Article pubs.acs.org/molecularpharmaceutics
Confocal UV and Resonance Raman Microscopic Imaging of Pharmaceutical Products Frederick G. Vogt* and Mark Strohmeier Product Development, GlaxoSmithKline plc., 709 Swedeland Road, King of Prussia, Pennsylvania 19406, United States S Supporting Information *
ABSTRACT: Chemical imaging using confocal Raman microscopy is a useful analytical tool in drug development because of its ability to spatially image active ingredients and excipients in dosage forms and relate their distribution to product performance. While Raman spectra are highly specific for individual components of a formulation, most Raman microscopic mapping experiments require extensive experimental time. Laser wavelengths in the near-infrared range are used to suppress fluorescence but reduce sensitivity because of the inverse quadratic dependence of Raman scattering on laser wavelength. Compact, simple ultraviolet (UV) laser designs now allow for confocal UV Raman microscopy to be performed using a versatile instrument also capable of conventional Raman microscopy and epifluorescence imaging analyses. This study presents the results of UV Raman microscopy analyses using 266 nm laser irradiation of four pharmaceutical compositions of interest, including two types of tablets containing low doses of active ingredients (in the 0.2% w/w range), an amorphous dispersion containing 1% w/w of a small molecule drug, and an enteric coated layered peptide formulation. Resonance Raman enhancements are observed for four of the active ingredients studied in these formulations. The spectroscopic properties of the materials used in this study are also assessed by diffuse reflectance UV−visible spectroscopy, fluorescence spectroscopy, and conventional bulk Fourier transform Raman spectroscopy using 1064 nm laser irradiation. Confocal UV Raman microscopy was found to offer good sensitivity and allowed for rapid microscopic mapping of drugs and excipients at low concentrations in pharmaceutical formulations. KEYWORDS: UV Raman spectroscopy, resonance Raman spectroscopy, chemical imaging, pharmaceutical analysis, solid state spectroscopy
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resolution can be limited and fiber cross-talk may cause interferences.1 Diode-pumped solid state (DPSS) continuouswave (CW) lasers have been extensively developed over the past ten years, and more recently DPSS lasers operating at ultraviolet (UV) wavelengths (e.g., 266 nm) have become available in a compact format suitable for confocal Raman microscopy in industrial laboratories.7 Raman spectroscopy using such a UV laser, here referred to as UV Raman spectroscopy, also can avoid fluorescence by acquiring the Raman spectrum in the wavelength region that is shorter than the wavelength where fluorescence emission occurs.8,9 The short wavelength (λ) of 266 nm radiation can yield signal enhancements compared to common lasers because of the λ−4 dependence of the scattering intensity.10 The use of 266 nm laser irradiation also offers the possibility of obtaining resonance Raman enhancements of Raman signals.9−13 Although Raman microscopy using UV laser irradiation has
INTRODUCTION Analytical techniques based on chemical or molecular imaging with Raman microscopy are increasingly useful in the study of pharmaceutical dosage forms.1−5 These techniques allow for study of the distribution of drugs and excipients in various types of formulations, including oral tablets and dry powders for inhalation.1−5 Key performance characteristics of pharmaceutical products can often be elucidated using Raman microscopic imaging techniques. The choice of laser in Raman microscopy of pharmaceutical materials is generally determined by the need to avoid fluorescence and/or sample burning. This is usually accomplished using lasers that emit in the red to near-infrared wavelength region, with wavelengths of 633, 785, and 1064 nm commonly encountered.1−6 Using these lasers, point mapping of components at the 1% w/w level in pharmaceutical tablets or similar materials normally requires 5 s or more of Raman acquisition time per map point, leading to an undesirable choice between long map acquisition times of a day or more or a reduction in the number of points sampled even using sensitive detectors.1,2 Alternative mapping techniques, such as fiber array Raman imaging, are able to greatly increase image acquisition rates in a non-confocal instrument, but spatial © 2013 American Chemical Society
Received: Revised: Accepted: Published: 4216
May 24, 2013 September 14, 2013 September 19, 2013 September 19, 2013 dx.doi.org/10.1021/mp400314s | Mol. Pharmaceutics 2013, 10, 4216−4228
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A direct compression tablet containing 7-methoxy-1-methyl5-(4-(trifluoromethyl)phenyl)-[1,2,4]triazolo[4,3-a]quinolin-4amine (I), a developmental Apo-A1 upregulator, and a similarly prepared placebo formulation were also used in this work.18 The preparation of the hydrated crystalline form of I used in this work as well as its chemical synthesis have been previously described.18,19 The active tablet contained 0.2% w/w of the hydrated form of I that was milled to a 50% cumulative undersize distribution of 1.5 μm (Micron Technologies, Inc., Malvern, PA, USA),18 50.0% w/w microcrystalline cellulose (FMC Biopolymer, Philadelphia, PA, USA), 43.1% w/w mannitol (Roquette Pharma, Lestrem, France), 5.0% w/w sodium starch glycolate (Roquette Pharma, Lestrem, France), 1.0% w/w magnesium stearate (Mallinckrodt, Hazelwood, MO, USA), 0.5% w/w sodium lauryl sulfate (Sigma-Aldrich, St. Louis, MO, USA), and 0.2% w/w colloidal silicon dioxide (Sigma-Aldrich, St. Louis, MO, USA). A placebo tablet was prepared using the same ingredients but without I and with the quantity of mannitol increased to 43.3% w/w. The tablets were prepared by blending I (except in the placebo), microcrystalline cellulose, mannitol, and sodium starch glycolate (SSG) for 15 min in a Turbula mixer (Glen Creston Ltd., London, U.K.) and then adding colloidal silicon dioxide prior to 15 min of additional blending, followed by addition of sodium lauryl sulfate (SLS) and magnesium stearate and blending for 3 additional minutes. From the blends, tablets of nominal mass of 500 mg were prepared by direct compression using 3.75 tons of pressure in a Manesty Betapress tablet press (Bosch Packaging Technology Ltd., Knowsley, U.K.). The amorphous dispersion was prepared using 6-(2-(5chloro-2-(2,4-difluorobenyzloxy)phenyl)cyclopent-1-enyl)picolinic acid (II), a developmental EP1 antagonist that has been previously described.20 The dispersion was prepared by dissolving 10 mg of a crystalline form of II and 990 mg of polyvinylpyrrolidone (PVP) (BASF GmbH, Ludwigshafen, Germany) in 25 mL of acetone and 2 mL of methanol. The solution was rapidly evaporated at 80 °C under vacuum, scraped, ground to a powder with a mortar and pestle, and dried under vacuum overnight to remove any remaining solvent. Confocal UV Raman Microspectroscopy. Confocal UV Raman microscopy was performed using a LabRam HR system (Horiba Jobin Yvon, Edison, NJ, USA) equipped with a Synapse charge-coupled device (CCD) detector and a BX41 confocal optical epifluorescence microscope fitted with a 20× UV objective (Olympus America, Inc., Chester Valley, PA, USA). The confocal hole was set to 500 μm for all experiments. A FQCW 266-50 DPSS laser with a wavelength of 266 nm and a maximum power of 50 mW (CryLas GmbH, Berlin, Germany) was used for UV Raman experiments. Laser power levels reported in this work for the UV laser were in the range of 20 to 25 mW and are measured at the laser head, not at the sample. The system is also equipped with a 633 nm HeNe laser (CVI Melles Griot, Albuquerque, NM, USA) with a maximum output power of 20 mW at the laser head that was also used in this work, as well as a 785 nm diode laser (Sacher Lasertechnik GmbH, Marburg, Germany). Holographic blazed diffraction gratings with 600 and 2400 grooves/mm were used for Raman and UV Raman spectra, respectively. The maximum spectral resolution for UV Raman studies was 1.5 cm−1, and data was smoothed using detector binning during acquisition to obtain higher signal-to-noise with effective resolutions of up to 12 cm−1. UV Raman spectra were acquired in the 3500 to 400
many advantages, the high energy of the beam can lead to sample burning and can necessitate the use of lower powers with a consequent reduction in Raman signal intensity. Another potential disadvantage is that electronic resonance Raman enhancements often result in Raman bands that are broadened by the superposition of multiple “hot” bands that result from transitions wherein the excited electronic state returns to different vibrational levels in the ground electronic state.10 Resonance Raman bands can exhibit additional line broadening if the excited electronic state relaxes faster than the states involved in nonresonance Raman scattering.10 This broadening can reduce the specificity of resonance Raman spectroscopy in studies of pharmaceutical products. This study evaluates the performance of confocal UV Raman microscopy and imaging through four applications to pharmaceutical formulations: (1) imaging of the distribution of an active ingredient in a commercial vitamin tablet at the 0.2% w/w level via a resonance Raman enhancement, (2) imaging of the distribution of a small molecule drug candidate at a similar level as well as a number of excipients in a direct compression tablet formulation, (3) imaging of another small molecule drug candidate at 1% w/w in an amorphous solid dispersion in a polymer, and (4) imaging of a peptide drug candidate within an enteric-coated formulation that has been previously studied using conventional Raman microscopy with a 785 nm laser to avoid fluorescence.14 The applications to tablets and an amorphous solid dispersion containing a low dose of an active ingredient were chosen to illustrate the applications of confocal UV Raman microscopy to formulations of high-potency actives that are frequently encountered in drug development.15−17 Excipient mapping over a wide range of concentrations is also demonstrated. Solid-state UV−visible and fluorescence spectra are also obtained for the materials used in this study to examine absorbance properties at 266 nm and the fluorescence emission caused by excitation at this laser wavelength. The applications illustrate the general performance and pharmaceutical applicability of confocal Raman microscopy and imaging with UV laser irradiation.
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EXPERIMENTAL METHODS Materials. Tablets were cross-sectioned by first embedding intact tablets within epoxy putty (Ultratech International, Inc., Jacksonville, FL, USA). After hardening, the tablet and block of putty were cross-sectioned using a stainless steel blade and a hand microtome (AO Scientific Instruments, Buffalo, NY, USA). The preparation of the layered sucrose sphere coated with peptide and an enteric polymer was described in detail in previous work.14 A commercial 1 mg cyanocobalamin tablet was purchased and used as received in this work (Nature Made Nutritional Products, Mission Hills, CA, USA). The commercial tablet included dibasic calcium phosphate, stearic acid, microcrystalline cellulose, magnesium stearate, and croscarmellose sodium as excipients. The concentrations of these excipients were not available from the manufacturer. The relative levels of these excipients were therefore estimated using conventional bulk Fourier transform (FT) Raman spectroscopy and 13C solidstate nuclear magnetic resonance (SSNMR) analysis of the tablet as described below. From these analyses, the primary components of the formulation were found to be dibasic calcium phosphate and stearic acid, with smaller amounts of microcrystalline cellulose detected. 4217
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cm−1 range using the 2400 grooves/mm grating by acquiring and combining two spectral windows. Optical images were captured with brightfield illumination using an Infinity 3 cooled CCD camera (Lumenera Corp., Ottawa, Ontario, Canada). Spectral baseline correction and despiking were applied to UV Raman map data. Spectral maps were produced using both band areas and direct classical least-squares (DCLS) analysis, which were performed using LabSpec version 5 (Horiba Jobin Yvon, Edison, NJ, USA). DCLS scores plots are shown using false colors with intensities given in response units to facilitate comparisons. Fourier Transform Raman Spectroscopy. FT Raman spectra of bulk samples were obtained using a MultiRAM Fourier transform spectrometer (Bruker Optics, Billerica, MA, USA). Spectra were obtained by collection of the 180° scattered signal. The detector was a long-hold nitrogen-cooled germanium diode detector (Bruker Optics, Billerica, MA, USA). Laser excitation was performed using a 1 W, 1.064 μm diode-pumped neodymium-doped yttrium aluminum garnet (Nd:YAG) laser that was attenuated to powers in the 0.2 to 0.8 W range measured at the laser output (Klastech GmbH, Dortmund, Germany). A spectral resolution of 2 to 4 cm−1 was used, and the number of scans collected ranged from 256 to 2048 scans as described in later sections. The duration of the FT Raman experiments varied from 10 min to several hours. The duration of the FT Raman experiments was generally ten to a hundred times that required to obtain UV Raman spectra using the confocal microscope. For example, spectra obtained with 256 scans using 2 cm−1 resolution required 18 min of acquisition time using the mirror velocity applied here. About 100 mg of each sample was loaded into 5 mm outer diameter borosilicate glass tubes for analysis. UV−Visible Spectroscopy. Diffuse reflectance (DR) UV− visible spectroscopy was employed to characterize the optical properties of many of the solids used in this work, particularly their absorption characteristics at 266 nm. UV−visible spectra were acquired using a Varian Cary 50 spectrometer (Agilent, Palo Alto, CA, USA) equipped with a Barrelino DR probe (Harrick Scientific Products, Pleasantville, NY, USA). Spectra were collected from 200 to 1100 nm using scan rates of 60 to 120 nm/min. For some measurements, samples were diluted by physically mixing into magnesium oxide powder as discussed below. The instrument was calibrated for wavelength using four maxima (in the range of 279.4 to 637.5 nm) in the spectrum of holmium oxide and calibrated for photometric accuracy using four maxima in the spectrum of K2Cr2O7. Diffuse reflectance calibration for 0% and 100% reflectance was achieved with Spectralon diffuse reflectance standards (Labsphere, North Sutton, NH, USA). Fluorescence Spectroscopy. Fluorescence experiments on bulk samples were performed using a Fluorolog 3 spectrometer equipped with double-grating Czerny−Turner monochromators and reflective optics (Horiba Jobin Yvon, Edison, NJ, USA). About 100 mg of sample was packed in a black anodized aluminum solids holder and covered with a quartz plate for analysis. Fluorescence was detected using frontfacing geometry at a 30° angle with the excitation light. Diffraction gratings were ruled at 1200 tr/mm and blazed at 330 and 500 nm for the excitation and emission monochromators, respectively. Slit widths of 1 to 5 nm were used for the monochromators as described in later sections and in the Supporting Information. Detection times of 1 to 5 s per point were employed. The excitation monochromator was calibrated
using the lamp emission line at 467 nm, and the emission monochromator was calibrated using 350 nm light to excite the Raman line from a water sample at 397 nm. Powder X-ray Diffraction and SSNMR Analysis. Powder X-ray diffraction (PXRD) experiments were used to identify several solid phases used in this study. PXRD patterns were obtained using a Panalytical X′Pert Pro diffractometer configured in the Bragg−Brentano reflection geometry with an X′Celerator real time multistrip detector (Panalytical, Eindhoven, The Netherlands). Samples were flattened onto zero-background silicon holders and analyzed at ambient temperature and humidity. Cu Kα radiation (1.54 Å) was used with a generator voltage and current of 40 kV and 40 mA, respectively. Samples were scanned in continuous mode from 2° to 40° θ with a 2θ step size of 0.0167°. The sample was rotated with a 1 s revolution time. 19 F SSNMR analysis of the amorphous dispersion of II was performed using an Avance II+ SSNMR spectrometer (Bruker Biospin, Billerica, MA, USA) operating at a 1H frequency of 500.08 MHz and field strength of 11.7 T, using a 4 mm magicangle spinning (MAS) HFX probe tuned to 1H, 19F, and 13C frequencies. A cross-polarization (CP) sequence using a ramped pulse on the 1H channel was used to record 1D 19F spectra with SPINAL-64 1H decoupling.21,22 2D 1H−19F CP heteronuclear correlation (CP-HETCOR) spectra were acquired using 1H homonuclear decoupling as described previously in other analyses of amorphous dispersions.23,24 13 C spectra of the cyanocobalamin tablet and reference materials and 19F spectra of I and the tablet of I were measured using an Avance SSNMR spectrometer operating at a 1 H frequency of 399.87 MHz and field strength of 9.4 T (Bruker Biospin, Billerica, MA, USA). Spectra were recorded using a 4 mm MAS HFX probe, CP, and SPINAL-64 decoupling, and for 13C, total sideband suppression (TOSS) was used. 25 13 C spectra were referenced to external hexamethylbenzene, and 19F spectra were referenced to CFCl3 using absolute frequency ratios.26,27
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RESULTS AND DISCUSSION Analysis of a Cross-Sectioned 1 mg Cyanocobalamin Tablet. The first system studied was a commercial 1 mg cyanocobalamin (vitamin B12) vitamin supplement tablet with a total tablet mass of 560 mg. The active ingredient thus comprises only 0.18% w/w of the tablet, which presents a challenging system for conventional confocal Raman microscopic analysis. Analysis of cyanocobalamin is further complicated by the potential presence of several known polymorphs and an amorphous phase of cyanocobalamin.28 In addition to cyanocobalamin, the commercial tablet formulation consisted of dibasic calcium phosphate, stearic acid, microcrystalline cellulose, magnesium stearate, and croscarmellose sodium at concentrations unreported by the manufacturer. For simplicity, only the two most concentrated excipients (dibasic calcium phosphate and stearic acid) that were found by SSNMR and FT-Raman analysis are discussed in this initial example (see Supporting Information). A more detailed study of excipients at a range of concentrations using confocal UV Raman microscopy is presented in the next section for a pharmaceutical tablet where the quantities of all excipients are known. The spectroscopic properties of cyanocobalamin relevant for UV Raman spectroscopy were examined prior to the tablet 4218
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(the latter wavelength corresponding to 3500 cm−1), this weak fluorescence does not lead to any significant effects on the UV Raman spectrum obtained for cyanocobalamin in Figure 1, which is presented after only minimal baseline correction. Rapid burning of cyanocobalamin powder was observed using the confocal Raman microscope with the 633 nm laser set at an output power of 25 mW. This burning is likely the result of lower energy electronic transitions excited by 633 nm irradiation that lead to more facile photodissociation, which does not occur as easily with the higher energy electronic transitions excited by the 266 nm laser. In spite of this, it was possible to obtain a quick spectrum with the 633 nm laser in about 10 s. This spectrum is shown in Figure 1 for comparison with the results obtained using the 266 nm laser. A much weaker resonance Raman enhancement is seen with irradiation at 633 nm despite the small absorbance at this wavelength (see Supporting Information). However, because of burning, attempts to map tablets using the 633 nm laser even at low power settings led to poor results. In Figure 2, the UV Raman mapped region of a portion of a microtomed cyanocobalamin tablet is shown superimposed on an optical brightfield image taken using a 20× UV objective. An image showing the position of the mapped region within the entire area of cross-sectioned tablet is given in the Supporting Information. The 50 × 50 map (2500 map points) shown in Figure 2 was sampled with 10 μm spatial resolution using 8 s per point (8 scans with a 1 s detector exposure) and a single spectral window of about 1870 to 190 cm−1. Using this approach required about 6 h of experimental time and was found to yield high sensitivity for low levels of cyanocobalamin (shown in the blue map in Figure 2) and two major excipients. The red map shows the distribution of stearic acid, a major excipient in the formulation. The green map shows distribution of the other major excipient, dibasic calcium phosphate. The signal of cyanocobalamin is strongly enhanced by the resonance Raman effect discussed above, allowing it to be mapped efficiently in spite of its low mass. The three maps show little spatial correlation, suggesting that these substances are distributed randomly in the tablet. Based on the map analysis, cyanocobalamin is likely spread throughout the two major excipients. In Figure 3, representative UV Raman spectra extracted from each of the three maps at the points marked in Figure 2 are shown in comparison to reference spectra of these components. The extracted spectrum for cyanocobalamin from the blue map shows a shift in the strongest peak, which has a maximum at 1619 cm−1 in the extracted map point compared to the peak at 1590 cm−1 in the primarily amorphous reference sample. Although signals from excipients may be partly responsible for the shift of this peak, it is more likely caused by crystalline forms of cyanocobalamin. An experiment was performed to determine if recrystallization of amorphous cyanocobalamin from methanol (known to produce a crystalline polymorph) would lead to a different UV Raman spectrum.28 The spectrum obtained for this recrystallized sample showed a clear shift in several bands, including the most intense band, which appeared at 1612 cm−1 (see Supporting Information). Although the use of UV Raman spectroscopy to study polymorphism is not the primary focus of this study, this result suggests that, in future investigations, resonance UV Raman spectra of polymorphs may become a useful tool for the study of materials only available in small quantities. However, less specificity is
analysis. The cyanocobalamin reference powder used here was assessed by PXRD and found to consist primarily of amorphous cyanocobalamin (see Supporting Information). Figure 1
Figure 1. Expanded regions of the Raman spectra of amorphous cyanocobalamin acquired using an FT Raman spectrometer with a 1064 nm laser (300 mW power, 256 scans, 2 cm−1 resolution) compared to the spectrum obtained using confocal Raman microscopy with laser irradiation at 266 nm (25 mW power, 128 scans with a 1 s exposure time). The spectrum obtained with 266 nm laser irradiation shows broadening and intensity changes indicative of a resonance Raman effect. The spectrum obtained with confocal Raman microscopy using the 633 nm laser (4 scans, 2 s exposure time) before burning is also shown for comparison.
compares the FT Raman spectrum of the amorphous cyanocobalamin solid obtained with a 1064 nm laser on bulk powder to the UV Raman spectrum obtained on a small portion of the same material with the 266 nm laser via the confocal microscope. The UV Raman spectrum shows strong enhancement of bands in the 1600 to 1200 cm−1 region and the characteristic broadening associated with electronic resonance Raman spectra.10 The resonance Raman enhancement primarily affects stretching vibrations involving aromatic, amide, and other unsaturated groups in cyanocobalamin. The cyanocobalamin powder was checked for burning by the UV laser using optical microscopy and by reacquisition of spectra over an extended period using different laser power levels. The UV Raman spectrum shown in Figure 1 was the result of experiments that were not influenced by burning, which could be avoided by using a laser power of 25 mW or less. The resonance Raman enhancement seen for the amorphous cyanocobalamin powder is related to a strong electronic absorbance at 266 nm observed in the DR UV−visible spectrum of this material (see Supporting Information). The fluorescence properties of cyanocobalamin are also amenable to UV Raman spectroscopy, because excitation of the amorphous solid powder with 266 nm light leads to an extremely weak fluorescence emission spectrum with a maximum at about 400 nm (see Supporting Information). Since the UV Raman spectrum is acquired in the region between 266 and 293 nm 4219
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Figure 3. Comparison of UV Raman (266 nm irradiation) spectra extracted from the map of the cross-sectioned cyanocobalamin tablet in Figure 2, shown in comparison to UV Raman reference spectra for the three major components. For comparison, FT Raman (1064 nm irradiation) spectra of the two excipients are also shown; the FT Raman spectrum of cyanocobalamin is shown in Figure 1
Representative UV Raman spectra extracted from the two other mapped components corresponding to the major excipients in the tablet, dibasic calcium phosphate and stearic acid, are also shown in Figure 3. These spectra agree with UV Raman reference spectra of the separate materials, although dibasic calcium phosphate is seen to be present in the extracted spectrum of stearic acid. Comparison of these spectra with the corresponding FT Raman spectra shows no indication of a significant resonance Raman effect for dibasic calcium phosphate and a minor effect for stearic acid. Overall, the results show that the use of UV laser excitation allowed for efficient mapping with excellent sensitivity for the low-dose active ingredient and two major excipients. Analysis of a Cross-Sectioned 1 mg Tablet of a Pharmaceutical Compound. The second system studied in this work is designed to further explore the ability of confocal UV Raman microscopy in the imaging of distributions of both low-dose drug (I) and a range of excipient concentrations in a typical direct compression oral tablet for drug delivery.
Figure 2. Band intensity plots from a UV Raman map (50 × 50 points) of a region of the cross-sectioned cyanocobalamin tablet. The map was recorded using 10 μm stage increments. Band intensity plots are shown superimposed on an optical brightfield image obtained with a 20× objective. The red, green, and blue plots show Raman band intensity in the 1490 to 1400 cm−1 (stearic acid), 1030 to 975 cm−1 (dibasic calcium phosphate), and 1725 to 1515 cm−1 regions (cyanocobalamin), respectively. Spectra extracted from the map points marked with yellow boxes are shown in Figure 3
expected compared to conventional Raman spectroscopy because of the broadening of peaks. 4220
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excipients are also given in the Supporting Information. Microcrystalline cellulose showed a tendency to burn (observed visually and via a band at 1650 cm−1) that was minimized by use of the lowest possible UV laser power of 20 mW, although a small amount of potential burning in the pure material was still detected through a minor level of residual band intensity (see Supporting Information). This residual band could also arise from resonance enhancement of impurities in the microcrystalline cellulose, and as it did not increase with repeated spectral measurements on the same area, it is not expected to significantly affect the analysis. Overall, the excipients showed little or no evidence of resonance Raman effects. This is attributable to the lack of chromophores in the excipients with absorption at 266 nm, as seen in the DR UV−visible spectrum of a crushed placebo tablet containing all of the excipients used in the 1 mg tablet of I (see Supporting Information). The fluorescence properties of the excipients are also favorable for UV laser irradiation, with an emission maximum at 430 nm observed using 266 nm excitation (see Supporting Information). In Figure 4b, a composite image of the microtomed 1 mg tablet of I embedded in putty is shown. An initial map was made of the expanded region shown in Figure 4c. This map was performed using 10 μm stage increments in both dimensions with a spectral range of 3500 to 500 cm−1 (from two combined spectral windows) and required 4 h of analysis time. DCLS analysis was employed to model the data because of spectral overlap among the large number of components in the tablet. The DCLS model included all of the excipients except for the 0.2% w/w colloidal SiO2, which was ignored for the reasons described above. The DCLS scores showing the distribution of I are shown in Figure 4d. A relatively even distribution of the drug over the mapped region is observed and is likely a consequence of the milling step used prior to formulation. A spectrum extracted from one of the stronger regions is compared to the UV Raman reference spectrum of I in Figure 4a. The spectrum obtained from the map in Figure 4a appears broader than that of the reference spectrum because a larger CCD binning factor was used to collect the map to optimize sensitivity. The spectrum obtained from the map also contains peaks from major excipients (green arrows) that do not experience resonance Raman enhancement in this region, such as a strong peak from mannitol at 878 cm−1, although the primary band from form B of I at 1610 cm−1 dominates the spectrum. The mapping performance of the 266 nm laser was compared to that of the 633 nm laser on a similar area of the tablet using comparable experimental conditions (see Supporting Information). Although the distribution of the major excipients such as mannitol could be mapped using the 633 nm laser, DCLS and direct spectral analysis was unable to detect any signal from I in this comparative experiment. Using information obtained from the first map using the 266 nm laser, which measured nearly the full Raman spectral region in two windows, the region between 1800 and 200 cm−1 was found to be most specific for the components of interest here and was mapped over the entire tablet using 50 μm stage increments. This large map involved 230 × 230 points (52900 points) and was performed using a single 1 s scan at each point, allowing for a complete map to be formed in approximately 18 h. The enhancement observed for I allowed it to be detected even under these conditions, again using DCLS analysis to analyze the components, with the resulting distribution of I shown via the scores plot in Figure 5. Scores plots for the other
Although excipients are inactive ingredients, the distribution of excipients can greatly impact the dissolution performance of oral tablets. This tablet contains a range of common excipients functioning as binders, dissolution aids, glidants, and lubricants and covering a wide range of concentrations: microcrystalline cellulose (50.0% w/w), mannitol (43.1% w/w), sodium starch glycolate (5.0% w/w), magnesium stearate (1.0% w/w), SLS (0.5% w/w), and colloidal silicon dioxide (0.2% w/w). The 500 mg active tablet contains 1 mg of I and thus has a drug loading of 0.2% w/w. The chemical structure of compound I, an upregulator of Apo-A1, is shown below.
The polymorphic form of this material is a nonstoichiometric hydrate with a variable water content known as form B.18 The crystal structure of this form has been previously reported and studied in detail.18 The materials used in the present study were representative of this crystal structure and contained amounts of water near to a monohydrate. The polymorphic form of I in the tablet was independently determined to be form B using 19F SSNMR, which is a challenging task given the low level of drug but is important because it simplifies the UV spectral analysis in the present study by obviating the need to consider polymorphism (see Supporting Information). As with the cyanocobalamin tablet, the spectroscopic properties of I and the excipients used in the tablet were measured to assess the applicability of confocal UV Raman microscopy before tablet mapping. The UV Raman spectrum of a reference sample of form B was obtained using the confocal microscope and compared to the FT Raman spectrum of a bulk sample as shown in Figure 4a. A resonance Raman effect is observed with enhancements and broadening of aromatic stretching vibrations seen in the region of 1620 to 1210 cm−1. Many of these bands are assigned to aromatic stretching vibrations that are also the strongest peaks in the FT Raman spectrum. The UV−visible absorption and fluorescence properties of form B were also characterized, and showed strong absorption below 380 nm and strong fluorescence emission at 404 nm (see Supporting Information). These properties arise from the aromatic chromophores present in I that are likely responsible for the observed resonance Raman enhancement. FT Raman spectra of the excipients in the tablet were obtained from pure reference materials and are compared in the Supporting Information. Colloidal silicon dioxide exhibits an extremely weak and highly broadened Raman spectrum (see Supporting Information) that lacks sensitivity and specificity and, as a result of this and its low level in the formulation, was not analyzed further. The UV Raman spectra of the remaining 4221
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Figure 4. (a) Comparison of the FT Raman spectrum of form B (1064 nm laser irradiation) with the confocal microscopic UV Raman spectrum of the same material (266 nm irradiation). The FT Raman spectrum was obtained in about 18 min using a laser power of 600 mW, and the UV Raman spectrum was obtained in about 128 s using a laser power of 20 mW. A tablet map point from the yellow point shown in (d) is also shown. (b) Composite brightfield optical image of the microtomed 1 mg tablet of I obtained with a 20× objective. The green box and blue box correspond to mapped regions. (c) Brightfield optical image showing the first mapped region. (d) DCLS scores for the distribution of I obtained from a 25 × 25 UV Raman map of the blue region in (c) with 10 μm stage increments (increasing red intensity indicates greater concentration of I). The yellow point denotes the map point from which the bottom spectrum in (a) was obtained, highlighting the strong response of the drug relative to the excipient bands (green arrows). Each spectrum in the map was obtained using 16 scans with a 1 s acquisition time in two windows covering 3500 to 550 cm−1, using a 266 nm laser power of 25 mW. Total map acquisition time was approximately 4 h.
obtained from a point containing a large amount of I is given in the Supporting Information. The scores plot showing SLS distribution in Figure 5 (yellow map) shows evidence of regions of high intensity for this excipient. This is likely caused by its addition to the formulation late in the blending process and the resulting lack of uniform mixing. A spectrum taken from one of the high-intensity SLS points and the DCLS fit is given in the Supporting Information. A microtomed placebo tablet was also mapped and analyzed in the same manner and also showed evidence of large regions of SLS in the UV Raman DCLS scores plot, confirming this finding (see Supporting Information). This example demonstrates that the use of confocal UV Raman microscopy on a typical oral tablet formulation not only allows for mapping of a drug at 0.2% w/w levels where resonance Raman enhancements are available but also allows for observation of improper
excipients in the formulation are also shown in Figure 5. The component for magnesium stearate was included in the DCLS model but led to very weak intensities in its corresponding scores plot and is omitted from Figure 5. The error from the DCLS model shows little residual intensity, with typical errors across the mapped region in the 2% range. The distributions of the excipients across the tablet can be seen to be relatively even, with the major excipients (mannitol and microcrystalline cellulose) appearing to be generally exclusive of each other in that where one is at a maximum, the other is less concentrated. The distribution of I (red map) correlates most clearly with the distribution of microcrystalline cellulose (blue map), suggesting the two may be physically interacting with one another. The mounting putty shows a single broad band at about 1610 cm−1, leading to the additional intensity around the circular tablet area, but is spatially resolved and does not affect the distribution analysis of I. An example spectrum and DCLS fit 4222
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Figure 5. DCLS scores plots of the components from a UV Raman map (230 × 230 points) of the 1 mg tablet of I. The map was acquired with 1 scan and a 1 s detector exposure time, and used a single spectral window centered at 1100 cm−1. The total map acquisition time was 18 h. The scores plots represent the distribution of the following components: I (red), mannitol (green), microcrystalline cellulose (blue), sodium starch glycolate (purple), and sodium lauryl sulfate (yellow). The orange map at the bottom right represents the error. The scores for magnesium stearate were minimal and are not shown, and colloidal silicon dioxide was not included in the DCLS model because of extremely weak Raman scattering from this material.
Analysis of an Amorphous Dispersion Containing 1% w/w Drug. Amorphous solid dispersions of a drug in a
mixing of a key excipient (SLS) at 0.5% w/w levels without the benefit of a resonance Raman enhancement. 4223
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Molecular Pharmaceutics
Article
ment occurs with this polymer. PVP shows moderate absorbance by DR UV−visible spectroscopy at 266 nm, but this electronic transition does not lead to significant resonance Raman enhancements. PVP also shows only weak fluorescence when excited at 266 nm (see Supporting Information). Although this initial assessment considered the crystalline form of II and not the amorphous form present in the dispersion, confocal Raman mapping of the dispersion using 266 nm laser irradiation was expected to yield good sensitivity for the distribution of II in the PVP if a resonance Raman effect also occurs for amorphous II (which could not be easily prepared as a separate phase). The UV Raman spectra of the components in Figure 7a suggests that Raman bands for each component can be observed in both the 3400 to 2800 cm−1 and 1800 to 500 cm−1 regions, although spectral overlap indicates that a multivariate or band ratio technique will be needed. UV−visible and fluorescence characterization of the 1% w/w dispersion of II in PVP was also performed to predict the effects of 266 nm laser irradiation. The 1% w/w dispersion of II in PVP showed evidence of an electronic absorption at approximately 320 nm that was not present in PVP alone by comparison of the similarly observed DR UV−visible spectra of the dispersion and PVP (see Supporting Information). The absorbance at 320 nm is similar to the most red-shifted absorbance observed for crystalline II. This absorbance is thus likely caused by the presence of II in the dispersion. The fluorescence emission spectrum of the bulk powdered 1% w/w dispersion of II in PVP exhibits little emission below 350 nm and suggests that UV Raman spectra will not be obscured even in the 3400 to 2800 cm−1 region (see Supporting Information). The emission maximum of the 1% w/w dispersion of II in PVP appeared at 428 nm with a shoulder at 397 nm. A 20 × 20 UV Raman map of the dispersion is shown in Figure 6b. The map required 64 s per point, and each point utilized two spectral windows covering most of the Raman spectrum from 3500 to 500 cm−1. The total acquisition time was approximately 7 h, and the laser power was 25 mW. Because homogeneity is being assessed in a two-component system, this map is presented as a band ratio plot where the color intensity refers to the ratio of the area between 3400 and 3100 cm−1 (specific for II) over the area of the region between 3090 and 2800 cm−1 (specific for PVP). A multivariate approach could also be used and may be preferable in more complex situations.14,30 The variation in the band ratio map in Figure 6b is amplified to show detail, but the actual effect is barely detectable, as illustrated in Figure 6c. Here the spectra extracted from the points of minimum and maximum band ratio intensity from Figure 6b are compared. The changes in band intensity are minor, indicating a highly uniform distribution of II in the polymer across the map region. This is consistent with the finding by 2D 1H-19F CP-HETCOR SSNMR that a molecular dispersion has formed. A FT Raman spectrum of the bulk dispersion in Figure 6d shows the low intensity of the bands of II in a conventional spectrum, highlighting the approximately 30- to 50-fold resonance enhancement obtained using UV irradiation. In Figure 6d, the spectral region between 1800 and 1150 cm−1 is shown, as only subtle differences could be observed in the FT Raman spectra in the region between 3400 and 2800 cm−1 (see Supporting Information), in contrast to the results using UV irradiation where the bands from II were sufficiently strong to be used for the map in Figure 6b. The strong band at 1612 cm−1 in crystalline II was observed to shift to 1615 cm−1 in the
hydrophilic polymer have become a common and useful formulation approach for active ingredients with undesirable properties in the crystalline state.15−17 Amorphous solid dispersions help promote supersaturated conditions and can improve the aqueous dissolution performance of poorly soluble drugs.15−17 The use of UV irradiation in Raman microscopy can enhance the analysis of these dispersions particularly when the drug is present at low levels. For example, compound II is an EP1 antagonist that exhibits low solubility of
