Enhanced Direct Ambient Analysis by Differential Mobility-Filtered

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Anal. Chem. 2010, 82, 9159–9163

Letters to Analytical Chemistry Enhanced Direct Ambient Analysis by Differential Mobility-Filtered Desorption Electrospray Ionization-Mass Spectrometry Asiri S. Galhena, Glenn A. Harris, Mark Kwasnik, and Facundo M. Ferna´ndez* School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, United States Desorption electrospray ionization (DESI) is rapidly becoming established as one of the most powerful ionization techniques allowing direct surface analysis by mass spectrometry (MS) in the ambient environment. DESI provides a significant number of unique analytical capabilities for a broad range of applications, both quantitative and qualitative in nature including biological tissue imaging, pharmaceutical quality control, in vivo analysis, proteomics, metabolomics, forensics, and explosives detection. Despite its growing adoption as a powerful high throughput analysis tool, DESI-MS analysis at trace levels often suffers from background chemical interferences generated during the electrospray ionization processes. In order to improve sensitivity and selectivity, a differential mobility (DM) ion separation cell was successfully interfaced to a custom-built DESI ion source. This new hybrid platform can be operated in two modes: the “DM-off” mode for standard DESI analysis and “DMon mode” where DESI-generated ions are detected after discrimination by the differential mobility cell. The performance of the DESI-DM-MS platform was tested with several samples typically amenable to DESI analysis, including counterfeit pharmaceuticals and binary mixtures of isobaric chemicals of importance in the pharmaceutical and food industries. In the DM-on mode, DESI-MS signal-to-noise ratios were improved by 70-190% when compared to the DM-off mode. Also, the addition of the DM cell enabled selective in-source ion activation of specific DESI-generated precursor ions, providing tandem MS-like spectra in a single stage mass spectrometer. Over the past five years, efforts toward performing mass spectrometry (MS) experiments under open-air configurations have been intensifying steadily due to the advancement of direct ionization techniques often referred to as “ambient” ionization.1-4 * Corresponding author. Phone: 404 385 4432. Fax: 404 385 6447. E-mail: [email protected]. Homepage: http://tinyurl.com/ fernandezgroup. (1) Chen, H.; Gamez, G.; Zenobi, R. J. Am. Soc. Mass. Spectrom. 2009, 20, 1947–1963. 10.1021/ac102340h  2010 American Chemical Society Published on Web 10/22/2010

These techniques have significantly advanced MS by minimizing upfront sample preparation, thus providing high-throuhput analysis capabilities to rapidly screen, quantify, and identify unknowns directly from the sample surface. Among the many different ambient ionization techniques, desorption electrospray ionization (DESI) has become popular due to many demonstrated advantages, including the ability of chemically imaging surfaces, rapid quantitative screening, and soft ionization to limit fragmentation of the target analytes.4 Although little to no sample pretreatment is required, DESI may suffer from limited sensitivity and/or selectivity due to less-than-optimal droplet/ion transmission and typical chemical interferences found also in electrospray ionization (ESI). For example, our efforts with counterfeit drug detection have shown the need for more advanced analytical technologies to aid in combating this ever growing public health problem.5,6 The presence of isobaric species and chemically interfering species is a common scenario in both pharmaceutical analysis7 and other quality control applications.8 In resource-limited environments without access to tandem MS instrumentation, new instrumental approaches that allow structural elucidation by selective in-source fragmentation without relying on chromatographic separations would be beneficial. Differential mobility spectrometry (DMS, also called field asymmetric-waveform ion mobility spectrometry, FAIMS) is a gasphase ion mobility separation technique based on differences between ion mobilities as a function of a time-dependent asymmetric high electrical field, operated at or near atmospheric pressure.9 DMS is orthogonal to MS which affords higher (2) Harris, G. A.; Nyadong, L.; Fernandez, F. M. Analyst 2008, 133, 1297– 1301. (3) Van Berkel, G. J.; Pasilis, S. P.; Ovchinnikova, O. J. Mass Spectrom. 2008, 43, 1161–1180. (4) Weston, D. J. Analyst 2010, 135, 661–668. (5) Fernandez, F. M.; Green, M. D.; Newton, P. N. Ind. Eng. Chem. Res. 2008, 47, 585–590. (6) Kaur, H.; Green, M. D.; Hostetler, D. M.; Fernandez, F. M.; Newton, P. N. Therapy 2010, 7, 49–57. (7) Nyadong, L.; Harris, G. A.; Balayssac, S.; Galhena, A. S.; Malet-Martino, M.; Martino, R.; Parry, R. M.; Wang, M. D.; Fernandez, F. M.; Gilard, V. Anal. Chem. 2009, 81, 4803–4812. (8) Keller, B. O.; Suj, J.; Young, A. B.; Whittal, R. M. Anal. Chim. Acta 2008, 627, 71–81. (9) Krylov, E. V.; Nazarov, E. G.; Miller, R. A. Int. J. Mass spectrom. 2007, 266, 76–85.

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Table 1. Optimized Operational Parameters of the DESI-DM-MS Platform

DESI

DM

MS

DESI-DM-MS parameter

DM-on modea

spray probe voltage ion transfer capillary voltage solvent (MeOH) flow rate desolvation gas (N2) flow tip-to-surface distance spray incident angle (R) collection angle (β) separation voltage (SV) compensation voltage (CV) CV scan steps CV step duration desolvation/transport gas (N2) flow rate dopant (isopropanol) concentration desolvation/transport gas (N2) temperature throttle gas (N2) flow rate orifice 1 voltage orifice 1 temperatureb ring electrode voltage orifice 2 voltage transfer hexapole bias

3.6 kV 128 V 5-8 µL min-1 150 L h11.5-3 mm ∼55° ∼30° 500-1500 V (-42)-15 V 100-150 100 ms 0.2 L min-1 ∼5000 ppm ∼80 °C 0.5 L min-1 128 V 5V 18 V 27 V

a For DM-off mode, no SV or CV is applied. b Not controlled (room temperature).

selectivity and specificity versus standalone MS.10 Key among the beneftis of DMS operation with MS detection are improved signalto-noise ratio (S/N) gains and separation of isobaric species and structural isomers without time-consuming chromatographic methods. DMS has been used in many analytical applications in conjuction with MS, yet there have been no reports on the coupling of DESI or any other ambient ionization techniques to a differential mobility (DM)-MS system.11 Herein, we report the first interfacing of a DESI-DM ionization/ion separation module to a commercial, medium resolution, time-of-flight (TOF) mass spectrometer. The DESI-DM-MS platform encompasses a custom-made DESI source,12 a modified Sionex Corporation (Bedford, MA) microDMX differential mobility sensor, and an orthogonal TOF mass spectrometer (JEOL AccuTOF, Tokyo, Japan). The DESI source was built around a joystick-controlled motorized microscope XY stage (Prior Scientific, Rockland, MA) for automatic and manual sample positioning in the x-y and z coordinates, respectively. The DESI sprayer was mounted on a MicroBlock 3-axis positioner (Thorlabs, Newton, NJ) for manual adjustment of the emitter position in the x-y-z coordinates with respect to the TOF orifice inlet and fitted with a high precision rotation mount (Thorlabs, Newton, NJ) for manual adjustment of the spray impact angle (Table 1). The DM unit consists of a desolvation chamber, DM electrode housing, and a DM assembly holder. The DESI-DM assembly holder (Figure 1) mounts the DM module to the orifice 1 plate of the AccuTOF instrument. A side port for throttle gas (ultra pure N2, Airgas, Atlanta, GA) supply at an optimum flow rate of 0.5 Lmin-1 is machined on the side of the assembly holder. Isopropanol is fed as a dopant (∼5000 ppm, V/V) into the DM desolvation chamber in combination with heated (∼80 (10) Schneider, B. B.; Covey, T. R.; Coy, S. L.; Krylov, E. V.; Nazarov, E. G. Int. J. Mass spectrom. 2010, in press, DOI: 10.1016/j.ijms.2010.01.006. (11) Kolakowski, B. M.; Mester, Z. Analyst 2007, 132, 842–864. (12) Nyadong, L.; Green, M. D.; De Jesus, V. R.; Newton, P. N.; Fernandez, F. M. Anal. Chem. 2007, 79, 2150–2157.

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°C) N2 at a flow rate of 0.2 L min-1. The DESI ion transfer capillary (250 mm long, OD: 1.58 mm, ID: 0.6 mm) is attached to the entrance of the DM desolvation chamber using a vacuum sealable 1/16 in. Swagelok fitting. Sufficient vacuum suction to enhance ion/droplet transport from the DESI sampling stage through the DM unit to the MS orifice 1 inlet is achieved by vacuum sealing each component of the DM unit with polytetrafluoroethylene (PTFE)-based Gore-Tex washers. Additional heating (∼250 °C) was provided to improve ion desolvation during transit through the ion transfer capillary via an external heater block. The DM electrodes consisted of two parallel, 10 mm × 8 mm plates, which are held a uniform distance apart (0.5 mm) to create the DM analytical gap. The high-frequency radio frequency (RF) (clipped sinusoidal waveform) operates at a fixed frequency of 1.25 MHz. The DC voltages required for DM operation were provided by a half-cycle flyback-type power generator independently controlled by the Sionex microDMX (v2.4.0) software interface, while MS data were collected by and analyzed with the JEOL AccuTOF Mass Center (v1.3.0) application software. The optimum operational parameters for DESI-DM-MS operation used in this study are shown in Table 1. The DESI-DM-MS platform can operate in two modes: fly through “DM-off” mode or “DM-on” mode. In DM-off mode, the voltages on the DM electrodes were turned off, and ions were allowed to fly through the DM module. This mode was exclusively used for the optimization of ion transmission through the DESIDM interface without mobility separation. Figure 2a shows the analysis of a counterfeit antimalarial pharmaceutical tablet in DMoff mode. The two predominant peaks in the spectrum (m/z 407.108 and 365.106) were assigned to [artesunic acid + Na]+ and [lactose + Na]+, respectively. Analysis of the same pharmaceutical tablet (previously labeled [formulation 4]) by DESI-MS7 revealed the presence of the same chemical components. The peak at m/z 443.233 corresponds to 50 µL of 1 mM rhodamine 6G (R6G), a chemical tag spotted on the tablet surface. R6G is a precharged ionic species that served as a simulated concomitant background ion and was used for ion transmission optimization and mass drift correction. In DMon mode, an asymmetric RF voltage, often referred to as separation voltage (SV) or dispersion voltage (DV), and a counterbalancing DC potential, called the compensation voltage (CV) are simultaneously applied on the DM unit electrodes. Figure 2b-d illustrates the separation of the three major chemical species, sodiated lactose (m/z 365.106), sodiated artesunic acid (m/z 407.108), and R6G (m/z 443.233), from the tablet surface. Mobility separation was performed at a fixed SV (1300 V), with three different CVs (-2.2, -0.7, and +1.7 V, respectively). In this mode, prior determination of SV and CV is required for the best differential mobility separation. For this purpose, the SV was first scanned between 500 and 1500 V, with no CV applied to identify the minimum SV required to minimize on transmission, which was 1200 V. The SV was then increased stepwise, and at each SV setting, the CV was ramped between -20 and 5 V. A SV of 1300 V was chosen on the basis of a compromise of maximum sensitivity and separation. On the basis of their ionic mobilities under low and high field conditions, these ions migrate toward the ground electrode but in three different trajectories. These ion

Figure 1. Schematic of the DESI-DM-MS interface.

Figure 2. Analysis of a counterfeit antimalarial pharmaceutical tablet by DESI-DM-MS: (a) DM-off mode (no voltages applied) and DM-on mode for the isolation of (b) sodiated lactose excipient (m/z ) 365.106, SV ) 1300 V, CV ) -1.7 V), (c) sodiated artesunic acid (m/z ) 407.108, SV ) 1300 V, CV ) -0.7 V), and (d) R6G (m/z ) 443.233, SV ) 1300 V, CV ) 1.7 V). The inset shows the normalized selected ion trace for each ion in the MS acquisition time scale with a CV sweep rate of 0.25 V/step. [Int], absolute intensity of the base peak.

trajectories are corrected when the appropriate CV is applied during the CV ramp. In the DM-on mode, only the ions of interest were allowed to transmit through the system, one at a time. The orthogonal separation capability of DMS allowed discrimination and simplification of the spectra by filtering the unrelated background chemical noise. This resulted in improved S/N (Figure 2b-d). The inset in Figure 2b shows the normalized selected MS ion chronograms. Although the ionic species were not baseline separated, they were easily distinguished from each other. The use of isopropanol as a polar modifier in the transport gas stream provided increased differences in CV values of the

detected ionic species compared to what was observed with pure N2 but at the expense of peak width (data not shown). A similar effect has been previously reported by other research groups.13,14 However, the gain in CV differences was greater than the increase in DM scan peak widths; thus, isopropanol was used for all experiments. In order to further improve DM resolution, a mixture of N2 (80%) and He (20%, V/V) was tested as an alternative transport gas.15,16 In the presence of He, peak widths were ∼30% narrower than in the case of pure N2 (Supplemental Figure S-1, (13) Krylov, E. V.; Nazarov, E. G. Int. J. Mass spectrom. 2009, 285, 149–156. (14) Rorrer, L. C.; Yost, R. A. Int. J. Mass spectrom., in press.

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Figure 3. Application of DESI-DM-MS to the analysis of chemical standards on PTFE surfaces. Mixture of 0.5 mM pyrimethamine (m/z ) 248.083) and 1 mM polyethylene glycol (PEG 400): (a) DM-off (no voltages applied) and (b) DM-on mode (SV ) 1300, CV ) 1.2 V). The inset of (b) shows the DESI-DM-CID-MS mode of operation at orifice voltage of 120 V. (c) The analysis of a binary mixture of pyrimethamine (100 µM) and 5-HMF (100 µM) in DM-on mode (SV ) 900 V, CV ) -15 to 5 V). Inset shows the marginally resolved TOF MS spectra of melamine and 5-HMF. *, ammonia adducts; [Int], absolute intensity of the base peak.

Supporting Information) but sensitivity was reduced by ∼30-40%. Furthermore, sporadic electrical discharging between the DM electrodes was observed when He was used along with chemical dopants. Because problems with electrical discharging with this % He have not been described before, it was assumed that they were due to the pressure reduction induced in the DM cell by the mass spectrometer gas inflow. Therefore, this approach was not further pursued. Additional characterization of the DESI-DMMS platform was performed by depositing chemical standards on PTFE surfaces. In Figure 3a, analysis of a mixture of 0.5 mM pyrimethamine (m/z ) 248.083) and 1 mM polyethylene glycol (PEG 400) in DM-off mode is illustrated. Higher concentration of PEG was deliberately added to the mixture in order to introduce a high chemical background. The peak at m/z ) 249.091 corresponds to protonated pyrimethamine with a S/N of 7. However, when analyzed in DMS-on mode (SV ) 1300 V, CV ) 1.2 V), the chemical background induced by PEG is completely eliminated and the pyrimethamine ion S/N increased from 7 to (15) Schneider, B. B.; Covey, T. R.; Coy, S. L.; Krylov, E. V.; Nazarov, E. G. Anal. Chem. 2010, 82, 1867–1880. (16) Shvartsburg, A. A.; Danielson, W. F.; Smith, R. D. Anal. Chem. 2010, 82, 2456–2462.

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20 (improvement of ∼2.9×). The chemical background prefiltering effect is highly encouraging, especially for future analysis of complex chemical mixtures and imaging purposes. Furthermore, the ability of performing collision induced dissociation (CID) of preselected ions leaving the DM unit was tested by activating the protonated pyrimethamine precursor ion. In this mode, ion dissociation is achieved by first selecting the ions of interest in the DM unit and then passing them into the mass spectrometer with additional activation energy provided by collisions in the first differentially pumped region. To induce ion dissociation, the orifice 1 voltage was raised while the other instrumental parameters were kept constant. The inset in Figure 3b shows the DESI-DM-CIDMS spectrum of the pyrimethamine [M + H]+ at an orifice 1 voltage of 120 V. Other collision energies are shown in Supplemental Figure S-2, Supporting Information. The extensive fragmentation observed is highly informative and similar to previously reported ESI-MS/MS spectra.17 DESI-DM-CID-MS, thus, provides higher selectivity and specificity than DESI-MS and can be viewed as a low-cost alternative to higher performance tandem mass spectrometers. (17) Storme, M. L.; Jansen, F. H.; Goeteyn, W.; Van Bocxlaer, J. F. Rapid Commun. Mass Spectrom. 2006, 20, 2947–2953.

Finally, the performance of the DESI-DM-MS platform for the separation and detection of near-isobaric compounds was tested. For example, 5-hydroxymethylfurfural (5-HMF), which is formed as a result of heating dried milk powder,18 produces a protonated ion (theoretical m/z ) 127.0389) that is indistinguishable from protonated melamine (theoretical m/z 127.0726) using lowresolution mass spectrometers. Recent attempts of detecting melamine in dairy products triggered the exploration of new, lowcost, and rapid analysis techniques for its detection. However, almost all of these detection methods required high-resolution MS and/or tandem MS to eliminate possible false positives.19 In this study, an equimolar mixture (50 µL of 100 µM solutions in methanol) of melamine and 5-HMF were deposited on a PTFE surface and analyzed by DESI-DM-MS. In DM-off mode, two species are marginally identified in the TOF mass spectrum (inset of Figure 3c). However, in DM-on mode, the two nearly isobaric compounds were successfully baseline resolved (SV ) 900) and positively identified on the basis of their appearance time in the CV spectrum (Figure 3c). CONCLUSIONS For the first time, a hybrid DESI-DM-MS platform was successfully implemented on a commercial mass spectrometer. (18) Groux, M. J. Dairy Sci. 1974, 57, 153–155. (19) Dane, A. J.; Cody, R. B. Analyst 2010, 135, 696–699.

DESI-DM-MS was found to be effective in suppressing chemical noise and separating interfering ions of similar mass-to-charge ratio produced by DESI. In-source ion activation was achieved by increasing the orifice 1 voltage, allowing additional tandem MSlike capabilities. This type of hybrid instrumentation could successfully be utilized in analyzing complex sample mixtures by DESI in their native states while improving S/N ratios for enhanced sensitivity and selectivity.

ACKNOWLEDGMENT This work was supported by the Bio-Imaging Mass Spectrometry (BIMS) Center at the Georgia Institute of Technology and by ARRA NSF MRI Instrument Development Grant #0923179 to F.M.F. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review September 2, 2010. Accepted October 19, 2010. AC102340H

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