Microchip Atmospheric Pressure Chemical Ionization Source for Mass

Oct 20, 2004 - Microelectronics Centre, Helsinki University of Technology, P.O. Box ... Faculty of Pharmacy, P.O. Box 56, FIN-00014 University of Hels...
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Anal. Chem. 2004, 76, 6659-6664

Microchip Atmospheric Pressure Chemical Ionization Source for Mass Spectrometry Pekka O 2 stman,† Seppo J. Marttila,‡ Tapio Kotiaho,§ Sami Franssila,*,‡ and Risto Kostiainen*,†,⊥

Viikki Drug Discovery Technology Center, Faculty of Pharmacy, P.O. Box 56, FIN-00014 University of Helsinki, Finland, Microelectronics Centre, Helsinki University of Technology, P.O. Box 3500, FIN-02015 HUT, Finland, Laboratory of Analytical Chemistry, Department of Chemistry, P.O. Box 55, FIN-00014 University of Helsinki, Finland, and Department of Pharmaceutical Chemistry, Faculty of Pharmacy, P.O. Box 56, FIN-00014 University of Helsinki, Finland

A novel microchip heated nebulizer for atmospheric pressure chemical ionization mass spectrometry is presented. Anisotropic wet etching is used to fabricate the flow channels, inlet, and nozzle on a silicon wafer. An integrated heater of aluminum is sputtered on a glass wafer. The two wafers are jointed by anodic bonding, creating a two-dimensional version of an APCI source with a sample channel in the middle and gas channels symmetrically on both sides. The ionization is initiated with an external corona-discharge needle positioned 2 mm in front of the microchip heated nebulizer. The microchip APCI source provides flow rates down to 50 nL/min, stable long-term analysis with chip lifetime of weeks, good quantitative repeatability (RSD < 10%) and linearity (r2 > 0.995) with linear dynamic rage of at least 4 orders of magnitude, and cost-efficient manufacturing. The limit of detection (LOD) for acridine measured with microchip APCI at flow rate of 6.2 µL/min was 5 nM, corresponding to a mass flow of 0.52 fmol/s. The LOD with commercial macro-APCI at a flow rate of 1 mL/min for acridine was the same, 5 nM, corresponding to a significantly worse mass flow sensitivity (83 fmol/s) than measured with microchip APCI. The advantages of microchip APCI makes it a very attractive new microfluidic detector. The new atmospheric pressure ionization techniques in mass spectrometry (MS), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo ionization (APPI), have key roles in life sciences, biopharmaceutical chemistry, and environmental research. One of the most significant breakthroughs in life sciences was the discovery, presented for the first time by Fenn et al. in 1988 and honored with the Nobel prize in chemistry in 2002, that ESI-MS provides an extremely sensitive and accurate method for measurements of large biomolecules, such as proteins, peptides, and oligonucleotides.1,2 The discovery of nano electrospray (nano-ESI) and its combination to nano liquid chromatography (nano-LC) extended * Corresponding authors. (Kostiainen) Phone: +358-9-191-59-134. E-mail: [email protected]. (Franssila) Phone: +358-9-451 2332. E-mail: [email protected]. † Viikki Drug Discovery Technology Center, University of Helsinki. ‡ Microelectronics Centre, Helsinki University of Technology. § Laboratory of Analytical Chemistry, University of Helsinki. ⊥ Department of Pharmaceutical Chemistry, University of Helsinki. 10.1021/ac049345g CCC: $27.50 Published on Web 10/20/2004

© 2004 American Chemical Society

bioanalysis to attomole sensitivity,3 establishing the basis for proteomics, system biology, and metabolomics. Nowadays, ESI is the most common ionization method in LC/MS and is widely applied in biochemistry, life sciences, drug research, and environmental research. The method is excellent for ionic and polar compounds, but the ionization efficiency for neutral and nonpolar compounds may be poor. Atmospheric pressure chemical ionization (APCI) offers an alternative ionization technique that is capable of ionizing with high efficiency both polar and ionic compounds in addition to neutral compounds.4,5 Other advantages of APCI over ESI include the possibility to use both polar and nonpolar solvents and the capability to tolerate higher electrolyte concentrations. Furthermore, suppression of ionization by the coeluting compounds is significantly less with APCI than with ESI.6 The ionization mechanism of APCI has been presented earlier.4,5 Recently introduced atmospheric pressure photo ionization has the same benefits as APCI, but it is even more suitable for nonpolar compounds than APCI.7-9 However, APCI and APPI are not suitable for large biomolecules, and the methods have been applied for small molecules with molecular weights below 1000 amu. The flow rates used with recent APCI ion sources are high, typically 50-1000 µL/min, excluding analysis in minimal sample volumes. A deeper understanding of the role of the nonvolatile neutral and nonpolar compounds in biological and environmental systems requires analytical techniques that are more sensitive than those currently available. Miniaturization has been found to be one efficient way to improve sensitivity and speed of analysis. Microfabrication techniques enable the fabrication of large numbers of miniaturized instruments at low cost. The possibility of integration of the whole (1) Meng, C. K.; Mann, M.; Fenn, J. B. Proc. 36th Annu. Conf., Am. Soc. Mass Spectrom., San Francisco, 5-10 June 1988 1988, 771-772. (2) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451-4459. (3) Mann, M.; Meng, C. K.; Fenn, J. B. Anal. Chem. 1989, 61, 1702-1708. (4) Horning, E. C.; Carroll, D. I.; Dzidic, I.; Haegele, K. D.; Horning, M. G.; Stillwell, R. N. J. Chromatogr. Sci. 1974, 12, 725-729. (5) Carroll, D. I.; Dzidic, I.; Stillwell, R. N.; Haegele, K. D.; Horning, E. C. Anal. Chem. 1975, 47, 2369-2373. (6) King, R.; Bonfiglio, R.; Fernandez-Metzler, C.; Miller-Stein, C.; Olah, T. J. Am. Soc. Mass Spectrom. 2000, 11, 942-950. (7) Robb, D. B.; Covey, T. R.; Bruins, A. P. Anal. Chem. 2000, 72, 3653-3659. (8) Kauppila, T.; Kuuranne, T.; Meurer, E., C.; Eberlin, M. N.; Kotiaho, T.; Kostiainen, R. Anal. Chem. 2002, 74, 5470-5479. (9) Saba, A.; Raffaelli, A.; Pucci, S.; Salvadori, P. Proceedings of the 50th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, June 3-7, 2002.

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analytical process in one chip has led to the concepts of “micro total analysis systems” (µTAS) and “lab-on-a-chip”. Microanalytical systems have been applied, for example, to bioanalysis of amino acids,10-13 proteins,14-18 and peptides;19 the analysis of drugs;20-22 and DNA sequencing and separations.23-26 Most microfluidic applications so far have relied on fluorescence detection,27 but much effort during the past few years has been put into miniaturizing of an ESI interface for MS. Microchip ESI is compatible with flow rates used in microfluidic separations (submicroliters/minute), being ideal for fast and highly sensitive analysis.27 Microfluidic separation systems have been previously coupled to MS with a fused-silica capillary or a nanospray needle,19,28-32 attaching a separate spraying capillary or a nanospray needle with liquid junction interface at the chip outlet33-37 or as an integral part of the microdevice.22,38-45 However, no (10) Harrison, D. J.; Fluiri, K.; Seiler, K.; Fan, Z. Science 1993, 261, 895-897. (11) Munro, N. J.; Huang, Z.; Finegold, D. N.; Landers, J. P. Anal. Chem. 2000, 72, 2765-2773. (12) Rocklin, R. D.; Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 2000, 72, 52445249. (13) Chen, X.; Wu, H.; Mao, C.; Whitesides, G. M. Anal. Chem. 2002, 74, 17721778. (14) Liu, Y.; Foote, R. S.; Jacobson, S. C.; Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 2000, 72, 4608-4613. (15) Badal, M. Y.; Wong, M.; Chiem, N.; Salimi-Moosavi, H.; Harrison, D. J. J. Chromatogr., A 2002, 947, 277-286. (16) Ekstro ¨m, S.; O ¨ nnerfjord, P.; Nilsson, J.; Bengtsson, M.; Laurell, T.; MarkoVarga, G. Anal. Chem. 2000, 72, 286-293. (17) Ekstro ¨m, S.; Ericsson, D.; O ¨ nnerfjord, P.; Bengtsson, M.; Nilsson, J.; MarkoVarga, G.; Laurell, T. Anal. Chem. 2001, 73, 214-219. (18) Laurell, T.; Nilsson, J.; Marko-Varga, G.; J. Chromatogr., B 2001, 752, 217232. (19) Li, J.; Thibault, P.; Bings, N. H.; Skinner, C. D.; Wang, C.; Colyer, C.; Harrison, J. Anal. Chem. 1999, 71, 3036-3045. (20) Tuomikoski, S.; Huikko, K.; Grigoras, K.; O ¨ stman, P.; Kostiainen, R.; Baumann, M.; Abian, J.; Kotiaho, T.; Franssila, S. Lab Chip 2002, 2, 247253. (21) Dethy, J.-M.; Ackermann, B. L.; Delatour, C.; Henion, J. D.; Schultz, G. A. Anal. Chem. 2003, 75, 805-811. (22) 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. (23) Shi, Y.; Simpson, P. C.; Scherer, J. R.; Wexler, D.; Skibola, C.; Smith, M. T.; Mathies, R. A. Anal. Chem. 1999, 71, 5354-5361. (24) Liu, S. R.; Shi, Y. N.; Ja, W. W.; Mathies, R. A. Anal. Chem. 1999, 71, 566573. (25) Koutny, L.; Schmalzing, D.; Salas-Solano, O.; El-Difrawy, S.; Adourian, A.; Buonocore, S.; Abbey, K.; McEwan, P.; Matsudaira, P.; Ehrlich, D. Anal. Chem. 2000, 72, 3388-3391. (26) Bakajin, O.; Duke, T. A. J.; Tegenfeldt, J.; Chou, C.-F.; Chan, S. S.; Austin, R. H.; Cox, E. C. Anal. Chem. 2001, 73, 6053-6056. (27) Huikko, K.; Kostiainen, R.; Kotiaho, T. E. J. Pharm. Sci. 2003, 20, 149171. (28) Figeys, D.; Gygi, S.; McKinnon, G.; Aebersold, R. Anal. Chem. 1998, 70, 3728-3734. (29) Lazar, I. M.; Ramsey, R. S.; Sundberg, S.; Ramsey, J. M. Anal. Chem. 1999, 71, 3267-3631. (30) Chartogne, A.; Tjaden, U. R.; van der Greef, J. Rapid Commun. Mass Spectrom. 2000, 14, 1269-1274. (31) Liu, H.; Felten, C.; Xue, Q.; Zhang, B.; Jedrzejewski, P.; Karger, B. L.; Foret, F. Anal. Chem. 2000, 72, 3303-3310. (32) Lazar, I. M.; Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 2001, 73, 17331739. (33) Zhang, C.-X.; Manz, A. Anal. Chem. 2001, 73, 2656-2662. (34) Deng, Y.; Zhang, H.; Henion, J. Anal. Chem. 2001, 73, 1432-1439. (35) Deng, Y.; Henion, J.; Li, J.; Thibault, P.; Wang, C.; Harrison, D. J.; Anal. Chem. 2001, 73, 639-646. (36) Kameoka, J.; Craighead, H. G.; Zhang, H.; Henion, J. Anal. Chem. 2001, 73, 1935-1941. (37) Xue, Q.; Foret, F.; Dunayevskiy, Y. M.; Zavracky, P. M.; McGruer, N. E.; Karger, B. L. Anal. Chem. 1997, 69, 426-430. (38) Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 1997, 69, 1174-1178.

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microchip realizations of ESI have yet been demonstrated for longterm routine analysis. In this work, we will present for the first time microchip APCI for connection of mass spectrometry to microfluidic systems or to nano and micro liquid chromatography (LC). There have been a few attempts to modify APCI sources for flow rates below 10 µL/min46,47 either by custom-made sources or modifications of commercial sources, but they lack the concept of microchip-based miniaturization, and they are incompatible with microfluidic devices. The performance of microchip APCI was carefully evaluated and was shown to be suitable for highly sensitive, quantitative, and long-term routine analysis. EXPERIMENTAL SECTION Fabrication Process of the Miniaturized Heated Nebulizer. Double-side polished (100) silicon wafers were used as substrates and Pyrex 7740 glass wafers as channel cover plates. Three photomasks were required for chip fabrication. The first mask defines the channel, mixer, and nozzle patterns in silicon, the second one defines the inlet holes on the backside of the silicon wafer, and the third one is used to fabricate the integrated microheater on the glass wafer. Two sets of alignment marks were used: one for photolithography and one for anodic bonding because bond alignment had to be done without a microscope. The silicon wafers were cleaned in ammonia/peroxide and hydrogen chloride/peroxide mixtures (RCA-1 & RCA-2) before thermal oxidation at 1000 °C, which resulted in a 600-nm-thick oxide.48 The channel and mixer pattern was printed on the wafer front side, and fluidic inlet holes on the back using double-sided lithography equipment (Electronic Visions AL6-2). Oxide was etched in a single step in buffered hydrofluoric acid (BHF). Anisotropic silicon etching was done in 25 wt % TMAH (tetramethylammonium hydroxide) solution at 80 °C. The etch rate was ∼0.5 µm/min. The etch time was minimized by etching from both sides of the wafer simultaneously. The width of the nebulizer gas and liquid sample channels were 300 µm and 120 µm, respectively. The channel depth was 85-190 µm, depending on the channel width. Pyrex glass wafers were cleaned with acetone and 2-propanol. After cleaning, a 300 nm aluminum layer was sputtered onto the glass wafer. Mask number three defined the microheater pattern. Aluminum was wet-etched in phosphoric acid solution (PES 8015-5 from Merck). The resistance of the aluminum heater at room temperature was ∼90 Ω. (39) Zhang, B.; Liu, H.; Karger, B. L.; Foret, F. Anal. Chem. 1999, 71, 32583264. (40) Schultz, G. A.; Corso, T. N.; Prosser, S. J.; Zhang, S. Anal. Chem. 2000, 72, 4058-4063. (41) Licklider, L.; Wang, X.-Q.; Desai, A.; Tai, Y.-C.; Lee, T. D. Anal. Chem. 2000, 72, 367-375. (42) Rohner, T. C.; Rossier, J. S.; Girault, H. H. Anal. Chem. 2001, 73, 53535357. (43) Yuan, C.-H.; Shiea, J. Anal. Chem. 2001, 73, 1080-1083. (44) Kim, J.-S.; Knapp, D. R. J. Am. Soc. Mass Spectrom. 2001, 12, 463-469. (45) Wen, J.; Lin, Y.; Xiang, F.; Matson, D. W.; Udseth, H. R.; Smith, R. D. Electrophoresis 2000, 21, 191-197. (46) Nyholm, L. M.; Sjo ¨berg, P. J. R.; Markides, K. E. J. Chromatogr., A 1996, 755, 153-164. (47) Tanaka, Y.; Otsuka, K.; Terabe, S. J. Pharm. Biomed. Anal. 2003, 30, 18891895. (48) Franssila, S. In Introduction to Microfabrication; John Wiley & Sons: Chichester, 2004; p 145.

Figure 1. Structures of the studied compounds.

Before bonding the mask oxide was removed from the silicon wafer, and the surface was hydrophilized in ammonia/peroxide solution (RCA-1) to make alignment easier and to get higher bond strength. After alignment, the wafers were joined together by anodic bonding. The wafer stack was heated to 350 °C, and a voltage of 300 V was applied for 15 min. Fluidic connectors (Nanoport, Upchurch Scientific, Oak Harbor, WA) were glued with epoxy after wafer dicing. Chip size (18 × 29 mm) was mainly determined by the size of the fluidic connectors. Samples. Propranolol hydrochloride, verapamil hydrochloride, and acridine were obtained from Sigma-Aldrich; testosterone and dopamine were from Fluka (Fluka Chemie, Buchs, Switzerland), and quinoline was from Acros (Acros Organics, Geel, Belgium). Structures of the compounds studied are presented in Figure 1. Stock solutions were prepared by dissolving the compounds in methanol (J. T. Baker, Mallinckrodt Baker B.V, Deventer, Holland) to a concentration of 100 mmol/L. Final working solutions of the analytes at concentrations of 1 nmol/L to 100 µmol/L were prepared by diluting stock solutions further with pure methanol or with mixtures of water/methanol (either 20/80 or 80/20, v/v) + 0.1% acetic acid. Mass Spectrometry. The mass spectrometer used was a PE Sciex API-300 triple quadrupole (Perkin-Elmer Sciex, Toronto, Canada) fitted with an x-y-z stage (Proxeon, Odense, Denmark) and equipped with a Teflon holder (The Finnish School of Watchmaking, Espoo, Finland) to facilitate the chip’s aligning close to the MS inlet. The optimal distance between the chip edge and the curtain plate was 0.5-1.0 cm. High-purity nitrogen (99.999%, Oy Woikoski Ab, Voikoski, Finland) was used as the nebulizer gas, and nitrogen produced by a Whatman 75-720 nitrogen

generator (Whatman Inc., Haverhill, MA) was used as the curtain gas. Nebulizer gas was introduced to the microchip APCI via a 510-µm-i.d. PEEK tubing (Upchurch Scientific, Oak Harbor, WA) with a back pressure of 0.5 bar. For sample injection, a liquid chromatograph with an autosampler (Hewlett-Packard HP 1050) was used with an injection volume of 10 µL. The flow was split using an Acurate AC-100-VAR splitter (LC Packings, Zurich, Switzerland). For the flow rates lower than 1 µL/min, a microsyringe pump (Harvard PHD 2000 Advanced Syringe Pump, Harvard Apparatus, Holliston, MA) combined with a Rheodyne 7725 injector (Rheodyne, Cotati, CA) with a 10 µL loop was used. The sample was introduced to the microchip APCI via 50-µm-i.d. PEEK tubing (Upchurch Scientific, Oak Harbor, WA). The temperature of the microchip heated nebulizer was measured with a Fluke 54 series II (Fluke Corporation, Everett, WA) with a K-type thermoelement. External power supply (GW GPS-3030, Good Will Instruments Co. Ltd, Taiwan) was used to adjust the temperature of the microchip APCI. The external corona-discharge needle was a knitting needle (John James size 3/9, Entaco Limited, Warwickshire, England) situated 2 mm in front of the chip. The needle current was set to 1 µA. The mass spectrometer voltages were optimized for each analyte for both MS and tandem mass spectrometric (MS/MS) measurements with the controlling software program. The scan range for MS measurements was m/z 100-500 (1.0 s/scan). MS/ MS measurements were carried out in the multiple reaction monitoring (MRM) mode. Reference data was collected using the same apparatus fitted with an APCI source (Perkin-Elmer Sciex, Toronto, Canada). The temperature of the nebulizer was 350 °C. Compressed air filtered by an Atlas Copco air dryer (Wilrijk, Belgium) was used as the auxiliary gas. Injection volume was 20 µL. Other parameters were the same as in the microchip APCI measurements. RESULTS AND DISCUSSION The microchip APCI (Figure 2) was tested with compounds (quinoline, propranolol, and verapamile) having different physical and chemical properties. To demonstrate the performance of the heated nebulizer, sample solutions of 100 µM concentration in water/methanol (20:80, v/v) at a flow rate of 1 µL/min were infused, and the abundances of protonated molecules of the test compounds as a function of temperature were measured (Figure 3). Temperature readings were taken at the silicon surface. Quinoline, with a lower boiling point (234 °C), was vaporized at lower temperatures than propranolol (453 °C) and verapamil (586 °C), indicating proper performance of the microchip heated nebulizer. The decreasing parts of the curves of propranolol and verapamil are due to fragmentation of the compounds, which is well-known in commercial APCI sources, too. The microchip APCI operating temperature can be stabilized in 1-2 min, and cooling takes only 10-20 s. The heat transfer in microchip APCI is at least 10 times faster than with commercial APCI, allowing change of temperature during a LC run and, therefore, optimal conditions for each compound in a sample mixture. Significant improvements in thermal speeds are well-known for scaled-down devices.49 The microchip APCI produces high quality MS spectra in both positive and negative ion modes (Figure 4). Acridine and vera(49) Madou, M. In Fundamentals of Microfabrication; CRC Press, Inc: Boca Raton, FL, 1997; p 429.

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Figure 2. (A) Design of the microchip APCI: gas channels, inlets, and nozzle in the silicon wafer and integrated heater in the glass wafer. (B) Close-up of the microchip APCI.

Figure 3. Absolute intensities of the protonated molecules of (b) quinoline (Tbp ) 234 °C), (9) propranolol (Tbp ) 453 °C), and (1) verapamil (Tbp ) 586 °C) as a function of chip outside temperature with water/methanol (80:20, v/v) solution. The concentrations were 100 µM.

pamil, as well as nonpolar testosterone, were efficiently ionized in positive ion mode, producing an intense protonated molecule (m/z 180, 455, and 289, respectively) with few fragment ions. Dopamine measured in negative ion mode was also efficiently

ionized, producing a deprotonated molecule (m/z 152) with some fragment ions. The spectra are very similar to those measured with a commercial macroscopic APCI source, as demonstrated with verapamil (Figures 4D and 4E). The microchip APCI was shown to be able to operate with low flow rates between 0.05 and 5 µL/min, which makes it compatible with nano-LC and microfluidic devices. Although attempts to adapt APCI sources for flow rates lower that 10 µL/ min have been reported,46,47 they lack the concept of microchipbased miniaturization, and they are incompatible with microfluidic devices. The ability to use flow rates below 1 µL/min with microchip APCI opens up new possibilities to analyze neutral compounds in the minimum amounts, which is of utmost importance for understanding the role of small nonpolar molecules in biological systems. Furthermore, 1 µL of sample infused at a flow rate of 0.1 µL/min is able to produce a signal for ∼10 min. This allows sequential mass analysis, including, for example, optimization of operation parameters, measurements of MS, and different kinds of MS/MS spectra in positive and negative ion mode. The performance of the microchip APCI was evaluated by introducing 10 µL of a 100 µM sample in water/methanol (80:20,

Figure 4. Mass spectra of (A) testosterone and (B) acridine in positive ion mode and (C) dopamine in negative ion mode with the microchip APCI. The concentrations were 100 µM. Comparison of positive ion mass spectra of 10 µM verapamil with the microchip APCI (D) and with commercial PE Sciex API-300 APCI source (E). 6662

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Figure 5. (A) Selected ion chromatogram of protonated acridine (100 µM) produced by direct infusion of water/methanol (80:20 v/v) sample solution at a flow rate of 1 µL/min. (B) Absolute intensity of protonated molecule of acridine (100 µM) as a function of sample concentration. Injection volume was 10 µL, flow rate was 2.5 µL/min, and eluent was water/methanol (20:80, v/v) solution.

v/v) with a flow rate of 2.5 µL/min. The microchip APCI produced a highly stable ion current of the protonated molecule of acridine and allowed continuous operation for many hours without any problems (Figure 5A). We have used the same chip for weeks without any signs of deterioration of the device. The relative standard deviation of the peak height of the ion current of the protonated acridine in five measurements was 7.2%. The linearity of the response was measured in the range of 0.2-100 µM of acridine in water/methanol (20:80, v/v) with a flow rate of 2.5 µL/min. Good linearity with a regression coefficient, r2, of 0.995 was obtained (Figure 5B). These results indicate a high degree of reliability and reproducibility and good potential for quantitative routine analysis. Unstable performance of ESI is partly due to redox reactions caused by a high electrostatic field at the tip of the microfluidic channel. These reactions may lead to bubble formation and physical changes at the surface of the outlet of the microfluidic channel and, therefore, to nonrobust analysis.22,51-55 These problems do not exist in micro APCI with an external corona discharge needle. This is an important step in development of a robust interface for connecting microfluidic devices to a mass spectrometer. The limit of detection with the microchip APCI was tested by introducing 10 µL of acridine sample at flow rate of 6.2 µL/min. The signal was recorded by triple stage quadrupole mass spectrometer (Sciex 300) using multiple reaction monitoring (MRM). Limit of detection with signal-to-noise ratio of 3 was 5 nM, corresponding to mass flow of 0.52 fmol/s, indicating excellent sensitivity (Table 1). It must be borne in mind that the microchip used in this work is an early prototype, and the sensitivity can be improved further by optimizing all the structures of the microchip and by using a mass spectrometer having better sensitivity. The LOD measured with the commercial macro APCI using the same mass spectrometer and conventional injection volume (20 µL) and flow rate (1000 µL/min) was 5 nM, corresponding to a mass flow of 83 fmol/s. When the flow rate was reduced to 6.2 µL/min using macro APCI, the limit of detection (50) Cao, P.; Moini, M. J. Am. Soc. Mass Spectrom. 1997, 8, 561-564. (51) Van Berkel, G. J.; Zhou, F.; Aronson, J. T. Int. J. Mass Spec. Ion Processes 1997, 162, 55-67. (52) Moini, M.; Cao, P.; Bard, A. J. Anal. Chem. 1999, 71, 1658-1661. (53) Moini, M.; Smith, A. D. Anal. Chem. 2001, 73, 240-246. (54) Zhang, T.; Palii, S. P.; Eyler, J. R.; Brajter-Toth, A. Anal. Chem. 2002, 74, 1097-1103. (55) Liu, S.; Griffiths, W. J.; Sjo ¨vall, J. Anal. Chem. 2003, 75, 1022-1030.

Figure 6. Absolute intensity of protonated molecule of acridine (100 µM) as a function of flow rate. Injection volume was 1 µL, flow rate was 2.5 µL/min, and eluent was water/methanol (20:80, v/v) solution. Table 1. Detection Limits of Microchip APCI and Macro APCI detection limit

microchip APCI macro APCI macro APCI

flow rate, (µL/min)

concn, (nmol/L)

mass flow, (fmol/s)

6.2 6.2 1000

5 75 5

0.52 14 83

was 75 nM, corresponding to a mass flow of 7.82 fmol/s. (Table 1). The comparison shows that the microchip APCI sensitivity in terms of concentration in the sample is thus comparable to macro APCI, but the mass flow sensitivity is ∼100-200 hundred times better with microchip APCI. The results also show that the sensitivity with macro APCI is more than 10 times worse than with microchip APCI, when both interfaces were operated at the same flow rate (6.2 µL/min). One reason for the more efficient ionization with microchip than with macro APCI may be the size of a plume generated by the APCI sources. The space where the ionization takes place can be assumed to be the same for both APCI sources, since nearly identical corona discharge needles are used with the same voltages. Because microchip APCI forms a smaller plume than macro APCI, a larger fraction of the neutral analytes can be ionized with microchip APCI, leading to improved ionization efficiency. In practice, this means that microchip APCI analysis can be carried out with significantly smaller sample volumes than macro APCI without decreasing sensitivity in terms of concentration in the sample. Analytical Chemistry, Vol. 76, No. 22, November 15, 2004

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Signal current dependence on the flow rate (0.05-5 µL/min) was studied by introducing 1 µL of 100 µM acridine and monitoring selected product ion currents. The intensity of the measured ion current is directly dependent on the flow rate, indicating that microchip APCI-MS is a mass-flow-dependent detector (Figure 6). CONCLUSIONS Microchip APCI, presented now for the first time, provides excellent sensitivity (below 1 fmol/s), flow rates down to 50 nL/ min, efficient ionization for neutral and nonpolar compounds as well as polar and ionic compounds, robust analysis, good reproducibility, and cost-efficient manufacturing. The use of an external corona-discharge needle provides an easy connection of the microchip APCI to any mass spectrometer equipped with atmospheric pressure ionization source. The microchip APCI is directly compatible with microfluidic systems as well as with nano and

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micro LC, and this opens new possibilities to analyze small nonpolar and neutral compounds, in addition to ionic compounds, with high sensitivity from minimal sample volumes. For these reasons, microchip APCI provides a very interesting alternative to microchip ESI as a MS detector for microfluidic separation systems and nano and micro LC. Moreover, the heated nebulizer can be used for various other applications in which samples have to be vaporized before analysis. ACKNOWLEDGMENT We gratefully acknowledge The Academy of Finland, The Finnish National Technology Center, and Environics Oy.

Received for review May 4, 2004. Accepted September 9, 2004. AC049345G