Methods of Analysis and Quality-Assurance Practices by the U.S. Geological Survey Organic Geochemistry Research Group--Determination of Four Selected Mosquito Insecticides and a Synergist in Water Using Liquid-Liquid Extraction and Gas Chromatography/Mass Spectrometry

A method of analysis and quality-assurance practices were developed for the determination of four mosquito insecticides (malathion, metho-prene, phenothrin, and resmethrin) and one synergist (piperonyl butoxide) in water. The analytical method uses liquid-liquid extraction (LLE) and gas chromatography/mass spectrometry (GC/MS). Good precision and accuracy were demonstrated in reagent water, urban surface water, and ground water. The mean accuracies as percentages of the true compound concentrations from water samples spiked at 10 and 50 nanograms per liter ranged from 68 to 171 percent, with standard deviations in concentrations of 27 nanograms per liter or less. The method detection limit for all compounds was 5.9 nanograms per liter or less for 247-milliliter samples. This method is valuable for acquiring information about the fate and transport of these mosquito insecticides and one synergist in water.

The persistence of organic pesticides in water is of great importance because of concerns over water quality. Pesticides that find their way into lakes, streams, or drinking-water supplies may pose a potential health threat to wildlife and humans. The U.S. Geological Survey (USGS), as part of the Toxic Substances Hydrology Program, has been studying the fate and transport of four mosquito insecticides and a synergist in the New York City metropolitan area.

Recently, there has been concern in the Northeastern United States about the appearance of the West Nile virus. The West Nile virus was first identified in Africa (Center for Disease Control, 2001a) and has since spread to temperate regions of Europe and North America. It is generally not dangerous to healthy humans but can develop into a deadly form of encephalitis (inflammation of the brain) in the elderly, children, and people with compromised immune systems. In the United States, West Nile virus is transmitted by infected mosquitoes, primarily members of the culex species (Center for Disease Control, 2001b).

A direct way to combat the spread or prevent a recurrent outbreak of West Nile virus is to control the mosquito population. One of the methodologies for control is the use of insecticides, either larvicides or adulticides.

Larvicides for mosquito control include metho-prene, an insect growth regulator. Methoprene controls mosquito larva populations by mimicking the natural juvenile growth hormone, JHIII. This hormone inhibits developing mosquito pupae from molting and passing into the adult stage where they could reproduce. Methoprene is available in suspension, emulsifiable, and soluble concentrate formulations, as well as in briquette, aerosol, and bait form. Methoprene was introduced in the late 1970s as a means of flea and mosquito control.

Adulticides, which may be used in the chemical control of mosquitos, include the organophosphate malathion and the pyrethroids phenothrin, also called sumithrin, and resmethrin. In addition, a synergist compound commonly is applied with pyrethroids to overcome resistance that pests develop with use of insecticides.

Malathion is a nonsystemic, wide-spectrum organophosphate insecticide. It was one of the earliest organophosphate insecticides developed (introduced in 1950). Malathion is used for the control of mosquitoes, flies, household insects, animal parasites (ectoparasites), and head and body lice.

Pyrethrins are natural insecticides in the flowers of certain species of the chrysanthemum plant. Semisynthetic derivatives of the chrysanthemumic acids have been developed as insecticides. These are called pyrethroids and tend to be more effective than natural pyrethrins, and they are less toxic to mammals. The most frequently used pyrethroids for adult mosquito control are phenothrin, also called sumithrin, and resmethrin.

Insects possess an enzyme system called the mixed-function oxidases (MFOs) that give them the ability to rapidly detoxify and become resistant to many insecticides, especially pyrethroids. Piperonyl butoxide (PBO) inhibits the action of MFOs, which allows the applicator to use less active ingredient to obtain the mortality rate desired or to prolong the usefulness of insecticides by overcoming MFO resistance. As is common with pyrethroid insecticides, the synergist compound piperonyl butoxide (PBO) is applied with phenothrin and resmethrin.

An analytical method and quality-assurance practices were developed for the determination of four mosquito insecticides and one synergist at nanogram-per-liter levels in water samples. The method involves using liquid-liquid extraction (LLE) to isolate the compounds from water samples and gas chromatography/mass spectrometry (GC/MS) to identify and quantify these compounds. Quality-control practices include evaluation of laboratory blank and spiked samples, instrument performance, and corrective actions. Method detection limits (MDLs) are calculated on the basis of procedures recognized by the U.S. Environmental Protection Agency (USEPA) (1992). Mean recoveries of the targeted insecticides and synergist from reagent, surface, and ground water also are presented.

The LLE-GC/MS method of analysis described in this report and used at the USGS Organic Geochemistry Research Laboratory in Lawrence, Kansas, has been assigned the method number "O-2134-01" by the USGS Office of Water Quality in Reston, Virginia. At the Organic Geochemistry Research Laboratory, the method of analysis described herein has been given the analysis code "GCM." This unique analysis code can be used to identify the method.

                                ¹ Tomlin (1997).
                                ² Nature Conservation Council of New South Wales (2001).

Compounds were selected because of their potential use in controlling mosquitoes in the New York City metropolitan area. The calibration range for the method is equivalent to concentrations from 5 to 100 ng/L without dilution.

Water samples are filtered at the collection site using glass-fiber filters with 0.7-mm nominal pore diameter to remove suspended particulate matter. In the laboratory, a surrogate compound is added, and a small volume of sample is removed from the bottle. Then hexane is added directly to the remaining sample in the bottle and mixed. The hexane extract is removed, spiked with an internal standard, and evaporated under nitrogen. The sample components are separated, identified, and measured by injecting an aliquot of the concentrated extract into a high-resolution, fused-silica capillary column of a GC/MS system under selected-ion mode (SIM). Compounds eluting from the GC column are identified by comparing their measured ions and retention times to reference ions and retention times obtained by the measurement of control samples under the same conditions used for the water samples. The concentration of each identified compound is measured by relating the MS response of the quantitation ion produced by that compound to the MS response of the quantitation ion produced by the surrogate standard.

Organic compounds having identical mass ions and GC retention times to those of the compounds of interest may interfere.

• Sample bottles-Baked 8-oz amber glass bottles (Boston round) with Teflon-lined lids.
• Reagent water-Generated by purification of tapwater through activated charcoal filtration and deionization with a high-purity, mixed-bed resin, followed by another activated charcoal filtration, and finally distillation in an autostill (Barnstead, or equivalent, Dubuque, IA).
• Analytical standards-Standards of the insecticides and synergist, surrogate standard, and the internal standard.
• Disposable serological pipette-25 mL (Kimble, or equivalent, Vineland, NJ).
• Bottle-top liquid dispensers-For measuring and dispensing 7 mL of hexane and 1 mL reagent water (BrandTech Scientific, Inc., or equivalent, Wertheim, Germany).
• Solvents-
• Hexane, American Chemical Society (ACS) and high-performance liquid chromatography (HPLC) grade.
• Ethyl acetate, HPLC grade.
• Methanol, ACS and HPLC grade.
• Disposable snap-cap finish centrifuge tubes-10 mL (Kimble, or equivalent, Vineland, NJ).
• Gas for evaporation-Nitrogen, ultrapure grade.
• Pasteur pipettes-(Kimble, or equivalent, Vineland, NJ).
• 0.1-mL autosampler vials-Amber plastic vial with glass cone insert and cap (Wheaton, Millville, NJ).
• GC carrier gas-Helium, ultrapure grade.

Following USGS protocol, sampling methods capable of collecting water samples that accurately represent the water-quality characteristics of the surface water or ground water at a given time or location are used. Detailed descriptions of sampling methods used by the USGS to obtain surface-water samples are given in Edwards and Glysson (1988) and Ward and Harr (1990). Similar descriptions of sampling methods for obtaining ground-water samples are given in Hardy and others (1989).

Briefly, sample-collection equipment is free of tubing, gaskets, and other components made of nonfluorinated plastic material that might leach interferences into water samples or sorb organic compounds from the water. The water samples from each site are composited in a single container and filtered through a 0.7-µm glass-fiber filter using a peristaltic pump (Sandstrom, 1995). Filters are leached with about 200 mL of sample prior to filtration of sample. The filtrate for analysis is collected in baked 8-oz amber glass bottles with Teflon-lined lids. Samples are chilled immediately and shipped to the laboratory via an overnight carrier. At the laboratory, samples are logged in, assigned identification numbers, and extracted on the day they arrive.

• Extraction set-up-An extraction set consists of as many as six samples. In addition to the samples, each extraction set has at least one replicate sample, a matrix spike control, one laboratory blank control, a high-concentration control, and a low-concentration control. All the bottles in the extraction set are processed identically.
• Sample preparation-Samples and controls are prepared in 8-oz amber glass bottles filled to the base of the neck of the bottle. The volume of sample processed is 247 mL. Should a sample contain less than 247 mL, distilled water is added to bring the volume to the required 247 mL. Any volume added is recorded.
• Spiking of surrogate standard-Each sample and control is spiked with 100 µL of surrogate standard (1.23 ng/µL terbuthylazine in methanol). Spiking is performed using a repeating pipetter with a dedicated syringe tip. All samples, the replicate sample, and controls then are capped and shaken by hand to assure that the surrogate standard is well mixed.
• Removal of excess liquid-Approximately 25 mL of sample are removed from each sample and control using a 25-mL disposable serological pipette. This allows space for the sample to be extracted in its original sampling bottle.
• Transferring of compounds to organic phase-7 mL of hexane are added to each sample and control using a bottle-top dispenser. Each sample bottle then is capped and agitated by vigorously shaking by hand for at least 30 sec. A mechanical wrist-action shaker may be used. Agitation then is repeated for an additional 10 sec two times to assure that there has been sufficient mixing to allow any insecticides and synergist to be transferred into the organic hexane phase.
• Removal of hexane-Distilled water is added to each bottle to bring the level of sample and hexane to the top of the bottle. This allows for easier removal of the hexane. Each bottle is allowed to stand for 10 min so that the organic hexane phase can separate from the aqueous phase. The organic hexane layer is removed from each sample and control using a pasteur pipette and transferred to a labeled test tube that has been pre-spiked with 100 µL of internal standard (0.2-ng/µL phenanthrene-d₁₀ in ethyl acetate).
• Evaporation-The spiked extracts are evaporated to a volume of approximately 60 µL using a solvent evaporator with 103 kPa nitrogen and a 45-°C water bath. Each extract then is transferred to a 0.1-mL autosampler vial using an autopipette with disposable tips and capped. The extracts are stored at less than 0 °C until analysis by GC/MS.

• Initial calibration curves are prepared using freshly prepared calibration standards that are extracted using the same procedure as samples (described previously).
• Data are acquired from a GC/MS that meets all performance criteria using the same procedure and method as samples.
• Calculate the relative retention time (RRTc) for each selected compound and the surrogate compound in the calibration solution or in a sample as follows:

(1),
RRT_c = RT_c/RT_i,
where RT_c = uncorrected retention time of the quantitation ion of the selected compound or surrogate compound, in minutes, and
where RT_i = uncorrected retention time of the quantitation ion of the internal standard (phenanthrene-d₁₀), in minutes.

Mass spectrometer performance is evaluated by assessing isotopic ratios, contamination, electron multiplier sensitivity, and abundance.

• Tune the mass spectrometer before each GC/MS sample set (approximately 43 injections or three extraction sample sets) using the procedure and software supplied by the manufacturer. Parameters in the tuning software are set to give &pulsmn;0.15-amu resolution at masses 69, 219, and 502 in the spectrum of perfluorotributylamine (PFTBA). With the resolution of the 69 ion at 100-percent abundance, the mass 219 ion should be 35 &pulsmn;20 percent, and the mass 502 ion should be more than 3 percent relative abundance; however, the relative abundances may vary depending on the mass spectrometer used. Check mass assignments to ensure accuracy to &pulsmn;0.15 amu and that mass peak widths measured at one-half the peak height range from about 0.50 to 0.60 amu.
• Also, during the tuning of the mass spectrometer, check the mass spectrometer for the presence of excessive water and air, which indicate leaks in the vacuum. If detected, locate and fix leaks.
• Initially adjust the electron multiplier of the mass spectrometer to ensure that the established reporting level for each selected compound can be achieved.

• The expected retention time (RT) of the peak of the selected insecticide or synergist of interest needs to be within &pulsmn;6 sec of the expected retention time on the basis of the RRT_c obtained from the internal-standard analysis. Calculate the expected retention time as follows:

(2),
RT = (RRT_c)(RT_i),
where RT = expected retention time of the selected compound, in minutes
where RRT_c = relative retention time of the selected compound, dimensionless; and
where RT_i = uncorrected retention time of the internal standard, in minutes.

• Mass-spectral verification for each selected compound is done by comparing the relative abundance values of the quantification and qualification ion(s) to the same values obtained from the control standard samples. The relative ratios of the ions need to be within &pulsmn;20 percent of the relative ratios obtained in the absence of any obvious interferences.

• Calculate the dilution factor to correct for the volume of sample processed as follows:

(3),
DF = (247 / (247 - V_a) ,
where DF = dilution factor, and
where V_a = volume added = milliliters of distilled water added to a sample that contains less than 247 mL.

The dilution factor is incorporated into the calculation for determining final concentrations of samples.

• If a selected insecticide or synergist has passed the aforementioned qualitative identification criteria, calculate the concentration in the sample as follows:

(4),
C = ((A_c/A_i)²+ (b) (A_c/A_i) + 0) x (DF) x (SC) ,
where C = concentration of the selected insecticide or synergist in the sample, in nanograms per liter;
where A = coefficient of x² in the quadratic curve fit;
where A_c= area of the quantitation ion for the selected insecticide or synergist identified;
where A_i = area of the quantitation ion of the surrogate standard, terbuthylazine;
where b = coefficient of x in the quadratic curve fit;
where DF = dilution factor as calculated in equation 3; and
where CF = slope correction.

The four insecticides and the synergist are reported in concentrations ranging from 5 to 100 ng/L. If the concentration is greater than 100 ng/L, the sample is reextracted with a 1:10 dilution (sample:distilled water) and reanalyzed for those compounds that were greater than 100 ng/L.

Quality-control data are produced to quantitatively check the measurement process for environmental samples. The types of quality-control data collected include results of the analysis of duplicate samples, matrix-spiked samples, laboratory blank samples, and controls of differing concentrations.

Each extraction set of as many as six samples contains a minimum of one duplicate sample. The duplicate samples are analyzed concurrently and reanalyzed if agreement of the calculated concentration for any detected insecticide or synergist is not within 40 percent, as determined by the relative percentage difference.

(6),
RPD = | X₁ - X ₂ / X | x 100 ,
where RPD = relative percentage difference;
where |X₁ - X ₂| = absolute value of the difference between the two values; and
where X = mean of the two values.

Recovery of all target compounds is determined for each matrix-spiked sample. After the water sample is received in the laboratory, 12.35 µL of the primary fortification standard are added prior to extraction. Any compounds present in the unspiked sample are subtracted from the matrix-spiked sample's values. These final concentration values are reported.

Laboratory blank samples are used to demonstrate that laboratory equipment or instruments are cleaned adequately and that no contamination is contributed by the laboratory procedures. A laboratory blank consists of reagent water that is processed exactly like samples. If any insecticide or synergist is detected at any concentration greater than the MDL in the laboratory blank control, the source of the problem is determined and corrected. Samples analyzed in that extraction set then are reevaluated for contamination.

Low and high concentration controls are used to verify the calibration curve being used for quantification. The recoveries for each insecticide and synergist are determined. A new calibration curve is prepared if the recovery is outside the control limits in two consecutive runs. Control limits are initially set at ±20 percent until an adequate number of controls have been analyzed to calculate a relevant standard deviation. Control warning limits are set at ±1.5 standard deviations from the mean and the control limits at ±2 standard deviations from the mean.

Recovery of the surrogate, terbuthylazine, is determined for each sample, including all control samples. Control charts for the terbuthylazine recovery are constructed using the mean; the warning limits are set at 1.5 standard deviations from the mean and the control limits at ±2 standard deviations from the mean. The control charts are constructed using all previous sample terbuthylazine recoveries. A sample is reextracted and reanalyzed on the GC/MS if the recovery is outside the control limits.

This report presents a method of analysis and quality-assurance practices for the determination of four mosquito insecticides and one synergist in natural water samples. From the data presented in this report, liquid-liquid extraction with gas chromatography/ mass spectrometry detection are shown to be a sensitive and reliable method for the determination of nanogram-per-liter concentrations. Good precision and accuracy were demonstrated. Method detection limits ranged from 1.7 to 5.9 ng/L. The mean accuracies of the mosquito insecticides and synergist from water samples spiked at 10 and 50 ng/L ranged from 68 to 171 percent, with relative standard deviations of 6 to 49 percent. Information about the fate and transport of the four mosquito insecticides and one synergist in water can be acquired from the analysis of surface- and ground-water samples. These methods also can be useful for water-quality determinations and analytical verification in toxicological studies.

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