Atmospheric Pressure Chemical Ionization Source. 2. Desorption

Anal. Chem. 2008, 80, 2654-2663

Atmospheric Pressure Chemical Ionization Source. 2. Desorption-Ionization for the Direct Analysis of Solid Compounds Francisco J. Andrade, Jacob T. Shelley, William C. Wetzel,† Michael R. Webb, Gerardo Gamez, Steven J. Ray, and Gary M. Hieftje*

Department of Chemistry, Indiana University, Bloomington, Indiana 47405

The flowing afterglow-atmospheric pressure glow discharge (APGD) ionization source described in part 1 of this study (in this issue) is applied to the direct analysis of condensed-phase samples. When either liquids or solids are exposed to the ionizing beam of the APGD, strong signals for the molecular ions of substances present on their surfaces can be detected without compromising the integrity of the solid sample structure or sample substrate. As was observed for gas-phase compounds in part 1 of this study, both polar and nonpolar substances can be ionized and detected by mass spectrometry. The parent molecular ion (or its protonated counterpart) is usually the main spectral feature, with little or no fragmentation in evidence. Preliminary quantitative results show that this approach offers very good sensitivity (detection limits in the picogram regime are reported for several test compounds in part 1 of this study) and linear response to the analyte concentration. Examples of the application of this strategy to the analysis of real-world samples, such as the direct analysis of pharmaceutical compounds or foods is provided. The ability of this source to perform spatially resolved analysis is also demonstrated. Preliminary studies of the mechanisms of the reactions involved are described. The transition from the condensed to the gas phase, a necessary step in the analysis of liquids or solids by mass spectrometry (MS), is very often a serious limitation. Although sample dissolution is usually employed in the case of solids, where this limitation is more severe, this step is not problem-free: solubility, stability of the dissolved species in solution, and solvent interference in the ionization process can all cause difficulties. For this reason, considerable effort has been expended on the development of MS procedures that permit the direct analysis of solid materials. Desorption-ionization techniques, such as plasma desorption, laser desorption-ionization, secondary ion mass spectrometry, and matrix-assisted laser desorption-ionization (MALDI),1-3 have been particularly successful. One of the key * Corresponding author. E-mail: [email protected]. Fax: (812) 855-0958. † Current address: Thomas More College, 333 Thomas More Parkway, Crestview Hills, KY 41017. (1) Vestal, M. L. Chem. Rev. (Washington, DC, U.S.) 2001, 101, 361-375. (2) Busch, K. L. J. Mass Spectrom. 1995, 30, 233-240.

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features of these techniques is the ability to ionize analyte species independent of their volatility. This capability boosted the application of MS techniques, particularly in the life-science domain. One limitation, however, is that many of these approaches require working at reduced pressure. When the source is at low pressure, the process of sample introduction becomes more involved. Also, problems of sample compatibility with vacuum and sample conditioning can adversely affect the results.2 Finally, ions of organic molecules generated under vacuum are more susceptible to fragmentation due to the reduced likelihood of collisional cooling. For these reasons, there has been growing interest in the development of MS techniques that can deal with solid samples at atmospheric pressure. Examples of this trend are the recent introduction of atmospheric pressure MALDI4-7 and the launch of a series of novel techniques for the direct MS analysis of solids at atmospheric pressure. An early atmospheric pressure desorption-ionization procedure was the “liquid ionization” technique reported more than two decades ago by Tsuchiya and co-workers.8,9 In this approach, a needle with the sample suspended in a suitable organic solvent (e.g., glycerol, nujol) was exposed to the gaseous effluent from a corona discharge in argon. When a bias voltage (usually between 200 and 1500 V) was applied to the sample-containing needle, signals for the molecular ions of nonvolatile components in the sample were observed. Some years later, Lubman et al.10 reported the desorption-ionization of organic substances from a probe inserted directly into an atmospheric pressure glow discharge (APGD). They found that a probe carrying solid materials introduced into the discharge chamber resulted in molecular ions of the substances of interest. These two approaches showed promise, although they never enjoyed widespread use, perhaps because of the difficulty of sample introduction. (3) Karas, M.; Bachmann, D.; Hillenkamp, F. Anal. Chem. 1985, 57, 29352939. (4) Erickson, B. Anal. Chem. 2000, 72, 186A. (5) Laiko, V. V.; Moyer, S. C.; Cotter, R. J. Anal. Chem. 2000, 72, 5239-5243. (6) Li, Y.; Shrestha, B.; Vertes, A. Anal. Chem. 2007, 79, 523-532. (7) Dreisewerd, K.; Draude, F.; Kruppe, S.; Rohlfing, A.; Berkenkamp, S.; Pohlentz, G. Anal. Chem. 2007, 79, 4514-4520. (8) Tsuchiya, M.; Kuwabara, H. Anal. Chem. 1984, 56, 14-19. (9) Tsuchiya, M.; Taira, T.; Kuwabara, H.; Nonaka, T. Int. J. Mass Spectrom. Ion Phys. 1983, 46, 355-358. (10) Sofer, I.; Zhu, J.; Lee, H. S.; Antos, W.; Lubman, D. M. Appl. Spectrosc. 1990, 44, 1391-1398. 10.1021/ac800210s CCC: $40.75

© 2008 American Chemical Society Published on Web 03/18/2008

A turning point in atmospheric pressure desorption-ionization was reached very recently, with the introduction of desorptionelectrospray ionization (DESI) by Cooks and co-workers.11 In this approach, charged droplets from an electrosprayed solution are directed toward a solid sample by means of a high-velocity (around 350 m/s) gas stream. The charged droplets ablate the exterior of the sample, removing and ionizing organic molecules present on the surface.12-14 Accordingly, DESI permits the direct analysis of condensed-phase samples with minimal or no sample preparation. It exhibits high sensitivity and fast response times and can be applied to a wide range of compounds, from small organic molecules to large biological and synthetic polymers. Although mass spectra produced by DESI are generally similar to those of electrospray ionization, several overlapping mechanisms allow the ionization of polar and nonpolar compounds in both the positive and negative ion mode.12 Thus, depending on the type of analyte and the chosen operating conditions, DESI spectra can also resemble those obtained with a conventional atmospheric pressure chemical ionization (corona) source. DESI has been applied to the analysis of explosives,15-17 chemical-warfare agents,18 illicit drugs,19-21 plant materials,22 pharmaceutical preparations,23-26 industrial polymers,27 and biological fluids and tissues.14 It has also been shown that real samples, such as office equipment28 and foods,11 can be directly analyzed without sample preparation. Furthermore, the technique can provide spatial information, a feature already used for the analysis of thin layer chromatographic plates,29,30 for the analysis of plant materials,22 and for the identification of tumor cells in tissues.14 (11) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science (Washington, DC, U.S.) 2004, 306, 471-473. (12) Takats, Z.; Wiseman, J. M.; Cooks, R. G. J. Mass Spectrom. 2005, 40, 12611275. (13) Venter, A.; Sojka, P. E.; Cooks, R. G. Anal. Chem. 2006, 78, 8549-8555. (14) Cooks, R. G.; Ouyang, Z.; Takats, Z.; Wiseman, J. M. Science (Washington, DC, U.S.) 2006, 311, 1566-1570. (15) Takats, Z.; Cotte-Rodriguez, I.; Talaty, N.; Chen, H.; Cooks, R. G. Chem. Commun. (Cambridge, U.K.) 2005, 1950-1952. (16) Cotte-Rodriguez, I.; Takats, Z.; Talaty, N.; Chen, H.; Cooks, R. G. Anal. Chem. 2005, 77, 6755-6764. (17) Mulligan, C. C.; Talaty, N.; Cooks, R. G. Chem. Commun. (Cambridge, U.K.) 2006, 1709-1711. (18) Cotte-Rodriguez, I.; Cooks, R. G. Chem. Commun. (Cambridge, U.K.) 2006, 2968-2970. (19) Leuthold, L. A.; Mandscheff, J.-F.; Fathi, M.; Giroud, C.; Augsburger, M.; Varesio, E.; Hopfgartner, G. Rapid Commun. Mass Spectrom. 2005, 20, 103110. (20) Leuthold, L. A.; Mandscheff, J.-F.; Fathi, M.; Giroud, C.; Augsburger, M.; Varesio, E.; Hopfgartner, G. Chimia 2006, 60, 190-194. (21) Rodriguez-Cruz, S. E. Rapid Commun. Mass Spectrom. 2005, 20, 53-60. (22) Talaty, N.; Takats, Z.; Cooks, R. G. Analyst (Cambridge, U.K.) 2005, 130, 1624-1633. (23) Kauppila, T. J.; Wiseman, J. M.; Ketola, R. A.; Kotiaho, T.; Cooks, R. G.; Kostiainen, R. Rapid Commun. Mass Spectrom. 2005, 20, 387-392. (24) Williams, J. P.; Scrivens, J. H. Rapid Commun. Mass Spectrom. 2005, 19, 3643-3650. (25) Weston, D. J.; Bateman, R.; Wilson, I. D.; Wood, T. R.; Creaser, C. S. Anal. Chem. 2005, 77, 7572-7580. (26) Chen, H.; Talaty, N. N.; Takats, Z.; Cooks, R. G. Anal. Chem. 2005, 77, 6915-6927. (27) Nefliu, M.; Venter, A.; Cooks, R. G. Chem. Commun. (Cambridge, U.K.) 2006, 888-890. (28) D’Agostino, P. A.; Hancock, J. R.; Chenier, C. L.; Lepage, C. R. J. J. Chromatogr., A 2006, 1110, 86-94. (29) Kauppila, T. J.; Talaty, N.; Salo, P. K.; Kotiaho, T.; Kostiainen, R.; Cooks, R. G. Rapid Commun. Mass Spectrom. 2006, 20, 2143-2150. (30) Van Berkel, G. J.; Ford, M. J.; Deibel, M. A. Anal. Chem. 2005, 77, 12071215.

Following DESI, several other desorption-ionization approaches appeared. Some of them, such as the desorption-sonic spray ionization (DeSSI),27,31 are offsprings of DESI. This alternative approach uses a sonic spray (instead of an electrospray) as a way of generating the stream of charged droplets. In electrosprayassisted laser desorption-ionization (ELDI)32 a laser produces the desorption of neutrals, while the ionization is performed by reaction with ions and charged droplets formed by an electrosprayed solution. The remarkable impact of DESI in such a short period reflects the interest in and importance of these kinds of techniques. Alternative ways of performing atmospheric pressure desorption-ionization by means of gas discharges have also been reported recently. Desorption-atmospheric pressure chemical ionization (DAPCI)14,33,34 uses a modified DESI source, in which the electrospray needle has been replaced by a corona source. As this approach uses gas-phase ions and a heated gas stream to perform desorption-ionization, it has been shown that it performs better than DESI with low to moderate polarity compounds. DART (direct analysis in real time)35 shows some similarities to DAPCI and the liquid ionization source proposed by Tsuchiya and coworkers.8 In DART, a corona discharge sustained in helium or nitrogen is used to produce ions and excited species, which are then transported to a secondary compartment fitted with an additional set of electrodes.36 The gaseous stream that leaves this compartment is then heated and used to produce desorption and ionization of organic substances in open air. Unfortunately, neither the exact operating conditions nor the optimization of the DART source have been published, which makes it difficult to compare directly with the others. It has been claimed that helium metastables (He*) are involved in the desorption-ionization process.35 However, there is yet no solid experimental evidence to support this assertion, and the mechanism of DART remains unclear. In any case, DART has proven able to ionize a wide variety of compounds with a very simple sample introduction scheme. Application of this source to several types of real samples,35 including detection of counterfeit drugs37 and characterization of inks,38 has been demonstrated. Another technique that has been recently reported is the atmospheric solids analysis probe (ASAP).39 In this approach, the target compounds are desorbed by a stream of heated nitrogen, and a conventional atmospheric pressure chemical ionization source (corona discharge) or an electrospray source is used for (31) Haddad, R.; Sparrapan, R.; Eberlin, M. N. Rapid Commun. Mass Spectrom. 2006, 20, 2901-2905. (32) 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. (33) Williams, J. P.; Patel, V. J.; Holland, R.; Scrivens, J. H. Rapid Commun. Mass Spectrom. 2006, 20, 1447-1456. (34) Song, Y.; Cooks, R. G. Rapid Commun. Mass Spectrom. 2006, 20, 31303138. (35) Cody, R. B.; Laramee, J. A.; Durst, H. D. Anal. Chem. 2005, 77, 22972302. (36) Cody, R. B.; Laramee, J. A. (Jeol U.S.A., Inc.). Atmospheric Pressure Ion Source. U.S. Patent Appl. 20050056775, March 17, 2005, Cont-in-part of U.S. Serial No. 732,205. (37) Fernandez, F. M.; Cody, R. B.; Green, M. D.; Hampton, C. Y.; McGready, R.; Sengaloundeth, S.; White, N. J.; Newton, P. N. ChemMedChem 2006, 1, 702-705. (38) Jones, R. W.; Cody, R. B.; McClelland, J. F. J. Forensic Sci. 2006, 51, 915918. (39) McEwen, C. N.; McKay, R. G.; Larsen, B. S. Anal. Chem. 2005, 77, 78267831.

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ionization of the resulting gas-phase compounds. Thus, in terms of mechanisms, ASAP appears to be closely related to DAPCI. Few studies comparing the different atmospheric pressure desorption-ionization approaches have been published.33,37 These preliminary reports seem to indicate that DESI provides some advantages in particular applications, although it appears that all the techniques are complementary. It is clear, however, that this new, active, and extremely attractive field of MS, now called “ambient mass spectrometry”,14 has room for new ideas and improvements. In the present study, we apply a new atmospheric pressure desorption-ionization source, the flowing afterglow-atmospheric pressure glow discharge (FA-APGD), to the direct analysis of solid samples. The resulting mass spectral features are similar to those observed for the ionization of gaseous compounds;40 that is, the molecular ion or protonated molecular ion usually dominates the mass spectrum, and little or no fragmentation is observed. When fragmentation does occur, the pattern is relatively simple and easy to interpret. In this way, the method resembles other gasdischarge based desorption-ionization approaches, such as DART or DAPCI; it also benefits from an extremely simple instrumental setup. Preliminary characterization of the analytical performance of this approach reveals that either solid or solution samples can be desorbed and ionized by the afterglow. Sensitivities in the tens of picogram range have been achieved for representative compounds. Preliminary results suggest the role of helium metastables in the generation of reagent ions. EXPERIMENTAL SECTION Reagents and Instrumentation. All reagents were analytical grade. High-purity He (99.999% ultrahigh purity helium, Airgas, Radnor, PA) was used in all experiments. A detailed description of the instrumental setup (APGD cell and ion sampling interface) can be found in part 1 of this work.40 In short, a helium atmospheric pressure glow discharge was formed between a tungsten-pin cathode and a brass-plate anode. The discharge was contained in a Teflon body. A 1 mm diameter hole in the anode allowed excited species formed in the discharge to issue into the atmosphere to either directly desorb/ionize a sample or to generate reagent ions, which would subsequently ionize the analyte of interest. The He gas flow was set and monitored by means of a mass flow controller (model FC-280-SAV, Tylan General, Carson, CA), and the discharge was operated with a direct current (dc) high-voltage power supply (model DRC-5-400R, Universal Voltronics, Mount Kisco, NY) in the current-controlled mode. The discharge cell was mounted on a home-built translation stage for alignment with the sampling orifice of the mass spectrometer. Ions were detected by a LECO Renaissance (LECO Corp., St. Joseph, MI) time-of-flight (TOF) inductively coupled plasma (ICP) mass spectrometer. The ICP source was disabled and moved aside, and the APGD cell was positioned facing the ion-sampling interface of the spectrometer, on axis with the ionsampling orifice. The original conical sampling plate of the Renaissance spectrometer was replaced by an ion-sampling interface made from a flat stainless steel plate. The front plate was insulated from the rest of the instrument, so its electrical (40) Andrade, F. J.; Wetzel, W. C.; Webb, M. R.; Gamez, G.; Ray, S. J.; Hieftje, G. M. 2008, 80, 2646-2653.


Figure 1. Experimental arrangements used for sample introduction in the desorption-ionization experiments. (a) Shelf on which samples can be placed: i, APGD cell (for more detail see part 1 of this study); ii, sample shelf; iii, MS interface. (b) Probe for solids and liquids: i, plastic probe; ii, sampling plate and probe holder. (c) Home-built autosampler: i, stepper motor; ii, rotating disk for carrying samples.

potential could be independently adjusted by means of a dc power supply (model IP-17, Heath Company Inc., Benton Harbor, MI). In this way, the ion-sampling interface mimics those commonly used with atmospheric pressure ionization sources. The integration time for each spectrum was 1 s unless otherwise noted. The only difference in the analysis of liquids and solids is that the APGD cell was placed farther from the mass spectrometer interface (usually 1 cm, and sometimes up to 5 cm away, instead of the 5 mm used during the ionization of gaseous compounds), in order to facilitate sample introduction (see Figure 1a). Samples can be inserted directly in the afterglow by using insulated pliers. This manual approach, however, leads to significant fluctuations of the ion signals, as will be discussed below. Accordingly, alternative sample-introduction strategies were tested. In the first approach, a probe consisting of a 1.6 mm diameter, 60 mm long Peek rod was employed (cf. Figure 1b). The sample was deposited on one end of the rod, and the probe was inserted into the afterglow with the help of a suitable holder attached to the mass spectrometer front plate (see Figure 1b). For some samples, a flat shelf made from Teflon was placed in front of the mass spectrometer front plate, as shown in Figure 1a. This shelf, on which samples can be placed, has an independent x,y,z stage that allows proper alignment of a particular sample size within the afterglow stream. Finally, a homemade autosampling device was used for sampling flat surfaces (paper, membranes, pharmaceutical tablets, etc.). This device (see Figure 1c) consists of a disk rotated by a stepper motor. Because the speed and position of the motor can be controlled, samples can be scanned in a reproducible way. Procedure. First, instrumental conditions were adjusted to maximize the background ion signals, as was described in part 1 of this work.40 The resulting conditions were APGD operating current ) 25 mA, anode potential (AP) ) 45 V, front-plate potential (FPP) ) 43 V, and ion-lens potential (ILP) ) 40 V. Next, signals for the solid organic samples were optimized. Different types of samples were tested: solutions, pure solids, and “real” (intact) samples (pills, foods, etc.). The use of dried aliquots of sample solutions was generally preferred for analytical characterization of the source, because it is then simpler to know or adjust the exact amount of substance that is present (particularly when very

Figure 2. Mass spectra of several organic compounds: (a) 7-diethylamino-4-methylcoumarin, (b) caffeine, (c) salicylic acid, (d) harmane, (e) 8-hydroxyquinoline, and (f) anthracene. The top three spectra were acquired using a flat membrane as a support; the bottom three spectra were acquired using a probe as shown in Figure 1b. The shadowed area indicates approximately where background ions exist.

low amounts are used). In this case, droplets of the sample solution were deposited on a solid substrate (either the probe or a flat surface) and dried at room temperature. Factors affecting signal stability and magnitude were then evaluated. Particular attention was paid to the study of different types of supporting materials, such as paper, glass, cloth, wood, and metal. Following this examination, quantitative aspects of the desorption-ionization approach were studied. The detection of organic molecules in real samples (pharmaceutical tablets, food, etc.) was then examined. Finally, studies of the spatial resolution capabilities of this source and its ionization mechanisms were pursued. RESULTS AND DISCUSSION Qualitative Aspects. Figure 2 shows the mass spectra obtained for several solid organic compounds exposed to the flowing afterglow-APGD. These spectra were obtained from dried droplets of methanolic solutions or from a small amount of the solid drug, using a flat membrane (Figure 2a-c) or the probe (Figures 2d,e) as a support surface for the sample. A wide variety of organic substances, including amines, acids, polyaromatic hydrocarbons, and different types of aromatic compounds were tested; only a few are shown here. The general observations are similar to those made for gaseous organic compounds.40 First, the mass spectra contain the molecular ion (or the protonated molecular ion) as the strongest peak, and little or no fragmentation is observed. A typical mass spectrum is Figure 2a, where two different portions are highlighted. At the low-mass end, the spectrum shows a sort of “fish backbone” formed by background ions (mostly water clusters of different sizes), as was already

shown in part 1 of this work.40 At the high-mass end of the spectrum, the protonated molecular ion of 7-diethylamino-4methylcoumarin (m/z ∼ 232) can be seen. The background ions are present in all the mass spectra, although their relative intensities depend on the sample introduction procedure, the type of sample, and instrumental conditions. For example, when flat membranes are employed as a sample support, the ionizing beam is less intercepted and the structure of the background is preserved (see Figure 2a-c). On the other hand, when a probe is used to support the sample, the ionizing beam might be disrupted depending on the probe position and the background can be distorted (the same is true when “real” samples are analyzed). For this reason, background peaks have been shadowed in Figure 2. In general, polar compounds yield the protonated molecular ion (MH+), while nonpolar compounds usually yield the molecular ion, M+. In some cases, however, mixtures of both, M+ and MH+ (indicating overlapping ionization pathways) are present. This occurs in the case of anthracene (Figure 2f). In some cases clustering, particularly with water molecules, is observed. A typical example is Figure 2c, where the addition of a water molecule to the protonated molecular ion of salicylic acid is apparent. An extreme case is shown in Figure 2f, where clusters of anthracene with several water molecules arise. This clustering is not surprising considering that, as stressed by Kebarle et al.,41-43 the proton(41) Nicol, G.; Sunner, J.; Kebarle, P. Int. J. Mass Spectrom. Ion Processes 1988, 84, 135-155. (42) Sunner, J.; Ikonomou, M. G.; Kebarle, P. Anal. Chem. 1988, 60, 13081313.


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transfer reaction involves a necessary step in which adducts with solvent (in this case water) are formed. The presence of adducts can be reduced somewhat by modifying the ion sampling interface.44,45 The spectral simplicity apparent in Figure 2 is an attribute of the new source that renders straightforward the determination of molecular weight. Even for relatively complex samples shown later, spectra are uncomplicated because of limited fragmentation and the preferential ionization of constituents having high gasphase basicity (in the case of proton-transfer ionization). The signal is generated immediately after the sample has been exposed to the afterglow. When small amounts of a substance (below the microgram range) are used, the signal peaks within the first few seconds and then decays as function of time. If large amounts (microgram range or higher) are used, the signal reaches a plateau and quickly disappears when the sample is removed. Optimization of the Ion Signal. The influence of the APGD and ion-sampling conditions on the total ion current is similar to that reported in part 1 of this study.40 The total ion current, however, is lower here because of the greater distance from the cell to the mass spectrometer. For this reason, the He gas flow was raised above that used in the ionization of gases. Typically, He flow rates in the range of 0.9-1.5 L/min were used. However, the APGD cell might have to be moved even farther from the spectrometer, for example, when large samples are used. In these cases, higher gas flows (up to 2 L/min) can be employed. The optimal value for the front-plate potential here was approximately 37 V, which is slightly lower than the value found optimal for gaseous compounds.40 Sample Introduction. The many ways in which liquid or solid samples can be introduced makes this approach extremely versatile, although at the same time they lead to the problem of developing standard (i.e., reproducible) sample introduction procedures. As a first approximation, generating a signal requires only exposure of the sample, either liquid or solid, to the afterglow. Depending on the sample size, solids can be introduced with pliers or, if the sample is too small, on a solid support (substrate). One of the simplest ways to introduce a sample is by using a commercial swab (cotton, foam, polyester, etc.). In this case, the swab is used to wipe a surface and is then exposed to the afterglow. Different types of surfaces, from concrete to human skin have been successfully tested by using this procedure. Different kinds of commercial swabs were employed, all yielding similar results. As an illustration of this approach, a fixed volume of a caffeine solution was placed in a rotating cylindrical container under conditions that allow the solution to uniformly evaporate on the container’s walls. In this way, the inner surfaces of various glass containers were coated with known amounts of caffeine. A fixed area (2 cm2) of the container walls was then wiped with polyester swabs, which were then introduced into the afterglow. Amounts as low as 5 ng/cm2 could be easily detected using this procedure (data not shown). The use of a swab is attractive because it provides a standardshaped support that can be reproducibly placed in the afterglow. However, virtually any kind of solid material that fits within the (43) Sunner, J.; Nicol, G.; Kebarle, P. Anal. Chem. 1988, 60, 1300-1307. (44) Bruins, A. P. TrAC, Trends Anal. Chem. 1994, 13, 37-43. (45) Bruins, A. P. Mass Spectrom. Rev. 1991, 10, 53-77.


Figure 3. Time profile for the desorption-ionization of a droplet of a methanolic solution of benzoic acid. The dashed line is the time trace of the protonated molecular ion of methanol (m/z ) 33). The solid line corresponds to the molecular ion of benzoic acid (m/z ) 122). Both spectral features were measured simultaneously with the TOFMS.

gap between the APGD cell and the mass spectrometer can be employed. Support materials that offer flat surfaces, such as filter paper, membranes (cellulose acetate, nylon, etc.), fabrics, glass slides, and cloth, were tested. Experiments with different kinds of plastics and synthetic materials (polyamide, Teflon, polystyrene, acrylic, nylon) having a range of shapes were conducted. In each experiment, a droplet of solution containing a chosen organic compound (e.g., benzoic acid in methanol) was dried on the surface of the substrate to be evaluated. After evaporation of the solvent, the material was exposed to the afterglow. In all cases, the organic molecules present on the substrate surface were detected. Qualitatively, the substrate material did not have a marked influence on the nature of the resulting mass spectrum. It should also be noted that there was no observable damage sustained by the sampling support. However, difficulties of reproducing the sample introduction scheme with different materials made quantitative comparisons difficult to establish. As a first approximation, it seems that desorption can be more easily performed on hydrophobic substrates, although this observation requires further investigation. Solutions can be analyzed directly by exposing them to the afterglow. Sample solutions can be wicked into an absorbent material (swab) or placed in a capillary tube. In these experiments, ions from the solvent (the molecular ion and several clusters) typically dominate the mass spectra initially. Then, once the solvent has evaporated, the less volatile solute components are observed. Figure 3 shows the results when a droplet of a methanolic solution of benzoic acid is exposed to the afterglow. The approximate linear velocity of the gas exiting the discharge cell is 8 m/s, which is much lower than the linear gas velocity in DESI (>350 m/s). Because this is a relatively low-velocity gas beam, liquid droplets remain stationary on either the end of the capillary or other solid platforms. In addition, the nature of the solvent influences the ionization process when this procedure is used. First, the gas-phase acid-base behavior of the evaporated solvent influences ionization, as happens in APCI sources.44,45 Second, the volatility of the solvent plays a role in the time of appearance of

Figure 4. Quantitative analysis: (a) calibration plot for caffeine obtained with a flat membrane (filter paper) as a desorption surface. The inset plot shows a calibration plot in the mass range 1-5 ng. (b) Transients obtained with the probe of Figure 1b for different amounts of harmane: i, 18 ng; ii, 9 ng; iii, 3.6 ng. In all cases, the signal of the protonated molecular ion is used for the calculations.

the solute species. To avoid this solvent effect, it was generally preferred to use previously dried droplets of solution. Quantitative Aspects. The foregoing experiments demonstrate the desorption-ionization capabilities of the FA-APGD from a qualitative perspective. However, the new source also performs well for quantitative analysis. Figure 4a shows the results of the analysis of dried droplets of caffeine solution using a filter paper as a support. The filter paper was manually introduced into the afterglow for subsequent desorption-ionization. The area of the resulting transient signals was used to construct the calibration curve. Similar experiments have been conducted with a range of substances and using a variety of sample introduction procedures. For example, Figure 4b shows the transient signals obtained for the ionization of harmane in the low nanogram range by means of the probe illustrated in Figure 1b. In general, linear ranges are between 2 and 3 orders of magnitude, and limits of detection run from subnanograms to tens of picograms. These results are comparable to those reported for DESI.12,14 No quantitative results have yet been reported for DART. One limitation of proper quantitative characterization of this new approach lies in the reproducibility of the signal, which is in turn linked to the reproducibility of the sample introduction procedure. As was mentioned before, when large amounts (microgram range or higher) of substances are introduced, the signal reaches a plateau. The stability of the signal in this plateau region is usually between 2 and 5% RSD (for 1 min readings, 500 ms integration time). These fluctuations, which can be ascribed to gas flow turbulence in the ion-sampling interface, can be regarded as a good estimate of the best attainable precision. If the sample is removed and repositioned, the RSD between the signals might worsen to as much as 50%. This is the case, for example, when samples are introduced manually on a flat support (membranes, etc.) and the sample positioning is not meticulously controlled. Such “flat” surfaces actually have wrinkles or waves that can markedly affect the ion signal. This drawback might be alleviated in the future by use of a capillary sampling interface, which could help to control the area being sampled. With the sampling probe, the reproducibility between signals was approximately 10% RSD, but small changes in the probe positioning can significantly worsen this value.

The strong dependence of the ion signal on sample positioning has also been reported for other desorption-ionization techniques, such as DESI and DART. In DESI, the dependence of the signal on the angle between the source and the sample is well known and was used to postulate the possible mechanisms involved in the functioning of that source.12,13 In DART it has been shown that the sample position can affect both the total magnitude and the relative abundances of the peaks.38 In the present study, sample positioning affects the overall height of the analyte peaks but changes in their relative magnitudes have not been observed. In any case, a reproducible sample-introduction procedure is required in order to perform quantitative analysis. Application of the Flowing Afterglow-APGD to the Analysis of Real Samples. One of the most straightforward applications of this new technique is the analysis of pharmaceutical compounds. As with other desorption-ionization approaches, pharmaceutical tablets can be directly introduced into the afterglow and the signals for the main components can be readily detected. Examples of this approach are given in Figure 5, which shows the mass spectra obtained when tablets of Tylenol (Figure 5a) and Ibuprofen (Figure 5b) were directly exposed to the afterglow. Many different kinds of tablets can be analyzed, and the only constraint found so far is the limited mass range of our mass analyzer. The sole required sample preparation is removal of the coating on some tablets. Once again, although the qualitative detection of the main constituents is relatively straightforward, the generation of quantitative results is considerably more difficult. The reproducibility of sample introduction or positioning has a marked influence on the RSD of the signal. The acetaminophen protonated molecular ion (m/z ) 152) was measured in Paracetamol tablets by two different procedures (data not shown). In the first, the pills were manually placed in a sample holder as illustrated in Figure 1a. Once the tablet was exposed to the afterglow, as in the earlier example, the signal reached a plateau and remained relatively stable, with RSD 2-5%. However, when the procedure was repeated and either the same or a different tablet was relocated in the afterglow, the RSD among the different signals is at least 1 order of magnitude higher (∼30%). Clearly, the difficulty of reproducing the point where the afterglow contacts the sample seems dominant in controlling the signal uncertainty. Analytical Chemistry, Vol. 80, No. 8, April 15, 2008

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Figure 5. Mass spectra obtained when pharmaceutical tablets are exposed to the flowing afterglow: (a) Tylenol tablet, showing the main component (acetaminophen), (b) Ibuprofen tablet, showing its protonated molecular ion.

Figure 6. Detail of the mass spectra obtained by exposing (a) a coffee bean and (b) a green tea leaf to the flowing afterglow. The peak marked with an arrow corresponds to m/z ) 195, likely the protonated molecular ion of caffeine (m/z ) 195).

Accordingly, the autosampler shown in Figure 1c improves the RSD between signals to 5-10%. Of course, this approach is more easily performed on samples with a standardized shape. It was noted that traces of pharmaceutical compounds can be detected on the hands of the operator after the tablets are manipulated. If the fingers of the analyst are wiped with a swab, the molecular ions of the compounds originally present in the tablet can be detected when the swab is exposed to the afterglow. The new source therefore has potential use for the detection of contaminants in the pharmaceutical or food industry, detection of illicit drugs, forensics, and other areas. A similar approach can be used for the detection of additives in foods. For example, two kinds of lemons were purchased, one identified as “normal” and the other as “organic”. The outer skin of each lemon was then gently rubbed with a clean polyester swab, which was then introduced into the afterglow. Interestingly, for the “normal” lemon a distinctive and very large signal at m/z ) 202 appeared, while 2660 Analytical Chemistry, Vol. 80, No. 8, April 15, 2008

the organic lemon did not show anything in this mass window (data not shown). The signal found likely corresponds to 2-(4thiazolyl)benzimidazole (theoretical MH+ ) 202), a compound used to prevent mold growth on lemons. As a matter of fact, although the organic lemon grew mold after 2 days in the lab, the “normal” lemon remained without mold for more than four weeks. Unfortunately, we do not have the necessary tools to confirm this supposition. In any case, the procedure is extremely simple and powerful, and with a suitable mass spectrometer, could be applied to the rapid, nondestructive monitoring of food products. Similar examples are shown in Figure 6, where the mass spectra resulting from the exposure of an intact coffee bean (Figure 6a) and a green tea leaf (Figure 6b) to the afterglow are shown. In both cases, a strong peak at m/z 195 can be observed. Very likely, this peak corresponds to caffeine (theoretical MH+ ) 195, see Figure 2b).

Figure 7. Experimental setup and results of the enclosed afterglow experiments: (a) experimental setup; top, schematic diagram; bottom, cutaway view. i, APGD cell; ii, sleeve added to the cell to enclose the afterglow; iii, MS front plate; iv, port for adding foreign gases to the afterglow; v, picoammeter. The gas flow follows the directions of the arrows. (b) Total ion current at the front plate as a function of the gas flow added to the afterglow-He flow (for the APGD cell) ) 3 L/min.

The sensitivity of the new source is important also in avoiding sample cross-contamination. Because so little of a sample needs to be removed, there appears to be no sample deposition on sampling surfaces or holders, so cross-contamination and memory effects are negligible. Direct analysis of beverages is also possible. When a swab is immersed in a vessel containing brewed coffee, for example, and is then introduced into the afterglow, the signal for the caffeine molecular ion is detected after the peaks corresponding to water clusters have disappeared (i.e., after evaporation of the solvent). In summary, the new source has potential use in a broad range of fields. A comprehensive coverage of these examples is beyond the scope of the present study. Rather, the intent is to stress the broad utility of the source and to point out the obstacles and uncertainties that must be overcome in order to fully exploit it as an analytical tool. One of these concerns, the importance of reproducible sample positioning, has already been stressed. Another is the dependence of the analyte signal on the sample matrix. This limitation is common in ionization sources at atmospheric pressure. Chemical ionization relies on the generation of a pool of reagent ions. If this reservoir is depleted by a major constituent of the sample or by one with a great affinity for the reagent ions, the signal for the minor components might be reduced or, in the worst case, completely disappear. In conventional chemical ionization sources, this problem is often overcome by the use of a separation procedure but even then the choice of the solvent is critical as the solvent itself might cause an interference. In short, any technique that claims to be sample-preparation free must first demonstrate that it is interference-free. Some of the problems mentioned above might in the future be overcome by the use of an internal standard. Selecting an appropriate internal standard, however, requires some knowledge of the mechanisms involved in the desorption-ionization processes. Unfortunately, there have not been any earlier reports regarding these mechanisms for other gas-discharge based approaches. The next paragraphs will offer some preliminary findings in this area.

Reaction Pathways in the Flowing Afterglow-APGD. Desorption-ionization mechanisms at atmospheric pressure are still a matter of discussion. In DESI, it has been proposed that two different pathways, droplet pick up and gas-phase ionization, are involved.12,13 In this mechanism, both the kinetic energy of the droplets, which ablate the sample, and the charging of the surface have an effect on the desorption-ionization process. In DART, on the other hand, it is not clear what the mechanism is, perhaps because of the lack of precise data regarding operating conditions. It has been repeatedly mentioned that helium metastables (He*) play a significant role in desorption-ionization.35,37 For example, direct Penning ionization of the analyte and Penning ionization of water molecules have been suggested. Clearly, in terms of its operating characteristics, the flowing afterglow-APGD shares more similarities with DART and DAPCI than with DESI. Interestingly, the background spectra of both DART and APGD are similar to those from conventional APCI sources. The APGD background spectrum is dominated by water clusters and, to a lesser extent, by NO+, O2+, H2O+ and other ionized atmospheric components. The origin of these species has been briefly discussed in part 1 of this work.40 In conventional APCI sources, the ionization of N2, which in turns leads to the generation of water clusters, arises from electron impact in the region close to one of the electrodes. However, in the APGD it is likely that N2+ is generated through Penning ionization with He*. In order to test this hypothesis, and at the same time to gain some insight into the nature of the afterglow, a series of experiments was designed in which the afterglow was enclosed (see Figure 7a). In these experiments, a Teflon sleeve isolated the gas stream that leaves the discharge chamber. This stream impacts the mass spectrometer sampling plate, where part of it is sampled into the mass spectrometer and part is released through a long tube. Additionally, a selected gas can be added to the afterglow through the port shown in Figure 7a. In this way, both mass spectra and the total ion current at the front plate can be monitored. In order to avoid diffusion into the APGD cell of any of the gases added to the afterglow, the sleeve was made relatively Analytical Chemistry, Vol. 80, No. 8, April 15, 2008

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long and the helium flow was raised above the usual operating values (up to 3 L/min). When no foreign gas is added to the afterglow, a total ion current on the order of 50 nA is measured at the mass spectrometer front plate and typical ions such as NO+, H2O+, N2+ (and some clusters of these ions with water) are observed. We reported the presence of some of these ions in an APGD in earlier spectroscopic studies.46 Presumably, these ions originate either from contaminants in the He supply or from atmospheric leaks. The effect of adding foreign gases to the afterglow can be seen in Figure 7b. The addition of Ne does not produce any significant change in the ion current, whereas the addition of either N2 or Ar produces a marked elevation in this current. At the same time, this increase in ion current is accompanied by the appearance of signals of either N2+ and or Ar+ in the mass spectrum. This behavior clearly indicates that energetic species present in the flowing afterglow have the ability to ionize these two gases, i.e., they have energies above 15.75 eV (the ionization potential of N2 is 15.6 eV and that of Ar is 15.7 eV) but below 21.5 eV (ionization energy of Ne). Very likely, helium metastables with energies of 19 eV are involved. It is significant that the plots in Figure 7b level off at very low flow rates of the added gas (i.e., for concentrations that are below 1% in the afterglow). This behavior suggests that whichever species is involved in the ionization, it is readily consumed by the presence of a reactive gas. Therefore, it would be expected that, in contact with ambient air, these species would be quickly depleted by the reaction with nitrogen, water (as moisture), or oxygen. It seems unlikely that any of the metastables reaches the surface of the sample. If this is the case, the desorption-ionization mechanism appears likely to have a thermal and a chemical component. Unlike a corona (i.e., DART), the APGD heats the support gas (He here). Although the temperature of the flowing afterglow has not been measured accurately, it would be expected to be well above room temperature, considering that the values reported for the gas temperature of a He APGD are between 700 and 1000 K.46 Further, the presence of ionized water clusters impacting the sample surface might play a role similar to what has been postulated in DESI.12,13 Further experiments and refinement of the measurements performed here should bring more light to this topic. Regarding ionization pathways, the results described above (proton transfer as the predominant reaction, background spectra dominated by water clusters, etc.) suggest that the flowing afterglow, when open to ambient air, behaves qualitatively like a conventional APCI source. A similar result was reported for the ionization of organic compounds introduced into the discharge chamber of an APGD by Lubman and co-workers.10 Therefore, the APGD seems to behave as an enhanced APCI source, with proton transfer from water clusters as the main ionization pathway for polar compounds and charge transfer, possibly from NO+, as the main ionization pathway for nonpolar substances. Unfortunately, because of limitations in the mass range of the spectrometer used in this work, it was not possible to perform an extensive comparison of the APGD with other desorptionionization strategies. The results shown here demonstrate that for small molecules (m/z < 300) this new source performs comparably with other reported methods. Moreover, the ion 2662 Analytical Chemistry, Vol. 80, No. 8, April 15, 2008

Figure 8. Spatial profile of the protonated molecular ion of β-alanine. The drawing on top represents four droplets of the substance dried on a filter paper.

transmission efficiency of the instrument used here is not optimal, since it was designed to work with a completely different ion source. Therefore, the performance demonstrated above should be taken as a low estimate of that expected for a more suitable mass spectrometer. In summary, the APGD shows promise in the field of ambient mass spectrometry. One of its key advantages is instrumental simplicity. Unlike other techniques, neither heating of the buffer gas nor additional ionization chambers are required. An additional strength of the APGD is that it can be easily miniaturized, without losing its most important features. In preliminary work we have developed a GD cell with a volume of less than 20 µL and a power consumption of less than 1 W, which also performs desorptionionization. Further work on this miniaturized source is currently being conducted. Finally, the flowing afterglow has the possibility of providing spatial resolution. In Figure 8, the results of some preliminary trials are shown. Four droplets (1 µL each) of a 1 mM β-alanine solution were dried on a Teflon membrane. The membrane was then scanned across the flowing afterglow using the autosampler depicted in Figure 1b at a speed of 120 µm/s, and the signal for the molecular ion of alanine was recorded. These results demonstrate that the source can provide spatial resolution in the sub-millimeter range, comparable to what has been reported for DESI.47,48 Although this value is considerably poorer than can be achieved with some low-pressure sources, it offers interesting promise, for example, for the reading of thin-layer chromatography plates. Also, by using a better nozzle at the end of the source, it would be expected that improved spatial resolution could be achieved. CONCLUSIONS The flowing afterglow-APGD can be considered as one more tool in the field of ambient mass spectrometry. The source is (46) Andrade, F. J.; Wetzel, W. C.; Chan, G. C. Y.; Webb, M. R.; Gamez, G.; Ray, S. J.; Hieftje, G. M. J. Anal. At. Spectrom. 2006, 21, 1175-1184. (47) Ifa, D. R.; Wiseman, J. M.; Song, Q.; Cooks, R. G. Int. J. Mass Spectrom. 2007, 259, 8-15. (48) Wiseman, J. M.; Ifa, D. R.; Song, Q.; Cooks, R. G. Angew. Chem., Int. Ed. 2006, 45, 7188-7192.

very simple and robust, and it performs extremely well in the mass range tested. Future work with a more suitable mass spectrometer will enable us to compare the performance in the higher mass range. Additionally, more fundamental work is required in order to evaluate the full capabilities and, in particular, the limitations of this and other gas-based desorption-ionization sources. ACKNOWLEDGMENT Supported in part by the U.S. Department of Energy through Grant DOE DE-FG02-98ER 14890 and by the Indiana University

Metacyt initiative. The mass spectrometer used in this study was provided by the LECO Corporation. The authors are grateful to the Edward G. Bair Mechanical Instrument Services at Indiana University for assistance in the construction of the instrumentation used in this investigation.

Received for review January 29, 2008. Accepted February 15, 2008. AC800210S


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Atmospheric Pressure Chemical Ionization Source. 2. Desorption

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