Mass Spectrometric Assessment and Analytical Methods for

Dec 8, 2008 - DuPont Crop Protection, Stine-Haskell Research Center, 1090 Elkton Road, Newark, Delaware 19714, and ABC Laboratories, Inc., 7200 East ...
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Anal. Chem. 2009, 81, 797–808

Mass Spectrometric Assessment and Analytical Methods for Quantitation of the New Herbicide Aminocyclopyrachlor and Its Methyl Analogue in Soil and Water Sergio C. Nanita,*,† Anne M. Pentz,† Joann Grant,‡ Emily Vogl,‡ Timothy J. Devine,†,§ and Robert M. Henze† DuPont Crop Protection, Stine-Haskell Research Center, 1090 Elkton Road, Newark, Delaware 19714, and ABC Laboratories, Inc., 7200 East ABC Lane, Columbia, Missouri 65202 Analytical methods have been developed for the detection and quantitation of a new herbicide active ingredient, aminocyclopyrachlor, and its analogue aminocyclopyrachlor methyl in environmental samples. The analytes were purified from soil extracts and water samples using solid phase extraction based on mixed-mode cation exchange/reverse phase retention. Analyte identification and quantitative analyses were performed by high performance liquid chromatography coupled to tandem mass spectrometry by an electrospray ionization source. External standards prepared in neat solvents were used for quantitation, providing acceptable accuracy, with no matrix effects observed during method validation. The method limits of quantitation (LOQ) were 0.10 ng/mL (ppb, parts-per-billion) in water and 1.0 ng/g in soil for both compounds. The limit of detection (LOD) in water was estimated to be 20 ng/L (ppt, parts-per-trillion) for aminocyclopyrachlor and 1 ng/L for aminocyclopyrachlor methyl, while LODs in soil were 100 ng/kg and 10 ng/kg for aminocyclopyrachlor and aminocyclopyrachlor methyl, respectively. The stability of both compounds in various solvents was evaluated as part of method development. Tandem mass spectrometry experiments were also conducted to investigate the gas-phase fragmentation of aminocyclopyrachlor and its methyl analogue, and the results are reported. A statistical analysis of method validation data generated at two laboratories by multiple chemists authenticates the ruggedness and good reproducibility of the analytical procedures tested. Vegetation management is a common practice by industries to improve their services and/or products while ensuring public safety and by wildlife organizations to maintain endangered ecosystems.1-3 Herbicides represent one of many tools employed * To whom correspondence should be addressed. E-mail: sergio.c.nanita@ usa.dupont.com. Phone: 1-302-451-5806. † Stine-Haskell Research Center. ‡ ABC Laboratories, Inc. § Current Address: DuPont Central Research & Development, Materials Science & Engineering, Experimental Station, Route 141 and Powder Mill Road, Wilmington, Delaware 19880. 10.1021/ac8020642 CCC: $40.75  2009 American Chemical Society Published on Web 12/08/2008

in vegetation management. They are often used to control vegetation along railroads and roadsides1 for transportation safety. Electric companies employ these chemicals to selectively keep unwanted vegetation from interfering with their electricity distribution systems.2 Herbicides are also an important tool for vegetation management in forestry to control the population of invasive species.3 Aminocyclopyrachlor and aminocyclopyrachlor methyl represent the first herbicides in the pyrimidine carboxylic acid class.4,5 They provide excellent control of broad-leaf weeds with low use rates and are currently experimental herbicides being developed by DuPont Crop Protection for weed control in turf, brush vegetation management, and other potential uses.4,5 The herbicidal activity is based on the auxin-mimic6 mode of action, and it continues to be investigated. Regulatory agencies require that registrants of new agrochemicals provide analytical methods that allow trace-level quantitation of the active ingredient and any relevant metabolites in environmental matrixes such as soil and water7,8 and food of plant and/ or animal origin,8,9 depending on the intended use of the product. The methods are required to meet quality standards for accuracy, precision, and reproducibility.7-9 The methods can be used in monitoring efforts to ensure the product is used safely and in compliance with local regulations once registration is granted. Rugged analytical methods are also needed to support studies during the development of experimental agrochemicals, often involving the quantitation of active ingredient(s) and metabolites (1) Luken, J. O.; Beiting, S. W.; Kareth, S. K.; Kumler, R. L.; Liu, J. H.; Seither, C. A. Environ. Manage. 1994, 18, 251–255. (2) Slaughter, D. C.; Giles, D. K.; Tauzer, C. J. Transp. Eng. 1999, 125, 364– 371. (3) Masters, R. A.; Nissen, S. J.; Gaussoin, R. E.; Beran, D. D.; Stougaard, R. N. Weed Technol. 1996, 10, 392–403. (4) Clark, D. A.; Finkelstein, B. L.; Armel, G. R.; Wittenbach, V. A. Patent Publication Number WO/2005/063721. (5) Finkelstein, B. L.; Armel, G. R.; Bolgunas, S. A.; Clark, D. A.; Claus, J. S.; Crosswicks, R. J.; Hirata, C. M.; Hollingshaus, G. J.; Koeppe, M. K.; Rardon, P. L.; Wittenbach, V. A.; Woodward, M. D. Proceedings of the 236th ACS National Meeting, Philadelphia, PA, August, 2008; AGRO No. 19. (6) Kelley, K. B.; Riechers, D. E. Pestic. Biochem. Physiol. 2007, 89, 1–11. (7) U.S. EPA Ecological Effects Test Guidelines, OPPTS 850.7100, Data Reporting for Environmental Chemistry Methods, April 1996. (8) EEC Directive 91/414/EEC, Annex IIA 4.2.2 as amended by EC Directive 96/46/EC; SANCO/825/00 rev.7.(17/03/2004). Guidance Document on Residue Analytical Methods. (9) U.S. EPA Residue Chemistry Test Guidelines, OPPTS 860.1340, Residue Analytical Method, August1996.

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in thousands of soil and crop samples. For example, environmental fate studies10 provide key information about the fate of the active ingredient (e.g., rate of degradation) once the product is applied in the open environment. Poorly performing analytical methods may lead to the generation of unreliable data and increased cost and duration of studies because of reanalysis of rejected sample sets. The complexity of environmental samples and the requirement to measure trace-level concentrations of small molecules create several analytical challenges. For example, small organic molecules like active ingredients in agrochemicals may bind to soil.10 As a result, rigorous extraction procedures are often needed,11 and extraction efficiency of the analytes must be demonstrated in soil methods to ensure acceptable accuracy of analytical determinations.7 This is often done by extracting aged soil samples that have been treated with the active ingredient of interest tagged with a radioactive isotope, like 14C, and comparing the radioactivity measured in the extract to that measured in the soil. This procedure is known as radiovalidation of analytical methods. Another challenge is the matrix present in environmental samples;12,13 thus, some analytical procedures may require extensive sample preparation and cleanup. In addition, the requirement of trace-level quantitation limits the technologies that can be used for instrumental analysis. Highly sensitive and selective techniques are desired since they allow simplification of sample preparation procedures. When high sensitivity is achieved, preconcentration of the analytes may be avoided, and in some cases dilution minimizes the need for sample purification. High selectivity translates to less interferences which allows the use of simple separation procedures. Electrospray ionization (ESI)14,15 is currently the most used atmospheric pressure ion source for mass spectrometry applications since it allows proteins,16,17 biomolecular complexes,18-22 pharmaceuticals,23-26 and other nonvolatile molecules (large and small) that exist in solution to be efficiently ionized and transferred (10) Wauchope, R. D.; Yeh, S.; Linders, J. B. H. J.; Kloskowski, R.; Tanaka, K.; ¨ el, W.; Gerstl, Z.; Lane, M.; Unsworth, J. B. Rubin, B.; Katayama, A.; Kord Pest Manag. Sci. 2002, 58, 419–445. (11) McNally, M. E. P.; Wheeler, J. R. J. Chromatogr. 1988, 447, 53–63. (12) Dijkman, E.; Mooibroek, D.; Hoogerbrugge, R.; Hogendoorn, E.; Sancho, J.-V.; Pozo, O.; Hernandez, F. J. Chromatogr. A 2001, 926, 113–125. (13) Kloepfer, A.; Quintana, J. B.; Reemtsma, T. J. Chromatogr. A 2005, 1067, 153–160. (14) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64–71. (15) Fenn, J. B. Angew. Chem., Int. Ed. 2003, 42, 3871–3894. (16) Mann, M.; Hendrickson, R. C.; Pandey, A. Annu. Rev. Biochem. 2001, 70, 437–473. (17) Van Den Heuvel, R. H. H.; Heck, A. J. R. Curr. Opin. Chem. Biol. 2004, 8, 519–526. (18) Loo, J. A. Int. J. Mass Spectrom. 2000, 200, 175–186. (19) Smith, R. D.; Light-Wahl, K. J.; Winger, B. E.; Loo, J. A. Org. Mass Spectrom. 1992, 27, 811–821. (20) Nanita, S. C.; Cooks, R. G. J. Phys. Chem. B 2005, 109, 4748–4753. (21) Nanita, S. C.; Takats, Z.; Myung, S.; Clemmer, D. E.; Cooks, R. G. J. Am. Soc. Mass. Spectrom. 2004, 15, 1360–1365. (22) Aggerholm, T.; Nanita, S. C.; Koch, K.; Cooks, R. G. J. Mass Spectrom. 2003, 38, 87–97. (23) Hattori, H.; Ito, K.; Iwai, M.; Arinobu, T.; Mizutani, Y.; Kumazawa, T.; Ishii, A.; Suzuki, O.; Seno, H. Forensic Toxicol. 2007, 25, 100–103. (24) Lee, X.-P.; Kumazawa, T.; Sato, J.; Shoji, Y.; Hasegawa, C.; Karibe, C.; Arinobu, T.; Seno, H.; Sato, K. Anal. Chim. Acta 2003, 492, 223–231. (25) Umezawa, H.; Lee, X.-P.; Arima, Y.; Hasegawa, C.; Marumo, A.; Kumazawa, T.; Sato, K. Rapid Commun. Mass Spectrom. 2008, 22, 2333–2341. (26) Gros, M.; Petrovie´, M.; Barcelo´, D. Anal. Bioanal. Chem. 2006, 386, 941– 952.

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to the gas phase. This capability has allowed ESI to serve as an interface for coupling high performance liquid chromatography to mass spectrometry. Highly selective detection can be achieved with two stages of mass analysis (MS/MS), which allows known product ions to be monitored, providing additional discriminative information. This requires mass spectrometers, such as those equipped with triple quadrupoles, which are capable of performing tandem MS experiments. These systems are quickly becoming the well-accepted standard for residue analysis27,28 in environmental, food produce, and biological29 samples because of their outstanding precision, accuracy, sensitivity, and specificity. They are excellent partners for automated online SPE purification30,31 and sample preparation techniques32,33 (e.g., QuEChERS)34 used in multiresidue methods, allowing these systems to quantify hundreds of active ingredients in a single analytical run.35-38 A major and perhaps the most significant disadvantage of ESI when employed for quantitative analysis is the effect of matrix components on the ionization efficiency of analytes; co-eluting matrix can increase or decrease the response of analytes of interest, affecting the accuracy of quantitative measurements.39,40 There are various mechanisms proposed to explain the effects of matrix in analyte ionization efficiency.41 For example, it is known that proton exchange or charge competition between matrix and analytes may lead to signal suppression.41 Analyte/matrix competition for droplet surface during the Coulombic fission42 and droplet evaporation processes may also lead to changes in ionization efficiency, especially if the analytes of interest are ionized through the ion evaporation model (IEM),43 which is the case for most small polar molecules. Recently developed ambient ionization techniques44 derived from electrospray and atmospheric pressure chemical ionization (27) Alder, L.; Startin, J. R.; Alonso, S.; Anspach, T.; Brewin, S.; Broekaert, C.; Christiansen, A.; Dekok, A.; Frase, U.; Fresvig, M.; Hemmerling, Ch.; Hermansson, E.; Hiemstra, M.; Hogendoorn, E.; Kolb, J.; Kombal, R.; Melk, Ch.; Polonji, B.; Quirijns, J. K.; Ross, L.; Saint-Joly, Ch.; Scherbaum, E.; Van Damme, D.; Welter, A.; Wus¨t, B. J. AOAC Int. 2005, 88, 1762–1776. (28) Zywitz, D.; Anastassiades, M.; Scherbaum, E. Dtsch. Lebensmitt. Rundsch. 2003, 99, 188–196. (29) Lee, X.-P.; Kumazawa, T.; Fujishiro, M.; Hasegawa, C.; Arinobu, T.; Seno, H.; Ishii, A.; Sato, K. J. Mass Spectrom. 2004, 39, 1147–1152. (30) Rodriguez-Mozaz, S.; Lopez De Alda, M. J.; Barcelo´, D. Anal. Chem. 2005, 76, 6998–7006. (31) Postigo, C.; Lopez De Alda, M. J.; Barceló, D. Anal. Chem. 2008, 80, 3123– 3134. (32) Lehotay, S. J.; O’Neil, M.; Tully, J.; Garcia, A. V.; Contreras, M.; Mol, H.; Heinke, V.; Anspach, T.; Lach, G.; Fussell, R.; Mastovska, K.; Poulsen, M. E.; Brown, A.; Hammack, W.; Cook, J. M.; Alder, L.; Lindtner, K.; Vila, M. G.; Hopper, M.; De Kok, A.; Hiemstra, M.; Schenck, F.; Williams, A.; Parker, A. J. AOAC Int 2007, 90, 485–520. (33) Anastassiades, M.; Lehotay, S. J.; Stajnbaher, D.; Schenck, F. J. J. AOAC Int. 2003, 86, 412–431. (34) Anastassiades, M.; Lehotay, S. J.; Stajnbaher, D. Proc. WTQA 2002, 231– 241. (35) Greulich, K.; Alder, L. Anal. Bioanal. Chem. 2008, 391, 183–197. (36) Lehotay, S. J.; De Kok, A.; Hiemstra, M.; Van Bodegraven, P. J. AOAC Int. 2005, 88, 595–614. (37) Alder, L.; Greulich, K.; Kempe, G.; Vieth, B. Mass Spectrom. Rev. 2006, 25, 838–865. (38) Klein, J.; Alder, L. J. AOAC Int. 2003, 86, 1015–1037. (39) Rogatsky, E.; Stein, D. J. Am. Soc. Mass Spectrom. 2005, 16, 1757–1759. (40) Dams, R.; Huestis, M. A.; Lambert, W. E.; Murphy, C. M. J. Am. Soc. Mass Spectrom. 2003, 14, 1290–1294. (41) Hajslova, J.; Zrostlıkova, J. J. Chromatogr. A 2003, 1000, 181–197. (42) Ferna´ndez De La Mora, J. J. Colloid Interface Sci. 1996, 178, 209–218. (43) Kebarle, P. J. Mass Spectrom. 2000, 35, 804–817. (44) Cooks, R. G.; Ouyang, Z.; Takats, Z.; Wiseman, J. M. Science 2006, 311, 1566–1570.

(such as DESI,45 DAPCI,46 DART,47 and ASAP48,49) require minimal sample preparation, no chromatography, and have been successfully employed for quantitative or semiquantitative analysis of several chemicals in complex matrixes.50-55 However, they sacrifice accuracy and precision because of matrix effects particularly if internal standards, such as isotopically labeled compounds, are not used. The rapid analysis achieved with ambientsampling MS44 significantly increases throughput, making these techniques very useful when the problem and/or studies addressed allow the use of analytical methods customized for specific situations. This is not the case in the regulated agrochemical industry since governmental agencies prefer methods that employ analytical technology commonly available, and that do not need specialized chemicals as internal standards. Nevertheless, future research may unleash the potential of these technologies for tracelevel quantitative analysis in highly regulated industries, as the fundamentals of ambient-sampling MS continue to be investigated and instrumentation becomes widely available. The utility of HPLC/ESI/MS/MS outweighs its drawbacks, making it a popular combination of technologies used for a variety of analytical applications, and the methods discussed in this paper cover one example: trace-level quantitation of active ingredients of agrochemicals in environmental samples. The analytical methods were originally developed according to the U.S. EPA Guidelines7 to fulfill product registration requirements and support product development efforts and are recommended by the authors for monitoring residues of aminocyclopyrachlor and aminocyclopyrachlor methyl in environmental samples. EXPERIMENTAL SECTION Step-by-step analytical procedures are available for this article as Supporting Information. Reagents and Standards. All reagents and solvents used were commercially available at the time of this study, with the exception of analytical standards. These reference substances were analytical standard grade reagents synthesized by DuPont Crop Protection, Global Technology Division, E. I. du Pont de Nemours and Company. Individual 100 µg/mL stock standard solutions of aminocyclopyrachlor and aminocyclopyrachlor methyl were prepared in methanol. A 10.0 µg/mL mixed stock standard of the (45) Takat´s, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science 2004, 306, 471–473. (46) Cotte-Rodrig´uez, I.; Hernan´dez-Soto, H.; Chen, H.; Cooks, R. G. Anal. Chem. 2008, 80, 1512–1519. (47) Cody, R. B.; Laramee´, J. A.; Durst, H. D. Anal. Chem. 2005, 77, 2297– 2302. (48) McEwen, C. N.; McKay, R. G.; Larsen, B. S. Anal. Chem. 2005, 77, 7826– 7831. (49) McEwen, C.; Gutteridge, S. J. Am. Soc. Mass Spectrom. 2007, 18, 1274– 1278. (50) Ifa, D. R.; Manicke, N. E.; Rusine, A. L.; Cooks, R. G. Rapid Commun. Mass Spectrom. 2008, 22, 503–510. (51) Chen, H.; Li, M.; Zhang, Y.-P.; Yang, X.; Lian, J.-J.; Chen, J.-M. J. Am. Soc. Mass Spectrom. 2008, 19, 450–454. (52) Nyadong, L.; Late, S.; Green, M. D.; Banga, A.; Fernan´dez, F. M. J. Am. Soc. Mass Spectrom. 2008, 19, 380–388. (53) Mulligan, C. C.; MacMillan, D. K.; Noll, R. J.; Cooks, R. G. Rapid Commun. Mass Spectrom. 2007, 21, 3729–3736. (54) Kpegba, K.; Spadaro, T.; Cody, R. B.; Nesnas, N.; Olson, J. A. Anal. Chem. 2007, 79, 5479–5483. (55) Pierce, C. Y.; Barr, J. R.; Cody, R. B.; Massung, R. F.; Woolfitt, A. R.; Moura, H.; Thompson, H. A.; Fernandez, F. M. Chem. Commun. 2007, 8, 807– 809.

analytes was prepared by transferring aliquots of the aminocyclopyrachlor and aminocyclopyrachlor methyl individual standards into a volumetric flask and diluting to volume with methanol. Additional mixed stock standard concentrations, ranging from 10 ng/mL to 1,000 ng/mL, were prepared by diluting from the 10.0 µg/mL mixed stock standard. Mixed stock standards were used for sample fortification. These solutions were validated to be stable for at least three months when prepared in methanol and stored at or below -20 °C. Degradation of aminocyclopyrachlor was observed in other solvents, that is, when standard solutions were prepared in acetonitrile or acetone; thus, those solvents should be avoided when making reference solutions. Chromatographic calibration standards were prepared by serially diluting a mixed stock solution. Calibration standards, ranging from 0.020 ng/mL to 2.0 ng/mL, were prepared in 0.01% formic acid (aq) and were stable for at least five days when stored refrigerated. Soil and Water Control Samples. The soil analytical method was validated on a control matrix obtained from a field dissipation study test site in Ontario, Canada. The soil matrix was characterized and found to be a silt loam soil with 15.2% clay, 26.7% sand, 58.1% silt, and an organic matter content of 1.36%. The pHw of the soil was 7.4. The entire soil sample was homogenized using a Hobart processor to obtain representative subsamples. The water analytical procedure was validated on water from four different sources: well water from Kemblesville, PA, U.S.A. collected on Nov 20, 2007; surface waters from White Clay Creek, Newark, DE, U.S.A. collected on Nov 21, 2007 and Lums Pond, Bear, Delaware, U.S.A. collected on Nov 21, 2007; drinking (tap) water from DuPont Stine-Haskell Research Center, Newark, DE, U.S.A. collected on Nov 29, 2007. A subsample of each type of water used for method validation was sent to Agvise Laboratories (Northwood, ND, U.S.A.) for characterization. A summary of the water characterization data is available as Supporting Information. All soil and water samples were stored frozen when not in use. Soil Sample Preparation. Aminocyclopyrachlor and aminocyclopyrachlor methyl were extracted from fortified soil samples twice with 80/20 ACN/0.2% formic acid (aq) for 30 min in a sonication water bath at 60 °C ± 5 °C. Samples were centrifuged for 10 min at 10,000 rpm, and extracts were combined in graduated mixing cylinders. The samples were then extracted twice with 70/30 ACN/0.15 M ammonium acetate (aq) for 30 min in a sonication water bath at 60 °C ± 5 °C. The samples were centrifuged again, and the extracts were combined in the corresponding graduated mixing cylinders. The extracts were brought to final volume with acetonitrile, capped, and mixed by inverting several times. Aliquots of the extracts (6.0 mL each) were taken and evaporated to 1 mL using a nitrogen evaporator with the water temperature set at 40 °C, and then diluted with 0.2% formic acid (aq) to a volume of 6 mL. The samples were loaded into Oasis MCX solid phase extraction (SPE) cartridges (Part No. 186000776, Waters, Milford, MA, U.S.A.), where the analytes were retained. After washing the cartridge with 10 mL of methanol, the analytes were eluted with 15.0 mL of 50 mM ammonium hydroxide in methanol into a tube containing 1.0 mL of 0.2% formic acid (aq). The samples were evaporated to approximately 1 mL using a nitrogen evaporator with the water bath temperature set at 40 °C and then diluted with 0.01% Analytical Chemistry, Vol. 81, No. 2, January 15, 2009

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Table 1. HPLC Gradient Program Used for Analyte Quantitationa time 0.00 5.00 8.00 10.00 10.10 14.50

flowrate (mL/min) %A, 0.1% formic acid (aq) %B, methanol 1.000 1.000 1.000 1.000 1.000 1.000

95 41 1 1 95 95

5 59 99 99 5 5

a Approximate analyte retention times: aminocyclopyrachlor ) 5.1 min, aminocyclopyrachlor methyl ) 8.8 min.

formic acid (aq). A portion of each purified extract was filtered through a 0.45 µm PTFE filter and analyzed by LC/MS/MS. Water Sample Preparation. Water aliquots were measured into propylene centrifuge tubes and fortified with known amounts of aminocyclopyrachlor and aminocyclopyrachlor methyl. The samples were acidified by adding 60 µL of concentrated formic acid. The samples were loaded into Oasis MCX solid phase extraction cartridges (Part No. 186000776, Waters, Milford, MA, U.S.A.), where the analytes were retained. The analytes were eluted with 15.0 mL of 75 mM ammonium hydroxide in methanol into a tube containing 1.0 mL of 0.2% formic acid (aq). The samples were evaporated under nitrogen gas flow to approximately 2 mL with a water bath at 40 °C and diluted with 0.01% formic acid (aq) to a final volume of 5.0 mL. A 10-fold dilution was made for each sample by mixing 900 µL of 0.01% formic acid (aq) with 100 µL of each purified water sample in a clean glass autosampler vial. The 10-fold diluted samples were then analyzed by LC/MS/MS. HPLC/MS/MS Conditions. An Agilent 1100 Series HPLC (Agilent Technologies, Wilmington, DE, U.S.A.) equipped with a Luna Phenyl-Hexyl, 4.6 mm × 150 mm, 3 µm diameter particulate column (Phenomenex, Torrance, CA, U.S.A.) and coupled to an Applied Biosystems API-5000 triple quadrupole mass spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA., U.S.A.) equipped with an ESI source was used for instrumental analysis. The HPLC system was equipped with a vacuum degasser, a binary pump, a temperature-controlled column compartment, and a refrigerated autosampler. The entire system and data acquisition were controlled by Analyst 1.4.1 software. The Applied Biosystems API5000 was operated in LC/MS/MS positive ion mode with MRM detector output for quantitative analysis. HPLC conditions were set as follows: injection volume 60 µL, temperature of column compartment 30 °C, 0.1% formic acid in HPLC-grade water was used as mobile phase A, while HPLC-grade methanol was used as mobile phase B. The post-column flow was split to allow ∼100 µL/min to the mass spectrometer and ∼900 µL/min to waste. A summary of the HPLC gradient is provided in Table 1. Mass

spectrometric detection was achieved using conditions described in Table 2. Additional parameters were set as follows: resolution Q1 ) Unit, resolution Q3 ) Unit, ESI source voltage 1.5-2.0 kV for both analytes. RESULTS AND DISCUSSION Single-Stage and MS/MS Spectra. Single-stage mass spectra were recorded for aminocyclopyrachlor and aminocyclopyrachlor methyl from solutions (50/50 methanol/0.2% formic acid, aq) containing both compounds at a concentration of 10 µg/ mL to assess their ionization under electrospray conditions. A representative mass spectrum is provided in Figure 1, recorded under typical ESI instrumental parameters, that is, a spray voltage of 4.5 kV and source temperature 275 °C. The data show a significant difference between the relative intensities obtained for aminocyclopyrachlor and aminocyclopyrachlor methyl. The latter is ionized with about 5 times greater efficiency under the conditions tested. This is expected because of the amino acid properties of aminocyclopyrachlor. Both functionalities must be protonated (as COOH and +NH3) for aminocyclopyrachlor to have a net positive charge. On the other hand, the protonated amino group readily gives a positive charge to aminocyclopyrachlor methyl (see structures in Figure 2), yielding higher response compared to the amino acid analogue. Tandem mass spectrometry experiments were conducted on protonated aminocyclopyrachlor and aminocyclopyrachlor methyl using a triple quadrupole mass spectrometer to study their gasphase dissociation reactions, and to develop selective MS/MS methods using the same instrument type to be employed for quantitative analysis. As shown in Figure 2, after isolation in Q1 and subsequent excitation in the collision cell, aminocyclopyrachlor and aminocyclopyrachlor methyl readily lose formic acid and methyl formate, respectively, to form a common product ion at m/z 168. They also form product ions at m/z 101, m/z 68 and m/z 41, and all fragments may correspond to identical gas-phase ionic species being generated from protonated aminocyclopyrachlor and protonated aminocyclopyrachlor methyl. Additional information about the fragmentation reactions was obtained by recording MS/MS spectra for the monoisotopic precursor ions containing a 37Cl. Protonated 37Cl -aminocyclopyrachlor (m/z 216) and protonated 37Cl-aminocyclopyrachlor methyl (m/z 230) generated fragment ions at m/z 170, m/z 103, m/z 68, and m/z 41. The data confirm the presence (m/z shift of two units) or absence (m/z unchanged) of the chlorine atom in fragment ions. Further details about the gas-phase dissociation mechanism of these new herbicides were sought in MS3 experiments, which are described below.

Table 2. Optimized Mass Spectrometer Acquisition Parameters Used for Analyte Quantitation during Method Validation AB Sciex API-5000 optimized parameters Q1 m/z

Q3 m/z

dwell (msec)

CUR (psi)

GS1 (psi)

GS2 (psi)

TEMa (°C)

CAD (psi)

CEa (V)

1 - aminocyclopyrachlor

214

100

20

50

50

325-350

8

35-40

2 - aminocyclopyrachlor methyl

228

68 101 68 168

100

20

50

50

325-350

8

38-40

period - analyte

a

800

Range indicates the minimum and maximum parameter values used at two laboratories.

Analytical Chemistry, Vol. 81, No. 2, January 15, 2009

Figure 1. Mass spectrum of a solution containing aminocyclopyrachlor and its methyl analogue at a concentration of 10 µg/mL in methanol/ 0.2% formic acid (aq) 50/50. The solution was infused directly into the ion source at 5.0 µL/min.

Figure 2. ESI tandem mass spectra recorded for protonated (a) aminocyclopyrachlor and (b) aminocyclopyrachlor methyl in a triple quadrupole mass spectrometer. The precursor ions were isolated in Q1, collisionally activated with nitrogen gas, and fragment ions scanned in Q3. The spectra were recorded using an entrance potential of 10 V and collision energy of 40 V for both compounds.

MS3 spectra. The smaller fragment ions could be products of sequential dissociation reactions where the ion at m/z 168 is formed as an intermediate. However, the triple quadrupole mass spectrometer is limited to MS/MS experiments, and the hypothesis cannot be confirmed or ruled out with the tandem mass spectra provided in Figure 2. In an attempt to elucidate the origin of the smaller fragment ions and the gas-phase dissociation

mechanism of the analytes, MS3 spectra of protonated aminocyclopyrachlor (m/z 214 f m/z 168 f O) and aminocyclopyrachlor methyl (m/z 228 f m/z 168 f O) were recorded using a Thermo Electron LTQ mass spectrometer equipped with a linear ion trap mass analyzer (Figure 3). Note that relatively large isolation windows were used (8 m/z units) to perform the MS3 experiments on the entire precursor ion isotopic distribuAnalytical Chemistry, Vol. 81, No. 2, January 15, 2009

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Figure 3. Comparison of MS3 spectra recorded to evaluate the dissociation of ion m/z 168 when formed from (a) aminocyclopyrachlor and (b) aminocyclopyrachlor methyl. Spectra were recorded under identical instrumental conditions: isolation window for all selected ions ) 8 m/z units, collision energy of 50% and 25% for the first and second ion activation events, respectively.

tions. As shown in Figure 3, the ions at m/z 101 and m/z 68 are products of the sequential dissociation of protonated aminocyclopyrachlor and aminocyclopyrachlor methyl, with the initial loss of formic acid or methyl formate to form a carbocation intermediate (m/z 168). The dissociation reactions presented in Scheme 1 have been proposed to explain the formation of all major fragments observed in the mass spectra. It is suggested that the signals at m/z 101 and m/z 68 are from protonated chloropropanedinitrile and protonated cyclopropanecarbonitrile, respectively. Note that, as shown in Scheme 1, these ions could also exist as their corresponding carbocation tautomers. The formation of m/z 41 802

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ion could not be confirmed by MS3 because of intrinsic mass range limitations of the linear ion trap mass spectrometer; as shown in Figure 3, m/z ) 50 was the lowest allowed by the instrument in the scan range of MS3 experiments. Nevertheless, it is reasonable to propose m/z 41 as cyclopropylium, which could be generated from protonated cyclopropanecarbonitrile by the loss of hydrogen cyanide (see Scheme 1). It is still possible that the smaller fragment ions, that is, protonated chloropropanedinitrile, protonated cyclopropanecarbonitrile, and cyclopropylium (m/z 101, m/z 68, and m/z 41, respectively), are also formed directly from the protonated

Scheme 1. Gas-Phase Dissociation Reactions of Protonated Aminocyclopyrachlor and Protonated Aminocyclopyrachlor Methyl Observed by Tandem Mass Spectrometry

analytes. If so, the amount formed through the direct reaction could not be quantified. Overall, the similar fragmentation pattern observed for the two herbicidally active ingredients tested (see Figures 2 and 3) supports that they undergo dissociation through the same mechanism, as proposed in Scheme 1. This can be expected since the compounds only differ by a methyl group located outside the pyrimidine ring. MS/MS Experiments for Quantitation. The tandem mass spectrometric experiments described above allowed the identification of several dissociation channels that could be used for selective quantitation. These are m/z 214 f m/z 168, m/z 214 f m/z 101, m/z 214 f m/z 68, and m/z 214 f m/z 41 for aminocyclopyrachlor; and m/z 228 f m/z 168, m/z 228 f m/z 101, m/z 228 f m/z 68, and m/z 228 f m/z 41 for aminocyclopyrachlor methyl. The dissociation of protonated aminocyclopy-

rachlor and aminocyclopyrachlor methyl to form the fragment ion at m/z 68 yielded the greater signal-to-noise ratio, thus the ion transitions m/z 214 f m/z 68 and m/z 228 f m/z 68 were selected for analyte quantitation in this study. Once the major fragmentation pathways were identified for the analytes of interest, the triple quadrupole mass spectrometric conditions were optimized for both compounds using the Automated Quantitative Optimization option available in the instrument software Analyst 1.4.1. Optimized conditions appear in Table 2. Note that slightly different conditions were used at ABC Laboratories and DuPont Stine-Haskell Research Center since the Automated Quantitative Optimization was performed independently in two different API5000 mass spectrometers, and optimum parameters obtained at each location were used. Analytical Chemistry, Vol. 81, No. 2, January 15, 2009

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Figure 4. Stability of aminocyclopyrachlor and aminocyclopyrachlor methyl in various solvents. Solutions containing 100 ng/mL of each compound were prepared in acetonitrile, methanol, water, and acetone and stored at room temperature and refrigerated. The results shown correspond to mass spectrometric analysis performed after 12 days of storage.

Stability Tests. The stability of aminocyclopyrachlor and aminocyclopyrachlor methyl in common solvents was evaluated prior to analytical method development. Solutions containing both compounds at a concentration of 100 ng/mL were prepared in water, methanol, acetone, and acetonitrile. Subsamples of each solution were stored at room temperature and refrigerated (ca. 4 °C) for 12 days, and then they were analyzed by mass spectrometry to determine the post-storage concentration of the analytes. As shown in Figure 4, aminocyclopyrachlor methyl is relatively stable in all solvents tested. Additional tests revealed that its stability in aqueous systems increases with lower pH (e.g., < 7), while demethylation to form aminocyclopyrachlor occurs and is faster at higher pH (e.g., > 7). Interestingly, aminocyclopyrachlor was found to degrade quickly in pure acetone and acetonitrile (see Figure 4). This reaction is currently being investigated. Further tests were performed with acetonitrile, since it is a common (and often preferred) solvent for chromatography and sample preparation. The degradation of aminocyclopyrachlor was found to be much slower in mixtures of acetonitrile/water and acetonitrile/ diluted formic acid (aq), that is, degradation of aminocyclopyrachlor was not measurable in these systems during a 24 h period at room temperature and about 2 weeks in solutions stored at about -20 °C. The stability tests allowed the selection of methanol, low pH aqueous solutions like formic acid (aq), and acetonitrile/aqueous 804

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mixtures as preferred solvents for sample preparation; preparation of solutions in pure acetonitrile or acetone was avoided. Chromatographic Method Development. Aminocyclopyrachlor has both amino and carboxylic acid functionalities. Amino acids are known to be difficult to analyze by reverse-phase HPLC since they may exist in different forms, for example, neutral, ionic, or zwitterionic, depending on the pH of the solvent, which can lead to peak broadening and significant variability in retention times. This was observed for aminocyclopyrachlor when analyzed using ammonium acetate (aq), ammonium formate (aq), or diluted acetic acid as the aqueous mobile phase. In some cases acceptable peak shape and retention time standard deviations were obtained for chromagraphic standards; however, shifts in aminocyclopyrachlor retention time were observed when matrix was introduced into the samples. The addition of formic acid to the aqueous mobile phase, chromatographic standards, and samples was necessary to obtain reproducible retention times and to minimize peak width. On the other hand, the carboxylic functionality is methylated in aminocyclopyrachlor methyl, making the compound ideal for reverse-phase HPLC analysis, yielding reproducible retention times and peak shapes under most conditions tested. After the optimum chromatographic conditions where achieved, the dynamic range of standards was evaluated to define suitable concentrations for calibration. Acceptable linearity (R2 > 0.99) was consistently observed for calibration standards from 0.020 ng/mL to 2.0 ng/mL for both compounds; thus, this range was selected and used for quantitation of the analytes in soil and water (representative calibration curves are available as Supporting Information). Typical chromatograms obtained for low, mid, and high calibration standards are provided in Figure 5. The data were recorded using the optimized HPLC and mass spectrometric conditions described in Tables 1 and 2. A short analytical column (Luna Phenyl-Hexyl, 4.6 mm × 50 mm, 3 µm diameter particulate) was tested in an attempt to shorten the HPLC/MS/MS analysis time prior to method validation. However, matrix effects were observed, indicating that the analyte purification obtained using a 150 mm column was necessary in the design of a reliable analytical method with external calibration standards prepared in neat solvents. Design of Sample Preparation Procedure. The soil extraction procedure described in the experimental section was developed to extract the analytes from samples generated in a soil metabolism study which used the active ingredient with a radioactive label. Since good extraction efficiency was demonstrated for the analytes of interest in that particular study, the same extraction procedure was used to avoid additional radiovalidation experiments. In attempts to design the simplest sample preparation procedures, the possibility of quantifying the analytes in soil extracts and water samples without any analyte purification/cleanup was evaluated. Aliquots of soil extracts were evaporated, reconstituted in formic acid (aq), and analyzed. Similarly, fortified water samples were directly analyzed without sample preparation, except for the addition of formic acid. Significant matrix effects were observed when water samples (especially tap, creek, and pond) and soil extracts were injected without any purification, thus affecting the accuracy of the analysis. The use of internal standards or calibration standards prepared in control matrix (also known as

Figure 5. Representative chromatograms obtained for external calibration standards of aminocyclopyrachlor and aminocyclopyrachlor methyl.

matrix-matched standards) represent possible solutions to correct the accuracy of the method when matrix effects are encountered. However, methods that provide acceptable accuracy with simpler external standard calibration are preferred by regulatory agencies7,9 over methods that need standards in matrix or specialized internal standards like isotopically labeled compounds commonly used in mass spectrometry. Consequently, sample purification alternatives were evaluated, including SPE. The following SPE packing materials were tested: C18, ENV, and Phenyl from Varian, Inc. (Lake Forest, CA, U.S.A.); ENV+ from International Sorbent Technology (Mid Glamorgan, U.K.); ENV-Carb from Supelco (Bellefonte, PA, U.S.A.); Oasis HLB and Oasis MCX from Waters (Milford, MA, U.S.A.). Aminocyclopyrachlor, because of its high polarity and presence of amino and carboxylic acid functionalities, was not retained in any of the materials tested under a variety of conditions, except the Oasis MCX SPE cartridges which retained the analytes by mixed-mode, that is, cation exchange and reverse phase. Both analytes were quantitatively retained in Oasis MCX cartridges when loaded in acidified aqueous solvents, that is, as protonated species.

Quantitative elution of the analytes was obtained with diluted solutions of ammonia in methanol. Formic acid (aq) and methanol were tested and selected as appropriate solvents for SPE purification based on results from the stability test, their use in the optimized chromatographic conditions, and to simplify the overall analytical method by minimizing the diversity of solvents and reagent solutions needed. Analytical Method Validation. The analytical methods described in the experimental section were validated in one representative soil matrix and four water types including surface and ground sources as required by regulatory agencies,7-9 and the data acceptance criteria described in the environmental methods guidelines from U.S. E.P.A.7 were followed. Briefly, acceptable analytical methods are expected to provide average recoveries, (analyte added/analyte found) × 100, between 70-120% for the individual analytes in each matrix tested, with relative standard deviation (RSD) no greater than 20%. External calibration standards must yield average response factors (peak area/analyte concentration) with RSD < 20% over the targeted calibration range.7 Analytical Chemistry, Vol. 81, No. 2, January 15, 2009

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Table 3. Analyte Recovery Results Obtained during Method Validation aminocyclopyrachlor methyl

aminocyclopyrachlor

fortification level, ppb (ng/g)

recovery (%) ± RSDa

range

Nb

recovery (%) ± RSDa

range

Nb

ontario soil

1.0 10 700 overall

96 ± 2 93c 86c 93 ± 5

94-97 90-96 82-91 82-97

4 2 2 8

79 ± 3 79c 79c 79 ± 2

74-82 79-79 77-81 74-81

4 2 2 8

creek water creek water pond water pond water drinking water drinking water well water well water surface water (creek and pond combined)

0.10 1.0 0.10 1.0 0.10 1.0 0.10 1.0 0.10 1.0 overall 0.10 1.0 overall

101 ± 2 98 ± 3 96 ± 3 92 ± 5 88 ± 12 84 ± 10 88 ± 12 87 ± 10 99 ± 3 95 ± 5 97 ± 4 93 ± 10 90 ± 9 92 ± 9

99-104 94-100 93-99 88-99 72-98 77-96 80-106 77-96 93-104 88-100 88-104 72-106 77-100 72-106

5 5 5 5 5 5 5 5 10 10 20 20 20 40

92 ± 4 87 ± 6 93 ± 9 87 ± 11 79 ± 13 76 ± 4 78 ± 12 80 ± 7 93 ± 7 87 ± 8 90 ± 8 86 ± 12 83 ± 9 84 ± 11

87-97 80-92 83-102 77-98 66-93 72-80 72-94 74-87 83-102 77-98 77-102 66-102 72-98 66-102

5 5 5 5 5 5 5 5 10 10 20 20 20 40

matrix

all water matrixes tested

a

RSD ) Relative standard deviation. b N ) number of samples. c Relative standard deviation not calculated for two values.

Figure 6. Representative chromatograms for creek control water samples, and samples of the sample matrix fortified with aminocyclopyrachlor and aminocyclopyrachlor methyl at 0.10 and 1.0 ppb, and taken through the entire analytical procedure.

The limit of quantitation (LOQ) of the multianalyte method was defined as the level where the chromatographic peak typically yields a signal-to-noise ratio of approximately 5-20 to 1 for the least responsive analyte (aminocyclopyrachlor), which is bracketed by a low calibration standard with peak signal-to-noise ratio of at least 3 to 1. The limit of quantitation (LOQ) was also the lowest level validated in this study: 0.10 ng/mL (ppb) and 1.0 ng/g (ppb) 806

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for water and soil, respectively. Water method validation trials consisted of 40 control samples fortified with the analytes of interest (5 at the method LOQ and 5 at a higher level for each matrix) and 8 control samples (2 per matrix); while validation of the soil method included the analysis of 8 fortified control samples (4 at the method LOQ, 2 at 10×LOQ, and 2 at 700×LOQ), and two control samples. Validation for each matrix was conducted

Figure 7. (a) Analyte recovery distributions obtained for aminocyclopyrachlor and aminocyclopyrachlor methyl from soil and water samples during validation trials by various chemists. Run charts for (b) aminocyclopyrachlor and (c) aminocyclopyrachlor methyl include validation data in chronological order.

in two analytical sets over the course of at least 2 days, which allowed the day-to-day reproducibility of the method to be tested. A summary of the validation data generated for the quantitation of aminocyclopyrachlor and aminocyclopyrachlor methyl in soil and water appears in Table 3. The average recoveries obtained for both compounds in all matrixes tested were within the acceptable range. Moreover, out of 96 analyte recovery measurements, only one value fell outside the acceptable range (66% recovery for aminocyclopyrachlor in a drinking water sample). Acceptable linearity (R2 > 0.99) was observed for calibration standards from 0.020 ng/mL to 2.0 ng/mL for both compounds, and the relative standard deviation for external standard responses ranged from 3% to 16% and 3% to 11% for aminocyclopyrachlor and aminocyclopyrachlor methyl, respectively, during soil and water validation trials. Example chromatograms recorded for creek water samples fortified with aminocyclopyrachlor and aminocyclopyrachlor methyl are provided in Figure 6. Chromatograms recorded for the other matrixes tested (i.e., pond, well, and tap water, and soil) were similar to those presented in Figure 6. The similarity observed in chromatograms recorded for standards (Figure 5) and fortified samples (Figure

6) is expected because of the high specificity of tandem mass spectrometry detection. An advantage of the analytical methods for soil and water described in this paper is that the chromatographic standards are compatible with both analytical procedures. Thus calibration standards can be used to analyze soil and water samples simultaneously. The limit of detection (LOD) was defined as the analyte concentration in matrix with an observed response that has a signal-to-noise ratio of approximately 3-to-1. The LOD was estimated to be 20 ng/L (ppt, parts-per-trillion) for aminocyclopyrachlor and 1 ng/L for aminocyclopyrachlor methyl in water, while LODs in soil were 100 ng/kg and 10 ng/kg for aminocyclopyrachlor and aminocyclopyrachlor methyl, respectively. The LOD was estimated for each analyte based on instrument response and the signal-to-noise ratio observed for samples fortified at the LOQ of the method. Process Analysis. Ruggedness and reproducibility are two important characteristics that must be tested during the development of analytical methods and monitored as the procedures are routinely used at different laboratories. The data obtained during validation trials for soil and water were used together with Minitab Analytical Chemistry, Vol. 81, No. 2, January 15, 2009

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15.1.1.0 software to asses the performance of the methods discussed in this paper. Since validation data were independently generated by three chemists, it is possible to group the results by operator to evaluate the reproducibility of the methods. The average and standard deviation were calculated for recoveries obtained by each chemist, and Gaussian curves were fitted to the data for illustration purposes (see Figure 7a). In addition, run charts were created for both compounds based on the chronological order of sample analysis (Figure 7b,c). The lower spec limit (LSL) and upper spec limit (USL) have been defined according to U.S. EPA guidelines7 for environmental methods. The validation data for soil were generated by operator A, who was not familiar with the soil extraction method; while operator C was inexperienced with the entire water analytical procedure during validation and the data shown in Figure 7 are from C’s first sample analyses. On the other hand, operator B performed most of the method development work, and therefore was familiar with the procedures at the time validation sets were analyzed. Thus, the average recoveries obtained for aminocyclopyrachlor (A ) 79.1%, B ) 87.3%, and C ) 74.1%) and aminocyclopyrachlor methyl (A ) 92.6%, B ) 95.8%, and C ) 79.5%) correlate with the chemists’ experience with the methodology. The data show that the methods still meet regulatory guidelines7 even if analyte recoveries obtained by each chemist are examined separately (not a requirement or standard practice), since all average recoveries shown in Figure 7 are within the 70-120% range and relative standard deviations are below 20%. Scientists at DuPont Stine-Haskell Research Center and ABC Laboratories, Inc. were involved in the development and validation of these analytical methods. The satisfactory and comparable performance obtained at two laboratories and by three chemists authenticates the good reproducibility and ruggedness of these environmental methods, and their suitability for monitoring aminocyclopyrachlor and aminocyclopyrachlor methyl in water and soil. Moreover, after initial validation trials (Figure 7), the methods continued to be used by additional personnel as part of studies in support of product development, yielding comparable analyte recovery results for fortified samples in further validation sets, as well as fortifications analyzed concurrently with field samples. CONCLUSIONS Mass spectrometry-based analytical methods have been developed for detection and quantitation of aminocyclopyrachlor and its methyl analogue in water and soil samples. The methods have

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been validated following U.S. EPA guidelines7 and tested for reproducibility at two laboratories. The results suggest that the methods are suitable for monitoring these new herbicide active ingredients in environmental samples. The good reproducibility and ruggedness of the methods may be attributed to thorough investigation of the analytes during method development, including tests on their stability, SPE and chromatographic behavior, and gas-phase fragmentation. The latter allowed the identification of four dissociation channels which could be used for selective quantitation. The validated methods employed solid phase extraction (SPE) for analyte purification, and HPLC coupled to tandem mass spectrometry for detection and quantitation. The method limits of quantitation (LOQ) were 0.10 ng/mL in water and 1.0 ng/g in soil for both compounds. The LOD in water was estimated to be 20 ng/L for aminocyclopyrachlor and 1 ng/L for aminocyclopyrachlor methyl, while LODs in soil were 100 ng/kg and 10 ng/kg for aminocyclopyrachlor and aminocyclopyrachlor methyl, respectively. Methanol, low pH aqueous solutions like formic acid (aq), and acetonitrile/aqueous mixtures were selected as preferred solvents for sample preparation based on results from analyte stability tests; preparation of solutions in pure acetonitrile or acetone should be avoided because of the degradation of aminocyclopyrachlor. The results discussed in this paper may be useful to other scientists and laboratories when adding aminocyclopyrachlor and aminocyclopyrachlor methyl into existing multiresidue methods for pesticide screening. ACKNOWLEDGMENT The authors are indebted to several DuPont colleagues for their assistance: thanks to James J. Stry, Richard A. Simmons, Elena Cabusas, Frederick Q. Bramble Jr., Kristin H. Milby, Steve F. Cheatham, and William T. Zimmerman for helpful discussions, and David L. Ryan for help with the ion trap MS3 experiments. Fruitful discussions with Clark Chickering and Del A. Koch (ABC Laboratories, Inc.) are also acknowledged. SUPPORTING INFORMATION AVAILABLE Experimental procedures for the quantitation of aminocyclopyrachlor and aminocyclopyrachlor methyl in soil and water. This material is available free of charge via the Internet at http://pubs. acs.org. Received for review September 30, 2008. Accepted November 11, 2008. AC8020642