Anal. Chem. 2000, 72, 3841-3846
An Efficient Liquid Chromatography-Mass Spectrometry Interface for the Generation of Electron Ionization Spectra Achille Cappiello,*,† Michael Balogh,‡ Giorgio Famiglini,† Filippo Mangani,† and Pierangela Palma†
Istituto di Scienze Chimiche, Universita` di Urbino, Urbino, Italy, and Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757
The use of a new LC-MS interface (cap-EI), part of a Waters Integrity system, capable of generating EI spectra at micro flow rates is presented. The cap-EI interface relies on the production of a fine aerosol by means of a nebulizer and supported by a nitrogen jet. Sensitivity, response linearity, reproducibility, and LC compatibility of the interface were thoroughly examined using testosterone, caffeine, a mixture of antiinflammatory drugs, and 3,4-dihydroxybenzoic acid as test compounds. The interface is fully compatible with LC requirements such as high-water- and/or -buffer-content mobile phases. Reproducibility, high sensitivity in scan mode, as well, to produce library-searchable EI spectra, 2 orders of magnitude linearity, together with an intrinsic simplicity of the entire system are the key features of cap-EI interface. Liquid chromatography (LC) and electron ionization mass spectrometry (EIMS) continue to play a major role, in their respective field, in solving a variety of analytical problems. Innovations and refinements over the last two decades have drastically improved performance and reduced costs so that they can approach the new millennium in very good shape. Liquid chromatography and electron ionization mass spectrometry behave rather differently: the first typically employs very high pressure for the efficient separation of analytes dissolved in a sometimes complex liquid phase; the second one operates at a very high vacuum, in a rarefied gas phase with no toleration for extraneous substances. Together in a single analytical system they can bring to the user the best of both worlds: unlimited separation capability for millions of different soluble compounds and reproducible, library-matchable mass spectra for an easier identification of volatile compounds. The two techniques that, in principle, appear fully incompatible may show an impressive number of overlapping applications. Small-medium molecules, approximately under 1000 u, amenable by LC, can generate, in suitable conditions, highly informative, reproducible, library-matchable EI mass spectra for an easier identification and characterization. A similar approach is described in the interesting work of Amirav and coworkers concerning the supersonic jet interface,1 capable of generating structural information through rich mass spectra similar to those obtained under EI conditions. * Corresponding author. e-mail:
[email protected]. † Universita ` di Urbino. ‡ Waters Corp. 10.1021/ac991493x CCC: $19.00 Published on Web 07/11/2000
© 2000 American Chemical Society
The first successful on-line combination of liquid chromatography and EI mass spectrometry dates back about fifteen years to when Willoughby and Browner proposed the first version of what was later called a “particle beam” interface.2 The performance of particle beam was affected by a few, but rather crucial, drawbacks: poor sensitivity especially with water-rich mobile phases, poor linearity especially with polar compounds, low tolerance for heat-sensitive compounds, and an unreliable optimization procedure. With the purpose of solving these drawbacks, our research group introduced, in 1993,3 an up-to-date version of the interface in which the operating mobile phase flow rate was reduced to the microliter-per-minute range. To achieve this limit, the nebulizer was entirely redesigned, replacing the one supplied by the manufacturer. The rest of the interface was left unmodified. The effect of the minimized solvent intake brought the particle beam interface to a superior level of performance: (1) Detection limit and sensitivity for several classes of analytes were improved. (2) The applicability of the interface toward sensitive compounds was extended. (3) Tuning procedures were simplified, and the general reliability was enhanced. The containment of the deleterious effects of mobile-phase solvents on the solute detection can be observed at any step of the interfacing mechanism, resulting in a cleaner signal. Kientz et al.4 proposed a different approach for generating electron ionization spectra from a HPLC effluent based on an eluent-jet formation by means of inductive heating of the micro-LC effluent, and momentum separation in a jet separator. Dijkstra et al.5 lately improved this technique with a particular emphasis on its use under CI/MS conditions. The list of positive changes in the interfacing mechanism linked to the solvent intake reduction is quite long, influencing different parts of the system, from the aerosol generation to the final ionization. An exhaustive discussion on this topic is reported in two recent review articles.6,7 The advantage offered by the new interface was exploited in several key applications. (1) Amirav, A.; Dagan, S.; Tzanani, N.; Granot, O. presented at ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, TX, June 13-17, 1999; p 14. (2) Willoughby, R. C.; Browner, R. F. Anal. Chem. 1984, 56, 2626. (3) Cappiello, A.; Bruner, F. Anal. Chem. 1993, 65, 1281. (4) Kientz, C. E.; Huist, A. G.; De Jong, A. L.; Wils, E. R. J. Anal. Chem. 1996, 68, 675. (5) Dijkstra, R.; Van Baar, B. L. M.; Kientz, C. E.; Niessen, W. M. A.; Brinkman, U. A. Th. Rapid Commun. Mass Spectrom. 1998, 12, 5. (6) Cappiello, A. Mass Spectrom. Rev. 1996, 15, 283. (7) Cappiello, A.; Famiglini, G. J. Am. Soc. Mass Spectrom. 1998, 9, 993.
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Figure 1. Scheme of the nebulizer.
However, the major limitation of this approach consists in the lack of modifications or adaptations of the preexisting interfacing mechanism to accommodate the needs of microscale nebulization. The full exploitation of the potential offered by the reduced-scale nebulization process can be only achieved through a complete re-engineering of the whole interface as a single, coordinated, working unit. In this paper, a new LC-MS coupling device, based on a soluteparticle transmission mechanism, is presented. The new interface is called capillary-EI (cap-EI) to emphasize the ability of generating electron ionization spectra at a capillary-scale flow rate. The aim of this project was to realize a simple, rugged interface, exploiting at its best the microflow nebulization technology7 into a new suitable interface. The new cap-EI interface is hosted into a Waters Integrity System where an enhanced microflow rate nebulizer can benefit from several modifications and upgrades all over the interface. The new cap-EI interface was designed around the lower incoming mobile-phase flow rate so that any piece of the system would contribute to the highest possible transmission. The performance of the cap-EI interface was thoroughly evaluated with different test compounds and over a long period of time. Sensitivity, intraday reproducibility, response linearity were considered and gave the best response reported for electron ionization to date. Detection limits were in the picogram range in selected ion monitoring (SIM) mode and in the low-nanogram range in scan mode for several analytes. The improved sensitivity of the cap-EI interface can be particularly appreciated especially when operating in scan mode, since its major appeal relies on the outstanding identification possibility of the full electron ionization spectrum. Linearity was observed for up to 2 orders of magnitude of sample concentration, even with very polar compounds. EXPERIMENTAL SECTION Liquid Chromatography-Mass Spectrometry Interface. N2 Nebulizer. The LC-MS cap-EI interface consists of three main parts: a microflow rate nebulizer, an expansion region, and a momentum separator. The interface, derived from a Waters Thermabeam unit, was modified to accomplish a liquid intake at a capillary-scale flow rate. The nebulizer is an improved version of the one realized by the authors a few years ago (Figure 1). It relies on a pneumatic nebulization mechanism accomplished by a high-velocity gas jet coaxial with the liquid stream. The gas orifice forms a sharp restriction just around the end of the capillary tubing where the mobile phase protrudes. The gas is thus forced through the ring-shaped passage, which sharply increases its speed. The high-velocity surrounding gas fractionates the emerg3842
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ing liquid into submicrometer liquid droplets and forms a very fine and homogeneous aerosol. One of the major improvements offered by the new nebulizer is full nitrogen compatibility. Furthermore, the highest aerosol production is obtained at a lower gas flow rate (0.1-0.2 mL/min), sensibly reducing costs over the expensive helium. Adaptation to a gas that is eight times heavier than helium is obtained through a series of adjustments in the nebulizer geometry. The higher viscosity reduces the speed of nitrogen through the orifice but its higher mass produces a suitable aerosol as well. To maximize the effect of a slower gas jet, the N2-ready nebulizer uses a narrower tolerance in the orifice compartment: the capillary tubing has an outside diameter of approximately 0.18 mm that entails, with a gas orifice of 0.4-mm, a restriction of only 0.1 mm for a closer contact with the outcoming liquid. As reported in the figure, a first expansion region, machined longer and narrower compared with the first prototype, is placed outside the orifice with the purpose of limiting the reduction in gas speed and directing the small droplets toward the middle of the contiguous desolvation chamber. It has been demonstrated,3 as a consequence of the drastic reduction of the liquid intake, that a capillary-scale nebulizer is no longer influenced by the volatility of the solvents used in the mobile phase. On the other hand, it has also been observed8 that strong polar compounds may increase water retention on the solute particle surface and slow complete desolvation. Such a behavior results in a secondorder concentration response during the calibration procedure, with loss of sensitivity at the lowest concentrations. This phenomenon was successfully opposed by reducing the size of the droplet and further retracting the nebulizer from the end of the expansion region. Both the nebulizer and its holder were made out of a solid piece of PEEK polymer (Figure 2). The new nebulizer, like its predecessor, does not require capillary-tubing position or gas-pressure adjustments and operates at ambient temperature. The initial set-up procedure is made during instrument installation and is valid for any analyte and for any chromatographic condition. Lack of any interface tuning procedure shortens, considerably, the start-up procedure and optimizes the overall instrument throughput. Solute Enrichment and Vaporization. Submicrometer dry particles are collected into a two-stage momentum separator where solvent vapors and nitrogen are removed from the solute path. The reduced amount of vapors admitted into the high-vacuum region allows the exclusion of an auxiliary pump from the first stage and the use of a single roughing pump for both solute enrichment and turbo needs. In this simplified configuration, the
Figure 2. The photograph represents the removable PEEK nebulizer holder connected into the system.
vacuum varies from 7 × 10-2 Torr in the first stage of the momentum separator to 2 × 10-5 Torr in the ion source region, when 2 µL/min of mobile phase and 0.2 mL/min of nitrogen are introduced into the system. Improved signal response (see Results and Discussion) indicates that most of the solute enters the ion source for ionization with a consistent enhancement of the interface transport efficiency. Since the cap-EI interface, like its predecessor, relies on electron ionization, a phase change from solid to gas, prior to the formation of any ion, is imposed. Ionization of the analytes is achieved upon vaporization of the solute particles onto the hot ion volume surface. Smaller particles, like those generated in the cap-EI system, allowed prompt vaporization with reduced exposure to heat prior to ionization. Because of the reduced size of the particles, a larger total surface will be exposed to the heat, accomplishing a rapid conversion of the analyte into the gas phase, smoothing down the effects of the temperature shock and thus reducing the chance of thermal decomposition. Of course, such a treatment is acceptable only for those HPLC amenable substances with a minimum of heat compatibility and vapor pressure, and this may extend the use of cap-EI toward new applications. Mass Spectrometry. Mass spectrometer tuning and calibration were performed automatically using perfluorotributylamine (PTA) as a reference compound and monitoring m/z 69, 219, 313, 414, 502, 614 for intensity and mass calibration. The final tuning was optimized for m/z 219, which is the closest value to the most common fragments generated by the compounds used in this work. The mobile phase was not allowed into the system during
the tuning procedure. Dwell and scan times were adjusted in order to obtain a mean of 10 acquisition samplings for each HPLC peak at unit resolution. The ion source temperature was adjusted for the maximum signal without inducing thermal decomposition in the selected compounds and kept constant at 200 °C. Liquid Chromatography. The liquid chromatograph, four solvents with an auto sampler, was part of the Integrity System (Waters, Milford, MA), which originally included the LC, a photodiode array detector (PDA), the interface, and the mass spectrometer. The PDA detector was temporarily excluded from the sample path for the incompatibility of the cell volume to capillary-scale flow rates. Microliter-per-minute flow rates were obtained with a laboratory-made splitter that was placed between the pumping system and the injector.9 This device allows conversion of almost-conventional flow rates (200 µL/min), generated by a conventional HPLC system, into capillary-scale flow rates. A two-step splitting of the main stream of solvents generates a 2 µL/min mobile-phase flow rate with a splitting ratio of 100:1. The splitting device accurately reproduces at lower scale any solvent concentration gradient generated at higher flow rate in the pumping system. A zero-volume connector linked a 60-500-nL internal loop injector (Valco Instruments Co., Inc., Houston, TX) to the splitter. The external injector replaced the one supplied by the manufacturer in order to accomplish the limits imposed by micro-HPLC. A 60-nL loop was used for all flow injection (FIA) experiments. Larger volumes were used only under specific solvent gradient conditions. A laboratory-made packed capillary column was used for the chromatographic separations. These columns are routinely made in our laboratory from 1/16 in o.d. 250-µm-i.d. PEEK tubing (Alltech Associates, Inc., Deerfield, IL) and are packed with C18 reversed-phase 5-µm particles purchased from Phase Sep (Queensferry, UK). A 25-cm-long column has a mean of 20 000 theoretical plates at a 1 µL/min flow rate, and no appreciable loss of efficiency is observed for flow rates up to 5 µL/min.10 Acetonitrile was used as organic solvent in the mobile phase. Acetonitrile was preferred to methanol because of its lower viscosity, a parameter that is crucial in micro-HPLC. Reagents and Sample Preparation. Reagents (97-99% purity) were purchased from Sigma Scientific (St. Louis, MO). All solvents were HPLC grade from Farmitalia Carlo Erba (Milan, Italy) and were filtered and degassed before use. Reagent water was obtained from a Milli-Q water purification system (Millipore Corp., Bedford, MA). Stock solutions (10 mg/mL) were prepared for all the selected compounds. Dilute solutions were prepared prior to LC-MS analysis. Caffeine determination in plasma was performed spiking 1 mL of human plasma with 20 µL of a 100 ng/mL solution of caffeine in acetonitrile. Proteins were precipitated with trifluoroacetic acid, and after centrifugation at 3000 rpm, the supernatant was collected. A solid-phase extraction procedure was performed for sample enrichment11 (8) Cappiello, A.; Famiglini, G.; Mangani, F.; Careri, M.; Lombardi, P.; Mucchino, C. J. Chromatogr., A 1999, 855, 515. (9) Berloni, A.; Cappiello, A.; Famiglini, G.; Palma, P. Chromatographia 1994, 39, 279. (10) Cappiello, A.; Palma, P.; Mangani, F. Chromatographia 1991, 32, 389. (11) Cappiello, A.; Palma, P.; Berloni, A.; Famiglini, G.; Mangani, F. On-column Solid-Phase Microextraction for HPLC. 23rd International Symposium on High Performance Liquid-Phase Separations and Related Techiniques HPLC’99, Granada, Spain, May 30-June 4, 1999; PA 14/8.
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Figure 4. HPLC-MS analysis of human plasma extract spiked with 2 ppb of caffeine obtained in SIM at m/z 194. HPLC conditions: flow rate, 2 µL/min; solvent A, water; solvent B, acetonitrile; solvent program, from 100% to 20% A in 30 min; 50-µL injection volume.
Figure 3. m/z 124 ion profile relative to three injections of 100 pg of testosterone in SIM mode (a). Mass chromatogram relative to two injections of 600 pg of testosterone in scan mode (b).
RESULTS AND DISCUSSION During the evaluation of the cap-EI interface several parameters were considered. Far from being exhaustive, we feel that the criteria described below are particularly important to appreciate the level of the progress made in this field. First of all, we have to consider the LC-MS interface, in general, and the capEI, in particular, as the fusion of two independent analytical tools with their advantages but also their limitations. In this specific case, capillary HPLC is coupled to electron ionization mass spectrometry. Capillary HPLC columns allow efficient separations at very low flow rate but pose restrictions in the total injectable volume. Detection of very dilute samples may be at risk when only submicroliter volumes are allowed and this has to be supported by an adequate increase in the detector sensitivity. To claim the effective advantage of a micro-LC system versus a conventional one, the gain in terms of sensitivity, obtained with the detector, should exceed the loss of analyte mass. On the other side, when pure sensitivity is concerned, electron ionization cannot win the battle in a direct comparison with the many soft ionization techniques, but by means of cap-EI interface may offer fullspectrum capability at high-picogram-level sensitivity. The evaluation of the performance of the cap-EI interface was based on the following parameters: sensitivity, response linearity, reproducibility, and LC compatibility. Sensitivity. Beside its significance in terms of response for unit of concentration, it is important to evaluate the minimum amount of material required to generate a distinguishable signal. This quantity is usually described as the limit of detection (LOD) of the instrument. In this context, LOD was calculated as the signal generated by the mass spectrometer which exceeds the mean value of the background noise of 6 - σ, where σ is the standard 3844 Analytical Chemistry, Vol. 72, No. 16, August 15, 2000
Figure 5. HPLC-MS analysis of four drugs in scan mode: prednisolone (a), bethamethasone (b), naproxen (c), ibuprofen (d). HPLC conditions: flow rate, 2 µL/min; solvent A, water + 10 mM ammonium acetate; solvent B, acetonitrile + 10 mM ammonium acetate; solvent program, from 90% to 0% A in 20 min; 100-nL injection volume.
deviation of the baseline. Electron ionization presents two independent levels of detection limits: one is obtained when operating in SIM mode and, since it is the lowest, it is considered mostly for quantitation purposes; the other one, calculated in full-scan acquisition, is evaluated mostly for qualitative investigations. But it is the last one to principally draw our attention. Any little gain in the full-spectrum signal obtained from the minimum amount of analyte is particularly welcome in this context. In fact, the peculiarity of this interface is that it offers the advantage of a characteristic mass spectral signal to the multitude of suitable LC applications. To define these limits, testosterone was selected as the test compound for FIA experiments, caffeine was selected for a realworld application, and a mixture of four drugs was considered for a column separation. Each experiment represents a different aspect of the interface sensitivity and may contribute to better focus its overall performance. In these conditions, the LOD for testosterone is approximately 100 pg in SIM and 600 pg in scan (m/z 50-300). Figure 3a reports the m/z 124 SIM ion profile relative to a sequence of three 100-nL injections of a 1 ppm solution of testosterone; Figure 3b shows the scan profile relative to a sequence of four injections of testosterone. The first two peaks correspond to the injection of 600 pg of testosterone (10 ppm solution, 60 nL injected); the two major peaks correspond to 6 ng of testosterone (100 ppm solution, 60 nL injected). The analyte was introduced directly into the system at 2 µL/min with a mobile phase composed of an equal concentration of water and acetonitrile. Although the smaller peaks are barely distinguishable from the background, the subtracted spectrum is very well character-
Figure 7. Linear and quadratic regression plots from concentration calibration data relative to 3,4-dihydroxybenzoic acid: capillary-scale PB interface (a), cap-EI interface (b).
Figure 6. Subtracted spectra recorded from Figure 5 analysis (Wiley search quality factors between 92 and 98).
ized especially considering the very low amount injected. Figure 4 shows the detection of a 2 ppb concentration of caffeine in human plasma (100 pg of caffeine injected). Fifty microliters of plasma extract was injected into the microcolumn in large-volume injection conditions. The sensitivity of the cap-EI interface can be particularly appreciated in this real-world application, where neither the matrix nor the column separation affected significantly the quality of the signal. Figure 5 shows the separation of a fourdrug mixture obtained in solvent gradient conditions for a lowconcentrated sample. The absolute amounts injected were 17 ng for prednisolone, 22 ng for bethametasone, 33 ng for naproxen and ibuprofen. The mass detector was scanning between m/z 50 and 450 during the chromatographic run, and the signal-to-noise ratio obtained for the four peaks was approximately 10:1. Although column separation affected slightly the overall sensitivity, increas-
ing LOD values to the low nanogram range, at this concentration, the recorded spectra were suitably library searchable as reported in Figure 6. These values represent at least a 10-fold improvement in the detection limit with respect to that for similar instrumentations. They may vary slightly from substance to substance but should give a practical idea of the level of sensitivity reached by the capEI interface. Response Linearity. The extension of the linearity response range is a weak point of several interfacing devices. In this case, the behavior of the capillary-scale particle beam interface, introduced a few years ago by our group, was a little contradictory. Compared with the original particle beam interface, the linearity range was at least an order of magnitude wider for most neutral or slightly polar compounds, but was still unsatisfactory for several polar substances.8 In our opinion, the poor performance was related to an unreliable transmission of strongly solvated particles through the interface. The cap-EI interface is entirely tuned for capillary-scale flow rates, and concentration calibration experiments show a remarkable improvement in this sensible area. Figure 7 shows an interesting comparison between two 3-ordersof-magnitude concentration calibration plots obtained for 3,4dihydroxybenzoic acid, a considerably polar analyte, with a capillary-scale particle beam (a) and a cap-EI interface (b). The six-point calibration was started at a concentration 5 times greater Analytical Chemistry, Vol. 72, No. 16, August 15, 2000
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Figure 9. Signal response of caffeine at different concentrations of water and acetonitrile in the mobile phase. Flow rate: 2 µL/min. Sixty-nanoliter injection volume.
Figure 8. Reproducibility graph with 95% confidence bands.
than the LOD, considering five replicates for each concentration. Both experiments were carried out at a 2 µL/min flow rate, using a mobile phase composed of water and acetonitrile at equal concentration and modified with trifluoroacetic acid (TFA) (0.025% in acetonitrile and 0.05% in water) and using the same standard solutions. The difference between the two plots is remarkable and can be easily valued as the concentration increases. Cap-EI interface clearly shows a very linear response along an impressive 2 orders of magnitude calibration, which is also demonstrated by a linear correlation coefficient r of 0.9996 (y ) 2450x + 248.1). Poor transmission at the lowest concentrations, as observed in the capillary-scale particle beam interface, demands a second-order correlation coefficient r2 of 0.9991 (y ) 0.3x2 + 21.7x + 1913.3), thus giving to this interface a limited range of linear response. Reproducibility and LC Compatibility. Intraday reproducibility has been evaluated for a given composition of the mobile phase in FIA conditions (water-acetonitrile, 1:1). The evaluation was performed with 24 consecutive injections of a 1000 ppm solution of 3,4-dihydroxybenzoic acid in SIM mode. A visual representation of the cap-EI reproducibility with 95% confidence bands is shown in Figure 8. A relative standard deviation (RSD) of 5.22% was obtained, demonstrating a good stability of the interfacing process during a sequence of identical injections. The reduced amount of mobile phase processed by the interface adds appreciably to the cleanness of the instrument, dropping the frequency of normal maintenance operations and cutting the effects of assorted deposits on critical parts of the instrument (particle path and ion optics). As previously discussed, the negative influence of water on the transport mechanism is contained, giving a reasonably good response, even at the highest (12) Balogh, M. P. LC-GC 1998, 16 (2), 135-144.
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concentrations of the solvent. Figure 9 reports the testosterone signal recorded in SIM (m/z 124) at different concentrations of water and acetonitrile in the mobile phase. Except for the highest signal, obtained at 90% acetonitrile, the interface showed a flat response for testosterone in the range of compositions considered, with a only slight increase of signal at 90% water. This behavior results in a reliable response during solvent gradient separations and in an appreciable detection at full-water mobile phase.7 CONCLUSIONS Although atmospheric introduction methodologies have revolutionized the utility of LC-MS over the past decade12 specific applications are either not amenable to API analysis, such as hydrocarbon-based synthetic intermediates in drug development, or the cost and complexity of the instrumentation needed to acquire an ‘information-rich’ spectrum for compound identification is not feasible. The improvements illustrated here provide access to classical, well-characterized electron ionization data for a variety of applications. Cap-EI provides necessary linearity, ruggedness and reproducibility of response for environmental analysis, easeof-use for broad pharmaceutical ‘open access’ applications, and readily interpretable spectra from commercially available sources (such as NIST or Wiley), literature generated over many years from a variety of instruments or even first-principles evaluation of novel data. ACKNOWLEDGMENT The authors would like to acknowledge Waters Corp. for providing the instrumentation and for their support in this research. We would also like to thank Pasquale Monaco for his valid contribution. Received for review December 30, 1999. Accepted May 2, 2000. AC991493X
