Methods of Analysis by the U.S. Geological Survey Organic Geochemistry Research Group-Determination of Glyphosate, Aminomethylphosphonic Acid, and Glufosinate in Water Using Online Solid-Phase Extraction and High-Performance Liquid Chromatography/Mass Spectrometry

An analytical method for the determination of glyphosate, its principal degradation compound, aminomethylphosphonic acid (AMPA), and glufosinate in water with varying matrices has been developed. Four different sample matrices fortified at 0.2 and 2.0 µg/L (micrograms per liter) were analyzed using precolumn derivatization with 9-fluorenylmethylchloroformate (FMOC). After derivatization, cleanup and concentration were accomplished using automated online solid-phase extraction followed by elution with the mobile phase allowing for direct injection into a liquid chromatograph/mass spectrometer (LC/MS). Analytical conditions for MS detection were optimized, and quantitation was carried out using the following representative ions: 390 and 168 for glyphosate; 332, 110, and 136 for AMPA; and 402, 180, and 206 for glufosinate. Matrix effects were minimized by utilizing standard addition for quantification and an isotope-labeled glyphosate (2-¹³C,¹⁵N) as the internal standard. Method detection limits (MDLs) were 0.084 µg/L for glyphosate, 0.078 µg/L for AMPA, and 0.057 µg/L for glufosinate. The method reporting limits (MRLs) were set at 0.1 µg/L for all three compounds. The mean recovery values ranged from 88.0 to 128.7 percent, and relative standard deviation values ranged from 5.6 to 32.6 percent.

Glyphosate [N-(phosphonomethyl)glycine] is a broad-spectrum, nonselective, postemergence herbicide that is used extensively in the United States in various applications for weed and vegetation control. Aminomethylphosphonic acid (AMPA) is a degradation product of glyphosate. Glufosinate [ammonium DL-homoalanin-4-(methyl)phosphinate] is similar to glyphosate in chemical structure and use.

The three compounds are very polar and highly soluble in water. The detection of these compounds requires the use of a derivatization step. Published methods outline the use of precolumn derivatization using 9-fluorenylmethylchloroformate (FMOC) coupled with high-performance liquid chromatography (HPLC) with fluorescence detection (Spark-Holland, 1996) or tandem mass spectrometry (MS/MS) detection (Vreeken and others, 1998). Fluorescence detection has sensitivity but lacks specificity, and the MS/MS method can be subject to matrix variation in derivatization and fragmentation.

Utilization of isotope-labeled (2-¹³C,¹⁵N) glyphosate as an internal standard carried through all steps of a method developed by the U.S. Geological Survey (USGS) Organic Geochemistry Research Group in Lawrence, Kansas, addresses the variations in derivatization and fragmentation for the analysis of glyphosate. Analysis of samples using the standard-addition method, adding a known amount of standard(s) to a matching replicate of each unknown sample further addresses matrix variations for glyphosate, AMPA, and glufosinate.

The method of analysis described in this report has been assigned the USGS method code "0-2136-01." This unique code represents the automated method of analysis as it is described in the report and can be used to identify the method.

This report provides a detailed description of the method, including the apparatus, reagents, instrument calibration, and the solid-phase extraction (SPE) procedure required for sample analysis. Method detection limits (MDLs), mean extraction recoveries, and relative standard deviations for the method also are presented.

DETERMINATION OF GLYPHOSATE, AMINOMETHYLPHOSPHONIC ACID, AND GLUFOSINATE IN WATER

Water samples are filtered at the collection site using glass-fiber filters with nominal 0.70-µm pore diameter to remove suspended particulate matter. In the laboratory, 10 mL of sample(s) are dispensed into two labeled, 19-mL, screw-capped plastic tubes. The sample in the tube labeled "standard addition" is fortified with 1 µg/L of each compound to be analyzed. Internal standard solutions are added to both tubes, the sample is buffered to pH 9.0 by adding borate buffer, and after mixing, a solution of FMOC is added to all tubes. Derivatization is carried out in the dark in a water bath at 40 °C. After 24 hours, the reaction is stopped and stabilized by adding 2-percent phosphoric acid. All tubes are stored in the dark until analyzed.

A 5.5-mL aliquot of each sample and the matching standard-addition sample are diluted 1:1 with reagent water in autosampler vials, capped, and placed in the tray of the autosampler. The autosampler provides the sample loading for the automated, online SPE system. The SPE cartridge is conditioned with methanol and reagent water. Ten milliliters (10 mL) of the diluted sample are loaded onto the cartridge. After a 500-µL reagent-water rinse, the cartridge is placed into the flow path of the liquid chromatograph (LC) preceding the column. The conditions and gradient of the mobile phase are set to elute the compounds of interest and leave the excess derivatization reagent on the cartridge.

The sample's compounds are separated by the LC column and detected by the mass spectrometer (MS). Compounds are identified by comparing retention times with the retention times of the standard-addition sample and further by comparision of the selected fragment ions. The concentration of each compound is calculated by determining the ratio of the compound to the internal standard to the ratio of the same compound in the standard-addition sample minus the ratio of the sample. The sample and standard-addition sample are analyzed sequentially, using the same method and instruments.

• Analytical balance--capable of accurately weighing 0.050 0g ±0.0001 g.
• Autopipettes--10- to 10,000-µL, variable-volume autopipettes with disposable plastic tips (Rainin, Woburn, Massachusetts, or equivalent).
• Autosampler--Triathlon, type 900 (Spark-Holland, The Netherlands) equipped with:
10-mL syringe,
10-mL sample loop, and
Type C sample trays (eight each, holding four 20-mm, 10-mL vials).
• Automated online SPE instrument--Prospekt, type 795/796-900 (Spark-Holland, The Netherlands).
• Mechanical vortex mixer.
• Water bath.
• Analytical column--Phenomenex Prodigy, 5-mm, 250- x 3-mm C-18 column (Torrance, California).
• HPLC/MS benchtop system--Hewlett Packard (Wilmington, Delaware), model 1100 HPLC with autoinjector and MS detector.
• LC oven conditions: constant 35 °C.
• LC mobile phase: A, 5 mM ammonium acetate in distilled water; B, acetonitrile. Gradient from 5-percent solvent to 17-percent solvent B over 8.5 minutes, 17- to 60-percent solvent B over 10 minutes; 100-percent solvent B for 4 minutes. Flow maintained at 0.5 mL/min.
• MS detector mode: electrospray in negative-ion mode.
• Drying gas flow was set at 9 L/min.
• Nebulizer gas pressure was set at 25 lb/in².
• Fragmentor voltage was set at 70 V.
• Drying gas temperature was set at 250 °C.
• Capillary voltage was set at 3,500 V.
• Data acquisition system--computer and printer compatible with the HPLC system.
• Software--LC/MSD ChemStation, ver. A.06.03 (Hewlett Packard, Wilmington, Delaware), was used to acquire and store data, for peak integration, and for quantitation of compounds.

• Sample bottles--baked 4-oz amber glass bottles (Boston round) with Teflon-lined lids.
• Sample filters--nominal 0.7-µm glass-fiber filters (Gilson, Middleton, Wisconsin, or equivalent).
• Reagent water--generated by purification of tap-water through activated charcoal filter and deionization with a high-purity, mixed-bed resin, followed by another activated charcoal filtration, and finally distillation in an autostill (Barnstead, Dubuque, Iowa, or equivalent).
• Analytical standards--standards for glyphosate, aminomethylphosphonic acid, glufosinate, cysteic acid, and isotope-labeled glyphosate.
• SPE cartridges--Waters Oasis HLB extraction cartridges, Prospekt (10 mm x 2 mm) (Waters, Milford, Massachusetts).
• Disposable plastic screw-capped tubes--Fisher 14-959-40B (Fisher Scientific, Pittsburg, Pennsylvania) or equivalent.
• Plastic rack for tubes.
• Solvents--
• Acetonitrile, American Chemical Society (ACS) and HPLC grade.
• Methanol, ACS and HPLC grade.
• Gas for mass spectrometer--nitrogen.
• Ammonium acetate--ACS grade.
• Phosphoric acid--ACS grade.
• Sodium borate--ACS grade.
• 9-fluorenylmethylchloroformate--ACS grade.
• 0.1-mL autosampler vials--plastic vial with glass-cone insert and cap (Wheaton, Millville, New Jersey).
• 10-mL autosampler vials--glass vial with Teflon-lined cap (Chromacol, Trumbull, Connecticut).
• Nebulizer gas--nitrogen.

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

Sample-collection equipment must be free of tubing, gaskets, and other components made of nonfluorinated plastic material that might leach interfering compounds into water samples or absorb the compounds from the water. The water samples from each site are composited in a single container and filtered through a nominal 0.7-µm glass-fiber filter using a peristaltic pump. Filters are preconditioned with about 200 mL of sample prior to filtration of the sample. The filtrate for analysis is collected in baked, 125-mL amber glass bottles with Teflon-lined lids. Samples are chilled immediately and shipped to the laboratory within 3 days of collection. At the laboratory, samples are logged in, assigned identification numbers, and refrigerated at 4 °C until derivatized and analyzed.

• Primary standard solutions--Glyphosate, AMPA, and glufosinate were obtained from Chem Service, Inc. (West Chester, Pennsylvania). A solution of 1 mg/mL (corrected for purity) is prepared by accurately weighing, to the nearest 0.0001 g, 50 mg of the pure material into a 50-mL volumetric flask and then diluting with reagent water. The solution is stored at 4 °C.
• Intermediate composite standard--A 10-µg/mL composite standard is prepared in a plastic container by combining 1 mL of each of the three stock solutions of the compounds with 97 g of reagent water. This composite standard is stored at 4 °C. The composite standard is prepared on a monthly basis.
• Standard-addition solution--A 100-µg/L solution is prepared in a plastic container by diluting the intermediate composite standard solution 1:100 with reagent water.
• Internal-standard solution--The isotope-labeled glyphosate (2-¹³C,¹⁵N) is purchased as a 100-mg/mL stock solution from Cambridge Isotope Laboratories, Andover, Massachusetts. A 100-µg/L solution is prepared in a plastic container by adding 20 µL of the stock solution to 20 mL of reagent water.
• Internal-standard-solution time reference--50 µg/L of cysteic acid from Sigma, St. Louis, Missouri, in acetonitrile.

• 5 mM ammonium acetate in reagent water (mobile-phase A).
• 2 mM 9-fluorenylmethylchloroformate in acetonitrile.
• 2 percent (volume/volume) phosphoric acid in reagent water.
• 0.1 percent (volume/volume) phosphoric acid in reagent water (rinse solution for autosampler).
• 5 percent (weight/volume) sodium borate in reagent water.
• Acetonitrile (mobile-phase B and rinse solution for autosampler).
• Methanol.
• Reagent water.

Evaluation of High-Performance Liquid Chromatograph/ Mass Spectrometer Performance

Background absorbance readings, peak shape, and system pressure are used to evaluate LC performance. Background absorbance readings should remain stable and low and indicate that the LC column has equilibrated with the mobile-phase flow. If peak shape deteriorates, the columns may need to be replaced. If the pressure reading is high, there may be a clog in the mobile-phase flow path, or the column-compartment thermostat may not have reached the required temperature.

Mass spectrometer performance is evaluated by assessing isotopic ratios and abundance. The MS is tuned in electrospray, negative-ion mode before each HPLC/MS analytical run using the solutions, procedure, and software supplied by the manufacturer.

A calibration table is prepared from an analyzed standard using the LC/MSD Chemstation software (Hewlett Packard, Wilmington, Delaware). Manufactures' instructions are followed for using the internal standards as time references and for quantitation.

The relative retention time (RRTc) is calculated for each selected 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 selected compound, and
where RT_i = uncorrected retention time of the internal standard.

The expected retention time (RT) of the peak of the selected compound needs to be within ±2 percent of the expected retention time on the basis of the RRT_c obtained from the internal-standard analysis. The expected retention time is calculated as follows:

The samples are derivatized upon arrival in the laboratory, then stored in a refrigerator in the dark until analyzed on the instruments.

The LC/MSD Chemstation software (Hewlett Packard, Wilmington, Delaware) is used with the previously prepared calibration table for identification of compounds.

If a selected compound has passed the qualitative identification criteria, the concentration in the sample is calculated as follows:

(3),
C = [ A_c / A_i / [ A_csa / A_isa - A_c/A_i ]] x SA_cx DF,
where C = concentration of the selected compound in the sample, in micrograms per liter;
where A_c = area of peak of the (molecular or fragment) ion for the selected compound;
where A_i = area of peak of the molecular ion for the internal standard;
where A_csa = area of peak of the (molecular or fragment) for the selected compound in the standard-addition sample;
where A_isa = area of peak of the molecular ion for the internal standard for the standard-addition sample;
where SA_c = concentration of standard addition; and
where DF = dilution factor, for samples that have exceeded upper range of method.

Glyphosate, AMPA, and glufosinate are reported in concentrations ranging from 0.1 to 2.0 µg/L. If the concentration is greater than 2.0 µg/L, a portion of the original sample is diluted appropriately with reagent water and reanalyzed through the entire procedure.

A reagent-water sample, a ground-water sample collected from a well in Sedgwick County, Kansas, a surface-water sample from the Kisco River below Mt. Kisco, New York, and a surface-water sample from the spillway below Clinton Lake in Kansas were used to test the performance of method 0-2136-01. All samples were filtered through a nominal 0.7-µm glass-fiber filter and stored at 4 °C.

An HPLC method utilizing online SPE and fluorescence detection was reported by Spark-Holland (1996) as being quite sensitive but lacking the confirmation available using mass spectrometry. Also, the mobile phase used was not readily compatible with most mass spectrometers currently (2001) in use. The use of disposable SPE cartridges, as outlined by Spark-Holland (1996), improves LC column life and reduces the possiblity of carryover.

An HPLC/MS/MS method utilizing online SPE was reported by Vreeken and others (1998). This HPLC/MS/MS method used the same column repeatedly for cleanup and lacked the accuracy afforded by the use of internal standards. Both of the HPLC and HPLC/MS/MS methods used precolumn derivatization with FMOC.

An GC/MS/MS method utilizng ion-exchange chromatography followed by derivatization was reported by Royer and others (2000). The GC/MS/MS method required elaborate preparation of columns and an elaborate derivatization procedure.

The incorporation of an isotope-labeled (2-¹³C,¹⁵N) glyphosate as an internal standard carried through the derivatization, extraction, separation, and detection steps enhanced the reproducibility and accuracy of the online SPE and HPLC/MS method described in this report. The use of standard addition for quantitation overcomes the variation in derivatization and fragmentation observed from analyzing compounds with different matrices. The use of the more readily available single quadrapole mass spectrometer allowed for simpler and less-expense operation.

The method described in this report provides for routine analysis of glyphosate, AMPA, and glufosinate in environmental water samples. Derivatization with FMOC, online SPE, and HPLC/MS are shown to be a reliable and sensitive method for low concentrations.

Good precision and accuracy for the analysis of glyphosate, AMPA, and glufosinate were demonstrated for reagent water, ground water, and surface water. Method detection limits were 0.084 µg/L for glyphosate, 0.078 µg/L for AMPA, and 0.057 µg/L for glufosinate. The mean recoveries of water samples spiked at 0.2 and 2.0 µg/L ranged from 88.0 to 128.7 percent with relative standard deviations ranging from 5.8 to 32.6 percent.

Information about the fate and transport of glyphosate, its degradation compound AMPA, and glufosinate in water can be acquired from the analysis of ground water and surface water. This method also can be used for water-quality determinations.

Edwards, T.K., and Glysson, G.D., 1988, Field methods for measurement of fluvial sediment: U.S. Geological Survey Open-File Report 86-531, 118 p.

Hardy, M.A., Leahy, P.P., and Alley, W.M., 1989, Well installation documentation and ground-water sampling protocols for the pilot National Water-Quality Assessment Program: U.S. Geological Survey Open-File Report 89-396, 36 p.

Spark-Holland, 1996, Automated determination of AMPA/Glyphosate: Aj Emmen, The Netherlands, Application Info 53, 4 p.

Royer, A., Beguin, S., Tabet, J.C., Hulot, S., Reding, M.A., and Communal, P.Y., 2000, Determination of glyphosate and aminomethylphosphonic acid residues in water by gas chromatography with tandem mass spectrometry after exchange ion resin purification and derivatization-application on vegetable matrixes: Analytical Chemistry, v. 72, p. 3826-3832.

U.S. Environmental Protection Agency, 1992, Guidelines establishing test procedures for the analysis of pollutants-appendix B, part 136, Definition and procedures for the determination of the method detection limit: U.S. Code of Federal Regulations, Title 40, revised as of July 1, 1992, p. 565-567.

Vreeken, R.J., Speksnijder, P., Bobeldijk-Pastorova, I., and Noij, Th.H.M., 1998, Selective analysis of the herbicides glyphosate and aminomethyl phosphonic acid in water by online solid-phase extraction/high-performance liquid chromatography-electrospray ionization mass spectrometry: Journal of Chromatography A, v. 794, p.187-199.

Ward, J.R., and Harr, C.A., 1990, Methods for collection and processing of surface-water and bed-material samples for physical and chemical analyses: U.S. Geological Survey Open-File Report 90-140, 71 p.

APPENDICES

Glyphosate method 0-2136-01

Basic system parameters:

Wash solution: 0.1-percent phosphoric acid in reagent water.
User program (only program available using 10-mL vials).
Loads 10 mL of sample on cartridge in online automated solid-phase extraction (SPE) instrument.
10-mL syringe; 10-mL sample loop; 15-mL buffer loop (sample loop calibrated at 9.9 mL).
Type C: four vials by eight sample tray segments.

Glyphosate method 0-2136-01

Sample preparation program

Solvent 1 Methanol
Solvent 2 Acetonitrile
Solvent 3 Distilled, deionized water

SP program # 12:

Elutes previously loaded cartridge to HPLC column.
Prepares the next cartridge.
Loads 10 mL from sample loop of autosampler onto the SPE cartridge.
Rinses line from autosampler to online SPE instrument.

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