Article pubs.acs.org/OPRD
Heart-Cutting Two-Dimensional Ultrahigh-Pressure Liquid Chromatography for Process Development: Asymmetric Reaction Monitoring Shengli Ma,*,† Nelu Grinberg,† Nizar Haddad,† Sonia Rodriguez,† Carl A. Busacca,† Keith Fandrick,† Heewon Lee,† Jinhua J. Song,† Nathan Yee,† Dhileepkumar Krishnamurthy,† Chris H. Senanayake,† Jing Wang,‡ Jeff Trenck,‡ Shaun Mendonsa,‡ Peter R. Claise,§ Roger J. Gilman,§ and Thomas H. Evers§ †
Department of Chemical Development, ‡Analytical Development, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877, United States § Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757-3696, United States S Supporting Information *
ABSTRACT: This contribution presents the first application of two-dimensional, ultrahigh-pressure liquid chromatography (2D-UHPLC) for monitoring asymmetric reactions in process development. Several asymmetric transformations were studied to illustrate the operation of the instrument and evaluate the performance of 2D-UHPLC. Two-dimensional UHPLC is particularly advantageous because it allows a simultaneous analysis of the reaction conversion and its enantiomeric excess. By employing UHPLC the analysis time can be reduced significantly, and the achiral−chiral 2D coupling approach allows for direct injection of the reaction mixture. This study demonstrates the utility of 2D-UHPLC in asymmetric transformations for drug development.
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INTRODUCTION Process analytical chemistry (PAC) is the application of analytical methodologies to the monitoring and control of industrial chemical processes. The information provided by PAC can be used for reaction monitoring, and for control and optimization of chemical processes in term of capacity, quality, cost, consistency, and waste reduction.1 The tasks of process development are becoming more important due to the fact that the synthesis should be economical, with high yield and efficiency. However, these requirements are complicated by the fact that the synthetic process involves screening hundreds of reaction conditions. In addition, such reactions involve complex mixtures of compounds with diverse structures. For this reason, process analytical technology (PAT) has been growing in importance by expanding the possibilities offered for in-process monitoring.2,3 As such, high-throughput screening (HTS) and experiments have drastically improved the speed and productivity of process research. In particular, a large number of compounds can be quickly synthesized and screened with automation technology.4 One essential requirement for effective HTS is the development of fast and efficient analytical methods. In this respect, high performance liquid chromatography (HPLC) in general, and ultrahigh-pressure liquid chromatography (UHPLC) in particular, are very important tools in process development due to their versatility in separating, identifying, and quantifying the end product. In addition, these techniques permit reaction screening of hundreds of analyses on the complex reaction mixture. Asymmetric synthesis is becoming the method of choice to produce enantiomerically pure drugs.5 The determination of conversion and enantiomeric excess (ee) for asymmetric transformations is a very important task for reaction screening. © 2013 American Chemical Society
Accurate determination of ee usually requires laborious and time-consuming analytical method development. For instance, when the analyte is present in complex samples containing other chiral and achiral compounds, these compounds can coelute with the enantiomeric compounds of interest. HPLC with chiral stationary phases has been the most extensively used method for separating the chiral compounds from the reaction mixture. Generally, monitoring such reactions implies two types of chromatography along with two instruments: one to monitor the reaction and another to monitor the enantiomeric excess. However, ee determination of the reaction mixture still can be problematic due to interference from the crude sample. Thus complex mixtures of compounds with diverse structures cannot be analyzed directly. From this point of view HPLC in a heart-cutting twodimensional (2D) arrangement can be advantageous for rapid monitoring of the organic reactions. Multidimensional HPLC has become increasingly popular in recent years.6−8 Due to its inherent high capacity, efficient ananlysis of drugs, metabolites, hormones, and other compounds in complex environments has been achieved using this technique.9−19 The heart-cutting 2DHPLC, in which only a part of the first dimension run is “heartcut” and introduced into the second dimension, is a very suitable technique for the analysis of complex samples. As such, analysis of asymmetric reactions could be solved by heartcutting 2D HPLC, where two columns are linked via a switching valve in a manner that the fractions of the desired enantiomers flowing through the first column (e.g., a reversed phase C18 column) can be directed into a second column (an enantioselective one) in which further enantiomeric resolution Received: September 21, 2012 Published: March 31, 2013 806
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Figure 1. Schematic diagram of 2D UHPLC setup. (a) In this diagram the blue fluid is running the initial separation. Then when the analytes of interest elute off the column, the valve switches. (b) With the valve position switched, analytes of interest are eluted off the first column and are trapped on the head of the second column.
Figure 2. Reaction scheme of starting material (s.m.) 1 and product 2 and corresponding chromatograms with and without heart-cutting. (a) Chromatogram of starting material 1 and product 2 on the first dimension without heart-cutting; (b) heart-cutting chromatogram of starting material 1 and product 2: fraction of reaction product peak sent to the second dimension; (c) chiral separation of product 2 on the second dimension.
can be obtained. Further, with the introduction of the first commercially available UHPLC instrumentation in 2006, UHPLC has emerged as the method of choice for HTS.20 The implementation of UHPLC with small particles (e.g., sub2-μm) has provided significant improvement in efficiency while shortening the analysis time. By combining the advantages of 2D-HPLC and UHPLC, the 2D-UHPLC technique provides high separation potential, reproducibility, robustness, and the possibility of monitoring high-throughput synthesis. In addition, this technique also provides orthogonal selectivity, as two different columns (e.g., achiral and chiral) are used, whereas only a portion of the crude sample is introduced into the chiral column (second column). Thus, it avoids sample pretreatment, maintains the second column’s performance for a longer duration, and shortens the sample analysis time while providing accurate determination of ee. In this contribution we report the development of a heartcutting, two-dimensional UHPLC method for monitoring asymmetric reactions in process development. Asymmetric hydrogenation/transformation is the cornerstone to obtaining high enantioselectivity in synthesis.21 Much progress has been
made in obtaining low catalyst loading and high enantioselectivity and yield during process development.22 To achieve such goals, numerous reactions with different conditions needed to be screened. As such, an efficient analytical method for accurate ee determination is critical. To evaluate the practicality and feasibility of 2D UHPLC for reaction monitoring, three of the most frequently used asymmetric hydrogenation/transformations were demonstrated as examples. These are the reduction of an olefin, a ketone, and an unsaturated ester. Furthermore, we also include the asymmetric transformation of phosphine borane as an example. Reaction monitoring of these processes applied different chromatographic modes in two-dimensional, heart-cutting configurations: UHPLC−UHPLC using a reversed phase−reversed phase mode in each dimension, and reversed phase−normal phase mode in the second dimension. These approaches provide a fast, direct, and easy way for reaction monitoring and enabled the simultaneous determination of both the reaction conversion and the enantiomeric excess. 807
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RESULTS AND DISCUSSION The design of the two-dimensional (2D) UHPLC system incorporating switching valves is illustrated in Figure 1a,b). The sample is first subjected to a reversed phase separation on a reversed phase column (e.g., C18) in the first dimension, where the product, starting material (s.m.), and impurities are separated. In this process, the fluid runs through the valve switch, the first column, and then the detector. The system behaves the same as a traditional 1D UHPLC (without cutting). Once the retention time of the peak of interest is determined, a second run of the sample is performed with the valve position switched at the retention time of the peak of interest. During the second run, analytes of interest are redirected from the first column to the second column by switching the valve position. Once the analytes of interest are transferred, the valve position switches back to its original starting position. The system can control the switch valve position precisely in a 1/100-min time frame (0.6 s), allowing small and quantifiable amounts of mobile phase from the first column into the second column. It will be shown later that such precise control is important for separation on the second column. The separations on the first dimension (d1) and the second dimension (d2) were first developed independently and then combined and tuned together. To demonstrate the feasibility and versatility of 2D UHPLC, four reactions were monitored under different chromatographic conditions. It needs to be pointed out that fast and efficient separations were achieved for all four reactions. High flow rate combined with a sub-2 μm UHPLC column was applied on d1, and chiral columns with 3μm particle size were applied on d2. Under these conditions, utilization of the UHPLC system is necessary, as the initial pressure on the column is above 10,000 psi. This 2D UHPLC system is displayed in Figure S1 in Supporting Information [SI]. 1. Reversed Phase × Reversed Phase (chiral mobile phase). The development of the asymmetric hydrogenation of allylamine derivatives is a very important reaction for the synthesis of chiral drug molecules. In this case, chiral amine 2 was synthesized from hydrogenation of starting material 1 using a variety of bisphosphine ligands (Figure 2). To screen the reaction, the reaction mixtures were first introduced into reversed phase (RP) chromatography. An HSS C18 1.7 μm column was chosen, with a chaotropic reagent (HClO4) as mobile phase additive, and assembled into a UHPLC system.23 The separation of the starting material and product is obtained within 1.5 min, as shown in Figure 2a (without cutting). Enantiomeric separation of chiral amine 2 was previously reported using sulfated cyclodextrin as a chiral mobile phase on a fused core particle column.24 Here we applied the same conditions on d2. In Figure 2b, a small part of the product peak from d1 was cut and introduced into d2; it can be seen that the center part of the product peak is missing (noted by dotted line). The enantiomeric separation was thus obtained on d2, as shown in Figure 2c. The two enantiomers of chiral amine 1 were well resolved within 6 min. It should be mentioned that such separations are comparable to the results obtained on d2 independently (a comparison is illustrated in Figure S2 in the SI). This method provides an effective means for simultaneously screening conversion and enantioselectivity of the reaction. In addition, as only the product peak is introduced into d2, it gives the advantage of eliminating the reaction
matrix. For instance, using d2 alone, the starting material is partially overlapped with the first enantiomer peak. Thus, accurate ee determination cannot be obtained on the reaction mixture by using only d2 in UHPLC due to the interference from the starting material in the reaction mixture. To avoid such interference, a tedious sample preparation is usually applied to remove the starting material from the reaction mixture. However, by using the 2D heart-cutting UHPLC method, the reaction mixtures can be diluted with MeOH (to avoid detector overload) and then used for analysis by 2D heart-cutting UHPLC without any other treatment. 2. Reversed phase × Reversed Phase (chiral stationary phase). Most enantiomeric separation using HPLC is achieved by using chiral stationary phases (CSPs). In this regard, we show here an example of the separation of enantiomers on d2 using CSPs under reversed phase conditions. Asymmetric reduction of ketones is a routine technology for small- to industrial-scale production of optically active compounds, including pharmaceuticals, agrochemicals, and perfumes.25,26 Analysis of the reaction mixtures often requires two chromatographic methods to determine conversion and selectivity. In this case, however, 2D UHPLC was used to achieve fast screening of reaction conditions. Figure 3a
Figure 3. Reaction scheme of starting material (s.m.) 3 and product 4 and corresponding chromatograms with and without heart-cutting. (a) Chromatogram of starting material 3 and product 4 on d1 without heart-cutting; (b) heart-cutting chromatogram of starting material 3 and product 4: fraction of product 4 peak sent to d2; (c) chiral separation of product 2 on d2. Detailed reaction conditions and structure of the ligand can be found in reference 25.
presents the chromatogram of starting material 3 and product 4 separated on d1. Under conditions similar to those for separation of compound 1, 3 and 4 were well separated within 1.5 min, where the conversion of the reaction is obtained. Product 4 was cut (Figure 3b) and introduced into d2, where an OJ-RH chiral stationary phase column was used. The two enantiomers of the product were baseline resolved within 10 min (Figure 3c). With the benefit of 2D UHPLC analytical methods, the optimal reaction condition was obtained in a timely manner with excellent yield. As a result, enantiomerically pure 2-phenethylalcohol (ee: >95%) was obtained through catalyzed transfer hydrogenation. 3. Reversed Phase (RP) × Normal Phase (NP). To further demonstrate the feasibility of 2D UHPLC for process screening, two different reactions monitored by the RP × NP setup are shown. In both cases, a chiral stationary phase (CSP) 808
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was applied on d2 under NP conditions. It should be noted that a large number of enantiomeric separations of pharmaceutical molecules have been achieved under NP conditions using CSPs. Combining RP with NP separation into 2D UHPLC is challenging due to the fact that the stronger eluting solvent in RP becomes the weaker eluting solvent in NP. Such a phenomenon may further cause solvent mismatching with distorted peaks in d2. To avoid these issues, we applied a short and precise cut of the required peak in d1. Due to the precise control of the valve switch on UHPLC, the cut in d1 can be obtained within 0.6 s, resulting in a very small aliquot of eluent (10 μL) injected onto the d2 stream. In Figure 4, the
Figure 5. Chromatograms of starting material 7 and product 8 with and without heart-cutting. (a) Chromatogram of starting material 7 and product 8 on d1 without heart-cutting; (b) heart-cutting chromatogram of starting material 7 and product 8; (c) chiral separation of product 8 on d2. Details about reaction conditions and structure of ligand can be found in reference 28.
preparation. The reaction conversion and ee % can be determined simultaneously for different reactions on different stationary phases and mobile phase conditions. The combined methodologies of heart-cutting method and UHPLC instrumentation thus provide a powerful tool for reaction monitoring of asymmetric transformations.
Figure 4. Reaction scheme s.m. 5 and product 6 and corresponding chromatograms with and without heart-cutting. (a) Chromatogram of s.m. 5 and product 6 on d1 without heart-cutting; (b) heart-cutting chromatogram of starting material 5 and product 6; (c) chiral separation of product 6 on d2. Details about reaction conditions and structure of ligand can be found in reference 27.
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chromatogram for the synthesis of ethyl 3-phenylbutanoate is presented. In the first dimension, the separation of starting material 5 and product 6 is obtained under 1.5 min. (Figure 4a). Further, a cutting of 10 μL of the product peak was introduced to d2 (Figure 4b). A baseline separation of the two enantiomers was achieved within 7 min on a 3-μm particle size cellulose benzoate column (OJ-3) with heptane/isopropanol as the mobile phase (Figure 4c). Another RP × NP 2D UHPLC is shown in Figure 5, where the synthesis of phosphine borane was screened for the best conversion.28 In this case, a 3-μm amylose carbamate column (AD-3) was used for d2 with heptane/isopropanol as the mobile phase. The separation of starting material 7, some unknown impurities, and product 8 is achieved within 1.5 min (Figure 5a). A small part (10 μL) of the peak of product 8 is cut and subjected to d2 (Figure 5b). The enantiomeric separation of product 8’s enantiomers is achieved on AD-3 CSPs (Figure 5c). This 2D UHPLC method provides a fast and effective method for simultaneous screening of reaction conversion and enantioselectivity.
EXPERIMENTAL SECTION
2D-UHPLC Instrument Setup. All 2D UHPLC measurements were performed on a 2D Waters Acquity UPLC system (Waters, Milford, MA) equipped with heart-cutting capability. The system consists of two sets of binary pumps (15000 psi), one photodiode array detector (DAD) (996), one UV variable wavelength detector, and an autosampler. The system is equipped with a column compartment. A HSS C18 50 mm × 4.6 mm column (Waters, Milford, MA) was used in combination with a chiral column. The volumes of the syringe and loop of the system were 250 and 200 μL, respectively. Mixtures of methanol−water (50/50 v/v) were used as needle wash solvent. The 2D-heart cutting was achieved using six-port, two-position switching valves (Figure 1). The system was controlled by MassLynx software (Waters, Milford, MA). HPLC Method. All HPLC methods for reaction monitoring (achiral) were performed on a Waters Acquity HSS C18 column with dimensions of 2.1 mm × 50 mm and 1.8-μm particle size. The method was kept the same for all the compounds in the first dimension: the flow was 1.00 mL/min; the mobile phase A was 0.1% H3PO4 and 0.05% HClO4 in water; the mobile phase B was acetonitrile; a linear gradient was used as 85−5% A (0−1.5 min) and then 5% A (1.5−2.1 min). The re-equilibration time was 0.5 min between injections. The columns on the second dimension were selected according to the corresponding product structure, and the details are shown in SI.
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CONCLUSIONS This report has described the application of the 2D UHPLC method for the reaction monitoring of asymmetric transformations. The combination of achiral and chiral separation provided the required orthogonality for the separation using a 2D system. As such, 2D UHPLC provided a fast and efficient analysis for complex matrices, with less effort for sample 809
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(27) Lipshutz, B. H.; Frieman, B. A. Angew. Chem., Int. Ed. 2005, 44, 6345−6348. (28) Busacca, C. A.; Farber, E.; DeYoung, J.; Campbell, S.; Gonnella, N. C.; Grinberg, N.; Haddad, N.; Lee, H.; Ma, S.; Reeves, D.; Shen, S.; Senanayake, C. H. Org. Lett. 2009, 11, 5594−5597.
ASSOCIATED CONTENT
S Supporting Information *
This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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