Anal. Chem. 1999, 71, 5165-5170
Monitoring Process-Scale Reactions Using API Mass Spectrometry Philip Dell′Orco,*,† Jeffrey Brum,† Richard Matsuoka,‡ Manish Badlani,§ and Kenneth Muske§
Analytical Sciences and Synthetic Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, and Department of Chemical Engineering, Villanova University, Villanova, Pennsylvania 19085
We present results using an unmodified nebulized assisted electrospray ionization (ESI) interface to observe a process-scale organic reaction (26 wt % in reactants) in real time. The approach offers distinct advantages over optical methods because unambiguous MW information can be obtained. The approach uses a series of pumps, which, after sampling the reactor, (1) quench the reaction, (2) reduce the concentration (3000×), and (3) add a proton-donating buffer for ionization. The approach is demonstrated with a piperidine-catalyzed Knoevenagel condensation reaction in toluene with informative results. The particular softness of ESI affords the formation of protonated parent ions almost exclusively. Reactions are tracked following [M + H]+ ion signatures as a function of time. In addition to providing information about kinetic rates, mechanistic information was obtained via the observation of the Mannich base intermediate of the reaction. Use of the data in regard to absolute ion intensities as well as future applications are discussed. Recent developments of atmospheric pressure ionization (API) interfaces capable of direct introduction of liquid-phase samples have significantly expanded the applications of mass spectrometry.1 Electrospray ionization (ESI) and related techniques have been particularly important in expanding the applications of mass spectrometry into new areas.2 However, the potential for real-time monitoring of liquid-phase chemical reactions remains largely unexplored. As a particularly soft form of ionization, ESI offers unique capabilities where the study of reaction mechanisms and kinetics is concerned. Previously, the use of an ionspray interface in conjunction with continuous sample introduction was used with a variety of model reactions to display the feasibility of the technique.3 Other recent examples include investigations of liquid-phase electrochemical4 * Corresponding author: (phone) 610-270-7316; (fax) 610-270-7510; (e-mail) philip_C_Dell’
[email protected]. † Analytical Sciences, SmithKline Beecham Pharmaceuticals. ‡ Synthetic Chemistry, SmithKline Beecham Pharmaceuticals. § Villanova University. (1) Niessen, W. M. A.; van der Greef, J. Liquid Chromatography-Mass Spectrometry; Marcel Dekker: New York, 1992. (2) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9 (1), 37-70. (3) Lee, E. D.; Muck, W.; Henion, J. D.; Covey, T. R. J. Am. Chem. Soc. 1989, 51, 4600. (4) Hambitzer, G.; Heitbaum, J. Anal. Chem. 1986, 58, 1067. 10.1021/ac990554o CCC: $18.00 Published on Web 10/12/1999
© 1999 American Chemical Society
and photochemical5 reactions in real time, again with customized interfaces. Recently, membrane introduction mass spectrometry (MIMS) interfaces have been used to quantify reaction rates and reaction equilibria, which have been used to optimize fermentation reactions.6 This technique, however, is limited to compounds with appreciable vapor pressure and favorable permiselectivity.7 Recently, we reported using unmodified API interfaces for the quantitative kinetic analysis of a photochemical reaction.8 In our laboratory we seek a simple and general method for the quantitative interrogation of reaction rates and reaction pathways for a variety of synthetic organic reactions and waste treatment systems. In this paper we describe a successful extension of our previous work by investigating process-scale chemical reactions (e.g., at high molar concentrations) in realtime, using standard interfaces. We have chosen to study a Knoevenagel condensation reaction. The interrogated reaction, between an aldehyde and an acid ester, is currently under evaluation for use in the commercial manufacture of the pharmaceutical eprosartansa nonpeptide angiotensin II receptor antagonist for the treatment of hypertension.9 The approach utilized to monitor this chemical reaction provides serial dilution steps which also quench the reaction removing the problem of dead time. These studies produce a wealth of kinetic and mechanistic insight into the reaction. The potential of a selective mass analyzer with near real-time analysis and minimal intrusiveness presents a powerful tool for the optimization of synthetic organic reactions and waste destruction processes, in addition to providing mechanistic interpretation of reaction paths. EXPERIMENTAL SECTION The experimental apparatus is outlined in Figure 1. The reaction mixture is sampled from a reaction flask using a highprecision, calibrated HPLC pump (Dionex) operating at a minimal flow rate (0.2-0.3 mL/min). A second HPLC pump (Altech) adds ambient temperature methanol (HPLC grade) at 5-10 mL/min through 0.030" i.d. tubing, which dilutes the reaction sample at a 1/16" stainless steel tee; this addition also thermally quenches the reaction. A third HPLC pump (Altech) samples this (5) Arakawa, R.; Lu, J.; Mizuno, K.; Inoue, H.; Doe, H.; Matsuo, T. Int. J. Mass Spectrom. Ion Processes 1997, 160, 371. (6) Hansen, K. F.; Lauritsen, F. R.; Degn, H. Biotechnol. Bioeng. 1994, 44, 347. (7) Cisper, M. E.; Hemberger, P. H. Rapid Commun. Mass Spectrom. 1997, 11, 1449. (8) Brum, J.; Dell′Orco, P. Rapid Commun. Mass Spectrom. 1998, 12, 741. (9) Mcclellan, K. J.; Balfour, J. Drugs 1998, 55 (5), 713.
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Figure 1. Schematic of apparatus for on-line MS interrogation of reaction. Pump no. 1 samples the reaction solution, pump no. 2 provides ambient temperature methanol to quench the reaction and provide primary dilution, pump no. 3 is used to meter solution to a flow splitter, and pump no. 4 adds dilute HCl for solute protonation.
stream at 2 mL/min into a 100:1 splitter (Accurate). A fourth HPLC pump (Hewlett-Packard) adds ionizing solution (0.01 M HCl in 50/50 v/v% H2O and MeOH) through a static mixing tee, at a flow rate of 0.25 mL/min. Through the use of a metering valve, the solution is pumped through the flow splitter at a higher pressure than in the circulating loop, to ensure accurate fluid metering. The configuration displayed in Figure 1 allows the introduction of sample to the mass spectrometer with an approximate delay time of 4-6 min, depending on specific flow conditions employed (the reactor and instrument are in separate rooms, with the reactor placed in a hood). However, since the reaction is quenched by dilution with methanol, this delay does not represent a true “dead” time. The data shown here were obtained using a Hewlett-Packard (Palo Alto, CA) 5989A quadrupole mass spectrometer equipped with an Analytica of Branford hexapole ion guide (Analytica of Branford, Branford, CT). The approach outlined above allows us to use an unmodified ESI interface (Hewlett-Packard 59987A). All data in this study were acquired in positive-ion mode. The exit capillary voltage (ECV) was set at 100 V for all experiments unless stated otherwise. This provided adequate signal with minimal fragmentation of [M + H]+ ions. The reaction was conducted by heating the two reactants in toluene (total volume ) 200 mL) to 70 °C in a 250-mL roundbottom flask equipped with a glycol condenser operating at 5 °C, a calibrated Type K thermocouple in contact with the fluid, a magnetic stir bar, and a bored-through Teflon coated stopper through which a 0.020" i.d. pump sample inlet tube was threaded. Reaction temperature control was achieved by using a controller with proportional, integral and derivative (PID) action, with the fluid thermocouple as the sensing instrument. The PID controller 5166
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(J-Kem) regulated a heating mantle. Temperature profiles were monitored using LabView (National Instruments). When the solution reached 70 °C, sampling of the solution was initiated in order to get a background sample prior to catalyst addition. When the MS signal was stable, the catalyst (piperidine) was added to the solution. Reaction time was monitored using a stopwatch, and time between MS scans was recorded, to correlate scan numbers with reaction times. Reactant concentrations investigated were 0.65 M SB-201999, 0.39 M SB-290161, and 0.125-0.50 M piperidine. RESULTS AND DISCUSSION Observed Reaction Products and Intermediates. Figure 2 displays the primary global reaction path for the piperidinecatalyzed condensation being monitored in these experiments. In addition to the primary reaction path illustrated in Figure 2, the carboxylic acid species also undergoes piperidine-catalyzed decarboxylation and is normally used as an excess reactant. In commercial manufacture, the reaction is carried out between 65 and 70 °C with a Dean-Stark trap for water removal. Figure 3 displays a typical raw data set obtained from monitoring this reaction via on-line mass spectrometry, the inset describing specific reaction conditions. Figure 3 is comprised of extracted ion data of [M + H]+ ions of interest over the time scale of the reaction. The data clearly display the progress of the reaction in the formation of the product diene (SB 290162) indicated by the signal at m/z ) 419 resulting from the condensation of the carboxylic acid (SB201999) and the aldehyde (SB 290161) functionalities. Of all the compounds present in this system, only SB 201999 (without a significant basic site) cannot be readily tracked. One interesting feature in the time-resolved data is the clear point of initiation of the reaction upon addition
Figure 2. Primary global reaction path for the Knoevenagel reaction of the Eprosartan intermediate.
Figure 3. Ion current of major observed species as a function of time. The reactant (m/z ) 253) is observed, as is the product (m/z ) 419), a reactive intermediate (m/z 320), and the catalyst (m/z ) 86). Reaction conditions: 0.63 M SB 201999, 0.38 M SB 290161, and 0.32 M Piperidine, 70 °C reaction temperature.
of 0.50 equivalents (relative to SB 201999) of piperidine (m/z ) 86) occurring at approximately 5 min and indicated by the immediate formation of the feature at m/z ) 320. The formation of the feature at m/z ) 320 occurs simultaneous with a similar drop in the intensity of m/z ) 253 (SB 290161). These observations are consistent with the addition of piperidine to SB 290161, followed by dehydration, to form the transient iminium ion intermediate (m/z ) 320). Figure 4 displays supporting experimental data using piperidine d11 as a reactant with SB 290161. As observed in Figure 4, piperidine d11 forms an adduct with a m/z = 330.2 and a m/z ) 348.2. The m/z of 330.2 is consistent with the iminium ion hypothesis, while the feature at m/z ) 338.2 could be either the hydrated precursor to m/z ) 330.2 or a water adduct. Using particularly soft source conditions (ECV ) 70) with ambient temperature drying gas, the hydrated piperidine addition product at m/z ) 338 is observed, with a corresponding decrease in intensity of m/z ) 320. This leads to speculation that this hydrated precursor is the actual stable solution species. This hydrated precursor is thermally dehydrated by the elevated temperature (200-300 °C) of the drying gas. The species at m/z ) 338 and 320 are expected intermediates in the Knoevenagel mechanism for this reaction. These two species would not be
Figure 4. Confirmation of m/z 320 structure using piperidine d11. The species at m/z 330.2 is believed to be the deuterated analog of the m/z 320 species, shown in Figures 3 and 4, and formed by reaction of piperidine with SB 290161. The species at 348.2 is either the hydrated precursor to m/z 320 or a water adduct.
observed if the Hann and Lapworth mechanism, which produces the same end product but through different intermediates, was involved.10 The single time point mass spectrum shown in Figure 5 demonstrates the wealth of chemical information extracted by this method, and in particular, the advantage that ESI affords in producing predominantly [M + H]+ ions. Figure 5 shows the total mass spectrum for this experiment at time t) 5 min (after piperidine addition). In addition to the ion features discussed above, minor features are observed at m/z ) 153, 405, 505, and 437. The feature at m/z ) 153 is DBU (1,8-diazabicyclo[5,4,0]undec-7-ene) which is a catalyst in solution from the previous reaction stage. The feature at 405 has been attributed to an addition product of DBU to SB 290161, which is analogous to the piperidine addition product. Similarly, we postulate that m/z ) 505 is a dimer of m/z ) 253. The feature at 437 is likely a water adduct of m/z ) 419. Other minor features have not been fully interrogated. The observance of these additional species (which are not all observable using the developed liquid chromatographic method for the reaction) indicates the advantage of this technique over in situ optical techniques. The m/z data is a specific fingerprint for a molecular species, providing data that can be more easily interpreted than absorption spectra. In complex (10) Tanaka, M.; Oota, O.; Hiramatsu, H.; Fujiwara, K. Bull. Chem. Soc. Jpn. 1988, 61, 2473.
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Figure 5. Total ion spectrum at t ) 5 minutes. In addition to the species mentioned in Figure 3, other minor features are observed, which provide additional information about the reaction chemistry. See text for details. Reaction conditions: 0.63 M SB 201999, 0.38 M SB 290161, and 0.32 M Piperidine, 70 °C reaction temperature.
sample matrixes such as this, such spectra often have severe interferences in absorbance regions of interest, requiring complementary methods and chemometric evaluation for hypotheses of existing molecular species. These advantages are somewhat offset by the requirement of intrusive sampling when using on-line MS. The primary assumption in these studies is that all chemical reactions occur primarily in the toluene phase and are not substantially transformed after methanol dilution. We acknowledge the possibility that the relative response of the reactive base intermediate is strongly influenced by the dilution solvent. Furthermore, it is possible that some features are affected by the thermal environment at the electrospray source; we have, for example, already discussed the possibility that m/z ) 320 is really a result of m/z ) 338 dehydration under specific thermal conditions. Reaction Kinetics Information from On-Line Mass Spectrometry. A mechanism consistent with the spectral observations in Figures 3, 4, and 5 is shown in Figure 6. Figure 6 also provides symbolic representation for the different species, and the m/z values of observed molecular intermediates are provided. Figure 6 shows that a plausible reaction path consistent with the Knoevenagel mechanism involves the formation of a piperidinium ion by extraction of the methylidine proton on SB 201999 (species A). This piperidinium ion reacts with SB 290161 to form a hydrated piperidine adduct (m/z ) 338, [BP+]), which, in turn, forms a salt with the deprotonated carboxylic acid [A-]. After dehydration of [BP+] to form iminium ion [L] (m/z ) 320), a single bond forms between the methylidine carbon and the aldehyde carbon [M]. This species loses piperidine and carbon dioxide in a concerted step to form the final product, [N] (m/z ) 419). In this mechanism, all species are presented as ion pairs; this seems a more likely representation than free ions, which are not easily solvated by toluene. A mechanistic kinetic analysis of this reaction is currently being constructed and is beyond the scope of this paper. However, some discussion of the kinetic information provided by the mass spectrometry data is appropriate here. For reaction kinetics, it is 5168
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important to note that most observed species will be difficult to quantify absolutely, as many reactants/products /intermediates will have not even been isolated, rendering calibration impossible. In addition, proton competition can possibly alter responses in concentrated solutions relative to pure standards, further complicating quantitative efforts. This inability to calibrate renders rigorous mechanistic kinetic analyses difficult on the basis of ion current data alone. Nonetheless, on-line data provide a sense of the relative reaction kinetics for a set of data and provide rate of change information that can be used to evaluate selectivities and reasonable estimates of reaction velocity over time. Figure 7 shows data collected using identical reactant concentrations while varying catalyst concentration at 70 °C. As observed in Figure 7, the online monitoring approach is able to discern differences in rates as a result of varying piperidine concentration. Examination of the data indicates that a doubling of the piperidine concentration approximately doubles the initial rate of product appearance, which can be inferred from the slope of temporal ion current data. Different piperidine concentrations also appear to result in different equilibrium concentrations of product. Once again, we note that liquid chromatography peak areas for the reaction product exhibit identical behavior. In Figure 7, the “reactants” are actually a summation of [M + H]+ ion responses for ions m/z ) 253, 320, and 338. Algebraic manipulation of the differential equations suggested by Figure 2 results in the relationship shown in Equation 1.
-d{[BP+] + [L] + [B]} d[N] ) dt dt
(1)
As observed in Figure 7, the rate of change of ion current for all imidazole species is equal to the negative of the rate of ion change of the product. Moreover, the product increases by the same amount of ion current as observed in the reactant decrease. Assuming that ion currents are linearly related to concentrations by a response factor, it is straightforward to show that, in order for the ion current slopes to match in this manner, the response factors for the individual species must be similar. This would indicate that the approach of using a strong proton donor somewhat mitigates pKa differences of individual species, due to both the strength of the proton donor and the excess of available protons. We do not completely discount the possibility that serendipitous values of response factors would give the same relative slope behavior. It is also possible that our projected mechanism may not be an exact representation of reaction events. However, we consider the possibility that response factors are similar to be an equally plausible explanation for the observed behavior. For many reactions a rigorous quantitative kinetic model may not be necessary to achieve goals of high yield and high selectivity. For example, we anticipate that the real-time advantage offered by this method will allow the detection of reaction end-points and reaction selectivities. Observing the reaction end-point is fairly straightforward; a single component can be monitored for its total ion response change. Assuming a linear ion response, as has been observed in this work, a yield can be estimated. Similarly, selectivities can be analyzed. In the reaction presented here, it might be useful to ascertain whether the aldehyde reactant
Figure 6. Suggested elementary pathway for the Knoevenagel condensation, based on mass spectrometry observations.
Figure 7. Disappearance of reactants and appearance of product as a function of time and piperidine concentration. [SB-201999] ) 0.64 M; [SB-290161] ) 0.39 M. Black lines represent reactants; gray represents product. The reactants (m/z ) 253 and m/z ) 320) disappear at the same rate as the product (m/z ) 419) appears, indicating that SB-290161 reacts only to produce product and that ion responses of reactants and products are similar. (a) 0.5 equiv. of piperidine. (b) 0.25 equiv. of piperidine. (c) 0.125 equiv. of piperidine.
undergoes any parallel and undesired reactions. Since selectivities are ratios of reaction rates, which can be inferred by ratios of ion current rate of change, relative selectivities can be observed, which display when impurities grow to an intolerable level. In Figure 7, it is straightforward to observe that the selectivity of reactant ion current to product ion current is approximately 1.0; thus, no undesired impurities are forming from parallel reactions of SB 290161. The data as discussed above show excellent agreement when compared with liquid chromatography area response data. This result occurs after adjustment of the H+ concentration in the ionizing solution such that the equivalent pH in aqueous solution is significantly below the pKa values of the basic sites which are
protonated. Whereas much remains to be learned in regard to ion formation in ES processes, it has been observed that analyte pKa can exhibit a predominant effect in ionization efficiency.11 By using the protic solution described above we essentially saturate all basic sites with protons prior to analysis. In doing so we obtain results from [M + H]+ ion intensities which are correlate at least qualitatively with the concentrations used in the reaction. Within the context of the extensive work of the Kerbarle group on the mechanistic aspects of ESI, we appreciate that the similarities observed in the response factors for the analytes in this reaction may indeed be fortuitous.12 We are currently performing detailed investigations in order to understand the applicability of the Kerbarle model to our complex multicomponent mixtures. We are interested in these questions particularly as they pertain to direct use of absolute ion intensities in kinetic models. Experiments performed in which a similar buffer solution with a pH of 4.5 was used did, in fact, result in data with no direct correlation to experimental concentrations. As expected, ionization efficiency in these data were dominanted by analyte pKa. For the purpose of discussion and analysis of these data we have assumed pKa to be the dominant parameter affecting overall ion response. We recognize this as a first-order approximation. We note these concerns regarding absolute vs relative signal are not unique to this approach and are, in fact, virtually universal to any analysis carried out in real time. This potential control over relative signal intensities by proper choice of buffer solutions, drying gas temperatures, and capillary voltages can be interpreted as a distinct advantage. CONCLUSIONS A Knoevenagel reaction was monitored in real time by direct injection electrospray mass spectrometry. Specifically, the utility of the method presented here is that the chemical reaction was examined at process conditions, rather than at extreme dilutions. The interrogation of the reaction by this method was useful in both the identification of important reaction intermediates and in (11) (a) Yen, T. Y.; Childs, M. J.; Voyksner, R. D. J. Am. Soc. Mass Spectrom. 1996, 7, 1106. (b) Banks, J. F.; Shen, S.; Whitehouse, C. M.; Fenn, J. B. Anal. Chem. 1994, 66, 406. (c) Keenan, R. M. et. al. J. Med. Chem. 1993, 36, 1880. (12) (a) Tang, L.; Kebarle, P. Anal. Chem. 1993, 65, 3654. (b) Kebarle, P.; Tang, L. Anal. Chem. 1993, 65, 972A.
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the identification of kinetic time scales. We found that the use of mineral acids was useful in reducing ion response effects due to proton competition; the use of strong acids at relatively high concentrations seems to lessen pKa effects on ion response. Through future research aimed at understanding specific effects on ion response, it is hoped that this method can be used to interrogate these reactions for actual kinetic parameters. In the
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meantime, it has been demonstrated that simple kinetic information, such as extent of reaction and reaction selectivities, can be inferred simply from ion current data. Received for review May 25, 1999. Accepted September 3, 1999. AC990554O
