Automated Tandem Mass Spectrometry by Orthogonal Acceleration

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Anal. Chem. 2001, 73, 3611-3623

Automated Tandem Mass Spectrometry by Orthogonal Acceleration TOF Data Acquisition and Simultaneous Magnet Scanning for the Characterization of Petroleum Mixtures Stilianos G. Roussis*

Research Department, Products and Chemicals Division, Imperial Oil, Sarnia, Ontario, Canada N7T 8C8

The automated acquisition of the product ion spectra of all precursor ions in a selected mass range by using a magnetic sector/orthogonal acceleration time-of-flight (oaTOF) tandem mass spectrometer for the characterization of complex petroleum mixtures is reported. Product ion spectra are obtained by rapid oa-TOF data acquisition and simultaneous scanning of the magnet. An analog signal generator is used for the scanning of the magnet. Slow magnet scanning rates permit the accurate profiling of precursor ion peaks and the acquisition of product ion spectra for all isobaric ion species. The ability of the instrument to perform both high- and low-energy collisional activation experiments provides access to a large number of dissociation pathways useful for the characterization of precursor ions. Examples are given that illustrate the capability of the method for the characterization of representative petroleum mixtures. The structural information obtained by the automated MS/MS experiment is used in combination with high-resolution accurate mass measurement results to characterize unknown components in a polar extract of a refinery product. The exhaustive mapping of all precursor ions in representative naphtha and middle-distillate fractions is presented. Sets of isobaric ion species are separated and their structures are identified by interpretation from first principles or by comparison with standard 70-eV EI libraries of spectra. The utility of the method increases with the complexity of the samples. Characterization of hydrocarbon mixtures and specialty chemicals (e.g., additives) are the two most common analytical applications in the petroleum industry. GC1,2 and GC/MS3,4 methods can separate almost all individual compounds in low-boiling petroleum * Corresponding author: (tel) 519-339-2441; (fax) 519-339-4436; (e-mail) [email protected]. (1) Canadian General Standards Board (CGSB) Method Can/CGSB-3.0 No. 14.399. Standard Test Method for the Identification of Hydrocarbon Components in Automotive Gasoline Using Gas Chromatography; Canadian General Standards Board: Ottawa, Canada, K1A 1G6, 1999. (2) ASTM Method D 2427-92. Annual Book of ASTM Standards; American Society for Testing and Materials: Philadelphia, PA, 2001. (3) ASTM Method D 5769-98. Annual Book of ASTM Standards; American Society for Testing and Materials: Philadelphia, PA, 2001. (4) Nero, V. P.; Drinkwater, D. E. Prepr. Pap.-Am. Chem. Soc., Div. Fuel Chem. 1996, 41, 992-928. 10.1021/ac0102260 CCC: $20.00 Published on Web 07/04/2001

© 2001 American Chemical Society

mixtures such as gasoline. However, such detailed analysis cannot be done for fractions boiling higher than ∼210 °C due to the extreme complexity of the mixtures. The complexity of hydrocarbon mixtures increases dramatically with boiling point due to the rapid growth of the number of possible isomers as a function of the number of atoms in the structure.5 Moreover, the use of GC is not suitable for the analysis of polar or ionic compounds commonly used as additives in the various petroleum processes. Liquid chromatographic separation methods coupled with soft ionization mass spectrometric methods are more suitable for the analysis of heavy petroleum mixtures 6-8 and nonboiling polar and ionic additives.9-12 Liquid chromatography (LC) is used to separate heavy hydrocarbon samples into fractions of different polarities. A normal-phase column can separate samples into fractions of saturated, aromatic, and polar heteroatom-containing hydrocarbons.7,8,13 No attempt is generally made to separate individual hydrocarbon molecules in the mixtures. Instead, the analysis targets the separation of groups of compounds (e.g., saturates, one- and two-ring aromatics).13 Detailed characterization of the LC-separated fractions is typically done by mass spectrometry (MS). Soft ionization (e.g., low electron energy,14,15 charge exchange chemical ionization16,17) MS methods are preferred because they produce simple mass spectra consisting predominantly of molecular ions with negligible contributions from fragment ions. High-resolution mass spectrometry is used to (5) Boduszynski, M. M. Energy Fuels 1988, 2, 597-613. (6) Anacleto, J. F.; Ramaley, L.; Benoit, F. M.; Boyd, R. K.; Quilliam, M. A. Anal. Chem. 1995, 67, 5-4154. (7) Qian, K. Hsu, C. S. Anal. Chem. 1992, 64, 2327-2333. (8) Qian, K.; Hsu, C. S. Energy Fuels 1993, 7, 268-272. (9) Jewett, B. N.; Ramaley, L.; Kwak, J. C. T. J. Am. Soc. Mass Spectrom. 1999, 10, 529-536. (10) Gough, M. A.; Langley, G. J. Rapid Commun. Mass Spectrom. 1999, 13, 227-236. (11) Roussis, S. G.; Fedora, J. W. Proceedings of the 47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, TX, June 13-17, 1999; MPG 158. (12) Roussis, S. G. Proceedings of the 48th ASMS Conference on Mass Spectrometry and Allied Topics, Long Beach, CA, June 11-15, 2000;1 WOB 3:40. (13) Hsu, C. S.; McLean, M. A.; Qian, K.; Aczel, T.; Blum, S. C.; Olmstead, W. N.; Kaplan, L. H.; Robbins, W. K.; Schultz, W. W. Energy Fuels 1991, 5, 395-398. (14) Field, F. H.; Hastings, S. H. Anal. Chem. 1956, 28, 1248-1255. (15) Lumpkin, H. E. Anal. Chem. 1958, 30, 321-325. (16) Allgood, C.; Ma, Y. C.; Munson, B. Anal. Chem. 1991, 63, 721-725. (17) Hsu, C. S.; Qian, K. Anal. Chem. 1993, 65, 767-771.

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separate isobaric molecular ion species and assign most probable chemical formulas to the measured masses.18-21 The technique provides information about the overall (percent) amount of each element (C, H, S, O, N, etc.) in the sample, the relative amounts of compound classes with different combinations of heteroatoms, and aromaticity (Z-series) distributions for each class, as well as molecular and carbon number distributions for each Z-series.21 Other, more recently developed, soft ionization methods such as electrospray ionization (ESI)22 and atmospheric pressure chemical ionization (APCI)23 coupled to reversed-phase10,12 liquid chromatography are used for the analysis of thermally labile, polar, and ionic specialty compounds. Although high-resolution MS methods can separate isobaric ion species and propose probable chemical formulas that fit the measured masses, they provide no information about the structures of the separated species. Most often, the presence of certain structures is assumed based on general knowledge about the nature of the sample and supplemental information from other analytical methods obtained for the entire sample (e.g., elemental analysis, IR). Although the assumed structures or mixtures of structures are generally correct for previously well-characterized types of samples, this is not the case for unknown samples. This is especially true for non-hydrocarbon, specialty chemical samples. The accurate mass of an ion, although powerful evidence, is not necessarily adequate to unequivocally identify the presence of a compound. Tandem mass spectrometry (MS/MS)24-26 allows the generation of an additional fingerprint for a selected precursor ion and offers additional information for the confirmation of the proposed chemical formula. Ideally, selection of precursor ions in MS/MS experiments and measurement of product ion masses should be done using a highresolving power instrument. Confidence in the interpretation results is considerably increased when overlapping isobaric precursor ions are separated and tandem mass spectra are produced from single ions with the precursor and product ion masses accurately known. Additionally, tandem mass spectra should ideally be produced with characteristic fragmentation patterns that uniquely reflect the structures of the precursor ions. High-energy collisional activation (CA) with inert target gases is capable of transferring high amounts of internal energy to precursor ions and thus produce tandem mass spectra with extensive fragmentation that can be used to deduce the structures of precursor ions.27,28 Low-energy collisional activation transfers smaller amounts of internal energy producing tandem mass spectra with less extensive but usually diagnostic fragmenta(18) Schmidt, C. E.; Sprecher, R. F.; Batts, B. D. Anal. Chem. 1987, 59, 20272033. (19) Hsu, C. S.; Qian, K.; Chen, Y. C. Anal. Chim. Acta 1992, 264, 79-89. (20) Guan, S.; Marshall, A. G.; Scheppele, S. E. Anal. Chem. 1996, 68, 46-71. (21) Roussis, S. G. Rapid Commun. Mass Spectrom. 1999, 13, 1031-1051. (22) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37-70. (23) Duffin, K. L.; Wachs, T.; Henion, J. D. Anal. Chem. 1992, 64, 61-68. (24) McLafferty, F. W., Ed. Tandem Mass Spectrometry; Wiley-Interscience: New York, 1983. (25) Busch, K. L.; Glish, G. L.; McLuckey, S. A. Mass Spectrometry/Mass Spectrometry: Techniques and Applications of Tandem Mass Spectrometry; VCH Publishers: New York, 1988. (26) de Hoffmann, E. J. Mass Spectrom. 1996, 31, 129-137. (27) Kolli, V. S. K.; Orlando, R. J. Am. Soc. Mass Spectrom. 1995, 6, 234-241. (28) Ciupek, J. D.; Zakett, D.; Cooks, R. G. Anal. Chem. 1982, 54, 2215-2219.

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tion.28,29 Due to the different types of dissociation pathways of precursor ions under high- and low-energy collisional activation,24-30 an ideal tandem mass spectrometer should be able to perform both types of experiments. Most commercially available tandem mass spectrometers using quadrupole filter and ion trap analyzers do not permit highresolving power precursor ion separation,31 and collisional activation with these instruments is limited to low-energy regimes.32 Fourier transform ion cyclotron resonance (FTICR) mass spectrometers can separate isobaric precursor ions and accurately measure product ion masses under high-resolving power conditions.33,34 However, collisional activation with FTICR instruments is limited to low-energy regimes although several methods are available for effective internal energy transfer leading to increased dissociation.30,33-35 Four-sector magnetic tandem mass spectrometers were originally used for high-energy collisions and accurate precursor ion selection.27,36 These instruments possess many properties of the ideal mass spectrometer; however, they are expensive and complex to operate. Additionally, calibration of the second stage of analysis (MS-2) is rather involved due to the nonlinear scanning of the magnet.36 Simpler hybrid magnetic sector-quadrupole tandem mass spectrometers were introduced that allowed high-resolving power separation of precursor ions; however, only low-energy collisional activation could be performed with the quadrupole analyzer.37 More recently, a hybrid magnetic sector orthogonal acceleration time-of-flight (oa-TOF) instrument has become available38 that has the capabilities of the ideal tandem mass spectrometer discussed above. Isobaric precursor ions can be accurately selected by the magnetic sector (within the limits of the experimental resolving power), and the oa-TOF design allows for both high- and low-energy collisional activation experiments. Most importantly, operation and calibration of the MS-2 stage is greatly simplified due to the properties of the time-offlight mass analyzers.39 In this study, we have used such a magnetic sector oa-TOF instrument for the characterization of complex petroleum mixtures. The current major limitation of the magnetic sector oa-TOF instrument for the analysis of complex mixtures is the need for manual selection of precursor ions under high resolving power conditions. Although, a voltage scan with a suitable lock mass can in principle be used for the automatic selection of precursor ions in a manner analogous to that used in high-resolution voltage (29) Summerfield, S. G.; Gaskell, S. J. Int. J. Mass Spectrom. Ion Processes 1997, 165, 509-521. (30) Shukla, A. K.; Futrell, J. H. J. Mass Spectrom. 2000, 35, 1069-1090. (31) Willoughby, R.; Sheehan, E.; Mitrovitch, S. A Global View of LC/MS: How to Solve Your Most Challenging Analytical Problems; Global View Publishing: Pittsburgh, PA, 1998. (32) Johnson, J. V.; Yost, R. A.; Kelley, P. E.; Bradford, D. C. Anal. Chem. 1990, 62, 2162-2172. (33) Horn, D. M.; Zubarev, R. A.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10313-10317. (34) Martin, S. E.; Shabanowitz, J.; Hunt, D. F.; Marto, J. A. Anal. Chem. 2000, 72, 4266-4274. (35) McLuckey, S. A.; Goeringer, D. E. J. Mass Spectrom. 1997, 32, 461-474. (36) Takao, T.; Gonzalez, J.; Yoshidome, K.; Sato, K.; Asada, T.; Kammei, Y.; Shimonishi, Y. Anal. Chem. 1993, 65, 2394-2399. (37) Bertrand, M. J.; Bott, P. A.; Porter, C. J.; Bateman, R. H. Proceedings of the 37th ASMS Conference on Mass Spectrometry and Allied Topics, Miami Beach, FL, 1989; pp 1019-1020. (38) Bateman, R. H.; Green, M. R.; Scott, G.; Clayton, E. Rapid Commun. Mass Spectrom. 1995, 9, 1227-1233. (39) Cotter, R. J. Anal. Chem. 1999, 71, 445A-451A.

Figure 1. Schematic diagram of the ZabSpec Ultima (Micromass Ltd.) double-focusing magnetic sector/orthogonal acceleration time-of-flight tandem mass spectrometer.

selected ion recording (SIR) experiments,40 the process is very involved and only applicable to precursor ions of known masses. If the masses of interest are not known or if there are too many to be manually selected, then accurate automatic selection of precursor ions using the voltage scan is not possible. In those cases, a continuous MS/MS scan is needed to automatically acquire, in an exhaustive fashion, the product ion spectra of all ions in a specified mass range. In this work, we have implemented a method to automatically acquire the product ion spectra of all ions in a selected mass range. An analog signal generator is used to accurately control the scanning of the magnet in a magnetic sector oa-TOF tandem mass spectrometer. Exhaustive product ion mapping is obtained by oaTOF data acquisition and simultaneous magnet scanning. Precursor ions can be selected under low or high resolving power conditions and there is no need for calibration of the magnet. The current system is not compatible with fast on-line chromatographic separation, but it is possible to further develop the magnet control system to perform data-dependent LC/MS/MS experiments. The principles and capabilities of the method are described in this report. EXPERIMENTAL SECTION Mass Spectrometer. A ZabSpec Ultima tandem doublefocusing magnetic sector oa-TOF instrument (Micromass Inc., Manchester, U.K.) was used for the experiments. Figure 1 shows a schematic diagram of the instrument. The first stage of analysis (MS-1) can be operated independently of the oa-TOF MS-2 stage and can perform all linked scan experiments possible with magnetic sector instruments.41 MS-1 consists of a magnet (B) and a split electrostatic analyzer (ESA) in the E1BE2 configuration (Figure 1). This design permits increased ion transmission and resolution.42 An extensive system of lenses provides higher order focusing. The upper mass limit of the instrument is m/z 4000 at full accelerating potential (8 kV). A mass resolving power of 120 000 (10% valley criterion) is attainable at the detector after ESA 2 (double focusing). (40) Autospec-TOF Users Manual, Code Number 6666576, Micromass Ltd., June 1997; Vol. 1. (41) Boyd, R. K. Mass Spectrom. Rev. 1994, 13, 359-410. (42) Nemirovskiy, O. V.; Gooden, J. K.; Ramanathan, R.; Gross, M. L. NATO ASI Ser., Ser. C 1997, 504, 183-211.

Precursor ions are selected by tuning the magnetic sector to transmit only ions of a unique mass-to-charge ratio (m/z). Massselected precursor ions entering the oa-TOF region are decelerated (10-2.5% of their original kinetic energies) and are focused into an approximately parallel beam by a two-stage deceleration system of electrostatic lenses prior to entrance into the collision cell. Two independent inlet lines permit the introduction of different gases into the collision cell (length of 5 cm). By operating the oa-TOF collision cell at 10% of the ionization source voltage, a maximum of 800-eV laboratory kinetic energy is available for collisional activation. Center-of-mass collision energy calculations have shown that by appropriate selection of target collision gases and laboratory kinetic energies both high- and low-energy collisional activation experiments can be performed by the instrument.38 The fragment ions produced in the collision cell and the surviving precursor ions enter a sampling region where a periodic voltage orthogonal to the direction of travel is applied as a sudden pulse that accelerates ions toward a microchannel plate (MCP) detector. The effective path length of the TOF analyzer is 0.44 m. Due to their forward velocity, component ions are displaced forward on reaching the TOF detector. To account for the forward ion displacement and permit the acquisition of the entire product ion spectrum, a long MCP detector is used consisting of 3 × 50 mm plates. The ion signal is acquired using a time-to-digital converter (TDC) with a 1-GHz acquisition rate permitting a maximum mass range of m/z 7200 at 8-kV accelerating voltage and 800-eV collision energy. The pulse repetition rate must be slow enough to allow adequate time for all ions in the mass range to reach the detector before the start of the next pulse. For the maximum mass range of m/z 7200, the maximum repetition rate is 30 kHz. A faster repetition rate (e.g., 100 kHz) is possible for smaller mass ranges (e.g., m/z 500). We have found that more abundant product ion spectra are produced at faster pulse repetition rates (smaller mass ranges) as expected, due to the accumulation of more ion signal at the MCP detector per unit time. Ions are transmitted from the detector located after ESA 2 to the conventional photomultiplier detector located in the oaTOF housing at high ion transmission efficiencies (20-40% at m/z 1000, 8-kV accelerating voltage, 800-eV collision energy, maximum oa-TOF mass range). Two-point calibration is sufficient to calibrate Analytical Chemistry, Vol. 73, No. 15, August 1, 2001

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the mass scale.43 Routine mass accuracies of (25 ppm are achievable for product ions with masses higher than m/z 200 while (10 ppm best case mass accuracies have been reported.44 Sample Introduction. Volatile hydrocarbon mixtures (i.e., boiling point less than ∼340 °C) were introduced into the mass spectrometer via a heated reservoir inlet. The maximum temperatures of the reservoir and interface lines were 300 °C. Heavier hydrocarbon mixtures (i.e., boiling point higher than ∼340 °C) were introduced via a direct liquid introduction (DLI) system.45 Dilute solutions (e.g., 1000 ppm) of hydrocarbons in suitable solvents (e.g., cyclohexane, toluene) were infused using a HewlettPackard 1090 HPLC unit. The HPLC unit was interfaced with the mass spectrometer via a 100-µm-i.d. fused-silica capillary transfer line. The length of the transfer line was adjusted to maintain pressures lower than 5 × 10-5 Torr in the ionization housing at flow rates of 2-6 µL/min. For the DLI experiments, the ionization source temperature was 280 °C and all interface thermal zones were maintained at 230 °C. The heated reservoir inlet and the DLI systems provided stable ion signal for the duration of the MS/MS experiments. Introduction of polar and ionic specialty chemicals was done via an ESI system coupled to the HP 1090 HPLC unit. Weak concentrations (i.e., less than 10 ppm) of samples in methanol were infused into the mass spectrometer using the HPLC unit at flow rates ranging between 5 and 50 µL/ min. Ionization. Chemical ionization (CI), low voltage (∼10 eV), and conventional (70 eV) electron ionization (EI) methods were used for the analysis of the hydrocarbon mixtures. For samples introduced via the heated reservoir inlet and the DLI system, the accelerating voltage was 8 and 4 kV, respectively. The use of small hydrocarbons as solvents in the DLI experiments enables chemical ionization experiments without the need of reagent gases. The solvents serve as reagent compounds when a CI plug-in source volume is used. Chemical ionization using small hydrocarbons as reagent compounds is a soft ionization technique leading to the production of mass spectra with abundant molecular ion species. Charge exchange chemical ionization with small aromatic compounds has two significant advantages over low-voltage ionization: (1) higher sensitivity and (2) uniform response factors for a wide range of compounds.21 The analysis of polar and ionic samples was done using electrospray ionization.22 The method does not normally permit the analysis of nonpolar hydrocarbons but is ideally suited for the analysis of trace amounts of polar and ionic compounds. Nitrogen was used as both bath and nebulizer gas. The ESI interface temperature was 90 °C. The needle, sampling cone, skimmer lens, and ring electrode voltages were tuned to optimize sensitivity. For some samples (e.g., acidic fractions) the formation of dimers, trimers, etc., was possible and could be controlled by the sampling cone voltage. The accelerating voltage for the ESI experiments was 4 kV. Poly(propylene glycol) (PPG) mixtures were used to calibrate the mass scale in positive ion mode experiments and a mixture of perfluorinated n-carboxylic acids (43) Medzihradszky, K. F.; Adams, G. W.; Burlingame, A. L.; Bateman, R. H.; Green, M. R. J. Am. Soc. Mass Spectrom. 1996, 7, 1-10. (44) Keough, T.; Lacey, M. P.; Ketcha, M. M.; Bateman, R. H.; Green, M. R. Rapid Commun. Mass Spectrom. 1997, 11, 1702-1708. (45) Gagne, J.-P.; Roussis, S. G.; Bertrand, M. J. J. Chromatogr. 1991, 554, 293304.

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Figure 2. Automated MS/MS mapping of all precursor ions in a selected mass range by continuous oa-TOF product ion acquisition and simultaneous slow scanning of the magnet.

was used in negative ion mode experiments.46 Selected precursor ions in the calibrant mixtures were used to tune the instrument for maximum MS-2 ion beam transmission and resolution. The product ion mass spectra of the precursor ions served as calibrants for the external calibration of the oa-TOF mass scale. MS/MS Experiments. An external analog signal generator unit (RP Analytical Services Ltd., Manchester, U.K.) was installed to scan the magnet independently of the existing SIOS data system interface. The original Micromass system did not allow for simultaneous magnet scanning and oa-TOF data acquisition. The magnet reference signal from the external generator (output port pins 2 and 3) was inserted into pins 2 (return) and 6 (reference) of the BN02 connector (9-pin D type). This connector is on the 3563-200 ZabSpec magnet control board. A breakout cable and switch system was constructed that permits the performance of experiments under standard SIOS control or optional magnet control by the external signal generator. The external signal generator unit (0-10-V output voltage) permits linear and exponential scans starting from low (scan-up) or high (scan-down) mass limits. The magnet can be scanned at variable rates ranging from 0.1 to 7 × 103 s/decade. For high-resolution precursor ion selection experiments, slower rates were made possible (i.e., 5.25 × 104 s/decade) by modification of the scanning rate control circuit. The simultaneous MS/MS experiment is illustrated in Figure 2. The entire MS/MS domain is mapped by continuously acquiring spectra with the oa-TOF while the magnet is slowly being scanned over the mass range of interest. The typical oaTOF acquisition rate was 0.2 s/mass range. Typical scan delay times ranged between 0.1 and 0.5 s. A plot relating the magnet reference voltage to the masses of a calibration mixture was used to select the upper and lower mass limits for the scan. Figure 3 shows such a plot obtained at 4000and 8000-V accelerating voltages. The plots are nonlinear due to the scanning properties of the magnet. The continuous scan permits the exhaustive acquisition of the product ion spectra of all precursor ions in a given mass range. Once the upper and lower mass range limits are selected, acquisition of all product ion spectra in the mass range is done automatically without the need for manual selection of individual precursor ions. This feature greatly simplifies the analysis of complex petroleum fractions that contain peaks at almost every nominal mass. Since product ion spectra are acquired by the oa-TOF, there is no need for calibration of the magnet in low resolving power (46) Haas, M. J. Rapid Commun. Mass Spectrom. 1999, 13, 381-383.

smaller losses due to scattering. The pressure in the MS-2 collision cell was set to attenuate the main beam of a standard precursor ion peak (e.g., m-xylene ion at m/z 106 in EI, or PPG ion at m/z 447, positive ion ESI) to ∼ 50% of its original value. Samples. Standard compounds were obtained from Aldrich and Sigma. Petroleum samples and specialty chemicals were obtained from Imperial Oil, Ltd., Products and Chemicals Division (Sarnia, ON, Canada).

Figure 3. Plot relating the magnet reference voltage to the precursor ion mass scale at 4000- and 8000-V accelerating potentials. The plot is prepared using mixtures of compounds with known masses and serves for the selection of precursor ion mass range limits in the automated magnetic sector/oa-TOF MS/MS experiment.

experiments. Calibration of the oa-TOF mass scale suffices for mass assignment of the peaks. For high resolving power experiments, a plot similar to that shown in Figure 3 that relates the mass scale to the magnet scan time can be used to accurately measure the masses of the precursor ions. The calibration plot is prepared from the exact masses of known peaks (internal standards) in the precursor ion spectrum. Accurate mass measurement of the product ion spectrum can be done from the known exact mass of the precursor ion and at least one additional peak in the spectrum. This is generally possible for hydrocarbon compounds that commonly generate alkyl fragments that can be used as internal calibrants. The presence of several fragment ions (e.g., a series of alkyl fragment ions) eliminates the need for calibration of the precursor ion spectrum since the mass of the precursor ion can be determined by using the alkyl fragments as internal mass calibrants. When the Micromass digital-to-analog converter (DAC) is used to select precursor ions, the ability to select ions with specific m/z ratios is limited by the resolution of the DAC. As discussed elsewhere,44 the 16-bit Micromass DAC that controls the magnet current is too coarse and cannot be used for the accurate selection of precursor ion beams at maximum ion currents (peak tops). Moreover, the number of product ion spectra obtained across the precursor ion peak is also limited by the resolution of the DAC. This becomes particularly problematic as the resolving power increases and the mass peak width is reduced. On the other hand, the number of product ion spectra obtained across the precursor ion peak using the analog magnet scan only depends on the scanning rate of the magnet and the data acquisition rate of the oa-TOF. This is an especially important feature of the analog scan because at slow magnet scan rates it permits the accurate profiling of precursor ion peak shapes and the acquisition of product ion spectra for all isobaric ion species. Collision energies of 800 and 400 eV were used for the MS/ MS experiments at accelerating potentials 8 and 4 kV, respectively. The smallest mass range possible was acquired by the oa-TOF in order to increase the pulse repetition rate and maximize the total ion current (TIC) of the product ion spectra. Helium, argon, and xenon were evaluated as target collision gases. Argon was selected as most suitable target gas for the current work for its ability to transfer adequate amounts of internal energy while causing

RESULTS AND DISCUSSION The current approach is related to methods originally developed for the automated mapping of metastable ions produced in the field-free regions of magnetic sector instruments.47 The mapping was originally done by fast magnetic field scanning while the electric sector field was stepped in small intervals47-49 or, alternatively, by fast electric field scanning combined with slow magnetic sector field scanning.50,51 These early attempts recognized the unique capabilities of the product ion mapping for the characterization of complex mixtures, but they did not permit accurate precursor ion selection and accurate product ion mass assignment due to the ion optics limitations of the linked scans.41,47 The current work presents an advancement over the early metastable ion mapping methods by using the double-focusing properties of the magnetic sector (MS-1) to accurately select precursor ions and the oa-TOF (MS-2) to acquire product ion spectra with better that unit mass resolution. The routine application of the approach and typical results obtained from the analysis of representative petroleum systems are presented below. Characterization of Simple Systems. (1) m-Xylene by CS2 Charge Exchange. The analysis of a simple mixture of compounds using the developed approach is demonstrated in Figure 4. Charge exchange (CE) chemical ionization with CS2 as reagent compound was used to analyze m-xylene. CE with CS2 is a soft ionization technique suitable for the analysis of aromatic hydrocarbon compounds.21 Both CS2 and m-xylene were introduced into the ionization source via the heated reservoir inlet. m-Xylene is a common calibrant used to tune low voltage ionization experiments for the analysis of petroleum mixtures.21 Figure 4A shows the total ion current of the spectra acquired by the oa-TOF. The low and high mass limits were selected using the plot shown in Figure 3. Accurate knowledge of the mass limits is not necessary since masses in the product ion mass spectra are assigned independently of the magnet by external mass calibration of the oa-TOF. The peaks in Figure 4A contain the total ion current from both surviving precursors and product ions. The magnet was scanned starting from the low mass limit in order to align the higher masses with the longer acquisition times. Figure 4A shows both scan and time x-axes. The scanning rate of the magnet depends on the number of product ion spectra required per precursor ion peak and the time required for acquisition of each product ion spectum by the oa-TOF. Typically the oa-TOF acquisition rate is (47) Farncombe, M. J.; Mason, R. S.; Jennings, K. R.; Scrivens, J. Int. J. Mass Spectrom. Ion Phys. 1982, 44, 91-107. (48) Warburton, G. A.; Stradling, R. S.; Mason, R. S.; Farncombe, M. J. Org. Mass Spectrom. 1981, 16, 507-511. (49) Farncombe, M. J.; Jennings, K. R.; Mason, R. S.; Schunegger, U. P. Org. Mass Spectrom. 1983, 18, 612-616. (50) Coutant, J. E.; McLafferty, F. W. Int. J. Mass Spectrom. Ion Phys. 1972, 8, 323-339. (51) Fraefel, A.; Seibl, J. Int. J. Mass Spectrom. Ion Phys. 1983, 51, 245-254.

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Figure 4. Characterization of m-xylene by CS2 charge exchange and automated magnetic sector/oa-TOF MS/MS scanning: (A) total ion current from both surviving precursor and product ions; (B) product ion spectrum (MS/MS) of m-xylene (m/z 106); (C) product ion spectrum of the m-xylene isotopic peak at m/z 107; (D) product ion spectrum of the (CS2)S+ ion at m/z 108. The magnetic sector is scanned slowly while the oa-TOF acquires data at an effective scan rate of 1.7 product ion spectra/s.

fixed (0.2 s/mass range, 0.1-0.5 s interscan delay time) and 1020 product ion spectra are obtained per precursor ion peak. For example, a total of 380 product ion spectra were obtained in the experiment of Figure 4A over a time period of 3:50 min with an effective rate of 1.7 spectra/s. The total number of product ion spectra and total time for the experiment depend on the resolving power used for the selection of the precursor ions. More peaks are resolved at higher resolving powers requiring longer overall experimental time to maintain the acquisition of 10-20 product ion spectra per precursor ion peak. We have found that precursor ions produced by electron and chemical ionization methods usually contain high ion current signals leading to the production of abundant product ion spectra. In that case, it is possible to reduce the number of product ion spectra acquired per precursor ion peak for the benefit of shorter experiments. Fast magnet scanning rates producing as few as 4 or 5 scans per precursor ion peak are commonly acceptable for ions produced by EI and CI. Conversely, for ionization techniques such as electrospray that are typically associated with low ion current signal for the precursor ions, slower magnet scanning rates (e.g., 20 or more 3616 Analytical Chemistry, Vol. 73, No. 15, August 1, 2001

scans per precursor ion peak) significantly improve the quality (S/N) of the product ion spectra. The product ion spectrum of m-xylene (m/z 106) is shown in Figure 4B. The spectrum shows the characteristic fragmentation pattern of alkylbenzenes. The spectrum can be interpreted from first principles or by comparison with the spectra in standard 70 eV EI libraries of spectra (e.g., NIST, Wiley). We have found that collisional activation with argon or xenon at multiple collision pressure regimes leads to extensive structure dissociation with fragmentation patterns similar to those produced by high energy processes (70 eV EI) in the ionization source of a mass spectrometer. The most important benefit of this feature is the rapid identification of common organic compounds by direct comparison of the product ion spectra with the spectra in existing libraries. For the larger polynuclear aromatic hydrocarbon structures, we have found that it is possible to produce more extensive fragmentation by collisional activation than it is possible by 70 eV EI. For example coronene (C24H12), a seven-ring aromatic structure produces a simple spectrum under 70 eV EI consisting mainly of peak clusters close to the molecular ion (m/z 300) and the doubly charged ion at m/z 150. Collisional activation using argon or xenon produces extensive fragmentation with abundant groups of peaks at every carbon number ranging from C2 to C22. The total effective energy acquired by the precursor and product ions in the multicollision activation process can be significantly higher than that transferred by the 70 eV EI process. In this case, identification of the structure by comparison with existing 70 eV EI libraries is not possible but the interpretation is greatly aided by the presence of the large number of fragment ions. The product ion spectrum shown in Figure 4B was produced by selection of a single precursor ion at m/z 106 and it does not therefore contain peak contributions from the isotope at m/z 107. However, identification of m-xylene by comparison with the library spectra is not affected due to the small overall contribution of the isotopic peaks. The product ion spectrum of the m-xylene isotope at m/z 107, obtained independently of the m/z 106 ion, is shown in Figure 4C. A composite product ion spectrum can be produced by the weighted addition of the m/z 106 and m/z 107 product ion spectra (Figures 4B, 4C) with overall isotopic distributions very similar to those in the conventional 70 eV EI m-xylene spectrum. Although the product ion spectra of isotopic peaks can be complex, the data shown here indicate that the isotopic product ion spectrum provides an additional fingerprint for the identification of unknown molecular ions. For example, the masses of the fragment ion peaks in the isotopic product ion spectrum (Figure 4C) can be used to validate the fragment ion structures proposed in the mono-isotopic product ion spectrum (Figure 4B). Shifts in the masses of fragment ions in the poly-isotopic product ion spectrum should be consistent with the proposed fragment ion structures in the mono-isotopic spectrum. The complexity of the product ion spectra can be controlled by experimental parameters such as the collision energy, target gas and collision cell pressure. The MS-1 spectrum shown in Figure 4A contains an additional abundant peak at m/z 108. As a first possibility this ion may be due to an impurity in the heated reservoir inlet or in the m-xylene sample. Its product ion spectrum is shown in Figure 4D. The abundant peaks at even masses (e.g., 32, 44, 64, 76) in the production spectrum can be rationalized by the precursor ion

Figure 5. Characterization of mixture of surfactants by negative ion ESI MS: (A) MS-1 spectrum of the mixture; (B) product ion spectrum of the peak at m/z 339; (C) product ion spectrum of the peak at m/z 325; (D) product ion spectrum of the peak at m/z 311. High resolution (∼10 000, 10% valley criterion) was used for accurate mass measurement (MS-1) of the components in the mixture. Automated magnetic sector/oa-TOF MS/MS scanning was used to acquire the product ion spectra of all precursor ions in the mass range m/z 295-370.

structure (CS2)S+. This structure is formed in the charge exchange chemical ionization process when CS2 is used as reagent compound17 and is not an impurity in the sample or sample inlet. (2) Mixture of Surfactants by Negative Ion ESI MS. The MS-1 spectrum of a polar extract from a refinery product analyzed by negative ion mode ESI MS is shown in Figure 5A. The spectrum consists of an abundant peak at m/z 325 and several other peaks of lower abundance at masses ranging from m/z 269 to 367. The masses differ by 14 mass units (CH2), indicating the presence of a distribution of compounds belonging to the same homologous series. Characterization of such mixtures of compounds represents one of the most common requests in the petrochemical analytical laboratory. The mixtures may be polar extracts from products not meeting specifications, residues collected from refinery filters, or mixtures of additives. The objective of the analysis is to determine the chemical compositions and structures of the individual components in the mixture. We have found that electrospray ionization is suitable for the analysis of nonboiling or thermally labile polar and ionic compounds that are not amenable to GC/MS analysis. Since the nature of the compounds in the mixture is completely unknown, the first experiment deals with the determination of the chemical formulas of the compounds. This was accomplished by accurate mass measurement (MS-1) at a resolution of ∼10 000 (10% valley criterion). For the analysis of unknown mixtures, accurate mass

measurement at high-resolving power is needed in order to separate isobaric peaks and reduce the total number of possible chemical formulas. The accurate mass measurement produced an experimental value of 325.1806 for the most abundant ion in the spectrum shown in Figure 5A. This experimental mass, within a 10 ppm margin of error, corresponds to two possible chemical formulas: (1) C21H25O3 (-0.8 ppm error) and (2) C18H29O3S (9.6 ppm error). The calculations were done by assuming the presence of a maximum number of five atoms of oxygen and one of sulfur. Isotopic pattern considerations indicated that most likely the compounds do not contain other polyisotopic elements. However, several monoisotopic elements could be present. Clearly, the accurate mass analysis results are not sufficient for the determination of the chemical composition of the unknown compounds. Additional information is needed to decide which of the two possible formulas is the formula of the unknown compounds. The additional information was obtained from the automated MS/MS experiment. The ability to perform an automated MS/MS experiment is particularly convenient and time-efficient for the analysis of mixtures of compounds because all compounds in the mixtures are analyzed without the need for manual positioning of the magnet to select precursor ion peaks on an individual basis. The present analysis was done by direct infusion of the sample into the mass spectrometer using the HP 1090 LC unit. The total product ion acquisition time for all precursor ions between m/z 295 and 370 was ∼10 min. The similarity of the product ion spectra obtained for the different precursor ions (Figure 5B-D) validated the expectations of structural similarity between the mixture components. The product ion peak at m/z 80 is characteristic of the SO3- moiety. This ion validates the presence of a sulfur atom in the chemical formula and reveals that the mixture components are homologues of the CnH2n-7SO3 compound series with n ranging from 14 to 21. The other peaks in the spectrum at m/z 183, 197, 211, etc., are also consistent with previously reported MS/MS spectra of alkylbenzenesulfonate surfactant molecules.52 The automated MS/MS experiment conveniently produced structural information about all mixture components in ∼10 min (75 mass units). In that fashion, the overall time needed for the MS/MS experiment was reduced as compared to manual precursor ion selection and more time was available for interpretation of spectra. The availability of all product ion spectra in a single file greatly facilitated spectral comparisons. Common product ions (e.g., m/z 80) could be extracted to identify their corresponding precursor ions (precursor scan). All relationships between precursor and product ions (product, precursor, neutral scans) can be elucidated by reduction of the data obtained in the automated MS/ MS experiment. By using a computer program it is possible to rapidly determine the presence or absence of selected compound types. Quantitative methods similar to those available for 70-eV hydrocarbon compound type analysis53 can be developed for the analysis of mixtures of compounds using the MS/MS data. The approach would be particularly useful for the analysis of compounds produced by selective soft ionization methods. For example, by using ammonia CI, it is possible to selectively ionize the nitrogen-containing compounds in hydrocarbon mixtures. The (52) Lyon, P. A.; Stebbings, W. L.; Crow, F. W.; Tomer, K. B.; Lippstreu, D. L.; Gross, M. L. Anal. Chem. 1984, 56, 8-13. (53) Roussis, S. G.; Cameron, A. S. Energy Fuels 1997, 11, 879-886.

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Figure 6. Three-dimensional plot obtained by the automated magnetic sector/oa-TOF MS/MS experiment for a naphtha sample.

Figure 7. Three-dimensional plot obtained by the automated magnetic sector/oa-TOF MS/MS experiment for a middle-distillate sample.

automated MS/MS experiment could then be used to provide quantitative information about the different nitrogen compound types by previous determination of the response factors using mixtures of known compositions. The current approach requires continuous infusion of the sample and is not compatible with on-line LC separation. This prevents the identification of all possible isomeric structures in the sample having identical precursor ion masses. However, this is not a major limitation of the method for the purposes of the analysis of completely unknown mixtures that mainly require identification of the chemical compositions of the mixture com3618

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ponents. Characterization of the various isomers could be done in a different LC/MS/MS experiment, by the monitoring of a list of precursor ions with known masses as a function of elution time. In that case, selection of precursor ions must be done by accurate voltage calibration in a manner analogous to SIR experiments.40 As discussed before, this experiment is rather involved to perform. A possible further improvement of the MS/MS approach developed here that would permit the performance of automated LC/MS/MS experiments involves a fast magnet prescan to first determine the mass ranges of interest in the MS-1 spectrum. Then the magnet can be scanned slowly over the mass ranges of interest

while the oa-TOF keeps acquiring data continuously. The total possible number of precursor ions selected by this approach will be, however, limited by the chromatographic peak width. The wider the LC peak width, the more precursor ions can be analyzed by the magnetic sector/oa-TOF system. The usefulness of such a data-dependent LC/MS/MS experiment is reduced as the complexity of the mixtures increases. For the analysis of common petroleum mixtures that contain peaks at almost every mass, the automated magnetic sector/oa-TOF MS/MS acquisition is much more suitable than a data-dependent LC/MS/MS experiment. The analysis of representative petroleum mixtures is illustrated in the sections below. Analysis of Complex Systems. Naphtha and Middle Distillate Cuts. A three-dimensional plot of a naphtha petroleum cut (maximum boiling point ∼210 °C) obtained by the automated magnetic sector/oa-TOF MS/MS system is shown in Figure 6. Ionization was done by CS2 charge exchange. The reagent compound and the naphtha sample were both introduced into the mass spectrometer via the heated reservoir inlet. The flow from the reservoir into the mass spectrometer remained approximately constant for the duration of the experiment. The precursor mass range m/z 104-145 was analyzed in ∼16 min. The magnet reference voltage scanning rate was 7000 s/decade. The most abundant peaks are at even masses corresponding to alkylaromatic and naphthenic hydrocarbons. Peaks at odd masses are primarily due to isotopes and low-abundance fragment ions. The plot obtained from the analysis of a middle-distillate cut (boiling range 200-343 °C) is shown in Figure 7. Low voltage (∼20 eV) was used for the ionization of the sample. The sample was introduced into the mass spectrometer via the heated reservoir inlet. Abundant peaks were obtained at both even and odd masses. The mass range m/z 104-218 was analyzed in ∼34 min. Magnet scanning was started, as in the naphtha analysis, at the low-mass limit in order to align the higher masses with the longer acquisition times. The resolving power for the separation of precursor ions was ∼2000 (MS-1). Approximately seven product ion spectra were obtained for each precursor ion peak across the scanned mass range. In Figures 6 and 7, the MS-1 axis (magnet scanning) is given in time units (min) showing the acquisition time required for the experiment. The oa-TOF acquisition axis (MS-2) is given in units of mass (m/z). This is because the data are acquired and mass-assigned by the oa-TOF system. Changes in the magnet scanning rate affect the number of product ion spectra acquired per precursor ion peak but do not affect the mass assignment of the data by the oa-TOF system. Similar but more complex plots are obtained from the analysis of heavier petroleum samples (e.g., boiling range 343-565 °C). Due to their lower volatility, these samples are introduced into the mass spectrometer using the DLI system. The increased complexity of the heavier samples necessitates slower magnet scanning rates in order to increase the number of product ion spectra obtained per precursor peak. High collision energy conditions typically lead to the production of abundant low-mass product ions providing characteristic information about the different hydrocarbon compound types in a fashion analogous to that obtained under 70-eV EI. Low collision energy conditions generate product ion spectra with limited fragmentation containing abundant higher mass product ions, which are frequently isomer-specific.

Figure 8. Isobaric peak component identification in naphtha fraction: (A) expanded view of total ion current at m/z 124; (B) product ion spectrum of lower-mass precursor ion (m/z 124.035); (C) product ion spectrum of higher mass precursor ion (m/z 124.125).

Separation and Identification of Mixture Components. A set of two interesting isobaric peaks was detected in the naphtha sample at masses m/z 124 and 138. An expanded view of the composite MS-1 spectrum at m/z 124 is shown in Figure 8A. Panels B and C of Figure 8 show the product ion spectra obtained for the two isobaric ion species. The product ion spectrum of the lower mass precursor ion is consistent with a structure having the C7H8S composition. The product ion mass at m/z 45 (CHS+) is a characteristic fragment ion of sulfur compounds.54 The product ion spectrum of the higher mass precursor ion (Figure 8C) is consistent with a structure having the C9H16 chemical composition. Further identification of a specific structure is not possible since the spectra are most likely produced from the dissociation of a mixture of possible isomeric structures. Comparison of the product ion spectrum containing the characteristic peak at m/z 45 (Figure 9A) with the NIST library of spectra produced good matches with the two benzenethiol structures shown in Figure 9B and C. The similarity with the library spectra validates the proposed chemical composition of the mixture component. Several other isobaric sets of compounds have been detected in the naphtha and middledistillate samples by similar comparisons with the NIST library of spectra. Contour Plots. We have found that of particular usefulness in the detection of isobaric ions is a contour plot obtained by (54) Gallegos, E. J. Anal. Chem. 1975, 47, 1150-1154.

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Figure 10. Contour plot of the isobaric peak set at m/z 138 in the naphtha sample (Figure 6). Figure 9. Peak component identification in naphtha fraction by comparison of product ion spectra with standard spectra in 70-eV EI library: (A) product ion spectrum of unknown component; (B) best match spectrum in 70-eV EI NIST library; (C) second-best match spectrum in 70-eV EI NIST library. The precursor ion at m/z 124 is not shown.

projection of the data in the 3-D plot. This becomes especially important as the precursor ion mass increases and higher resolving powers are needed for peak separation. The usefulness of the contour plot is illustrated in Figures 10 and 11. Figure 10 shows the contour plot obtained for a set of isobaric ions at mass m/z 138 in the naphtha sample, shown in Figure 6. The product ion spectra of the two ions are consistent and the C8H10S and C10H18 compositions, which are the homologues to the compounds at m/z 124 shown in Figure 8. The contour plot clearly shows the precursor and product ions produced by the dissociation of the two isobars. Figure 11 shows a similar contour plot obtained for the set of isobars at m/z 170 in the 3-D plot of the middle-distillate sample. The product ion spectra of the two isobars are consistent with the C13H14 and C12H26 structures. The method provides for the separation of ions requiring resolving powers higher than those used or possible. Although the instrument resolving power may not be adequate to separate overlapping precursor ions, the contour plot of the product ion spectra can indicate the presence of the different isobars provided that enough product ion spectra are acquired across the precursor ion peaks. A computer program such as AMDIS could be used to automatically separate the overlapping product ion spectra.55 Characteristic Product Ion Spectra of Isobaric Hydrocarbons. The 20-eV EI allowed the ionization of both saturated and (55) Stein, S. E. J. Am. Soc. Mass Spectrom. 1999, 10, 770-781.

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Figure 11. Contour plot of the isobaric peak set at m/z 170 in the middle-distillate sample (Figure 7).

aromatic hydrocarbons in the middle-distillate sample (Figure 7). A resolving power of ∼2000 is adequate for the separation of compound types with the general formulas CnH2n+2 (paraffins) and CnH2n-12 (naphthalenes) at m/z 200 (10% valley criterion).21 These compound types are present in the middle-distillate sample.

Figure 12. Product ion spectra of selected paraffins in the middledistillate sample: (A) precursor ion at m/z 142, C10H22+; (B) precursor ion at m/z 156, C11H24+; (C) precursor at m/z 170, C12H26+.

Characteristic product ion spectra of some paraffins and naphthalenes are shown in Figures 12 and 13, respectively. Collisional activation using argon led to extensive fragmentation for both types of molecules. For example, the spectra in Figure 12 contain abundant low-mass alkyl fragments, characteristic of paraffinic structures. Similarly, extensive fragmentation is shown in the product ion spectra of the naphthalenes in Figure 13. As discussed previously, more fragmentation is possible for the aromatic structures by collisional activation than by 70-eV electron ionization. Depending on the application, it is possible to obtain less fragmentation, for example, by reduction of the collision energy, use of helium as target gas, or reduction of the collision cell pressure. The main benefit of such experiments would be the generation of simpler, low-energy product ion spectra. Frequently, low-energy spectra contain structure-specific product ions that can be used to differentiate isomeric structures. Although experiments with model compounds are required to predetermine the fragmentation patterns of the different isomeric structures, low-energy MS/MS experiments can be very powerful since they have the potential to characterize both isobaric and isomeric structures. Ion Current Signal and Resolving Power Considerations for ESI MS/MS. We have found that conventional electron and chemical ionization methods are ideally suited for the automated MS/MS experiment because they generate precursor ions with abundant ion current signals. The ion current in these ionization sources increases with compound concentration. The ability to increase the ion current signal as a function of compound concentration is very important for magnetic sector instruments that permit higher resolving powers at the expense of ion signal.

Figure 13. Product ion spectra of selected naphthalenes in the middle-distillate sample: (A) precursor ion at m/z 142, C11H10+; (B) precursor ion at m/z 156, C12H12+; (C) precursor at m/z 170, C13H14+.

That is, performing the automated MS/MS experiments at higher resolving power conditions using a magnetic sector instruments requires the production of abundant precursor ions in the ionization source so that there is enough ion current to be collected by the oa-TOF system. We have found that the electrospray ionization source, although suitable for the analysis of compounds in very weak concentrations, has the limitation that the ion current signal does not increase above a threshold amount that is determined by the nature and concentrations of the different compounds in the sample. We have examined the practical limitations of the automated MS/MS experiments when electrospray is used as ionization source. Figure 14 shows a set of isobaric peaks consisting of two model compounds (decylamine, 2,6-dimethylquinoline) at m/z 158. A mixture of the two compounds was prepared in methanol (∼1 ppm each compound). The solution was infused into the mass spectrometer at a flow rate of ∼5 µL/min. An exponential downscan was used for the scanning of the magnet (i.e., highmass peak is acquired first). The total ion current of the precursor ions analyzed at ∼4000 fwhh resolution (∼2500 10% valley criterion) is shown in Figure 14A. The total ion current of the precursor ions analyzed at ∼8000 fwhh resolution (∼5000 10% valley criterion) is shown in Figure 14B. The magnet was scanned at the same rate for both experiments (5.25 × 104 s/decade). This scanning rate produced ∼10 product ion spectra for the 8000 resolution experiment and ∼20 for the 4000 resolution experiment. The product ion spectra obtained for the two compounds at the 4000 fwhh resolution experiment are shown in Figure 15. Extensive fragmentation is obtained in the spectrum of decylamine Analytical Chemistry, Vol. 73, No. 15, August 1, 2001

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aliphatic amine structure but not for the quinoline. The difference in the detection limits is only due to the ion structures of the two molecules. Argon transfers sufficient amounts of internal energy for the dissociation of both structures, but losses due to scattering and the other collision processes are more significant for the more stable aromatic structure. The use of xenon as target gas did not greatly enhance the extent of fragmentation, but it reduced the overall product ion signal obtained for both structures. With the conventional ionization sources, lower detection limits can be obtained by increasing the precursor ion signal. Unfortunately, even a 10-fold increase in the model compound concentrations did not increase the precursor ion signal in ESI MS. We found that lower detection limits for the 2,6-dimethylquinoline structure were possible at 8000 and higher resolving powers by acquiring more product ion spectra (e.g., 30 or more). It may be necessary to decrease the magnet scanning rate as the resolving power increases in order to lower the detection limits of precursors ions formed by electrospray ionization. Figure 14. Precursor ion signal and resolving power considerations in ESI MS: (A) total ion current of decylamine (C10H24N) and 2,6dimethylquinoline (C11H12N) precursor ions obtained at ∼4000 (fwhh) precursor ion resolution (1 ppm of each compound in methanol); (B) total ion current of decylamine and 2,6-dimethylquinoline precursor ions obtained at ∼8000 (fwhh) precursor ion resolution.

Figure 15. ESI MS at ∼4000 (fwhh) precursor ion resolution: (A) 2,6-dimethylquinoline product ion spectrum; (B) decylamine product ion spectrum.

(Figure 15B) using argon as target gas at collision pressures leading to ∼50% main beam attenuation. Extensive fragmentation is also observed in the product ion spectrum of 2,6-dimethylquinoline (Figure 14B). The product ion spectra shown in Figure 15 were obtained by the summation of ∼20 spectra acquired in the automated experiment at ∼4000 precursor ion fwhh resolution (Figure 14A). The limit of the automated MS/MS method using electrospray ionization was reached in the acquisition of the product ion spectra at 8000 fwhh precursor ion resolution (Figure 14B). The composite product ion spectrum for the decylamine precursor ion was abundant enough for spectral interpretation but not the spectrum of the 2,6-dimethylquinoline structure. The ion signal obtained by summation of the ∼10 product ion spectra in the 8000 fwhh experiment was enough for the detection of the 3622

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CONCLUSION We have demonstrated the value of an automated magnetic sector/oa-TOF MS/MS method for the analysis of complex petroleum samples. The usefulness of the method increases with the complexity of the samples. The method permits the exhaustive acquisition of the product ion spectra of all precursor ions in a selected mass range by continuous oa-TOF data acquisition and simultaneous scanning of the magnet. Precursor ions can be selected under high resolving power conditions using the doublefocusing properties of the magnetic sector, and product ion spectra with accurate masses can be obtained by the oa-TOF system. The automated MS/MS approach greatly simplifies the original selection of precursor ions that was done by manual adjustment of the magnet current for the transmission of the precursor ions on an individual basis. The use of an external analog signal generator overcomes the limitations of mass selection by a digital-to-analog converter and permits the accurate profiling of precursor ion peaks. In that manner, the characterization of isobaric precursor ions is possible. The automated MS/MS method can provide critical additional information to that obtained from accurate high-resolution mass measurement experiments for the characterization of completely unknown systems. Such an example was given where the automated MS/MS method helped elucidate the nature of the components in a polar extract of a refinery product. The ability of the method to acquire the product ion spectra of all precursor ions in the selected mass range led to the rapid identification of all compounds in the sample. Further developments of the method could permit the performance of data-dependent MS/MS experiments; however, the utility of such experiments diminishes as the complexity of the mixtures increases. Characterization of simple molecules using the automated MS/ MS method was possible by simple comparison of the product ion spectra with the spectra in standard 70-eV EI libraries. For the heavier aromatic structures, the use of argon as target gas and high collision energy regimes led to more extensive fragmentation than that possible by 70-eV EI. The results obtained from the analysis of common petroleum fractions indicated that it is possible to develop novel MS/MS compound-type analysis

methods for the analysis of selected groups of compounds in complex mixtures by selective soft ionization techniques (CI, ESI, etc.). The use of low-energy collisional activation may further enhance the capabilities of the method and permit the differentiation of isomeric structures. Conventional ionization techniques such as EI and CI form precursor ions with abundant ion current signals and permit the performance of the automated MS/MS experiments at high magnetic sector resolving powers. However, the maximum resolving power for the separation of precursor ions formed by ESI is limited by the ion current signal, which does not increase above a maximum value as a function of concentration. Improvements in the detection limits of precursor ions formed by ESI are possible

by decreasing the magnet scanning rate as the resolving power increases. ACKNOWLEDGMENT The author thanks M. R. Green of Micromass, Inc., for his helpful discussions and recommendations about the external signal generator, B. J. Young for the installation of the external signal generator, and Dr. D. J. Bristow for his useful comments and review of the manuscript. Received for review February 26, 2001. Accepted June 1, 2001. AC0102260

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