10-mg/mL gulonolactone and sorbitol.
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7. Internal standard stock solution—A 25-ng/µL stock solution is prepared by adding 125 µL of 1-mg/mL acenaphthene-d10, bifenthrin-d5, clothianidin-d3, myclobutanil-d4, and oxyfluorfen-d5 to a 5-mL volumetric flask and bringing to volume with acetonitrile.
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8. Internal standard spike solution—A 2.5-ng/µL stock solution is prepared by diluting 500 µL of the 25-ng/µL stock solution to 5 mL in a volumetric flask by bringing to volume with the analyte protectant stock solution.
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9. Calibration internal standard solutions—A 2.5-ng/µL internal standard solution is made for the LC-MS/MS ESI(+), LC-MS/MS ESI(−), and GC-MS/MS calibration curves. For LC-MS/MS ESI(+), 62.5 µL of 1-mg/mL clothianidin-d3, myclobutanil-d4, and oxyfluorfen-d5 are added to a 25-mL volumetric flask and brought to volume with acetonitrile. For LC-MS/MS ESI(−), 62.5 µL of 1-mg/mL clothianidin-d3 are added to a 25-mL volumetric flask and brought to volume with acetonitrile. Lastly, for GC-MS/MS, 62.5 µL of 1-mg/mL acenaphthene-d10 and bifenthrin-d5 are added to a 25-mL volumetric flask and brought to volume with acetonitrile.
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10. Dilute calibration internal standard solutions—A 0.25-ng/µL dilute calibration internal standard solution is made for the LC-MS/MS ESI(+), LC-MS/MS ESI(−), and GC-MS/MS calibration curves. For the LC-MS/MS ESI(+) and ESI(−) solutions, add 5 mL of the 2.5-ng/µL calibration internal standard solution to a 50-mL volumetric flask and bring to volume with acetonitrile. For the GC-MS/MS solution, add 5 mL of the 2.5-ng/µL calibration internal standard solution to a 50-mL volumetric flask, add 5 mL of the analyte protectant stock solution, and bring to volume with acetonitrile.
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11. Surrogate standard stock solution—A 10-ng/µL stock solution is prepared by adding 1,000 µL of 50-ng/µL cis-permethrin-13C6, 500 µL of 100-ng/µL atrazine-13C3, fipronil-13C4,15N2, imidacloprid-d4, metolachlor-13C6, p,pʹ-DDE-13C12, and trifluralin-d14, and by adding 50 µL of 1-mg/mL tebuconazole-13C3 to a 5-mL volumetric flask and bringing to volume in acetone.
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12. Surrogate standard spike solution—A 1-ng/µL stock solution is prepared by diluting 500 µL of the 10-ng/µL stock solution to 5 mL in a volumetric flask by bringing to volume with acetone.
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13. Matrix spike solution—A 1-ng/µL matrix-spike solution is prepared by adding 1 mL of the 5-ng/µL LC-MS/MS ESI(+) and LC-MS/MS ESI(−) stock solutions and 500 µL of the 10-ng/µL GC-MS/MS stock solution to a 5-mL volumetric flask and by bringing to volume with acetone.
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14. Calibration solutions—Calibration solutions are prepared for LC-MS/MS ESI(+), LC-MS/MS ESI(−), and GC-MS/MS analyses. Calibration solutions contain all pesticides and surrogates at nine concentrations (0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, and 1.0 ng/µL). Internal standards are maintained at the same concentration (0.25 ng/µL) in all calibration solutions. The calibration solutions are made by adding the appropriate amount of intermediate stock solutions to 5-mL volumetric flasks and bringing to volume with dilute calibration internal standard solutions. Formic acid (0.1 percent) is added to prevent degradation of base-sensitive analytes in acetonitrile.
Sample Collection, Shipment, and Holding Times
Samples were collected following methods described in the National Field Manual (NFM) for the Collection of Water-Quality Data (U.S. Geological Survey, variously dated). Dip samples were collected into 1-L, narrow-mouthed, baked, amber-glass bottles. Integrated samples were collected using equipment that was cleaned following methods described in NFM chapter A3 (U.S. Geological Survey, variously dated). Samples were shipped or transported to the USGS OCRL within 24 hours of collection. Samples have a 7-day maximum holding time before processing. Holding-time studies revealed that nearly all pesticides were stable at 14 days in reagent water (Sandstrom and others, 2016). A 7-day holding time was chosen because this interval provided sufficient and reasonable time for sample processing. Specific projects may have shorter or longer holding times as described by internal quality assurance project plans.
Sample Preparation
Steps for the extraction of pesticides and pesticide degradates from filtered water and suspended sediment are outlined below. All laboratory materials must be properly cleaned (as described in the “Equipment and Supplies” section) before sample preparation to avoid contamination.
Filtered Water Extraction
-
1. Log project and sample information, including site, sampling date, sampling time, and QC are entered into a laboratory notebook or laboratory information management system (LIMS) database. Label each sample with a unique identifier. Throughout the sample preparation procedure, record any important comments, such as “added surrogate twice or lost x mL of sample when extracting” in the notebook or database.
-
2. If suspended sediment is being analyzed, before filtering, weigh a 142-mm, 0.7-µm pore size glass-fiber filter in a piece of clean foil large enough to envelop the filter for storage, and record the weight (in grams, g) of the foil + filter. If suspended sediment is not being analyzed, the filter does not need to be weighed.
-
3. Filter the sample through a 142-mm, 0.7-µm glass-fiber filter into a 1-L graduated cylinder using a diaphragm pump and a filter manifold.
-
a. For each water sample, record the extraction procedure (SPE[HLB] + bottle wash [BW]), extraction date, and volume (to the nearest hundredth of a L).
-
b. After filtration, the water sample will be transferred from the graduated cylinder back into its original sample bottle. If analyzing suspended sediment, carefully transfer the filter back to the pre-weighed foil in which the filter was originally weighed using stainless-steel tweezers or a spatula. Allow the filter to air-dry overnight by folding the foil over the filter to cover, but not touch, the collected sediment. Suspended sediment may need to be air-dried further if it is still wet upon return. If suspended sediment is not being analyzed, the filters may be discarded.
-
c. Once the suspended sediment is dried, weigh the filters in their foil and record the weight (in g) of the foil + filter + dry sediment. Calculate the total dry weight of suspended sediment (g) by subtracting the original foil + filter weight (g) from the new foil + filter + dry sediment weight (g). After the weight is recorded, store the filters in their original foil and place them in a −20 °C freezer until extraction.
-
d. Continue onward for the water-extraction procedure or proceed to step 14 for the suspended sediment filter extraction procedure.
-
-
4. Before processing the next sample or after the last sample, clean the tubing and filter manifold by pumping through 25–50 mL of methanol followed by 200–500 mL of organic-free water. Once the filter manifold is cleaned, continue with filtering the next water sample or leave the manifold open on a clean total wipe to dry for storage.
-
5. Remove the surrogate standard spike solution from the freezer and bring it to room temperature on the laboratory bench. The surrogate standard spike solution contains 1 ng/µL of atrazine-13C3, fipronil-13C4,15N2, imidacloprid-d4, metolachlor-13C6, cis-permethrin-13C6, p,pʹ-DDE-13C12, tebuconazole-13C3, and trifluralin-d14 in acetone. Using the 10–100 µL electronic pipette, add 50 µL of the surrogate solution to each water sample. Shake the sample well following the addition of the surrogate.
-
6. If the water sample is a matrix spike for quality control, add 50 μL of the matrix spike solution using a 10–100 µL electronic pipette. The matrix spike solution contains 1 ng/µL of all unlabeled analytes (listed in tables 1–3) in ethyl acetate. Samples may be spiked before filtration to meet cooperator requirements for specific projects.
-
7. Water samples are extracted by SPE using 6 mL, 500-mg Oasis HLB cartridges. Before extraction, condition the SPE cartridges using the manifold by passing through one column volume (approximately 6 mL) of dichloromethane, followed by one column volume (approximately 6 mL) of acetone, and two column volumes (approximately 12 mL) of organic-free water. Leave a few centimeters (cm) of water above the top frit (porous polyethylene disks at the top and bottom of SPE media) in the final conditioning step. Label cartridges with a unique identifier before performing SPE. Connect water samples to their respective cartridges using SPE tubing and perform extraction under vacuum (not to exceed −20 millimeters of mercury [mmHg]), drawing samples through the cartridge at a flow rate of approximately 10 mL/min.
-
a. Monitor the SPE cartridge for clogs. If the cartridge becomes clogged and the sample will not pump through, you will need to use another conditioned cartridge for the sample. The new cartridge will be processed in series with the previously clogged cartridge using the same unique identifier.
-
b. Once the water sample has passed through the SPE cartridge, the cartridge is either dried under vacuum on the manifold or stored at −20 °C. The SPE cartridges that were stored wet are returned to room temperature on the laboratory bench and placed on the manifold under vacuum until fully dried. Dried SPE cartridges are either immediately eluted or put in a zip-top bag, labelled with the date and project identifier, and stored at −20 °C until elution. Proceed to step 9 for elution procedure.
-
c. Empty bottles are retained for a bottle wash to ensure complete recovery of compounds that tend to adsorb to glass. Proceed to step 8 for bottle wash procedure.
-
d. Following extraction, SPE tubing must be cleaned by pulling through 10–15 mL of methanol followed by 20–50 mL of organic-free water under vacuum. Discard methanol into the organic solvent waste container before water is pulled through SPE tubing.
-
-
8. For bottle wash, add a small amount of sodium sulfate (about 10–20 g) to each bottle and gently shake the bottle to ensure that all water is removed. Using acetone, rinse the bottle three times with about 2–4 mL per rinse, and empty each rinse into one labeled concentrator tube. After all bottle wash rinses, place the tubes on the N-Evap and evaporate down to around 1 mL. The SPE cartridges will be eluted into these tubes in step 9.
-
9. Before elution, the ports on the vacuum manifold must be cleaned by pulling through a small amount (about 1–3 mL) of dichloromethane and acetone under vacuum. Discard solvents into the organic solvent waste container. Bring dried SPE cartridges to room temperature on the laboratory bench if they were previously stored in the freezer. Place labeled concentrator tubes (from step 8) into the elution rack within the vacuum manifold. Place labeled dry SPE cartridges in ports above their matching concentrator tube. Elute each cartridge with 10 mL of 1:1 (v:v) acetone:dichloromethane. If multiple SPE cartridges were used during sample extraction, cartridges are stacked using SPE tube adapters and eluted in series.
-
10. Following elution, samples must be gently evaporated under nitrogen to approximately 0.5 mL using the N-Evap. Solvent exchange the samples into acetonitrile by adding approximately 1.5-mL acetonitrile to the sample and evaporate to approximately 0.2 mL. Final volume may vary slightly but is corrected through the addition of the internal standard solution.
-
11. Remove the internal standard spike solution from the freezer and bring to room temperature on the laboratory bench. The internal standard spike solution contains 2.5-ng/µL acenaphthene-d10, bifenthrin-d5, clothianidin-d3, myclobutanil-d4, and oxyfluorfen-d5 in a solution of analyte protectants: ethylglycerol (100 mg/mL), gulonolactone (10 mg/mL), and sorbitol (10 mg/mL) dissolved in acetonitrile. Using the 10–100 µL electronic pipette, add 20 µL of the internal standard solution to each sample.
-
12. Transfer each sample to a 2-mL amber vial containing a 250-µL glass insert and cap. The samples can be stored for up to 30 days before analysis.
-
13. Analysis of water samples are first completed using the LC-MS/MS in positive (table 1) and negative (table 2) mode. After successful analyses, samples are recapped and analyzed using the GC-MS/MS (table 3). After successful analyses using GC-MS/MS, samples are capped with solid caps and sorted into numerical vial files for long-term storage in a freezer at −20 °C.
Suspended Sediment—Filter Paper Extraction
-
14. For extraction of suspended sediment on filter papers, fold and insert weighed filter into a 250-mL Erlenmeyer flask.
-
15. Remove the surrogate standard spike solution from the freezer and bring to room temperature on the laboratory bench. The surrogate standard spike solution contains 1 ng/µL of atrazine-13C3, fipronil-13C4,15N2, imidacloprid-d4, metolachlor-13C6, cis-permethrin-13C6, p,pʹ-DDE-13C12, tebuconazole-13C3, and trifluralin-d14 in acetone. Using the 10–100 µL electronic pipette, add 50 µL of the surrogate solution to each filter sample.
-
16. If the filter sample is a matrix spike for quality control, add 50 μL of the matrix spike solution using a 10–100 µL electronic pipette. The matrix spike solution contains 1.0 ng/µL of all unlabeled analytes (listed in tables 1–3) in ethyl acetate. Samples may be spiked before filtration to meet cooperator requirements for specific projects.
-
17. Filter samples are extracted in dichloromethane using sonication.
-
a. Using a solvent dispenser, dispense 10-mL aliquots of dichloromethane into the Erlenmeyer until the filter is submerged (about 50–80 mL) and cover it with foil.
-
b. Sonicate for 10 minutes and then decant the solvent into a 200-mL TurboVap tube through a funnel containing about 15–20 g of sodium sulfate held in place by a glass wool plug.
-
c. Repeat step 17a and step 17b with fresh dichloromethane and decant the solvent through the same funnel and into the same TurboVap tube.
-
d. Rinse the sodium sulfate in the funnel with approximately 3 mL of dichloromethane.
-
e. Once the solvent has completely dripped through the funnel, remove the funnel from the TurboVap tube and set it on a large piece of foil in the hood to dry before discarding sodium sulfate and glass wool.
-
-
18. After extraction and filtration, the sample is evaporated under nitrogen to about 5–10 mL using a TurboVap tube.
-
a. Transfer the sample from the TurboVap tube into a 15-mL concentrator tube and evaporate down under nitrogen to approximately 0.5 mL using the N-Evap. Solvent exchange the samples into acetonitrile by adding approximately 1.5-mL acetonitrile to the sample and evaporate to approximately 0.2 mL. Final volumes may vary slightly but are corrected through the addition of an internal standard solution.
-
-
19. Remove the internal standard spike solution from the freezer and bring it to room temperature on the laboratory bench. The internal standard spike solution contains 2.5-ng/µL acenaphthene-d10, bifenthrin-d5, clothianidin-d3, myclobutanil-d4, and oxyfluorfen-d5 in a solution of analyte protectants: ethylglycerol (100 mg/mL), gulonolactone (10 mg/mL), and sorbitol (10 mg/mL) dissolved in acetonitrile. Using the 10–100 µL electronic pipette, add 20 µL of the internal standard solution to each sample.
-
20. Analyses of filter samples are first completed using the LC-MS/MS in positive (table 1) and negative (table 2) mode. After successful analyses, samples are recapped and analyzed using the GC-MS/MS (table 3). After successful analyses using GC-MS/MS, samples are capped with solid caps and sorted into numerical vial files for long-term storage in a freezer at −20 °C.
Analysis by Liquid Chromatography-Tandem Mass Spectrometry
The LC-MS/MS must be tuned using LC-MS calibration standard in positive and negative ion modes following ESI if the instrument has not been tuned within the last 30 days. Checktunes must be completed weekly. The tuning solution contains multiple components for tuning throughout a large mass range in both ionization modes. Before analyzing samples using the LC-MS/MS, the following maintenance procedures must be completed to ensure optimal performance:
-
1. Check mobile phase and needle wash solvent levels and ensure there is enough solvent to complete the worklist run. If solvent is added, set solvent levels in MassHunter acquisition software.
-
2. Open purge valve and purge solvent lines at 2.5 mL/min for 5 minutes.
-
3. Rinse ESI spray chamber with isopropanol.
-
4. Wipe interior surfaces of spray chamber with task wipe and isopropanol.
-
5. Wipe off spray shield with task wipe and isopropanol. If surface contamination is still visible, remove the spray shield and sand in a figure eight motion with 4,000 grit or higher sandpaper.
-
6. Open the ballast on the rough pump for 5 minutes if oil is present in oil mist filter.
-
7. Equilibrate LC-MS/MS system with starting mobile phase composition for 15 minutes.
-
8. Check solvent waste bottles.
The LC-MS/MS analysis is completed on an Agilent Technologies 1260 infinity bio-inert high-performance liquid chromatograph coupled to a 6430 triple quadrupole mass spectrometer. An Agilent Technologies Zorbax Eclipse XDB-C18 column (2.1 mm×150 mm, 3.5 µm) preceded by a Zorbax Eclipse XDB-C8 guard cartridge (2.1 mm×12.5 mm, 5 µm) is used for separation. Analyses are completed in positive and negative ion modes following ESI. For ESI(+) analysis, the mobile phase consists of (A) 0.1 percent formic acid in water and (B) acetonitrile. The gradient starts at 98 percent A and 2 percent B and is held for 2 minutes before ramping up to 50 percent B in 2 minutes, followed by a 6-minute ramp to 60 percent B, and a final 2-minute ramp up to 100 percent B. The mobile phase is kept at 100 percent B for 2 minutes before being brought back to initial conditions in 1 minute and being given 5 minutes to re-equilibrate (20-minute total run time). For ESI(−) analysis, the mobile phase consists of (A) 0.1 percent formic acid in water and (B) methanol. The gradient starts at 98 percent A and 2 percent B and is held for 2 minutes. The gradient is ramped up to 100 percent B in 3 minutes and is held for 2 minutes before returning to initial conditions in 1 minute and being given 5 minutes to re-equilibrate (13-minute total run time). For all analyses, the injection volume is 10 µL, the column flow rate is 0.6 mL/min, the column temperature is 40 °C, the drying gas temperature is 350 °C, the gas flow is 10 liters per minute (L/min), the nebulizer pressure is 40 pounds per square inch (psi), and the capillary voltage is plus or minus 4,000 volts (V). Data are collected in the multiple reaction monitoring (MRM) mode. Retention times, instrument parameters, and MRM transitions of target compounds for ESI(+) and ESI(−) modes are reported in tables 1 and 2, respectively (Gross and others, 2023).
Analysis by Gas Chromatography-Tandem Mass Spectrometry
The GC-MS/MS must be tuned using calibration compound FC-43 (perfluorotributylamine) in positive mode following AEI if the instrument has not been tuned within the last 30 days. Checktunes must be completed weekly. Fragmentation of perfluorotributylamine in the ion source allows for the instrument to be tuned throughout a large mass range. Before running samples on the GC-MS/MS, the following maintenance procedures must be completed to ensure optimal performance:
-
1. Check carrier gas (helium) and ensure there is enough to complete the worklist run.
-
2. If necessary, perform inlet maintenance by changing inlet liner, septum, inlet ferrule, and cutting approximately 10 cm off the injector end of the analytical column.
-
3. Fill wash solvent vials for GC autosampler syringe.
-
4. Empty wash solvent waste vial.
-
5. Check GC autosampler syringe for clogged needle or seized plunger by pulling up solvent from the wash solvent vials. Change syringe if necessary.
The GC-MS/MS analysis is completed on a Trace 1310 gas chromatograph coupled to a TSQ 9000 triple quadrupole mass spectrometer (Thermo Scientific). The instrument is equipped with a programmable temperature vaporizing (PTV) inlet and an AEI source. Sample injection volume is 1 µL. The initial temperature of the PTV is 110 °C, which is increased at a rate of 5 °C per second (°C/s) to 290 °C and held for 3 minutes for transfer. Then, the temperature is increased at a rate of 14.5 °C/s to 320 °C and held for 10 minutes for cleaning. The inlet has a split flow of 50 mL/min and a splitless time of 3 minutes. Separation is performed on a DB-5MS analytical column (30 m×0.25 mm×0.25 μm; Agilent Technologies) with helium as the carrier gas at a flow rate of 1.2 mL/min. The oven is initially held at a temperature of 65 °C for 2 minutes, increased to 150 °C at a rate of 25 °C/minute, held for 1 minute, increased to 215 °C at a rate of 25 °C/minute, held for 2 minutes, increased to 280 °C at a rate of 5 °C/minute, and increased to 300 °C at a rate of 10 °C/minute. The oven is held at 300 °C for 5 minutes (31-minute total run time). The mass transfer line is held at 250 °C, and the ion source is held at 320 °C. Data are collected in the selected reaction monitoring (SRM) mode. Retention times, instrument parameters, and SRM transitions of target compounds are reported in table 3 (Gross and others, 2023).
Quality Assurance and Quality Control Criteria
The instruments are calibrated with each new sample batch by running a nine-point calibration curve (0.0025–1.0 ng/µL) at the beginning and end of the worklist. Sample extracts and QA/QC samples are analyzed in an instrument sequence to provide additional information to facilitate corrective actions that might be required if performance criteria are not met. The QA/QC samples include CCV standards, instrument blanks, laboratory blanks, matrix spikes, field blanks, and field replicates. Additionally, surrogate compounds are added to every sample before extraction, and internal standard compounds are added to every sample before instrumental analysis. Frequency of analysis and acceptance criteria for QA/QC checks are reported in table 4. These checks represent the minimum QA/QC and can be augmented based on project specific needs. Definitions for QA/QC sample types are below:
-
1. Calibration standards—Calibration standards are solutions of all analytes and surrogates at a range of concentrations (0.0025–1.0 ng/µL) with consistent internal standard concentrations (0.25 ng/µL). Calibration standards are used to calibrate the instrument and quantify results.
-
2. Continuous calibration verification (CCV)—The CCV solutions (0.05 ng/µL) are standard solutions of the target analytes prepared in a manner similar to the calibration standards. The CCVs are used to monitor the method stability throughout the batch in comparison to the calibration curves.
-
3. Instrument blanks—Instrument blanks are solvents (acetonitrile) injected onto the instruments to determine if there is carryover of target analytes between sample injections.
-
4. Laboratory blanks—A laboratory blank is organic-free water (1 L) that is processed through the entire sample preparation and analytical procedure to monitor for possible laboratory contamination.
-
5. Matrix spikes—A matrix spike is a duplicate environmental sample to which known quantities of the target analytes are spiked before sample processing. The matrix spike is processed exactly like an environmental sample and is used to determine if the sample matrix contributes bias to the analytical results and the degree to which the method is successful in recovering the target analytes. Background concentrations of the analytes are determined from the duplicate environmental sample that is not spiked with method analytes before analysis. Background concentrations are subtracted from spiked concentrations before calculating percentage recoveries.
-
6. Field blanks—Field blanks are samples of organic-free water (1 L) that are brought into the field and handled following field protocols. Organic-free water is taken from the laboratory to the field and handled in such a way that the water is sampled to monitor for possible contamination during sample collection.
-
7. Field replicates—A field replicate is a duplicate environmental sample used to monitor method reproducibility. Field replicates are analyzed together to monitor percentage differences in target analytes.
-
8. Surrogate standards—Surrogate standards are compounds with similar physicochemical properties to the target analytes that are not expected to be present in the environment. These compounds are added to every sample before processing to evaluate overall method performance through their recovery.
-
9. Internal standards—Internal standards are compounds not expected to be present in the environment that have similar physicochemical properties to the target analytes. Internal standards are added at the same level to every sample before instrumental analysis to correct for quantitative differences in extract volume and to compensate for instrument differences in injection volume. Internal standards are also used to monitor instrument conditions, such as injection errors, retention time shifts, and instrument abnormalities or malfunctions. Internal standard concentrations are the same between calibration standards, CCVs, and samples.
Table 4.
Quality assurance/quality control (QA/QC) performance checks, frequencies, and acceptance criteria.[≥, greater than or equal to; ng/µL, nanogram per microliter; %, percent; <, less than; RL, reporting limit]
If QA/QC samples fall outside of their respective acceptance criteria reported in table 4, sample handling, instrument performance, and data are reviewed further. Corrective actions are taken depending upon QA/QC type, as listed below:
-
1. Calibration standards—If calibration standards have a coefficient of determination (R2) less than 0.99, the data from the analysis will be rejected, and samples must be reanalyzed. If calibration standards continue to fail, instrument maintenance (cleaning, tuning, replacing consumables) or preparation of a new calibration curve may be necessary.
-
2. Continuous calibration verification (CCV)—If a CCV standard falls outside the accepted percentage recovery range (75 to 125 percent), data will be rejected from the last successful CCV standard forward in the worklist. Samples analyzed after the last successful CCV standard must be reanalyzed and instrument maintenance (cleaning and replacing consumables) may be necessary. Data are only reported when samples fall between two acceptable CCV standards.
-
3. Instrument blanks—If instrument blanks have concentrations above the RL, data will be rejected from the last successful instrument blank forward in the worklist. Samples ran after the last successful instrument blank must be reanalyzed. Check concentrations of analytes in samples analyzed before the contaminated instrument blank for high values. This sample may need to be diluted to prevent carryover. Ensure that the needle wash solution is filled to prevent carryover and prepare a new instrument blank with fresh solvent.
-
4. Laboratory blanks—If a laboratory blank has concentrations above the RL, samples will be reanalyzed. Ensure that the needle wash solution is filled to prevent carryover. If the laboratory blank continues to have detections above the RL, samples will be flagged for the analytes detected in the laboratory blank, and potential contamination sources will be examined. Sample processing will halt until the contamination source is discovered and eliminated as determined by a successful laboratory blank.
-
5. Matrix spikes—If a matrix spike recovery is not within the accepted percent recovery range (70 to 130 percent), the sample will be reanalyzed. Ensure no issues occurred in sample processing and re-extract a new matrix spike sample if errors occurred.
-
6. Field blanks—If a field blank has concentrations above the RL, samples will be reanalyzed. Ensure that the needle wash solution is filled to prevent carryover. If the field blank continues to have detections above the RL, samples will be flagged for the analytes detected in the field blank, and potential contamination sources will be examined.
-
7. Field replicates—If a field replicate is not within the acceptance criteria (relative percent difference [RPD] less than 25 percent), the replicate samples are reanalyzed. Ensure no issues occurred in sample processing, and re-extract new field replicates if errors occurred.
-
8. Surrogate standards—If surrogate-standard recoveries are outside the accepted percent recovery range (70 to 130 percent), ensure no issues occurred with that sample while processing. Samples must be reanalyzed, and if the surrogate standard is outside the acceptance criteria, the sample will be flagged. Overall surrogate recovery is evaluated for every batch, and if all recoveries are trending low or high, the surrogate will be evaluated to determine if a new solution must be made.
-
9. Internal standards—If internal standards fall outside the accepted percent recovery range (70 to 130 percent), the samples will be reanalyzed. Overall internal standard peak area is evaluated for every batch, and if the value is trending low or high, the internal standard will be evaluated to determine if a new solution must be made. Internal standard recoveries may fall outside of acceptable range due to variability in final sample volume or matrix effects. Samples may need to be diluted or concentrated to the correct volume. Corrective action may not be necessary if surrogate standards meet QA/QC objectives.
Further training may be necessary if errors occurred in sample preparation protocols. Samples must be reanalyzed if instrument performance was unsatisfactory. Lastly, samples with poor QC performance (surrogate recovery less than 70 percent) will be flagged. For example, in the LIMS database, if data quality objectives are not met upon review, the sample will be flagged. Use of matrix spike, surrogate, and internal standard verification vials will aid in diagnosing QA/QC failures.
Quantitation and Calculation of Results
Identification and quantification of analytes are completed from raw data files using instrument software (Agilent MassHunter v. 10.1 and Thermo TraceFinder v. 4.1). Before quantitative results are reported, each compound first needs to meet qualitative criteria. An analyte is not considered to be identified correctly unless the correct quantitation ion(s) of the peak are detected, the relative area ratios of the confirmation ions are within plus or minus 25 percent of the average ratio obtained from the calibration samples, and the relative retention time of the peak is within 5 percent of the expected retention time.
Samples are quantified using a nine-point external calibration curve (0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, and 1.0 ng/µL), where at least five concentrations must be used in quantitation (which standards are used depends on sample concentrations and instrument performance). Concentrations from the middle of the calibration curve must never be removed during quantification. Only calibration levels at the low or high end of the curve may be removed. Low-end concentrations (0.0025 and 0.005 ng/µL) may be removed if no response is observed for the analyte (the measured concentration is below the instrument detection limit). High-end concentrations (0.5 or 1.0 ng/µL) may be removed if they are outside the linear range for the analyte. Removing these calibration concentrations but maintaining at least five concentration levels will result in more accurate quantitation. The calibration curve is analyzed at the beginning and end of a sample worklist (table 5). The initial calibration curve is used for quantification. Analyzing the calibration curve at the beginning and end of a worklist run provides further confirmation, beyond CCV injections, that instrument performance is satisfactory throughout the range of concentrations. Regression analysis is completed on the calibration curves for each analyte, and the R2 for each standard curve must be greater than or equal to 0.99 to be accepted. If the R2 for each standard curve is not acceptable, calibration standard corrective actions may be necessary.
Table 5.
Example analytical sequence for liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) analyses.[Quality assurance/quality control (QA/QC) samples that are not specifically identified in the sequence (for example, laboratory blanks, field blanks, field replicates, and matrix spikes) are represented by samples in the sequence as they are given a unique sample identifier. Abbreviation: ng/µL, nanogram per microliter]
The calibration curve points are plotted as a relative response versus the analyte concentration. The relative response is calculated as follows:
whereRelative Response
is the internal standard normalized peak area,
AreaA
is the area of the quantitation ion peak for the specific analyte, and
AreaIS
is the area of the quantitation ion peak for the internal standard.
Analyte concentrations are known for each level of the standard curve and span 0.0025–1.0 ng/µL. Quantification of analytes in sample extracts are then completed from the curve-fit equations determined following regression analysis for each analyte. As an example, linear regression of the calibration curve results in the following equation:
whereRelative Response
is the internal standard normalized peak area (eq. 1),
m
is the slope of the line,
CE
is the analyte concentration (ng/µL) in the sample extract, and
b
is the y-intercept.
The analyte concentration (ng/µL) in the sample extract can then be calculated by rearranging the linear regression equation, as an example:
To calculate the concentration (ng/L) of the analyte in the original water or suspended sediment sample (CS,V), the following calculation is necessary:
whereCS,V
is the concentration of the analyte in the original water or suspended sediment sample on a volume basis,
CE
is the analyte concentration (ng/µL) in the sample extract,
VE
is the volume of the sample extract (200 µL), and
VS
is the volume (L) of the water sample.
Concentrations are reported from 0.5 to 200 ng/L. If the concentration exceeds 200 ng/L, a part of the sample extract is diluted appropriately with a dilute internal standard solution (0.25 ng/µL) in acetonitrile and reanalyzed.
Concentrations of analytes on suspended sediment samples also can be computed in nanograms per gram (ng/g) dry weight of suspended sediment. The dry weight of the suspended sediment is determined via the following equation:
whereWS
is the dry weight (g) of the suspended sediment,
Wf
is the dry weight (g) of the foil, filter, and dry suspended sediment following filtration, and
Wi
is the dry weight (g) of the foil and filter taken before filtration.
The concentration (ng/g dry weight) of the analyte on the suspended sediment (CS,W) can then be determined via the following equations:
whereCS,W
is the concentration of the analyte in the original suspended sediment sample on a weight basis,
CE
is the analyte concentration (ng/µL) in the sample extract,
VE
is the volume of the sample extract (200 µL),
CS,V
is the concentration (ng/L) of analytes on the suspended sediment,
VS
is the volume (L) of the water sample that passed through the filter, and
WS
is the weight (g) of the dried suspended sediment collected on the filter.
Method Performance
Recoveries were calculated from spiked samples (50 ng/L, n=3 and 15 ng/L, n=9) using the following equation:
whereCA
is the analyzed concentration calculated from the calibration curve following sample preparation and instrumental analysis of a spiked sample,
CB
is the background concentration from an unspiked replicate of the spiked sample, and
CS
is the spiked concentration added to the sample.
Environmental samples (not spiked) were analyzed concurrently with spiked samples to monitor background concentrations of analytes. Initial average recoveries and relative standard deviations (RSDs; 50 ng/L) are reported in table 6. Performance-based MDLs and RLs were determined for each analyte. The MDLs were calculated following procedures similar to the EPA determination of MDLs (U.S. Environmental Protection Agency, 2016). In short, 10 whole-water samples were collected from the American River (near Guy West Bridge). Water from the American River was used in place of laboratory reagent water because this water better represents real-world conditions. The American River carries snowmelt and drainage from the Sierra Nevada, and the water is detained by a series of dams upstream of the collection point, which makes this matrix water consistent in composition (Hladik and Calhoun, 2012). The river has low suspended sediment and low dissolved organic carbon and has not had any pesticide detections of the target compounds in blank samples during the development of this method. Before sample processing, nine water and nine filter samples were individually spiked to a concentration of 15 ng/L of all analytes, while one water and filter sample were left blank to monitor for background concentrations, contamination, or both. Samples were processed following protocols in the standard operating procedure (SOP) and analyzed by LC-MS/MS (ESI[+] and ESI[−]) and GC-MS/MS. Background concentrations or contamination were not detected in blank samples.
Table 6.
Recoveries and relative standard deviations (RSDs) for target analytes and surrogate compounds from initial recovery study (spiked concentration=50 nanograms per liter; sample size, n=3).[CAS, Chemical Abstract Service; NWIS P code, National Water Information System parameter code; %, percent; LC-MS/MS, liquid chromatography-tandem mass spectrometry; GC-MS/MS, gas chromatography-tandem mass spectrometry; —, no data]
The MDLs were computed using the following equation:
whereMDLS
is the method detection limit of an analyte,
t(n−1, 1−α=0.99)
is the Student’s t-value appropriate for a single-tailed 99th percentile t statistic and a standard deviation estimate with n−1 degrees of freedom, and
Ss
is the sample standard deviation of the replicate spiked sample analyses.
For the MDL test (15 ng/L) average recoveries, RSDs, MDLs, and RLs for water and filter fractions are reported in table 7 for all target analytes.
Table 7.
Water and filter recoveries, relative standard deviations (RSDs), method detection limits (MDLs), and reporting limits (RLs) for target compounds in MDL spike samples (spiked concentration=15 nanograms per liter; sample size, n=9).[%, percent; NR, not reported]
Data Handling
All samples must be tracked using a LIMS or laboratory notebook throughout sample preparation and analysis. Sample collection information, laboratory sample manipulations (filtration, SPE, and addition of solutions), and physical sample measurements are recorded. Once samples are analyzed by the instruments, raw data are checked before quantification. After quantification, the data are reviewed by the project lead, the lead chemist, or both, and any suspected data errors are investigated and resolved or verified. If data quality objectives are not met, sample handling, instrument performance, and data will be further reviewed. Further training may be necessary if errors were observed in sample preparation protocols. Samples must be reanalyzed if instrument performance was unsatisfactory. Lastly, samples with poor QC performance (surrogate recovery less than 70 percent) will be flagged in the LIMS database or final reports if data quality objectives are not met upon further review. Sample analytical results are then uploaded to the USGS National Water Information System (NWIS; U.S. Geological Survey, 2023b) based on project specific needs and timelines.
Summary
This method provides details for analysis of 183 pesticides and pesticide degradates in whole-water samples, through the analysis of filtered water and suspended sediment (183 analytes in filtered water; 178 analytes in suspended sediment). Following filtration, water is extracted via solid-phase extraction (SPE), combined with bottle washes, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS). Suspended sediment is extracted using an ultrasonication, solid-liquid extraction with organic solvent, passed through sodium sulfate, and analyzed by LC-MS/MS and GC-MS/MS. Recoveries of pesticides and pesticide degradates spiked at 50 nanograms per liter (ng/L) in water collected from the American River, California, ranged from 59.5 to 108.9 percent. Recoveries of pesticides and pesticide degradates in test water (183 analytes) and suspended sediment filters (178 analytes) fortified at 15 ng/L ranged from 70.1 to 121.0 percent and 71.1 and 117.0 percent in water and suspended sediment filter samples, respectively. Method detection limits (MDLs) ranged from 0.5 to 10.6 ng/L in water and 0.7 to 11.8 ng/L in suspended sediment filters. The reporting limits were 1.1–21.1 ng/L and 1.5–23.7 ng/L in water and filter samples, respectively. The developed method is applied to surface-water samples for the analysis of pesticides, pesticide degradates, and other agrochemicals. Support for this report was provided by the California Water Science Center.
References Cited
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Conversion Factors
International System of Units to U.S. customary units
Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:
°F = (1.8 × °C) + 32.
Supplemental Information
Concentrations of chemical constituents in water are given in nanograms per liter (ng/L).
Concentration equivalent units (assuming 1 liter equals one kilogram):
parts per thousand: mg/mL
parts per million (ppm): ng/µL
parts per billion (ppb): ng/mL
parts per trillion (ppt): ng/L
megaohm-centimeters (MΩ∙cm): resistivity of water, used in the assessment of ultrapure water
volt (V): unit of electric potential difference
Abbreviations
AEI
advanced electron ionization
CCV
continuous calibration verification
EPA
U.S. Environmental Protection Agency
ESI
electrospray ionization
GC
gas chromatography
GC-MS/MS
gas chromatography-tandem mass spectrometry
HLB
hydrophilic-lipophilic balance
LC
liquid chromatography
LC-MS
liquid chromatography-mass spectrometry
LC-MS/MS
liquid chromatography-tandem mass spectrometry
LIMS
laboratory information management system
MDL
method detection limit
OCRL
organic chemistry research laboratory
PTV
programmable temperature vaporizing
QA/QC
quality assurance/quality control
RL
reporting limit
RSD
relative standard deviation
SPE
solid-phase extraction
USGS
U.S. Geological Survey
For more information concerning the research in this report, contact the
Director, California Water Science Center
U.S. Geological Survey
6000 J Street, Placer Hall
Sacramento, California 95819
https://www.usgs.gov/centers/california-water-science-center
Publishing support provided by the U.S. Geological Survey
Science Publishing Network, Sacramento Publishing Service Center
Disclaimers
Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government.
Although this information product, for the most part, is in the public domain, it also may contain copyrighted materials as noted in the text. Permission to reproduce copyrighted items must be secured from the copyright owner.
Suggested Citation
Gross, M.S., Sanders, C.J., De Parsia, M.D., and Hladik, M.L., 2024, Methods of analysis—Determination of pesticides in filtered water and suspended sediment using liquid chromatography- and gas chromatography-tandem mass spectrometry: U.S. Geological Survey Techniques and Methods, book 5, chap. A12, 33 p., https://doi.org/10.3133/tm5A12.
ISSN: 2328-7055 (online)
| Publication type | Report |
|---|---|
| Publication Subtype | USGS Numbered Series |
| Title | Methods of analysis—Determination of pesticides in filtered water and suspended sediment using liquid chromatography- and gas chromatography-tandem mass spectrometry |
| Series title | Techniques and Methods |
| Series number | 5-A12 |
| DOI | 10.3133/tm5A12 |
| Year Published | 2024 |
| Language | English |
| Publisher | U.S. Geological Survey |
| Publisher location | Reston, VA |
| Contributing office(s) | California Water Science Center |
| Description | Report: vi, 33 p.; Data Release |
| Online Only (Y/N) | Y |
| Google Analytic Metrics | Metrics page |