Analysis of Pesticides by LC− Electrospray-MS with Postcolumn

Anal. Chem. 2000, 72, 4659-4666

Analysis of Pesticides by LC-Electrospray-MS with Postcolumn Removal of Nonvolatile Buffers Michael S. Gardner,† Robert D. Voyksner,*,‡ and Carol A. Haney†

Department of Chemistry, North Carolina State University, Box 8204, Raleigh, North Carolina 27695, and Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709

Liquid chromatographic (LC) separations for pesticides and many other compounds make use of nonvolatile buffers in the mobile phase. The coupling of LC with mass spectrometry (MS) does not allow the use of nonvolatile buffers. Substitution with volatile buffers is possible, but changes in chromatographic retention and resolution can result even if pH is held constant. The postcolumn removal of nonvolatile buffers using a commercially available ion suppressor is evaluated for the analysis of carbamate pesticides. The suppressor efficiently removes phosphate anions from an LC mobile phase. Most compounds show an increased signal by factors of 2-7 after postcolumn phosphate removal. The suppressor has little effect on the chromatographic parameters of some compounds, while serious negative effects are noted for others. Some compounds will give poor results due to adsorption or retention by the suppressor. The results indicate that such a device may be useful for the LC-MS analysis of some pesticides using nonvolatile buffers. Liquid chromatography-mass spectrometry (LC-MS) has become a popular technique for the analysis of pesticides.1-18 The

thermal lability of many pesticides precludes a simple gas chromatographic (GC) analysis unless derivitization is undertaken.19-23 HPLC with UV detection can be used but may not provide the specificity required for a multiresidue method.19,24-30 With the advent of modern, pneumatically assisted electrospray systems, the use of HPLC-MS with standard-bore columns has become routine. In addition to molecular weight information, electrospray can provide fragments via collision-induced dissociation (CID) mechanisms.1,6 This added information is valuable given the legal implications of environmental analyses. The carbamate pesticides, being thermally unstable and moderately polar, are good candidates for HPLC-ESI-MS, and are used as model compounds in this work. Analysis of polar pesticides by reversed-phase LC often requires the use of nonvolatile buffers and/or ion-pairing reagents. Liquid chromatographic analysis of carbamates and phenylureas using phosphate and other nonvolatile buffers has been reported.31-33 Additives such as phosphoric acid and its salts, alkanesulfonic acids and their salts, alkylammonium salts, and trifluoroacetic acid (TFA) are used to improve peak shape, retention, and chromatographic resolution on the lipophilic stationary phase, by promoting the formation of neutral ion pairs, covering active sites, and buffering the LC mobile phase.34-36

†

North Carolina State University. Research Triangle Institute. (1) Slobodnı´k, J.; van Baar, B. L. M.; Brinkman, U. A. J. Chromatogr., A 1995, 703, 81-121. (2) Rodriguez, M.; Orescan, D. B. Anal. Chem. 1998, 70, 2710-2717. (3) Lagana`, A.; Fago, G.; Marino, A. Anal. Chem. 1998, 70, 121-130. (4) Ferrer, I.; Barcelo´, D. J. Chromatogr., A 1999, 854, 197-206. (5) Sabik, H.; Jeannot, R. J. Chromatogr., A 1998, 818, 197-207. (6) D’Ascenzo, G.; Gentili, A.; Marchese, S.; Perret, D. J. Chromatogr., A 1998, 813, 285-297. (7) D’Ascenzo, G.; Gentili, A.; Marchese, S.; Perret, D. J. Chromatogr., A 1998, 800, 109-119. (8) Taguchi, V. Y.; Jenkins, S. W. D.; Crozier, P. W.; Wang, D. T. J. Am. Soc. Mass Spectrom. 1998, 9, 830-839. (9) D’Ascenzo, G.; Gentili, A.; Marchese, S.; Marino, A.; Perret, D. Chromatographia 1998, 48, 497-505. (10) Lagana`, A.; Fago, G.; Marino, A.; Mosso, M. Anal. Chim. Acta 1998, 375, 107-116. (11) Stout, S. J.; daCunha, A. R.; Picard, G. L.; Safarpour, M. M. J. AOAC Int. 1998, 81, 685-690. (12) Bossi, R.; Koppen, B.; Spliid, N. H.; Streibig, J. C. J. AOAC Int. 1998, 81, 775-784. (13) Volmer, D. A.; Hui, J. P. M. Arch. Environ. Contam. Toxicol. 1998, 35, 1-7. (14) Stout, S. J.; daCunha, A. R.; Picard, G. L.; Safarpour, M. M. J. Agric. Food Chem. 1996, 44, 2182-2186. (15) Marek, L. J.; Koskinen, W. C. J. Agric. Food Chem. 1996, 44, 3878-3881. (16) Volmer, D. A.; Vollmer, D. L.; Wilkes, J. G. LC-GC 1996, 14, 216-224. (17) Volmer, D.; Wilkes, J. G.; Levsen, K. Rapid Commun. Mass Spectrom. 1995, 9, 767-771. ‡

10.1021/ac0003302 CCC: $19.00 Published on Web 08/26/2000

© 2000 American Chemical Society

(18) Shalaby, L. M.; Bramble, F. Q., Jr.; Lee, P. W. J. Agric. Food Chem. 1992, 40, 513-517. (19) Lisˇka, I.; Slobodnı´k, J. J. Chromatogr., A 1996, 733, 235-258. (20) Stout, S. J.; daCunha, A. R.; Allardice, D. G. Anal. Chem. 1996, 68, 653658. (21) Klaffenbach, P.; Holland, P. T. Biol. Mass Spectrom. 1993, 22, 565-578. (22) Klaffenbach, P.; Holland, P. T. J. Agric. Food Chem. 1993, 41, 396-401. (23) Cotterill, E. G. Pestic. Sci. 1992, 34, 291-296. (24) Hogendoorn, E. A.; Dijkman, E.; Baumann, B.; Hidalgo, C.; Sancho, JuanVicente; Hernandez, F. Anal. Chem. 1999, 71, 1111-1118. (25) Kiso, Y.; Li, H.; Shigetoh, K.; Kitao, T.; Jinno, K. J. Chromatogr., A 1996, 733, 259-265. (26) Lian, H.-Z.; Zhang, W. B.; Jiang, Q.; Miao, J. J. Liq. Chromatrogr. Relat. Technol. 1996, 19, 207-216. (27) Helling, C. S.; Doherty, M. A. Pestic. Sci. 1995, 45, 21-26. (28) Hiemstra, M.; Joosten, J. A.; de Kok, A. J. AOAC Int. 1995, 78, 12671274. (29) Hogendoorn, E. A.; Th. Brinkman, U. A.; van Zoonen, P. J. Chromatogr. 1993, 644, 307-314. (30) Slobidnik, J.; Groenewegen, M. G. M.; Brouwer, E. R.; Lingeman, H.; Th. Brinkman, U. A. J. Chromatogr. 1993, 642, 359-370. (31) Vassilakis, I.; Tsipi, D.; Scoullos, M. J. Chromatogr., A 1998, 823, 49-58. (32) Somsen, G. W.; Jagt, I.; Gooijer, C.; Velthorst, N. H.; Th. Brinkman, U. A.; Visser, T. J. Chromatogr., A 1996, 756, 145-157. (33) Rodrı´guez, E.; Barrio, R. J.; Goicolea, A.; Go´mez de Balugera, Z. Anal. Chim. Acta 1999, 384, 63-70. (34) Electrospray Ionization Mass Spectrometry; Cole, R. B., Ed.; John Wiley and Sons: New York, 1997; pp 323-340.

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These-ion-pairing reagents are known to suppress the formation of ions in the electrospray source. However, electrospray is optimal when ions are formed in solution, and ion pairing neutralizes these precharged ions resulting in drastically decreased sensitivity.34,35 Also, many of these additives are nonvolatile, causing fouling and subsequent failure of the electrospray source.35 The usual practice is to substitute volatile, weaker ion-pairing buffers (such as acetic acid, formic acid, and their ammonium salts) for the nonvolatile ones or to eliminate all mobile-phase additives. Crescenzi et al.37 used only a small amount of TFA in the LC-ESI-MS analysis of 45 pesticides which included carbamates and phenylureas. Honing et al.38 analyzed a mixture of 10 carbamates by LC-ESI-MS using no mobile-phase additives. Since many separations have been developed using phosphates, substitution with another buffer may require a time-consuming redevelopment of the separation or may result in a less desirable separation. Postcolumn removal is a potential solution. Ion-exchange resins have been used for the on-line removal of sodium for the ESI-MS analysis of oligonucleotides,39 of sulfate anion for analysis of small polar molecules by HPLC-ESI-MS,40 and of sodium for the ion chromatography-ESIMS analysis of polar pollutants.41 These resins, however, have a finite capacity and eventually must be replaced or regenerated offline. On-line removal of the buffer ions is possible using a commercially available membrane-based ion suppressor. The ions are exchanged across a membrane with ions present in a regenerant solution. Membrane suppressors have been used on-line to remove sodium cation, alkylammonium cation, and alkanesulfonate anion from chromatographic eluent for thermospray,42 moving-belt,36 and ESI-MS.43,44 The membrane suppressors used in these studies required a constant supply of an aqueous chemical regenerant, such as tetrabutylammonium hydroxide. The self-regenerating suppressor, an improvement over the standard membrane suppressors,45 has been used on-line to remove phosphate anion for HPLC-particle beam-MS.46 Strong ion-pairing anions (e.g., phosphate), which would cause a decrease in analyte signal, are electrolytically replaced with weak ion-pairing anions (e.g., hydroxide) supplied in a regenerant solution. A major advantage of this device is the use of deionized water as a regenerant. The goal of this research is to evaluate the effectiveness of self-regenerating ion suppressor technology for the on(35) Crescenzi, C.; Di Corcia, A.; Marchese, S.; Samperi, R. Anal. Chem. 1995, 67, 1968-1975. (36) Escott, R. E. A.; McDowell, P. G.; Porter, N. P. J. Chromatogr. 1991, 554, 281-292. (37) Crescenzi, C.; Di Corcia, A.; Guerriero, E.; Samperi, R. Environ. Sci. Technol. 1997, 31, 479-488. (38) Honing, M.; Riu, J.; Barcelo´, D.; van Baar, B. L. M.; Th. Brinkman, U. A. J. Chromatogr., A 1996, 733, 283-294. (39) Huber, C. G.; Buchmeiser, M. R. Anal. Chem. 1998, 70, 5288-5295. (40) Forngren, B. H.; Samskog, J.; Gustavsson, S. Å.; Tyrefors, N.; Markides, K. E.; Bengt Långstro ¨m. J. Chromatogr., A 1999, 854, 155-162. (41) Bauer, K.-H.; Knepper, T. P.; Maes, A.; Schatz, V.; Voihsel, M. J. Chromatogr., A 1999, 837, 117-128. (42) Simpson, R. C.; Fenselau, C. C.; Hardy, M. R.; Townsend, R. R.; Lee, Y. C.; Cotter, R. J. Anal. Chem. 1990, 62, 248-252. (43) Conboy, J. J.; Henion, J. D.; Martin, M. W.; Zweigenbaum, J. A. Anal. Chem. 1990, 62, 800-807. (44) Wachs, T.; Conboy, J. C.; Garcia, F.; Henion, J. D. J. Chromatogr. Sci. 1991, 29, 357-366. (45) Rabin, S.; Stillian, J.; Barreto, V.; Friedman, K.; Toofan, M. J. Chromatogr. 1993, 640, 97-109. (46) Debets, A. J. J.; Mekes, T. J. L.; Ritburg, A.; Jacobs, P. L. J. High Resolut. Chromatogr. 1995, 18, 45-48.

4660 Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

Table 1. Instrumental Conditions ionization mode Vcyl Vend Vcap CapEx skimmer 1 drying gas flow drying gas temp

positive -5500 V -4500 V -5500 V 110 V 14.0 V 3.7 L/min 250 °C

rf bias voltage rf exit lens quad ent lens electron multiplier HED quad. temp scan range nebulizer pressure

8.1 V -30 V dynamic ramp 2386 V 9120 V 100 °C 150-500 m/z 85 psig

line removal of phosphate buffer in HPLC-ESI-MS. We will also report on the conditions that affect suppressor performance and apply this technique to the analysis of pesticides. EXPERIMENTAL SECTION A carbamate pesticide mixture was obtained from Ultra Scientific. Lincomycin was obtained from Sigma Chemical (St. Louis, MO). Other pesticides were obtained as neat standards from the U.S. EPA Repository (Ft. Mead, MD). Ammonium acetate and tetrabutylammonium hydroxide were obtained from Aldrich (Milwaukee, WI). Ammonium phosphate (monobasic) was obtained from EM Science (Cherry Hill, NJ). HPLC grade water was obtained from an in-house Picotech water purification system. HPLC grade acetonitrile and methanol were obtained from Fisher Scientific (Pittsburgh, PA). All weighing of compounds was performed on a Mettler AE100 balance. Measurement of pH during mobile-phase preparation was performed using a ColeParmer Benchtop meter. Colorphast indicator strips for measuring eluent pH were obtained from EM Science. All HPLC mobile phases were degassed with helium prior to use. Data were collected using a Hewlett-Packard 5989A mass spectrometer with HP 59987 electrospray interface with orthogonal spray, Analytica Iris hexapole ion guide, HP 1090 series II liquid chromatograph, and PC data system. Mass spectrometer conditions are shown in Table 1. The ion suppressor was a Dionex Cation Self-Regenerating Suppressor-Ultra (2 mm) with control unit. Regenerant solutions were delivered to the suppressor with a Perkin-Elmer series 200 LC pump at a flow rate of 6.0 mL/min. For flow injection analysis (FIA) experiments, injections were 25 µL. For gradient elution HPLC experiments, an HP Eclipse XDB-C18, 2.1 × 50 mm, 3.5-µm particle column was used. A 10 ng/µL carbamate mixture was used with a 10-µL injection. Three different sets of experiments were used to evaluate the effects of phosphate buffer and the suppressor on analyte signal and chromatographic resolution. Conditions of the experiments are shown in Table 2. RESULTS AND DISCUSSION The pesticide carbofuran was initially evaluated under FIA conditions to optimize the mode of operation of the suppressor, regenerant solutions, and suppressor flow rates for use with 10100 mM levels of phosphate. Analysis was by positive ion electrospray, full-scan mode, which is optimum for the carbamate pesticides. All buffered mobile phases were prepared in a 1:1 (v/v) acetonitrile/water mixture. A phosphate-free mobile phase and 100 mM ammonium phosphate mobile phase were used with the 1090 gradient pump to deliver a mobile phase containing varying concentrations of ammonium phosphate. The former contained an acetic acid/ammonium acetate buffer at pH 4.9, and

Table 2. Chromatographic Conditions set 1

set 2

set 3

mobile phase A (no phosphate)

5% acetonitrile (v/v) 95% water (v/v) 0.5% acetic acid (v/v) 10 mM ammonium acetate

5% acetonitrile (v/v) 95% water (v/v) 0.5% acetic acid (v/v) 10 mM ammonium acetate

5% methanol (v/v) 95% water (v/v) 0.5% acetic acid (v/v) 10 mM ammonium acetate

mobile phase B (no phosphate)

95% acetonitrile (v/v) 5% water (v/v) 0.5% acetic acid (v/v) 10 mM ammonium acetate

50% acetonitrile (v/v) 50% water (v/v) 0.5% acetic acid (v/v) 10 mM ammonium acetate

80% methanol (v/v) 20% water (v/v) 0.5% acetic acid (v/v) 10 mM ammonium acetate

gradient (no phosphate)

linear 0-87% B in 25 min

linear 0-100% B in 15 min hold 14 min

linear 0-100% B in 20 min hold 4 min

nobile phase A (with phosphate)

5% acetonitrile (v/v) 95% water (v/v) 0.05% acetic acid (v/v) 25 mM ammonium phosphate

5% acetonitrile (v/v) 95% water (v/v) 0.05% acetic acid (v/v) 100 mM ammonium phosphate

5% methanol (v/v) 95% water (v/v) 0.05% acetic acid (v/v) 25 mM ammonium phosphate

mobile phase B (with phosphate)

83% acetonitrile (v/v) 17% ammonium phosphate (25 mM in water) (v/v) 0.05% acetic acid (v/v)

50% acetonitrile (v/v) 50% water (v/v)

80% methanol (v/v) 20% water (v/v)

0.05% acetic acid (v/v) 100 mM ammonium phosphate

0.05% acetic acid (v/v) 25 mM ammonium phosphate

linear 0-100% B in 25 min

linear 0-100% B in 15 min. hold 14 min

linear 0-100% B in 20 min hold 4 min

gradient (with phosphate)

the latter contained ammonium phosphate buffered to pH 4.9 with acetic acid. Each experiment consisted of a series of injections at five phosphate concentrations, from 0 to 100 mM. To compensate for long-term instrument variation during the evaluation, an external standard, lincomycin, was prepared in and injected in phosphate-free mobile phase prior to each experiment. For each injection of carbofuran, the signal level was normalized to the signal level of the lincomycin injection. Carbofuran standards, 100 ng/µL, were prepared separately in mixtures of the two mobile phases corresponding to the five phosphate concentrations. The API source was cleaned with water and methanol between each experiment. The experiments were carried out both with and without the suppressor in-line. The suppressor was tested in an electrolytic mode, a chemical mode, and a combined chemical-electrolytic mode. The suppressor regenerant solutions evaluated include tetrabutylammonium hydroxide, boric acid, acetic acid, and acetate over a concentration range of 1-100 mM at flow rates of 3-9 mL/min. In the electrolytic modes, the behavior was also studied at varying applied currents. System back pressure is an additional consideration. The back pressure on the suppressor, mainly due to the ESI source, must not exceed 100 psi. While pressures in excess of this were not observed at flow rates of 0.20 mL/min, an excessive back pressure would exist for flow rates much larger than 0.25 mL/min. Methods that used a 4.6-mm-i.d. column would have to be adapted to use of a smaller bore column to make use of the suppressor. Strong and Dasgupta47 first reported the use of electrodialysis to suppress sodium ions in ion chromatographic eluent using only water as a regenerant. The suppressor used in the present work operates on similar principles. It consists of a platinum anode and cathode, regenerant, and eluent screens which provide the flow channels and are separated by anion-exchange membranes. The (47) Strong, D. L.; Dasgupta, P. K. Anal. Chem. 1989, 61, 939-945.

eluent flows between the two membranes and a regenerant solution flows countercurrent to the eluent on either side. The membranes and screens are functionalized with positively charged sites to increase the efficiency of ion transfer between the electrodes. The components are stacked together inside a case that has entry and exit ports for eluent and regenerant.45,48 In the electrolytic mode, deionized water is used as a regenerant. Electrolysis of water in the regenerant solution produces hydronium ions at the anode and hydroxide ions at the cathode. In the electric field, the hydroxide ions migrate from the cathode regenerant channel to the eluent stream. The hydroxide ions undergo neutralization with the hydronium ions in the acidic eluent. To maintain charge balance, anions in the eluent (e.g., H2PO4-) migrate through the membrane into the anode regenerant chamber and are flushed to waste by the regenerant stream45,48 (see Figure 1). A control unit applies constant current on the electrodes at a level set by the user, with four different current levels being allowed. Too low of a current will not remove all of the desired anions, and higher currents may cause unnecessary wear on the suppressor shortening its life span. In the chemical mode, hydroxide ions are supplied in the regenerant solution as tetrabutylammonium hydroxide. No current is applied, and the hydroxide migrates through both membranes to undergo neutralization with hydronium ions already present in the eluent. To maintain charge balance, other anions in the eluent (e.g., H2PO4-) migrate into the regenerant stream and are flushed to waste.45,49 The combined mode is similar to the electrolytic mode except that an additive such as boric acid is used in the regenerant. The borate (or other anion) in part displaces some of the eluent anions, in addition to or in place of some of the hydroxide.45 (48) Pohl, C.; Slingsby, R. W.; Stillian, J. R.; Gajek, R. U.S. Patent 4,999,098, 1991. (49) Stevens, T. S.; Davis, J. C.; Small, H. Anal. Chem. 1981, 53, 1488-1492.

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4661

Figure 1. Redox and anion exchange within the suppressor. Modified from Figure 3 in CSRS-ULTRA instruction manual. Used by permission of Dionex Corp., Sunnyvale, CA.

Figure 2. Comparison of signals with suppressor in electrolytic mode: (b) no suppression, (9) suppressor at 50 mA, and (2) suppressor at 100 mA.

Carbofuran was used as a model pesticide for optimization of the suppressor. The (M + H)+ ion at m/z 222 and fragment ion at m/z 165 are used together as a measure of the total signal. Results are reported as the ratio of analyte peak area to external standard peak area, plotted versus the phosphate concentration. For experiments using no suppression, the suppressor was removed from the eluent flow path. With no suppression, a consistent drop in signal by a factor of 2-9 was observed when the mobile phase contained ammonium phosphate. The source also became visibly contaminated with a white powder residue during and after the experiment. In the electrolytic mode, the suppressor caused slightly decreased signal when no phosphate was used in the mobile phase, as compared with no suppression (Figure 2). At the 50mA current level, the suppressor resulted in a consistent carbofuran signal at 25 and 50 mM phosphate. Without the suppressor, the carbofuran signal decreased by a factor of 1.3 at 25 mM and by more than a factor of 3 when 50 and 75 mM phosphate were 4662

Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

present. At 100 mM phosphate concentration, the carbofuran signal is significantly suppressed regardless of the experimental conditions. The best performance in the electrolytic mode was achieved at the 100-mA current setting. Signal actually improved by a factor of 1.8 and remained consistent at 50 and 75 mM phosphate concentrations. No visible contamination of the ESI source was observed when the suppressor was used in electrolytic mode at the studied current levels. Use of the suppressor at the 300- and 500-mA current levels was also attempted in electrolytic mode. At the 300-mA level, the suppressor controller would intermittently enter an “alarm” state, indicating that it was not possible for the controller to produce the requested current. At the 500-mA level, the controller would continuously remain in the alarm state. This problem was likely caused by the high concentration of acetonitrile (50% v/v) in the eluent, which would result in lower conductivity of the eluent. Since most reversed-phase gradient elutions equal or exceed 50% organic during the course of a run, the FIA evaluation was not run at lower organic concentrations. Data for the 300- and 500-mA current levels are therefore not available. The suppressor was evaluated in the combined mode using various regenerants. The data from these experiments are summarized in Table 3. Boric acid (10 mM) is recommended by the suppressor manufacturer as a regenerant for the combined mode. While this regenerant is not specifically mentioned in the literature for membrane suppressors, regenerants other than water can be used in the electrolytic mode with varying amounts of success.47,48 When the boric acid regenerant was used, the suppressor caused a decreased signal level when no phosphate was present, as compared with no suppression. With no suppressor in-line, the carbofuran signal drops steadily at increasing phosphate concentrations of 25 and 50 mM and levels off at very low signal at 75 and 100 mM phosphate. At the 50-mA current level, carbofuran signal remains constant up to 25 mM phosphate but drops to the level with no suppression at higher phosphate concentrations. At the 100-mA current level, signal increases slightly or remains level at phosphate concentrations of 25 and 50 mM, while lesser decreases were noted at the 75 and 100 mM phosphate concentrations. Visible contamination of the ESI source by a white powder residue was observed at the 50- and 100-mA settings. The contamination was possibly due to the presence of nonvolatile borate salts in the postsuppressor eluent. In an attempt to reduce this contamination while evaluating the potential benefits of boric acid, a 10-fold reduction of the boric acid concentration in the regenerant was studied at the 100-mA level. Lower boric acid concentrations result in slightly poorer performance at 25 and 50 mM phosphate but an improved signal at 75 and 100 mM phosphate concentrations. Contamination of the ESI source was still observed and did not appear to be lessened by the lower boric acid concentrations. At the 300- and 500-mA current levels, the same problems were encountered as with deionized water regenerant, and these data are therefore not available. Reduced signal observed at high levels of phosphate when the suppressor is in-line may have been caused by incomplete removal of the phosphate at these levels. However, incomplete removal would have likely resulted in some contamination of the ESI source, which was not observed. Data from direct measurement of dihydrogen phosphate, to be discussed subsequently, also

Table 3. Effect of Suppressor on Analyte Signala [PO43-] (mM)

no suppression

50 mA, 10 mM boric acid

10 mM boric acid

1 mM boric acid

0 25 50 75 100

2.630 1.543 0.705 0.420 0.272

1.810 1.881 0.622 0.413 0.361

2.386 3.167 2.229 0.778 0.460

2.363 2.643 1.604 0.987 1.328

a

1.675 1.661 1.749 2.010 1.613

1.129 1.300 1.204 1.184 1.092

10 mM acetic acid

100 mM acetic acid

1.424 1.148 1.015 0.986 0.878

1.726 1.723 1.505 1.097 0.789

Suppressor mode indicated by current level and regenerant.

Table 4. Postsuppressor Eluent pH as Measured by pH Stripsa regenerant mobile-phase phosphate concn (mM)

deionized H2O

boric acid (10 mM)

ammonium acetate (10 mM)

0 25

4.5 9.5

4.0 8.0

4.0 5.0

a

current level, 100 mA 10 mM 100 mM ammonium acetate ammonium acetate

Suppressor current, 50 mA.

suggest that incomplete removal is not the cause of the problem. In addition, reduced signal was observed with the suppressor inline when no phosphate was present. Replacement of acetate, phosphate, and mono- and dihydrogen phosphate anions with hydroxide by the suppressor would have yielded ammonium hydroxide, a stronger base. The corresponding increase in pH could suppress positive ESI signal. Therefore, the use of an anion other than hydroxide to replace phosphate is desirable. Borate salts are nonvolatile and result in decreased signal when compared with the deionized water regenerant, as discussed previously. Ammonium acetate and acetic acid are volatile compounds commonly used to adjust mobile-phase pH in ESI-MS, so the use of acetate as a counterion is a logical choice. Indeed, Table 4 shows that while pH is affected very little when no phosphate is present, it is increased by the suppressor when phosphate is present. This increase is less drastic when boric acid is used as a regenerant. When ammonium acetate is used as a regenerant, the pH change is minimized. Ammonium acetate was evaluated as a regenerant at 10 and 100 mM concentrations, with the suppressor operated at the 100mA current setting. With either the 10 or 100 mM ammonium acetate regenerant, the suppressor caused a decrease in carbofuran signal when no phosphate was present, as compared with no suppression. At a 25, 50, 75, and 100 mM phosphate concentration, the carbofuran signal dropped significantly when no suppressor was used while signal remained constant for both 10 and 100 mM ammonium acetate concentrations. At concentrations of 75 and 100 mM phosphate, the in-line suppressor increased signal by a factor of 4 compared to no suppressor. The signal was consistently higher with 10 mM ammonium acetate compared to 100 mM ammonium acetate. At 50-100 mM phosphate concentrations, signal is improved compared with no suppression. This is the first example where phosphate suppression at higher concentrations is eliminated. Acetic acid was also evaluated as a regenerant at 10 and 100 mM concentrations, with the suppressor operated at the 100-mA

current setting. Similar to the findings with ammonium acetate, the suppressor, in the absence of phosphate, caused a decreased signal with either concentration of acetic acid compared to no suppressor and no phosphate. With 10 mM acetic acid, the signal remained constant at phosphate concentrations of 25-100 mM. Using 100 mM acetic acid as a regenerant, the signal was higher compared to the 10 mM acetic acid at 25 and 50 mM phosphate. At 75 and 100 mM phosphate, the signals for 10 and 100 mM acetic acid regenerants were the same. Signal with in-line suppression was increased by a factor of 2 at a phosphate concentration of 50 mM and by a factor of 2.5 at the 75 and 100 mM concentrations compared to no suppression. Contamination of the source was not observed when either acetic acid or ammonium acetate regenerants were used. It should be noted that good results were achieved even when pH values were elevated by the suppressor (Table 4). This indicates that the presence of ammonium phosphate at acidic pH is more detrimental to performance in positive ESI than raising pH in the absence of phosphate. The use of ammonium acetate as a regenerant did prevent the mobile-phase pH from being increased by the suppressor and gave a consistent signal for carbofuran across the phosphate concentrations range studied. However, at low phosphate concentrations (0 and 25 mM), signal intensity is greater without suppression. One possibility is that while pH was not increased, phosphate removal by the suppressor was less efficient in the combined chemical-electrolytic mode when ammonium acetate is used as a regenerant.47 Since contamination of the source was not observed under these conditions, this possibility is in doubt. Another possibility is that ammonium acetate was introduced by the suppressor at concentrations high enough to suppress ionization. Last, the suppressor was evaluated in the chemical (nonelectrolytic) mode. Tetrabutylammonium hydroxide, 35 mM, was used as a regenerant with the current turned off. In this mode, the suppressor caused greatly decreased signal for carbofuran at all measured phosphate levels, and when no phosphate was present, as compared with no suppression (data not shown). In addition, a large background signal at m/z 242 was observed, corresponding to the tetrabutylammonium ion. This large background was likely suppressing the ionization of the carbofuran and, as a result, canceling any benefit derived from removal of phosphate. Phosphate removal by the suppressor was verified by direct measurement of the H2PO4- anion by negative ion electrospray. Figure 3 shows that some of the phosphate was removed using a deionized H2O regenerant and 50-mA current, and nearly all was removed using 100 mA. Boric acid (10 mM) as a regenerant Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

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Table 5. Effect of Ammonium Phosphate Buffer on Chromatographic Parameters without phosphate pesticide

M + H ret time (min) width (min)

area

with phosphate k′

R

Rs

ret time (min) width (min)

aminocarb methomyl monuron mexacarbate propoxur carbofuran carbaryl diuron methiocarb linuron neburon

209 163 199 223 210 222 202 235 226 249 275

4.96 5.60 13.12 13.47 14.37 14.66 15.53 16.21 18.59 18.89 21.70

0.29 0.25 0.18 0.28 0.20 0.13 0.16 0.19 0.13 0.10 0.16

112874607 32483753 7794214 109998355 25393648 23898577 3996554 1871101 4357036 1667477 7341780

Set 1 5.20 6.00 1.15 2.34 15.40 2.56 34.55 15.83 1.03 1.50 16.96 1.07 3.73 17.33 1.02 1.79 18.41 1.06 6.03 19.26 1.05 3.92 22.24 1.15 15.19 22.61 1.02 2.65 26.12 1.16 22.24

aminocarb methomyl monuron mexacarbate propoxur carbofuran carbaryl diuron methiocarb linuron neburon

209 163 199 223 210 222 202 235 226 249 275

5.11 5.72 13.10 13.61 14.34 14.65 15.45 16.10 18.16 18.49 21.51

0.31 0.26 0.15 0.29 0.16 0.18 0.18 0.16 0.15 0.14 0.24

90993177 20251813 17703726 123368295 33737126 43147549 7242527 3578315 4649443 1448100 12663922

Set 2 5.39 5.63 6.15 1.14 2.13 6.07 15.38 2.50 36.46 13.25 16.01 1.04 2.34 14.25 16.93 1.06 3.29 14.55 17.32 1.02 1.84 14.89 18.31 1.06 4.42 15.69 19.13 1.04 3.94 16.28 21.70 1.13 13.41 not observed 22.11 1.02 2.23 not observed 25.89 1.17 15.87 21.79

95940436 23167338 72366380 65133273 100001397 175669824 38473071 21459683 19380697 49076874 44750893

Set 3 8.40 8.63 1.03 0.47 18.22 2.11 22.00 19.26 1.06 2.38 19.53 1.01 0.64 19.80 1.01 0.48 20.52 1.04 1.34 22.61 1.10 5.94 24.15 1.07 4.67 24.48 1.01 1.09 27.20 1.11 9.27

aminocarb methomyl monuron propoxur carbofuran mexacarbate carbaryl diuron linuron methiocarb neburon

209 163 199 210 222 223 202 235 249 226 275

7.52 7.71 15.37 16.21 16.42 16.64 17.22 18.88 20.12 20.38 22.56

0.27 0.20 0.21 0.20 0.19 0.34 0.17 0.16 0.15 0.13 0.15

Figure 3. Direct measurement of H2PO4- anion: (b) no suppression, (9) suppressor at 50 mA with deionized water, (2) suppressor at 100 mA with deionized water, and ([) suppressor at 100 mA with 10 mM boric acid.

appeared to be equally efficient at 100 mA. Note that a phosphate background signal was present in some experiments even when a phosphate-free mobile phase is run. This is likely caused by contamination of the HPLC lines or ESI source from prior runs using ammonium phosphate. 4664 Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

5.34 6.82 13.48 12.67 14.59 14.84 15.66 16.18 18.21 18.45 21.03

8.03 8.54 15.59 16.29 16.54 16.00 17.28 18.99 20.23 20.54 22.75

R

area

k′

0.25 0.24 0.16 0.22 0.16 0.15 0.14 0.09 0.12 0.10 0.16

18041248 3635435 10644643 42303353 9252791 16230778 3014636 2467000 3216153 1316558 6267815

5.68 7.52 15.85 14.84 17.24 17.55 18.58 19.23 21.77 22.06 25.29

1.32 6.08 2.11 33.57 0.94 -4.30 1.16 10.09 1.02 1.59 1.06 5.76 1.03 4.55 1.13 19.26 1.01 2.18 1.15 20.08

0.19 0.12 0.13 0.17 0.10 0.13 0.11 0.11

5478589 399333 3006305 7597681 656275 2968811 383282 482609

6.04 6.59 15.56 16.82 17.18 17.61 18.61 19.35

1.09 2.36 1.08 1.02 1.02 1.06 1.04

0.16

1323545 26.23

0.21 0.10 0.14 0.14 0.16 0.25 0.17 0.14 0.15 0.15 0.13

14904299 2735504 14936906 5831362 15333815 21952134 5726237 3912947 3531538 6425484 9002095

9.04 9.67 18.49 19.36 19.68 19.00 20.60 22.74 24.28 24.68 27.44

Rs

2.91 57.84 6.58 2.12 2.94 6.55 5.37

1.07 1.92 1.91 35.32 1.05 2.94 1.02 1.01 0.97 -1.60 1.08 3.67 1.10 6.68 1.07 5.04 1.02 1.23 1.11 9.40

To demonstrate the utility of this methodology, three sets of chromatographic experiments were performed, under the conditions shown in Table 2. A mixture containing 19 carbamate pesticides was used. Twelve of the compounds gave strong (M + H)+ ions. Since fluometuron and diuron should both give (M + H)+ ions at m/z 233, diuron was identified with its ((M + 2) + H)+ chlorine isotope. Two peaks at m/z 233 in addition to diuron were observed. Fluometuron could therefore not be identified in the mixture and so was excluded from the data set. Six other compounds that gave weak (M + H)+ ions were also excluded. The remaining compounds are shown in Table 5, along with chromatographic parameters. In set 1, the addition of ammonium phosphate increased retention (k′) for earlier eluting peaks, while decreasing it for later eluting peaks. The largest effects on selectivity (R) were observed in earlier eluting peaks. Resolution between adjacent peaks is improved in only half of cases, while it is degraded for the remaining peaks. Sensitivity is reduced in the presence of phosphate in every case except 2 (monuron and diuron). In set 2, retention is increased slightly for all peaks. Selectivity changes more for earlier eluting peaks. Resolution is enhanced for nearly all of the peaks observed; however, two peaks were not observed when ammonium phosphate was present, making resolution measurements impossible for some peaks. Sensitivity decreased

Figure 4. Extracted ion chromatograms of 11 carbamate pesticides: Peaks: (1) aminocarb, m/z 209 (M + H)+; (2) methomyl, m/z 163 (M + H)+; (3) monuron, m/z 199 (M + H)+; (4) propoxur, m/z 210 (M + H)+; (5) carbofuran, m/z 222 (M + H)+; (6) mexacarbate, m/z 223 (M + H)+; (7) carbaryl, m/z 202 (M + H)+; (8) diuron, m/z 235 (M + H)+; (9) linuron, m/z 249 (M + H)+; (10) methiocarb, m/z 226 (M + H)+; (11) neburon, m/z 275 (M + H)+. (A) no suppressor, no phosphate; (B) no suppressor, with phosphate; (C) with suppressor, no phosphate; (D) with suppressor, with phosphate.

substantially in every case in the presence of phosphate. In set 3, retention increased for all peaks except mexacarbate, which showed a decrease. Selectivity changes were greater in the earlier eluting peaks but essentially unaffected in the later eluting peaks. Resolution was improved between all peaks and sensitivity decreased for all peaks. It should be noted that all mobile phases were buffered to a constant pH and that in each set of gradient elutions the organic/water gradient was not varied. This indicates that a chemical interaction between the ammonium phosphate and either the stationary phase or the analyte is responsible for the changes in retention and selectivity observed. The most dramatic effect was observed with aminocarb and mexacarbate, which have amine functionality (the other carbamates in the data set do not). This suggests an ion-pairing interaction, since amines are protonated at acidic pH. The water/methanol gradient elution in set 3 gave the most significant chromatographic improvement with the use of phosphate buffer and so is evaluated using the ion suppressor. In addition to the data already shown, the analysis was run with the ion suppressor in-line both with and without ammonium phosphate in the mobile phase. Extracted ion chromatograms are shown in Figure 4, and relevant chromatographic data are included as Supporting Information. The effect of ammonium phosphate on the separation is further illustrated by comparing the chromato-

grams in Figure 4A and B. Peak 1, aminocarb, shows tailing when no phosphate is used, but the tail was eliminated when phosphate was used. Aminocarb and the adjacent methomyl show narrower peak width and improved resolution. Monuron, propoxur, carbofuran, and mexacarbate are also narrowed. Carbofuran and mexacarbate coelute when phosphate is absent, but are better resolved when phosphate is present. Due to changes in selectivity, monuron and mexacarbate show decreased resolution. Little effect was noted on the later eluting peaks, except that the peaks were narrowed, which increased the observed values for resolution. It is clear that sensitivity was decreased for all peaks. Figure 4A is the control (no suppressor, no phosphate). Figure 4B (no suppressor, 25 mM phosphate) shows the improvement in chromatographic performance but reduced sensitivity. The effect of the suppressor on the compounds under phosphate-free conditions can be seen by comparing Figure 4A and C. Aminocarb, methomyl, propoxur, and carbofuran show small increases in peak width (average 0.05 min) which are likely due to the added dead volume of the suppressor. Peak 3, monuron, shows a large 0.66min increase in width, indicating that this compound may be strongly retained or adsorbed by active sites in the suppressor. Diuron, linuron, and neburon (late-eluting peaks) also show large increases in peak width as well as substantial loss of intensity. It should be noted that these compounds, which are adsorbed or retained by the suppressor, contain one or more chlorine atoms attached to an aromatic ring. The remaining compounds contain no chlorine. Peak 6 shows a slight 0.05-min decrease in width. Six of the compounds show large decreases in sensitivity (see Supporting Information). Comparison of panels B and D of Figure 4 shows the effect of the suppressor on the separation using phosphate. Sensitivity for methomyl, monuron, propoxur, and carbofuran was increased by a factor of 4.6 on average. Methomyl and monuron peaks were both broadened, and monuron’s broadening is severe. Diuron, linuron, and neburon also show increases in sensitivity, but all show broadening and increased tailing. Sensitivity for aminocarb, mexacarbate, and carbaryl is decreased. Decreases in resolution are observed between all pairs of peaks, except for carbofuran, mexacarbate, and carbaryl. These compounds show an increase in resolution. The overall effect of combining the use of a phosphate buffer with the suppressor in Figure 4D should be compared with the use of ammonium acetate buffer with no suppressor in Figure 4A, to evaluate the usefulness of the suppressor in this application. With the phosphate and suppressor, aminocarb shows decreased peak width. Methomyl, propoxur, carbofuran, mexacarbate, carbaryl, and methiocarb show only small increases in width compared with the acetate/no suppressor separation. Monuron, diuron, linuron, and neburon show large increases in peak width. Four pairs of peaks show improvements in resolution. The remaining pairs have lower resolution. Decreased peak area is observed for all peaks except monuron, diuron, and linuron, which show an increased peak area. Again it should be noted that the chlorine-containing compounds showed large increases in peak width, while the others did not. As compared with the separation without phosphate, again it appears that most of the chromatographic advantages of using phosphate are lost when the suppressor is placed in-line. Yet, the Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

4665

suppressor enhances the electrospray signal in most cases and reduces contamination of the ESI source, enabling the detection of the pesticides. While only a few compounds dramatically lost signal in the presence of the suppressor, most lose chromatographic resolution. This loss is in part due to the 20-µL dead volume of the device but cannot completely be attributed to such a small dead volume. This suggests that analytes may interact, to a greater or lesser degree, with the anion-exchange sites on the membrane surface, or with the surface material itself, thus causing changes in chromatographic peak shape for the compounds. CONCLUSIONS The operation of the suppressor was optimized using the pesticide carbofuran. The suppressor was tested in electrolytic mode using deionized water as a regenerant; in chemical mode using tetrabutylammonium hydroxide as a regenerant; and in combined chemical/electrolytic mode using boric acid, ammonium acetate, and acetic acid as regenerants. In the electrolytic and combined modes, the current level was also evaluated. The best performance as defined by signal intensity was obtained in electrolytic mode using deionized water at 100-mA current, except at the highest phosphate levels. The combined mode using ammonium acetate or acetic acid at 100-mA current gave the best performance at the highest phosphate concentrations. In the absence of phosphate, the suppressor causes a decrease in analyte

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Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

signal. The removal of phosphate by the suppressor is verified by direct measurement of the dihydrogen phosphate anion. The suppressor was also evaluated for use in a chromatographic separation. While the suppressor increases sensitivity for some analytes when phosphate is present, it causes chromatographic resolution to be lost. While ammonium acetate can offer good separation efficiency for the carbamates, the relative retention times for the compounds are shifted compared to the phosphate mobile phase. Therefore, the use of the suppressor can prove quite useful for analysis of some pesticides, when MS identification of peaks occurring in a phosphate system (e.g., HPLC/UV) is required. Some compounds appear to be adsorbed or retained by the suppressor, which results in poor chromatographic performance for these compounds. The suppressor must be evaluated for this effect for each compound to be analyzed. SUPPORTING INFORMATION AVAILABLE A table of relative peak areas and chromatographic parameters for the data shown in Figure 4 . This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review March 20, 2000. Accepted July 13, 2000. AC0003302