Rapid Structure Determination of Ranitidine Hydrochloride API in Two Crystal Forms Using Microcrystal Electron Diffraction

Abstract

The solid-state properties of drug candidates play a crucial role in their selection. Quality control of active pharmaceutical ingredients (APIs) based on their structural information involves ensuring a consistent crystal form and controlling water and residual solvent contents. However, traditional crystallographic techniques have limitations and require high-quality single crystals for structural analysis. Microcrystal electron diffraction (microED) overcomes these challenges by analyzing difficult-to-crystallize or small-quantity samples, making it valuable for efficient drug development. In this study, microED analysis was able to rapidly determine the configuration of two crystal forms (Forms 1, 2) of the API ranitidine hydrochloride. The structures obtained with microED are consistent with previous structures determined by X-ray diffraction, indicating microED is a useful tool for rapidly analyzing molecular structures in drug development and materials science research.

Introduction

The solid-state properties of a drug candidate compound are selected through the evaluation of solid-state properties such as crystal polymorphism, salt formation, and solvation, as well as physicochemical properties such as solubility and stability. This information enables a development risk assessment, called a “development assessment,” of a drug candidate compound.¹⁾ For quality control of the drug substance of candidate compounds, it is necessary to ensure constancy of the crystal form and control contents of water and residual solvents. In cases where the compound forms a salt crystal or co-crystal, contents of acidic and basic counterions or co-formers are also evaluated, respectively. The validity of this specification range is ensured by the chemical structure of the drug substance, i.e., its crystal structure. Therefore, the crystal structure of a drug candidate compound is essential information in determining the scientific relevance of a quality control strategy. Single-crystal X-ray diffraction (scXRD) structural analysis and solid-state NMR are frequently used to analyze the solid state of drug candidate compounds. However, scXRD structural analysis requires high-quality single crystals, which can sometimes impede efficient drug development, and while solid-state NMR excels at measuring atomic interactions within a crystal lattice, it cannot determine the precise atomic arrangement in a crystal.

The electron crystallography overcomes these challenges by analyzing difficult-to-crystallize or small-quantity samples, making it valuable for efficient drug development.

Microcrystal electron diffraction (microED) overcomes these challenges by analyzing difficult-to-crystallize or small-quantity samples, making it valuable for efficient drug development.

In recent years, a cryo-electron microscopy technique designated as electron crystallography has attracted attention as a new structural analysis tool for drug candidates.^2–6) There are several methods to obtain electron diffraction patterns from microcrystals with continuous stage rotation such as microED, three dimensional (3D) electron diffraction (3D ED), and continuous rotation electron diffraction (cRED). MicroED can offer structural information at a molecular level with high resolution.^7–12) Whereas conventional scXRD crystallography has limitations for measurable crystal shapes and sizes, microED can analyze crystals of various shapes, leading to expansion of X-ray crystallography for structural analysis of very small crystal samples. Indeed, scXRD crystallography requires large crystals (approximately 1 µm), whereas microED can be applied to nanocrystals (approximately 100 nm).^5,13) Moreover, microED can be used to analyze nano- to microcrystalline powder samples, unlike the requirement for single crystals for conventional X-ray crystallography. Therefore, microED can be applied for structural analyses constrained by crystallization difficulties or an insufficient sample volume. In addition, samples are not required to be pure and mixtures can be employed for structural analysis of their various component compounds. Compared with scXRD methods, structural analysis by microED can be performed rapidly (2–3 min) and obtained diffraction images/patterns can be analyzed in a relatively short time with automated analysis software. Thus, microED is a highly flexible method that overcomes the hurdle of crystallization and allows molecular structures to be directly obtained from nano- to microcrystalline powder samples quickly and with simple operation. Accordingly, microED is useful for analysis of nano- and/or microcrystals of proteins, peptides, and small organic molecules including active pharmaceutical ingredients (APIs).^14–23)

Recently, we used solid-state NMR for structural analysis of crystal polymorphs of ranitidine hydrochloride (Forms 1, 2) as model APIs.²⁴⁾ Intermolecular correlation of spatially close ¹H and ¹⁴N was detected in this analysis of the powdered form of ranitidine hydrochloride, allowing for the rapid discrimination of spatial atomic arrangements. However, this method is limited to specific atomic arrangements previously revealed by scXRD analysis to be in close spatial proximity. Thus, it is difficult to apply solid-state NMR to APIs whose detailed structures are unknown due to differences in drug storage environments and manufacturing methods. Here, we performed microED and investigated atomic arrangements of two ranitidine hydrochloride crystals (Forms 1, 2), which clarifies the strong potential of microED as an atomic level structural analysis method for nano- to microcrystalline powder APIs (Fig. 1).

Fig. 1. MicroED Analysis of Ranitidine Hydrochloride

Whereas scXRD crystallography requires large crystals, microED can analyze nano- to microcrystalline powder sample. The picture was ranitidine hydrochloride polymorphs of form 1 and form 2 observed by scanning electron microscopy (SEM).²⁵⁾ The crystal structure of Form 1 and 2 obtained by microED was a cis conformation and a mixture of cis and trans conformations of nitro groups, respectively.

Results and Discussion

MicroED is a convenient method for characterizing crystal structures at the molecular level using nano- to microcrystalline powder samples, without the need for preparation of single crystals. In this study, we utilized two crystal polymorphs (Forms 1, 2) of ranitidine hydrochloride that were prepared using ethanol as the crystallization solvent. These crystal polymorphs have been previously described in the literature and served as samples for our microED analysis.²⁶⁾ The polymorphic form of obtained crystals was confirmed by powder XRD (pXRD).^25,27–30) The HPLCpurity of each crystal form exceeded 99.6% at a UV wavelength of 235 nm. Form 1 is composed of very small crystals ranging from 1–5 µm in size, which tend to agglomerate and form large fluffy particles. In contrast, Form 2 consists of larger individual crystals with sizes ranging from 10–100 µm.²⁹⁾

The crystalline form of obtained crystals was measured by microED (XtaLAB Synergy-ED, Rigaku Corporation, Tokyo, Japan). Measurement conditions included an accelerating voltage of 200 kV, dose rate of 0.01 electrons per Ų per second, and tilt speed of 1°/s. The measurement itself was very quick, taking only 80 s per crystal. Subsequently, automated structural analysis was performed with Olex2 software (OlexSys, Durham, U.K.) to obtain results quickly and easily. The total measurement and analysis time was 1 h, and high-resolution data were obtained. The measurements were performed more quickly. The three-dimensional structures of crystals obtained through microED were compared with known structures determined by scXRD analysis. The real-space images of Forms 1 and 2 were obtained by transmission electron microscope (TEM) (Fig. 2). For Form 1, our comparison confirmed that the structure obtained through microED had a cis conformation, consistent with the previously reported crystal structure³¹⁾ (Fig. 3). The reliability factor of Form 1 was R₁ = 19.14%. Notably, the R value of XRD is a few percent and that of the previously reported structure was R = 4.16%.³¹⁾ Diffraction data were collected from ten individual ranitidine hydrochloride Form 1 crystals, with each covering approximately 80° of the reciprocal space. To remove diffractions with a low signal-to-noise ratio, the resolution was truncated to 0.8 Å, which was considered adequate. The merged data set has 12023 total diffractions and 3482 unique diffractions with 91.8% data completeness and an R_int value of 0.1632. The observed 2/m Laue symmetry of diffraction intensities shows that the ranitidine hydrochloride Form 1 crystal belongs to a monoclinic crystal system. Average unit-cell constants of Form 1 are a = 12.39(11) Å, b = 6.7(2) Å, c = 22.55(19) Å, α = 90°, β = 93.83(11)°, and γ = 90° with the P2_1/n space group. These values were reasonable comparing those of the previously reported space groups, a = 12.1918(6) Å, b = 6.5318(3) Å, c = 22.0382(3) Å, α = 90°, β = 93.985(3)°, and γ = 90°, respectively.³¹⁾

Fig. 2. Real-Space Image of Ranitidine Hydrochloride Forms 1 (a) and 2 (b) Measured by TEM

Fig. 3. MicroED Structure of Ranitidine Hydrochloride Form 1

In contrast to Form 1, the crystal structure of Form 2 has only one published structure³²⁾ and no comprehensive analysis has been performed. The previous report suggests that ranitidine hydrochloride Form 2 may exhibit a mix of cis and trans conformations for the nitro group.³²⁾ In this study, we conducted the first comprehensive structural analysis of Form 2 by utilizing microED to measure nano- to microcrystalline powdered samples of ranitidine hydrochloride.

Diffraction data were collected from ten individual ranitidine hydrochloride Form 2 crystals, with each covering approximately 40° of the reciprocal space. The reliability factor achieved by microED was R₁ = 18.86%, similar to that of Form 2, whereas the literature value measured by XRD was R = 4.4 Notably, the R value of XRD is a few percent and that of the previously reported structure was R = 4.16%.³²⁾ To remove diffractions with low signal-to-noise ratio, the resolution was truncated to 0.8 Å. The merged data set has 19529 total diffractions and 3229 unique diffractions with 91% data completeness and an R_int value of 0.1791. The observed 2/m Laue symmetry of diffraction intensities shows that the ranitidine hydrochloride Form 2 crystal belongs to a monoclinic crystal system. Average unit-cell constants for Form 2 are a = 7.33(15) Å, b = 13.27(13) Å, c = 19.2(2) Å, α = 90°, β = 95.3(3)°, and γ = 90° with the P2_1/n space group. Notably, the obtained space groups were reasonable comparing the previously reported space group (P2_1/n) with average unit-cell constants a = 18.798(3) Å, b = 12.980(3) Å, c = 7.204(1) Å, α = 90°, β = 95.09(1)°, and γ = 90°.³²⁾ The potential around the nitro group of ranitidine hydrochloride Form 2 indicates that the orientation of the nitro group of ranitidine hydrochloride Form 2 is a mixture of cis (Fig. 4a) and trans (Fig. 4b).

Fig. 4. MicroED Structure of Ranitidine Hydrochloride Form 2, a) cis Conformation and b) trans Conformation

Comparison of the obtained microED structures with reported XRD structures demonstrates a high level of agreement for both Form 1 and Form 2 (Supplementary Figs. S1–S3, Tables S1, S2), indicating that microED can accurately measure these respective forms. The results obtained from microED analysis suggest that it has the potential to overcome challenges associated with scXRD, which often involves a trial-and-error process.

Conclusion

In this study, we utilized microED to rapidly analyze two crystal polymorphs (Forms 1, 2) of ranitidine hydrochloride. The crystal structure was quickly measured by microED; the measurement itself was completed in 1–2 min per crystal and the analysis was quickly and easily performed by automated structural analysis software. MicroED measurements and structural analyses were swiftly completed within a few hours for both Forms 1 and 2, generating high-resolution data in each case. Therefore, microED holds the potential to offer a faster approach for API measurements compared with conventional XRD. The resolution of obtained microED structures was sufficiently high and diffraction data were collected from ten individual crystals. For Form 1, the crystal structure obtained through microED was consistent with the previously reported X-ray structure, confirming a cis conformation. In the case of Form 2, structural analysis using microED revealed a mixture of cis and trans conformations of nitro groups. Structures measured by microED were consistent with the reported X-ray structures for both Form 1 and Form 2, indicating the accuracy of microED in determining these crystal forms. Overall, this study highlights the potential of microED as a rapid and efficient method for analyzing crystal structures such as APIs. MicroED offers the advantage of analyzing nano- to microcrystalline powdered samples instead of single crystals, making it a promising tool for structural analysis in drug development and materials science research.

Experimental

Preparation of Ranitidine Hydrochloride Polymorphs

Ranitidine hydrochloride crystal polymorphs (Forms 1, 2) were prepared using ethanol as a crystallization solvent, as previously described.²⁶⁾ The obtained crystal was analyzed using pXRD to confirm the polymorphic form.^25,27–30) The HPLC purity of each crystal form exceeded 99.6% at a UV wavelength of 235 nm.

MicroED Measurement of Ranitidine Polymorphs

The crystals for measurement were manually searched. The series of selected-area electron diffraction (SAD) pattern from Ranitidine microcrystal were quickly taken by XtaLAB Synergy-ED (Rigaku) with accelerating voltage of 200 kV. The electron source was the filament of lanthanum hexaboride (LaB6). By heating the filament, thermoelectrons were emitted from the tip of LaB6. The detector was HyPix-ED, which was a hybrid pixel array detector provided by Rigaku. In order to reduce the electron radiation damage, all of the measurements were performed and at a low electron dose of less than 1.1 e⁻/Ų cooling by liquid nitrogen. The camera length (608 mm) was calibrated by using a gold polycrystals specimen. The beam size and SA size were 30 microns and 3 microns in the sample position, correspondingly. The tilt series of ED pattern were obtained by using SAD. The series of ED pattern was automatically collected under continuous rotation from −40 to +40 degree with a rate of 1°/s with an acquisition time of 80 s using CrysAlis^pro for ED software. The ED patterns were obtained every 0.5 degree. The initial structure was solved by using direct method, and refined by using SHELXL program with Olex2 (OlexSys Ltd., U.K.) interface. Multiple datasets (automatically obtained 41 data sets from Ranitidine form 1 (23 data sets) and form 2 (18 data sets)) were merged to obtain 3D crystal structure with high completeness (2 and 3 data sets used for structural analysis of Ranitidine forms 1 and 2, respectively). Total acquisition time was about 8 h. Thirty-six data sets did not be used for structural analysis. Two data sets from Ranitidine form 1 and three data sets from Ranitidine form 2 were merged correspondingly. The cell constants of Ranitidine form 1 were a = 12.39 Å, b = 6.7 Å, c = 22.55 Å, β = 93.83°, and the space group was P2_1/n. Total of 12023 reflections were measured and merged. Finally, 3482 independent reflections were obtained. In the case of Ranitidine form 2, the constants were a = 7.33 Å, b = 13.27 Å, c = 19.2 Å, β = 95.3°, with the space group of P2_1/n. Total and independent reflections were 19529 and 3229, respectively. CCDC-2289944 for Form 1 and CCDC-2289945 for Form 2 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge at https://www.ccdc.cam.ac.uk/, and the raw diffraction images at Zenodo (https://doi.org/10.5281/zenodo.10445933).

Acknowledgments

The authors would like to express their deepest appreciation to Prof. Mitsunobu Doi and Dr. Takuma Kato (Faculty of Pharmacy, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569–8686, Japan) for their assistance in microED analysis. The pictures obtained by SEM in Fig. 1 were reproduced from the reference 7 with permission from Elsevier. This study was supported in part by AMED under Grant numbers 23mk0101208 (to E.Y., N.U., and Y.D.) and 23ak0101190 (to E.Y. and N.U.).

Conflict of Interest

Yoshitaka Aoyama is an employee of JEOL Ltd. Takashi Matsumoto is an employee of Rigaku Corporation. Hidetomo Yokoo, Eiichi Yamamoto, Nahoko Uchiyama, and Yosuke Demizu have no conflict of interest.

Supplementary Materials

This article contains supplementary materials. The crystallographic data are available free of charge via the Internet at http://pubs.acs.org.

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