Choosing between Atmospheric Pressure Chemical Ionization and

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Anal. Chem. 2001, 73, 5441-5449

Choosing between Atmospheric Pressure Chemical Ionization and Electrospray Ionization Interfaces for the HPLC/MS Analysis of Pesticides E. M. Thurman,† Imma Ferrer,‡ and Damia Barcelo´§

U.S. Geological Survey, 4821 Quail Crest Place, Lawrence, Kansas 66049, and National Water-Quality Laboratory, U.S. Geological Survey, 25046 Denver Federal Center, MS 407, Denver, Colorado 80225

An evaluation of over 75 pesticides by high-performance liquid chromatography/mass spectrometry (HPLC/MS) clearly shows that different classes of pesticides are more sensitive using either atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI). For example, neutral and basic pesticides (phenylureas, triazines) are more sensitive using APCI (especially positive ion). While cationic and anionic herbicides (bipyridylium ions, sulfonic acids) are more sensitive using ESI (especially negative ion). These data are expressed graphically in a figure called an ionization-continuum diagram, which shows that protonation in the gas phase (proton affinity) and polarity in solution, expressed as proton addition or subtraction (pKa), is useful in selecting APCI or ESI. Furthermore, sodium adduct formation commonly occurs using positive ion ESI but not using positive ion APCI, which reflects the different mechanisms of ionization and strengthens the usefulness of the ionization-continuum diagram. The data also show that the concept of “wrongway around” ESI (the sensitivity of acidic pesticides in an acidic mobile phase) is a useful modification of simple pKa theory for mobile-phase selection. Finally, this finding is used to enhance the chromatographic separation of oxanilic and sulfonic acid herbicides while maintaining good sensitivity in LC/MS using ESI negative. The newer interfaces for high-performance liquid chromatography/mass spectrometry (HPLC/MS), atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI) have been important tools in environmental analytical chemistry1-4 during the last five years, especially in the area of pesticide analysis in soil and water.2-3 The sensitivity, ruggedness, and ease of use given by the newer interfaces over thermospray, particle beam, and fast atom bombardment (FAB) have made methods development rapid, sensitive, and reliable.1-4 During these past * Corresponding author: (e-mail) [email protected]; (fax) 785-832-3500. † U.S. Geological Survey, Lawrence, KS. ‡ U.S. Geological Survey, Denver, CO § Current address: Department of Environmental Chemistry, IIQAB-CSIC c/Jordi Girona 18-26, 08034 Barcelona, Spain. (1) Voyksner, R. D. Environ. Sci. Technol. 1994, 28, 118A. (2) Barcelo, D. Applications of LC-MS in Environmental Chemistry; Elsevier: Amsterdam, 1996. (3) Ferrer, I.; Barcelo´, D. Analusis 1998, 26, M118. (4) Niessen, W. M. A. J. Chromatogr., A 1999, 856, 179. 10.1021/ac010506f Not subject to U.S. Copyright. Publ. 2001 Am. Chem. Soc.

Published on Web 10/09/2001

five years, methods have been developed for many pesticides including triazine, phenylurea, organophosphate, carbamate, chlorophenoxy acid, and sulfonylurea pesticides3,5-37 using both APCI and ESI. Despite the methods now described in the literature, there is not an exact procedure for choosing the most sensitive interface or the best mobile phase when new analytical methods are being developed for pesticides and their degradation products. There are several reasons for the confusion that exists concerning the selection of an interface or mode of ionization for pesticide analysis. First, there are a number of studies published in the recent literature that use both APCI and ESI and a variety of mobile phases for the analysis of the same compound. For example, triazine herbicides may be analyzed by either APCI or ESI with either methanol/water, acetic acid, or trifluoroacetic acid added to the mobile phase.5-9,14,15,23,25,26 The fact that multiple interface and mobile-phase combinations work well only complicates the selection of the most sensitive method. Second, a number of papers have reported methods without summaries of the strength and weaknesses of APCI and ESI for pesticide analysis. There are, in fact, only a limited number of studies that compare the best conditions for optimal sensitivity for a small number of pesticides.3,12 Third, although theoretical explanations (5) DiCorcia, A.; Nazzari, M.; Rao, R.; Samperi, R.; Sebastiani, E. J. Chromatogr., A 2000, 878, 87. (6) Curini, R.; Gentili, A.; Marchese, S.; Marino, A.; Perret, D. J. Chromatogr., A 2000, 874, 187. (7) Geerdink, R. B.; Kooistra-Sijpersma, A.; Tiesnitsch, J.; Kienhuis, P. G. M.; Brinkman, U. A. Th. J. Chromatogr., A 1999, 857, 147. (8) Steen, R. J. C. A.; Hobenboom, A. C.; Leonards, P. E. G.; Peerboom, R. A. L.; Cofino, W. P.; Brinkman, U. A. Th. J. Chromatogr., A 1999, 857, 157. (9) Di Corcia, A.; Crescenzi, C.; Samperi, R.; Scappaticcio, L. Anal. Chem. 1997, 69, 2819. (10) Marr, J. C.; King, J. B. Rapid Commun. Mass Spectrom. 1997, 11, 479. (11) Hogenboom, A. C.; Slobodnik, J.; Vreuls, J. J.; Rontree, J. A.; van Baar, B. L. M.; Nissen, W. M. A.; Brinkman, U. A. Th. Chromatographia 1996, 42, 506. (12) Slobodnik, J.; Hogenboom, A. C.; Vreuls, J. J.; Rontree, J. A.; van Baar, B. L. M.; Nissen, W. M. A.; Brinkman, U. A. J. Chromatogr., A 1996, 741, 59. (13) Spliid, N. H.; Koppen, B. J. Chromatogr., A 1996, 736, 105. (14) Cai, Z.; Cerny, R. L.; Spalding, R. F. J. Chromatogr., A 1996, 753, 243. (15) Crescenzi, C.; Di Corcia, A.; Marchese, S.; Sameri, R. Anal. Chem. 1995, 67, 1968. (16) Stout, S. J.; daCunha, A. R.; Picard, G. L.; Safarpour, M. M. J. Agric. Food Chem. 1996, 44, 2182. (17) Barnes, K. A.; Fussell, R. J.; Startin, J. R.; Pegg, M. K.; Thorpe, S. A.; Reynolds, S. L. Rapid Commun. Mass Spectrosc. 1997, 11, 117. (18) D′Ascenzo, G.; Gentili, A.; Marchese, S.; Marino, A.; Perret, D. Environ. Sci. Technol. 1998, 32, 1340. (19) Castro, R.; Moyano, E.; Galceran, M. T. J. Chromatogr., A 1999, 830, 59. (20) Castro, R.; Moyano, E.; Galceran, M. T. J. Chromatogr., A 2000, 869, 441.

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of ionization using APCI and ESI have been given in a series of papers,38-59 and the differences between them are clear, there is no well-defined procedure to select the most effective interface for pesticide analysis on the basis of chemical structure. For these reasons, it is a common procedure to try both APCI and ESI on a new pesticide in both positive and negative modes and “see what works best”. In an effort to eliminate some of this confusion between theory and practice of LC/MS for pesticide analysis, this paper discusses three aspects of methods development for LC/MS. First, an evaluation of more than 75 pesticides and their degradation products from 10 classes using APCI and ESI is presented along with a simple diagram that considers the type of interface and (21) Lacorte, S.; Barcelo´, D. Anal. Chem. 1996, 68, 2464. (22) Pleasance, S.; Anacleto, J. F.; Bailey, M. R.; North, D. H. Am. Soc. Mass Spectrom. 1992, 3, 378. (23) Banoub, J.; Gentil, E.; Kiceniuk, J. Int. J. Environ. Anal. Chem. 1995, 61, 11. (24) Rodriguez, M.; Orescan, D. B. Anal. Chem. 1998, 70, 2710. (25) Crescenzi, C.; Di Corcia, A.; Guerriero, E.; Samperi, R. Environ. Sci. Technol. 1997, 31, 479. (26) Di Corcia, A.; Crescenzi, C.; Guerriero, E.; Samperi, R. Environ. Sci. Technol. 1997, 31, 1658. (27) Baltusen, E.; Snijders, H.; Janssen, H. G.; Sandra, P.; Cramers, C. A. J. Chromatogr., A 1998, 802, 285. (28) Koppen, B.; Spliid, N. H. J. Chromatogr., A 1998, 803, 157. (29) D’Ascenzo, G.; Gentili, A.; Marchese, S.; Perret, D. J. Chromatogr., A 1998, 800, 109. (30) D’Ascenzo, G.; Gentili, A.; Marchese, S.; Perret, D. J. Chromatogr., A 1998, 813, 285. (31) Bossi, R.; Koppen, B.; Spliid, N. H.; Streibig, J. C. J. AOAC 1997, 69, 283. (32) Aguilar, C.; Ferrer, I.; Borrull, F.; Marce´, R. M.; Barcelo´, D. J. Chromatogr. A 1998, 794, 147. (33) Ferrer, I.; Hennion, M.-C.; Barcelo´, D. Anal. Chem. 1997, 69, 4508. (34) Puig, D.; Silgoner, I.; Grasserbauer, M.; Barcelo´, D. J. Mass Spectrosc. 1996, 31, 1297. (35) Vreeken, R. J.; Speksnijder, P.; Bobeldijk-Pastorova, I.; Noij, Th. H. M. J. Chromatogr., A 1998, 794, 187. (36) Lacorte, S.; Molina, C.; Barcelo´, D. J. Chromatogr., A 1998, 795, 13. (37) Molina, C.; Grasso, P.; Benfenati, E.; Barcelo´, D. J. Chromatogr., A 1996, 737, 47. (38) Dole, M.; Mach, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. P.; Alice, M. P. J. Chem. Phys. 1968, 49, 2240. (39) Dole, M.; Mach, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. P.; Alice, M. P. J. Chem. Phys. 1970, 52, 4977. (40) Simons, D. S.; Colby, B. N.; Evans, C. A., Jr. Int. J. Mass Spectrom. Ion Phys. 1974, 15, 291. (41) Iribarne, J. V.; Thomson, B. A. J. Chem. Phys. 1976, 64, 2287. (42) Thomson, B. A.; Iribane, J. V. J. Chem. Phys. 1979, 71, 4451. (43) Yamashita, M.; Fenn, J. B. Phys. Chem. 1984, 88, 4451. (44) Yamashita, M.; Fenn, J. B. Phys. Chem. 1984, 88, 4671. (45) Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B. Anal. Chem. 1985, 57, 675. (46) Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem. 1987, 59, 2642. (47) Fenselau, C.; Cotter, R. J. Chem. Rev. 1987, 87, 501. (48) Covey, T. R.; Bonner, R. F.; Sushan, B. I.; Henion, J. D. Rapid Commun. Mass Spectrom. 1988, 2, 249. (49) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64. (50) Huang, E. C.; Henion, J. D. J. Am. Soc. Mass Spectrom. 1990, 1, 158. (51) Ikonomou, M. G.; Blades, A. T.; Kebarle, P. Anal. Chem. 1991, 63, 1989. (52) Hiraoka, K.; Kudaka, I. Rapid Commun. Mass Spectrom. 1992, 6, 265. (53) Kebarle, P.; Tang, L. Anal. Chem. 1993, 65, 972A. (54) Straub, R. F.; Voyksner, R. D. Am. Soc. Mass Spectrom. 1993, 4, 578. (55) Gatlin, C. L.; Turecek, F. Anal. Chem. 1994, 66, 712. (56) Volmer, D.; Levsen, K.; Honing, M.; Barcelo´, D.; Abian, J.; Gelpı´, E.; van Baar, B. L. M.; Brinkman, U. A. Th. Am. Soc. Mass Spectrom. 1995, 6, 656. (57) Fenn, J. B.; Rosell, J.; Meng, C. K. Am. Soc. Mass Spectrom. 1997, 8, 1147. (58) Kebarle, P.; Ho, Y. iN Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation and Applications; Cole, R. B., Ed.; Wiley-Interscience: New York, 1997; p 3. (59) Zhou, S.; Edwards, A.; Cook, K. D.; van Berkel, G. J. Anal. Chem. 1999, 71, 769.

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polarity of the molecule. The diagram is called an ionizationcontinuum diagram because it considers positive and negative species across the continuum of charge and various acid dissociation constants (pKas) that may occur. The rationale behind the diagram is discussed in light of the basic theory of ionization for APCI and ESI. Second, from the discussion of this diagram, the paper proceeds to the discrepancies and exceptions of the modes of ionization and to what is sometimes called “wrong-way-around” electrospray60 and how it may be applied to improve chromatography of herbicide degradates, such as the oxanilic acids of the chloroacetanilide herbicides. The term “wrong-way-around” electrospray refers to the observation of intense (M + H)+ ions that occur when basic solutions are nebulized and (M - H)- ions that occur when acidic solutions are nebulized.60 Finally, the paper deals with sodium adduct formation and final interface and mobilephase selection for the analysis of pesticides. EXPERIMENTAL CONDITIONS Equipment and Reagents. A liquid chromatograph model HP 1090 (Hewlett-Packard, Palo Alto, CA) was connected to a Hewlett-Packard mass spectrometer, model HP 1100, equipped with APCI and ESI interfaces. The operating parameters included drying gas (N2) flow rates of 4 L/min for APCI and a drying gas flow rate of 12 L/min for ESI positive and negative. The drying gas temperature was 350 °C for both APCI and ESI. The nebulizing gas pressure was 60 psi for APCI positive and negative and 40 psi for ESI. The capillary voltage was 2000 V for both APCI and ESI positive and 3000 V for APCI and ESI negative. The fragmentor voltage was 70 V for both APCI and ESI. For APCI, the vaporizer temperature was 350 °C. The corona current was 8 µA for APCI positive and 70 µA for APCI negative. The gain setting was at 3 for APCI and ESI positive and negative. The instrument control and data-processing utilities included the use of a Hewlett-Packard application software installed in a Pentium computer (Hewlett-Packard). Chromatograms were recorded under full-scan conditions (from m/z ) 100-400), and raw peak area per nanogram of analyte was used to determine sensitivity. The variability of the instrument from day-to-day running was also considered by analysis of three compounds daily as internal standards. These compounds were deuterated atrazine for positive ion APCI and ESI and fluometuron for negative ion APCI. Metolachlor ethanesulfonic acid was used for negative ion ESI. The second criterion for reproducible results was the number of counts that were obtainable from the tuning solutions supplied by the manufacturer, Hewlett-Packard. The abundance range was 2 million counts for APCI positive (10%, 1.0 million counts for APCI negative (10%, 0.5 million counts for ESI positive (10%, and 0.25 million counts for ESI negative (10%. These conditions were adjusted daily for reproducible comparisons of APCI and ESI positive and negative. HPLC-grade acetonitrile, methanol, and water were purchased from Merck (Darmstadt, Germany). Acetic acid, trifluoroacetic acid, and ammonium hydroxide were also purchased from Merck. Pesticide standards were obtained from ChemService (Westchester, PA) for acetanilide, bipyridylium, chlorophenoxyacid, phenylurea, sulfonylurea, and triazine herbicides. The isoprenoid, orga(60) Mansoori, B. A.; Volmer, D. A.; Boyd, R. K. Rapid Commun. Mass Spectrom. 1997, 11, 1120.

nophosphate, and organochlorine insecticides were also purchased from ChemService. The chloroacetanilide ESA and oxanilic acid metabolites were obtained from Monsanto (St. Louis, MO). Flufenacet ESA and oxanilic acid metabolites were obtained from Bayer Corp. (Stillwell, KS). Phenylurea herbicide metabolites were obtained from the Agricultural Research Service Stoneville, MS, laboratory. Stock standard solutions of 500 ng/µL were prepared by weighing the solutes and dissolving them in methanol. A stock standard solution of 10 ng/µL for each pesticide was used to inject into the LC/MS system for sensitivity analysis. Methods. For the study of the effect of organic modifier and electrolyte additives on analyte response, a series of test eluents were prepared. The test eluents consisted of methanol/water (50: 50) and acetonitrile/water (50:50) that contained the following additives: acetic acid, trifluoroacetic acid, and ammonium hydroxide. From our past publications3,21,32-34,36,37,61,64.65 and experience with pesticide analysis by LC/MS, these were effective mobile phases across a wide pH range and solvent selections for sensitivity comparisons between APCI and ESI. Pesticide standard solutions were analyzed by flow injection and by chromatography using a Phenomenex (Torrance, CA) C-18 column, 5 µm, 250 × 3 mm, in the LC/MS system. The response of all analytes was calculated as a function of the signal obtained under full-scan conditions using raw counts per nanogram of injected standard, as previously described. Separation of the acetanilide and chloroacetanilide herbicides was accomplished with a mobile phase of 0.3% acetic acid, 24% ethanol, 35.7% water, and 40% acetonitrile. The analytical columns consisted of two Phenomenex C18 columns (Torrance, CA) that were 5 µm, 250 × 3 mm, coupled to a Keystone (Bellefonte, PA) 3-µm, 250 × 4.6-mm C18 column. Column temperatures were set at 70 °C to achieve better separation and peak shapes for the metabolites. The flow rate was 0.3-0.4 mL/min, depending on back pressure (maximum of 400 bar). RESULTS AND DISCUSSION Comparison of APCI and ESI. More than 75 pesticides and degradation products from 10 general classes were analyzed by HPLC/MS using APCI (+ and -) and ESI (+ and -) to determine response factors and selectivity of the two interfaces. A partial summary of results is shown in Table 1 after optimization of operating conditions for maximum sensitivity as described under Experimental Conditions. The general response for the HP 1100 LC/MS varied from ∼250 pg for the most sensitive compounds (see Table 1), such as the triazines (atrazine, propazine, etc.) and the phenylurea herbicides (diuron, fluometuron), to the least sensitive compounds by APCI+ with no response, the bipyridylium herbicides. While the general sensitivity of the ESI+ was 5-10 times less sensitive for the triazines and phenylurea herbicides than APCI+, ESI+ was considerably more sensitive for the bipyridylium compounds than APCI+ (Table 1). Thus, the first general conclusion that resulted from the comparison of the (61) Hostetler, K. A.; Thurman, E. M. Sci. Total Environ. 2000, 248, 147. (62) Thurman, E. M.; Goolsby, D. A., Meyer, M. T.; Mills, M. S.; Pomes, M. L.; Kolpin, D. W. Environ. Sci. Technol. 1992, 26, 2440. (63) Ferrer, I.; Ballesteros, B.; Marco, M. P.; Barcelo, D. Environ. Sci. Technol. 1997, 31, 3530. (64) Harrison, A. Chemical Ionization Mass Spectrometry, 2nd ed.; CRC Press: Boca Raton, FL, 1992. (65) Santos, T. C. R.; Rocha, J. C.; Barcelo, D. J. Chromatogr., A 2000, 879, 3.

Table 1. Response Factors of Various Classes of Pesticides in Optimized Mobile Phases Using LC/MS in Full-Scan Modea compound phenylurea herbicides chlorotoluron diuron fluometuron isoproturon linuron triazine herbicides atrazine deisopropylatrazine didealkylatrazine cyanazine irgarol propazine simazine terbuthylazine bipyridylium herbicides chlormequat mepiquat diquat paraquat acetanilide metabolites acetochlor ESA alachlor ESA metolachlor ESA acetochlor OXA alachlor OXA metolachlor OXA chlorophenoxyacid herbicides dicamba 2,4-D 2,4-DP 2,4,5-T chlorophenols phenol 3-nitrophenol 2,4-dinitrophenol 2,3,6-trinitrophenol 4-chloro-3-methylphenol 2,4,5-trichlorophenol alkyl sulfate surfactant sodium dodecyl sulfate

APCI+

APCI-

ESI+

ESI-

64 57 64 100 14

0 10 7 0 7

9 7 8 14 7

4 5 6 0 3

100 50 25 71 100 100 71 100

0 0 0 0 0 0 0 0

7 3 2 7 14 9 5 9

0 0 0 0 0 0 0 0

0 0 0 0

0 0 0 0

8 10 7 7

0 0 0 0

0 0

0 0

0 0

0 0 0

0 0 0

0 0 0

11 11 14 14 14 14

0 0 0 0

14 14 14 14

0 0 0 0

50 50 50 50

0 0 0 0 0 0

0 43 14 14 14 50

0 0 0 0 0 0

0 11 14 14 6 7

0

0

0

30

a The numbers are the response from 0 to 100%. Response factors are based on an injection of 25 µL of standard containing 250 pg of analyte with a LOD defined as a signal-to-noise ratio of 10:1.

data in Table 1 was that APCI+ was more sensitive than ESI+ for nonionic basic pesticides and that ESI+ was more sensitive for positively charged pesticides, such as the bipyridylium compounds, than APCI+. On the other hand, the acidic pesticides gave more response in negative ion mode, and the ESI- interface was generally more sensitive than the APCI- interface, by a factor of 10-100 times or even more. For example, the ESI- interface gave a response of 11-14% (Table 1, which is for a 250-pg injection) for the ESA metabolites of the chloroacetanilide herbicides, but the APCIsource did not respond at all for these compounds. The APCIsource gave a similar response to the ESI- for the chlorophenoxy acid herbicides of 14% (Table 1) but no response to the SDS surfactants (Table 1). The chlorinated and nitrated phenols were equal to or slightly less sensitive with the ESI- versus the APCIsource. Thus, the second general conclusion from the data in Table 1 is that for acidic pesticides the negative ion source of ESI is more sensitive. Analytical Chemistry, Vol. 73, No. 22, November 15, 2001

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Table 2. Detection of Various Classes of Pesticides by LC/MS in Full-Scan Modea compound acetanilide herbicides acetochlor alachlor dimethamide flufenacet metolachlor phenylurea herbicides chlorotoluron diuron demethyldiuron didemethyldiuron fluometuron demethylfluometuron didemethylfluometuron isoproturon linuron triazine herbicides atrazine deethylatrazine deisopropylatrazine didealkylatrazine deethylhydroxyatrazine deisopropylhydroxyatrazine hydroxyatrazine ametryn cyanazine deethylcyanazine cyanazine acid deethylcyanazine acid irgarol decyclopropane-irgarol propazine prometon prometryn deisopropylprometryn simazine terbutryn terbuthylazine phenolic compounds phenol 3-nitrophenol 2,4-dinitrophenol 2,3,6-trinitrophenol 4-chloro-3-methylphenol 2,4,5-trichlorophenol

APCI+

APCI-

ESI+

ESI-

+ + + + +

+ + + + +

+ + + + +

0 0 0 0 0

++ ++ ++ ++ ++ ++ ++ ++ ++

+ + 0 0 + 0 0 0 +

++ ++ ++ ++ ++ ++ ++ ++ ++

+ + 0 0 + 0 0 0 0

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

++ ++ ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0

0 ++ + + + ++

0 0 0 0 0 0

0 + + + + +

compound bipyridylium herbicides chlormequat mepiquat diquat paraquat acetanilide degradates acetochlor ESA alachlor ESA dimethanamide ESA flufenacet ESA metolachlor ESA acetochlor OXA alachlor OXA dimethanamide OXA flufenacet OXA metolachlor OXA chlorophenoxy acid herbicides dicamba 2,4,5-T 2,4-DP 2.4-D organophosphate insecticides acephate azinophos-ethyl fenitrothion fenthion fensulfoton vamidothion chloropyrifos organochlorine insecticides dieldrin aldrin endosulfan carbamate insecticides carbofuran aldicarb methomyl methiocarb sulfonylurea herbicides metasulfuron methyl bensulfuron alkyl sulfate surfactant sodium dodecyl sulfate

APCI+

APCI-

ESI+

ESI-

0 0 0 0

0 0 0 0

++ ++ ++ ++

0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

++ ++ ++ ++ ++ ++ ++ ++ ++ ++

0 0 0 0

+ + + +

0 0 0 0

++ ++ ++ ++

++ ++ ++ ++ ++ ++ ++

+ + + + + + +

++ ++ ++ ++ ++ ++ ++

0 0 0 0 0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

++ ++ ++ ++

0 0 0 0

++ ++ ++ ++

0 0 0 0

++ ++

+ +

++ ++

+ +

0

0

0

++

a The 0 means no response (LOD >1 µg on column), + means method possible (LOD 10 ng-1 µg on column), ++ means sensitive method possible (LOD 1 µg on column, shown as a “0” in Table 2), a method possible (LOD 10 ng-1 µg on column, shown as a +), or a sensitive method possible (LOD 100 µg/L for no method possible. These concentrations are based on typical procedures that use a 100-mL water sample and a final concentration of analyte in a volume of 100 µL with an injection volume of 10 µL.61 These 5444

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concentration ranges are the detection limits necessary for monitoring pesticides in surface water and groundwater.62 Three important general observations are now immediately obvious from Table 2. The first is that APCI+ continues to be more sensitive than ESI+ for the analysis of triazine and phenylurea herbicides and their degradates, which are not charged in solution and slightly basic. Although both interfaces worked in positive ion mode, the detection limit was ∼2-3 times more sensitive for the APCI+ interface. This result sometimes is important in trace analysis of pesticides in soil and water extracts where extremely low detection limits result in simpler methods of sample handling. For example, Ferrer et al.63 reported that the detection limit for irgarol (a triazine antifouling agent) is 0.005 µg/L with only 50 mL of seawater using APCI+ with selected ion monitoring and on-line solid-phase extraction (in this case, 250 pg of irgarol is injected onto the column). In Europe, where reporting limits must be less than 0.1 µg/L, it is quite important to have methods that easily reach this reporting limit. Therefore, the difference between APCI+ and ESI+ for triazine and phenylurea herbicides is important for analytical methods development

for pesticides in groundwater, where concentrations are often considerably less than in surface water. Because of the volatility and ease of protonation of the triazine and phenylurea herbicides, they have excellent sensitivity using APCI+. Thus, APCI+ is effective for those stabile, neutral pesticides that are weakly basic and easily volatilized. The literature supports this conclusion also with many successful results on the triazines and phenylurea herbicides using APCI+.3,11-13,17,21,22,32-34,36 Because the solutes must first be vaporized into the gaseous state before ionization, it is important that the solutes be nonionic in solution and nonlabile to thermal degradation according to this theory of ionization. Thus, the triazine and phenylurea herbicides fit this model well. Both classes of compounds are generally nonionic in solution and are stable at the temperature of the interface (generally ∼350 °C). Other classes of pesticides that are sensitive by APCI+ are the organophosphate and carbamate insecticides and the sulfonylurea herbicides (Table 2). These compounds are generally neutral solutes with basic sites that can easily be protonated in the vapor state and are stable to the temperature of the interface. It is interesting to note that the ESI+ interface works well for these compounds also. In fact, it is a general conclusion of Tables 1 and 2 that whenever a compound works well by APCI+ it also works well by ESI+ but not necessarily vice versa. This result relates to the fact that liquid-state basicity in ESI is related to proton affinity for APCI in the vapor state. However, positively charged solutes in solution, such as the bipyridylium herbicides, are not volatile in the APCI source. In APCI, chemical ionization occurs in the vapor state.2,64 Neutral solutes must first vaporize from the neubulized droplet and then be protonated in the vapor state to form the [M + H]+ ion. The APCI+ source has a corona discharge needle, which creates a stream of electrons that serve to ionize the solvent of the mobile phase, which is typically methanol/water or acetonitrile/methanol/water mixed with the nitrogen nebulizing gas. Thus, according to the current theory of APCI ionization,2,64 the CH3OH2+ and H3O+ are protonated species present in the vapor state and transfer protons to the weakly basic pesticides that are present in the vaporized state according to their proton affinity.64 Proton affinity refers to the acidity or basicity of a compound in the vapor state.64 A second observation from Table 1 that is verified in Table 2 is that ESI- is more sensitive than APCI- for the chlorophenoxyacid herbicides, alkyl sulfates, and acetanilide herbicide metabolites (which are all anionic solutes in solution). In fact, APCI- only worked for the chlorophenoxyacid herbicides (see Tables 1 and 2) and no response was observed for the alkyl sulfates and acetanilide herbicide degradates (Tables 1 and 2). This observation is generally consistent with the published literature. For example, APCI- has been attempted on chlorophenoxyacid herbicides with limited success.7,8,65 Instead, ESI- has been chosen by several researchers for the analysis of these compounds.3,15,28,30 In general, APCI- is seldom used for pesticide analysis, whereas ESI- is used frequently.2,3,28,30 One explanation for why APCI- is not as sensitive as ESI- is presented by Hiraoka and Kudaka52 and by Straub and Voyksner54 in a discussion of factors that affect sensitivity in negative ion ESI. They noted that a corona discharge may occur in ESI- at the

electrospray needle, which reduces the effectiveness of ESI-. The continuous corona discharge occurring in APCI- could similarly act as a source of electrons that will diminish sensitivity of the APCI- interface. Simply, negative ions are being consumed by protonation by the positively charged solvent, which is being formed from the corona discharge of the APCI source, with a net lower negative ion intensity in the source. However, if electron capture can occur, as can happen with chlorinated aromatic acids such as 2, 4-D, then the response may be satisfactory in APCI.64 Thus, a major difference between APCI negative and ESI negative is the concept that APCI works on compounds that capture electrons and will lose a proton to become negatively charged. ESI negative, on the other hand, works well on any compound that may lose a proton in solution and become negatively charged, such as the chlorophenoxyacid herbicides, sulfonic acid degradates of the chloroacetanilide herbicides, anionic surfactants, and other herbicides with an acidic pKa. For ESI, the current model of ionization is summarized from several references.51,53,57,58,60 In positive ion mode in the HP 1100 (a typical source configuration), the HPLC effluent is pumped through a nebulizing needle that is at ground potential. The spray goes through a semicylindrical electrode that is at high potential. The potential difference between the needle and the electrode produces a strong electrical field that charges the surface of the liquid and forms a spray of charged droplets. The charged droplets are attracted toward the capillary sampling orifice where a counterflow of heated nitrogen gas shrinks the droplets and carries away uncharged solvent. Positive ions migrate downfield toward the surface of the microdroplet as the microdroplet moves toward the metal cap of the capillary, and the negative ions migrate toward the other end of the microdroplet. Because the electrical current is carried by the charged droplets from the ESI needle to the metal cap at the capillary tube, electrons flow in the external circuit of the electrospray power supply.58,60 Thus, in positive ion mode, oxidation occurs at the needle, and reduction of the positively charged species occurs at the metal cap of the capillary.51,53,60 During the time that migration is occurring, there is also the evaporation of the solvent and droplet shrinkage taking place as the heated nitrogen gas (300 °C) is blown into the cloud of charged droplets. It is in this stage of droplet shrinkage that fission occurs, which is the process where a large droplet (20-50 µm) expels a small microdroplet of ∼1 µm.53 The large droplet is thought to be misshapen and elongated toward the metal end of the capillary where the excess positive charge has accumulated by electrophoretic migration (∼2% of the original mass, but ∼15% of the charge, resides in this microdroplet). The offspring droplets also can go through this same process53 until its size reaches between 0.08 and 0.03 µm. These smaller sized droplets are considered to be a second-generation offspring.53 At this size, the surrounding electric field becomes strong enough to lift a solute ion over the energy barrier and to ionize it before the complete evaporation of the solvent, a process called field-assisted desorption.54,57 It is assumed that the molecule is either positively or negatively charged before final formation of the gas-phase ion and that the charging of the analyte has occurred in solution. These charged ions are solvated by water during the ionization process, which Analytical Chemistry, Vol. 73, No. 22, November 15, 2001

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Figure 1. Ionization-continuum diagram for perticides. Key: ESA, ethanesulfonic acid degradate of the chloroacetanilide herbicides; PU, phenylurea herbicides; OP, organophosphate insecticides; Carb, carbamate insecticides; OC, organochlorine insecticides; SU, sufonylurea herbicides.

Figure 2. Ionization-continuum diagram showing the regions of effective usefulness of the various interfaces for LC/MS, including atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), and electron impact (EI) ionization.

lowers the energy of ionization that is required. For example, a naked sodium ion requires 98 kcal/mol to form a gas-phase ion and a sodium ion with seven water molecules clustered around it requires only 56 kcal/mol.53 Considerably more discussion on this topic is presented by Kebarle and Tang.53 The formation of solvent clusters no doubt plays an important role in the final gas-phase ionization step. The results for the ionic species presented here, including the chlorophenoxyacids, acetanilide oxanilic acids, sulfonic acids, and fixed positive ions such as monoquat and diquat, fit this model of electrospray ionization (Tables 1 and 2). All of these solutes are either anions or cations in solution and are vaporized effectively from the ionic state. Thus, bipyridylium herbicides, which are positive ions in solution, work well using ESI+ but have almost no response using APCI+. Likewise, the pesticides that are negative ions in solution, such as the chlorophenoxyacid herbicides, the acetanilide sulfonic acids, and sodium dodecyl sulfate (SDS), have excellent sensitivity and response using ESI- but have less or no response using APCI-. These pesticide data fit well the model of ionization for electrospray and APCI. The third observation from data in Tables 1 and 2 is that there are compounds that work approximately equally well in either APCI+ or ESI+, such as the organophosphate and carbamate insecticides. In these cases, the molecules are apparently protonated either in the gas phase in APCI+ or in the final droplet in ESI+. These compounds all contain either nitrogen or oxygen atoms that have a reasonably high proton affinity. Interestingly, the organophosphate insecticides as well as the phenylurea herbicides will sometimes work in APCI- or in ESI- and the results are variable (Table 2). This result reflects the electron capture potential of APCI negative and the potential for proton loss in ESI negative. The fact that APCI- may not give a molecular ion adduct, but fragments with losses that resemble EI fragmentation such as the loss of a methyl group (-15 amu), is evidence of electron impact ionization occurring. Ionization-Continuum (IC) Diagram. The data in Tables 1 and 2 for the detection of pesticides using APCI and ESI also can 5446

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be depicted in a diagram that extends from positive ion to negative ion in what is being called an “ionization-continuum diagram for pesticides” (Figure 1). The various classes of pesticides are plotted on the IC diagram according to whether they ionize in positive ion, in negative ion, or in both modes. Note that the bipyridylium herbicides (permanently positively charged) plot on the positive end of the diagram and that the ESA degradates of the chloroacetanilide herbicides plot on the negative side of the diagram. These compounds work only using the ESI interface. The phenylurea and sulfonylurea herbicides and the organophosphate insecticides will ionize in both positive and negative ion modes using both APCI and ESI, but more sensitively using APCI. The triazine herbicides work well in positive ion mode by using either APCI or ESI. These results (Tables 1 and 2 and Figure 1) are consistent with the literature as cited earlier. The organochlorine insecticides do not ionize readily in either APCI or ESI and are drawn in the center of the diagram (data from Table 2). In fact, these insecticides are relatively easily ionized with electron impact using GC/MS. Thus, there is an association between the response factors of nonionic compounds that respond in APCI (+ and -) and compounds that respond by GC/MS electron impact ionization. This similarity reflects the concept of volatilization of the solute before ionization in both APCI/MS and GC/MS, which does not occur in ESI/MS, as well as the fact that some electron impact ionization and electron capture may be occurring in APCI-. Figure 2 is a generic version of the ionization-continuum diagram and is derived from Figure 1 and Tables 1 and 2. Figure 2 shows that ESI works well in ionic regions and also works well on polar species where there is acidity or basicity in solution. Whereas APCI works on both polar and nonpolar species but usually does not work on ionic species in solution. APCI and electron impact (EI) work on the compounds near the center of the diagram, such as the carbamate and organophosphate insecticides. APCI- also works in electron capture or in electron impact mode. The usefulness of Figure 2 is that an interface may be chosen for pesticides or their degradates a priori knowing that

Table 3. Formation of Sodium Adducts by APCI+ and ESI+ Using a HP 1100 Mass Spectrometera compound phenylurea herbicides chlorotoluron diuron fluometuron isoproturon linuron triazine herbicides atrazine cyanazine irgarol propazine simazine terbuthylazine bipyridylium herbicides chlormequat mepiquat diquat paraquat organophosphate insecticides acephate azinophos-ethyl fenitrothion fenthion fensulfoton vamidothion chloroacetanilide herbicides acetochlor alachlor metolachlor sulfonylurea herbicides nicosulfuron flumetsulam chlorsulfuron carbamate insecticides carbofuran aldicarb methomyl methiocarb

APCI+

ESI+

0 0 0 0 0

+ + + + +

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0 0 0

+ + + + + +

0 0 0

+ + +

0 0 0

+ + +

0 0 0 0

+ + + +

aThe 0 means no response, and the + means that formation of sodium adducts commonly occurs.

the interface has a good probability of working and one’s time and effort may be spent in optimizing a technique. Furthermore, the diagram is a useful conceptual framework to explain when to use either of the two interfaces for pesticide analysis using LC/ MS or for teaching methods development for pesticides. Sodium Adduct Formation in ESI+. A trend was observed for the formation of strong signals for sodium adducts among the various pesticide groups (Table 3). First, from Table 3 it is obvious that sodium adducts were common using ESI+ for the phenylurea and sulfonylurea herbicides and the carbamate and organophosphate insecticides. The APCI+ interface did not form sodium adducts for any of the pesticides shown in Table 3 when the HP 1100 mass spectrometer was used. Because protonation occurs in the vapor phase when APCI is used, sodium addition should also occur in the vapor state. There are at least two possible explanations for why this does not occur to a significant extent. First, sodium ions are not readily produced in the vapor state using the APCI source, because sodium ions cannot be easily vaporized as ions. A second possibility is that the sodium adduct is a weak interaction with the pesticide and the heated interface of APCI destroys any sodium adducts that might form, which is a less likely explanation than the former.

On the other hand, sodium adducts formed readily using ESI+ for a number of pesticides, including phenylurea and chloroacetanilide herbicides and organophosphate insecticides, but not triazine and bipyridylium herbicides. A common functional group in the first group of pesticides is the carbonyl group, which may donate a lone pair of electrons to form stable sodium adducts. The carboxyl group is also important for uncharged adduct formation and is commonly seen. Thus, sodium adducts may be used for structural elucidation in LC/MS indicating either carbonyl or carboxyl functional groups. Formation of the sodium adducts in ESI may follow two pathways. First, the adduct forms in solution during the final phases of microdroplet formation, and the ionization of the adduct occurs from the solution phase. Fenn et al.57 hypothesized this as the likely scenario for the formation of sodium adducts of a polyglycol ether. A second possibility is that hydrated sodium atoms, with a much lower energy of ionization than a bare sodium atom (56 versus 98 kcal/mol, respectively), are readily volatized and react with analyte molecules in the vapor state, forming sodium adducts. Sodium then adds to the oxygen of the carbonyl or carboxyl in the vapor state. Given that the APCI+ source does not commonly form sodium adducts, this second mechanism of formation seems unlikely. Eliminating sodium in the mobile phase or in the matrix of the sample is important in diminishing the sodium adduct. Furthermore, sodium adducts are often more abundant when flow injection analysis is used because of sodium contamination in the standard and the lack of separation of the sodium ion from the pesticide. Injecting the pesticide through the chromatographic column when full mass spectral scans are being obtained for fragmentation studies will separate the excess sodium and may reduce sodium adducts in ESI+. “Wrong-Way-Around” Results for ESI. Mansoori et al.60 coined the term wrong-way-around electrospray for both the observation of intense [M + H]+ ions that occurred in spraying basic solutions and the observation of intense [M - H]- ions sprayed from acidic solutions. They hypothesized that acidification is occurring at the surface of the microdroplet and that pH may be decreased by several orders of magnitude. The wrong-wayaround ESI+ results shown in Table 4 for atrazine and irgarol fit the definition of Mansoori et al.60 First, both atrazine and irgarol gave strong ESI+ ion signals at a pH above their pKa when they were not ionized (atrazine has a pKa of 1.7 and irgarol has a pKa of 4.9). The data in Table 4 show that the response of atrazine and irgarol is significant using ESI+ with a methanol/water solution and no acidic additives at pH 6.0, although irgarol is twice as sensitive as atrazine (2500 versus 1250 pg, with a signal-tonoise ratio of 10:1). These results suggest that protonation and ionization of the triazine herbicides are occurring during final droplet formation. Second, atrazine showed no difference in response factors between pH 2.5 and 9.0 using mobile phase A, which contained an acetic acid additive and pH adjustment with ammonium hydroxide. Irgarol showed only a slight difference in response at the acidic pH near its pKa of 4.9. Two other triazines, cyanazine acid and prometryn (pKa of 1.7 and 4.9 for cyanazine acid and 4.9 for prometryn) gave results similar to those of atrazine and irgarol, showing that wrong-way-around results are common for the triazine herbicides. Analytical Chemistry, Vol. 73, No. 22, November 15, 2001

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Figure 3. Chromatogram of the chloroacetanilide and acetanilide herbicides using wrong-way-around electrospray and mobile-phase conditions of 50% water with 0.3% acetic acid and 50% of a mixture of 24:40 methanol/acetronitrile with 0.3% acetic acid. Chromatogram shows 20 ng on column. The limit of detection for this method is 500 pg on column.

One process that might explain this result is the accumulation of protons at the surface of the microdroplet, decreasing the pH such that protonation could occur even for atrazine with a pKa of ∼1.7. Several recent papers have addressed the phenomenon of acidification of the droplet. Mansoori et al.60 did collect the sprayed liquid and found that pH had decreased 0.2 pH unit using ESI+. This value of 0.2 pH unit is similar to the value reported by Zhou et al.59 for in situ measurements of pH using a pH-sensitive fluorophore. In fact, many studies summarized by Mansoori et al.60 have reported this wrong-way-around result, chiefly for amino acids. The results described herein show for the first time a similar result for pesticides. The ESI- also showed wrong-way-around results. For example, the weak carboxylic acid compounds responded in ESI- in acidic solutions in which they were theoretically protonated on the basis of their pKa. For example, oxanilic acids of the chloroacetanilides were effectively ionized in 0.1% acetic acid.66 Negative ions, therefore, were forming in solution despite the pKa calculation that concluded that the carboxyl group should be nonionic in solution. Again, it is hypothesized that at the droplet surface the concentration of negative charge (hydroxide ion) is removing protons from the weak acids despite the pH of the bulk solution, producing negative ions in solution at the surface of the microdroplet at the last moment before ionization. (66) Ferrer, I.; Thurman, E. M.; Barcelo, D. Anal. Chem. 1997, 69, 4547.

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Table 4. Limit of Detection, Defined as a Signal-to-Noise Ratio of 10:1, of Triazine Herbicides under Both APCI and ESI Positive Conditions Using LC/MS in Full-Scan Mode at Various Buffer Conditions Using a 25-µL Sample Injectiona compd

atrazine, pg

irgarol, pg

cyanazine acid, pg

prometryn, pg

pH 8.0 pH 6.0 pH 5.0 pH 4.0 pH 2.5

250 250 250 250 250

APCI+ Mobile Phase A 250 750 250 750 250 750 250 750 250 750

nd nd nd nd nd

pH 6.0

250

APCI+ with Mobile Phase B 250 750

nd

pH 9.0 pH 8.0 pH 7.0 pH 6.0 pH 5.0 pH 4.0 pH 3.0 pH 2.5

1250 1250 1250 1250 1250 1250 1250 1500

ESI+ with Mobile Phase A 1000 3000 1000 3000 1000 3000 750 3000 750 5000 750 5000 750 5000 1000 7500

1000 1000 1000 750 750 750 750 1000

pH 6.0

2500

ESI+ with Mobile Phase B 1250 nd

nd

a nd, not determined. Mobile phase A was methanol/water/acetic acid (50:50:0.1%). Mobile phase B was methanol/water (50:50) and no acetic acid.

Regardless of the exact mechanism, this is a predictable process and an important detail to consider when the correct mobile phase for LC/MS analysis is being prepared. It is especially important in that an acidic mobile phase is quite useful in liquid chromatographic analysis of acidic compounds because retention times are considerably longer and separation is easier to accomplish. Thus, these acidic mobile phases do not need to be neutralized by the postcolumn addition of ammonium hydroxide for increased sensitivity, despite the advice offered in some manuals on LC/MS (see Hewlett-Packard booklet on HPLC/MS). Interface and Mobile-Phase Selection. Examination of the data in Tables 1 and 2 reveals several salient points regarding the structure of the molecule that may be used for selection of the most sensitive interface. The molecules containing triazine rings or basic nitrogen, such as phenylurea, sulfonylurea, and triazine herbicides, all ionize well using APCI+ or ESI+. Generally, however, APCI+ is most sensitive. Fixed positively charged herbicides, such as diquat and paraquat, ionize only through an electrospray mechanism. Pesticides containing carbonyl groups such as phenylureas, sulfonylureas, and organophosphates work well in APCI+. Pesticides containing carboxyl groups, sulfate, and sulfonate pesticides ionize well in ESI-. Mobile-phase selection is simplified by the findings of wrongway-around ESI. Pesticides containing carboxylic acid groups and sulfonic acid groups ionize readily using negative ion ESI with an acidic mobile phase. APCI+, of course, also produces ions from basic pesticides in an acidic mobile phase because mobile-phase effects are minor in APCI. An acidic mobile phase, thus, is recommended for a generic mobile phase for ESI (+ and -) HPLC/MS (0.1% acetic acid or 0.1% formic acid), especially as a first starting point for methods development. Although it is true that sensitivity may be enhanced by the addition of a postcolumn

base for acidic compounds using ESI-, this is often not necessary. An example of the sensitivity and usefulness of the acetic acid buffer is shown in Figure 3, which shows the chromatographic separation of a series of acidic pesticides including 2,4-D, flufenacet, alachlor, acetochlor, and metolachlor oxanilic acids and flufenacet, alachlor, acetochlor, dimethamide, and metolachlor ethanesulfonic acids. The excellent separation is achieved with an acidic mobile phase using 0.3% acetic acid and a 50% water and 50% mixture of 24:40 methanol/acetonitrile in 0.3% acetic acid. Twenty nanograms of each compound is injected on column in this chromatogram with a LOD of 500 pg on column and a signalto-noise ratio of 20:1. Thus, wrong-way-around results can often make reversed-phase chromatography simpler for negatively charged species. Finally, for basic solutes, the addition of an ammonium formate buffer at pH ∼3-4 may help chromatography and still allow good sensitivity using either APCI+ or ESI+. ACKNOWLEDGMENT The use of brand, trade, or firm names in this paper is for identification purposes only and does not constitute endorsement by the U.S. Geological Survey. This work was partly supported by the Commission for Cultural, Educational and Scientific Exchange between the United States and Spain (Contract HNCCT 98148) and by the Toxic Substances Hydrology Program of the U.S. Geological Survey. Special thanks to colleagues for discussions at Menorca, Montreux, Tenerife, Tuixen, and Eminence and the ideas from good Catalan coffee, “Ensaimada amb nata i cafe` amb llet”.

Received for review May 3, 2001. Accepted July 25, 2001. AC010506F

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