Ambient Mass Spectrometry with a Handheld Mass Spectrometer at

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Ambient Mass Spectrometry with a Handheld Mass Spectrometer at High Pressure Adam Keil, Nari Talaty, Christian Janfelt, Robert J. Noll, Liang Gao, Zheng Ouyang, and R. Graham Cooks*

Chemistry Department, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907

The first coupling of atmospheric pressure ionization methods, electrospray ionization (ESI) and desorption electrospray ionization (DESI), to a miniature hand-held mass spectrometer is reported. The instrument employs a rectilinear ion trap (RIT) mass analyzer and is batteryoperated, hand-portable, and rugged (total system: 10 kg, 0.014 m3, 75 W power consumption). The mass spectrometer was fitted with an atmospheric inlet, consisting of a 10 cm × 127 µm inner diameter stainless steel capillary tube which was used to introduce gas into the vacuum chamber at 13 mL/min. The operating pressure was 15 mTorr. Ions, generated by the atmospheric pressure ion source, were directed by the inlet along the axis of the ion trap, entering through an aperture in the dcbiased end plate, which was also operated as an ion gate. ESI and DESI sources were used to generate ions; ESIMS analysis of an aqueous mixture of drugs yielded detection limits in the low parts-per-billion range. Signal response was linear over more than 3 orders of magnitude. Tandem mass spectrometry experiments were used to identify components of this mixture. ESI was also applied to the analysis of peptides and in this case multiply charged species were observed for compounds of molecular weight up to 1200 Da. Cocaine samples deposited or already present on different surfaces, including currency, were rapidly analyzed in situ by DESI. A geometry-independent version of the DESI ion source was also coupled to the miniature mass spectrometer. These results demonstrate that atmospheric pressure ionization can be implemented on simple portable mass spectrometry systems. Mass spectrometry (MS) continues to develop rapidly; applications in the life sciences, especially in areas like protein sequencing,1 the identification of metabolic products, discovery of new biomarkers,2 and MS-imaging of tissue sections,3,4 are particularly notable. Other application areas include public safety screening, * Corresponding author. (1) Eng, J. K.; McCormack, A. L.; Yates, J. R. J. Am. Soc. Mass Spectrom. 1994, 5, 976-989. (2) Chen, H. W.; Pan, Z. Z.; Talaty, N.; Raftery, D.; Cooks, R. G. Rapid Commun. Mass Spectrom. 2006, 20, 1577-1584. (3) Wiseman, J. M.; Ifa, D. R.; Song, Q. Y.; Cooks, R. G. Angew. Chem., Int. Ed. 2006, 45, 7188-7192. (4) Stoeckli, M.; Chaurand, P.; Hallahan, D. E.; Caprioli, R. M. Nat. Med. 2001, 7, 493-496.

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use by first responders in characterizing an incident site, industrial hygiene, and environmental monitoring. These applications are enabled by innovations in both instrumentation and methodology. In the former category, the miniaturization of mass spectrometers5,6 now allows in situ measurements.7 In the latter category, ambient ionization techniques, such as desorption electrospray ionization and other ambient ionization methods,8-10 permit the examination of samples in their native environments. There is an obvious complementarity between these two topics, since the ideal instrument for in situ applications must be small and selfcontained, yet take measurements in the environment without any special sample preparation. An instrument which satisfies both these requirements is described here. It is a handheld tandem mass spectrometer fitted with an atmospheric pressure interface, allowing implementation of such useful ionization methods as desorption electrospray ionization (DESI), atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI). The miniature mass spectrometer used for these experiments is a custom-built engineering prototype.11 This instrument is the latest in a series of miniature instruments5 developed in our laboratory over the past decade, based initially on the cylindrical ion trap12 and now on the rectilinear ion trap (RIT)13 mass analyzer. These simplifiedgeometry analyzers were optimized through multiparticle simulations.14 The Mini 10 is a fully autonomous tandem mass spectrometer. The total system weighs 10 kg and has a power consumption of 75 W which allows 2-4 h of field battery operation. All ancillary components are also reduced in size and weight. Under routine conditions, the instrument provides unit mass (5) Badman, E. R.; Cooks, R. G. J. Mass Spectrom. 2000, 35, 659-671. (6) Pau, S.; Pai, C. S.; Low, Y. L.; Moxom, J.; Reilly, P. T. A.; Whitten, W. B.; Ramsey, J. M. Phys. Rev. Lett. 2006, 96 (12) 120801. (7) Short, R. T.; Toler, S. K.; Kibelka, G. P. G.; Roa, D. T. R.; Bell, R. J.; Byrne, R. H. TrAC, Trends Anal. Chem. 2006, 25, 637-646. (8) Takats, Z.; Wiseman, J. M.; Cooks, R. G. J. Mass Spectrom. 2005, 40, 12611275. (9) Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2006, 17, 1712-1716. (10) Shiea, J.; Huang, M. Z.; Hsu, H. J.; Lee, C. Y.; Yuan, C. H.; Beech, I.; Sunner, J. Rapid Commun. Mass Spectrom. 2005, 19, 3701-3704. (11) Gao, L.; Song, Q. Y.; Patterson, G. E.; Cooks, R. G.; Ouyang, Z. Anal. Chem. 2006, 78, 5994-6002. (12) Patterson, G. E.; Guymon, A. J.; Riter, L. S.; Everly, M.; Griep-Raming, J.; Laughlin, B. C.; Zheng, O. Y.; Cooks, R. G. Anal. Chem. 2002, 74, 61456153. (13) Ouyang, Z.; Wu, G. X.; Song, Y. S.; Li, H. Y.; Plass, W. R.; Cooks, R. G. Anal. Chem. 2004, 76, 4595-4605. (14) Wu, G. X.; Cooks, R. G.; Ouyang, Z.; Yu, M.; Chappell, W. J.; Plass, W. R. J. Am. Soc. Mass Spectrom. 2006, 17, 1216-1228. 10.1021/ac071114x CCC: $37.00

© 2007 American Chemical Society Published on Web 09/15/2007

resolution, an upper mass-to-charge limit of 600, a linear dynamic range of at least 3 orders of magnitude, and low ppb detection limits for many compounds. In parallel with the work leading to miniaturization of the ion trap mass spectrometers, an ambient ionization method, desorption electrospray ionization (DESI), has been operationally optimized.8 The DESI experiment has been extended to include selective ionization by adding appropriate reagents into the spray solvent15 and its fundamentals have begun to be elucidated.16 In the DESI experiment, charged micron-sized droplets of moderate velocity (100 m/s) are directed at the surface to be analyzed. Droplets colliding with the surface fragment to smaller progeny droplets at the same time picking up analyte molecules. The charged secondary droplets are converted into gas-phase ions during transport through the atmospheric inlet of the mass spectrometer. The DESI experiment is compatible with sampling rates of 1 Hz and generally requires no sample pretreatment. It is applicable to a wide range of large and small molecules present in an array of sample types. DESI has been implemented so far with conventionally sized commercial laboratory instruments,17,18 FTICR,19 a prototype orbitrap,20 as well as in our version 8 miniature transportable,21 but not hand portable, atmospheric inlet mass spectrometer.22 These recent advances in portable MS instrumentation, coupled with ambient ionization techniques, have the potential to greatly enhance in situ analysis. Applications of miniature mass spectrometers in the biological and medical sciences are likely to be numerous. Their portability suggests that such instruments could be ubiquitously deployed in clinical settings. The fact that DESI can be used to detect a variety of analytes present in complicated biological matrixes, such as blood and urine, also means that results can be obtained nearly instantaneously for a variety of patient-generated samples.2 Application to bacterial infections and cultures is possible.23 Drug discovery in remote settings also becomes possible, with instant DESI-MS analysis of plants and their extracts in the field.24 Also possible is factory production line monitoring with portable DESI-MS or ESI-MS monitoring.25,26 Finally, application to public safety has been one of the key demonstrations of the capabilities of the DESI method.8,27 (15) Chen, H.; Cotte-Rodriguez, I.; Cooks, R. G. Chem. Commun. 2006, 597599. (16) Venter, A.; Sojka, P. E.; Cooks, R. G. Anal. Chem. 2006, 78, 8549-8555. (17) Cooks, R. G.; Ouyang, Z.; Takats, Z.; Wiseman, J. M. Science 2006, 311, 1566-1570. (18) Van Berkel, G. J.; Ford, M. J.; Deibel, M. A. Anal. Chem. 2005, 77, 12071215. (19) Bereman, M. S.; Nyadong, L.; Fernandez, F. M.; Muddiman, D. C. Rapid Commun. Mass Spectrom. 2006, 20, 3409-3411. (20) Hu, Q. Z.; Talaty, N.; Noll, R. J.; Cooks, R. G. Rapid Commun. Mass Spectrom. 2006, 20, 3403-3408. (21) Laughlin, B. C.; Mulligan, C. C.; Cooks, R. G. Anal. Chem. 2005, 77, 29282939. (22) Mulligan, C. C.; Talaty, N.; Cooks, R. G. Chem. Commun. 2006, 17091711. (23) Song, Y. S.; Talaty, N.; Tao, W. A.; Pan, Z. Z.; Cooks, R. G. Chem. Commun. 2007, 61-63. (24) Talaty, N.; Takats, Z.; Cooks, R. G. Analyst 2005, 130, 1624-1633. (25) Chen, H. W.; Talaty, N. N.; Takats, Z.; Cooks, R. G. Anal. Chem. 2005, 77, 6915-6927. (26) Williams, J. P.; Lock, R.; Patel, V. J.; Scrivens, J. H. Anal. Chem. 2006, 78, 7440-7445. (27) Cotte-Rodriguez, I.; Takats, Z.; Talaty, N.; Chen, H.; Cooks, R. G. Anal. Chem. 2005, 77, 6755-6764.

A considerable challenge in coupling atmospheric pressure ionization methods to miniature MS systems is the reduced pumping capacity of a portable MS system, due to size and power consumption restrictions. Elegant future solutions might be found in high-performance miniature turbo pumps, but currently operation at elevated pressure and/or reduced inlet flow is required. By fitting an atmospheric pressure interface to a handheld mass spectrometer, we have taken the first steps toward a handheld miniature mass spectrometer capable of atmospheric pressure ionization. Simple addition of a long capillary to the Mini 10 allows sampling directly from the atmosphere by providing the needed pressure differential. This general approach has been used before in much larger fieldable instruments for sampling.28 The atmospheric pressure interface allows sampling of the ions/droplets generated by atmospheric pressure ionization methods, including APCI, ESI, and DESI. The instrument modifications are simple and are described briefly in the experimental section. The results showing analytical performance data for a variety of sample types ionized by ESI and DESI are reported and discussed. EXPERIMENTAL SECTION Reagents. Methanol was obtained from Mallinckrodt Baker, Inc. (Phillipsburg, NJ). Tributylamine and dibutylamine were purchased from Sigma-Aldrich (St. Louis, MO). All solutions were prepared in a 50/50 MeOH/H2O solvent mixture unless otherwise noted. The drug mixture which contained 250 µg/mL each of methamphetamine, cocaine, and heroin in acetonitrile solvent was purchased from Cerilliant (Round Rock, TX) and diluted with methanol/water for ESI experiments and with pure methanol for DESI experiments. Bradykinin was purchased from Sigma-Aldrich and the synthetic peptide KGAILKGAILR from Synpep. A Millipore Milli Q system provided 18.2 MΩ cm-1 conductivity water for dilution. Instrumentation. The Mini 10 mass spectrometer (10 kg, 0.014 m3, 75 W) was used to collect the data presented here. Mass analysis was performed in its rectilinear ion trap mass analyzer (xo ) 5.0 mm, yo ) 4.0 mm, axial length zo ) 43.2 mm, rf ) 970 kHz).11 For these experiments, the scan function had three periods: ion injection into the rectilinear ion trap (generally 200 ms), cooling (10 ms), and scan out (20 ms). Ions were scanned out to the detector using resonance ejection (345 kHz), with an ac voltage ramp (∼1-3 V) applied in conjunction with the rf voltage ramp (∼200-1500 V) to effect ion ejection over the mass range of m/z 72-600. Ion detection used an electron multiplier (model 2312, Detector Technology) biased to -1300 V (5 × 107 gain at -2 kV). The instrument was controlled and data were processed using software (version 2.3) from Griffin Analytical Technologies (West Lafayette, IN). The vacuum system is comprised of a microturbopump (10 L/s, Pfeiffer TPD 011) and oil-free 2-stage diaphragm pump (5 L/min, KNF Neuberger model 1091-N84.0-8.99). For the electrospray (ESI) experiments, ions were generated using a home-built source held at 4 kV potential. Spray solvent was 50/50 methanol/water with a flow rate ) 10 µL/min (unless otherwise noted). The nebulization (sheath) gas was nitrogen at (28) Wise, M. B.; Thompson, C. V.; Merriweather, R.; Guerin, M. R. Field Anal. Chem. Technol. 1997, 1, 251-276.

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Figure 1. (a) Schematic of the handheld mass spectrometer with atmospheric inlet added; (b) a view of the components inside the manifold. (c) Typical ESI mass spectrum obtained of a 1/1 mixture of dibutylamine (M + H)+, m/z 130, and tributyl amine (M + H)+, m/z 186. Inset: MS/MS spectrum of tributyl amine.

40 pounds per square inch (psi). A stainless steel capillary (10 cm × 127 µm i.d.) served as the atmospheric inlet; it was held at ground potential and not heated. This capillary has a smaller inner diameter than those used on commercial instruments or our previous miniature mass spectrometer with atmospheric inlet (254 µm),22 and this was necessitated by the pumping speed of the Mini 10’s miniature turbopump. Gas flow through the inlet capillary, due entirely to the pressure difference between the vacuum chamber and ambient atmosphere, transported the ions into the ion trap. The gas flow rate through the inlet capillary was measured as 13.1 mL/min, resulting in an indicated pressure of 15 mTorr (Pirani gauge, MKS Instruments, uncorrected reading). Figure 1a shows the Mini 10 and the atmospheric pressure ion source schematically. Both the flow rate and vacuum chamber pressure are consistent with simple gas throughput calculations. Inside the vacuum chamber, the capillary ended about 1 mm before the end plate lens of the RIT, which was used for ion gating. No tube lens or ion guide was employed. The capillary was centered on the 5 mm diameter aperture in this electrode, and thus ion injection occurred along the z-axis of the mass analyzer. The end plate electrode was used for gating ions into the RIT. During ion injection, the end plate electrode was held at -1 V dc; during mass analysis the electrode was biased at +300 V dc. As noted above, ions were usually collected for 200 ms prior to mass analysis. At 15 mTorr pressure, significant peak broadening was observed in the mass spectra compared to those recorded at manifold pressures of 10-5 Torr. Typical peak widths, measured at full-width-half-maximum (fwhm), were about 3 mass-to-charge units. However, the increased pressure did not affect mass analysis 7736 Analytical Chemistry, Vol. 79, No. 20, October 15, 2007

nor hinder ion detection. Tandem mass spectrometry experiments could be carried out without any added buffer gas, using instead the background air present in the manifold. Severe degradation of the multiplier’s lifetime and performance was anticipated at this pressure; however, no significant change in sensitivity or loss of signal was observed over the course of the experiments presented here. The instrument was operated in the fashion described here on a near-daily basis for several months while the electron multiplier was changed just once, due to diminished gain. DESI. The DESI source employed consists of a pneumatically assisted electrospray capillary mounted near the capillary inlet on a movable bracket. Sprayer-to-inlet and sprayer-to-surface distances can be changed, as can the relative orientation of both the spray and inlet capillaries to the analysis surface and to each other. The conditions used were much milder compared to previous DESI experiments.8,29 The nebulizing gas flow (N2) was maintained at 60 psi, much lower than the regular 120-150 psi as previously reported. The incident angle was anywhere from 40 to 50° (larger for peptides) and the take-off angle was maintained at 10°, consistent with earlier reports. The spray voltage was 5.5 kV for the peptides and 5 kV for smaller molecules. The capillary used for the atmospheric inlet was not heated. The distance between the spray capillary and the inlet capillary was ∼5 mm, similar to previous DESI experiments on conventional instruments. In some of the DESI experiments, a new “geometry independent” enclosed DESI source was implemented to eliminate ion signal dependence upon the placement of the spray and (29) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science 2004, 306, 471-473.

Figure 2. (a) ESI mass spectrum of a 1/1/1 mixture of methamphetamine, cocaine, and heroin at three concentrations examined in 50/50 methanol/water. Inset shows the lower end of a calibration curve obtained for the compounds in the mixture. (b) Tandem mass spectra for individual drugs in the mixture.

collection capillaries.30 Samples containing 5 µL of cocaine solution were spotted onto two different surfaces so that the final amount of cocaine was 50 ng over the area deposited. RESULTS AND DISCUSSION ESI. A typical spectrum recorded by electrospraying a mixture of 100 nM dibutylamine and 100 nM tributyl amine is shown in Figure 1. This spectrum was obtained at a pressure of ∼15 mTorr and the peaks have widths (fwhm) of 3 m/z units. The loss of resolution can be compensated for by the use of tandem mass spectrometry. The inset is a product ion MS/MS spectrum of protonated tributyl amine. The broadened peak centered at m/z 143 is consistent with both propene loss (m/z 144) and propane loss (m/z 142) from the precursor protonated molecule. Both alkane and alkene loss are well-known fragmentation processes for protonated alkyl amines.31 (30) Venter, A.; Cooks, R. G. Anal. Chem., 2007, 79, 6398-6403.

The ESI mass spectra of solutions resulting from serial dilutions of a mixture of drugs of abuse are shown in Figure 2. A combined solution of methamphetamine, cocaine, and heroin (100 µg/mL each, protonated molecules at m/z 150, 304, and 370, respectively) was diluted using methanol/water (1/1) to yield total concentrations of 6.25 µg/mL, 2.5 µg/mL, and 625 ng/mL (depicted in Figure 2a). The tandem mass spectrum (MS/MS) for each compound is shown in Figure 2b. Precursor ion isolation was accomplished with a SWIFT waveform;32 ions were then excited with the appropriate low amplitude ac frequency to cause collisional dissociation in the RIT. The methamphetamine MS/MS spectrum shows a characteristic product ion at m/z 119 corresponding to a loss of CH3NH2. In the cocaine MS/MS spectrum, the product ion observed at (31) Sigsby, M. L.; Day, R. J.; Cooks, R. G. Org. Mass Spectrom. 1979, 14, 556561. (32) Guan, S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1996, 157/ 158, 5-37.

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Figure 3. (a) ESI mass spectrum of an equimolar mixture of bradykinin and the synthetic peptide KGAILKGAILR; (b) ESI mass spectrum of bradykinin; (c) ESI mass spectrum of synthetic peptide KGAILKGAILR.

m/z 182 corresponds to loss of tropane methyl formate (C10H15O2N). In the case of the heroin MS/MS spectrum, product ions were obtained at m/z values of 328, 310, 268, and 211, all of which have been observed previously.33,34 The product ions of all the drugs were consistent with data reported in the literature. Plots of ion abundance for each analyte were linear over 3 orders of magnitude. Calibration curves are shown in the inset to Figure 2. Detection limits correspond to parts per billion (ng/mL) levels in solution. Solutions containing the peptide bradykinin, the synthetic peptide KGAILKGAILR, or their mixture at 1 µg/mL were subjected to ESI, and the spectra obtained are shown in Figure 3. These spectra look very similar to those obtained on conventional commercial instruments, showing a multiply charged ion distribution as expected from an ESI experiment. Both +2 and +3 charges for each peptide were observed in the mixture. The mass range was comfortably extended to m/z 600 and included ions with a +3 charge state, demonstrating the system’s usefulness for the analysis of biomolecules with molecular weights of high as 1800 Da. Systematic studies are now underway to apply this methodology and instrumentation to small proteins in an attempt to extend the observable mass range even further. Mass analysis with resonance ejection at lower frequencies should provide an extended mass range.35 This result could extend the potential range of bioapplications for this handheld mass spectrometer if coupled to an HPLC device in the future. (33) Jackson, A. U.; Talaty, N.; Cooks, R. G.; Van Berkel, G. J. J. Am. Soc. Mass Spectrom., in press. (34) Kauppila, T. J.; Talaty, N.; Kuuranne, T.; Kotiaho, T.; Kostiainen, R.; Cooks, R. G. Analyst 2007, 132 (9), 868. (35) Kaiser, R. E.; Cooks, R. G.; Stafford, G. C.; Syka, J. E. P.; Hemberger, P. H. Int. J. Mass Spectrom. Ion Processes 1991, 106, 79-115.

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DESI. The operating conditions for DESI on the handheld instrument were deliberately kept milder than conventional experiments (see Experimental Section). The use of relatively high pressures in a small low rf voltage ion trap is also likely to minimize the internal energies deposited in ions generated in these experiments. The DESI setup is schematically depicted in Figure 4a. A DESI spectrum, obtained by using this setup, of 50 ng of cocaine deposited onto Teflon is also shown in Figure 4a. The peak at m/z 304 is the protonated cocaine molecule, and there are no observable fragment ions. The inset shows a spectrum of the same amount of cocaine deposited on a $50 bill. The sample spread over an area on the $50 bill of roughly twice the area on the Teflon surface. (This was verified by adding a dye to the solution.) For both surfaces, the DESI spray samples the same area, about 4 mm2. Yet, the concentration of analyte over this surface is roughly half for the bill, consistent with a total ion signal of 1200 counts, while that for the Teflon was almost twice the amount at 2600. DESI is known to be surface dependent with Teflon being the best surface for analysis of drugs of abuse as observed during a previous study.34 The amount of cocaine detected (∼nanogram amounts) is typically within the range of the amount of drugs found on the surface of currency contaminated by drugs.36 Such an application emphasizes the potential to carry rapid in situ analysis for a wide variety of applications. A new version of the DESI experiment which eliminates the ion signal’s strong dependence on the exact position and angles of the spray and collection capillaries, was carried out (schematically shown in Figure 4b).30 The spray capillary and collection capillary were placed at a 5-10° angle relative to each other, with (36) Sleeman, R.; Burton, R.; Carter, J.; Roberts, D.; Hulmston, P. Anal. Chem. 2000, 72, 397A-403A.

Figure 4. (a) Conventional DESI experiment and corresponding DESI mass spectrum of 50 ng of cocaine from a Teflon surface; inset DESI spectrum of cocaine deposited on a U.S. $50 bill. (b) Geometry independent DESI experiment and a corresponding DESI mass spectrum of 50 ng of cocaine from a Teflon surface.

both capillaries approximately normal to the analysis surface. Both capillaries were further enclosed in a small common volume (approximately 1 mL, Swagelok nut). This design causes the DESI spray to be relatively contained within the volume and have a greater chance of being drawn into the sampling capillary than in the conventional DESI experiment. The DESI spray was aimed at 50 ng of cocaine residue on a Teflon surface. The spectrum (Figure 4b) obtained is identical to the conventional DESI spectra already shown (Figure 4a). This method may also allow sampling a larger area of the surface as collection of scattered droplets may be more efficient. The ruggedness of this method should also allow it to be attached to the instrument for portable use. Current experiments are also underway to further miniaturize the DESI setup and to analyze mixtures in real life settings. CONCLUSIONS The use of a narrower capillary than is normally used for atmospheric pressure ionization allowed direct atmospheric sampling using the Mini 10 mass spectrometer. Both DESI and ESI were possible, and quantitative results were generated by ESI-

MS for drugs of abuse and peptides. Moreover, DESI-MS was also implemented using two different types of sources. These results show that atmospheric pressure ionization can be implemented on simple portable MS systems. The current system is not yet fully optimized in two important respects: first, the supplementary components of the ionization method, i.e., gas supply and solution spray system for DESI, ESI, and APCI, are still to be miniaturized. Second, the atmospheric interface is rudimentary, consisting of a capillary connection between the ambient ion source and the mass spectrometer, and is also subject to improvements. ACKNOWLEDGMENT The authors thank Jason Duncan and Dr. Andre Venter for technical assistance. Support from the Office of Naval Research, Grant No. N00014-05-10454, is gratefully acknowledged. Received for review May 28, 2007. Accepted July 20, 2007. AC071114X

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