Crystallization Monitoring by Raman Spectroscopy: Simultaneous

Oct 16, 2004 - Figure 10 Comparison of FBRM and Raman spectroscopy for detecting nucleation: (a) comparison of solution concentration estimated from R...
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Ind. Eng. Chem. Res. 2005, 44, 1233-1240

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Crystallization Monitoring by Raman Spectroscopy: Simultaneous Measurement of Desupersaturation Profile and Polymorphic Form in Flufenamic Acid Systems Yuerong Hu,† Jessica K. Liang,‡ Allan S. Myerson,‡ and Lynne S. Taylor*,† Department of Industrial and Physical Pharmacy, School of Pharmacy, Purdue University, West Lafayette, Indiana 47907, and Department of Chemical and Environmental Engineering, Illinois Institute of Technology, Chicago, Illinois 60616

In this study, the application of Raman spectroscopy to quantitative in-line monitoring of the crystallization from solution of an enantiotropic polymorphic system, flufenamic acid, is explored. Separate feasibility studies were performed to evaluate the utility of Raman spectroscopy to quantitatively monitor changes in solution concentration and to measure polymorphic composition. The crystallization of flufenamic acid was then monitored as a function of temperature and time using an immersion probe coupled to a Raman spectrometer. The spectral data contained information about both the solution phase and the crystallized solids. Using standard data analysis techniques, solution and solids information could be deconvoluted. Changes in solute concentration as a function of time were quantified from the Raman data and showed good agreement with results from other techniques used for corroboration. The polymorphic forms initially crystallizing from solution, in the presence and absence of seeds, could be clearly identified. Furthermore, the kinetics of the conversion of the metastable form to the stable form could be readily followed. This study illustrates the complex nature of the crystallization process, the ability of Raman spectroscopy to monitor this process, and the potential for this technique to aid in process optimization and control. Introduction Most pharmaceutical manufacturing processes involve batch crystallization of the active pharmaceutical ingredient (API) from solution as a process of purification, final crystal form selection, and/or particle size control. Crystallization is an extremely complex process, particularly when multiple crystal forms (polymorphs) can be produced, as is common with pharmaceutical materials. Crystallization process conditions in batch systems such as the rate and method of supersaturation generation (through cooling, antisolvent addition, reaction, or evaporation), temperature range, solvent system, and agitation rate can influence the crystal size distribution, crystal shape, and crystal form (polymorph) obtained. The physical properties of the drug substance can have a profound effect on the manufacturing of the final dosage form and ultimately the therapeutic efficacy. With the aim of improving the understanding of pharmaceutical manufacturing processes and better controlling product quality, the U.S. Food and Drug Administration (FDA) has launched an initiative to encourage the pharmaceutical industry to implement process analytical technologies (PAT).1 The scientific concept of PAT was introduced by Callis et al. as process analytical chemistry in 1987.2 With the advance of computer technology, optronics, and improvements in instrument portability, various analytical techniques * To whom correspondence should be addressed. E-mail: [email protected]. Tel.: 765 496 6614. Fax: 765 494 6545. † Purdue University. ‡ Illinois Institute of Technology.

have demonstrated utility for on-line or in-line monitoring of different manufacturing processes.3,4 Raman spectroscopy is one such technique.5 Raman spectroscopy has many advantages including that (1) no special sample preparation is required,6 (2) the technique is noninvasive,7 and (3) it is relatively insensitive to aqueous media.8 Another highly desirable characteristic of Raman spectroscopy is that it can be used for remote detection through fiber-optic coupling of sampling probes, facilitating the in-line monitoring of chemical reactions and crystallization processes whereby data are generated in real time by direct analysis of the process. Raman spectroscopy is a well-established technique for quantitative analysis. It has been used for the qualitative monitoring of chemical reactions in solution, and chemometrics has been used to follow reaction progress.9,10 Within the pharmaceutical arena, Raman spectroscopy has been applied extensively for the characterization of polymorphic systems. Szelagiewicz et al. combined Raman spectroscopy with thermal techniques to identify and characterize a new polymorphic form of lufenuron,11 and Taylor and Zografi used Raman spectroscopy to quantitatively analyze the crystallinity of indomethacin systems.12 Auer et al. used FT-Raman spectroscopy to investigate the polymorphic forms present in a number of commercial pharmaceutical products.13 Mixtures of polymorphic forms in the product were also quantatively determined. Some of the disadvantages of Raman spectroscopy include problems with fluorescence for some compounds and the relatively low sensitivity of the technique. Currently, in-line monitoring of crystallization with Raman spectroscopy is still in a relatively early stage. Schwartz14 et al. have described the use of Raman

10.1021/ie049745u CCC: $30.25 © 2005 American Chemical Society Published on Web 10/16/2004

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Figure 1. Molecular structure of flufenamic acid.

spectroscopy to monitor in situ lysozyme concentration changes in hanging drop crystallizations. Wang et al. have used in-line Raman spectroscopy to quantitatively determine the polymorphic transformation of progesterone in a slurry.8 Other polymorphic systems have also been measured in situ using Raman spectroscopy and the polymorphic conversions quantified from peak position shifts.15,16 The ability to monitor concentration during batch crystallization allows for the determination of the desupersaturation curve and calculation of the mass of solid produced at any point in the process. The ability to monitor the crystal form present and its transformation during crystallization is crucial in understanding and controlling polymorphism during the crystallization process. To the best of our knowledge, there have been no reports describing the simultaneous monitoring of solution concentration and polymorphic identity using Raman spectroscopy. The aim of this study is to investigate the utility of this technique for in-line monitoring of solute concentration as a function of time in conjunction with obtaining information about the polymorphic outcome of the crystallization event and any subsequent solvent-mediated transformations. Flufenamic acid (FFA), a potent antiinflammatory drug, was used as the model compound (Figure 1). It has been reported that FFA has up to seven different crystal forms,17 although forms III and I are the most commonly encountered. Form III is the stable form at room temperature, and forms III and I are enantiotropic, with a transition temperature of 42 °C.17 Materials and Methods Materials. FFA was purchased from Aldrich (Milwaukee, WI). Pure FFA form III was obtained by dissolving an appropriate amount of FFA (as received) in toluene at about 80 °C and crash-cooling to 0 °C with vigorous stirring. FFA form I crystals were acquired by storing dry form III crystals in an oven overnight at 115 °C18 and then slowly cooling them to room temperature. The polymorphic purity of form I was greater than 97%, and that of form III was greater than 99% as determined by X-ray powder diffraction. All FFA samples were sieved, and only particles smaller than 177 µm were used. Absolute ethanol (Aaper Alcohol and Chemical Company, Shelbyville, KY), analytical-grade toluene (Mallinckrodt Chemical Inc, Paris, KY), and doubly distilled water were used as solvents. Sodium dodecyl sulfate (SDS, 95%) was purchased from Mallinckrodt Chemical Inc. Instrumentation. Raman spectra were collected using a Raman Rxn1 System from Kaiser Optical System, Inc. (Ann Arbor, MI) equipped with a 450-mW external cavity stabilized diode laser operating at 785

nm. Backscattered radiation was collected from the sample via a 1/4-in. immersion probe sealed with a sapphire window coupled to the spectrometer with a fiber-optic cable. The power at the sample was approximately 100 mW, and the focal point was approximately 100 µm from the window. As a safety measure, a mechanical shutter was activated to prevent light from entering the probe until the measurement was initiated, at which point the probe was optically isolated in the reactor vessel. The acquisition conditions were optimized so that a spectrum was captured with an exposure time of 5 s and 10 accumulations unless otherwise stated. Chord length distributions of the FFA crystals were obtained in situ using a Lasentec (Redmond, WA) focused beam reflectance measurement (FBRM) S400 probe with 5-s measurement duration for unseeded crystallizations and a 15-s measurement duration for seeded experiments. Lasentec FBRM Data Review was used to analyze the data. Crystallization experiments were carried out in a Mettler Toledo (Millersville, MD) Multimax Multiple Reactor system. A 50-mL round-bottom glass reaction vessel was used, and 30 mL of solution was added to the vessel for all experiments. The temperature in the reactor was controlled to an accuracy of 0.1 °C by manipulating the set-point temperature of the heating coils in the MultiMax reactor box, and cooling was provided by a cooling coil connected to a Neslab circulating bath. Absolute ethanol was used as the refrigerant, and the temperature of the ethanol was maintained at 0 °C. A glass PT100 probe was placed in the reactor through a vertical opening parallel to the impeller shaft and used to monitor the solution temperature. A 45° pitched-blade impeller was used to maintain good homogeneity of particles. The Raman and FBRM probes were inserted into the vessel through separate 45° angled ports in the glass cover such that each probe tip was pointing at the impeller blades (a few millimeters above the blade tips) at 45° to ensure good mixing around the probe window. At the end of each experiment, the probes were visually examined for signs of crystallized material. No crystallization was observed to occur on the probes. The concentration of FFA was determined at λmax ) 288 nm with a UV/visible spectrometer19 (DU 7400, Beckman, Irvine, CA), and an X-ray powder diffractometer (D500, Siemens, Germany) was used to identify the polymorphic forms. Solubility Measurements. The solubilities of the two forms of FFA were measured over the temperature range 25-62 °C in ethanol/water (70:30 v/v). Batch experiments were performed in a tightly sealed 50-mL jacketed glass vessel shielded from light. Excess solid was added to 35 mL of solvent, and a magnetic stirrer was used to provide agitation. A circulating water bath was used to control the temperature of the solution ((1 °C). After equilibration for 24 h, agitation was halted, and excess solid was allowed to settle. The supernatant was filtered and diluted to a concentration between 10 and 20 µg/mL, and the absorbance at 288 nm was determined using a UV spectrometer. A standard curve, prepared over the concentration range 0-25 µg/mL, was used to determine the concentration. It was obvious from these experiments that form III rapidly converted to form I at temperatures above 42 °C; hence, only solubility data obtained at low temperatures are pre-

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sented for this polymorph. Below 42 °C, form I is metastable, and XRPD was used to confirm that no conversion had occurred at the end of the solubility experiment. The solubility data for form I were used to calculate the degree of supersaturation in the crystallization experiements. Standard Curves. Raman spectra were collected from a series of FFA ethanol/water (70:30 v/v) solutions over the concentration range 0-270 mg/mL. Measurements were made at 58 °C using a jacketed glass vessel controlled by a Fisher 910 water circulation bath. A Digi-Thermo temperature sensor (accurate to 0.1 °C) (Fisher Scientific, Pittsburgh, PA) was used to monitor solution temperature. Spectra were smoothed using a second-degree polynomial and a window of 51 data points (approximately 17 cm-1) as described by Savitzky and Golay.20- 24 The first derivative was then computed using the SavitzkyGolay method. The smoothing and derivative procedures were repeated to produce the second derivative spectrum. The intensity difference between the peak at around 1710 cm-1 and the valley at approximately 1690 cm-1 was used as a measure of solution concentration. Spectra were normalized to the intensity difference of the solvent peak and valley at 895 and 882 cm-1, respectively. Powder blends consisting of various ratios of forms I and III were dispersed in 0.5% SDS (w/w) aqueous solutions and stirred at 500 rpm in the MultiMax 50mL reactor. Raman spectra of the aqueous slurries were collected at 25.0 °C. For each sample, five Raman spectra were collected consecutively. Thirteen samples with varying ratios of forms I and III were analyzed. SDS was added to wet the hydrophobic FFA particles and to ensure a homogeneous distribution of the form I and form III crystals in the slurry. No solvent-mediated polymorphic conversion was observed over the time scale of the experiments, presumably because of the low solubility of FFA in the dispersing medium. The intensities of the peaks at 615 cm-1 (form III) and 685 cm-1 (form I) were determined following peak fitting with a Lorentzian function.25 Microcal Original 7.0 was used to perform the peak fitting. A standard curve was constructed by plotting the ratio of the intensity at 615 cm-1 to the sum of the intensities at 615 and 685 cm-1 against the known composition. Feasibility Studies on FFA solutions. A supersaturated solution (σ ) 4.0, where σ is the ratio of the solubility of form I to its equilibrium solubility minus 1) of FFA in ethanol/water (70:30 v/v) was prepared by heating. Following complete dissolution, the solution (20 mL) was transferred to a clean light-resistant container and cooled to room temperature. The Raman immersion probe was inserted into the vial, and spectra were collected at 5-min intervals during the crystallization process until the intensity of the peak at 1682 cm-1 remained constant. Immediately after collection of each Raman spectrum, 50 µL of solution was extracted, diluted, and analyzed by UV spectroscopy. For this experiment, no stirring was used so that only the spectrum of the solution would be analyzed (quiescent conditions were employed whereby the crystallized solid partitioned to the sides and bottom of the container and was not detected by the short-focal-length Raman probe). To study the effect of temperature on the Raman spectrum, a 10 mg/mL solution of FFA in ethanol/water

Figure 2. Solubilities of forms I and III flufenamic acid in ethanol/ water (70:30 v/v) determined using UV spectroscopy (n ) 5).

(70:30 v/v) was heated between 20 and 75 °C. The apparent concentration as a function of temperature was estimated from a standard curve constructed using the Raman response at 30 °C for solutions ranging from 0 to 20 mg/mL. Crystallization Experiments. A mixture of 7.5 g of FFA and 30 mL of ethanol/water (70:30 v/v) was added to a 50-mL Multimax reactor. The slurry was initially stirred at 500 rpm and equilibrated at 25 °C to ensure particle wetting, before being heated linearly to 62 °C over 10 min and then equilibrated isothermally for 20 min to ensure that all particles were dissolved. For seeding experiments, FFA form III seeds (375 mg) were added into the vessel at 54 °C and cooled according to the temperature profiles described in Figures 8a and 9a below. For unseeded experiments, the cooling regimen is illustrated in Figure 10a below. The Raman spectra were collected at 1-min intervals, and the chord length distribution was collected as described earlier. Spectra were analyzed using the peak fitting routine to extract information about each polymorph and the second derivative method for solution information, as described above. These data analysis techniques were necessary to deconvolute overlapping solution and solid peak information. Results and Discussion The solubility profiles of forms I and III as a function of temperature are shown in Figure 2. Because of the rapid conversion of form III above the transition temperature, only low-temperature data are available for this polymorph. It can be seen that, in the temperature range 25-38 °C (Figure 2 inset), the solubility difference between the two polymorphs is very small (approximately 1 mg/mL) with form III having the lower stability, consistent with this polymorph being the stable form over this temperature range. Raman spectra of pure forms I and III as well as FFA in solution are shown in Figure 3. The spectra were carefully scrutinized to identify peaks characteristic to each phase of FFA. The solution spectrum has a broad peak centered at around 1680 cm-1 (not present in the solvent), form III has a distinct peak at 615 cm-1, and form I has a peak at 685 cm-1 with minimal interference from either solute or form III. These peaks were used to develop calibration models and to interpret events during the in-line monitoring of crystallization. Linear relationships between both solution concentration and

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Figure 3. Raman spectra of various forms of FFA. From top to bottom: FFA 270 mg/mL solution (EtOH/H2O ) 70:30 v/v), form I, and form III. Peaks characteristic of each phase are identified by arrows.

Figure 4. Solution calibration curve showing the Raman response as a function of FFA concentration (EtOH/H2O 70:30 v/v). The peak at 1682 cm-1 was used to monitor the FFA concentration (n ) 5).

Figure 5. Calibration curve for the relative concentration of form III in a mixture of forms I and III slurried in water (n ) 5).

polymorphic ratio with Raman response were observed, as shown in Figures 4 and 5, respectively. Feasibility Studies. It was of interest to see whether Raman spectroscopy could be used to monitor concentration changes during the crystallization of FFA. Figure 6 shows the intensity of the solute-specific peak at 1682 cm-1 as a function of time for an initially supersaturated solution (σ ) 4.0) of FFA. The readings were initially constant and decreased with time, indi-

Figure 6. Comparison of Raman spectral information, obtained in situ, and UV data from samples extracted at various times intervals during the quiescent crystallization of FFA in EtOH/ H2O (70:30 v/v). t ) 0 is when the Raman probe was inserted.

cating a reduction in solution concentration consistent with crystallization. Also shown in Figure 6 are solution concentration values obtained by withdrawing aliquots of the solution at the same time points as the Raman spectra were collected and performing UV analysis on these aliquots. The agreement between the trend in the Raman spectral intensity as a function of time and the measured concentration is excellent. This result provides an indication of the potential for monitoring desupersaturation profiles in real time using in-line Raman spectroscopy, at least under ideal experimental conditions (quiescent, isothermal). However, in most crystallization processes, agitation is typical, and upon crystallization, both solution and solid phases are sampled by the probe, and the resulting Raman spectrum contains contributions from each component. This aspect is discussed in more detail below. Another important consideration is the influence of temperature on the Raman spectra. During crystallization processes, supersaturation is commonly induced through cooling. Solvent density normally increases with decreasing temperature, leading to a change in volume. In addition, variations in intermolecular interactions with temperature can lead to changes in the intensities and positions of peaks that can lead to complications with calibrations. This effect has been observed for infrared spectroscopy and necessitates complicated calibration regimens.26 Results from this study, shown in Figure 7, clearly demonstrate that temperature has only a small effect on the Raman signal for this system and hence should not significantly influence solution concentration measurements during cooling. Over this temperature range, a variation of around 5% in solution concentration is seen, which is acceptable for systems for which the main objective is to monitor the desupersaturation profile as crystallization proceeds. In-line Monitoring of Seeded Crystallization. After the fact that a linear Raman response could be obtained as a function of either solution concentration or polymorphic composition had been established, it was of interest to investigate how much data could be extracted from an agitated crystallization experiment, where information on both solution and solids is present in the spectra and needs to be deconvoluted. To achieve crystallization in a controlled manner, a supersaturated solution (σ ) 0.2, form I), held isothermally at 54 °C, was seeded with form III FFA. In addition, to confirm

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Figure 7. Effect of temperature on concentration readings as estimated from the solution standard curve (n ) 5).

the accuracy of solution concentrations determined from Raman data, aliquots of solution were withdrawn before and after crystallization and analyzed using UV spectroscopy. The change in solution concentration as a function of time is shown in Figure 8a and the temperature-time profile is overlaid. The initial increase in concentration is due to dissolution of solid material as the temperature is raised. The Raman response then plateaus upon completion of dissolution. As the temperature is lowered to produce supersaturation, there is minimal change in concentration. At this point, there is an excellent agreement between the concentrations determined by UV spectroscopy and by the in-line Raman measurement. Addition of form III seeds results in an abrupt decrease in solution concentration, consistent with crystal growth of the seeds. The concentration slowly decreases and reaches a plateau value, indicating that growth has finished. This is consistent with the data from Figure 6, which show that desupersaturation in this system takes only a few minutes. During the polymorphic transformation (discussed below), the solution concentration is constant, as expected. There is a further slight decrease in the Raman signal at t ) 90 min, corresponding to the end of the polymorphic transformation. This change can be associated with a decrease in the solution concentration occurring because of the solubility of form I or some spectral interference by the new solid form. Once again, the signal stabilizes rapidly. This is consistent with the solid-state spectra in Figure 8b. There is a small but significant discrepancy between the UV and Raman measurements at t ) 135 min, suggesting an effect due to either temperature or the solid present. The next stage of data analysis was to determine whether solids information could be extracted in the presence of solute. It should be noted that, for these crystallization conditions, only a 20% reduction in solution concentration was achieved; hence, the solids concentration will be low relative to amount of FFA still present in solution. Figure 8b shows data extracted from the spectral region containing information characteristic to the solid forms. Using the previously described standard curve for polymorphic ratio, the percentage of form III (relative to form I) was tracked as a function of time. Following seeding with form III, this polymorph initially predominated. Interestingly, some form I also crystallized upon seeding with form III (around 20-30% relative to form III, see Figure 8b). Because form III is metastable at 54 °C, conversion to form I occurred, and the relative percentage of form III decreased. The

Figure 8. (a) FFA concentration changes during isothermal crystallization at 54 °C induced by seeding with form III seeds. Points (∆) indicate UV spectroscopy determination of concentration using aliquots of solution withdrawn just prior to seeding and at the end of the experiment. (b) Concentration of form III relative to form I. (c) Concentration profiles of all phases. The arrows indicate when form III seeds were added. The batch temperature profile is overlaid.

conversion to form I was confirmed by XRPD analysis at the end of the experiment. The solution and solid information is summarized in Figure 8c, where the concentration of each phase was calculated from the Raman data assuming conservation of mass. In-line Monitoring of Multistage Crystallization. To further investigate the use of Raman spectroscopy for crystallization monitoring, a multistage crystallization experiment was performed. Starting from the same initial concentration of 250 mg/mL of flufenamic acid, a supersaturated solution was again seeded with form III and cooled to 25 °C in a stepwise manner. At each stage, solution and solids information was extracted

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Ind. Eng. Chem. Res., Vol. 44, No. 5, 2005 Table 1. Comparison of Solubility Values Generated from in Situ Raman Data with Those Determined from a Solubility Study (Figure 2) and Measured by UV Spectroscopy solubilitya (mg/mL) form I

temperature (°C)

UVb

Ramanc

25 35 38 54

54.3 (0.5) 67.6 (0.7) 183.5 (3.1)

58 (2) 73 (2) 181 (3)

form III UVb Ramanc 34.4 (0.2)

33 (3)

a Values in parentheses are the standard deviations. b For the UV results, n ) 5. c For the Raman data, the values were generated from the final 25 spectra of each temperature stage.

Figure 9. (a) FFA concentration changes during multistage crystallization induced by seeding with form III seeds. (b) Concentration of form III relative to form I. (c) Concentration profiles of all phases. The arrows indicate when form III seeds were added. The batch temperature profile is overlaid.

from the spectral data. Figure 9a shows the solution concentration as a function of time and temperature. Once again, an abrupt decrease in solution concentration is induced upon addition of seeds as a result of crystallization. The concentration decreases with time as crystallization progresses. Each stage of temperature lowering results in a decrease in concentration, as expected. Furthermore, at each temperature, the solution concentration tends toward a plateau value, consistent with the attainment of saturation. A comparison of solubility values determined from the in situ Raman measurements with the solubility data generated in this study (Figure 2) is provided in Table 1. Although the Raman data exhibit a higher variability, in general, there is reasonable agreement between the two sets of values, providing further confirmation of our ability to monitor concentration with Raman spectroscopy.

Information about the polymorphic form of the crystallizing solids was also extracted from the spectra and is shown in Figure 9b. Following seeding with form III, a mixture of polymorphs was again observed, with form III initially dominating before undergoing a solventmediated transformation to form I. When the temperature was lowered below the transition temperature to 38 °C (rendering form I metastable) and held at this value, form I was observed to persist over the duration of the isothermal holding period (about 16 h). Conversion to the stable form was eventually induced by lowering of the temperature to 25 °C. The kinetics of the conversion from form I to form III at this temperature can clearly be followed (Figure 9b). Following completion of the conversion, a drop of the slurry was removed and checked under a light microscope; all crystals observed had the color and morphology characteristic of form III.18 Information on both solution and solids is summarized in Figure 9c. Once again, the actual concentration of solids was calculated from the solution information assuming conservation of mass. Comparison of Focused Beam Reflectance Measurements and Raman Spectroscopy for Detection of Nucleation. As a final part of the investigation into Raman spectroscopy as a technique for in-line crystallization monitoring, this technique was compared with focused beam reflectance measurement (FBRM). FBRM is widely used for detection of the onset of nucleation and for particle size profiling during crystal growth. Although Raman spectroscopy does not usually provide easily interpretable information about particle characteristics, it might be sensitive to the onset of nucleation, either by probing changes in solute peaks or by detecting the appearance of solid-specific peaks. For this experiment, spontaneous primary nucleation of a supersaturated solution at 54 °C was monitored simultaneously with both probes. The temperature was then rapidly reduced to 25 °C, which resulted in secondary nucleation and further crystal growth. In Figure 10a, particle count readings and solute concentration (extracted from the Raman spectra) are shown as a function of time. The onset of crystallization is unambiguously shown by the FBRM data as a sharp increase in the particle count. Interestingly, nucleation is also clearly detectable from the Raman data as a decrease in the intensity of the solute peak at 1682 cm-1, which translates into a decrease in solution concentration. Indeed, within the resolution of the experiment, there is no discernible difference in the time points for nucleation detected by FBRM and Raman spectroscopy, although the physical parameters being measured are quite different and specific to the two techniques.

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form at the nucleation temperature, but not at the harvesting temperature. Thus the conversion kinetics to the room temperature stable form become of interest. Our results indicate that the transformation from form I to form III is slow (>16 h). Attempts to obtain the room-temperature-stable form III by seeding above the transition temperature initially resulted in a mixture of polymorphs followed by conversion of the metastable form. In this instance, conversion of form III to form I is complete within 1 h when the system is held above the transition temperature. The difference in the observed transformation kinetics for the two forms can most likely be explained by the presence or absence of the stable form. When form III was seeded above the transition temperature, form I nucleated concomitantly. This might be due to heterogeneous nucleation of form I on the structurally related surfaces of the form III seeds. The presence of the stable phase (form I) should facilitate the conversion of form III by eliminating the nucleation stage of the solvent-mediated transformation. Similarly, below the transition temperature, form I should convert to form III; however, a long induction time was observed at 38 °C (Figure 9c). As of result of a further decrease in the temperature to 25 °C, nucleation of form III occurred, and form I converted to form III in about 100 min. These results strongly suggest that interconversions between form I and form III are probably nucleation controlled. Further studies are under way to better understand the interconversion between the two polymorphic forms. Figure 10. Comparison of FBRM and Raman spectroscopy for detecting nucleation: (a) comparison of solution concentration estimated from Raman data with FBRM results and (b) comparison of solids information as elucidated from Raman data with FBRM results. The concentration of form I was calculated from the solution data shown in Figure 10a. The batch temperature profile is overlaid.

One would expected that, simultaneous with a decrease in solution concentration, solid-phase information would appear in the spectral data. For these crystallization conditions, form I crystallized spontaneously at 54 °C and persisted over the time course of the experiment. However, as seen from Figure 10b, there is a time delay (relative to the FBRM data) before form I could be detected, presumably as a consequence of detection limits for this polymorph. Interestingly, in contrast with the previous experiment, no conversion of form I was seen at 25 °C. Clearly, in this case, “aging” of the slurry at 38 °C for a number of hours had some influence on the tendency for conversion. These results show the potential of Raman spectroscopy for process optimization and control, although the applicability of this technique at larger scales needs to be established. Because only a small sample volume is probed, the position of the probe in a large reactor is likely to be critical. It might be possible to overcome the risk of localized sampling by employing multiple probes at different locations in the reactor and ensuring that the probes are not located in stagnant regions of the reactor. The model compound employed in these studies displays a complex crystallization behavior and therefore is an ideal candidate for applying in-line monitoring techniques to understand the relationship between process parameters and final crystal form. Because the transition temperature is close to room temperature, the polymorph initially crystallized during cooling of a supersaturated solution is the thermodynamically stable

Conclusions It has been demonstrated that Raman spectroscopy can be used to measure solute concentration and polymorphic form as a function of time and temperature in batch crystallization operations. The ability to obtain concentrations along with crystal forms allows for the extraction of data related to the growth rates and conversion rates of polymorphic forms during crystallization operations. In addition, temperature-induced interconversions between enantiotropically related polymorphs can readily be identified and rates can be quantified. This type of in-line monitoring can provide crucial information in the development and operation of batch crystallization processes for substances with multiple crystal forms that have differentiable Raman spectra. Acknowledgment Dongmao Zhang is thanked for help with data analysis. We are grateful for the financial support provided by the Particle Technology and Crystallization Consortium, which has enabled this collaboration between Illinois Institute of Technology and Purdue University. Kaiser Optical Systems is acknowledged for support with instrumentation. Literature Cited (1) U.S. Food and Drug Administration (FDA). FDA Process Analytical Technology (PAT) Initiative. See http://www.fda.gov/ cder/OPS/PAT.htm. (September, 2004). (2) Callis, J. B.; Illman, D. L.; Kowalski, B. R. Process Analytical Chemistry. Anal. Chem. 1987, 59, 624A. (3) Fevotte, G. New Perspectives for the On-line Monitoring of Pharmaceutical Crystallization Processes Using in Situ Infrared Spectroscopy. Int. J. Pharm. 2002, 241, 263.

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(4) Davis, T. D.; Morris, K. R.; Huang, H.; Peck, G. E.; Stowell, J. G.; Eisenhauer, B. J.; Hilden, J. L.; Gibson, D.; Byrn, S. R. In Situ Monitoring of Wet Granulation Using Online X-ray Powder Diffraction. Pharm. Res. 2003, 20, 1851. (5) Folestad, S.; Johansson, J. Raman Spectroscopy: A Technique for the Process Analytical Technology Toolbox. Am. Pharm. Rev. 2004, 7, 82. (6) Taylor, L. S. Raman Spectroscopy as a Tool to Probe Solid State Form in Tablets. Am. Pharm. Rev. 2001, 4, 60. (7) Skoulika, S. G.; Georgiou, C. A. Rapid, Noninvasive Quantitative Determination of Acyclovir in Pharmaceutical Solid Dosage Forms Through Their Poly(vinyl chloride) Blister Package by SolidState Fourier Transform Raman Spectroscopy. Appl. Spectrosc. 2003, 57, 407. (8) Wang, F.; Wachter, J. A.; Antosz, F. J.; Berglund, K. A. An Investigation of Solvent-Mediated Polymorphic Transformation of Progesterone Using in Situ Raman Spectroscopy. Org. Process Res. Dev. 2000, 4, 391. (9) Shackman, J. G.; Giles, J. H.; Denton, M. D. Pharmaceutical Reaction Monitoring by Raman Spectroscopy. In Further Developments in Scientific Optical Imaging; Special Publication 254; Denton, M. B., Ed.; Royal Society of Chemistry: London, 2000; pp 186-201. (10) Failloux, N.; Bonnet, I.; Baron, M.; Perrier, E. Quantitative Analysis of Vitamin A Degradation by Raman Spectroscopy. Appl. Spectrosc. 2003, 57, 1117. (11) Szelagiewicz, M.; Marcolli, C.; Cianferani, S.; Hard, A. P.; Vit, A.; Burkhard, A.; von Raumer, M.; Hofmeier, U. Ch.; Zilian, A.; Francotte, E.; Schenker, R. In Situ Characterization of Polymorphic Forms: The Potential of Raman Techniques. J. Therm. Anal. Calorim. 1999, 57, 23. (12) Taylor, L. S.; Zografi, G. The Quantitative Analysis of Crystallinity Using FT-Raman Spectroscopy. Pharm. Res. 1998, 15, 755. (13) Auer, M. E.; Griesser, U. J.; Sawatzki, J. Qualitative and Quantitative Study of Polymorphic Forms in Drug Formulations by Near Infrared FT-Raman Spectroscopy. J. Mol. Struct. 2003, 661-662, 307. (14) Schwartz A. M.; Berglund K. A. The Use of Raman Spectroscopy for in Situ Monitoring of Lysozyme Concentration during Crystallization in a Hanging Drop. J. Cryst. Growth 1999, 203, 599.

(15) Starbuck, C.; Spartalis, A.; Wai, L.; Wang, J.; Fernandez, P.; Lindemann, C. M.; Zhou, G.; Ge, Z. Process Optimization of a Complex Pharmaceutical Polymorphic System via in Situ Raman Spectroscopy. Cryst. Growth Des. 2002, 2, 515. (16) Zhou, G.; Wang, J.; Ge, Z.; Sun, Y. Ensuring Robust Polymorph Isolation Using in Situ Raman Spectroscopy. Am. Pharm. Rev. 2002, 5, 74. (17) Krc, J., Jr. Crystallographic Properties of Flufenamic Acid. Microscope 1977, 25, 31. (18) Galdecki, Z.; Glowka, M. L.; Gorkiewicz, Z. Examination of the Polymorphism of 2-[(3-Trifluoromethyl)phenyl]aminobenzoic Acid. Acta Pol. Pharm. 1978, 35, 77. (19) Abignente, E.; de Caprariis, P. Flufenamic Acid. In Analytical Profiles of Drug Substances; Florey, K., Ed.; Academic Press: New York, 1982; Vol. 11, pp 313-346. (20) Savitzky, A.; Golay, M. J. E. Smoothing and Differentiation of Data by Simplified Least Squares Procedures. Anal. Chem. 1964, 36, 1627. (21) Steinier, J.; Termonia, Y.; Deltour, J. Comments on Smoothing and Differentiation of Data by Simplified Least Square Procedure. Anal. Chem. 1972, 44, 1906. (22) Madden, H. H. Comments on the Savitzky-Golay Convolution Method for Least-Squares Fit Smoothing and Differentiation of Digital Data. Anal. Chem. 1978, 50, 1383. (23) Cameron, D. G.; Moffatt, D. J. A Generalized Approach to Derivative Spectroscopy. Appl. Spectrosc. 1987, 41, 539. (24) Zhang, D.; Ben-Amotz, D. Enhanced Chemical Classification of Raman Images in the Presence of Strong Fluorescence Interference. Appl. Spectrosc. 2000, 54, 1379. (25) Pelletier, M. J. Quantitative Analysis Using Raman Spectrometry. Appl. Spectrosc. 2003, 57, 20A. (26) Wu¨lfert, F.; Kok, W. Th.; Smilde, A. K. Influence of Temperature on Vibrational Spectra and Consequences for the Predictive Ability of Multivariate Models. Anal. Chem. 1998, 70, 1761.

Received for review March 31, 2004 Revised manuscript received July 9, 2004 Accepted August 20, 2004 IE049745U