Anal. Chem. 2010, 82, 9336–9343
Resistive Glass IM-TOFMS Kimberly Kaplan,† Stephan Graf,‡ Christian Tanner,‡ Marc Gonin,‡ Katrin Fuhrer,‡ Richard Knochenmuss,‡ Prabha Dwivedi,† and Herbert H. Hill, Jr.*,† Department of Chemistry, Washington State University, Pullman, Washington 99164, United States, and Tofwerk AG, Thun, Switzerland The design of a new ion mobility mass spectrometer (IMMS) is presented. This new design features an ambientpressure resistive glass ion mobility drift tube (RGIMS) coupled to a high-resolution time-of-flight mass spectrometer (TOFMS) by an enhanced interface that includes two segmented quadrupoles. The interface design demonstrates an increase in sensitivity while maintaining high resolving power typically achieved for ambient-pressure IMS drift tubes. Performance of the prototype instrument was evaluated and the analytical figures of merit for standard solutions as well as complex samples such as human blood were determined. For a 3 µM solution of caffeine, the peak was collected in 36 s and gave a response of 10 counts/s. The detection limit (defined as 1 count/s) was calculated to be 300 nM concentration of caffeine from the response rate from the 36 s run. Controlled fragmentation of caffeine was achieved through adjustment of voltages applied on the interface lenses. Over 300 tentative metabolites were detected in human blood along with 80 isomers/isobars with ion counts >5. Isotope ratios from extracted mass spectra of selected mobility peaks were used to identify selected metabolite compounds. High separation power for both IMS (resolving power, td/∆tw1/2, was 85) and MS (mass resolving power, m/∆m, maximum was 7000 with a mass accuracy between 2 and 10 ppm) was measured. Developed software for data acquisition, control and display allowed flexibility in instrument control, data evaluation and visualization. Ion mobility mass spectrometry (IM-MS) has increased in popularity as an analytical technique over the past decade. The mobility advantage over mass spectrometry (MS) is multifaceted. Rapid separation of complex mixtures prior to mass spectral analysis, isomer separation, chemical noise reduction, isotope analysis in complex mixtures, and mass-mobility correlation for compound class identification1-6 are some of the value-added advantages that IMS brings to MS. All of these characteristics * To whom correspondence should be addressed: Tel 509-335-5648; e-mail
[email protected]. † Washington State University. ‡ Tofwerk AG. (1) Karimi, A.; Alizadeh, N. Talanta 2009, 79, 479–485. (2) Clowers, B.; Dwivedi, P.; Steiner, W.; Hill, H. H.; Bendiak, B. J. Am. Soc. Mass Spectrom. 2005, 16, 660–669. (3) Jackson, S.; Wang, H.-Y.; Woods, A. S.; Ugarov, M.; Egan, T.; Shultz, A. J. Am. Soc. Mass Spectrom. 2005, 16, 133–138. (4) Berant, Z.; Karpas, Z. J. Am. Chem. Soc. 1989, 111, 3819–38924.
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have resulted in the emerging use of IM-MS covering applications in a wide range of analytical fields such as peptide identification and interaction,7-10 drug analysis,11 chemical warfare degradation products,12 and metabolomics.13 In a recent review article, various types of ion mobility mass spectrometers were described and the “workhorse” of IM-MS was defined as a drift tube IMS coupled with a time-of-flight mass spectrometer (IM-TOFMS).14 IM-TOFMS is a comprehensive analysis technique that enables the recording of the complete mass spectra of all components separated by the IMS.15,16 Conventional IMS drift tubes are constructed in a stacked ring design and can be either in vacuum (∼1.3-13 mbar9,10 to 1.33 × 10-5 mbar8) or at ambient pressure (∼920 mbar17). The stacked ring IMS (SRIMS) consists of alternating electrodes and insulators with the electrodes connected by a resistor chain. This construction of a SRIMS is often tedious. The analytical parameters of an ion mobility spectrometry measurement include resolving power (the drift time of the ion, td, divided by the width of the ion peak at half height, ∆t), sensitivity, and selectivity. As a stand-alone system, the SRIMS design has been shown to produce variation in the electric field near the edge of the electrodes that contribute to the deterioration in resolving power.17 However, when SRIMS are operated at ambient pressure, resolving powers are near or above the theoretical resolving power. In general, the resolving power of an ambient-pressure SRIMS will improve when it is interfaced to a mass spectrometer since the pinhole interface (5) Kim, H.; Kim, H. I.; Johnson, P. V.; Beegle, L. W.; Beauchamp, J. L.; Goddard, W. A.; Kanik, I. Anal. Chem. 2008, 80, 1926–1936. (6) Henderson, S.; Valentine, S.; Counterman, A.; Clemmer, D. Anal. Chem. 1999, 71, 291–301. (7) Gillig, K.; Ruotolo, B.; Stone, E.; Russell, D.; Fuhrer, K.; Gonin, M.; Schultz, A. Anal. Chem. 2000, 72, 3965–3971. (8) Valentine, S. J.; Kulchania, M.; Srebalus Barnes, C. A.; Clemmer, D. E. Int. J. Mass Spectrom. 2001, 212, 97–109. (9) Ruotolo, B. T.; Gillig, K. J.; Stone, E. G.; Russell, D. H.; Fuhrer, K.; Gonin, M.; Schultz, J. A. Int. J. Mass Spectrom. 2002, 219, 253–267. (10) Woods, A. S.; Kommen, J. M.; Ruotolo, B. T.; Gillig, K. J.; Russel, D. H.; Fuhrer, K.; Gonin, M.; Egan, T. F.; Schultz, J. A. J. Am. Soc. Mass Spectrom. 2002, 13, 166–169. (11) Steiner, W.; Clowers, B.; Matz, L.; Siems, W.; Hill, H. H., Jr. Anal. Chem. 2002, 74, 4343–4352. (12) Steiner, W.; Clowers, B.; Fuhrer, K.; Gonin, M.; Matz, L.; Siems, W.; Schultz, A.; Hill, H. Rapid Commun. Mass Spectrom. 2001, 15, 2221–2266. (13) Dwivedi, P.; Wu, P.; Klopsch, S.; Puzon, G.; Xun, L.; Hill, H. H., Jr. Metabolomics 2008, 4, 63–68. (14) Kanu, A.; Dwivedi, P.; Tam, M.; Matz, L.; Hill, H. H., Jr. J. Mass Spectrom. 2008, 43, 1–22. (15) Borsdorf, H.; Eiceman, G. A. Appl. Spectrosc. Rev. 2006, 41, 323–375. (16) Collins, D. C.; Lee, M. L. Anal. Bioanal. Chem. 2002, 372, 66–73. (17) Wu, C.; Siems, W.; Asbury, R.; Hill, H. H., Jr. Anal. Chem. 1998, 70, 4929– 4938. 10.1021/ac1017259 2010 American Chemical Society Published on Web 10/22/2010
Figure 1. (A) Schematic diagram of a resistive glass ion mobility spectrometer interfaced to a time-of-flight mass spectrometer via a 300 µm orifice pinhole leak, followed by two segmented quadrupole ion guides and a set of focusing ion lenses. (B, C) Three-dimensional schematic of the resistive glass tube ion mobility spectrometer (B) coupled with high-resolution time-of-flight (TOF) mass spectrometer (C).
is only sampling ions from the center of the drift tube where the electric field is the most uniform. A common challenge when an ambient-pressure IMS is coupled to a MS is the loss of sensitivity at the interface. When the IMS is at ambient pressure and the MS is at ∼10-6 mbar, the ions have to survive the pressure difference through ion guides and lenses, and often ions are lost along the way. The objective of this project was to evaluate the analytical figures of merit including the detection limits, resolving power, sensitivity, and ease of construction for a new IM-TOFMS. The IM-TOFMS consisted of an ambient-pressure resistive glass ion mobility spectrometer (RGIMS) coupled to a high resolution timeof-flight mass spectrometer (TOFMS) by an enhanced interface that includes two segmented quadrupoles. The alternative RGIMS design has been used as a stand-alone system and has demonstrated resolving powers comparable to those of ambient-pressure SRIMS systems.18-20 The highlight of this study is the simple (18) Kwasnik, M.; Fuhrer, K.; Gonin, M.; Barbeau, K.; Fernandez, F. Anal. Chem. 2007, 79, 7782–7791. (19) Kwasnik, M.; Fernandez, F. Rapid Commun. Mass Spectrom. 2010, 1911– 1918. (20) Spangler, G. E.; Vora, K. N.; Carrico, J. P. J. Phys. E: Sci. Instrum. 1986, 19, 191–198.
construction of the RGIMS, unlike SRIMS. For this study, there were two main objectives: (1) determine whether the resolving power of an RGIMS is improved when interfaced to a mass spectrometer and (2) increase the sensitivity of the interface by adding a segmented rf trap to the interface. With the addition of this trap, we investigated the ion transmission efficiency, resolving power, and induced fragmentation after ion mobility separation. In addition to the evaluation of new hardware, new software was evaluated for this RGIM-TOFMS instrument. Evaluation of both hardware and software was accomplished by comparing the response of standards between the SRIM-TOFMS and the RGIMTOFMS. EXPERIMENTAL METHODS Resistive Glass Ion Mobility Mass Spectrometry. Initial experiments with standards and samples were conducted by direct infusion methods. The schematic for the overall instrument is shown in Figure 1A. There were four main sections to the RGIM-TOFMS: (1) electrospray ionization source, (2) ion mobility spectrometer, (3) pressure-vacuum interface, and (4) highresolution time-of-flight mass spectrometer. Electrospray Ionization. A 2 cm long fused silica capillary with internal diameter of 50 µm served as the electrospray needle. One Analytical Chemistry, Vol. 82, No. 22, November 15, 2010
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end of this fused silica needle was the exit where the electrospray ionization occurred, and the other was connected to a low dead volume Valco capillary connector. A fused silica transfer line of approximately 30 cm length and 50 µm ID was used to transfer the sample from the Harvard Apparatus 22 syringe pump, which was used for the direct infusion experiments. The capillary connector was housed in a machined Teflon insulator. The flow rate through the electrospray needle ranged from 1 to 3 µL/min. Resistive Glass Ion Mobility Spectrometer. The ion mobility spectrometer was made from two monolithic resistive glass tubes (Burle Industries Inc., Sturbridge, MA), shown in Figure 1B. The IMS consisted of two regions: reaction region (10 cm) and drift region (20 cm) with an ID of 38 mm and an OD of 47 mm. Metal washers were used as contact rings for the high voltage supply to the resistive glass tubes. The IMS tube was divided into two regions by a Bradbury-Nielson (BN) ion gate. The BN gate was made from 38 µm gold-plated tungsten wires spaced 0.5 mm apart to form a grid across the ion drift space. When the gate was closed, ±50 V was applied to adjacent gate wires to create a 2000 V/cm electric field perpendicular to the 370 V/cm electric field of the drift region. When the gate was opened, the gate wires were held at the potential appropriate for its position along the drift tube, allowing ions to travel into the drift region. The ion gate’s typical pulse width range was 0.2-0.5 ms. Ambient Pressure-Vacuum Interface. After separation in the ion mobility spectrometer, ions entered the mass spectrometer through a 300 µm pinhole leak and were focused by a series of lenses and two segmented quadrupole ion guides. The first segmented quadrupole was operated with a radio frequency (rf) of 5.7 MHz at ∼2 mbar and the second segmented quadrupole had a rf of 4.7 MHz at ∼5 × 10-3 mbar. The DC ion optics was a series of ion lenses that operated at ∼5 × 10-6 mbar. Pumps used for the TOF included a turbo molecular split flow with three stages, one with 10 L/s and the other two with 200 L/s. The 10 L/s stage was pumping the second stage of the interface and the two 200 L/s stages were pumping the ion optics chamber and the TOF chamber. The pumps were purchased from Varian and the models were IDP-3, SH-110, Triscroll-300, and Triscroll-600. Time-of-Flight Mass Spectrometer. The time-of-flight mass spectrometer (TOFMS) used in these studies was a Tofwerk highresolution time-of-flight (HTOF) shown in Figure 1C. Extractor float voltage was 40 V and the acceleration voltage was 3500 V. Ions were detected with a Photonis multichannel plate (MCP). The potential applied to the MCP plates was 2.5 kV. The data acquisition system consists of a timing generator, preamp/ discriminator, a time-to-digital converter (TDC), and a PC. The timing generator triggers the IMS gate, the TOFMS extraction, and the TDC. The TDC was typically operated at 400-800 ps time resolution. The TOFMS extraction frequency was set to 60 kHz with an IMS frequency of 50 Hz; therefore, there were 1200 mass spectra for every IMS scan. Control and Data Acquisition Software. The software used to control both the IMS and TOF timing was developed by Tofwerk. Two different programs were used: (1) IMS-TOF voltage control and (2) timing control, data acquisition, and data evaluation. A multidimensional data set was recorded by the data acquisition system that included mass spectra, mobility spectra, and 9338
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sequences thereof. The latter could be used in combination with additional separation techniques before the IMS-TOF, such as liquid chromatography (LC)-IMS-TOF or gas chromatography (GC)-IMS-TOF. For visualization and data evaluation, different data reduction schemes could be applied to calculate, for example, mass-selected mobility spectra or mobility-selected mass spectra. As the high-resolution mass spectrum data were stored during data acquisition, different masses could be selected in postprocessing. Stacked Ring Ion Mobility Mass Spectrometry. Initial experiments with standards were conducted by direct infusion methods. There were four main sections to the SRIM-TOFMS: (1) electrospray ionization source, (2) ion mobility spectrometer, (3) pressure-vacuum interface, and (4) time-of-flight mass spectrometer. Description of the SR-IMMS system is found in the Supporting Information and described elsewhere.12 Experiments Performed. Analytical Figures of Merit. Solutions of caffeine purchased from Sigma-Aldrich (Switzerland) were prepared in 50:50 2-propanol/water solvent and directly electrosprayed into the RGIM-TOFMS and the SRIM-TOFMS. A 3 µM solution was directly infused into the RGIM-TOFMS at 1 µL/min for 36 s with a 0.2 ms gate pulse width. The temperature of the RGIMS tube was 150 °C, and the maximum voltage applied to the drift tube was 7.4 kV to give an electric field of 370 V/cm in the drift tube at a pressure of ∼950 mbar. In the SRIM-TOFMS experiment, a 3 µM solution was directly infused at 3 µL/min for 30 min with a 0.2 ms gate pulse width. The temperature of the SRIMS was set to 200 °C and the electric field was 453 V/cm in the drift tube at a pressure of ∼920 mbar. Human Blood. Two droplets of human blood were used to assess the capability of the RGIM-TOFMS with a complex sample. Human blood was from one anonymous donor. These data were compared with data obtained for human blood with the SRIMTOFMS that were reported earlier.21 The anonymous donor initially sterilized a finger with an alcohol wipe and then pierced the finger with a sterilized lancet (One Touch Ultrasoft). Two drops of blood were collected in 400 µL of 50:50 2-propanol/water with 0.1% formic acid solvent and centrifuged for 10 min at 13K rpm. The supernatant was then decanted and directly infused for 20 min at a flow rate of 3 µL/min into the IM-TOFMS for a total electrosprayed volume of 60 µL. Metabolite Identification. In order to identify metabolites from these data, the spectral peak list was calibrated. The TOF used in this investigation was capable of 2-10 ppm mass accuracy. For this set of experiments, sodium, potassium, and poly(ethylene glycol) polymer ions were used for internal calibration. Once the spectrum was calibrated, the Human Metabolome database (HMDB)22 was searched by m/z values to obtain a list of potential compounds. The search criteria for tentative metabolite identification included a mass tolerance of ±0.1 Th. By searching only m/z values, multiple compounds were matched as possible metabolites. (21) Dwivedi, P.; Schultz, A.; Hill, H. H. Int. J. Mass Spectrom. 2010 (in press). (22) Wishart, D. S.; Tzur, D.; Knox, C.; Eisner, R.; Chi Guo, A.; Young, N.; Cheng, D.; Jewell, K.; Arndt, D.; Sawhney, S.; Fung, C.; Nikolai, L.; Lewis, M.; Coutouly, M.-A.; Forsythe, I.; Tang, P.; Shrivastava, S.; Jeroncic, K.; Stothard, P.; Amegbey, G.; Block, D.; Hau, D. D.; Wagner, J.; Miniaci, J.; Clements, M.; Gebremedhin, M.; Guo, N.; Zhang, Y.; Duggan, G. E.; MacInnis, G. D.; Weljie, A. M.; Dowlatabadi, R.; Bamforth, F.; Clive, D.; Greiner, R.; Li, L.; Marrie, T.; Sykes, B. D.; Vogel, H. J.; Querengesser, L. Nucleic Acids Res. 2007, 35, D521–D526.
Calculations. To compare measured mobility values for caffeine, the reduced mobility equation was applied:
K0 )
( )(
L2 273.15 P Vtd T 1013
)( )
(1)
where L is the length (cm) of the drift tube, V is the applied voltage across the tube, td is the drift time (s) of the ion, T is the temperature of the drift region (kelvins), and P is the operational pressure (millibars). Conditional resolving power was also calculated for the two IM-TOFMS systems shown in eq 2:23
Rc )
L2 273.15 P VK0 T 1013.24 16kBT ln 2 L2 273.15 P + Vez VK0 T 1013.24
[ ( tg2
)(
)]
2 0.5
(2)
where V is the gate voltage, L is the ion drift tube length, K0 is the reduced mobility, P is the pressure in millibars, T is the temperature in kelvins, td is the measured ion drift time, tg is the gate pulse width, kB is the Boltzmann constant, e is the elementary charge, and z is the charge state of the ion. RESULTS AND DISCUSSION Analytical Figures of Merit Comparison. Analytical figures of merit (FOMs) were compared between the RGIM-TOFMS and the SRIM-TOFMS. The FOMs that were used for this comparison were reduced mobility (K0), IMS resolving power (Rp), and limit of detection (counts per second). A 3 µM standard solution of caffeine was used for the evaluation of the two instruments. Both instruments were operated under similar conditions: ion gates were operated with a 0.2 ms injection pulse and the ratio of IMS scan frequency to TOF scan frequency was 1:1000. The drift gas temperature was 200 °C for the SRIM-TOFMS instrument and 150 °C for the RGIM-TOFMS instrument. The electric fields for the SRIM-TOFMS and RGIM-TOFMS were 453 and 370 V/cm, respectively. The pinhole leading into the TOFMS was 250 µm for the SRIM-TOFMS and 300 µm for the RGIM-TOFMS. The RGIM-TOFMS response rate for detecting a 3 µM solution of caffeine was 10 counts/s, in comparison with 0.1 count/s for the SRIM-TOFMS. The collection time to obtain a response for caffeine was significantly different between the two instruments. The RGIM-TOFMS was able to detect caffeine in 36 s with 360 counts, whereas the SRIM-TOFMS was run for 30 min to accumulate ∼20 counts. By use of the response rate from the 36 s and 30 min runs, the detection limit (defined as 1 count/s) was calculated to be 300 nM in the RGIM-TOFMS and 30 µM in the SRIM-TOFMS for caffeine. The RGIM-TOFMS was found to be 2 orders of magnitude greater in sensitivity for caffeine detection. This increase in sensitivity for the RGIM-TOFMS could have been due to several factors: better transmission through the RGIMS drift tube due to a more homogeneous electric field, the larger pinhole at the interface, or the use of rf fields to more efficiently trap the ions and focus them into them into the TOF. Overall, (23) Kanu, A. B.; Gribb, M. M.; Hill, H. H. Anal. Chem. 2008, 80, 6610–6619.
however, this 2 order increase in sensitivity permitted the collection of spectra more rapidly with the RGIM-TOFMS than with the SRIM-TOFMS. The reduced mobility (K0) was calculated by use of eq 1 for caffeine in both instruments and was 1.54 cm2 · V-1 · s-1 for the SRIM-TOFMS and 1.51 cm2 · V-1 · s-1 for the RGIM-TOFMS. The reported K0 for caffeine from the literature ranges from 1.5324 to 1.5425 cm2 · V-1 · s-1. The standard deviations for K0 measurements are generally considered as ±0.02 cm2 · V-1 · s-1.26 The difference in the mobility may have been lower in the RGIM-TOFMS due to problems with contamination. When low levels of contaminates or modifiers are present in the drift region, drift times of analyte ions can be shifted often to a longer drift time than those expected in a pure drift gas, leading to a smaller reduced mobility value.27 In general, the RGIM-TOFMS appeared to be more contaminated than those obtained with the SRIM-TOFMS, especially at higher temperatures. The source of the contamination was not clear but may be due to low levels of outgassing from the resistive treatment of the glass. While the contamination was at very low levels, it has been found to be a problem with resistive glass tubes.18,19 Thus, to date resistive glass tubes have been operated at lower temperatures than stacked ring tubes. The measured IMS (Rm) resolving power for the 3 µM caffeine standard was 75 and 94 for the SRIM-TOFMS and RGIM-TOFMS, respectively. These measured resolving powers were compared to the calculated conditional IMS resolving powers (Rc as shown in eq 2). Conditional resolving powers for SRIM-TOFMS and RGIM-TOFMS were 61 and 89, respectively. Note that the measured resolving powers obtained for both the SRIM-TOFMS and the RGIM-TOFMS exceeded the calculated conditional resolving powers. Measured resolving power that exceeds conditional resolving power is often due to the gate depletion effect of BN gates.17 This effect is due to the fact that the ions are depleted around the gate wires and the actual ion pulse is always slightly smaller than expected from the open/ close timing of the ion gate. These results indicate that both the stacked ring design and the resistive glass design provided uniform electric fields along the central axis of the drift tube. The efficiency in ion transmission through the new interface in the RGIMTOFMS was demonstrated by the increase in sensitivity. When monolithic glass was evaluated as a stand-alone IMS, the conditional resolving power was 186 and the measured resolving power was 76 for reserpine dimer.18 Thus, the percent efficiency of the stand-alone instrument was only 41%. According to the method of Wu et al.,17 the lack of 100% efficiency is primarily caused by the gate and pulsing electronics for the gate. Collision-Induced Fragmentation of Caffeine by RGIMTOFMS. The ability of IMS-TOFMS to provide mobility-selected MS/MS-like data is demonstrated in Figure 2. Collision-induced dissociation (CID) of (M + H)+ ion of caffeine was controlled by adjusting the voltages on the ion optical elements between the two quadrupole stages. IM-TOFMS plot for a 3 µM solution (24) Dwivedi, P.; Herbert, H. H. Int. J. Ion Mobil. Spectrom. 2008, 11, 61–69. (25) Waltman, M. J.; Dwivedi, P.; Hill, H. H., Jr. ; Blanchard, W. C.; Ewing, R. G. Talanta 2008, 77, 249–255. (26) Tam, M.; Hill, H. H., Jr. Anal. Chem. 2004, 76, 2741–2747. (27) Fernandez-Maestre, R.; Harden, C. S.; Ewing, R. G.; Crawford, C.; Hill, H. H., Jr. Analyst 2010 (in press).
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Figure 2. Two-dimensional IM-TOFMS plots for 3 µM caffeine with m/z on the x-axis and drift time on the y-axis. IM-TOFMS spectra were collected for 36 s. (A) IM-TOFMS plot with no fragmentation of caffeine with the collision-induced dissociation (CID) turned off. (B) Mobility selected in blue mass spectrum of caffeine associated with panel A; no fragmentation is observed. (C) Fragmentation when the CID is turned on for the mobility-selected caffeine peak in blue. (D) Mass spectrum of the fragmentation pattern. The fragments are numbered and identified in Supporting Information.
of caffeine is shown in Figure 2A. In Figure 2A the 2D IM-TOFMS plot has mass/charge (Th) on the x-axis and IMS drift time (microseconds) on the y-axis. The fragmentation pattern of the (M + H)+ ion of caffeine in the 2D IM-TOFMS plot is shown in Figure 2C. The mass spectra for the mobility-selected peak in Figure 2C is shown in Figure 2D. All of the fragment ions detected had the same drift time as that of the precursor ion, indicating
that fragmentation occurred at the IM-TOFMS interface and not in the IMS region or in the ionization source. An overview of the proposed fragments28 and the corresponding fragmentation path is given in Supporting Information, Figure S1. Metabolites in Blood. Figure 3 is the IM-TOFMS spectrum of a complex biological sample. This spectrum was obtained for 20 min and a volume of 10 µL of extract from 2 drops of
Figure 3. Two-dimensional IM-TOFMS plot for 2 drops of human blood in 400 µL of solvent [50:50 2-propanol/water, formic acid (0.1%)]. The IM-TOFMS spectrum was collected for 20 min and ∼300 metabolites were detected, with the simultaneous separation of 80 isomeric/isobaric compounds. The red box highlights metabolites identified on the basis of isotope pattern and m/z as shown in Figure 4. 9340
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Figure 4. (A, E) Ion mobility spectra for 2 drops of human blood. (B) Mass spectrum of blood metabolome in the mass range of 410 and 417 Th. (C) Mass-selected ion mobility spectrum m/z 413 Th. (D) Mobility-selected mass spectrum displaying the isotope pattern for the metabolite in the drift time range of 44 and 45 ms. (F) Mass spectrum of blood metabolome for m/z values between 605 and 625. Th. (G, I) Mass-selected ion mobility spectrum m/z 616 Th showing the mobility of two isomers. (H) Mobility-selected mass spectrum showing isotope pattern for the metabolite with a drift time between 44 and 45 ms. (J) Mobility-selected mass spectrum showing the isotope pattern for the metabolite with a drift time between 48.5 and 50 ms.
human blood was used. Each point shown in the IM-TOFMS plot represents a metabolite ion. In the RGIM-TOFMS plot, ∼300 metabolite ions were detected with ion counts >5 and 80 isomers and/or isobars were simultaneously separated. Metabolites that have similar structures (i.e., amino acids, carbohydrates, fatty acids, etc.) formed specific mobility-mass correlation lines (also known as trend lines) that were unique for each class of compound. In this initial study, the mobility-mass
correlation (MMC) lines have not been validated with standards, so identifications of metabolites here must be considered tentative. Nevertheless, this spectrum is illustrative of the type of data that can be obtained from complex samples. One of the primary advantages of IM-TOFMS is that the ions are separated in mobility space such that isotope patterns can be (28) Tuomi, T.; Johnsson, T.; Reijula, K. Clin. Chem. 1999, 45, 2164–2172.
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Table 1. Metabolite Assignments for 616.29 Th from the Human Metabolome Database and Expected Isotope Patterns for Each Assignment predicted isotope HMDB ID HMDB00668 HMDB05774 HMDB01045 HMDB10401 HMDB04995 HMDB06888
common name hematoporphyrin IX endomorphin-2 enkephalin L lysoPC(22:4(7Z,10Z, 13Z,16Z)) codeine 5b-cyprinol sulfate
chemical formula
MW [adduct matching HMDB MW]
MW difference
C34H38N4O6 C32H37N5O5 C28H37N5O7 C30H54NO7P
616.312 927 [598.279 114] 616.250 610 [571.279 480] 616.334 595 [555.269 287] 616.334 961 [571.363 770]
0.02 0.04 0.04 0.04
M M M M
C18H21NO3 C27H48O8S
616.338 074 [299.152 130] 616.362 122 [532.307 007]
0.05 0.07
2M + NH4 [1+] M + 2-prop + Na + H [1+] 2M + NH4 [1+] M + K [1+]
HMDB02044 8-hydroxyguanosine C10H13N5O6 616.206 970 [299.086 578] HMDB10399 lysoPC(22:1(13Z)) C30H60NO7P 616.373 901 [577.410 767]
used to identify compounds. For example, in Figure 3 there are two boxes around three metabolite peaks: one at 413.47 Th and two at 616.29 Th. Figure 4A-D shows the mass spectrum of the metabolite at 413.47 Th with and without mobility separation. The new software, developed for this instrument, provides the ability to view the data as total IMS spectra, total mass spectra, 2-D IMMS plots, mass-selected mobility spectra, and mobility-extracted mass spectra. The average IMS is shown in Figure 4A, and the average m/z between 410 and 417 Th is in Figure 4B. An example of an extracted mobility for a selected ion (413 Th) is shown in Figure 4C. To obtain accurate isotope patterns for identification, the mass spectrum for a specific drift time is extracted. An example of an isotope pattern mass spectrum of a mobility-selected peak can be observed in Figure 4D. Though Figure 4 panels B and D have similar isotope ratios, the signal-to-noise is substantially improved when an m/z spectrum is extracted from a mobility-selected peak. The peak at 413.47 Th has an isotope peak at 414.47 Th that is ∼25% of the height of the base peak. There were multiple metabolite assignments based on m/z alone, which included 24-methylenelophenol, avenasterol, ∆7-avenasterol, or stigmasterol. All four tentative matches have the chemical formula C29H48O. The isotope distribution for this chemical formula as [M + H]+ ion is 413.378 (100%), 414.382 (31.3%), and 415.385 Th (0.8%). As noted earlier, the measured 413.47 and 414.47 peaks have similar isotope distribution as those expected for the steroids. To verify the steroid assignment, standards would have to be analyzed. Figure 4E-J shows the mass and mobility spectra for the two metabolites separated at 616.29 Th (see boxed ions in Figure 3). On the left-hand side of the figure (Figure 4E,G,I) are ion mobility spectra and on the right-hand side (Figure 4F,H,J) are mass spectra. Figure 4E is the total ion mobility spectrum of the entire blood sample showing mobility peaks for all of the metabolites in the sample. Figure 4F is the mass spectrum for the non-mobilityselected ions in the m/z range of 610-625 Th. Peaks at nominal masses of 615, 616, 617, and 618 Th were observed. Figure 4G is the mass-selected ion mobility spectrum at 616.29 Th. When the mobility of the faster-eluting ion is selected to produce the mobility-selected mass spectrum of Figure 4H, peaks at 615.07 (100%), 616.07 (80%), and 617.07 Th (30%) were observed. When the slower-eluting peak was selected as shown in Figure 4I, the mobility-selected mass spectrum in Figure 4J showed isotope ratios for 616.29 (100%), 617.29 (33%), and 618.29 (14.5%). Thus, 9342
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0.08 0.08
adduct + + + +
NH4 [1+] 2Na - H [1+] 2-prop + H [1+] 2Na - H [1+]
616 Th 617 Th 618 Th 100 100 100 100
39.5 37.2 36.4 33.9
8.8 7.7 8 7
100 100
41 34.5
9.4 12
100 100
26.6 34
5.8 14.2
the primary ion for the faster-eluting metabolite was 615.07 and the primary ion for the slower-eluting metabolite was 616.29, indicating that the metabolites were not isomers but had overlapping isotope ions. Table 1 lists the singly charged ions reported in the Human Metabolome database for m/z 616.26 Th, which represent possibilities of metabolite assignments based on mass matching alone. Isotope patterns are also provided in the table for further identification. Many of the ions reported in the Human Metabolome database (HMDB) can be eliminated simply by inspection. For example, ions such as [M + 2Na - H]+ are not common ions produced from the electrospray process. All ions listed in the Human Metabolome database that were singly charged and matched the mass within ±0.1 Th were included in Table 1 to illustrate the use of isotopic analysis for compound identification. The metabolite assignment that had the closest match to the isotope pattern was lysophospholipid (LysoPC)(22:1), a phospholipid. According to the HMDB, LysoPC(22:1) has been quantified in blood of normal adults (>18 years) with a concentration of 0.569 ± 0.260 µM, well within the response range of the RGIM-TOFMS. SUMMARY AND CONCLUSIONS Evaluation of an ambient-pressure monolithic resistive glass IMS tube interfaced to a TOFMS demonstrates that the RGIMS performs as well as the SRIMS and is much easier to construct since the RGIMS eliminates most resistor connections. Unlike the stand-alone RGIMS tubes reported in the literature, the RGIMS tube when interfaced to a mass spectrometer provides a resolving power which matches that predicted from theory, indicating that the drift field inside a resistive glass tube is homogeneous and that ions are efficiently transferred through the rf quads to the mass spectrometer. The addition of the rf quads in the vacuum interface improves sensitivity over 2 orders of magnitude. In addition, the IM-TOFMS interface provides a voltage-controllable CID cell for mobility-selected MS2 data. New software developed for this instrument provides the ability to present the data as total ion mobility spectra, total mass spectra, two-dimensional mobility-mass spectra, mass-selected mobility spectra, and mobility-selected mass spectra. These spectra can be monitored as a function of time so that gas or liquid chromatography can be used to separate analytes before introduction of the sample into the IM-TOFMS.
RGIM-TOFMS is capable of measuring multiple metabolites from a single drop of human blood and provides 2D data to identify metabolites on the basis of their unique mobility and m/z values. Hundreds of metabolic features belonging to diverse classes of endogenous metabolites were detected. These metabolites were grouped in the 2D IM-TOFMS space, which allows identification of isomeric and isobaric compounds. The addition of the IMS to MS reduces the chemical noise and allows the isotope ratios for compounds to be accurately measured. However, for absolute metabolite identification, there is a need to develop an ion mobility database for metabolites. This unique IM-TOFMS instrument provides value-added advantages to mass spectrometry. These include (1) rapid ion separation prior to mass spectrometry, (2) isomer separation, (3) increase in signal-to-noise ratio, and (4) isotope evaluation for complex mixtures.
ACKNOWLEDGMENT This project was supported in part by a research grant from the Department of Health and Human Services, Public Health Services organization (Road Map Grant R21 DK070274). We also acknowledge Novartis Pharma Services AG (Basel, Switzerland) for partial support of this project. SUPPORTING INFORMATION AVAILABLE Text and one figure describing the stacked ring ion mobility mass spectrometer experimental setup and proposed fragmentation mechanism for caffeine. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review July 14, 2010. Accepted September 29, 2010. AC1017259
Analytical Chemistry, Vol. 82, No. 22, November 15, 2010
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