Four solid forms of tauroursodeoxycholic acid and solid-state transformations: effects of temperature and milling

Abstract

Four solid forms of tauroursodeoxycholic acid (TUDCA) were investigated by X-ray diffraction, morphological analysis, thermogravimetry, differential scanning calorimetry, and near-infrared (NIR) spectroscopic analysis. Results indicated that TUDCA existed as dihydrate (Form I), anhydrate (Form II), amorphous dehydrate (Form III), and amorphous anhydrate (Form IV). Solid-state transformations of TUDCA were extensively investigated and shown to be significantly affected by temperature and milling. Direct transformation of Form I into Form II occurred at high temperatures (100 °C and 150 °C), whereas that of Form II into Form I easily occurred at 25 °C. During milling, Form II was initially transformed into Form I within 10 min and then completely transformed into Form III after 100 min upon exposure to air. Moreover, the transition time from Form I to Form III was shorter than that of the separate milling process of Form I. However, Form II was directly transformed into Form IV when milled in the absence of air. In-line NIR spectroscopy was successfully applied for the rapid analysis of TUDCA solid-state transformations (Form II to Form I and Form IV to Form III) at ambient temperature. Stability investigation showed that the stability of the four TUDCA solid forms varied under different conditions.

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1. Introduction

The solid phase diversity of pharmaceutical molecules, including pharmaceutical polymorphs,^1–6 pseudo polymorphs,^7,8 salts,^9,10 co-crystals,^11–13 and amorphous solids,^14–16 has become a major hotspot in contemporary drug research, because of the unique physicochemical properties, efficacy, and toxic side effects existing in different solid forms. Pharmaceutical solid form screening has rapidly developed into a general approach to enhance the physicochemical properties of drugs,^17,18 which can remarkably affect their solubility^3,19,20 bioavailability,^21–23 hygroscopicity,^20,24 melting point,^3,25 stability,^26,27 and mechanical properties,²⁸ especially their compressibility,^13,20 and tabletability.^29,30 Modification of the critical parameters, including temperature,^31–34 milling,^35–37 moisture,³⁶ and solvent-triggered crystallization,^38,39 to induce the formation of solid phases of various drugs is very significant to develop an optimal drug crystal and control the final quality of drugs. Moreover, to examine the kinetics and thermodynamic stabilities of crystal forms, investigating the solid-state transformation of drugs in varying conditions is highly important.

Recently, solid-state transformation induced by various solvent-free technologies, as environmental-friendly methods to acquire relevant solid forms, including temperature-triggered transformation,^34,40,41 milling,^42,43 thermo-mechanical technology,³¹ extrusion,⁴⁴ and pressure-induced transformation,⁴⁵ has rapidly developed and is attracting increasing attention from researchers. High-temperature-induced solid-state transformation has been reported in several pharmaceuticals. High temperature may alter the structural state of materials, causing morphological changes in crystals,⁴⁶ complete amorphization,^47–49 and solid-state transformation.⁵⁰ Mechanical milling is frequently used in manufacturing procedures in the pharmaceutical industry to reduce particle sizes of drugs and excipients. Additionally, the high levels of mechanical energy generated during milling affect the structure of milled materials, including changes in crystal morphology, increases in the number of crystal defects, alteration in chemical stability, solid-state transformation, and partial or complete amorphization.^37,51,52 Therefore, determining the physicochemical stabilities of different forms and the process of their transformations at various conditions is essential. Moreover, special attention should be given to the possible solid-state conversion of drugs induced by high temperature and milling.

Tauroursodeoxycholic acid (TUDCA; Fig. 1), an endogenous hydrophilic bile acid normally produced at very low levels in humans,⁵³ is widely used to treat cholelithiasis and cholestatic liver diseases,^54,55 because TUDCA can attenuate endoplasmic reticulum stress.⁵⁶ Many detailed reports on the pharmacological effects of TUDCA have demonstrated that this drug exerts potential therapeutic effects against various diseases, including Huntington’s disease,⁵⁷ type 2 diabetes,⁵⁸ and inflammatory diseases of the central nervous system.⁵⁹ Solid forms of drugs can significantly affect the clinical efficacy,⁶⁰ toxic side effects,⁶¹ and quality of medicines.⁶² Hence, the solid forms and solid-state transformation of TUDCA should be investigated. The crystal structure of one solid form of TUDCA (denoted as Form I) was first reported in 2000.⁶³ We previously studied two TUDCA solid forms, namely, Form I and its amorphous form (denoted as Form III).⁶⁴ The molecular structure of TUDCA indicated that the conformationally flexible TUDCA molecules may exhibit multiple crystal forms and the ability for conversion among solid forms.

Fig. 1 Molecular structure of TUDCA.

In the present study, four solid forms of TUDCA were characterized by a variety of analysis methods, providing a way to examine the solid forms of TUDCA. Solid-state transformations of TUDCA under varying conditions were extensively investigated, and two new solid forms (II and IV) were observed. The mechanisms of solid-state transformations of Form II into Form I and Form IV into Form III at room temperature were investigated using in-line near-infrared (NIR) spectroscopy combined with time-resolved X-ray powder diffraction (XRPD) and morphological analysis. Furthermore, the effects of temperature and milling on the solid-state conversion of TUDCA were investigated in detail, and the relative stability mechanism of the four TUDCA solid forms was discussed.

2. Experimental section

2.1 Materials

TUDCA (Form I) with a purity of 98% was purchased from Meryer Co., Ltd. (Shanghai, China). Ultrapure water (18 MΩ resistivity obtained from a Millipore system) was used throughout the experiment. All reagents were of analytical grade.

2.2 Methods

2.2.1 Preparation of TUDCA solid forms. Form I (single crystals) was obtained through slow crystallization of an aqueous solvent of TUDCA at 4 °C as described in our previous work.⁶⁴ Form II was obtained by heating Form I (from the supplier) for 4 h at 100 °C. Form III was prepared by drying a viscous high concentration of TUDCA methanol solution in a vacuum oven at 50 °C; Form III can also be obtained by mechanical grinding Form I for 12 h.⁶⁴ Form IV was obtained by milling Form II in a sealed ball milling jar for 2 h.

2.2.2 Solid-state transformation experiments under varying temperatures. To investigate the effect of temperature on solid-state transformation, Form I, Form II, and the amorphous forms (approximately 100 mg each) were initially deposited on the XRPD holder, pressed with a glass slide to avoid the influence of sample preparation on the data about solid-state conversion, and then subjected to varying temperatures. The samples were monitored every 20 or 30 min through XRPD detection. The transformation of Form II into Form I and Form IV into Form III at room temperature was investigated using in-line NIR spectroscopy combined with time-resolved XRPD and morphological analysis.

2.2.3 Milling experiments. Milling experiments were performed using a planetary mill with our previously described methods.⁶⁴ Each milling of Form II (2 g) was performed in a sealed jar at room temperature. The samples were milled for 10, 20, 30, 40, 50, and 60 min to evaluate the effect of milling on Form II.

To investigate the effect of the existence of water molecules in the air on the milling of Form II, Form II was manually milled. Manual milling experiments of Form II (2 g) were performed for 10–100 min using an agate mortar and pestle (160 mm) at room temperature.

2.3 Analytical techniques

Optical microscopy, scanning electron microscopy (SEM), XRPD, single-crystal X-ray diffraction (SXRD), thermogravimetry (TGA), differential scanning calorimetry (DSC), and NIR spectroscopy were performed in accordance with our previously described methods.⁶⁴ For the qualitative analysis of NIR spectra of the four TUDCA solid forms, powder samples were loaded in glass vials (15 mm × 45 mm) for detection, and each spectrum was collected with interleaved scans at 10 000–4000 cm⁻¹ range and 8 cm⁻¹ resolution by using 32 co-added scans. Each sample was measured in triplicate, and the mean spectrum was used in the final analysis. Moreover, for in-line and real-time monitoring of the solid-state transformations of Forms II and IV, the samples were loaded in a sample cup with a diameter of 2.8 cm; spectra were continuously collected in a similar wave-number range and resolution. The solid-state transitions of Forms II and IV were monitored for 50 min and 100 min, respectively. For off-line characterization, the samples were detected once every 10 min using XRPD and an optical microscope.

Data analysis of NIR spectra was performed using the TQ Analyst 8.0 chemometric software (Thermo Nicolet, USA). To qualitatively analyze the four forms of TUDCA, the 160 spectra obtained from 40 samples of each TUDCA form were split into a calibration set (120) and a prediction set (40). Discriminant analysis (DA) combined the principal component analysis (PCA) and Mahalanobis distance (MD) for the qualitative analysis of Forms I–IV of TUDCA. Moreover, the spectra acquired from the in-line process of Form II and Form IV’s solid-state transformations were analyzed using PCA.

3. Results and discussion

3.1 Characterization of TUDCA solid forms

3.1.1 Shape and surface morphology. The photomicrographs and SEM micrographs of the four TUDCA solid forms are shown in the lower images of Fig. 2, where the tabular, prismatic, and transparent crystal structure of Form I (Fig. 2a) can be observed. Form I gradually transformed into Form II with extended heating time at 100 °C; in addition, the transparency of the crystal granules gradually disappeared. The opaque Form II (Fig. 2b) ultimately formed after 3 h of heating. From the bottom half of Fig. 2c, the Form III obtained from methanol (left) has an irregular thin shape and transparent flakiness, and the Form III obtained by mechanical milling (right) is irregular white powder. Form IV is also irregular white powder (Fig. 2d).

Fig. 2 Crystal morphologies (the lower images, the scale bar in the bottom right corner of photomicrographs represents 200 μm, and the SEM micrographs of the amorphous TUDCA obtained through mechanical milling were magnified by 12 000×) and XRPD patterns (upper images) of TUDCA: (a) Form I, (b) Form II, (c) Form III, and (d) Form IV.

3.1.2 XRPD analysis. Accurate identification of the solid forms of TUDCA is very crucial in this study. Fig. 2 (upper images) shows the XRPD patterns of the solid forms of TUDCA. Form II showed major 2θ peaks at 5.68°, 11.30°, 13.84°, 15.10°, 17.41°, 18.58°, 21.23°, 23.85°, and 27.13°, which are specific for Form II and different from Form I; Form IV showed major 2θ wide peaks at 6.20° and 15.91°, which are also different from Form III.⁶⁴ The positions of the peaks in the XRPD patterns of the crystal form correspond to the periodic spacing of atoms in the solid state, and varying lattice constants will in turn generate varying peak positions. These results indicate that Forms I and II are the different crystal forms of TUDCA on the basis of the varying and observable sharp peaks in their XRPD patterns, whereas Forms III and IV are the amorphous forms of TUDCA on the basis of the absence of any sharp peaks in their XRPD patterns. Moreover, the distinct differences of the four powder diffractograms indicate that the four solid forms of TUDCA have distinctive spatial arrangement, allowing for their rapid identification.

3.1.3 Single-crystal structure of Form I. Form I exhibits a monoclinic P2₁ space group with the following unit-cell parameters: a = 17.2850 Å, b = 8.3429 Å, c = 9.6537 Å, β = 91.1450°, unit-cell volume V = 1391.85 ų, and Z = 2.⁶⁴ The crystal structure was assembled through various complex hydrogen bonds (indicated by blue dashed lines) (Fig. 3a and c). One TUDCA molecule and two water molecules comprise the asymmetric unit (Fig. 3b). Two states of water molecules (hydronium ion (H₃O⁺) and water monomer (H₂O)) are present in the crystal structure. Water plays an important role in the formation of supramolecular architectures and bridges among the neighboring TUDCA molecules. Every hydrogen atom of the H₃O⁺ is engaged in O–H⋯O hydrogen bonding, that is, it associates with the oxygen atom of amide in the TUDCA molecule through O–H⋯O3, forms O–H⋯O6 hydrogen bonds via an oxygen from the sulfonic group, and links the oxygen atom of a hydroxy to form O–H⋯O1 hydrogen bonds, respectively. Likewise, the two hydrogen atoms on the H₂O molecule, which serves as hydrogen bond donor to the sulfonic oxygen atom (hydrogen bond acceptor), connect the two neighboring TUDCA molecules (O–H⋯O4 and O–H⋯O5 hydrogen bonds) forming an opposite zigzag arrangement; the oxygen in the water molecule associates with hydroxyl hydrogen to form the O–H⋯O8 hydrogen bond. In addition, the adjacent and opposite arrangement of TUDCA molecules assemble through N–H⋯O6 interactions in an opposite zigzag fashion along the ac plane. Unfortunately, the detailed structure of Form II was not obtained because Form II was unstable.

Fig. 3 Crystal structure of TUDCA Form I as revealed by SXRD. (a) Packing diagram. (b) Asymmetric unit with segmental atom-numbering scheme. (c) Magnified portion of the packing diagram (boxed region of (a)) showing the local hydrogen-bonded motifs (indicated by blue dashed lines).

3.1.4 TGA and DSC analyses. Fig. 4 shows the TGA and DSC thermograms of Forms I–IV of TUDCA. The TGA thermograms (Fig. 4a) show that the mass reductions of the first period of Forms I and III are 6.75% and 6.67%, respectively. These results are consistent with the theoretical mass reduction of 6.72% caused by the loss of two water molecules from the dihydrate (Form I) of TUDCA.⁶⁴ Furthermore, the two water molecules in Forms I and III were simultaneously lost. The changes in mass with increasing temperature in Forms I and III are nearly the same across the temperature range. Therefore, combining with the results of the single crystal structure analysis of Form I, the same shape of the mass change of Forms I and III demonstrates that Form III also exists as dihydrate. Moreover, the mass change temperature of Form III in the first period is significantly lower than Form I, elucidating that water molecules are more stable in Form I than in Form III. The total change trend of the TGA thermogram of Form II is similar to those of the thermograms of Forms I and III except for the mass reduction at the first stage, indicating that Form II is the anhydrated form of TUDCA. Form IV was obtained by milling Form II in a hermetically sealed agate ball milling jar; thus, Form IV is also an anhydrated form of TUDCA. However, the mass reduction in the first period of Form IV was 2.78% (a small part of Form IV rapidly transformed into Form III when the test Form IV was sampled and weighed). Moreover, the mass reductions in the first period of Form IV would increase to 4.64%, 5.56%, and 6.62% (similar to the TGA thermograms of Form III) when exposed to air for 10, 30, and 60 min, respectively. This phenomenon indicates that Form IV is considerably unstable and easily transforms into Form III. Furthermore, the mass reductions in the first period of Form IV did not change after exposure to air for over 60 min, demonstrating that Form IV completely transformed into Form III after 60 min. Fig. 4b shows the DSC thermograms of the four solid forms of TUDCA. Compared with Forms I and III, the DSC thermograms of Forms II and IV did not display any obvious characteristic endotherm peaks below 190 °C. By contrast, Form I exhibited a characteristic endotherm at 150.8 °C and Form III displayed an extremely broad endotherm at 93.3 °C. The endotherm peaks at approximately 190 °C, which are present in all solid forms of TUDCA, belong to the characteristic endotherm of non-aqueous TUDCA. The results of DSC analysis are consistent with those of TGA analysis.

Fig. 4 (a) TGA and (b) DSC curves of Forms I, II, III, and IV of TUDCA.

3.1.5 NIR spectroscopy analysis. NIR spectroscopy is widely used in the chemical and pharmaceutical industries to assess the quality of various products; this analytical technique is also crucial in the current drug research. We observed a few differences in the raw NIR spectra of the four solid forms of TUDCA (Fig. 5a). The spectral absorbency is significantly higher in Form I than in the three other forms; Form II comes second. The bands at 7131, 7047, 6560, 6510, 5813, 5708, 5130, and 4883 cm⁻¹ are characteristic of the raw spectra of Form I, whereas the bands at 7124, 7035, 6561, 6507, 5797, and 5728 cm⁻¹ that are shifted to lower wavenumbers are characteristic for Form II. Moreover, the strong peaks at 5130 cm⁻¹ (Form I) were lost in the raw NIR spectrum of Form II. Compared with that of Forms I and II, the spectral characteristic absorbency of the amorphous forms is extremely scarce, and some differences in the raw NIR spectra of the amorphous forms also exist, especially for the bands at 5126 and 7174 cm⁻¹ of Forms III and IV, respectively.

Fig. 5 (a) NIR raw spectra of the four solid forms of TUDCA and (b) the various solid forms projected onto PC1 and PC2 scores computed by PCA.

DA was combined with PCA and MD to qualitatively analyze the four solid forms of TUDCA. The samples of Forms I, II, III, and IV in the calibration set were assigned as 1, 2, 3, and 4, respectively, which represent four classifications. The developed discriminant models were applied to assign the prediction set samples to their classifications. The result indicates 100% prediction accuracy in 40 external samples in the prediction set. Fig. 5b shows the spectra of four forms projected onto PC1 and PC2 scores computed by PCA. The four forms of TUDCA were clustered using two PCA factors that explained 99.41% of the variability within these samples (93.40% by PC1 and 6.01% by PC2), indicating the feasibility of the model for the characterization of various solid forms of TUDCA. Therefore, NIR spectroscopy is suitable for qualitative analysis of the solid forms of TUDCA.

3.2 Effect of temperature on TUDCA solid forms

3.2.1 Effect of temperature on Form I. The effect of temperature on the solid-state transformation of Form I was investigated. Form I is relatively stable because no solid-state changes were observed when subjected to 50 °C for up to 24 h. However, when the temperature was raised to 100 °C and 150 °C, solid-state transformation of Form I was observed and the transformation rate increased with increasing temperature.

Temperature-triggered solid-state conversion was investigated in detail every 20 min for a total of 180 min to study whether Form I solid-state transformations are direct crystal–crystal transformations, an intermediate stage of transient amorphization, or a transient unstable crystal phase generated at temperatures of 100 °C and 150 °C. XRPD patterns shown in Fig. 6b show that the clear characteristic diffraction peaks of Form II at 5.68° and 11.30° 2θ appeared in the first 20 min and the intensity of peaks increased with time. The major diffraction peaks of Form I at 5.14° and 10.25° 2θ gradually decreased with increasing time and completely disappeared after 180 min of heating time at 150 °C. This phenomenon indicates that Form I gradually transformed to Form II over an extended time at 150 °C and was almost completely converted after 160 min. Moreover, the rate of transformation was very rapid in the first 80 min and no new diffraction peaks were observed arising throughout the remainder of the process. These results indicate that an unstable intermediate crystal form is not observed in the temperature-triggered solid–solid conversion, and the solid-state transformation of Form I is a direct crystal–crystal transformation. The process of solid–solid transformation was also investigated at a temperature of 100 °C as shown in Fig. 6a, and the result indicated that the conversion is also a direct crystal–crystal transformation process of Form I into Form II. However, an extended heating time was necessary for the generation of Form II, which began to appear at 80 min and was almost completely converted after 180 min. Furthermore, Form II did not undergo any change with extended heating time at 100 °C and 150 °C after 180 min. Conversion of Form I to an amorphous form is quite possible in the transformation process from Form I into Form II because water loss from Form I potentially results in the continuous disruption of crystal lattice, destruction of periodicity, and production of a disordered state in the transition process of Form I into Form II; however, generation of an amorphous form did not occur in the process. Moreover, based on the differences of the two water molecules in the crystal structure of Form I, in theory, water molecules would be lost from the sample of Form I one by one. Counterintuitively, two water molecules in Form I are simultaneously lost at high temperatures, because of the absence of an intermediate metastable state in the transformation from Form I to Form II. The monohydrate of TUDCA was also not observed in the conversion procedure of Form I to Form II. Thus, Form I solid-state transformation represents a direct crystal–crystal transformation at high temperature.

Fig. 6 XRPD patterns of the transformation of Form I into Form II at various time points (the curves from bottom to top: Form I–Form II, detected once every 20 min with time ranging from 0 min to 180 min) in different temperature conditions. (a) 100 °C and (b) 150 °C.

3.2.2 Effect of temperature on Form II. The influence of temperature on Form II was also investigated to understand the mechanism of Form II solid-state transformation. The results indicate that Form II is observed to be relatively stable when placed at 100 °C and 150 °C because no solid-state changes were observed. However, the solid-state transformation of Form II into Form I was clearly observed at 50 °C and at room temperature, as shown in Fig. 7 and 8.

Fig. 7 XRPD patterns of the transformation of Form II into Form I at various time points (the curves from bottom to top: Form II to Form I) when Form II was placed at 50 °C.

Fig. 8 (a) Overall changes in the raw NIR spectra of Form II during the 0–40 min transition process at room temperature; (b) peak intensity changes for the NIR bands at 5130 cm⁻¹ specific to Form I at different time points; (c) PCA plot for the whole conversion process of Form II (the red points represent the position of every photomicrograph) and photomicrographs (the scale bar in the bottom right corner represents 200 μm) obtained from the off-line characterizations using optical microscopy; (d) XRPD patterns of samples during the Form II transformation process (the curves from bottom to top: detected once every 10 min).

The XRPD patterns in Fig. 7 show that the characteristic diffraction peaks of Form I at 5.14° and 10.25° 2θ gradually appeared and the intensity of these peaks increased with time. Conversely, the major diffraction peaks of Form II at 5.68° and 11.30° 2θ gradually decreased with increasing time and completely disappeared after 150 min at 50 °C. This phenomenon indicates that Form II gradually transformed to Form I with extended time at 50 °C. Moreover, no new diffraction peaks appeared through the entire solid-state transformation process. Two water molecules in Form II are simultaneously connected to the anhydrate form of TUDCA (Form II) from the transformation of Form II into Form I at 50 °C. Thus, the monohydrate of TUDCA was also not observed in the conversion of Form II into Form I. This result indicates that the unstable intermediate crystal form is not observed in the conversion, and the solid phase transformation of Form II is a direct crystal–crystal transformation. Furthermore, Form I did not undergo any change with extended time at 50 °C after 150 min. The solid-state transformations of Form II and Form IV were also investigated at room temperature, as shown in the following Sections 3.2.3 and 3.2.4.

3.2.3 In-line NIR spectra and off-line characterization of Form II to Form I. In-line NIR spectroscopy was found to be an effective technology for solid-state transformation research in pharmaceutical production, specifically in monitoring the detailed conversion procedure of solid drug forms.^65–67 In the present study, in-line NIR spectroscopy in combination with off-line XRPD characterization and morphology analysis was used to monitor solid-state conversion of Form II at room temperature, considering that the transformation rate of Form II into Form I is rapid, and that the fast and non-destructive NIR spectroscopy analytical technique exhibits good capability in detecting and analyzing the detailed process of rapid conversion. The overall changes of untreated NIR spectra at every minute from 1 min to 40 min are shown in Fig. 8a, which indicated that the spectrograms were significantly altered as a result of increasing conversion time and upward shifting. In addition, the NIR spectra were nearly invariable after 40 min. The NIR band at 5130 cm⁻¹, which is a characteristic absorbency for Form II, was also used to monitor the transformation of Form II into Form I, as shown in Fig. 8b, which clearly shows that the final solid form (Form I) content of the mixture rapidly increased in the first 35 min and that after 40 min, the starting solid form (Form II) was completely transformed to Form I. Moreover, the absorbency intensity at 5130 cm⁻¹ remained constant after 40 min, further showing that the transformed solid form (Form I) did not undergo any change with extended time.

PCA was used to discriminate the transformation of Form II into Form I based on conversion time. The typical PCA score plots of transformation of Form II into Form I are shown in Fig. 8c, which shows that different transformation rates were clustered using 2 PCA factors that explained 99.06% of the variability within the samples, 96.18% by PC1 and 2.88% by PC2, indicating that the model for the characterization of different Form II transformation contents of TUDCA was feasible. For the transformation process of Form II into Form I, data points initially moved rapidly from the top right to the bottom center with large interval distance, and then changed with a slight spatial variation, in which the speed of conversion from Form II to Form I gradually decreased, and finally clustered and reached the end point after 40 min, which is consistent with the results shown in Fig. 8a and 7b.

For the off-line characterization using optical microscopy and XRPD, the samples were detected at designated time points at 0, 10, 20, 30, 40, and 50 min. Overall, the results were reproducible. Fig. 8c shows the photomicrographs of transformation from Form II to Form I versus process time. The starting solid form (Form II) was a tabular, prismatic, and opaque crystal structure, as shown in the top right of Fig. 8c. With extended time, the opaque crystal granules gradually transformed into a transparent solid form step by step and the shape and surface morphology detected at 40 min was similar to 50 min, which indicates that Form II increasingly changed into Form I and was completely transformed after 40 min. As shown in Fig. 8d, the peak strength of samples at 5.68° and 11.30° 2θ characteristic of Form II decreased with prolonged time and the diffraction peaks almost completely disappeared after 40 min. The major diffraction peaks of Form I at 5.14° and 10.25° 2θ gradually appeared with increasing time and became completely invariant after 40 min. These results are consistent with the results of the in-line NIR spectra analysis and morphology analysis. Thus, Form I was generated from Form II by an extended time at room temperature and Form II was almost completely converted to Form I after 40 min. Moreover, no new diffraction peaks were observed to arise in the entire transition process, which indicated that the unstable intermediate crystal form was not observed in the process and the transformation of Form II to Form I is a direct crystal–crystal transformation process.

3.2.4 In-line NIR spectra and off-line characterization of Form IV to III. The clear transformation of amorphous to crystal TUDCA was not observed within a short period of time (24 h) when Forms IV and III were placed at 50 °C, 100 °C, and 150 °C. The thermodynamically unstable amorphous forms of TUDCA exhibit a crystallization tendency of transition, for example, Form III (amorphous) is converted to Form I (crystal) in long-term storage stability tests.⁶⁴ In the TGA characterization of TUDCA solid forms (Section 3.1.4), Form IV was very unstable, and easily and rapidly converts to another solid form at room temperature. Moreover, DA combined with PCA and MD is suitable for qualitative analysis of TUDCA solid forms. Based on preliminary analysis, the final solid form transformed from Form IV at room temperature was Form III. In general, XRPD is considered as the standard technique for the analysis of different solid forms. However, the differences between Form IV and Form III were very small, and were difficult to distinguish (Fig. 9d). In addition, the transformation rate is very fast in the conversion of Form IV into Form III at room temperature so the whole transformation process was not monitored. Therefore, the XRPD technique was not able to detect and analyze the detailed transformation of Form IV into Form III because of the small differences and rapid conversion. Alternatively, in-line NIR spectroscopy is an effective technology for solid-state transformation research, specifically in monitoring the detailed and fast conversion process of solid drug forms. To understand the transformation mechanism of Form IV at room temperature, a fast and non-destructive in-line NIR spectroscopy analytical technique was used to detect and analyze the detailed process of rapid conversion.

Fig. 9 (a) Overall changes in raw NIR spectra of Form IV during the 0–100 min transition process at room temperature; (b) peak intensity changes for the NIR bands at 5129 cm⁻¹ specific to Form IV samples at different time points; (c) PCA plot for the whole conversion process from Form IV into Form III; (d) XRPD patterns of samples during the transformation process (the curves from bottom to top: detected once every 10 min).

All NIR spectra of Form IV for different periods of time for a total of 100 min were obtained in the wavenumber range of 4000 to 10 000 cm⁻¹ to monitor physicochemical changes during the whole solid form conversion process at room temperature (Fig. 9a). First, the spectrograms were altered as a result of increasing conversion time and upward shifting. On the other hand, NIR spectra were nearly invariable after 60 min. The NIR band at 5129 cm⁻¹, which is a characteristic absorbency for Form III, was also used to monitor the transformation of Form IV into Form III. As shown in Fig. 9b, the conversion rate was very fast in the first 13 min because of the considerable increase in intensity at 5129 cm⁻¹. With prolonged time, the changes in peak intensity eased up slowly. However, absorbency intensity at 5129 cm⁻¹ remained constant after 60 min. This phenomenon indicates that Form III content in the sample rapidly increased in the first 13 min, the rate of transformation of Form IV into Form III gradually decreased from 13 to 60 min, and that after 60 min, the starting solid form (Form IV) was almost completely transformed to Form III. Moreover, the absorbency intensity at 5129 cm⁻¹ remained almost constant after 60 min, further illustrating that Form III did not change with extended time at room temperature.

Typical PCA score plots of the transformation of Form IV into Form III are shown in Fig. 9c. Each data point signifies a single NIR spectrum obtained every 1 min. NIR spectra from 5850 cm⁻¹ to 5050 cm⁻¹ were selected to perform the conversion process analyses. The results showed that different transformation contents of Form IV were clustered using 2 PCA factors that explained 94.50% of the variability within the samples, 75.95% by PC1 and 18.55% by PC2, indicating that the model for the characterization of the different contents of Form III in the mixture of Forms IV and III and the process of monitoring the transformation from Form IV into Form III was feasible. Within 0 min to 60 min, data points initially moved quickly from the top right to the bottom center with large interval distance in the first 30 min (especially in the first 10 min), and then changed with a slight spatial variation and gathered into one stack, in which the speed of conversion from Form IV into Form III gradually decreased. In addition, plot points also changed slowly after 60 min, and clustered in the range of 60 min to 100 min, which indicated that Form III was almost fully formed after 60 min. This observation exactly matches the results shown in Fig. 9b. Moreover, the results were consistent with the TGA analysis in Section 3.1.4.

For the off-line characterization using XRPD, the samples were detected at designated time points at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 min. The results were clearly reproducible. As shown in Fig. 9d, sharp peaks did not appear in the whole transformation of Form IV into Form III. This phenomenon indicated that crystallization of amorphous TUDCA did not occur in the conversion. Thus, the conversion of Form IV into Form III is a direct amorphous–amorphous transformation.

3.3 Effect of milling on Form II

To understand mechanically activated solid-state conversion, sealed milling experiments on Form II were performed at room temperature for different periods of time for a total of 60 min. Approximately 2 g of Form II was milled using a planetary mill in the absence of air, and change in milling was monitored using XRPD. The results of the XRPD analysis are shown in Fig. 10a. Peak strength of the samples decreased with prolonged milling duration, and the diffraction peaks almost completely disappeared after 40 min (Fig. 10a). This result indicates that the Form II content was reduced by extended milling time, and Form II was almost completely converted to Form IV after 40 min. Moreover, as milling duration was extended to 60 min, the diffraction peaks became nearly invariable, and no new diffraction peaks appeared through the entire milling process. These results signify that an unstable intermediate crystal form was not observed in this mechanical process, and that the solid-state transformation of Form II is a direct crystal–amorphous transformation.

Fig. 10 XRPD patterns of TUDCA solid-state conversion by (a) ball-milling with a planetary mill in the absence of air, and (b) hand milling with exposure to air at various milling times (the curves from bottom to top: detected once every 10 min).

To investigate the influence of water molecules in air on the process of milling Form II, hand milling of Form II was performed and examined at room temperature, and monitored using XRPD. The results of XRPD analysis for samples at various milling times are shown in Fig. 10b. Form II quickly converted into Form I in 10 min. The results indicate that Form II is more easily transformed to Form I within a short period of time after milling. This phenomenon is probably because milling causes a decrease of sample particle size and increase of surface area of the milled sample, resulting in the increase of contact area of Form II and water molecules in air, speeding up the rate of transformation of Form II into Form I. Clearly, the peak strength of the samples decreased with prolonged milling time after 10 min, and the diffraction peaks almost completely disappeared after 100 min of milling time. This observation indicated that Form II can translate to an amorphous form with an intermediate stage wherein Form II initially changed to Form I in a short span of time (10 min), and then transformed into Form III after 100 min of milling. The water molecules in air resulted in the transformation of Form II to Form I. Moreover, the transition time from Form I to amorphous form in the milling process was significantly shorter than the time of generating the amorphous form by separately milling Form I. This phenomenon is probably because the intermediate stage of Form II to Form I accelerated formation of the disordered state. Thus, the transformation of Form II via hand milling at room temperature is a crystal–crystal–amorphous transformation.

3.4 Stability study of TUDCA solid forms

Temperature-induced solid-state transitions among the four TUDCA solid forms were investigated. The results revealed that Form II is the most stable form at high temperature. However, Form II is the most unstable form at room temperature when the samples were exposed to air and transformed into Form I within a short period. The stability of Form II was also investigated when the samples were sealed to remove the influence of air. The results indicate that the transition of Form II into Form I did not occur, suggesting that the reason for the transition of Form II into Form I in air is that the water molecules in air led to the formation of Form I and two water molecules were added into the crystal structure of Form II simultaneously because of the absence of an intermediate solid state. The storage stability of Form III was studied in a previous work.⁶⁴ Results showed that Form III will convert to Form I by extending storage time, and the stability of Form III decreased and the rate of transformation increased with rising temperature, indicating that Form III is not stable and will transform to the crystal state of TUDCA (Form I). The amorphous forms did not change to crystal forms in a short period (24 h) under high temperatures (50 °C, 100 °C, and 150 °C). However, Form IV transformed into Form III at room temperature, which is a direct amorphous–amorphous transformation. Milling produced different amorphous solid forms from Forms II and I at room temperature, and new solid forms were not observed with prolonged milling time after formation of the amorphous solid forms, which suggests that the amorphous forms of TUDCA were induced and remained stable through the mechanical process. Moreover, Form II converted to a dihydrate amorphous form (Form III) when exposed to air, whereas the anhydrate amorphous form (Form IV) was obtained when kept and milled in the absence of air. The conversion of Form II into Form III via hand milling may occur faster through the formation of Form I, in which generation of Form I accelerated formation of the disordered state. Consequently, the stability order via milling at ambient temperature was Form II < Form I < amorphous forms. Theoretically, the amorphous solid-state forms are thermodynamically unstable systems, and will transform to the crystal form. An amorphous–crystal transformation process occurred when the amorphous forms were stored at a certain temperature for long periods of time, such as the transformation of Form III into Form I, whereas mechanical action (milling) can induce formation of amorphous solid forms and prevent transformation from amorphous form to crystal state.

4. Conclusions

In this study, temperature-induced solid-state transformations of TUDCA were observed, and a new solid form (Form II) was produced at high temperature. A new amorphous form (Form IV) was also obtained when Form II was milled under isolated air. In combination with Forms I and III, Forms II and IV were systematically characterized and examined using optical microscopy, SEM, XRPD, DSC, TGA, and NIR spectroscopy. The results indicated that the new solid forms were anhydrate forms of TUDCA.

Milling and temperature can significantly influence the transformation of TUDCA solid forms. Solid-state transformation of Form I into Form II was observed at 100 °C and 150 °C; clearly, the rate of transformation increased with rising temperature. Furthermore, Form II was easily converted into Form I at room temperature after 40 min upon exposure to air, and this process presented a direct crystal–crystal transformation. Hence, Forms I and II of TUDCA can be converted into one another under different conditions. The transformation of Form II into Form I was monitored and analyzed in detail via in-line NIR spectroscopy combined with off-line morphology and XRPD analyses. Two water molecules in Form I were simultaneously lost during the transformation of Form I into Form II at high temperature because of the absence of an intermediate metastable state. Similarly, two water molecules in Form II were simultaneously connected to Form II (anhydrate form of TUDCA) from the transformation of Form II into Form I at 50 °C and room temperature. Thus, the monohydrate of TUDCA was not observed in these conditions. In the solid-state transformation of Form IV into Form III at room temperature, a direct amorphous–amorphous transformation occurred. Additionally, the conversion from Form IV into Form III at room temperature was very fast. Milling induced solid-state transformation of Form II into amorphous form, and this transformation was monitored by XRPD. The milling results indicated that Form II first transformed into Form I during the first 10 min and then changed into Form III upon exposure to air, whereas it would directly convert into Form IV when isolated from air exposure. Form II was relatively stable under high temperature, as well as isolation from air exposure, but it rapidly changed into Form I when exposed to air at room temperature.

Acknowledgements

This work was supported by the Applied Basic Research Project of Sichuan Province (Grant No. 2014JY0042), the Testing Platform Construction of Technology Achievement Transform of Sichuan Province (Grant No. 13CGPT0049), and the National Development and Reform Commission and Education of China (Grant No. 2014BW011). We thank the College of Polymer Science and Engineering, Sichuan University, for providing instrumentation and infrastructure for the thermal analysis.

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