High-Throughput Mass Spectrometer Using Atmospheric Pressure

Dec 3, 2004 - Latest Advancements and Trends. Petra S. Dittrich, Kaoru Tachikawa, and ... Ion Traps for Miniature, Multiplexed, and Soft-Landing Techn...
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Anal. Chem. 2005, 77, 459-470

High-Throughput Mass Spectrometer Using Atmospheric Pressure Ionization and a Cylindrical Ion Trap Array Alexander S. Misharin, Brian C. Laughlin, Andrey Vilkov,† Zolta´n Taka´ts, Zheng Ouyang, and R. Graham Cooks*

Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-2084

The analytical performance of an atmospheric pressure sampling, multiple-channel, high-throughput mass spectrometer was investigated using samples of a variety of types. The instrument, based on an array of cylindrical ion traps, was built with four independent channels and here is operated using two fully multiplexed channels (sources, ion optics, ion traps, detectors) capable of analyzing different samples simultaneously. Both channels of the instrument were incorporated within the same vacuum system and operated using a common set of control electronics. A multichannel electrospray ionization source was assembled and used to introduce samples including solutions of organic compounds, peptides, and proteins simultaneously into the instrument in a highthroughput fashion. Cross-talk between the channels of the instrument occurred in the detection system and could be minimized to 1-2% using shielding between detector channels. In this initial implementation of the instrumentation, an upper mass/charge limit of ∼1300 Th was observed (+13 charge state of myoglobin) and unit mass/ charge resolution was achieved to ∼800 Th. The rather limited dynamic range (2-3 orders of magnitude for lowconcentration analytes) is due to cross-talk contributions from more concentrated species introduced into a different channel. Analysis of mixtures of alkylamines and peptides is demonstrated, but analysis of mixtures with a wide spread in mass/charge ratios was not possible due to mass discrimination in the ion optics. Further refinement of the vacuum system and ion optics will allow the addition of more channels of parallel mass analysis and facilitate applications in fields such as proteomics and metabolomics. High-throughput analysis is important in many areas of research, including combinatorial library screening,1 proteomics,2 and metabolomics.3 In these research areas, many of the samples * To whom correspondence should be addressed. Telephone: (765) 494-5262. Fax: (765) 494-9421. E-mail: [email protected]. † Current address: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory. P.O. Box 999, Richland, WA 99352. (1) Enjalbal, C.; Martinez, J.; Aubagnac, J. Mass Spectrom. Rev. 2000, 19, 139161. (2) Aebersold, R.; Goodlett, D. R. Chem. Rev. 2001, 101, 269-296. (3) Yanagida, M. J. Chromatogr., B 2002, 771, 89-106. 10.1021/ac048983w CCC: $30.25 Published on Web 12/03/2004

© 2005 American Chemical Society

to be analyzed are similar in composition so traditional analysis by mass spectrometry, viz. introduction of one sample into one mass spectrometer at one time, involves repeating the same operation over and over again, often with detectable signals for analytes of interest being observed only occasionally. In such situations, it is desirable to multiplex as much of the experiment as possible in order to allow the analysis of many samples simultaneously. These factors have led a number of groups to develop multiplexed mass spectrometry experiments of various types. In one type of experiment, multiple electrospray ionization (ESI) emitters on either the macroscale4,5 or microscale6,7 are interfaced to a single mass spectrometer. Rapid switching between the channels can be controlled through independent access to each channel in the case of the microscale devices or through the control of ion transmission using ion optics in the case of the macroscale devices. In a second type of experiment, parallel chromatographic separations are employed and interfaced to a single mass spectrometer through the use of a sequential ion sampling interface.8-12 These experiments show the ability to use a single mass spectrometer to rapidly change between individual sample streams and capture events occurring on a chromatographic time scale. However, these valuable methods do not address the simultaneous mass analysis of multiple samples in a truly multiplexed mass spectrometry experiment, which is the objective of the instrumental developments described here. The principles of the approaches described above have been used to provide commercial solutions to dealing with large numbers of samples in a mass spectrometer. The use of a single (4) Tang, K. Q.; Tolmachev, A. V.; Nikolaev, E.; Zhang, R.; Belov, M. E.; Udseth, H. R.; Smith, R. D. Anal. Chem. 2002, 74, 5431-5437. (5) Schneider, B. B.; Douglas, D. J.; Chen, D. D. Y. Rapid Commun. Mass Spectrom. 2002, 16, 1982-1990. (6) Xue, Q.; Foret, F.; Dunayevskiy, Y. M.; Zavracky, P. M.; McGruer, N. E.; Karger, B. L. Anal. Chem. 1997, 69, 426-430. (7) Lui, H.; Felten, C.; Xue, Q.; Zhang, B.; Jedrzejewski, P.; Karger, B. L.; Foret, F. Anal. Chem. 2000, 72, 3303-3310. (8) Feng, B.; McQueney, M. S.; Mezzasalma, T. M.; Slemmon, J. R. Anal. Chem. 2001, 73, 5691-5697. (9) Lee, H.; Griffin, T. J.; Gygi, S. P.; Rist, B.; Aebersold, R. Anal. Chem. 2002, 74, 4353-4360. (10) Xu, R.; Wang, T.; Isbell, J.; Cai, Z.; Sykes, C.; Brailsford, A.; Kassel, D. B. Anal. Chem. 2002, 74, 3055-3062. (11) Xu, R.; Nemes, C.; Jenkins, K. M.; Rourick, R. A.; Kassel, D. B.; Liu, C. Z. C. J. Am. Soc. Mass Spectrom. 2002, 13, 155-165. (12) Van Pelt, C. K.; Corso, T. N.; Schultz, G. A.; Lowes, S.; Henion, J. Anal. Chem. 2001, 71, 582-588.

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mass analysis channel with provisions for time sharing of the mass spectrometer between several LC columns13,14 is commercially available in the form of the MUX system (Micromass, Waters, Milford, MA) in which the effluents from up to eight parallel LC columns can be analyzed using a single mass spectrometer operated using multiple electrospray ionization sources. The NanoMate system (Advion BioSciences, Ithaca, NY) provides the second commercial approach to high-throughput analysis. In this system, rapid robotic sample changing at the inlet of a mass spectrometer is coupled to an array of micromachined silicon nanospray tips.15,16 The micromachined nanospray ionization source has been developed to provide quick sample changes and reduced dead time between analyses while providing a new tip to each sample analyzed. Although both systems provide an increase in throughput over the traditional one sample per mass spectrometer approach, the increase in sample throughput is limited, and there remains a need for multiplexed analysis systems that extend scalability beyond the limitations of currently available technology. While the experiments described here clearly do not achieve this objective fully, they do represent steps along a path that may lead to this goal. The aim of this approach is to build a mass spectrometer that is multiplexed in a manner that allows each channel to be operated in a truly parallel and independent fashion. This means multiplexing the ionization source, ion-transfer optics, mass analyzer, and detector in such a way that each channel is a complete mass spectrometer. Through the use of a common vacuum system, shared control electronics, and a common data acquisition system, multiplexing of the largest and most expensive portions on the instrument is avoided and a relatively small instrument can be developed provided that one chooses a suitable mass analyzer. Ion trap mass spectrometers have the necessary performance characteristics to cover many application areas in mass spectrometry as well as the capability to be miniaturized. The cylindrical ion trap17-22 (CIT) was selected for this purpose. The ease with which the simplified geometry of the CIT can be machined when compared to the hyperbolic cross-section electrodes of the Paul ion trap provides an easy path to miniaturizing and multiplexing the device. Development and optimization of the CIT for use as a mass spectrometer began with experiments on the full-sized device18 (inscribed radius 1 cm) operated in massselective instability mode23 and progressed to miniaturized de(13) de Biasi, V.; Haskins, N.; Organ, A.; Bateman, R.; Giles, K.; Jarvis, S. Rapid Commun. Mass Spectrom. 1999, 13, 1165-1168. (14) Yang, L.; Mann, T. D.; Little, D.; Wu, N.; Clement, R. P.; Rudewicz, P. J. Anal. Chem. 2001, 73, 1740-1747. (15) Schultz, G. A.; Corso, T. N.; Prosser, S. J.; Zhang, S. Anal. Chem. 2000, 72, 4058-4063. (16) Van Pelt, C. K.; Zhang, S.; Henion, J. D. J. Biomol. Technol. 2002, 13, 7284. (17) Langmuir, D. B.; Langmuir, R. V.; Shelton, H.; Wuerker, R. F. Containment Device. U.S. Patent 3,065,640, 1962. (18) Wells, J. M.; Badman, E. R.; Cooks, R. G. Anal. Chem. 1998, 70, 438-444. (19) Badman, E. R.; Johnson, R. C.; Plass, W. R.; Cooks, R. G. Anal. Chem. 1998, 70, 4896-4901. (20) Kornienko, O.; Reilly, P. T. A.; Whitten, W. B.; Ramsey, J. M. Rapid Commun. Mass Spectrom. 1999, 13, 50-53. (21) Moxom, J.; Reilly, P. T. A.; Whitten, W. B.; Ramsey, J. M. Rapid Commun. Mass Spectrom. 2002, 16, 755-760. (22) Danell, R. M.; Ray, K. L.; Glish, G. L. Abstr, Pap, Am, Chem, Soc. 2001; 49-Anyl part 41. (23) Stafford, G. C.; Kelley, P. E.; Syka, J. E. P.; Reynolds, W. E.; Todd, J. F. J. Int. J. Mass Spectrom. Ion Processes 1984, 60, 85-98.

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vices.19 Before this time, the CIT had been used exclusively as an ion storage device24-27 or as a mass spectrometer operated in the archaic mass-selective stability scan mode.28-30 The full-sized CIT exhibited performance characteristics similar to that of a standard (Paul) quadrupole ion trap in terms of resolution, mass range, mass accuracy, and mass linearity, as well as tandem mass spectrometric capabilities. The miniaturized CIT has also shown performance comparable to full-sized devices, both in our studies and in those performed by others.19-21,31 Tandem mass spectrometric capabilities of the reduced size device were demonstrated by Lynn,32 and miniaturization of the CIT has led to the development of a field-portable mass spectrometer33 in which the first tandem mass spectrometry experiments in a miniature mass spectrometer were performed.34 Multiplexing of the CIT analyzer has been demonstrated for two types of arrayssthose in which the individual mass analyzers are assembled in parallel35 and those in which this is done in series.36 It has also been shown that it is also possible to construct arrays of mass analyzers of different dimensions that are operated using identical voltages and in which mass selection is a consequence of the varying dimensions of the different ion traps within the same parallel array.37 The parallel arrays suited to highthroughput experiments are easily constructed through machining multiple ion traps, which are spatially distributed within the same electrode structure. It should be noted that miniature linear quadrupole mass analyzers have also been made into arrays,38-40 not for the purpose of high-throughput, parallel sample analysis but for increased signal in conventional single-sample analysis. The present experiment grew out of earlier related experiments in this laboratory in which an array of cylindrical ion traps was fitted with an electron ionization (EI) or chemical ionization (CI) source.41,42 The external EI/CI source used on this earlier multiplexed instrument limited its operation to volatile or semi(24) Mikami, N.; Sato, S.; Ishigaki, M. Chem. Phys. Lett. 1991, 180, 431-435. (25) Mikami, N.; Sato, S.; Ishigaki, M. Chem. Phys. Lett. 1993, 202, 431-436. (26) Ji, Q.; Davenport, M. R.; Enke, C. G.; Holland, J. F. J. Am. Soc. Mass Spectrom. 1996, 7, 1009-1017. (27) Grebner, T. L.; Neusser, H. J. Int. J. Mass Spectrom. Ion Processes 1994, 137, L1-L6. (28) Benilan, M. N.; Audoin, C. Int. J. Mass Spectrom. Ion Phys. 1973, 11, 421432. (29) Bonner, R. F.; Fulford, J. E.; March, R. E.; Hamilton, G. F. Int. J. Mass Spectrom. Ion Phys. 1977, 24, 255-269. (30) Mather, R. E.; Waldren, R. M.; Todd, J. F. J.; March, R. E. Int. J. Mass Spectrom. Ion Phys. 1980, 33, 201-230. (31) Kornienko, O.; Reilly, P. T. A.; Whitten, W. B.; Ramsey, J. M. Rev. Sci. Instrum. 1999, 70, 3907-3909. (32) Meaker, T. F.; Lynn, B. C. In Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, 2001; TOC pm. (33) Patterson, G. E.; Guymon, A. J.; Riter, L. S.; Everly, M.; Griep-Raming, J.; Laughlin, B. C.; Ouyang, Z.; Cooks, R. G. Anal. Chem. 2002, 74, 61456153. (34) Riter, L. S.; Peng, Y.; Noll, R. J.; Patterson, G. E.; Aggerholm, T.; Cooks, R. G. Anal. Chem. 2002, 74, 6154-6162. (35) Badman, E. R.; Cooks, R. G. Anal. Chem. 2000, 72, 3291-3297. (36) Ouyang, Z.; Badman, E. R.; Cooks, R. G. Rapid Commun. Mass Spectrom. 1999, 13, 2444-2449. (37) Badman, E. R.; Cooks, R. G. Anal. Chem. 2000, 72, 5079-5086. (38) Boumsellek, S.; Ferran, R. J. J. Am. Soc. Mass Spectrom. 2001, 12, 633640. (39) Ferran, R. J.; Boumsellek, S. J. Vac. Sci. Technol., A 1996, 14, 1258-1265. (40) Orient, O. J.; Chutjian, A.; Garkanian, V. Rev. Sci. Instrum. 1997, 68, 13931397. (41) Tabert, A. M.; Griep-Raming, J.; Guymon, A. J.; Cooks, R. G. Anal. Chem. 2003, 75, 5656-5664. (42) Tabert, A. M.; Misharin, A. S.; Cooks, R. G. Analyst 2004, 129, 323-330.

volatile compounds. The need to perform high-throughput experiments on nonvolatile compounds is commonly met using one of the many atmospheric pressure ionization techniques, from which electrospray ionization was chosen for the instrument described here. The significant differences in vacuum pumping and ion optical requirements for ESI compared to EI/CI meant that an entirely new high-throughput instrument was built in order to implement the ESI experiment. One of the major concerns for high-throughput mass spectrometers in which ions travel in generally parallel paths between the ionization source and mass analyzer is cross-talk between adjacent channels of mass analysis. That is, the presence of signals due to compounds that are introduced into one channel appearing in another channel. There are two possible sources of cross-talk; in the first, neutral molecules pass from one channel of mass analysis (channel A) to another channel (e.g., channel B) resulting in ion/molecule reactions involving the neutrals from channel A and ions in channel B. In the second type, ions cross channels either before or after mass analysis. These processes can occur in different regions of a multiplexed mass spectrometersfrom the ion sources to the detectors or anywhere in between. The first type of cross-talk will be referred to as a “neutral cross-talk” and the latter type as “ionic cross-talk”. Ions crossing from one channel of mass analysis into another can either simply be detected in that channel or they can undergo reactions with neutrals in the new channel. Ionic cross-talk can be caused by poor ion optics and can even occur in the detection system when ions ejected from one CIT reach the detector of a different channel. During experiments performed using the multichannel mass spectrometer with an EI/CI ionization source,41 the major source of cross-talk between channels was shown to be neutral cross-talk associated with ion/molecule reactions in the ion trap analyzer array. The objectives of the present research were to build and characterize a four-channel CIT array instrument equipped with electrospray ionization sources to allow analysis of different types of compounds including drug molecules, peptides, and large biomolecules (e.g., proteins). In this paper, the instrument is operated using only two of the four channels as a safeguard against damage to the turbomolecular pumps used on the vacuum system. Throughout this paper, mass/charge ratios for ions will be expressed in Thomsons, where 1 Thomson (Th) ) 1 Da/atomic charge.43 EXPERIMENTAL SECTION The high-throughput CIT array mass spectrometer is equipped with ESI sources and consists of four complete parallel channels of mass analysis, two of which were operated during the experiments described here. All components of the instrument, with the exception of the ion sources, are housed in a common vacuum manifold and operated using common control electronics. A schematic representation of the major components and their interconnections is shown in Figure 1. The instrument consists of a two-stage, differentially pumped vacuum system through which ions created in the ESI sources are sampled and transferred into the CIT array. The vacuum interface is based on stainless steel sampling capillaries that serve (43) Cooks, R. G.; Rockwood, A. L. Rapid Commun. Mass Spectrom. 1991, 5, 83.

Figure 1. Schematic of the major components of the highthroughput mass spectrometer depicting their interconnections.

Figure 2. Schematic of four-channel high-throughput cylindrical ion trap mass spectrometer equipped with electrospray ionization sources. The vacuum manifold is not shown.

to sample ions from the electrospray plume while limiting the gas flow into the vacuum system. The capillaries, which measure 254 µm inner diameter by 80 mm in length, are held in position by a copper block, shown in Figure 2, in a coaxial arrangement with the apertures of an array of skimmers serving to limit gas transfer into the second vacuum stage. The copper block is used both to position the capillaries relative to each other and to provide heat to cause desolvation of clusters, although the block was not heated in these experiments. During initial experiments to characterize the instrument using a single channel, an oil-sealed rotary vane pump (model EM230, BOC Edwards, Wilmington, MA), providing a nominal pumping speed of 30 m3/h, was used to evacuate the first vacuum chamber. In subsequent multichannel experiments, a Roots vacuum pump (model RUVAC WSU1001, Leybold Vacuum USA, Export, PA) backed by a single-stage oil-sealed rotary vane pump (model SV 630, Leybold Vacuum USA) was used for this purpose. The Roots pumping system is capable of providing up to ∼1100 m3/h pumping speed at 1 Torr but is reduced to ∼500 m3/h at the instrument’s operating pressure. The design of the first stage of the instrument, implemented during the early stages of instrument development, does not allow the use of the full pumping speed of the Roots pumping system due to conductance limitations arising from the size of the pumping port on the chamber. Typical indicated pressures in the first stage of the Analytical Chemistry, Vol. 77, No. 2, January 15, 2005

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instrument were ∼330 mTorr as measured using a Pirani gauge (model TR 211, Leybold Vacuum USA) when the Edwards rotary vane pump was used to evacuate the first stage and only one inlet capillary was opened to the atmosphere. With the Roots pumping system, this pressure was ∼90 mTorr, as measured using the same gauge. The second vacuum stage is evacuated using two 500 L/s turbomolecular pumps (model TPH 510, Pfeiffer Vacuum, Nashua, NH), which are backed using oil-sealed rotary vane pumps. A stainless steel plate separating the first and second vacuum stages was fitted with skimmers to sample ions transmitted through the capillary and to limit gas flow into the second vacuum stage. The diameter of each skimmer in the array is 0.50 mm. During operation of the instrument with the Roots pumping system and two operating channels of ionization and mass analysis, the indicated pressure in the second vacuum stage was ∼5 × 10-4 Torr, as monitored by a Bayard-Alpert type ion gauge and controller (series 307, Granville-Phillips, Longmont, CO). The pressure in the second stage was dependent on the separation and the coaxial alignment of the capillaries relative to the skimmers. The distance between inlet capillaries and skimmers was adjusted in the course of the experiments in order to maximize the signal intensity in each channel while keeping the pressure in the second vacuum stage in a range that is safe for the turbomolecular pumps. A separation of 1.5-2.0 mm with the capillary coaxial to the skimmers was found to provide the best tradeoff between ion transmission and the pressure of the second stage. Placing the capillaries off-axis with respect to the skimmer has been shown to reduce the gas transmitted to the second stage due to the free jet expansion occurring at the end of the capillary when used with a tube lens44 to provide focusing of the ion beam; however, this arrangement was not used in these experiments due to the difficulty of precisely controlling the off-axis displacement of all capillaries simultaneously relative to the skimmers. Since the copper block holding the inlet capillaries is free to rotate around the central axis of the array, two polyetheretherketone (PEEK, Victrex USA Inc., Greenville, SC) locating pins were placed between the skimmer plate and the copper block to maintain a coaxial alignment. A dual-channel electrospray ion source was designed for use during these experiments. Each electrospray channel consists of a fused-silica capillary (Agilent Technologies, Palo Alto, CA) with an inner diameter of 0.100 mm by ∼50 cm long, fixed inside a 1/ -in. Swagelok tee. The tee is held in position by an acetal 16 (Delrin, DuPont Co., Wilmington, DE) holder, placed on an XYtranslational stage that allows one to adjust the position of the silica capillary relative to the stainless steel inlet capillary in the horizontal (XY) plane. It is also possible to adjust the vertical position of the holder on the XY stage, thus regulating the position of the silica capillary in the Z direction. Both electrospray ionization sources were placed on a single moving stage, allowing both channels to be positioned with respect to the inlet simultaneously. During these experiments, the electrospray sources were operated without nebulizing gas assistance. Solutions containing the analytes used in these experiments were delivered through (44) Mylchreest, I. C.; Hail, M. E.; Herron, J. R. Finnigan Corp., U.S. Patent 5,157,260, 1992.

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Figure 3. Photograph of four-channel CIT array of mass analyzers. The inner area of array has been removed to reduce total capacitance of the device.

the fused-silica capillaries using a syringe pump (model PhD 2000, Harvard Apparatus Inc., Holliston, MA) at flow rates of ∼3.0 µL/ min. Typically, a bias of 4.5 kV was applied directly to the needles of the syringes using a high-voltage power supply (model 205B10R, Bertan High Voltage, Valhalla, NY). The ion optics of the second stage consists of an electrostatic einzel lens (see Figure 2). The lens focuses the ions transmitted through the skimmer into the entrance end cap aperture of the CIT. The lens is of a thick-thin-thick design in which the first and last lens elements are 13.4 mm thick and the second element is 1.22 mm thick. All three elements are constructed of stainless steel and separated from each other using 2.0-mm-thick poly(tetrafluoroethylene) (Teflon, DuPont Co., Wilmington, DE) spacers. Each element of the lens has four 10-mm-diameter holes for ion transfer, one corresponding to each channel of the array. The entire assembly, including the CIT array, is mounted on four PEEK rods, which provide a coaxial alignment to all of the elements of the lens, the CIT array, and the skimmers. A ninechannel, bipolar ((500 VDC) power supply (model TD-9500HV, Spectrum Solutions, Inc., Russellton, PA) was used to supply voltages to the skimmer and the elements of the einzel lens. The biases used in these experiments were 0, 0, and -8.6 V on lens elements 1, 2, and 3, respectively, with 0 V on the skimmers. To transfer higher mass ions, the bias on the skimmer, and that on lens elements 2 and 3 was changed to -1.0, 0.5, and -1.5 V, respectively. The lens power supply used has a specified stability of 0.1% over 8 h and over the course of this study has proven to be stable at the low potentials used. Gating of the ion beam is possible through the modulation of the bias placed on the second element of the einzel lens, but was not used during these experiments. The CIT array used in these experiments is constructed of stainless steel and consists of four identical CITs, positioned symmetrically on a 28-mm-diameter circle as pictured in Figure 3. Each CIT has an inscribed radius (ro) of 5.00 mm and a centerto-end cap distance (zo) of 4.93 mm. The apertures in the end caps for ion entrance and egress are 2.0 mm in diameter. The end caps are spaced from the ring electrode using 1.04-mm Delrin spacers with the entire trap assembly being held together using PTFE screws. The geometry chosen for the CITs in the array was the result of extensive studies using the multiple particle ion

trap simulation program, ITSIM 5.0.45 The CIT array has been machined to remove most of the unnecessary metal from both the ring and the end cap electrodes, in an effort to reduce the total trap capacitance and thus the capacitive load on the rf drive electronics. Instrument control is achieved through the use of modified ThermoFinnigan LCQ Duo electronics and Xcalibur software (Version 1.2, ThermoFinnigan, San Jose, CA). The system consists of the instrument’s main control board, its rf electronics subsystems, the detector high-voltage power supply, and embedded computer. The main rf drive waveform for the ion trap array is generated by the LCQ electronics at 760 kHz and amplified using the LCQ power rf amplifier. A second stage of amplification occurs in a tuned inductor/capacitor tank circuit common in ion trap instruments. The difference in capacitance between the standard ThermoFinnigan ion trap and the CIT array leads to a different resonance point in the circuit. To bring the circuit back into resonance at 760 kHz, the air core, solenoid-type inductor was retapped to provide slightly reduced inductance. Due to the relatively high pressure in the region of the trap array and the geometry of a CIT ring electrode, which has sharp edges that can result in high field strength, it was not possible to set the high-mass limit of the scan above ∼1500-1600 Th. End cap waveforms for resonance ejection are taken from the LCQ electronics and applied directly to the CIT array end caps in a dipolar fashion without modification. In most of the experiments described here, resonance ejection at the nonlinear resonance point βz ) 0.7 (qz ) 0.81), corresponding to an octapolar resonance line, was chosen to improve the mass/charge resolution.46 The amplitude of the signal generated for resonance ejection was ramped during the mass scan from 1.4 Vp-p at the start of a mass scan to 3.0 Vp-p at the end in order to maintain improved resolution through the mass/charge range. During experiments performed under boundary ejection conditions, the end caps of the array were grounded. Detection of the ions ejected from the ion trap array is performed using two K and M electron multipliers (model TX 7505, Springfield, MA), one for each channel used in the course of these experiments. The multipliers are mounted on an adjustable mount such that the position of the multipliers relative to the ion traps can be adjusted in all directions. The mount also allows for the angle of the face of the multipliers relative to the ion trap array to be changed. A metal plate is mounted between the two detector channels to physically shield each channel from the other in an attempt to minimize cross-talk occurring in the detection system. Electron current collected on the anode of the electron multiplier is fed into the LCQ electrometer where it is amplified and digitized and then saved and displayed using the Xcalibur instrument control software. Since the LCQ provides only one channel of current amplification and data acquisition, the twochannel experiments required the electrometer input be changed to record the channel of interest. The anode of the multiplier in the nonactive channel was grounded during the data acquisition. Switching between channels of the instrument for data acquisition requires only swapping the electrometer input to the anode of (45) Bui, H. A.; Cooks, R. G. J. Mass Spectrom. 1998, 33, 297-304. (46) Franzen, J.; Gabling, R. H.; Schubert, M.; Wang, Y. In Practical Aspects of Ion Trap Mass Spectrometry; March, R. E., Todd, J. F. J., Eds.; CRC Press: Boca Raton, FL, 1995; Vol. 1, pp 49-167.

the electron multiplier in the desired channel and grounding the anode of the multiplier in the opposite channel. The switch is performed in less than 5 s such that the experiment does not need to be interrupted to change between channels. Future versions of this instrument will have the capability to simultaneously detect the electron multiplier from multiple channels through the use of a current preamplifier array and multiple channel data acquisition software already implemented in the other high-throughput instrument developed in this laboratory.41 It is important to note, although no signal was being collected in the nonactive channel, ions were still being generated in that channel. All analytes used in these experiments were obtained from Sigma Aldrich (Milwaukee, WI) and used without further purification except for the tetrapeptide L-Met-Arg-Phe-Ala (MRFA), which was obtained from Research Plus, Inc. (Manasquan, NJ) and also used without further purification. Solutions of the compounds used were prepared using HPLC grade solvents and deionized water at concentrations ranging from 10 mM to 10 µM. High-throughput multichannel experiments were performed using the two-channel mass spectrometer, each fitted with an electrospray ionization source. During these experiments, the first compound (or mixture of compounds) was analyzed in channel A of the instrument while another compound (or mixture of compounds) was analyzed simultaneously in channel B. Spectra of the analyzed compounds were also recorded using singlechannel experiments. In the single-channel experiments, each analyte was electrosprayed and mass analyzed in the same channel that was used for the two-channel experiment but no sample was introduced into the other channel of the instrument. The results of these single-channel and two-channel experiments are presented in the figures. During the acquisition of data from each channel of the instrument in the two-channel experiments, the experimental conditions were kept constant. The conditions used in the two-channel experiments were identical to those used in the single-channel experiments. To ensure the stability of the signal and consistency of the data during two-channel experiments, the data acquisition was repeated several times while switching between the two channels of the instrument. RESULTS AND DISCUSSION Analysis of Amino Acids and Instrument Cross-Talk. To investigate the performance of the multiplexed instrument in the case of small molecules, solutions of arginine (C6H14N4O2, molecular weight 174.20) and glutamine (C5H10N2O3, molecular weight 146.14) were prepared and electrosprayed from the multichannel source into the instrument operated in both singlechannel and multichannel modes. Figure 4a shows a mass spectrum recorded during a single-channel experiment in which a 10 mM arginine solution in methanol/water (1:1 v/v) containing 1% acetic acid was electrosprayed into channel A of the instrument. Correspondingly, Figure 4b shows the spectrum obtained during a single-channel experiment in which a 10 mM solution of glutamine in methanol/water (1:1 v/v) containing 1% acetic acid was electrosprayed into channel B. Both spectra are dominated by the protonated amino acids, 175 Th for arginine and 147 Th for glutamine. Figure 4b also shows the formation of the protonbound dimer of glutamine at 293 Th. Panels c and d of Figure 4 show spectra obtained from channels A and B of the instrument, respectively, during the corresponding two-channel experiment. Analytical Chemistry, Vol. 77, No. 2, January 15, 2005

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Figure 4. Spectra of 10 mM solutions of arginine and glutamine during one- (a, b) and two-channel (c, d) experiments obtained using resonance ejection at βz ) 0.7. In (c, d) the positions are marked at which the ∼1% cross-talk is observed from the opposite channel.

In this experiment, the same solutions of arginine and glutamine as used in the single-channel experiments were introduced using the same channels of the instrument as were used in the singlechannel experiments. The ion injection time during both the single- and the twochannel experiments was 2 s. The electron multipliers were biased at -1200 V. Long ion injection time and high concentrations of analytes were chosen because one of the purposes of these experiments was to characterize the extent of a cross-talk between channels of the instrument; i.e., it was desirable to maximize the ion signal intensity in each channel and maximize the signal-tonoise ratio. Relatively high biases on the multipliers were chosen for the same reason. It can be seen that, during the two-channel experiments, although very small (∼1% relative abundance), signals corresponding to the amino acid being sprayed into the opposite channel can be seen. To elucidate the sources of cross-talk present in both channels of mass analysis during the two-channel experiment, an additional single-channel experiment was performed. In this single-channel experiment, arginine was sprayed into channel A of the instrument and detected in both channels, while nothing was sprayed into channel B. Table 1 shows the dependence of the level of crosstalk on the bias on multipliers during this experiment. The level of cross-talk is given as the ratio of the peak height for the [arginine + H]+ ion signal in channel B of the instrument (due to cross-talk) expressed as a percentage of the peak height for the [arginine + H]+ ion signal intensity in channel A. The same dependence was investigated during the two-channel experiment described above, with the levels of cross-talk also shown in Table 1. 464 Analytical Chemistry, Vol. 77, No. 2, January 15, 2005

Table 1. Amino Acid Cross-Talk in Channel B Due to Ions Electrosprayed into Channel A level of cross-talk, % multiplier bias, V

injection time, ms

one-channel experiment

two-channel experimentt

-1000 -1100 -1200

2000 2000 2000

0.3 0.8 1.7

0.3 0.9 1.6

Table 2. Amino Acid Cross-Talk in Channel B Due to Ions Electrosprayed into Channel A with the Shield Plate Installed Closer to the Ion Traps level of cross-talk, % multiplier bias, V

injection time, ms

one-channel experiment

two-channel experimentt

-1000 -1100 -1200 -1300

2000 2000 2000 2000

0.8 2.7 6.1 10.1

0.8 2.4 5.8 10.4

In all experiments described in this paper, a metal plate was placed between detectors to physically shield the two channels from each other and help prevent ion cross-talk in the detection system, after ions have been ejected from the ion trap array. The exit holes in the end caps of two ion traps that were not used in experiments were blocked by a conductive adhesive-backed copper tape (3M, St. Paul, MN) in order to eliminate the possible contribution of these traps to the cross-talk. Table 2 shows the

Table 3. Cross-Talk in Channel B Due to Ions Electrosprayed into Channel A Normalized to the Analyte Signal in Channel B

Figure 5. Dependence of the level of cross-talk vs injection time during one- and two-channel experiments. The data from the twochannel experiment represents two replicates of the same experiment.

results of the same single- and two-channel experiments described to acquire the data for Table 1, performed with smaller separation distance (∼1 mm) between shield plate and exit end caps of the ion traps. The experiments described in Table 1 used a separation distance of ∼4 mm. Although the intent of moving the shield plate closer to the ion traps was to further reduce ionic cross-talk due to ions exiting one trap and entering the electron multiplier of the other channel, the actual effect observed was the opposite. The dependence of the level of cross-talk on the injection time at a constant multiplier bias was also investigated in the course of these experiments. This dependence is shown in Figure 5 for the single-channel experiment and for two replicates of the twochannel experiment described above while the multiplier bias was held at -1100 V. In no experiments was additional cooling time programmed into the scan function; thus, only the injection time determines the average time ions spend inside the traps prior to mass analysis. The observation of the same type of behavior in the level of cross-talk versus injection time for both the one- and two-channel experiments over a wide range of injection times (100-5000 ms) suggests that the cross-talk is not due to neutral cross-talk followed by ion/molecule reactions, i.e., proton transfer from glutamine (PA ) 224.1 kcal/mol47) to arginine (PA ) 251.20 kcal/mol47). The thermochemical data suggest that the presence of [glutamine + H]+ signal in channel A of the instrument, in which arginine was analyzed during a two-channel experiment, could be caused only by ionic cross-talk in which ions from channel B are detected in channel A, since proton transfer from [arginine + H]+ to glutamine is endothermic by 27 kcal/mol. The possible roll-off of the curve in Figure 5 suggests that the traps reach their ion capacity limit at long injection times. The space charge effects due to the large number of ions in the trap can influence the spread of ion trajectories during ejection and hence the amount of the ionic cross-talk seen in the detection system, i.e., the portion of ions that will reach a detector in another channel. The ionic cross-talk observed could take place before the CIT array, i.e., in the ion sources or associated optics, or may take place upon ejection of the ions from the array in the ion detection system. Table 3 shows the dependence on multiplier bias of the ratio of the peak height in channel B of the instrument for [arginine + H]+, present due to a cross-talk, to the peak height (47) Hunter, E. P.; Lias, S. G. J. Phys. Chem. Ref. Data 1998, 27, 713-656.

bias on multipliers, V

injection time, ms

ratio of ion signal intensities [arginine + H]+/ [glutamine + H]+

-1000 -1100 -1200 -1300

2000 2000 2000 2000

0.2 0.8 2.3 5.9

of the [glutamine + H]+ being analyzed in that channel. The data presented in Tables 2 and 3 were acquired during the course of the same experiment. Although no attempt was made to balance the gain of the electron multipliers in the two channels of the instrument, the magnitude of the change of cross-talk with multiplier bias again indicates that the majority of the cross-talk is in the detection system of the instrument, i.e., occurs after ions leave the ion trap array. The increase in the cross-talk when the shield plate was moved closer to the ion trap array can be explained by electron cross-talk at the anodes of the individual electron multiplier assemblies. The unshielded anodes of the multiplier assemblies could have become more exposed to each other as the plate was moved forward, resulting in secondary electrons produced by the multiplier that were not collected on the anode of the native channel being collected as cross-talk in the opposite channel. No attempt was made to distinguish between true ionic cross-talk and that produced though electron crosstalk in the detection system. Further reduction in the cross-talk to levels below that seen at the detectors as shown in Table 1 will require detailed electric field calculation and ion motion simulation in this region to ensure proper shielding between channels. The other possible sources of cross-talk in electrostatic ion optics and atmospheric interface of the instrument play minor a role compared to the cross-talk in the detection system. The cross-talk in this instrument is much lower in level and has a origin different from that observed in the multichannel mass spectrometer fitted with EI/CI sources.41 The cross-talk in the multichannel mass spectrometer with EI/CI ion sources was caused by ion/molecule reactions between neutrals of one channel and ions contained within the ion trap of a different channel. Crosstalk between channels where the observed cross-talk is the result of a thermodynamically unfavorable ion/molecule reaction is presumed to have arisen from the exchange of neutrals between channels in the ion source array. In contrast, the cross-talk in the instrument described in this paper is small and has been shown to occur mainly in the detection system. The fact that ion/molecule reactions do not contribute to the cross-talk in this instrument the same way as they do in the case of a EI/CI mass spectrometer can be explained by more efficient ionization and discrimination of ions against neutrals in the electrospray ion source compared to the EI and CI sources. Also the relatively high volatility of the analytes used in EI and CI leads to the mixing of compounds from different channels of analysis in a common vacuum system, which can lead to subsequent ion/ molecule reactions that will result in a cross-talk between channels. The cross-talk present in the detection system during these experiments, although small, should be reduced further, and this Analytical Chemistry, Vol. 77, No. 2, January 15, 2005

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Figure 6. Spectra of 1 mM solutions of ephedrine and lomefloxacin during one- (a, b) and two-channel (c, d) experiments obtained using resonance ejection at βz ) 0.7.

Figure 7. Mass spectra of a mixture of n-octadecylamine and di-n-dodecylamine and a mixture of tri-n-dodecylamine, MDA, and tri-n-butylamine obtained during one- (a, b) and two-channel (c, d) experiments obtained using boundary ejection. Peaks labeled by an / are due to an impurity in the mixture solution. 466 Analytical Chemistry, Vol. 77, No. 2, January 15, 2005

Figure 8. Mass spectrum of 5 µM MRFA and 20 µM myoglobin tuning mixture obtained using boundary ejection.

might be possible through proper separation and shielding of ion traps and detectors in different channels of the instrument. Analysis of Small Drug Molecules. Small drug molecules were chosen as analytes to further explore the performance of the multiplexed ESI instrument in the analysis of organic molecules. Ephedrine (C10H15NO, molecular weight 165.23) is a weak partial agonist of β-1 and β-2 adrenoreceptors in humans, popular in the treatment of several disorders including asthma and rhinitis. Lomefloxacin (C17H19F2N3O3, molecular weight 351.8) is a second-generation quinolone antibiotic, used as a treatment for a variety of bacterial infections such as bronchitis and urinary tract infections. Single- and double-channel experiments were performed by electrospraying 1 mM solutions of ephedrine and lomefloxacin hydrochloride in methanol/water (1:1 v/v) into channels A and B of the instrument, respectively. Figure 6a shows a mass spectrum of ephedrine recorded during a single-channel experiment from channel A of the instrument. Figure 6b shows a mass spectrum of lomefloxacin obtained during a separate singlechannel experiment using channel B. As is expected, the mass spectra from both compounds are dominated by the protonated molecules at 166 and 352 Th for ephedrine and lomefloxacin, respectively. The formation of a low relative abundance protonbound dimer of ephedrine is also seen at 331 Th in Figure 6a and c. Panels c and d of Figure 6 show spectra obtained from channels A and B of the instrument during a two-channel experiment in which the same solutions of ephedrine and lomefloxacin were used as in the single-channel experiments. The ephedrine solution was

analyzed in channel A of the instrument while the solution of lomefloxacin was simultaneously analyzed in channel B. To characterize the dynamic range of the instrument, an additional two-channel experiment was performed using different concentrations of ephedrine and lomefloxacin. In this experiment, a 1 mM solution of ephedrine in methanol/water (1:1 v/v) was analyzed in channel A of the instrument, and a 10 µM solution of lomefloxacin in methanol/water (1:1 v/v) was simultaneously analyzed in channel B. Note the factor of 100 difference in concentration of the two analytes. In this experiment, the intensity of the lomefloxacin signal in channel B of the instrument was ∼2.5 times higher than the signal intensity of ephedrine in this channel, present due to cross-talk. When using large differences in concentrations of analytes, the signal intensity due to cross-talk from a high-concentration component in another channel can became comparable to the signal intensity due to a low-concentration analyte of interest in a given channel, imposing limitations on the dynamic range of the instrument. From this experiment, the dynamic range of the instrument was shown to be 100 while maintaining a 2.5 times higher signal intensity for the low-concentration analyte relative to the cross-talk due to the higher concentration analyte in the other channel. Analysis of Alkylamines. Further studies demonstrating the high-throughput capabilities of this instrument for the analysis of small organic molecules were performed using mixtures of alkylamines. In these experiments, a mixture of n-octadecylamine Analytical Chemistry, Vol. 77, No. 2, January 15, 2005

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Figure 9. Mass spectra of the 5 µM MRFA and 20 µM myoglobin tuning mixture and a 100 µM cytochrome c during one- (a, b) and twochannel (c, d) experiments obtained using boundary ejection.

(C18H39N, molecular weight 269.5) and di-n-dodecylamine (C24H51N, molecular weight 353.7) was analyzed in channel A of the instrument while a mixture of tri-n-dodecylamine (C36H75N, molecular weight 521.99), n-methyldiethylamine (C5H13N, MDA, molecular weight 87.16), and tri-n-butylamine (C12H27N, molecular weight 185.35) was simultaneously analyzed in channel B. The mass spectrum shown in Figure 7a was obtained during a singlechannel experiment from channel A of the instrument by spraying a mixture of n-octadecylamine and di-n-dodecylamine from an acetonitrile/water (7:3 v/v) solution containing 1% acetic acid. Figure 7b shows the mass spectrum recorded during a separate single-channel experiment employing channel B by spraying a mixture of tri-n-dodecylamine, n-methyldiethylamine, and tri-nbutylamine from an acetonitrile/water (7:3 v/v) solution containing 1% acetic acid. The single-channel experiments show the generation of the protonated amines as expected. Some fragmentation of the longer chain alkylamines is also observed. Specifically, the loss of an alkene from the tri-n-dodecylamine is observed, a wellknown fragmentation process for a protonated amine.48 Fragmentation by the loss of shorter alkenes from the protonated amines also occurs. The extent of the fragmentation seen is similar to that seen when the same solutions are analyzed using a ThermoFinnigan LCQ Classic mass spectrometer. The spectra shown in Figure 7c and d were acquired during a two-channel high-throughput experiment in which a mixture of n-octadecylamine and di-n-dodecylamine was sprayed and mass analyzed in (48) Sigsby, M. L.; Day, R. J.; Cooks, R. G. Org. Mass Spectrom. 1979, 14, 556561.

468 Analytical Chemistry, Vol. 77, No. 2, January 15, 2005

channel A of the instrument and a mixture of tri-n-dodecylamine, n-methyldiethylamine, and tri-n-butylamine was simultaneously sprayed and mass analyzed in channel B. The solutions were the same as those used during the single-channel experiments described above. In the single- and two-channel experiments on the amines, the mass spectra were obtained using boundary ejection. As a consequence, broadening of the peaks of the mass spectra in Figure 7 is observed when compared to the other mass spectra obtained using resonance ejection during the course of these studies. The broadening of the low-mass peaks is more significant than that for the high-mass peaks and can be explained by the influence of the larger space charge in the ion traps early in the scan. Analysis of Peptides/Proteins. To determine the mass range of the instrument and demonstrate its performance for peptides and proteins, a single-channel experiment was performed in which a tuning solution of myoglobin (equine heart, molecular weight 17 603), present as apomyoglobin (molecular weight 16 951) under the denaturing conditions in solution, and MRFA (molecular weight 523.7) consisting of 5 µM myoglobin and 20 µM MRFA in methanol/water (1:1 v/v containing 0.5% acetic acid) was analyzed in channel B of the instrument. The mass spectrum obtained during this experiment under boundary ejection conditions is shown in Figure 8. The ion injection time was 10 ms. At longer ion injection times (>100 ms), space charging effects become important and lead to the significant broadening of peaks of different charge states of the protein. The highest mass-to-charge

degraded when compared to the later-ejecting peaks. This is again presumed to be a result of greater space charge in the ion trap during ejection of the lower mass ions.

Figure 10. Mass spectrum obtained using resonance ejection at βz ) 0.7 for the analysis of a mixture of 100 µM bradykinin (531 Th), 75 µM substance p (675 Th), and 150 µM angiotensin (433, 649 Th) in channel A of the instrument.

ratio detected in this experiment corresponds to the +13 charge state of myoglobin at 1305 Th. The data show the ability of the instrument to analyze peptides and proteins and to yield a chargestate distribution for myoglobin that is representative of that observed under denaturing conditions. As mentioned earlier, discharge between the ring electrode and the end caps of the CIT array impose an upper limit on the rf amplitude and hence the mass range achievable under these conditions. The upper mass limit of the instrument is estimated to be 1500-1600 Th due to available rf voltage for ion ejection from the trap. In a high-throughput experiment, myoglobin/MRFA mixture and cytochrome c were analyzed simultaneously using two channels of the instrument. The myoglobin/MRFA mixture was the same solution that was used in the mass range experiment described above. A 100 µM solution of cytochrome c (equine heart, molecular weight 12 384) in methanol/water (1:1 v/v) was prepared and used in the experiment. The injection time during this experiment was 10 ms with the mass spectra obtained under boundary ejection conditions. Figure 9a shows a mass spectrum obtained during a single-channel experiment from channel A of the instrument in which myoglobin/MRFA mixture was analyzed while Figure 9b shows the mass spectrum obtained during another single-channel experiment in which cytochrome c was mass analyzed in channel B. Panels c and d of Figure 9 show spectra obtained from channels A and B of the instrument during twochannel experiments in which the same solutions of myoglobin/ MRFA mixture and cytochrome c as used in the single-channel experiments were analyzed simultaneously. Myoglobin/MRFA mixture was analyzed in channel A while cytochrome c was analyzed simultaneously in channel B. The high levels of background early in the mass scans result from ion injection while the mass scan is occurring. This occurs simply because the ion beam generated in the ESI souce is not gated to prevent ion injection during the mass scan. The data collected show the applicabiltiy of this instrument to protein analysis. Further investigation into mixture analysis was performed with the instrument operating under higher performance resonance ejection conditions (βz ) 0.70). In this single-channel experiment, a solution of 100 µM bradykinin (molecular weight 1060.2), 75 µM substance P (molecular weight 1347.6), and 150 µM angiotensin I (molecular weight 1296.5) in methanol/water/acetic acid (49:49:2 v/v) was electrosprayed into channel A of the instrument. The resulting mass spectrum, shown in Figure 10, demonstrates the ability to analyze mixtures of similar compounds. The resolution when using resonance ejection improves to nearunit resolution for all four peaks in the spectrum. The resolution of the 433 Th ion from triply charged angiotensin is slightly

CONCLUSIONS A novel, four-channel CIT array mass spectrometer with electrospray ion sources has been designed, built, and characterized using two channels of parallel mass spectrometry. The capabilities and limitations of this instrument have been assessed in one- and two-channel experiments using several different types of compounds and mixtures of compounds. An upper mass to charge limit of 1300 Th was demonstrated with unit mass/charge resolution to ∼800 Th. During the simultaneous analysis of different compounds in different channels of the instrument, small amounts of cross-talk were observed. Though the sources of a cross-talk were identified and minimized, some cross-talk between parallel channels (∼1-2%) still remains and it limits dynamic range. Further reductions in the cross-talk will be achieved through additional refinement in the ion optics and particularly the ion detection systems. Although a zero cross-talk system is preferred, small amounts of cross-talk can be corrected numerically through postacquisition data processing, as the information necessary to make the correction is available in the channels from which the cross-talk originated. Effects of mass discrimination in the ion optics have been observed and will be minimized through the addition of rf-only ion guides and the removal of the einzel lens. Additional future efforts will be focused on extending the performance of the instrument through the use of the rectilinear ion trap49 a device that has been shown to provide a significant increase in ion trapping efficiency and ion storage capacity. Finally, the possibility of using multiple ion source types in a multiplexed fashion to increase the specificity in the detection of a specific analyte as described by Tabert et al.42 remains of interest. Note that, in these experiments, each trap in the array receives the same electrical potential and hence must perform the same experiment. It should be possible to obtain independent access to each channel by constructing each trap with its own set of end caps, at significant cost in terms of instrument complexity. This would require the use of independent signal generation equipment to drive the end caps of each channel of the instrument. The lack of independent access to each channel in the present instrument may be perceived as a weakness, but in situations where similar samples are being analyzed in the same manner, this instrument offers the potential for a great time savings over the use of a single-channel instrument. As a result of the high gas load on the vacuum system, only two channels were operated in the experiments described here. Future efforts will address the limitations of the vacuum system through a redesign of the instrument. The addition of extra pumping stages and the associated ion optics will allow for the operation of the instrument with more channels while still using relatively small turbomolecular pumps. The expansion of this concept to a greater numbers of channels is currently being pursued together with many of the improvements noted above (49) Ouyang, Z.; Wu, G.; Song, Y.; Li, H.; Plass, W. R.; Cooks, R. G. Anal. Chem. 2004, 76, 4595-4605.

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and with the ultimate goal of producing an instrument capable of processing 96 channels in parallel. ACKNOWLEDGMENT The authors acknowledge ThermoFinnigan and The Procter & Gamble Co. for providing funding for this research. Additional funding was received from NASAs Jet Propulsion Laboratory under the Planetary Instrument Definition and Development Program (PIDDP), and the Astrobiology Science and Technology Instrument Development (ASTID) program. B.C.L. acknowledges

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Phillips Petroleum Co. for fellowship support. The authors also thank Garth Patterson and Yanan Peng for important contributions early in this work and Andrew Guymon for his continuing support in the development of this instrumentation.

Received for review July 12, 2004. Accepted October 18, 2004. AC048983W