Original Paper. The role of UHPLC in pharmaceutical development. i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. 1 Introduction

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1 J. Sep. Sci. 2007, 30 S. M. Chesnut et al. Stephen M. Chesnut John J. Salisbury Analytical Research and Development, Pfizer Global Research and Development, Groton/New London Laboratories, Groton, CT, USA Original Paper The role of UHPLC in pharmaceutical development Pharmaceutical separations can be divided into three categories: high throughput, high productivity, and high resolution. These categories contain specific pharmaceutical applications, each of which has distinct separation goals. Traditionally, these goals have been achieved utilizing conventional HPLC with typical column dimensions and particle sizes. The recent introduction of ultra-hplc (UHPLC) has provided a new potential for method development and analysis. Pharmaceutical chemists must determine the impact of this emerging technology. UHPLC is achieved by using sub-2 lm particle size column packing at increased linear velocities. In order to utilize this technology, mobile phase viscosity must be minimized or the chromatography system must be redesigned to withstand an increased backpressure. Today, there are many commercially available UHPLC systems capable of exceeding conventional pressure limits of 400 bar. The advantage of UHPLC over conventional HPLC is the capability to increase the speed without sacrificing efficiency. In comparison to traditional HPLC, our research showed that UHPLC can decrease run times up to 76. In addition, for high resolution applications, UHPLC achieved significant efficiency advantages over traditional HPLC. This paper will evaluate the potential roles for utilizing UHPLC in the pharmaceutical industry. Keywords: Acquity UPLC TM / HPLC / Pharmaceutical / Sub-2 lm / Ultrahigh performance / Received: December 5, 2006; revised: March 2, 2007; accepted: March 5, 2007 DOI /jssc Introduction Correspondence: John J. Salisbury, Analytical Research and Development, Groton/New London Laboratories, Pfizer Inc., Eastern Point Road, MS 4015, Groton, CT 06340, USA Fax: john.j.salisbury@pfizer.com Abbreviation: UHPLC, ultra-hplc Chromatographic methods in the pharmaceutical laboratory fall into three categories: high throughput, high productivity, and high resolution (maximum efficiency; adopted from Pfizer Analytical Research Center (PARC), University of Ghent, Belgium). Each category has different requirements that define the goals of the separation. These requirements are defined by the intended use of the method, phase of development, and customer needs. The goals of the separation include run time, efficiency, and resolution. In pharmaceutical development, HPLC is the most widely used technique for determining the assay and impurity profile of drug substances and dosage forms. It is a versatile technique with a wide range of applications and is compatible with numerous detection techniques. Recently, there has been a trend to improve the quality of the chromatographic separation through the utilization of columns with smaller diameter particles. Initially, this required the use of shorter columns due to the pressure limitations of conventional HPLC equipment. This trend led to decreased analysis time with minimal improvement in chromatographic efficiency. More recently, sub-2 lm particle size packed columns with typical column lengths have been used to increase the chromatographic efficiency and the decrease run time [1]. This required improved chromatographic systems engineered to accommodate the increased backpressures. The result was a new tool for the analytical chemist, known as ultrahigh performance (or pressure) LC. Analytical chemists need to determine what the role of ultra-hplc (UHPLC) will be in pharmaceutical laboratories. As this technology is phased into development, it may become the preferred approach to fast LC method development. It may be able to decrease the analysis time or increase the resolution of critical pairs. UHPLC has proven capable of passing typical system suitability and validation criteria, but the transferability to customers in quality control laboratories remains largely unknown [2 4]. This paper will compare this new technology to traditional HPLC and evaluate the advantages and disadvantages for chemists developing analytical methods for pharmaceutical compounds.

2 S. M. Chesnut et al. J. Sep. Sci. 2007, 30 2 Experimental 2.1 Chemicals All the chemicals were of ACS reagent grade or HPLC grade. ACN was purchased from JT Baker. Milli-Q (Mosheim, France) purified water (A10 mx, a99 ppb TOC) was used throughout the analyses. Chemical Research and Development, Pfizer, Groton, CT, USA provided samples of active pharmaceutical ingredient (API) and impurities. Impurities consisted of the diastereomer of the bulk material, impurities with strong polar differences, and structurally similar impurities to the bulk material. Sample mixtures were prepared at concentrations of 0.6 mg/ml for the main component with relevant impurities spiked at 0.1%. 2.2 van Deemter plots Comparisons of peak efficiencies as a function of linear velocity were evaluated on an Acquity UPLC TM and 2695 Separations Module (Waters Corporation, Milford, MA, USA). The range of linear velocities for the HPLC was mm/s while the linear velocity range was mm/s for the UHPLC. The following columns were analyzed: Acquity UPLC BEH C18, 100 mm62.1 mm, 1.7 lm (Waters Corporation), XBridge C18, 150 mm64.6 mm, 5 lm (Waters Corporation), and XBridge C18, 150 mm64.6 mm, 3.5 lm (Waters Corporation). The analyte was a 0.6 mg/ml solution of a Pfizer proprietary compound (Pfizer) that eluted with a retention factor of approximately 8 in a mobile phase consisting of 30% water and 70% ACN. Efficiencies were calculated based on the USP tangent plate calculations. Table 1 lists the applicable equations for the van Deemter analysis. 2.3 UHPLC-UV and HPLC-UV system conditions All the chromatographic separations were performed using an Acquity UPLC system (Waters Corporation) and a 2695 Separations Module (Waters Corporation). In each case, mobile phase A was deionized water using a Millipore system and mobile phase B was ACN (JT Baker, HPLC grade). For each experiment, the column heater was maintained at 358C. The output signal was monitored and processed by Empower chromatography manager software (Waters Corporation). A mobile phase of 30:70 water/acn was utilized for all the isocratic runs. Water was obtained using a Millipore system and ACN was purchased from JT Baker (HPLC grade). For conventional HPLC, a 10 ll injection of the 0.6 mg/ml sample mixture was injected using a flow rate of 1.0 ml/min on an XBridge C mm64.6 mm id, 3.5 lm column. Data were collected at 210 nm at the rate Table 1. van Deemter calculation and variable definitions Calculation V M = 0.5 L c d c 2 t 0 = V M /F l = L/t 0 Term definitions V M = volume of mobile phase in column (mm 3 ) [5] L c = length of column (mm) d c = internal diameter of column (mm) t 0 = column dead time (min) F = flow rate (mm 3 /min) l = velocity of the mobile phase (mm/s) of 2 Hz. Three isocratic runs were performed on the Acquity UPLC. The first utilized a 1 ll injection of the 0.6 mg/ml sample mixture injected at a flow rate of 0.6 ml/min. The second used a 1.4 ll injection of the 0.6 mg/ml sample injected at a flow rate of 0.2 ml/min. The third used a 1.4 ll injection of the 0.6 mg/ml sample injected at a flow rate of 0.2 ml/min. In each case, the data were collected at 210 nm at a rate of 20 Hz. The first experiment utilized an Acquity UPLC BEH C mm62.1 mm id, 1.7 lm column. The second experiment utilized an Acquity UPLC BEH C mm62.1 mm id, 1.7 lm column. The third used three Acquity UPLC BEH C mm62.1 mm id, 1.7 lm columns in series. For all gradient analyses, the mobile phase A was water and the mobile phase B was ACN. For conventional HPLC, a 10 ll injection of the 0.2 mg/ml sample mixture was injected using a flow rate of 1.0 ml/min on an XBridge C mm64.6 mm id, 3.5 lm column. The gradient program utilized an initial hold of 5 min minus the dwell of the system at 30:70 water/acn followed by a linear ramp to 10:90 water/acn over 20 min. The dwell volume of the 2695 Separations Module utilized was measured to be 1.1 ml. For the Acquity UPLC a 1 ll injection of the 0.6 mg/ml sample mixture was injected using a flow rate of 0.6 ml/min on an Acquity UPLC BEH C mm6100 mm id, 1.7 lm column. The gradient program used an initial hold of 0.5 min at 30:70 water/acn followed by a linear ramp to 10:90 water/acn over 2.8 min. In each case, the data were collected at 210 nm at a rate of 20 Hz. The dwell volume of the Acquity UPLC utilized was measured to be 0.2 ml. The gradient programs utilized by the UPLC were reduced to keep the percent column volume change constant in order to maintain the selectivity. 3 Results and discussion For any chromatographic separation, it is important to first determine an essential peak set or key predictive sample set (KPSS) [6]. This is the set of samples that most accurately represents the impurity profile of the pharmaceutical compound and may include precursors from the

3 J. Sep. Sci. 2007, 30 Liquid Chromatography Figure 1. H uplots obtained for a Pfizer proprietary compound on Acquity and XBridge columns. Columns: Acquity UPLC BEH C18, 1.7 lm, 100 mm62.1 mm id; XBridge C18, 3.5 lm, 150 mm64.6 mm id; XBridge C18, 5 lm, 150 mm64.6 mm id. synthetic route, process-related impurities, and potential degradation products. Typically, initial method development for a late stage compound would focus on screening these samples using a variety of chromatographic parameters on a conventional HPLC. Over the last several years, ultrahigh pressure LC has been introduced to overcome the pressure limitations that small particles impose on conventional pumping systems [7, 8]. As a result, chromatographers in the pharmaceutical industry now have a new alternative to the traditional approach to the method development. After the goal of the separation is defined, a suitable technique (UHPLC or HPLC) can be chosen, based on the appropriate situation. The goal of the separation can be defined, as high throughput, high productivity, or high resolution. The conventional separation method might be a compromise between all the three factors, but to simplify this discussion, each factor will be discussed separately. In each case, the application helps drive the desired efficiency and analysis time for the separation. It is important to recognize the fundamental understanding of efficiency with respect to a given column length and particle size. Using the theoretical equation for efficiency, the plate number (N) can be calculated as per Eq. (1), where L is the column length and d p the particle diameter. N ¼ L 2d p ð1þ Using this calculation, the desired column length and particle size can be determined for a given efficiency. For example, if a particular separation requires theoretical plates, then one can either choose a 250 mm length column with 5 lm particle size or a 150 mm length column with 3 lm particle size. Both columns yield equivalent efficiency yet using the 150 mm length column with 3 lm particle size can reduce analysis time. Since the maximum plates allowed for these columns are 25000, it is clear that you cannot achieve a separation greater than plates, which is required by most of the high resolution applications. But if you were to connect two of these columns in series, the generation of theoretical plates can be achieved. Graphically, the benefits of UHPLC can be represented from the van Deemter curves in Fig. 1 for columns consisting of 5, 3.5, and 1.7 lm particle diameters. The relative flat nature of the 1.7 lm column from approximately 1.5 to 6 mm/s is clearly evident. By running separations at higher linear velocities on sub-2 lm columns, run times can be dramatically reduced without sacrificing efficiency [9]. To understand the effect of chromatographic separations utilizing sub-2 lm columns at increased linear velocities, it is essential to understand the fundamental van Deemter equation and subsequent plots. Though a complete discussion of the van Deemter curve is not presented in this analysis, thorough reviews can be found in the literature [10 12]. 3.1 High throughput applications High throughput applications are defined as any separation where a reduced run time is the most important factor driving the chromatography. In this case, resolution is not the major criterion, but for pharmaceutical applications, a resolution of at least 1.5 for all the components from the main band is still required. Some applications include dissolution tests, main band assays, reaction completions, and in process controls. These applications generally require less than 5000 plates and run times less than 5 min. Conventional HPLC will typically work for these types of applications unless there is a critical pair which includes the main band. In these cases, UHPLC may be required to maintain both resolution of the critical pair and achieve run times less than 5 min.

4 S. M. Chesnut et al. J. Sep. Sci. 2007, 30 Figure 2. Comparison of isocratic elutions utilizing conventional HPLC and Acquity UPLC. (A) Conventional HPLC isocratic 30:70 water/acn, 1.0 ml/min, 10 ll injection, XBridge C mm64.6 mm id, 3.5 lm, pressure = 1900 psi. (B) Acquity UPLC isocratic 30:70 water/acn, 0.6 ml/min, 1 ll injection, Acquity UPLC BEH C mm62.1 mm id, 1.7 lm, pressure = psi. (C) Acquity UPLC isocratic 30:70 water/acn, 0.2 ml/min, 1.4 ll injection, Acquity UPLC BEH C mm62.1 mm id, 1.7 lm, pressure = 4944 psi. Yang found that by using UHPLC to optimize a quality control assay, both higher sample analysis throughput and better assay sensitivity were observed [3]. 3.2 High productivity applications The majority of pharmaceutical analyses tend to fall in the high productivity category. These applications generally require decent efficiency ( plates), resolution of at least 1.5 between all the components and run times less than 30 min. Currently, for run times less than 30 min conventional HPLC typically achieves plate numbers in the range. Therefore, for high productivity separations, the utilization of ultrahigh pressure LC may be the best approach. Our work shows that for a difficult separation run times of 5 min can generate approximately plates utilizing this technology. Currently, conventional HPLC does not typically

5 J. Sep. Sci. 2007, 30 Liquid Chromatography Table 2. Summary of data for isocratic optimization from HPLC to UHPLC for high productivity applications Conventional HPLC Acquity UPLC (run time) Acquity UPLC (efficiency) Run time (min) Theoretical plates (Peak C) Resolution (Peaks C and D) Injection load (lg) Linear velocity (mm/s) achieve these results. As a result, UHPLC is the preferred technique for high productivity applications. Optimization of current HPLC methods to high productivity methods utilizing UHPLC can be accomplished through several routes. One approach is to reduce the particle size and column length while increasing the linear velocity. The result is a dramatically reduced run time without sacrificing efficiency. For the purpose of this discussion, we will call this approach as high productivity (run time). Based on our research, the run time of a typical isocratic elution was reduced by 56 without further method development. This is clearly observed in Fig. 2B. For this sample matrix, there are several impurities spiked in at a 0.1% level relative to the main band. A summary of these results can be found in Table 2. This type of separation would be considered as high productivity due to its efficiency and run time. It is important to note that these optimizations are quickly accomplished by geometric scaling of the flow rate and injection volume through an Excel spreadsheet or computer program. A second approach to optimizing a conventional HPLC method to UHPLC is to reduce the particle size while keeping both the column length and linear velocity constant. Through this approach, a gain in efficiency should be observed while maintaining run time. We will call this approach as high productivity (efficiency) and the results are summarized in Table 2 and a chromatogram can be found in Fig. 2C. Looking at Table 2, our research showed an approximately 30% increase in the efficiency compared to the conventional HPLC method with equivalent run time. As in the high productivity (run time) approach, this optimization was achieved through simple geometric scaling without further method development. The advantages of gradient elution must be considered for any separation that contains compounds with a wide range of polarities or wide retention range. The development of gradient methods with conventional HPLC can be complex and often requires time-consuming gradient screens with computer-assisted development. Molnar discusses the general strategy for computer-supported gradient method development in DryLab [13]. It is important to recognize that the geometric scaling of conventional gradient methods to UHPLC methods is more cumbersome than isocratic scaling. As in the geometric scaling of isocratic methods to UHPLC, the flow rate and injection volume must be scaled for gradient methods. Further the gradient segments must be scaled. This can be performed by expressing the gradient duration in percent change per column volume units, which needs to be calculated for each step of the gradient. Therefore, the user can calculate the time required to deliver the same number of column volumes to the UPLC column at a given flow rate. Fortunately, software programs that perform these calculations are readily available by UHPLC vendors. When converting conventional methods to UHPLC methods, if the percent change per column volume is not maintained then selectivity changes may occur. However, if the percent change per column volume is held constant, it is possible to capitalize on UHPLC columns and dramatically speed up the separation while maintaining selectivity. This can be observed in Fig. 3. A conventional HPLC method was converted to a UHPLC method by increasing the linear velocity and keeping the percent change per column volume constant. The run time was reduced from 35 to 5 min without significant selectivity differences. System dwell volume can have a significant effect on transferring gradient methods to UHPLC. Conventional HPLC systems have typical dwell volumes of 1000 ll or more. The Acquity UPLC dwell volume is marketed at 109 ll when a standard mixer is used [11]. The Acquity UPLC used for this analysis utilized a column switcher component and was found to have a system dwell close to 200 ll. Because of these low dwell volumes compared to conventional HPLCs, selectivity can change when transferring a chromatographic method from conventional instrumentation to the UHPLC. It is therefore recommended that when developing any gradient separation that the gradient program allows for a system dwell correction. This can typically be performed by putting an initial isocratic hold that corrects for such differences. 3.3 High resolution applications Any separation where resolution is of the utmost importance can be deemed a high resolution (or efficiency) application. In these cases, run time can be sacrificed

6 S. M. Chesnut et al. J. Sep. Sci. 2007, 30 Figure 3. Comparison of gradient elutions utilizing conventional HPLC and Acquity UPLC. (A) Conventional HPLC gradient: initial 30:70 water/acn, final: 10:90 water/acn, 1.0 ml/min, 10 ll injection, XBridge C mm64.6 mm id, 3.5 lm, pressure = 1900 psi (B) Acquity UPLC gradient, initial: 30:70 water/ ACN, final: 10:90 water/acn, 0.6 ml/min, 1 ll injection, Acquity UPLC BEH 150 mm62.1 mm id, 1.7 lm, pressure = psi. since peak capacity is the most important factor. Such applications may include early development screenings, forced degradation analysis, and low-level impurity quantitation among others. For these separations, efficiency of greater than theoretical plates may be desired. Based on Eq. (1), high theoretical plates can be obtained in LC by increasing the column length and/or reducing the particle size. In both cases, there are limitations. The limiting factor for increasing column length is run time whereas the limiting factor for reducing the particle size is the large pressure increase. This is due to the pressure being inversely proportional to the square of the particle size. It is important to understand that either HPLC or UHPLC may be used for these applications. Due to the pressure limitations of small particles, recent work has suggested that one approach is to couple conventional columns at higher temperatures on conventional HPLC. Lestremau et al. [14] have shown that coupling eight 25 cm64.6 mm id columns packed with 5 lm particles at 808C generated effective plates at only 400 bar. The limiting factor in this approach is run time. In order for Lestremau to achieve this separation, a run time of greater than 300 min was required. This is a viable approach for separations which require a high efficiency, but are limited to conventional instrumentation. More recently, our group investigated the use of UHPLC to achieve excellent separation (A plates) in a reasonable time frame (40 min). Early analysis shows the capability of achieving greater than plates by using three 2.1 mm6100 mm, 1.7 lm columns in series without the use of high temperature. A comparison of both the high productivity separation (Fig. 2) and high resolution separation (Fig. 4) is summarized in Table 3. In this chromatogram, the run time increased from 5 to 40 min but the resolution doubled and the theoretical plates increased by For pharmaceutical applicai 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

7 J. Sep. Sci. 2007, 30 Liquid Chromatography Figure 4. Example of high resolution application using Acquity UPLC isocratic 30:70 water/acn, 0.2 ml/min, 1 ll injection, 35C, Acquity UPLC BEH C mm62.1 mm id, 1.7 lm, pressure = 9820 psi. Table 3. Comparison of high productivity and high resolution applications tions that require high resolution, UHPLC may be the best compromise to achieve high resolution separations in run times that are less than 60 min. 4 Concluding remarks High productivity High resolution Column dimensions 2.1 mm6100 mm 2.1 mm6300 mm Run time (min) 4 40 Theoretical plates (Peak C) Resolution (Peaks C and D) Linear velocity (mm/s) It is clear that UHPLC has a role in pharmaceutical analysis, and is an emerging technology. UHPLC can improve the traditional pharmaceutical method development and optimization through the utilization of sub-2 lm column particle sizes at high linear velocities. It can be used in a variety of pharmaceutical separations, notably, applications concerning high throughput, high productivity and high resolution. It will decrease the run time and the solvent usage, while maintaining or increasing the efficiency and resolution. In addition, conversions of conventional HPLC methods to UHPLC methods can be done quickly without a further method development but separate instrumentation is required. The Acquity UPLC costs approximately 25% more than a conventional HPLC. Yet, the benefits of solvent consumption and waste disposal charges may offset this cost increase. Further, as the number of commercially available column chemistries increase, UHPLC will become a common tool in all the method development laboratories. More importantly, as users become comfortable with this tool, chromatographic methods will begin to be transferred to manufacturing and contract facilities. MS is an additional important tool utilized by the pharmaceutical industry. Though not discussed in this paper, the sensitivity of MS can be significantly enhanced by UHPLC. By using smaller diameter particles and smaller id columns (i. e., 2.1 mm), reduced chromatographic dispersions occur at lower flow rates which result in increased source ionization efficiencies because flow splitting is not necessary. There are several applications found in the literature. For rapid throughput analysis of amphetamine substances in toxicological studies, UHPLC-MS achieved a separation of 5 11 peaks in 1.75 min [15]. UHPLC-MS/IPC was successfully applied for rapid quantitative analyses of bromine-containing preservatives [16]. For the analysis of peptides and proteins, UHPLC increased the resolution and achieved higher peak capacity, which are of particular importance to these applications [17]. UHPLC-MS/MS was successfully used to quantitate low-level impurities in pharmaceutical drug substances [18]. Finally, Plumb et al. [19] investigated the use of UHPLC-MS for the analysis of metabolites. For all the cases, it is important to achieve high data collection rates with the MS to be able to detect the narrow peaks achieved by the UHPLC. 5 References [1] Wu, N., Lippert, J. S., Lee, L., J. Chromatogr. A 2001, 911, [2] Wren, S., Tchelitcheff, P., J. Chromatogr. A 2006, 1119, [3] Yang, Y., Hodges, C., LCGC North America 2005, Suppl., [4] Jerkovich, A. D., LoBrutto, R., Vivilechhia, R. V., LCGC North America 2005, Suppl., [5] Snyder, L. R., Kirkland, J. J., Glajch, J. L., Practical HPLC Method Development, Wiley, New York 1997.

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