Anal. Chem. 2007, 79, 3519-3523
Microchip Sonic Spray Ionization Jaroslav Po´l,*,† Tiina J. Kauppila,† Markus Haapala,† Ville Saarela,§ Sami Franssila,§ Raimo A. Ketola,† Tapio Kotiaho,‡ and Risto Kostiainen*,†
Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014 University of Helsinki, Finland, Micro and Nanosciences Laboratory, Helsinki University of Technology, P.O. Box 3500, FI-02015 TKK, Finland, and Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki, P.O. Box 55, FI-00014 University of Helsinki, Finland
The first microchip version of sonic spray ionization (SSI) as an atmospheric pressure ionization source for mass spectrometry (MS) is presented. The microchip used for SSI has recently been developed in our laboratory, and it has been used before as an atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) source. Now the ionization is achieved simply by applying high (sonic) speed nebulizer gas, without heat, corona discharge, or high voltage. The microchip SSI was applied to the analysis of tetra-Nbutylammonium, verapamil, testosterone, angiotensin I, and ibuprofen. The limits of detection were in the range of 15 nM to 4 µM. The technique was found to be highly dependent on the position of the chip toward the mass spectrometer inlet, and on the gas and the sample solution flow rates. The microchip SSI provided dynamic linearity following a pattern similar to that used with electrospray, good quantitative repeatability (RSD ) 16%), and longterm signal stability. Numerous applications of life sciences, biochemistry, and environmental, food, and drug analysis employ liquid chromatography-mass spectrometry (LC-MS). Ionization of analytes prior to MS is usually performed by one of the atmospheric pressure ionization (API) techniques, electrospray ionization (ESI),1-3 atmospheric pressure chemical ionization (APCI),4-7 or atmospheric pressure photoionization (APPI).8,9 The use of ESI is * To whom correspondence should be addressed. Phone: +358-9-191 59 134 (R.K.); +358-9-191 59169 (J.P.). E-mail:
[email protected] (R.K.);
[email protected] (J.P.). † Division of Pharmaceutical Chemistry, University of Helsinki. ‡ Department of Chemistry, University of Helsinki. § Helsinki University of Technology. (1) Dole, M.; Mack, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. D.; Alice, M. B. J. Chem. Phys. 1968, 49, 2240-2249. (2) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451-4459. (3) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4671-4675. (4) Horning, E. C.; Horning, M. G.; Carroll, D. I.; Dzidic, I.; Stillwell, R. N. Anal. Chem. 1973, 45, 936-943. (5) Carroll, D. I.; Dzidic, I.; Stillwell, R. N.; Horning, M. G.; Horning, E. C. Anal. Chem. 1974, 46, 706-710. (6) Horning, E. C.; Carroll, D. I.; Dzidic, I.; Haegele, K. D.; Horning, M. G.; Stillwell, R. N. J. Chromatogr. Sci. 1974, 12, 725-729. (7) Horning, E. C.; Carroll, D. I.; Dzidic, I.; Haegele, K. D.; Horning, M. G.; Stillwell, R. N. J. Chromatogr. 1974, 99, 13-21. (8) Robb, D. B.; Covey, T. R.; Bruins, A. P. Anal. Chem. 2000, 72, 36533659. (9) Syage, J. A.; Evans, M. D.; Hanold, K. A. Am. Lab. 2000, 32, 24-29. 10.1021/ac070003v CCC: $37.00 Published on Web 03/24/2007
© 2007 American Chemical Society
limited to the analysis of polar and ionic compounds, whereas APCI and APPI can also be used to ionize less polar and even neutral compounds, respectively. Sonic spray ionization (SSI) is a less commonly used API technique, which was first introduced in 1994.10 Unlike the previously listed ionization techniques, it does not require the use of high voltage, corona discharge, photons, or heat to aid the ionization of solutes. Instead, the ions are produced merely by a nebulizer gas at a speed close to the speed of sound (i.e., sonic speed). Similarly to ESI, the ionization efficiency of SSI is usually highest when the analytes are already ionized in solution, and therefore the ionization efficiency of nonpolar compounds may be poor. However, SSI is a very robust and soft ionization technique, which can be employed in the ionization of thermolabile compounds. The mechanism of ion formation by SSI has not yet been well understood, and, so far, two different mechanisms have been proposed. Experiments with amino acid clusters11 have suggested that the formation of charged droplets follows Dodd’s statistical charging model,12 whereas the ion formation is based on the charged residue model introduced by Kebarle et al.13 On the other hand, a more recent study with 2-aminopyridine-derivatized oligosaccharides as model compounds has suggested another model focusing on counter (electrolyte) ion distribution surrounding the solvated analyte.14 SSI has been used with MS for direct analysis of neurotransmitters10,15 and proteins.16 Further, SSI-MS has been utilized in connection with both liquid chromatography (LC)14,17-28 and (10) Hirabayashi, A.; Sakairi, M.; Koizumi, H. Anal. Chem. 1994, 66, 45574559. (11) Takats, Z.; Nanita, C. S.; Cooks, R. G.; Schlosser, G.; Vekey, K. Anal. Chem. 2003, 75, 1514-1523. (12) Dodd, E. E. J. Appl. Phys. 1953, 24, 73-80. (13) Kebarle, P.; Tang, L. Anal. Chem. 1993, 65, 972A-986A. (14) Takegawa, Y.; Deguchi, K.; Ito, S.; Yoshioka, S.; Nakagawa, H.; Nishimura, S. Anal. Chem. 2005, 77, 2097-2106. (15) Hirabayashi, A.; Sakairi, M.; Koizumi, H. Anal. Chem. 1995, 67, 28782882. (16) Hirabayashi, Y.; Hirabayashi, A.; Takada, Y.; Sakairi, M.; Koizumi, H. Anal. Chem. 1998, 70, 1882-1884. (17) Mortier, K. A.; Zhang, G.-F.; Van Peteghem, C. H.; Lambert, W. E. J. Am. Soc. Mass Spectrom. 2004, 15, 585-592. (18) Arinobu, T.; Hattori, H.; Ishii, A.; Kumazawa, T.; Lee, X.-P.; Kojima, S.; Suzuki, O.; Seno, H. Anal. Chim. Acta 2003, 492, 249-252. (19) Arinobu, T.; Hattori, H.; Ishii, A.; Kumazawa, T.; Lee, X. P.; Suzuki, O.; Seno, H. Chromatographia 2003, 57, 301-307. (20) Dams, R.; Benijts, T.; Gunther, W.; Lambert, W.; De Leenheer, A. Anal. Chem. 2002, 74, 3206-3212.
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capillary electrophoresis (CE).29 The LC-MS and CE-SSI-MS applications include the analysis of tolterodine drug,22 neuroleptic drug haloperidol and its metabolites,23 morphine,30 parametoxyamphetamine,24 mixture of local anesthetics,19 endocrine-disruptingchemicals (estrogenic hormones, n-alkylphenols, bisphenols, parabens, dihydroxybenzophenones),25 midazolam together with its metabolites,26 paclitaxel,17,28 derivatized oligosaccharides,14,31 and terpene trilactones,27 as well as the profiling of impurities in heroin.20 Often the performance of SSI has been compared to that of conventional ionization sources like ESI and APCI. Depending on the compound, the performance of SSI has proven to be similar to or even better than the conventional ionization techniques. The higher sensitivity of SSI applies, for example, to tetracaine, benzoxinate, dibucaine, bupivacaine, mepivacaine,19 serine,11 haloperidol,23 and morphine.20 Of the applications listed, only a few10,11,15,32 have employed the original design without applying high voltage. On the contrary, in most of the applications, high voltage has been applied to the source housing (i.e., to the analyzed solution) to aid ionization. This arrangement does not differ substantially from conventional ESI, where a high nebulizer gas flow rate has been added. One way of improving sensitivity and speed of the analysis is the miniaturization of the analytical systems. Interfaces of microfluidic separation techniques to MS have been extensively studied. Microchip ionization devices have become popular for their compactness and low costs when manufactured in high numbers. In addition to this, the sensitivity and efficiency of the analysis have often been improved with miniaturization. Recently, microchip ESI,33-38 ESI tips,39 microchip APCI,40 and APPI41 have been introduced and employed as ionization sources for LC-MS42 and (21) Hirabayashi, Y.; Takada, Y.; Hirabayashi, A.; Sakairi, M.; Koizumi, H. Rapid Commun. Mass Spectrom. 1996, 10, 1891-1893. (22) Bjo ¨rkman, H. T.; Edlund, P.-O.; Jacobsson, S. P. Anal. Chim. Acta 2002, 468, 263-274. (23) Arinobu, T.; Hattori, H.; Seno, H.; Ishii, A.; Suzuki, O. J. Am. Soc. Mass Spectrom. 2002, 13, 204-208. (24) Mortier, K. A.; Dams, R.; Lambert, W. E.; De Letter, E. A.; Van, Calenbergh, S.; De Leenheer, A. P. Rapid Commun. Mass Spectrom. 2002, 16, 865870. (25) Benijts, T.; Gunther, W.; Lambert, W.; De Leenheer, A. Rapid Commun. Mass Spectrom. 2003, 17, 1866-1872. (26) Kanazawa, H.; Okada, A.; Igarashi, E.; Higaki, M.; Miyabe, T.; Sano, T.; Nishimura, R. J. Chromatogr., A 2004, 1031, 213-218. (27) Chen, E.; Ding, C.; Lindsay Robert, C. Anal. Chem. 2005, 77, 2966-2970. (28) Green, H.; Vretenbrant, K.; Norlander, B.; Peterson, C. Rapid Commun. Mass Spectrom. 2006, 20, 2183-2189. (29) Hirabayashi, Y.; Hirabayashi, A.; Koizumi, H. Rapid Commun. Mass Spectrom. 1999, 13, 712-715. (30) Dams, R.; Benijts, T.; Gunther, W.; Lambert, W.; De Leenheer, A. Rapid Commun. Mass Spectrom. 2002, 16, 1072-1077. (31) Deguchi, K.; Takegawa, Y.; Hirabayashi, A.; Nakagawa, H.; Nishimura, S.I. Rapid Commun. Mass Spectrom. 2005, 19, 2325-2330. (32) Takats, Z.; Nanita, S. C.; Schlosser, G.; Vekey, K.; Cooks, R. G. Anal. Chem. 2003, 75, 6147-6154. (33) Huikko, K.; O ¨ stman, P.; Grigoras, K.; Tuomikoski, S.; Tiainen, V. M.; Soininen, A.; Puolanne, K.; Manz, A.; Franssila, S.; Kostiainen, R.; Kotiaho, T. Lab Chip 2003, 3, 67-72. (34) Rohner, T. C.; Rossier, J. S.; Girault, H. H. Anal. Chem. 2001, 73, 53535357. (35) Kim, J. S.; Knapp, D. R. J. Am. Soc. Mass Spectrom. 2001, 12, 463-469. (36) Yuan, C.-H.; Shiea, J. Anal. Chem. 2001, 73, 1080-1083. (37) Schultz, G. A.; Corso, T. N.; Prosser, S. J.; Zhang, S. Anal. Chem. 2000, 72, 4058-4063. (38) Licklider, L.; Wang, X.-Q.; Desai, A.; Tai, Y.-C.; Lee, T. D. Anal. Chem. 2000, 72, 367-375. (39) Tuomikoski, S.; Sikanen, T.; Ketola, R. A.; Kostiainen, R.; Kotiaho, T.; Franssila, S. Electrophoresis 2005, 26, 4691-4702.
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GC-MS.43 The design of SSI has been miniaturized by applying tapered fused silica capillary for the solvent spray.44 This has made possible the use of lower solvent flow rates (20-100 nL/min) as compared to the conventional designs where the common flow rates are 30-1000 µL/min. We introduce a novel microchip SSI employing the same microchip previously reported42 for APCI and APPI. The chip utilizes etched channels for the solvent and gas, but employs neither a heater, nor a corona discharge, nor photons in the ionization. Only nebulizer gas flow at high speed is needed for the ionization. The microchip SSI source is optimized in terms of solvent and nebulizer gas flow rates, and position of the microchip toward the MS orifice. Finally, the analytical performance of the technique is studied with a group of test compounds. EXPERIMENTAL SECTION Reagents. Tetra-N-butylammonium iodide (Lancaster Synthesis, Morecambe, Great Britain), testosterone (Fluka Chemie, Buchs, Switzerland), ibuprofen (University Pharmacy, Helsinki, Finland), verapamil (Sigma-Aldrich, Steinheim, Germany), and angiotensin I trifluoroacetate (Bachem, Weil am Rhein, Germany) were used as model compounds. Methanol and ammonium hydroxide were from J.T. Baker (Deventer, Holland), and formic acid was from Riedel-de-Hae¨n (Seelze, Germany). Water was purified using a Milli-Q-Plus system (Millipore, Molsheim, France). Sample Delivery. For the dilution of the samples, a mixture of 50% methanol/50% water (v/v) was prepared with the addition of either formic acid for positive ion mode or ammonium hydroxide for negative ion mode. The final concentration of each additive was 0.1% (v/v). The sample solution was pumped via direct infusion with an electronically controlled (Harvard Apparatus, MA) syringe (1 mL, Hamilton, Bonaduz, Switzerland) directly to the SSI microchip using a flow rate of 0.5-15 µL/min. Microchip SSI. Nebulizer microchip, which has been described previously,42,45 was used for SSI without any hardware modifications. The microchip consisted of a silicon wafer with an etched inlet for nebulizer gas, a housing for fused silica capillary, a vaporizing channel, and an exit nozzle. It was joined to a Pyrex glass wafer by anodic bonding. Nebulizer gas entered through a Nanoport connector (glued on the nebulizer inlet) to the vaporizer channel where it mixed with a sample that was introduced via fused silica capillary (glued inside the channel housing). The twocomponent high temperature epoxy glue Duralco (Cotronics Corp., Brooklyn, New York) was hardened by exposure to heat (125 °C for 2 h). The glue is heat resistant up to 350 °C. The glue was applied to the outer edge of the Nanoport/microchip so that it did not penetrate inside the channel. Moreover, the Nanoport was equipped with a seal that protected the channel. Tests performed with MS did not shown any increase of the background (40) O ¨ stman, P.; Marttila, S. J.; Kotiaho, T.; Franssila, S.; Kostiainen, R. Anal. Chem. 2004, 76, 6659-6664. (41) Kauppila, T. J.; O ¨ stman, P.; Marttila, S.; Ketola, R. A.; Kotiaho, T.; Franssila, S.; Kostiainen, R. Anal. Chem. 2004, 76, 6797-6801. (42) O ¨ stman, P.; Ja¨ntti, S.; Grigoras, K.; Saarela, V.; Ketola, R. A.; Franssila, S.; Kotiaho, T.; Kostiainen, R. Lab Chip 2006, 6, 948-953. (43) O ¨ stman, P.; Luosuja¨rvi, L.; Haapala, M.; Grigoras, K.; Ketola, R. A.; Kotiaho, T.; Franssila, S.; Kostiainen, R. Anal. Chem. 2006, 78, 3027-3031. (44) Hirabayashi, A. Rapid Commun. Mass Spectrom. 2003, 17, 391-394. (45) Franssila, S.; Marttila, S.; Kolari, K.; O ¨ stman, P.; Kotiaho, T.; Kostiainen, R.; Lehtiniemi, R.; Fager, C.-M.; Manninen, J. J. Microelectromech. Syst. 2006, 15, 1251-1259.
Figure 1. Schematic view of the microchip SSI arrangement. (a) Different angles of the sonic spray versus MS orifice plate were tested: (1) perpendicular, (2) 45°, and (3) parallel. (b) Optimum positions of the microchip SSI toward the MS orifice in distance (x) and in height (y) were for both 4 mm.
Figure 2. Intensity of the M+ signal of tetra-N-butyl ammonium at m/z 242 as a function of sample solution flow rate. The sample concentration was 10 µM, and the solvent was water/methanol (50/ 50) with 0.1% of formic acid.
noise. The vaporizer channel (length 10 mm, width 800 µm, depth 250 µm) ends with a nozzle that forms the spray. Dimensions of the chip are 18 mm length, 10 mm width, and 1 mm height. Because this is the same chip as the APCI chip, an integrated heater is present, but it was not used in this application. Nitrogen (99.9995%, Woikoski, Vuohija¨rvi, Finland) or synthetic air (Woikoski, Vuohija¨rvi, Finland) was used as the nebulizer gas at pressures of 5-25 bar. Mass Spectrometer. The mass spectrometer was a PE Sciex API 300 triple quadrupole (Sciex, Concord, Canada). It was operated in both positive and negative modes. The mass spectrometer parameters (electrooptical voltages) were optimized for each compound separately using the standard ESI source (Sciex, Concord, Canada). The scan range (scan rate 1 s with a step size of 0.1 m/z) was 50-500 amu for tetra-N-butylammonia, testosterone, and ibuprofen, 150-600 amu for verapamil, and 400-1300 amu for angiotensin I. The data were collected using PE Sciex MassChrom software (version 1.1.1). RESULTS AND DISCUSSION Position of the Microchip. The position of the SSI microchip relative to the MS orifice was found to be critical for efficient
Figure 3. Absolute signal intensity of SSI-MS as a function of concentration for tetra-N-butyl ammonium. The sample solution flow rate was 5 µL/min, and the solvent was water/methanol (50/50) with 0.1% of formic acid.
Figure 4. Stability of the M+ signal of tetra-N-butylammonium (500 nM) at m/z 242. The sample solution flow rate was 5 µL/min, and the solvent was water/methanol (50/50) with 0.1% of formic acid.
ionization and transport of ions into the MS, and it was optimized by adjusting two parameters: (i) angle and (ii) distance. The optimization was done by infusing a 100 µM solution of tetra-Nbutylammonium in water/methanol/formic acid (50/50/0.1%) at a flow rate of 10 µL/min and by monitoring the resulting M+ signal of tetra-N-butylammonium at m/z 242 in positive ion mode. (i) Analytical Chemistry, Vol. 79, No. 9, May 1, 2007
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Figure 5. The SSI mass spectra of 1 µM verapamil (a), 500 nM tetra-N-butylammonium (b), 100 µM testosterone (c), 100 µM angiotensin I (d), and 100 µM ibuprofen (e). The sample solution flow rates were 5 µL/min of water/methanol (50/50) with 0.1% of formic acid in positive ion mode (a-d) and 10 µL/min of water/methanol (50/50) with 0.1% of ammonium hydroxide in negative ion mode (e). Ten spectra were accumulated using a dwell time of 1 s.
Three different angles of the chip were tested (Figure 1a). The best signal was obtained when the microchip sprayed a jet perpendicularly toward the MS orifice (case 1 in Figure 1). The position of the microchip was adjusted so that the jet hit just the edge of the MS curtain plate. Direct spraying into the orifice would result in higher MS response, but, on the other hand, impurities would intensively accumulate at the MS inlet optics and deteriorate the reproducibility and sensitivity in longer use. At the 45° angle of the microchip (case 2 in Figure 1a), the signal decreased to approximately one-fifth, and when the microchip was at the angle 90° (parallel, case 3 in Figure 1a), the signal disappeared entirely. (ii) The distance of the microchip from the orifice was also observed to influence the intensity of the signal significantly. The closer the microchip was to the orifice, the more intensive was the signal obtained. The optimized distance (x-direction) and height (y-direction) of the microchip nozzle toward the MS orifice were both 4 mm (Figure 1b). Gas Velocity. Velocity of the nebulizer gas has been reported to have a significant effect on the ionization in SSI, and the best ionization efficiency is achieved at the sonic speed.10 In this study, the velocity of the nebulizer gas was controlled by adjusting its inlet pressure. The maximum signal for the M+ of tetra-Nbutylammonium (concentration 10 µM, sample flow rate 10 µL/ min) at m/z 242 was reached at 10 bar (1.1 L/min), after which increasing the pressure up to 30 bar had only a minor effect on the ionization efficiency. A similar trend has been reported for SSI employing tapered fused silica capillary for the solvent spray.44 On the basis of this comparison and the phenomena that the best ionization efficiency is gained at the sonic speed,10 we estimate that in our microchip SSI the sonic speed was reached at a pressure of 10 bar and a flow rate of 1.1 mL/min. Both nitrogen and synthetic air were tested as nebulizer gases with the same 3522 Analytical Chemistry, Vol. 79, No. 9, May 1, 2007
result. Synthetic air represented a more economical alternative and was thus used in the rest of the experiments. Sample Solution Flow Rate. Correlation between the sample flow rate and the analyte signal helps to determine the application range of the method and to characterize the ionization mechanism. To test this, a 10 µM solution of tetra-N-butylammonium was infused into the microchip SSI employing flow rates of 0.5-10 µL/min while the pressure of the nebulizer gas was kept constant at 10 bar. The signal of the M+ of tetra-N-butylammonium was observed to increase linearly (r2 ) 0.9995) with rising flow rate within the tested range (Figure 2). Hirabayashi et al.16,46 have demonstrated that high-speed gas in SSI produces droplets with small diameter (∼1 µm) whose formation is almost independent of the solvent flow rate. This makes SSI a mass-flow-dependent ionization technique, which is in agreement with our results. A different mechanism of droplet formation takes place in ESI where an increased flow rate forms larger droplets. The emission of ions from large droplets becomes limited, and therefore the signal does not increase when the sample flow rate is higher.47 Thus, ESI is a concentration-dependent ionization technique. Linearity. Linearity, repeatability, and stability were tested with tetra-N-butylammonium as the test compound. Linearity was examined for concentrations from 50 nM (limit of quantification, where S/N ) 10) to 5 µM. The response of SSI followed the same pattern that has previously been described for ESI.48,49 The increasing concentration of the sample in the solution was (46) Hirabayashi, A.; Fernandez de la Mora, J. Int. J. Mass Spectrom. 1998, 175, 277-282. (47) Cole, B. R. Electrospray Ionization Mass Spectrometry; John Wiley & Sons, Inc.: New York, 1997. (48) Tang, L.; Kebarle, P. Anal. Chem. 1993, 65, 3654-3668. (49) Kostiainen, R.; Bruins, A. P. Rapid Commun. Mass Spectrom. 1996, 10, 1393-1399.
Table 1. Limits of Detection (S/N ) 3) for the Studied Compounds compound verapamil testosterone angiotensin I tetra-N-butylammonium ibuprofen
monitored ion
m/z
LOD (µM)
[M + H]+ [M + H]+ [M + 2H]2+ M+ [M - H]-
455 289 649 242 205
0.1 1.7 4 0.015 1
observed to cause a proportional increase of the signal until a plateau was reached. The response was linear (r2 ) 0.9999) for 1 order of magnitude, 50-500 nM, after which an increase in the concentration of the analyte caused a nonlinear response (Figure 3). It is possible, however, that the linearity in SSI-MS depends on the ionized compound and the solvent composition, becuase linear ranges within about 1 order of magnitude have been reported for triterpene dilactones (10-80 ng, i.e., 10-6-10-4 M)27 and midazolam26 (0.1-5.0 µg/mL, i.e., 0.3 × 10-6-1.5 × 10-5 M), and 4 orders of magnitude for endocrine-disrupting chemicals25 (10-10 000 ng/L, i.e., 5 × 10-11-5 × 10-8 M) and for serine octamer11 (10-7-10-3 M). Repeatability. The quantitative repeatability of the microchip SSI was tested with 50 nM (S/N ) 10) tetra-N-butylammonium solution infused at 10 µL/min. The six times repeated measurement of the peak height of the M+ of the analyte at m/z 242 gave a relative standard deviation (RSD) of 16.3%. Thus, the microchip SSI was shown to be a robust and stable ionization source when considering that the theoretical RSD at S/N ) 10 is 10%. Stability. To test the stability of microchip SSI-MS, the 500 nM solution of tetra-N-butylammonium was infused to the microchip SSI at a flow rate of 5 µL/min for 2 h. Microchip SSI-MS produced a stable ion current throughout the measurement, which was only limited by the volume of the 1 mL syringe (Figure 4). Spectra Obtained with Microchip SSI. Finally, the performance of microchip SSI was demonstrated by collecting the spectra of five compounds possessing different polarities and sizes: verapamil, tetra-N-butylammonium, testosterone, and angiotensin I in positive ion mode, and ibuprofen in negative ion mode. Samples detected in positive mode were infused at a flow rate of 5 µL/min, and the solution of ibuprofen in negative ion mode was infused at a flow rate of 10 µL/min. The abundances of protonated molecules (verapamil, testosterone, and angiotensin I), positively charged ion (tetra-N-butylammonium), or deproto-
nated molecule (ibuprofen) were measured. Quality spectra were produced in both positive and negative ion modes. In positive ion mode, tetra-N-butylammonium showed the M+ ion at m/z 242 as the most abundant ion, whereas verapamil and testosterone produced [M + H]+ ions at m/z 455 and 289, respectively. In addition to this, testosterone showed also intense [M + Na]+ and [M + Na + H2O]+ adducts in the spectrum, due to its lower proton affinity. Angiotensin I, an oligopeptide, showed singly, doubly, and triply protonated molecules in the spectrum at m/z 1297, 649, and 433, respectively. In negative mode, ibuprofen showed a deprotonated molecule at m/z 205 and a deprotonated dimer at m/z 411 (Figure 5). The appearance of adducts and dimers and the fact that none of the spectra showed any fragments prove the soft nature of the microchip SSI. Limits of Detection. Limits of detections (LOD) for the test compounds were determined by analyzing sample solutions in different concentrations at a flow rate 10 µL/min. The concentration that resulted in S/N ) 3 of the most abundant m/z peak of the analyte in the full scan mass spectrum was chosen as the LOD (Table 1). The LODs were found to be in the range of 15 nM to 4 µM, being lowest for tetra-N-butylammonium and highest for angiotensin I. CONCLUSIONS We have presented a novel application of our versatile API microchip: sonic spray ionization. With minor instrumental changes, the same microchip can be used as APCI, APPI, or SSI interface for the connection of microfluidic separation techniques or micro-LC to mass spectrometry. The microchip SSI was shown to provide soft and efficient ionization of polar compounds in both positive and negative ion modes with excellent stability. Because the microchip employed was originally designed for APCI, the sensitivity and the sample solution flow rate dependence of the SSI technique are expected to be further improved by redesigning the chip specifically for SSI. ACKNOWLEDGMENT Financial support from the Academy of Finland (Project # 1209189) and CHEMSEM graduate school is gratefully acknowledged. Received for review January 2, 2007. Accepted February 8, 2007. AC070003V
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