Direct Analysis in Real Time for Reaction Monitoring in Drug

Jun 2, 2007 - The operating conditions of the DART ion source in the positive and negative ion modes were optimized by varying the discharge needle vo...
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Anal. Chem. 2007, 79, 5064-5070

Direct Analysis in Real Time for Reaction Monitoring in Drug Discovery Chris Petucci,*,† Jason Diffendal,‡ David Kaufman,‡ Belew Mekonnen,‡ Gene Terefenko,‡ and Brian Musselman§

Discovery Analytical Chemistry and Medicinal Chemistry, Wyeth Research, Collegeville, Pennsylvania 19426, and IonSense, Saugus, Massachusetts 01906

Direct analysis in real time (DART) is a novel ionization technique that provides for the rapid ionization of small molecules under ambient conditions. In this study, several commercially available drugs as well as actual compounds from drug discovery research were examined by LC/UV/ESI-MS and DART interfaced to a quadrupole mass spectrometer. For most compounds, the molecular ions observed by ESI-MS were observed by DART/MS. DART/MS was also studied as a means to quickly monitor synthetic organic reactions and to obtain nearly instantaneous molecular weight confirmations of final products in drug discovery. For simple, synthetic organic transformations, the trends in the intensities of the mass spectral signals for the reactant and product obtained by DART/ MS scaled closely with those of the diode array or the total ion chromatogram obtained by LC/UV/ESI-MS. In summary, DART is a new tool that complements electrospray ionization for the rapid ionization and subsequent mass spectral analysis of compounds in drug discovery. High throughput mass spectral analyses are becoming progressively more important in the pharmaceutical industry as hundreds of thousands of compounds are screened each year.1-3 To meet this demand, rapid analytical methods employing mass spectrometry have been used over the past several years that include multiplexed electrospray (MUX),4,5 electrospray chips,6,7 * To whom correspondence should be addressed. Phone: (484) 865-8377. Fax: (484) 865-9397. E-mail: [email protected]. † Discovery Analytical Chemistry, Wyeth Research. ‡ Medicinal Chemistry, Wyeth Research. § IonSense. (1) Oezbal, C. C.; LaMarr, W. A.; Linton, J. R.; Green, D. F.; Katz, A.; Morrison, T. B.; Brenan, C. J. H. Assay Drug Dev. Technol. 2004, 2, 373381. (2) Loo, J. A.; DeJohn, D. E.; Du, P.; Stevenson, T. I.; Loo, R. R. O. Med. Res. Rev. 1999, 19, 307-319. (3) Triolo, A.; Altamura, M.; Cardinali, F.; Sisto, A.; Maggi, C. A. J. Mass Spectrom. 2001, 36, 1249-1259. (4) Fung, E. N.; Chu, I.; Li, C.; Liu, T.; Soares, A.; Morrison, R.; Nomeir, A. A. Rapid Commun. Mass Spectrom. 2003, 17, 2147-2152. (5) Yuzhong, D.; Wu, J.-T.; Lloyd, T. L.; Chi, C. L.; Olah, T. V.; Unger, S. E. Rapid Commun. Mass Spectrom. 2002, 16, 1116-1123. (6) Zhang, L.; Laycock, J. D.; Miller, K. J. JALA 2004, 9, 109-114. (7) Zhang, S.; Van Pelt, C. K.; Henion, J. D. Electrophoresis 2003, 24, 36203632.

5064 Analytical Chemistry, Vol. 79, No. 13, July 1, 2007

fast LC/MS,8,9 and AP/MALDI.10,11 Recently, new ionization techniques such as DESI (desorption electrospray ionization),12-37,46 (8) O’Connor, D.; Mortishire-Smith, R. Anal. Bioanal. Chem. 2006, 385, 114121. (9) Castro-Perez, J.; Plumb, R.; Granger, J. H.; Beattie, I.; Joncour, K.; Wright, A. Rapid Commun. Mass Spectrom. 2005, 19, 843-848. (10) Wang, Y.; Schneider, B. B.; Covey, T. R.; Pawliszyn, J. Anal. Chem. 2005, 77, 8095-8101. (11) Moyer, S. C.; Cotter, R. J. Anal. Chem. 2002, 74, 468A-476A. (12) Taka´ts, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science 2004, 306, 471-473. (13) Wiseman, J. M.; Taka´ts, Z.; Gologan, B.; Davisson, V. J.; Cooks, R. G. Angew. Chem., Int. Ed. 2005, 44, 913-916. (14) Myung, S.; Wiseman, J. M.; Valentine, S. J.; Taka´ts, Z.; Cooks, R. G.; Clemmer, D. E. J. Phys. Chem. B 2006, 110, 5045-5051. (15) Chen, H.; Talaty, N. N.; Taka´ts, Z.; Cooks, R. G. Anal. Chem. 2005, 77, 6915-6927. (16) Cotte-Rodriguez, I.; Taka´ts, Z.; Talaty, N.; Chen, H.; Cooks, R. G. Anal. Chem. 2005, 77, 6755-6764. (17) Talaty, N.; Taka´ts, Z.; Cooks, R. G. Analyst 2005, 12, 1624-1633. (18) Wiseman, J. M.; Puolitaival, S. M.; Taka´ts, Z.; Cooks, R. G.; Caprioli, R. M. Angew. Chem., Int. Ed. 2005, 44, 7094-7097. (19) Kauppila, T. J.; Wiseman, J. M.; Ketola, R. A.; Kotiaho, T.; Cooks, R. G.; Kostiainen, R. Rapid Commun. Mass Spectrom. 2006, 20, 387-392. (20) Taka´ts, Z.; Wiseman, J. M.; Cooks, R. G. J. Mass Spectrom. 2005, 40, 12611275. (21) Chen, H.; Pan, Z.; Talaty, N. N.; Cooks, R. G.; Raftery, D. Rapid Commun. Mass Spectrom. 2006, 20, 1577-1584. (22) Nefliu, M.; Venter, A.; Cooks, R. G. Chem. Commun. 2006, 888-890. (23) Chen, H.; Cotte-Rodrı´guez, I.; Cooks, R. G. Chem. Commun. 2006, 597599. (24) Cooks, R. G.; Ouyang, Z.; Taka´ts, Z.; Wiseman, J. M. Science 2006, 311, 1566-1570. (25) Wiseman, J. M.; Ifa, D. R.; Song, Q.; Cooks, R. G. Angew. Chem., Int. Ed. 2006, 45, 7188-7192. (26) Ifa, D. R.; Wiseman, J. M.; Song, Q.; Cooks, R. G. Int. J. Mass Spectrom. 2007, 259, 8-15. (27) Hu, Q.; Talaty, N.; Noll, R. J.; Cooks, R. G. Rapid Commun. Mass Spectrom. 2006, 20, 3403-3408. (28) Venter, A.; Sojka, P. E.; Cooks, R. G. Anal. Chem. 2006, 78, 8549-8555. (29) Song, Y.; Talaty, N.; Tao, A. W.; Pan, A.; Cooks, R. G. Chem. Commun 2007, 61-63. (30) Williams, J. P.; Patel, V. J.; Holland, R.; Scrivens, J. H. Rapid Commun. Mass Spectrom. 2006, 20, 1447-1456. (31) Leuthold, L. A.; Mandscheff, J.-F.; Fathi, M.; Giroud, C.; Augsburger, M.; Varesio, E.; Hopfgartner, G. Rapid Commun. Mass Spectrom. 2006, 20, 103110. (32) Jackson, A. T.; Williams, J. P.; Scrivens, J. H. Rapid Commun. Mass Spectrom. 2006, 20, 2717-2727. (33) Williams, J. P.; Scrivens, J. H. Rapid Commun. Mass Spectrom. 2005, 19, 3643-3650. (34) Rodriguez-Cruz, S. E. Rapid Commun. Mass Spectrom. 2006, 20, 53-60. (35) Ford, M. J.; McNaney, C.; Drexler, D. M.; Sanders, M. Presented at the 54th Meeting of the American Society for Mass Spectrometry, Seattle, WA, May 28-June 1, 2006. 10.1021/ac070443m CCC: $37.00

© 2007 American Chemical Society Published on Web 06/02/2007

DART (direct analysis in real time),30,36-48 DAPCI (desorption atmospheric pressure chemical ionization),30,49 ELDI (electrosprayassisted laser desorption/ionization),50 ASAP (atmospheric solids analysis probe),51 and MALDESI (matrix-assisted laser desorption electrospray ionization)52 have been developed to rapidly ionize a variety of compounds under ambient conditions with MS detection. Of these techniques, both DESI and DART have been the most widely used for the analysis of a multitude of nonpolar and polar analytes without prior sample preparation. DESI has been coupled to mass spectrometry to analyze a wide variety of compounds and media including enzyme-substrate complexes,13 proteins,14 pharmaceuticals,15,19,30,36,37 explosives,16 plant alkaloids,17 biological tissues,18,25,26 metabolites,19,35 forensic samples,20 metabolomic samples,21 polymers,22 chiral molecules,23 and intact bacteria.29 The DESI technique involves production of a fine spray of charged droplets directed onto a surface containing the analyte of interest. Desorbed analytes are ionized and then introduced as desolvated ions into a mass spectrometer. Several mechanisms for ion formation have been proposed such as chemical sputtering, gas-phase ionization, shockwave models, and a droplet pick up mechanism.24,28 Recently, a study of the droplet dynamics of DESI supported a droplet pick up mechanism whereby analytes on a surface are solvated by electrosprayed droplets resulting in ion formation by typical electrospray ionization processes.28 DART has been interfaced to mass spectrometry for the analysis of counterfeit antimalarials,36,46 formulated products,37 (36) Nyadong, L.; Hampton, C.; Leung, H.; Newton, P.; Green, M.; de Jesus, V.; Fernandez, F. M. Presented at the 54th Meeting of the American Society for Mass Spectrometry, Seattle, WA, May 28-June 1, 2006. (37) Marcus, A.; New, A. Presented at the 54th Meeting of the American Society for Mass Spectrometry, Seattle, WA, May 28-June 1, 2006; poster. (38) Morlock, G.; Schwack, W. Anal. Bioanal. Chem. 2006, 385, 586-595. (39) Cody, R. B.; Larame´e, J. A.; Durst, H. D. Anal. Chem. 2005, 77, 22972302. (40) Wu, J.-T.; Musselman, B. Presented at the 54th Meeting of the American Society for Mass Spectrometry, Seattle, WA, May 28-June 1, 2006. (41) Gomez, M. Presented at the 54th Meeting of the American Society for Mass Spectrometry, Seattle, WA, May 28-June 1, 2006. (42) Lochansky, M. I.; Gomez, M. A.; Williams, J. D.; Johnson, R. L.; Miller, L. A. Presented at the 54th Meeting of the American Society for Mass Spectrometry, Seattle, WA, May 28-June 1, 2006; poster. (43) JEOL AccuTOF mass spectrometer with DART application notes, chemicals on dollar bills, counterfeit drugs, pharmaceutical tablets and formulations, explosives, trace herbicide detection, fungicides, triglycerides, biological fluids, amino acids, polymers, chemical warfare agents, metabolites, peptides, oligosaccharides, organometallics, drugs of abuse, explosives, toxic industrial chemicals, at http://www.jeol.com/HOME/tabid/36/Default.aspx. (44) Zhang, Z.; Morris, R.; Kadar, E.; Sullivan, D.; Cody, R. Presented at the 54th Meeting of the American Society for Mass Spectrometry, Seattle, WA, May 28-June 1, 2006; poster. (45) Morlock, G.; Yoshihisa, U. J. Chromatogr. A 2007, 1143, 243-251. (46) Fernandez, F. M.; Cody, R. B.; Green, M. D.; Hampton, C. Y.; McGready, R.; Sengaloundeth, S.; White, N. J.; Newton, P. N. Chem. Med. Chem. 2006, 1, 702-705. (47) 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, 807809. (48) Haefliger, O. P.; Jeckelmann, N. Rapid Commun. Mass Spectrom. 2007, 21, 1361-1366. (49) Taka´ts, Z.; Cotte-Rodriguez, I.; Talaty, N.; Chen, H.; Cooks, R. G. Chem. Commun. 2005, 1950-1952. (50) Shiea, J.; Huang, M.-A.; HSu, H.-J.; Lee, C.-Y.; Yuan, C.-H.; Beech, I.; Sunner, J. Rapid Commun. Mass Spectrom. 2005, 19, 3701-3704. (51) McEwen, C. N.; McKay, R. G.; Larsen, B. S. Anal. Chem. 2005, 77, 78267831. (52) Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2006, 17, 1712-1716.

Figure 1. Diagram of the DART ion source, adapted from ref 39, coupled to a Waters ZQ quadrupole mass spectrometer.

bioanalytical samples,40 chemical reactions,41 metabolic stability,42 fatty acid methyl esters,47 flavors and fragrances,48 and a wide variety of other samples (i.e., metabolites, counterfeit drugs, pharmaceutical tablets and formulations, explosives, herbicides, fungicides, triglycerides, biological fluids, amino acids, polymers, chemical warfare agents, peptides, oligosaccharides, and organometallics).43 Ionization of analytes using DART begins with an electric discharge in a stream of nitrogen or helium gas that produces a plasma of electrons, ions, and metastable species.39 When helium is used, the ionization mechanism involves the reaction of excited-state helium with water in the atmosphere to produce protonated water clusters followed by proton transfer to the analyte. Another potential ionization mechanism that has been reported involves Penning ionization in which a metastable species ionizes a neutral to produce a radical cation and an electron.39 In the negative ion mode, negative ions of analytes have been proposed to form from reactions with negatively charged oxygenwater cluster ions.39 In this report, we demonstrate the application of DART interfaced to a quadrupole mass spectrometer for the rapid analysis of small molecules in drug discovery. Commercially available drug compounds were analyzed by LC/UV/ESI-MS and DART/MS to characterize the similarities and differences between these two ionization techniques. In addition, several medicinal chemistry samples were analyzed by DART/MS to demonstrate the utility of the technique for fast reaction monitoring and characterization of the final products in drug discovery. EXPERIMENTAL SECTION The details of the geometry of the DART ion source have been described previously by Cody and co-workers.39 Figure 1 shows the configuration of the DART ion source (IonSense, Saugus, MA) coupled to a Waters ZQ quadrupole mass spectrometer. The DART ion source was interfaced to the mass spectrometer by mounting it to the vacuum block after removing the existing orthogonal electrospray source. The orifice of the DART ion Analytical Chemistry, Vol. 79, No. 13, July 1, 2007

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source was positioned so that the stream of helium exiting the source was orthogonal to the inlet orifice of the mass spectrometer. The distance between the outlet of the DART ion source and the inlet orifice was approximately 5 cm. This distance was fixed due to constraints between the geometry of the DART ion source and the vacuum block of the mass spectrometer. The sample holder consisted of a metal rod mounted onto the DART ion source with an alligator clip at one end for insertion of melting point tubes into the center of the helium gas stream at the outlet of the source. Variations in the position of the sample in the DART ion source can give rise to large variations in signal intensities by as much as an order of magnitude or more. The precision of sample placement was controlled by manually setting a bolt on the sample holder rod so that the sample was placed in the DART source at nearly the same position for each sample. Samples were analyzed by dipping the end of a melting point tube into a solution of an analyte in an organic solvent such that approximately 2 µL of sample was sampled. The concentrations of the analyte solutions were 0.1 mg/mL for standard drugs and 0.1-1 mg/mL for drug discovery compounds in methanol. The best sensitivity was obtained by positioning the end of the melting point tube in the center of the exit orifice of the DART ion source by using the sample holder. After volatilization and ionization of the analytes, positively or negatively charged ions were directed toward the inlet orifice of the mass spectrometer by the helium gas flow. The electrospray needle was operated at 3 kV with a cone voltage of 25 V and a cone gas flow of 50 L/min of nitrogen. The quadrupole was scanned from 100 to 1000 Da in continuum mode and run in either positive or negative mode with a scan time of 0.2 s and an interscan delay of 0.3 s. The photomultiplier was set at (500 V. Mass spectra of standard drugs, for comparison with ESI-MS, were obtained by selecting the maximum signal at the peak in the extracted ion signal. For reaction monitoring experiments, mass spectra were obtained by averaging spectra across the peak of interest in the entire total ion signal with baseline subtraction. The mass spectrometer was tuned and calibrated with sodium cesium iodide from 18 to 1000 Da. The operating conditions of the DART ion source in the positive and negative ion modes were optimized by varying the discharge needle voltage, perforated disk electrode 2 voltage (perforated disk electrode 1 is kept at ground), grid electrode voltage, helium flow, temperature, and sample position until the maximum signal intensity for the test compound warfarin (2 µL of 0.1 mg/mL in methanol) was reached without appreciable sample degradation. Warfarin was used as an initial test molecule for DART ionization because it ionizes strongly by positive/ negative electrospray ionization and is used to test the performance of our open access LC/UV/MS systems. The parameters for the positive and negative ion modes were a discharge needle voltage of 5235 V, perforated disk electrode 2 at (100 V, grid electrode at (200 V, helium gas flow at 3.5 L/min, and temperatures of 150-350 °C depending on the mp of the compound. An ion signal intensity of approximately 2 × 107 was observed for the molecular ion of warfarin ([M + H]+ ) 309) via DART at 150 °C which is an order of magnitude less than the typical signal intensity of the same warfarin standard using our LC/UV/ESIMS. In addition, the first pass screen of the commercial drugs, drug discovery compounds, and reaction mixtures revealed that 5066

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temperatures up to 350 °C were necessary to desorb all the molecules in the DART source and observe a sufficient molecular ion for higher mp compounds. A Waters Alliance 2790 interfaced to a Waters ZQ quadrupole mass spectrometer and Waters photodiode array detector was used for the automated LC/UV/ESI-MS experiments. Flow injection experiments (1 min injection to injection) were performed with 2 µL injections of standard drugs by the HPLC at 0.8 mL/ min of 50/50 methanol/water containing 0.1% formic acid each. The gradient HPLC method consisted of a 2 min linear gradient through a Waters Atlantis MS C18, 5 µm column beginning with 90% of 0.1% formic acid in water/10% of 0.1% formic acid in acetonitrile to 95% of 0.1% formic acid in acetonitrile/5% of 0.1% formic acid in water with a hold for 1.25 min and a recycle time of 0.25 min. A 5 µL injection was used with sample concentrations of 0.1 mg/mL for standard drugs and 0.1-1 mg/mL for drug discovery compounds in methanol. The HPLC flow rate was 0.8 mL/min split equally between the PDA and mass spectrometer to give approximately half of the 5 µL injection entering the electrospray source. The electrospray needle was operated at 3 kV with a cone voltage of 25 V and a cone gas flow of 50 L/min of nitrogen. The desolvation temperature was 350 °C with a desolvation flow of 500 L/min of nitrogen and a source temperature of 120 °C. The quadrupole was scanned from 100 to 1000 Da in continuum mode and run in either positive or negative ion mode for flow injection experiments and in positive/negative switching mode for gradient HPLC experiments with a scan time of 0.2 s and an interscan delay of 0.3 s. The photomultiplier was set at (500 V. Mass spectra were obtained by selecting the maximum signal at the peak apex in the total ion chromatogram for the flow injection experiments or by averaging spectra for 0.05 s on both sides of the peak apex of the total ion chromatogram peaks (gradient LC/UV/ESI-MS experiments) with baseline subtraction. Standard drug compounds (warfarin, terfenadine, propranolol, labetolol, piroxicam, pyrimethamine, sotalol, loperamide, lomefloxacin, β-estradiol, chlorthalidone, sulconazole, terbutaline, oxyphencyclimine, penacillamine, hydroxyzine, proglumide, 5-(4hydroxyphenyl)-5-phenylhydantoin, 4-cyano-4-phenylpiperidine, glucuronic acid, atropine, cimetidine, clenbuterol, and promethazine) were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. Drug discovery compounds were obtained from the medicinal chemistry department at Wyeth. RESULTS AND DISCUSSION Solvent Effects. Solvent effects on DART ionization at 250 °C were examined by dissolving 0.1 mg of warfarin (mp 162 °C) in 1 mL of common solvents used in discovery synthetic chemistry such as dimethylformamide (bp 153 °C), tetrahydrofuran (bp 65 °C), dimethylsulfoxide (bp 189 °C), acetonitrile (bp 81 °C), ethyl acetate (bp 76.5 °C), and methanol (bp 64.7 °C). Mass spectra were recorded for warfarin in each solvent by loading melting point tubes with 2 µL of sample and placing them in the DART source. A histogram plot (Figure 2), with error bars (n ) 3), of the average ion signal intensity of the molecular ion of warfarin, [M + H]+ ) 309, plotted against each solvent shows signal intensities (approximately (1.5-3) × 107) at least 3 times higher for the molecular ion of warfarin compared to that for warfarin in DMSO (approximately 0.5 × 107). The error bars in

Figure 2. Comparison of the intensities of the molecular ion of warfarin, [M + H]+ ) 309, dissolved in different solvents after DART ionization at 250 °C.

the plot demonstrate that the observed differences between the total ion signals for the molecular ion of warfarin in each solvent compared to DMSO are significant. In addition, the ion signal intensity of warfarin in most other solvents was nearly the same as that for the same amount of solid warfarin, which is to be expected since these solvents will most likely flash evaporate in the DART source at 250 °C. Furthermore, the reproducibility of the warfarin signals was sufficient for the qualitative reaction monitoring experiments reported below. Since DMSO is the highest boiling point solvent, it evaporated off the slowest and most likely competes with warfarin for protontransfer reactions with protonated water clusters in the gas phase for the first few seconds of the total ion signal until it completely evaporated away. This was evidenced by the presence of a protonated dimer of DMSO, [M + H]+ ) 157, in the first few mass spectra of warfarin obtained from the total ion signal. Since synthetic medicinal chemistry is typically not sample limited for mass spectral analysis (∼0.1-2 mg/mL), samples were sufficiently concentrated so that the total ion signal for analyte ions lasted as long as 30 s while that for solvents like DMSO lasted for only 6 s. Under these conditions, the data show that solvent choice is not a significant factor for typical medicinal chemistry compounds that may be sampled from NMR samples in deuterated DMSO or a variety of other solvents for fast MW screening of synthetic products. Temperature Effects. Temperatures up to 350 °C were not a significant factor with regard to sample degradation for the DART ionization of solutions of standard drugs (0.1 mg/mL in methanol, Figure S-1, Supporting Information) and drug discovery compounds (0.1-1 mg/mL in methanol) used in this study. The greatest extent of sample degradation was observed for glucuronic acid and estradiol at 350 °C where the [M + H - H2O]+ ion was approximately the same intensity as the parent ion after exposure of the sample in the DART ion source for a few seconds. Proglumide was the only other compound that showed appreciable degradation with a few fragment ions of equal intensity as the molecular ion at 350 °C. Two examples of the typical effects of temperature of the DART ion source gas on representative drug molecules are shown in Figure 3. The DART mass spectrum of lomefloxacin at 150 °C

(Figure 3a) and 350 °C (Figure 3b) are essentially identical with a molecular ion of [M + H]+ ) 352, while that for sulconazole, [M + H]+ ) 399, at 150 °C (Figure 3c) and 350 °C (Figure 3d) shows a small amount of fragment ions at m/z 183 and 329 which was typical for many of the compounds studied with some acyclic functionalities. However, for almost all of the compounds in this study, the molecular ion was the predominate ion and the intensities of most fragment ions were approximately 10-15% or less of the intensity of the molecular ion in the first few seconds of the total ion signal. Oxidation in DART. Cody and co-workers have briefly reported on the presence of oxidation products in DART.39 In this study, small amounts of ions resulting from oxidation of the corresponding parent compounds were observed in nearly all of the standard drugs and some drug discovery compounds. The intensities of these ions were no more than 10% of the intensities of the molecular ions in the first few seconds of the total ion signals for most compounds. An example of a typical oxidation via DART at 150 °C is shown for 4-cyano-4-phenylpiperidine (0.1 mg/mL in methanol) in Figure 4 revealing the presence of a proposed ion of [M + O + H]+ ) 203 and fragment ion of [M + O + H H2O]+ ) 185. DART and ESI of Standard Drugs. Several commercially available drugs (2 µL of 0.1 mg/mL in methanol) were analyzed by DART/MS and flow injection LC/UV/ESI-MS (Table 1) to compare the differences between the mass spectral data obtained with both ionization techniques. The DART ion source was kept at 150 °C for most compounds except for loperamide, chlorthalidone, terbutaline, 5-(4-hydroxyphenyl)-5-phenylhydantoin, atropine, and cimetidine which where ionized at 350 °C to obtain a significant signal. Nearly all of the compounds ionized in either the positive and negative ion modes by DART and ESI except for β-estradiol and terbutaline which did not give signals in the negative ion mode via DART. Table 1 lists the maximum molecular ion signal intensities observed for DART and ESI normalized to ESI signals. In the positive ion mode, the signal was greater for ESI than for DART ionization with the signal intensities of nearly all molecular ions produced by DART no more than 1 order of magnitude less than that for ESI. This difference could be due to several factors including the difference in distance between the point where ions are created in both ionization techniques and the inlet orifice of the mass spectrometer or the fact that more protonated dimers and oxidation products are observed for DART compared to ESI. In the experimental configurations used, the tip of the ESI needle was 1 cm away from the inlet orifice while the outlet of the DART ion source was 5 cm away from the inlet orifice which could not be varied because of the fixed design of this particular DART/ MS configuration. In the negative ion mode, the signal for molecular ions observed by ESI was up to 2 orders of magnitude greater than that for molecular ions detected by DART. The absence of signal for β-estradiol and terbutaline in the negative ion mode may be due to neutralization reactions that occur in the atmosphere between the point of ionization and transfer into the vacuum region of the mass spectrometer. In order to examine this effect, stearic acid was ionized by DART (at IonSense) in the negative ion mode while increasing the distance between the point of ionization and Analytical Chemistry, Vol. 79, No. 13, July 1, 2007

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Figure 3. Mass spectra of lomefloxacin, [M + H]+ ) 352, at (a) 150 °C and (b) 350 °C and sulconazole, [M + H]+ ) 397/399/401 (3 Cl), at (c) 150 °C and (d) 350 °C via DART/MS. A protonated dimer, [2M + H]+ ) 793/795/797 (3 Cl), and fragment ions of m/z 183/185 (1 Cl) and 329/331/333 (3 Cl) for sulconazole are present in the spectra in parts c and d.

Figure 4. DART mass spectrum (150 °C) of 4-cyano-4-phenylpiperidine, [M + H]+ ) 187, showing the proposed oxidation product, [M + O + H]+ ) 203, of the parent compound and a fragment ion of [M + O + H - H2O]+ ) 185 from the loss of water from m/z 203.

the inlet orifice of the mass spectrometer. Increasing the distance resulted in a reduction in the relative abundance of the [M - H]molecular ion by an order of magnitude per each 2 cm of distance, and at 6 cm, no [M - H]- was observed. As a result, later source interface designs have been changed to reduce the potential for interaction of gas-phase ions with components from the atmosphere. These designs were not evaluated during this study. Representative DART and ESI mass spectra for warfarin, glucuronic acid, cimetidine, and lomefloxacin are shown in Figure

S-2 (Supporting Information). The DART and ESI mass spectra for these compounds are essentially the same except for warfarin and glucuronic acid. Warfarin shows a proposed ion of [M + O - H]- ) 323 resulting from oxidation of warfarin via DART and a proposed fragment of this ion, [M + O - H2O H]- ) 305, in Figure S-2a that is not present in the ESI mass spectrum in Figure S-2e. The fragmentation seen in the DART mass spectrum of glucuronic acid (Figure S-2b) is likely due to a combination of thermal degradation at 150 °C and collisions with metastable helium gas. Another common difference between both ionization techniques was that more protonated dimers were produced by DART, especially for solutions of standard compounds with concentrations of 1 mg/mL or greater. Analysis of compounds by DART/MS is also advantageous to rapidly obtain molecular weight confirmations rather than running flow injection analysis by LC/UV/ESI-MS. To demonstrate the potential for fast analyses by DART/MS to analyze final products in medicinal chemistry, solutions of standard drugs (2 µL of 0.1 mg/mL in methanol) were placed on the tip of melting point tubes and introduced one at a time in the DART ion source. The

Table 1. Comparison of Molecular Ions of Commercial Drugs Observed in a Quadrupole Mass Spectrometer after DART and Electrospray Ionization DART

warfarin terfenadine propranolol labetalol piroxicam pyrimethamine sotalol loperamide lomefloxacin β-estradiol chlorthalidone sulconazole terbutaline oxyphencyclimine penacillamine hydroxyzine proglumide 5-(4-hydroxyphenyl)5-phenylhydantoin 4-cyano-4-phenylpiperidine glucuronic acid atropine cimetidine clenbuterol promethazine a

ESI

[M + H]+

intensitya

[M - H]-

intensitya

[M + H]+

intensity

[M - H]-

intensity

309 472 260 329 332 249 273 477 352 273 339 397 226 345 150 375 335 269

0.2 0.4 0.3 0.2 0.5 0.4 0.1 0.4 0.07 0.1 0.9 0.1 0.6 0.9 0.6 0.1 0.1 0.9

307 ndb ndb 327 330 ndb 271 ndb ndb ndb 337 ndb ndb ndb 148 ndb 333 267

0.7

309 472 260 329 332 249 273 477 352 273 339 397 226 345 150 375 335 269

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

307 ndb ndb 327 330 ndb 271 ndb ndb 271 337 ndb 224 ndb 148 ndb 333 267

1.0

187 ndb 290 253 277 285

0.3

ndb 193 ndb 251 ndb ndb

187 ndb 290 253 277 285

1.0

ndb 193 ndb 251 ndb ndb

0.1 0.7 0.4 0.4

0.5 0.4 0.1

0.2

0.01 0.03 0.05 0.7 0.1

1.0 1.0 1.0 1.0

Maximum ion signal intensity normalized to the maximum ion signal obtained by ESI. b nd, not detected.

5068 Analytical Chemistry, Vol. 79, No. 13, July 1, 2007

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Figure 5. Extracted and total ion signals in the positive ion mode generated via DART/MS at 350 °C: (a) extracted ion signal for loperamide, [M + H]+ ) 477, (b) phenylhydantoin, [M + H]+ ) 269, and (c) terfenadine, [M + H]+ ) 472 and (d) total ion signal (in minutes) resulting from the sequential analysis of 24 standard drug compounds via DART/MS listed in Table 1.

total ion signal for the analysis of the 24 standard drug compounds analyzed sequentially (Table 1) at 350 °C by DART/MS is shown in Figure 5 along with extracted ion signals for loperamide, phenylhydantoin, and terfenadine. The mass spectra for these compounds (Figure S-3, Supporting Information) were identical to that obtained by electrospray ionization. The 24 compounds were analyzed manually within 13 min as opposed to 24 min (1 min injection-injection) using the flow injection LC/UV/ESI-MS method. The DART analysis could possibly be decreased further by introduction of samples into the source with robotics. DART and ESI of Drug Discovery Compounds. Final drug discovery compounds ranging from approximately 0.1-1 mg/mL

(2 µL) in methanol with 90% or greater purity were analyzed by DART/MS and LC/UV/ESI-MS in both the positive and negative ion modes. DART ionized 22 out of 27 of these compounds at 350 °C while all of the compounds ionized by electrospray ionization. The compounds that did not ionize by DART included four N-substituted indoles and one ester that had weak ESI signals. Overall, the mass spectra for nearly all of the drug discovery compounds analyzed by DART/MS were very similar to that for the mass spectra in the electrospray total ion chromatogram. Representative DART and ESI mass spectra for some of the discovery compounds are shown in Figure S-4 (Supporting Information). The main differences between the ESI (Figure S-4a) and DART (Figure S-4d) mass spectra for one of the discovery compounds are the absence of an ammonia fragment ion, [M + H - NH3]+ ) 217, and presence of a protonated dimer, [2M + H]+ ) 467, via DART/MS. The spectrum for the compound shown in Figure S-4e generated via DART/MS also shows a protonated dimer, [2M + H]+ ) 959, that is not present in the corresponding ESI mass spectrum shown in Figure S-4b. Reaction Monitoring. DART/MS was also examined for its ability to be used for reaction monitoring compared to using LC/ UV/ESI-MS in drug discovery. The synthetic transformations that were monitored include the N-methylation of an indole and a debenzylation reaction by analyzing 2 µL of a diluted reaction mixture in 1 mL of MeOH. The mass spectra obtained for the reactant indole, [M + H]+ ) 254, and product N-methyl indole, [M + H]+ ) 268, were monitored up to the final reaction time of 16 h via DART/MS at 350 °C (Figure 6). The DART mass spectra show that the trends in the ratios of reactant and product ion signal intensities scale close enough to those of the total diode array signals to be used for qualitative reaction monitoring. In addition, the trends in

Figure 6. Representative total diode array chromatograms for the reaction monitoring of an indole converting to an N-methylindole via LC/ UV/ESI-MS: UV spectra at (a) 0 min, (b) 20 min, (c) 40 min, (d) 80 min, (e) 140 min, and (f) 16 h; mass spectral data for the indole, [M + H]+ ) 254, and N-methylindole, [M + H]+ ) 268, generated via DART/MS (350 °C) at (g) 0 min, (h) 20 min, (i) 40 min, (j) 80 min, (k) 140 min, and (l) 16 h.

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reactant and product ion signal intensities and magnitude of the ESI signal in the ESI-MS mass spectra (not shown) closely matched those obtained by DART/MS. These results demonstrate that mass spectrometry may be sufficient for qualitative monitoring of simple reactions with structurally similar reactants and products instead of reaction monitoring by UV. Another simple reaction monitoring experiment involved a debenzylation of a heterocyclic compound analyzed via DART/ MS at 350 °C. The mass spectral data for the reactant, [M + H]+ ) 274/276 (1 Cl), and product, [M + H]+ ) 184/186 (1 Cl), are shown in Figure S-5 (Supporting Information) after the completion of the reaction in 1 h. The corresponding LC/UV/ESI-MS experiments (not shown) revealed one UV peak in each of the separations of the product and starting material with similar mass spectra for each component as those obtained by DART/MS. These two experiments demonstrate that when the chemistry of a reaction can be easily predicted, mass spectrometry may be used to monitor reactions without the need for chromatography. CONCLUSIONS DART has been demonstrated to be a complementary tool to electrospray ionization in drug discovery. Mass spectra of standard drug compounds generated by DART/MS were nearly identical to those obtained by LC/UV/ESI-MS. However, ion signal intensities for molecular ions in the positive ion and negative ion

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modes were approximately 1-2 orders of magnitude lower for DART compared to ESI for the particular instrument configuration reported here. This effect has been partially eliminated by changes in the DART ion source design (not reported here) to minimize the effect of potential neutralization reactions in the metastable helium gas stream, especially in the negative ion mode. DART/ MS was also useful for the rapid molecular weight confirmation of final products in drug discovery and in cases where HPLC may not be necessary prior to mass spectrometry to determine the MW of a purified fraction. As a result, DART/MS may also be advantageous for the rapid molecular weight screening of compound libraries and for obtaining rapid accurate mass measurements. In addition, simple organic transformations with few reaction by-products could be adequately monitored by DART/ MS as opposed to using LC/UV/ESI-MS which is the traditional means of reaction monitoring in drug discovery. SUPPORTING INFORMATION AVAILABLE Figures S-1 through S-5 with additional information noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for April 30, 2007. AC070443M

review

March

5,

2007.

Accepted