Identification and Characterization of Isomeric Intermediates in a

High-throughput metal screening in pharmaceutical samples by ICP-MS with automated flow injection using a modified HPLC configuration. Qiang Tu , Tieb...
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Anal. Chem. 2006, 78, 1282-1289

Identification and Characterization of Isomeric Intermediates in a Catalyst Formation Reaction by Means of Speciation Analysis Using HPLC-ICPMS and HPLC-ESI-MS Qiang Tu,*,† Tiebang Wang,*,† Christopher J. Welch,*,† Peng Wang,† Xiujuan Jia,† Conrad Raab,† Xiaodong Bu,† Darren Bykowski,‡ Benjamin Hohenstaufen,‡ and Michael P. Doyle‡

Merck Research Laboratories, Merck & Co., Inc., P.O. Box 2000, Rahway, New Jersey 07065, and Department of Chemistry & Biochemistry, University of Maryland, College Park, Maryland 20742

Information on chemical speciation is much needed in mechanistic and kinetic studies on catalyst formation processes in pharmaceutical research. Speciation analysis was applied to the identification and quantification of various rhodium species involved in a ligand exchange process leading to formation of catalyst dirhodium(II) tetrakis[methyl 2-oxopyrrolidin-5(S)-carboxylate]. Inductively coupled plasma mass spectrometry (ICPMS) was used as an element-specific detector following species separation by reversed-phase high-performance liquid chromatography (RP-HPLC), and electrospray ionization mass spectrometry (ESI-MS) was used for species identification and confirmation. A novel interface between the HPLC and ICPMS, which consisted of an eluent splitter, a desolvation unit, and the ICPMS built-in peristaltic pump, enabled the use of RP-HPLC with gradient elution and up to 100% organic components in the LC eluent without organic loading in the plasma. A variety of reaction intermediates were identified and quantified along the pathway to formation of the desired product, including isomeric di-, tri-, and tetrasubstituted species previously believed to be absent. This has provided new insights into the mechanism and kinetics of the reaction. The combination of HPLC-ICPMS and HPLC-ESI-MS has proven to be a valuable tool for the investigation of species evolution in catalyst formation process. Catalytic processes have been expanding into diverse areas of pharmaceutical applications and are playing increasingly important roles in the synthesis of pharmaceutical candidates and commercial products. Understanding the mechanism and kinetics of reactions leading to the catalyst formation makes a significant impact on the development of novel synthetic routes for pharmaceutical products. Although the reaction media are normally complex systems consisting of different metal species, studies in this field generally employ conventional tools for organic analysis, such as nuclear magnetic resonance (NMR), ultraviolet/visible * To whom correspondence should be addressed. Tel.: (732) 594-5928. Fax: (732) 594-6645. E-mail: [email protected]. † Merck & Co., Inc. ‡ University of Maryland.

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(UV), infrared spectroscopy, molecular mass spectrometry (MS), etc. These techniques are responsive to many other compounds beyond a particular metal species because none of them is a metalspecific detector. Even where detection and identification of the species is possible, quantification is more challenging as the response from detectors such as UV or MS may differ with different species and suffers from matrix effects. This has to a large extent hampered the investigation of the fine details of reaction chemistry that are crucial for understanding and characterization of the catalyst formation process. Therefore, it can be assumed that a species-specific detection technique will become an invaluable tool for fundamental research in this field. Speciation analysis is undergoing continuous development in many fields, with the majority of applications in environmental and life sciences.1,2 However, to our knowledge, no literature can be found employing this powerful analytical tool to look into individual metal species in metal-containing organic reactions in pharmaceutical process research or other related fields. Speciation analysis normally involves on-line coupling of a selective separation technique, such as liquid chromatography (LC),3,4 gas chromatography,5 capillary electrophoresis (CE),6 and field flow fractionation,7 with an element-specific detector. Among many different separation-detection combinations attempted for speciation analysis, the coupling of LC and inductively coupled plasma mass spectrometry (ICPMS) has emerged as the dominating system.2,4 This is mainly because of the variety of separation mechanisms available from LC, the sensitive and robust element (or isotope)specific detection from ICPMS. This combination allows unambiguous identification and accurate quantitation of the amount of specific metals within each resolved species. (1) Caruso, J. A.; Montes-Bayon, M. Ecotoxicol. Environ. Saf. 2003, 56, 148163. (2) Waddell, R.; Lewis, C.; Hang, W.; Hassell, C.; Majidi, V. Appl. Spectrosc. Rev. 2005, 40, 33-69. (3) Sarzanini, C.; Mentasti, E. J. Chromatogr., A 1997, 789, 301-321. (4) Montes-Bayon, M.; DeNicola, K.; Caruso, J. A. J. Chromatogr., A 2003, 1000, 457-476. (5) Lobinski, R.; Adams, F. C. Spectrochim. Acta, Part B 1997, 52, 1865-1903. (6) Kannamkumarath, S. S.; Wrobel, K.; Wrobel, K.; B’Hymer, C.; Caruso, J. A. J. Chromatogr., A 2002, 975, 245-266. (7) Al-Ammar, A.; Siripinyanond, A.; Barnes, R. M. Spectrochim. Acta, Part B 2001, 56, 1951-1962. 10.1021/ac051679y CCC: $33.50

© 2006 American Chemical Society Published on Web 01/13/2006

The majority of LC-ICPMS applications so far focus on ion exchange and size exclusion chromatography.3,4 Only a few examples of reversed-phase (RP) or normal-phase (NP) highperformance liquid chromatography (HPLC) for speciation analysis of organic compounds have been reported.4,8,9 Although the liquid flow rates used in most LC techniques are comparable to suitable liquid flow rates for direct aspiration of solutions into the ICP, the direct coupling of the two techniques is problematic when large amounts of organic solvents are present in the LC mobile phase. Organic solvents have higher vapor pressures than aqueous solutions, which leads to increased solvent loading in the ICP and in turn results in an unstable or even extinguished plasma. Decreased sensitivity, carbide polyatomic ion interferences, carbon deposit on the ICP sampler and skimmer cones, and the resulting signal drift are some of the other problems related to the use of mobile phases with large organic content. Micronebulizers operated at lower flow rates (e.g., microconcentric, direct injection, or oscillating nebulizers) have been used for the introduction of mobile phases high in organic solvents into the ICP. Microbore or capillary LC columns are often used in combinations with these nebulizers.4,10 Cooled spray chambers and the addition of a small amount of oxygen to the auxiliary gas are also common approaches to reduce the organic loading. A more promising approach for interfacing LC, especially RP/NP-HPLC, with ICPMS is to produce a desolvated aerosol for sample introduction, as long as the element species are not removed with the solvent. Not only is the solvent loading to the plasma minimized with this technique but the sensitivity can also be largely improved owing to increased sample transport efficiency. The use of desolvation apparatus such as a Nafion membrane drier with a cryogenic condenser10 and ultrasonic nebulizer with built-in desolvation system9 has been reported. However, there is still a need for simpler, more robust, and flexible interface for RP/NP-HPLCICPMS applications. As the ICPMS only provides elemental information, speciation by LC-ICPMS is generally achieved by matching the retention times of the samples with those of the standards. When peaks are observed that do not correspond to a standard, however, complimentary information is needed for the identification of unknown species or the confirmation of the actual presence of species in a given sample. This is often attempted by the use of molecular mass spectrometry techniques such as electrospray or atmospheric pressure chemical ionization mass spectrometry (ESIMS or APCI-MS) in an off-line mode or on-line coupling with LC or CE.11,12 However, this effort has so far been used only to a much lesser extent for speciation analysis in environmental and life science research. Insufficient sensitivity, complex spectra, and instability of the species are some of the constraints that need to be carefully addressed in using the technique for practical speciation studies.11-13 (8) Michalke, B. Trends Anal. Chem. 2002, 21, 142-153. (9) Axelsson, B.-O.; Jornten-Karlsson, M.; Michelsen, P.; Abou-Shakra, F. Rapid Comm. Mass Spectrom. 2001, 15, 375-385. (10) Cairns, W. R. L.; Ebdon, L.; Hill, S. J. Fresenius J. Anal. Chem. 1996, 355, 202-208. (11) Schramel, O.; Michalke, B.; Kettrup, A. J. Chromatogr., A 1998, 819, 231242. (12) Rosenberg, E. J. Chromatogr., A 2003, 1000, 841-889. (13) Sharp, B. L.; Sulaiman, A. B.; Taylor, K. A.; Green, B. N. J. Anal. At. Spectrom. 1997, 12, 603-609.

The aim of this research is to bring the resolving power of speciation analysis to pharmaceutical process research. Studies were arranged to explore the feasibility of using HPLC-ICPMS and HPLC-ESI-MS for the study of reaction mechanism and kinetics of a catalyst formation process. Special attention was paid to the identification and quantification of reaction intermediates as well as their possible coexisting structural isomers. We have shown how this approach can lead to a deeper understanding of mechanistic and kinetic aspects of the reaction under study.14 Herein the analytical development of this research is reported. EXPERIMENTAL SECTION Catalyst Formation Reaction. The model catalyst, dirhodium(II) tetrakis[methyl 2-oxopyrrolidin-5(S)-carboxylate], or Rh2(5SMEPY)4, is a dirhodium(II) complex with chiral carboxamidate ligands developed by Doyle and co-workers.15-17 It has shown remarkable capabilities for highly enantioselective catalytic metal carbene transformations, especially enantioselective cyclopropanation and C-H insertion processes involving formation of transient metal carbenoid intermediates, as well as for heteroDiels-Alder reactions.18,19 The target catalyst has a (cis-2,2) configuration, where two oxygens and two nitrogens are bound to each rhodium, with the two nitrogens or two oxygens are oriented cis to one another. The reaction is carried out by treating rhodium acetate with excess chiral ligands methyl pyroglutamate (MEPY) in refluxing chlorobenzene in a Soxhlet extractor. Formation of the catalyst is believed to involve the successive displacement of acetate ligands around a dirhodium core from the rhodium acetate precursor,20-22 as illustrated below.

Theoretically, as many as 15 rhodium species could exist in the course of reaction, which include Rh2(OAc)4, Rh2(OAc)3(MEPY), five isomeric species of Rh2(OAc)2(MEPY)2, four isomeric species of Rh2(OAc)(MEPY)3, and four isomeric species of (14) Welch, C. J.; Tu, Q.; Wang, T.; Raab, C.; Wang, P.; Jia, X.; Bu, X.; Bykowski, D.; Hohenstaufen, B.; Doyle, M. P. Submitted to Angew. Chem., Int. Ed. (15) Doyle, M. P.; McKervey, M. A.; Ye, T. Modern Catalytic Methods for Organic Synthesis with Diazo Compounds; John Wiley & Sons: New York, 1998. (16) Doyle, M. P. In Chiral Dirhodium(II) Carboxamidates for Catalytic Asymmetric Synthesis, in New Methodologies in Asymmetric Catalysis; Malhotra, S., Ed.; Oxford University Press: Oxford, England, 2004. (17) Timmons, D.; Doyle, M. P. In Chiral Dirhodium(II) Catalysts and their Applications, in Catalysis by Di- and Polynuclear Metal Clusters, 3rd ed.; Cotton, F. A., Murillo, C. A., Walton, R. A., Eds.; Springer Science: New York, 2005. (18) Doyle, M. P.; Phillips, I. M.; Hu, W. J. Am. Chem. Soc. 2001, 123, 53665367. (19) Doyle, M. P.; Valenzuela, M.; Huang, P. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 5391-5395. (20) Doyle, M. P.; Winchester, W. R.; Hoorn, J. A. A.; Lynch, V.; Simonsen, S. H.; Ghosh, R. J. Am. Chem. Soc. 1993, 115, 9968-9978. (21) Doyle, M. P.; Winchester, W. R.; Protopopova, M. N.; Kazala, A. P.; Westrum, L. J. Org. Synth. 1996, 73, 13-24. (22) Doyle, M. P.; Raab, C. E.; Roos, G. H. P.; Lynch, V.; Simonsen, S. H. Inorg. Chim. Acta 1997, 266, 13-18.

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Figure 1. Schematic diagram of the interface to couple HPLC to ICPMS.

Rh2(MEPY)4. More detailed information about the reaction can be found elsewhere.15-22 Sample Collection and Preparation. Heat was applied to start the reaction, and the samples were collected by removing aliquots from the reaction mixture every 6 min within the first hour, every 10 min within the second hour, and every 20 min until the fifth hour. The samples were immediately cooled to room temperature, evaporated to near dryness at or below 40 °C to avoid further ligand exchange, and then transferred to the analytical laboratory over a period of a few days. Before analysis, the samples were dissolved in 2 mL of acetonitrile. To ensure the stability of the species, semi-real-time samples were collected by carrying out the synthesis reaction within the research facility where the analysis was performed. The samples were collected every 40 min within 5 h, immediately diluted with methanol, and subjected to analysis. The results from these samples were compared to those obtained from using the more “aged” samples prepared elsewhere. For the HPLC-ICPMS test, preliminary determination of the total Rh concentrations of the samples was made by flow injection using the HPLC autosampler followed by ICPMS determination. The samples were further diluted with methanol to a total rhodium concentration of ∼20 ng mL-1 before being injected into the HPLC. Recoveries of the species were determined by comparing the total Rh concentrations of the samples with the summation of individual species after separation. A sample collected at 80 min after the start of the reaction was used for the HPLC-ESIMS test. For this test, the sample was diluted two times with methanol and had a total Rh concentration of 150 µg mL-1. Instrumentation. An 1100 HPLC system (Agilent Technologies, Wilmington, DE) was coupled to an Agilent 7500cs ICPMS through a custom-built interface. The ICPMS provided elementselective detection of rhodium (m/z 103). The HPLC system included a binary pump, a vacuum degasser, a thermostated autosampler, a thermostated column compartment, and a diode array detector. The interface consisted of a PEEK Tee (0.50 mm, Upchurch Scientific, Oak Harbor, WA) serving as an eluent splitter, an Aridus desolvation sample introduction system (CETAC Technologies, Omaha, NE), and the ICPMS built-in peristaltic pump (see Figure 1). A portion of the eluent from the LC outlet was nebulized into a heated PFA spray chamber using a PFA microconcentric nebulizer and transported to a heated microporous PTFE tubular membrane. Solvent vapor passed through the membrane and was removed by a stream of Ar gas, while the 1284

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analyte continued through the membrane tube to the ICPMS. Chromatographic data analysis was made by using the Agilent Plasma Chromatographic software. The instrument settings were checked daily and optimized when necessary. For HPLC-ESI-MS, an Agilent 1100 series HPLC system equipped with a photodiode array UV-visible detector was coupled to a Finnigan LCQ (San Jose, CA) mass spectrometer with an ESI source for LC-MS analysis. ESI positive mode was used for MS detection with full scan from m/z 100 to 2000. Helium was used as damping gas in the ion trap and as sheath and auxiliary gas. The spray voltage was tuned to 5 kV with sheath and auxiliary gas at 80 and 60 units, respectively. The heated capillary temperature was set at 250 °C. Xcalibur software was used for instrumental control, data acquisition, and data analysis. The injection volume for the analysis was set at 10 µL. The rhodium species involved in the reaction were separated by RP-HPLC with a Cadenza C18 column (Silvertone Sciences, Philadelphia, PA). For HPLC-ICPMS, the mobile phases consisted of 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in 50:50 (v/v) acetonitrile/methanol. Gradient elution was employed to resolve all the species. The same chromatographic conditions were applied to the HPLC-ESI-MS analysis. In addition, mobile phases with the addition of 0.05% formic acid instead of 0.1% TFA were also employed in the HPLC-ESI-MS analysis, and a better sensitivity was achieved. The gradient elution was the same as that of the HPLC-ICPMS experiment. Operating conditions of all the instrumentation are summarized in Table 1. Reagents. A standard of dirhodium tetraacetate (99.99+%) was obtained from Sigma-Aldrich (Milwaukee, WI). Dirhodium(II) tetrakis[methyl 2-oxopyrrolidin-5(S)-carboxylate] was acquired from Fisher Scientific (Pittsburgh, PA). Methanol and acetonitrile, LC-MS Chromasolv grade, were also supplied by Sigma-Aldrich (Seelze, Germany). TFA, p.a. grade, was obtained from Fluka (Steinheim, Germany). A 1000 µg mL-1 Rh stock standard solution used for total Rh determinations was purchased from High-Purity Standards (Charleston, SC). The water used in the experiments was prepared by passing distilled water through a Hydro Ultrapure water system (Hydro Service and Supplies, Garfield, NJ). Detailed information about the reaction conditions leading to the formation of Rh2(5S-MEPY)4 can be found elsewhere.15-20

Table 1. Operating Parameters for HPLC, Aridus, ICPMS, and ESI-MS column mobile phase

flow rate injection volume

HPLC Conditions Cadenza CD-C18 (250 × 4.6 mm, 3 µm), 40 °C for ICPMS and ESI-MS: (A) H2O + 0.1% TFA (B) methanol-acetonitrile (50:50 v/v) + 0.1% TFA also for ESI-MS: (A) H2O + 0.05% formic acid (B) methanol-acetonitrile (50:50 v/v) + 0.05% formic acid gradient: 20% B 0-12 min, 25% B 13-16 min, 30% B 17-30 min 1.0 mL min-1 2 µL for ICPMS, 10 µL for ESI-MS

Aridus Conditions sweep gas flow setting N2 gas flow carrier gas flow spray chamber temp desolvator temp

7.2 10 mL min-1 0.88 L min-1 110 °C 160 °C

ICPMS Conditions rf power 1100 W plasma gas flow 14.9 L min-1 auxiliary gas flow 0.90 L min-1 sampling depth 13 mm peristaltic pump speed 0.22 rps acquisition mode time-resolved analysis, m/z 103 integration time 1.00 s ESI-MS Conditions spray voltage sheath gas flow auxiliary gas flow capillary voltage capillary temp tube lens offset

5 kV 80 unit 60 unit 18 V 250 °C -5 V

RESULTS AND DISCUSSION HPLC-ICPMS Interface. As discussed earlier, the organic solvents present in the LC mobile phase would increase solvent loading in the ICP. Micronebulizers, microbore or capillary LC columns, cooled spray chambers, and oxygen addition are some of the approaches used in order to reduce this effect.4,10 When these techniques are used for RP/NP-HPLC with gradient elution, however, the sensitivity of the ICPMS may change with the eluent composition over a chromatographic run. This may result in difficulties in calibration and quantification. The use of a desolvation apparatus may be a better approach for interfacing RP/ NP-HPLC with ICPMS. As the sample is introduced into the ICPMS as a desolvated aerosol, problems related to organic solvent loading can be minimized. The HPLC-ICPMS interface we developed is much simpler and highly flexible compared to those reported in the literature9,10 and is ideally suitable for the coupling of RP/NP-HPLC with ICPMS. Eluent from the LC outlet reaches a Tee, which serves as a splitter, before it is connected to the peristaltic pump tubing on the ICPMS. The split ratio is determined by the size of the tubings used and by the speed of the peristaltic pump, and a fixed split ratio can be obtained in continuous chromatographic runs

regardless of the change of the composition in the mobile phase. By adjusting the peristaltic pump speed and the carrier gas flow rate while monitoring reflected power of the ICP, an appropriate split ratio for a specific LC eluent can be selected such that a good sensitivity is attained while the membrane desolvator is not overloaded. The plasma and the ion lens settings required major reoptimization when the membrane desolvator was used for the first time, because the optimized parameters were largely different from those used for conventional wet sample aerosols. The addition of a small amount of N2 to the carrier gas also resulted in significant enhancement of sensitivity. The mechanism of this effect was unclear. Under optimized conditions, the sensitivities of 7Li+, 89Y+, and 205Tl+ used for tuning were enhanced by as much as 2 orders of magnitude compared with conventional pneumatic nebulization. The addition of a small amount of oxygen to the auxiliary gas is a common approach for a solvent desolvator, as is also recommended by CETAC. This was found necessary when the nebulizer was operated in self-aspiration mode. The developed interface enabled the handling of organic solvents without the addition of oxygen, thanks to the use of a splitter by which the organic loading in the plasma was minimized. No visible carbon buildup was observed after prolonged use of the system. Running without oxygen addition was advantageous because problems such as the increase of the oxygen-containing polyatomic ion interferences and accelerated degradation of the sampler and skimmer cones could be avoided. Only a slight increase in the baseline was experienced with the use of solvent gradients. When the peristaltic pump was operated at its maximum speed, an HPLC flow rate of up to 2 mL min-1 with 100% methanol or acetonitrile in the mobile phase could be applied to the system without affecting the stability or the performance of the plasma. No carryover or loss of analyte was observed with the desolvation unit by comparing results with the conventional sample introduction system. Optimization of Separation Conditions. RP-HPLC with the Cadenza CD-C18 column enabled the differentiation of species including the starting material, intermediates, product, and their coexisting structural isomers. The LC flow rate was kept at 1 mL min-1 throughout the experiments. Neither methanol-water nor acetonitrile-water mixtures as mobile phase resulted in satisfactory resolution of all the peaks, especially for the isomers. Consequently, mixtures of methanol-acetonitrile with various ratios were tested as the organic component of the mobile phase for separation. The best separation was achieved when the methanol-acetonitrile mixture had a ratio of 50:50 (v/v) and was used in combination with water as the mobile phase. The addition of 0.1% TFA to both solutions resulted in improved peak resolution, and thus, it was used as a modifier throughout this study. The separation was also influenced by the sample preparation method. Peak fronting was observed when the samples were diluted in acetonitrile. In comparison, sharp and symmetric peaks were obtained for samples diluted in methanol. Therefore, all the samples were diluted in methanol before the LC separation. A slight change of the organic component in the mobile phase resulted in a significant variation of the peak resolution. For the species involved in the reaction leading to Rh2(MEPY)4 formation, gradient elution was found necessary to separate the species with Analytical Chemistry, Vol. 78, No. 4, February 15, 2006

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Figure 2. Comparison of HPLC-ICPMS chromatogram (A) and total ion chromatogram of HPLC-ESI-MS (B) for a sample collected at 80 min after the start of reaction. Sample concentration (as Rh): 150 µg mL-1 for (A) and 20 ng mL-1 for (B). Operating conditions can be found in Table 1. A-to L refer to the respective species listed in Table 2. Table 2. Number of Theoretically Possible Species and Identified Species through Successive Displacement of Acetate Ligands from Rhodium Acetate (Rh2(OAC)4) by Enantiopure Methyl Pyroglutamate (MEPY) Ligands en Route to Dirhodium(II) Carboxamidate Compounds (Rh2(MEPY)4) compound

number of possible isomers

species identified (Figures 2, 3, 5)

Rh2(OAC)4 Rh2(OAC)3(MEPY) Rh2(OAC)2(MEPY)2 Rh2(OAC)(MEPY)3 Rh2(MEPY)4

1 1 5 4 4

A B C, D, E F, G, H I, J, K, L

the organic component increased from 20 to 30% in 25 min. After careful optimization of reversed-phase HPLC conditions, 12 out of 15 theoretically possible Rh species were separated and then detected by ICPMS (Figure 2A and Table 2). The undetected two Rh2(OAc)2(MEPY)2 isomers and one Rh2(OAc)(MEPY)3 isomer may be either absent from the reaction or present but unresolved by the present method. Species Identification/Confirmation. Unlike speciation analysis in environmental research where many standards of the species of interest are available, standards of the intermediates and different structural isomers of the same metal species in the catalyst formation reactions rarely exist. Therefore, the common 1286 Analytical Chemistry, Vol. 78, No. 4, February 15, 2006

approach of comparing LC retention times of unknown peaks with those of the standards is often not feasible. In this case, the only commercially available standards are the starting material Rh2(OAc)4 and the desired product Rh2(5S-MEPY)4 in the (cis-2,2) configuration. Comparing peak retention times in the HPLCICPMS chromatograms in Figure 2A with those of the two standards, peaks A and K can be identified as Rh2(OAc)4 and Rh2(5S-MEPY)4, respectively. For the identification or confirmation of other species, HPLC-ESI-MS became a compulsory part of the entire analysis scheme in providing molecular mass information of the species. As mentioned previously, it is believed that Rh2(5S-MEPY)4 is formed through successive displacement of the acetate ligand from Rh2(OAc)4.20-22 As a result, many reaction intermediates with various degrees of substitutions could be present. Considering the possible formation of isomers, there might be as many as 15 Rh species present in the sample (Table 2). A sample collected 80 min after the reaction was used for ESI-MS analysis since this fraction was found to be one of those that contained the maximum number of species of interest. Similar HPLC conditions were applied for separation prior to the structural identification of each resolved peak by ESI-MS. Both 0.1% TFA and 0.05% formic acid were tested as modifiers in the mobile phases for ESI-MS. Though similar total ion chromatograms (TICs) were observed in both cases, the mobile phases containing 0.05% formic acid resulted in much better sensitivity. The obtained TIC is illustrated in Figure 2B. A comparison of chromatograms shown in Figure 2 demonstrated unique advantages of using HPLC-ICPMS for this application. First, there was a huge difference in sensitivity with ICPMS and ESI-MS detection, considering the difference in sample concentration was 7500 times. Second, Figure 2A presented a clear picture of the number of Rh-containing species in the sample as a result of element-specific detection by ICPMS. By comparison, the TIC from ESI-MS was rather complex. Finally, ICPMS detection enabled accurate quantitation of individual species, while quantitation by ESI-MS was very difficult, especially without a standard of each species. Some possible effects that may have impact on ESI-MS detection include matrix-related ion suppression or enhancement, generation of new species in the ionization-extraction process, and sample carryover. Although the TIC from ESI-MS analysis showed limited capability of providing species identification for this application, extracted ion chromatograms (EICs) could be used as an easy and explicit way to map out the distributions of all Rh-containing species detected by HPLC-ICPMS. The M + 1 ions for each sought-for species [m/z 526 for Rh2(OAc)3(MEPY), m/z 609 for Rh2(OAc)2(MEPY)2, m/z 692 for Rh2(OAc)(MEPY)3, and m/z 775 for Rh2(MEPY)4] were monitored. For Rh2(OAc)4, however, the M + 1 ion had a very weak signal, and the dominating ion with a m/z of 865 (which could be attributed to the dimer ion of this molecule with the loss of an acetate ligand) was therefore used to monitor Rh2(OAc)4 in the EIC. As shown in Figure 3, the EICs clearly demonstrate the distribution of each species and their corresponding isomers in a single chromatographic run. Comparing with the EICs, all the Rh-containing species illustrated in the HPLC-ICPMS chromatogram (Figure 2A) can be easily identified. The single peak in Figure 3 with a m/z of 865 corresponding to

Figure 3. Extracted ion chromatograms for the same sample as in Figure 2 by HPLC-ESI-MS. Operating conditions can be found in Table 1. Species identified are listed in Table 2.

peak A in Figure 2A can be attributed to Rh2(OAc)4, and the dominating peak with a m/z of 526 corresponding to peak B can be attributed to Rh2(OAc)3(MEPY). The three major peaks with a single m/z of 609 in Figure 3 corresponding to peaks C-E can be assigned to be the three structural isomers of Rh2(OAc)2(MEPY)2 with confidence. Similarly, peaks F-H can be identified as the three isomers of Rh2(OAc)(MEPY)3 with a m/z of 692, and the tetrasubstituted product was found as four structural isomers (peaks I-L) with a m/z of 775. It can also be noted that different species had significantly different sensitivity by ESI-MS detection when compared with

ICPMS. Even the isomers might show a large disparity in sensitivity (e.g., EIC at m/z 692). This probably indicated that each species/isomer was sensitive to the operating conditions employed in the HPLC-ESI-MS analysis. A summary of the identified species can also be found in Table 2. The assignment of exact structure of each of the isomers can be further examined by other techniques, e.g., NMR, since they have the same m/z using ESI-MS detection. Analytical Figures of Merit. The detection limits were calculated as three times the standard deviation (n ) 10) of the integrated peak areas obtained with a mixed standard solution of Analytical Chemistry, Vol. 78, No. 4, February 15, 2006

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Figure 4. HPLC-ICPMS chromatograms for samples collected at different time frames, showing the evolution of species during the course of the reaction.

Figure 5. Change of species as percentage of the total Rh content over the course of reaction leading to formation of Rh2(5S-MEPY)4. A-L refer to the respective species listed in Table 2.

Rh2(OAc)4 and Rh2(5S-MEPY)4 (100 pg mL-1 as Rh for each species). An injection volume of 10 µL was used for this experiment, although larger volumes can be used to increase the detection limit if necessary. The detection limits (as Rh) were calculated to be 11 pg mL-1 for Rh2(OAc)4 and 19 pg mL-1 for Rh2(5S-MEPY)4, which corresponded to an absolute detection limit of 0.11 and 0.19 pg, respectively. Repeatability was determined by 10 replicative 10-µL injections of a mixed standard solution containing both Rh2(OAc)4 and Rh2(5S-MEPY)4 (10 ng mL-1 as Rh for each species). The relative standard deviations (RSDs) of the peak area for the two species 1288 Analytical Chemistry, Vol. 78, No. 4, February 15, 2006

were 3.7 and 3.6%, and the RSDs of the retention times were 0.1 and 0.2%, respectively. The average recovery calculated from three collected samples was 94%, indicating that there were nearly no species left in the HPLC column. Reaction Mechanism and Kinetics. HPLC-ICPMS chromatograms obtained for various time-course aliquots are illustrated in Figure 4, which vividly depict the species evolution during the course of the reaction. For the first time, the starting material, the mono-, di-, and trisubstituted intermediates as well as the tetrasubstituted product along with their isomers can not only be

identified but also be quantified. This has made it possible to study the fine details of reaction mechanism as well as kinetics of ligand exchange. Thanks to the excellent quantitation capability of the ICPMS detector, the change in concentration for each species over time can be easily determined and is illustrated in Figure 5. The established presence of mono, di-, tri-, and tetrasubstituted products confirms the mechanism of this reaction as ligand exchange as proposed in the literature.15-21 Whereas, previous work suggested that the third and fourth ligand substitution would be much slower than the first two,18-21 the current evidence from this study suggests that there is no significant rate differences in proceeding from the first through the fourth ligand exchange. Moreover, prior to this investigation, the (cis-2,2) isomer was the only known species in the literature;15-22 however, all four Rh2(MEPY)4 isomers were detected and identified in the current study, suggesting the possible isomerization of this catalyst compound under the reaction conditions used. The capability of being able to monitor the changes of various product isomers over time is of great value in catalyst forming reactions, since the reaction conditions for maximum yield of the desired product can thus be easily achieved and optimized. The current research also demonstrates the need of a purification step in order to acquire a desired single isomeric product because other isomeric products may have different enantioselectivity properties. More detailed discussion on the new understanding of mechanism and kinetics of the reaction as a result of this analytical endeavor can be found elsewhere.14 The stability of the species over time was also evaluated by comparing the HPLC-ICPMS chromatograms obtained from analyzing freshly prepared samples and samples aged for up to one month. No significant differences were observed for samples from the two experiments. The ligand exchange reaction was shown to have been “stopped” or extremely slowed when heat was removed. Being able to “freeze” these reactions in a certain time frame makes it easier to study the species evolution and reaction mechanism. This also confirmed the stability of these

species when exposed to oxygen as reported previously,18-21 and the interconversion of the species over the period of the study was negligible. CONCLUSION In summary, the combination of HPLC-ICPMS with HPLCESI-MS has been shown to be a powerful tool in separation, identification, and quantitation of various rhodium species during the course of the model reaction leading to the formation of Rh2(5S-MEPY)4. It is especially useful for identifying and quantifying intermediate species including presumed isomeric materials with great selectivity and sensitivity, thus providing insight into the fine details of the mechanism and kinetics of the catalyst formation. These technical developments will surely contribute to a deeper understanding of catalyst synthesis in pharmaceutical process research and other related fields. Care must be exercised when employing the same analytical tool to the study of other reactions. The use of HPLC-ICPMS and HPLC-ESI-MS for the study of other metal-containing reaction intermediates might be less straightforward than the present example, with potential pitfalls such as reactivity with solvent or oxygen, or fast ligand exchange on the “HPLC time scale” being easily imagined. Nevertheless, simple modifications such as oxygen exclusion, the use of nonreactive eluents, and the use of fast chromatography or low-temperature chromatography may possibly be suitable for expanding the utility of the approach. ACKNOWLEDGMENT The authors express their gratitude to Ivan Santos, Vincent Antonucci, and Zhihong Ge of Merck & Co., Inc. for their helpful review of the manuscript.

Received for review September 20, 2005. Accepted December 15, 2005. AC051679Y

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