3.1. Visual Evaluation of LVODT and LVT Formulations by Microscopic IR Spectroscopy
The distributions of LV, the API in LV
ODT and LV
T formulations, were visually evaluated by microscopic IR spectroscopy.
Figure 1 shows the standard IR spectra of LV and MCC. The LV standard (LV
ST) showed a characteristic peak at 3200–3500 cm
−1 derived from the hydroxyl groups of the carboxylic acids and crystalline water. In addition, a sharp peak that was observed between 1700 cm
−1 and 1800 cm
−1 for LV
ST derived from carbonyl group was completely absent in the MCC spectrum. Therefore, we determined that the distribution of LV could be visually evaluated by creating a mapping image using the values within the peak area from 1700 cm
−1 to 1800 cm
−1 (PA
1700–1800) as an index.
Figure 2a shows the microscopic image, and
Figure 2b,c show the mapping images of the LV
ODT, respectively. To confirm the distribution of API in LV
ODT, we created a mapping image using the PA
1700–1800 values as an index (Map 1,
Figure 2b). High- and low-area regions (red and blue, respectively) were observed in Map 1 of LV
ODT and were clearly distinguishable from each other (
Figure 2b). Furthermore, the distributions of the red region and that of the white granules in the microscopic image (
Figure 2a,b) matched perfectly. We also created a mapping image using the correlation to the MCC standard spectrum (Map 2;
Figure 2c). In Map 2, a clear separation of the highly correlated (red) and low-correlated (blue) regions was obtained, similar to Map 1, but their distributions was in complete contrast to that of Map 1. Furthermore, the distribution of the red regions in Map 2 matched perfectly with the areas other than the white granules in the microscopic images (
Figure 2a,c). The spectrum obtained from the red regions of Map 1 was similar to the standard spectrum of LV (
Figure 2d), and the spectrum obtained from the blue regions was similar to the standard spectrum of MCC (
Figure 2e). These results suggest that the structure of LV
ODT was such that the API-containing granules with particle sizes of several hundred micrometers are distributed within the formulation.
However, in LV
T_TOWA, although red and blue region distributions were observed in both Map 1 and Map 2, there was no correlation with the microscopic images (
Figure 3a–c). In both spectra obtained from the pixels in the red and blue regions of Map 1 (
Figure 3b), a clear peak in the range of 1700–1800 cm
−1 was observed, which was different from that of LV
ODT (
Figure 3d,e). These results suggest that the LV
T_TOWA spectra contained information on both the API and non-APIs at all measurement points and that LV and non-API particles were uniformly distributed; however, slight differences in the ratios of API and non-API particles at different measurement points were seen. These results were also observed for the other LV
T formulations used in this study (LV
T_CRAVIT, LV
T_NIPRO, and LV
T_SAWAI;
Figures S1–S3, respectively).
Comparing the spectra obtained from the red regions in the mapping images (Map 1) of LV
ODT and LV
T_TOWA revealed differences in the peak shapes in the range of 3200 cm
−1–3500 cm
−1 (
Figure 2d and
Figure 3d). In particular, a clear peak was observed for LV
T_TOWA at approximately 3250 cm
−1, which was also seen in LV
ST (
Figure 1) but not in LV
ODT. This suggests differences in the crystal forms or environments around the API between LV
ODT and LV
ST or LV
T_TOWA.
3.2. DSC and TG-DTA Measurements in Lightly Crushed LVODT and LVT Formulations
Microscopic IR spectroscopy revealed that in LV
ODT, the API-containing granules were well distributed within the formulation. Therefore, we attempted to physically separate the regions of granules containing the API from other than granules containing non-APIs to examine the crystalline forms of the API granules in greater detail. For LV
ST, an endothermic peak was observed at approximately 70 °C. The accompanying mass reduction (
Figure S4a) suggested that this peak was due to the release of crystalline water. An endothermic peak without mass reduction was observed at approximately 235 °C (
Figure 4a,b and
Figure S4a), indicating that it was caused by the decomposition of the API [
9,
10,
11,
12].
For the large particles of LV
ODT, an endothermic peak was observed at approximately 220 °C, which was likely due to the decomposition of API and was approximately 10 °C lower than that of LV
ST; the shape of the peak was also apparently different (
Figure 4a). These differences suggest that the crystalline form or the environment around the API in LV
ODT was different from those of LV
ST determined by thermal analyses. Furthermore, this endothermic peak was observed for large and medium particles but not for small particles (
Figure 4a). The enthalpies of melting calculated from the peak areas for the large and medium particles were 30.4 and 16.1 J/g, respectively, and unquantifiable for the small particles. These results suggest the presence of the API in large and medium particles and that the granules with diameters of several hundred micrometers observed in the microscopic images were contained in these particles. In this formulation, endothermic peaks at approximately 170 °C and 290 °C were observed, which were absent in the LV
ST data (
Figure 4a). The peak at 170 °C was not accompanied by a mass reduction (
Figure S4b–d), indicating an endothermic reaction associated with the melting of mannitol, which is a non-API in LV
ODT (
Table 1). In contrast, the peak at 290 °C was accompanied by a mass reduction (
Figure 4a and
Figure S4b–d), suggesting an endothermic reaction associated with the thermal decomposition of non-APIs containing MCC. These endothermic peaks increased with decreasing particle diameters (
Figure 4a and
Figure S4b–d), suggesting that mannitol and MCC were originally distributed outside of the granules and were present in higher concentrations around particles with smaller diameters due to milling.
For LV
T_TOWA, the onset temperatures of the peaks associated with the release of crystalline water and the decomposition of the API were almost identical from those of LV
ST, with only small differences in the enthalpies of the API for each particle size (32.8, 49.5, and 45.0 J/g for large, medium, and small particles, respectively) (
Figure 4b and
Figure S5). These results suggest that the API was homogeneously distributed in LV
T_TOWA regardless of the particle size and existed in the same crystalline form as LV
ST, which was consistent with the results obtained by microscopic IR spectroscopy.
3.3. PXRD of the LVODT and LVT Formulations
Since results of previous measurements (
Section 3.1 and
Section 3.2) suggested that the crystalline form of the API or the environment around the API differed between LV
ODT and LV
T_TOWA, we used PXRD to obtain thermal measurements of particles in each diameter class (large, medium, and small particles). The spectra of LV
ODT did not match those of LV
ST, with the peak intensities varying with diameter (
Figure 5a). Combined with the thermal analysis results, this suggested that the peak at approximately 7.5°, which became smaller as the particle size decreased, reflected the presence of the API, while the peak at approximately 24°, which became larger as the particle size decreased, reflected the presence of some non-APIs. The absence of the diffraction peak at approximately 7.5° in LV
ST confirmed that the crystal form and the surrounding environment of the API in LV
ODT were different from those of LV
ST. In contrast, the spectra of LV
T_TOWA were almost identical to those of LV
ST at all particle diameters, confirming that the crystal form of the API in LV
T_TOWA was consistent with that of LV
ST, and that the API was uniformly distributed (
Figure 5b). According to the interview forms for each formulation used in this study [
9,
10,
11,
12], the API contained in LV
T_TOWA was LV
0.5, suggesting that LV
ST was also LV
0.5. PXRD measurements of experimentally prepared LV
0.5 and LV
1.0 were performed and compared with the spectra of the LV
ODT particles at different diameters, which revealed diffraction peaks at similar angles of incidence as those of the LV
1.0 for the large and medium particles (
Figure 5a). A specific peak observed for the large and medium particles of LV
0.5 at approximately 6° (
Figure 5a) suggested that the part of the API used in LV
ODT was a transition state from LV
0.5 to LV
1.0.
3.4. Visual Evaluation of LVODT and LVT Formulations by Conventional and LF Raman Microspectroscopy
Next, we tested whether microscopic Raman spectroscopy could provide additional useful information.
Figure 6 shows a mapping image drawn using conventional Raman spectroscopy data of LV
ODT and LV
T_TOWA. For LV
ODT, characteristic spectra were obtained from the regions of white granules and brown nongranular regions in the microscopic images (
Figure 6a).
Figure 6b shows the average spectra obtained from part of the nongranular region and the granular region (areas 1 and 2 of
Figure 6a, respectively). Specific peaks were detected at approximately 1600 cm
−1 and 2900 cm
−1 for the regions with and without granules (areas 2 and 1), respectively (
Figure 6b); Raman images were drawn in red and blue using the intensity of each peak as an indicator. The regions of high intensity at 1600 and 2900 cm
−1 perfectly coincided with the regions with and without granules in the microscopic image, respectively (
Figure 6a). The average Raman spectra obtained from the granule regions (area 2) were almost identical to those of LV
ST (
Figure 6b). However, there was no difference in the spectra of the LV
T_TOWA for various sites, with all of the spectra being almost identical to those of LV
ST (
Figure 6c,d). The results obtained by conventional Raman spectroscopy were similar to those obtained by microscopic IR spectroscopy, with no new information obtained using this technique.
We then performed similar measurements using microscopic LF Raman spectroscopy. For LV
ODT, characteristic peaks were obtained from a thin layer (approximately 20 µm) at the outer edge of the granules in addition to the peaks form the white granules and nongranular regions that were also observed in the conventional Raman spectra (
Figure 7a). The average spectra obtained from the nongranular region (area 1), granules (area 2), and the thin layer at the outer edge of the granules (area 3) had specific peaks at 55, 20, and 150 cm
−1, respectively (
Figure 7b); Raman imaging was, therefore, performed using the intensities of these wavenumbers as indicators, marked in green, red, and blue, respectively. The red region corresponded to the white granules in the microscopic image and the blue layer, which was approximately 20 µm thick, was found on the outer edge of the red region (
Figure 7a). The API-derived spectrum from the red region was similar but not a perfect match to that of LV
ST (
Figure 7b).
The LF Raman spectra of experimentally prepared LV
1.0 included peaks at approximately 25 and 40 cm
−1. The peak at 40 cm
−1 was specific to LV
1.0 (
Figure S6). Moreover, the average spectrum obtained from area 2 included a peak at 52 cm
−1 that appeared to be derived from LV
0.5 (
Figure 7b), strongly suggesting that part of the API used in LV
ODT transition from the LV
0.5 to the LV
1.0, as observed using other methods. In contrast, the shape of the peak at 25 cm
−1 (
Figure S6) differed from that of the spectrum obtained from the red region (
Figure 7b), which was attributed to differences in instrument resolutions.
Furthermore, the spectrum of the blue layer indicated not only the presence of API due to the peak below 50 cm
−1 being consistent with that of the red region, but also the strong characteristic peak around 150 cm
−1. This striking peak was assigned with that of titanium dioxide, the non-API mainly used as mostly sunscreen in tablet coatings (
Figure 7b and
Figure S7). It is notable that the peak was recognized solely in the measurement of LF region, since the peaks of titanium dioxide could be overlapped with other ingredients in conventional region. LV itself is a very bitter compound, making it necessary to mask this bitterness for easy administration as an orally disintegrating tablet. Aminoalkyl methacrylate copolymer E is used as a bitterness-masking agent along with this drug; the granules would be coated with this polymer. The titanium dioxide is a common pharmaceutical ingredient widely used with various polymers for film coating. Therefore, titanium dioxide is likely to coexist with this polymer and could visualize the coating layer in LF Raman measurement.
We showed that the shape of the endothermic peak due to LV melting observed in LV
ODT during the thermal measurements was significantly different from those of LV
ST and LV
T_TOWA (
Figure 4a). This may have been due to the heat transfer to API being not as smooth as that of LV
T_TOWA because of the coating of the granules. In contrast, no obvious differences were observed between the spectra from any of the LV
T_TOWA sites, with all spectra consistent with LV
ST (
Figure 7c,d). These results for LV
T_TOWA by conventional and LF Raman spectroscopy (
Figure 6c,d and
Figure 7c,d) were also observed for other LV
T formulations (LV
T_CRAVIT, LV
T_NIPRO, and LV
T_SAWAI). This demonstrated that the LV
T formulations contain LV
0.5, with a uniform distribution of the API and non-APIs, which was consistent with the results of previous studies using other methods.
The microscopic LF Raman spectroscopy used in this study allowed us to discriminate between the crystalline forms of API in LVODT and LVT, as well as visually analyze the distribution of the approximate 20 µm thickness film coating that covers the outer surface of the granules in LVODT. The results showed that microscopic LF Raman spectroscopy can detect changes in the physical properties of generic formulations, which represents the novel effectiveness of this method in the field of pharmaceutical science.