Anal. Chem. 2003, 75, 973-977
Combined Electrospray Ionization-Atmospheric Pressure Chemical Ionization Source for Use in High-Throughput LC-MS Applications Richard T. Gallagher,* Michael P. Balogh,† Paul Davey, Mike R. Jackson,† Ian Sinclair, and Lisa J. Southern‡
AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, U.K.
Fast and accurate analytical methods are essential to keep pace with sample libraries produced from combinational chemistry and high-throughput biological screening. Many laboratories now use a combination of ionization techniques for the characterization of these samples, including atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), and photoionization (PI). Data are shown here from the analysis of a compound collection plate containing a variety of sample structures. ESI will normally analyze around 80% of these samples, necessitating a source change to analyze a further 10%. In this work, we have developed a new combined ESIAPCI source (ESCi) for use in on-line HPLC applications. The combined source allows alternate on-line ESI and APCI scans with polarity switching within a single analysis. The ESCi source has been designed to be a simple replacement for the existing mass spectrometer interfaces. Each ionization method is optimized independently using separate tuning parameters. Instrument electronics can readily switch between the two ionization methods and polarities within normal interscan time periods. The new source has reduced the analysis time of sample plates by eliminating the need for a source hardware change, source optimization, and repeat analyses. Liquid chromatography-mass spectrometry (LC-MS) is widely used for the analysis of compound collections or libraries because of its high speed, sensitivity, and specificity. The majority of potential drug candidates are polar molecules with molecular weights from 200 to 700 amu. Many laboratories use a combination of mass spectrometry ionization techniques for sample characterization, including atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), and photoionization (PI). For the analysis of drug candidate synthetic intermediates, which may be less polar species, other techniques, such as electron impact (EI) and chemical ionization (CI), may additionally be employed. Previous experience in this laboratory shows that that ∼80% of the compound collection candidates ionize readily by a generic electrospray ionization method.3 To analyze an additional * Corresponding author. Phone: +44 (0) 1625 513341. Fax: +44 (0) 1625 517436. E-mail:
[email protected]. † Waters Inc., 34 Maple Street, Milford, MA 01757. ‡ Waters Ltd., Centennial Park, Elstree, Hertfordshire, WD6 3SZ, U.K. 10.1021/ac0205457 CCC: $25.00 Published on Web 01/10/2003
© 2003 American Chemical Society
10% of samples, the analyst has two main options: either the pH of the liquid chromatography mobile phases may be altered to aid ion formation or, alternatively, the ionization technique may be changed. This latter choice requires a change in instrument hardware and reoptimization of the ion source parameters. Both of these options result in lost analysis time. To overcome this problem, some new instrument configurations have been employed. Byrdwell has used a dual parallel mass spectrometer system to acquire ESI and APCI data simultaneously from a single LC system.4,5 This method, although successful, ties up two mass spectrometers for a single analysis. Recent developments in ion source design have tackled this problem by opting to produce multiple ionization techniques within a single mass spectrometer source so as to remove the need for ion source changes. A combined atmospheric pressure photoionization (APPI)-APCI source has been developed by Hanold et al.6 This source is most suited for the analysis of pharmaceutical drug synthesis intermediates that are normally less polar than the target drug candidates. Following on from a simultaneous ESI/APCI source developed by Siegel et al.,7 Castoro8 has utilized the features of a commercial system. In this latter approach, the source is a standard electrospray interface with the addition of an APCI corona discharge needle that is permanently powered on. This results in mass spectra exhibiting enhanced pseudo-molecular ions with some fragmentation giving limited structural information. Simultaneous ESI with APCI does not easily allow controlled experiments to determine if ion suppression effects are created in this combined mode. However, this source was able to detect compounds in LC-MS analyses that may not have been detected by a single ionization approach. Another direction is the dual source approach. Here, two or more ion sources are essentially combined in a single unit that (1) This reference was removed on proof. (2) This reference was removed on proof. (3) Gallagher, R. T.; Davey, P.; Sinclair, I.; Balogh, M. P.; Jackson, M. R.; Southern, L. Proceedings of the Am. Soc. for Mass Spectrom., 50th ASMS Conference, Orlando, FL, June 2-5, 2002. (4) Byrdwell, W. C. Rapid Commun. Mass Spectrom. 1998, 12, 256-272. (5) Byrdwell, W. C.; Neff, W. E. Rapid Commun. Mass Spectrom. 2002, 16, 300-319. (6) Horner, J. A.; Hanold, K. A.; Thakur, R. A. Proceedings of the Am. Soc. for Mass Spectrom., 50th ASMS Conference, Orlando, FL, June 2-5, 2002. (7) Siegel, M. M.; Tabei, K.; Tong, H.; Lamber, F.; Candela, L.; Zoltan, B. J. Am. Soc. Mass Spectrom. 1998, 9, 1196-1203. (8) Castoro, J. A. Proceedings of the Am. Soc. for Mass Spectrom., 50th ASMS Conference, Orlando, FL, June 2-5, 2002.
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allows multiple analyses by selected sampling of each source.7,9-12 Siegel et al. have developed a dual ESI-APCI source7 that exhibited many benefits but focused on flow injection rather than chromatographic separations. This source operated at low flow rates and used compromised operating conditions. Later work has shown chromatographic applications of this source.9,10 Siegel et al. also recently modified a multiple spray system (MUX) to provide a high-throughput ESI and APCI analysis system.11 This system has eight spray nozzles to allow high sample throughput but is hindered by high system maintenance and an overall system sensitivity drop. A polarity switching capability was demonstrated in Siegel’s work, although it was not fully optimized and showed arcing in negative ionization mode.7,9,10 Although these source designs go some way toward improving the limitations of traditional single mode ion sources, they still lack the ability to maintain consistently high throughput. In this work, we have developed a new combined ESI-APCI source (ESCi13) for use in high-speed on-line LC-MS applications. The combined source allows alternate on-line electrospray and APCI scans within a single analysis with polarity switching resulting in a higher sample throughput. The ESCi design is characterized by clearly defined boundaries between dissimilar ionization modes, yielding high spectral fidelity. EXPERIMENTAL SECTION Instrumentation. Experiments were conducted on a Waters ZQ single-quadrupole mass spectrometer (Waters, Elstree, U.K.). HPLC equipment used was the Waters Alliance HT 2795 LC system with a Waters 2996 diode array detector scanning 225320 nm. The ESCi source uses the existing Waters ESI source hardware with the addition of the APCI discharge needle. A highvoltage switching unit and higher specification power supply were used to power the capillary and discharge needle. During ESCi operation, the high-voltage power supply can alternatively switch from either the electrospray capillary (voltage regulated) to the APCI discharge needle (current regulated) or polarity switch, within a 100-ms interscan delay time. The power supply uses MOS FETs (metal oxide semiconductor field effect transistor) to switch both polarity and mode. Mode is defined as voltage (ESI) or current (APCI,) and the software recognizes each separately, diverting power to the appropriate device. The key to the success of the ESCi source is in this switching at high speeds with good fidelity, thus allowing extremely high power demands to be repeatedly dissipated in millisecond time scales. HPLC Methods. Flow Rate Optimization Experiment. Symmetry C18 column, 10 mm × 2.1 mm i.d., 3.5 µm (Waters, Elstree, U.K.); 5-µL injection of diphenhydramine solution (100 ng on column) into isocratic flow, water/acetonitrile (50:50 v/v) at various flow rates from 50 to 1000 µL/min. (9) Tabei, K.; Siegel, M. M. Proceedings of the Am. Soc. for Mass Spectrom., 47th ASMS Conference, Dallas, TX, June 13-17, 1999. (10) Tabei, K.; Hausner B.; Lambert, F.; Maccio, M.; Siegel, M. M. Proceedings of the Am. Soc. for Mass Spectrom., 48th ASMS Conference, Long Beach, CA, June 11-16, 2000. (11) Siegel, M. M.; Truebenbach, C. S. Proceedings of the Am. Soc. for Mass Spectrom., 50th ASMS Conference, Orlando, FL, June 2-5, 2002. (12) Liu, C.; Kovarik, P.; LeBlanc, Y.; Sakuma, T.; Garofolo, F.; Marland, A.; Pang, H.; McIntosh, M.; Wong, E.; Kennedy, M.; Covey, T. Proceedings of the Am. Soc. for Mass Spectrom., 50th ASMS Conference, Orlando, FL, June 2-5, 2002. (13) ESCi is a registered trademark for Waters Inc.
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Figure 1. Optimum flow rate evaluation for the ESCi Source. Trend lines for the peak area average from 10 repeat injections of diphenhydramine in APCI and ESI modes vs eluant low rate.
Figure 2. LC-ESCi-MS analysis on a mixture of daidzein and acetophenone. Total ion chromatogram (TIC) traces for ESI and APCI positive ion modes.
Qualitative Performance Experiment. Xterra MS 50 mm × 2.1 mm i.d. column, 3 µm (Waters, Elstree, U.K.); 5-µL injection volume into an isocratic flow of 0.1% aq formic acid/acetonitrile (70:30 v/v) at 0.5 mL/min. Compound Collection Sample Analysis. Xterra MS 30 mm × 2.1 mm i.d. column (Waters, Elstree, U.K.). Mobile phases: A, 0.1% aqueous formic acid; B, acetonitrile. Flow rate, 0.7 mL/min. Samples were analyzed using a linear gradient varying B from 5% to 95% over 2 min, with a total run time of 3 min. Injection volume for all samples was 1.0 µL. Mass Spectrometry Methods. Each ionization method was optimized independently using separate source-tuning parameters (capillary voltage or APCI pin voltage, sample cone, and extraction cone). A modified source-tuning parameters software page for the Waters ZQ was produced to allow the separate ionization mode optimization, MASSLYNX version 4.0 (Waters UK Limited, Atlas Park, Simonsway, Manchester, U.K.). Common tuning parameters include all gas flows and interface temperatures (Source temperature, 120 °C; desolvation temperature, 350 °C in these experiments). Ionization sources used were the ESI, APCI, and the combined ESCi source. For the ESI and APCI source, the polarity was switched on alternate scans. For the ESCi source, either the ionization mode or the polarity were switched from scan to scan in the following order: positive ESI, negative ESI, negative APCI, positive APCI. Cone voltage was set to 20 V throughout all experiments.
Figure 3. Example data from compound collection 1. Comparison of mass spectral data obtained for a compound of 319 amu analyzed by ESCi source (left column), ESI source (upper right), and APCI source (lower right).
Flow Rate Optimization Experiment. The mass spectrometer was scanned from 150 to 1000 amu at 0.3 s/scan with a 0.1-s interscan time. Qualitative Performance Experiment. The mass spectrometer was scanned from 150 to 500 amu at 0.2 s/scan with a 0.1-s interscan time. Compound Collection Sample Analysis. The mass spectrometer was scanned from 150 to 800 amu at 0.2 s/scan with a 0.1-s interscan time. Total run time per sample was 3 min. Materials. All solvents were obtained from Fisher Scientific UK Ltd., Loughborough, U.K. Diphenhydramine, daidzein, and acetophenone were obtained from Sigma-Aldrich, Gillingham, U.K. Flow rate optimization experiments used diphenhydramine dissolved in water/acetonitrile (50:50 v/v) at a concentration of 20 µg/mL. For the qualitative performance experiment, daidzein and acetophenone were each dissolved in water/acetonitrile (50:50 v/v) to a concentration of 50 µg/mL. A 50:50 (v/v) mixture was then analyzed. Sample collection samples were dissolved from solid in methoxyethanol at a concentration of 1 mg/mL. RESULTS AND DISCUSSION ESCi Source Optimum Flow Rate Evaluation. Diphenhydramine (Ph)2CHOC2H4N(Me)2 will ionize by both ESI and APCI and fragments readily to a base peak at m/z 167 for the (Ph)2CH+ ion. The performance of the ESCi source was evaluated in each ionization mode by observing this base peak at various flow rates from 50 to 1000 µL/min. These experiments were performed at a source temperature of 120 °C, a value that falls within the optimum region determined by Siegel et al.8,9 The average peak area response from 10 injections was plotted against eluant flow rate (µL/min), and the trend lines obtained are shown in Figure 1. The APCI ion signal response from the ESCi source varies from
Figure 4. Example data from compound collection 2. This example shows the photodiode array and mass chromatogram traces for an LC-ESCi-MS analysis. The major peak (RT 1.52 min) is ionized in APCI+ mode only; the minor impurity (RT 1.79 min) ionizes in both APCI+ and ESI+ modes.
4.5 × 107 counts at 50 µL/min rising to 6.8 × 107 at 1000 µL/min, an increase of ∼50%. The ESI signal has greater flow-rate dependence from 4.5 × 109 counts at 50 µL/min, falling to 5.0 × 108 at 1000 µL/min, a near order of magnitude signal fall. The trend lines shown in Figure 1 indicate that the behaviors are Analytical Chemistry, Vol. 75, No. 4, February 15, 2003
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Figure 5. Example data from compound collection 3. This example of an LC-ESCi-MS analysis for a compound of mass 226 amu shows the molecular ion mass chromatogram traces (left) and the corresponding mass spectra molecular ion regions (right).
consistent with expectations14 for ESI and APCI, even though coaxial heating is limited in APCI mode to that provided by the ESI probe. Most likely, the geometry of this source along with gas convection turbulence, heat distribution effects, and plasma propagation by the liquid promote the ability to create ions by chemical ionization without additional heat. These response curves are comparable to standard ESI, and APCI systems14 showing optimal performance of the APCI mode are not significantly compromised in the combined source at extremes of flow rate. ESCi Source Qualitative Performance. From the early days of discovering the fundamentals of atmospheric ionization processes, there are clearly multiple behaviors present in these sources, which are documented in the literature.12 These include thermospray occurring along with, and as a component of, APCI and even, in some cases, clearly APCI behaviors occurring with electrospray.15-17 These are dependent on the mobile phase and other conditions.18-19 A consequence in switching between ionization modes very rapidly is that there is always a possibility of some “cross talk” of ionization processes occurring. To test the ability of the ESCi source to produce “pure” ESI and APCI spectra, a test mixture of daidzein and acetophenone was analyzed by LCESCi-MS. Figure 2 shows the total ion chromatograms (TIC) for (14) Kebarle, P.; Ho, Y. Electrospray Ionisation Mass Spectrometry, Fundamentals, Instrumentation and Applications; Cole, R. B., Ed.; Wiley-Interscience: New York, 1997, Chapter 1. (15) Sakairi, M.; Kambara, H. Anal. Chem. 1988, 60, 774-780. (16) Sakairi, M.; Kambara, H. Anal. Chem. 1989, 61, 1159-1164. (17) Brown, P.; Nachi, R.; Julian, T.; Dzerk, A.; Lin, P. Proceedings of the Am. Soc. for Mass Spectrom., 48th ASMS Conference, Long Beach, CA, June 11-15, 2000. (18) Sjo ¨berg, P. J. R.; Bo¨kman, C. F.; Bylund, D.; Markides, K. E. J. Am. Soc. Mass Spectrom. 2001, 12, 1002-1010. (19) Balogh, M. P.; Jackson, M. R. Waters Inc., 34 Maple Street, Milford, MA 01757; unpublished results.
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the ESI and APCI positive ion modes and the photodiode array trace (PDA). Daidzein will ionize by all four modes, but it is more sensitive to ESI positive ion, and a protonated molecular ion was observed at m/z 255 at a retention time of 6.95 min. Acetophenone dominates the APCI+ trace relative to daidzein and produced a MH+ ion at m/z 121 at a retention time of 5.47 min. The single peak in the ESI+ TIC trace shows no that no cross talk between the APCI+ and ESI+ modes is occurring. This ability to switch the power supplies and ionization modes rapidly is fundamental in improving the data quality as more of the scan duty cycle is spent on data acquisition and less is sacrificed to switching. Compound Collection Analysis. To test the performance of the ESCi ion source, a 96-well plate was randomly selected from a compound collection and analyzed using a short gradient LCMS method. The plate contained a variety of known samples covering molecular weights from 150 to 500 amu. Initially, this plate was analyzed using the standard APCI and ESI sources and then reanalyzed on the same instrument using the combined ESCi source. In all cases, the HPLC analysis conditions remained the same. Example data obtained from the LC-MS analysis are shown in Figures 3 to 5. In Figure 3, a comparison of the data obtained by the ESCi source to the single mode sources is shown for a compound of molecular weight 319 amu that ionizes in all four modes. In these experiments, the same source temperatures (source temperatures, 120 °C; desolvation temperature, 350 °C) and gas flows were used for the three sources. In this example, the pseudo-molecular ions (MH+ m/z 320 or [M - H]- m/z 318), adduct ions (e.g., MNa+ m/z 342, MK+ m/z 358) and major fragment ions are equivalent in the ESI source and the ESCi source operating in ESI mode for both polarities (top four spectra in Figure 3). Some additional fragment ions are produced in the ESI source for both polarity
modes. The APCI data obtained does show significant differences. The major peaks in positive ion APCI mode are similar for both sources. In negative ion mode, the APCI source produced only fragment ions (m/z 155 and 164), as compared to the ESCi source, which produced a deprotonated molecular ion (m/z 318 [M-H]-) as the base peak. This is most likely due to the standard APCI source’s producing more thermal degradation, which is a disadvantage in this application in which a pseudo-molecular ion is desired for initial identification purposes. In Figure 4, a compound eluting at a retention time of 1.52 min is shown to ionize only under positive APCI conditions. An impurity in this sample, at retention time 1.79 min, ionizes by both positive ion APCI and ESI in the ESCi source. Traditionally, this sample would have been initially analyzed using ESI and then repeated under APCI conditions. This example highlights the strength of the ESCi source in that a reanalysis to detect the molecular ion was not required. The full results obtained for the plate analysis show that the ESCi source correctly identified ∼10% more samples (80 hits) than the standard ESI or APCI sources (72 and 71 hits, respectively). A hit was deemed correct if the UV purity assessment (>85%) and the molecular mass measurement were both correct.3 ESCi has great potential for use in open access LC-MS systems. Synthetic chemists require fast LC-MS analysis of synthesis products to aid structural confirmation. Measurements of purity, molecular weight, and the correct molecular ion isotope distribution are required. The importance of having separate APCI and ESI mass spectra is highlighted by the example shown in Figure 5. This example shows the molecular ion mass chromato-
gram traces obtained from an LC-ESCi-MS analysis. The major peak in each trace corresponds to a compound of molecular weight 226 amu. The molecular ion region of the mass spectra, obtained from the major chromatographic peak, shows that for positive ion mode, a protonated molecular ion is formed by ESI and APCI. Under negative ion ESI, a deprotonated molecular ion is produced, but by negative ion APCI conditions, a deprotonated and a radical cation are both produced. Having obtained separate ion mode traces greatly simplified the interpretation of these data. A full quantitative evaluation of the ESCi system is currently ongoing. However, this preliminary data has shown no noticeable degradation of the ESCi source performance, as compared to standard ESI and APCI interfaces. CONCLUSIONS This combined electrospray-atmospheric pressure chemical ionization source (ESCi) can generate clearly differentiated and reproducible electrospray and APCI spectra while polarity-switching. The qualitative performance of the combined source has been compared to the existing electrospray and APCI interfaces and found to be equivalent in ESI mode and produce less thermal fragmentation in APCI mode. This data quality can be maintained over a flow rate from 50 to 1000 µL/min. This new source has greatly reduced the analysis time of sample plates by eliminating the need for repeat analysis. Received for review August 23, 2002. Accepted November 19, 2002. AC0205457
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