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Chlorine K-Edge X-Ray Absorption Near-Edge Structure Analysis of Clarithromycin Hydrochloride Metastable Crystal
Journal of Pharmaceutical Sciences xxx (2020) 1-5
Contents lists available at ScienceDirect
Journal of Pharmaceutical Sciences journal homepage: www.jpharmsci.org
Note
Chlorine K-edge X-ray absorption near-edge structure analysis of clarithromycin hydrochloride metastable crystal Masataka Ito, Rika Shiba, Hironori Suzuki, Shuji Noguchi* Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi-City, Chiba 274-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 January 2020 Accepted 18 March 2020
X-ray absorption fine structure (XAFS) spectra, especially X-ray absorption near-edge structure (XANES), show unique features depending on the chemical states and structures in the vicinity of the X-ray absorbing elements. As such, they can be used to identify the chemical environment of elements. XAFS spectroscopy was applied to investigate the chemical environment of chloride ions in a metastable form A crystal of the antibiotic clarithromycin hydrochloride hydrate. The XANES of the form A crystals showed low similarity to that of the active pharmaceutical ingredient (API) hydrochloride salt crystals, but showed a high similarity to that of a hydrochloride aqueous solution. This indicated that the chloride ion in the form A crystals predominantly interacted with water molecules and was fully hydrated, which was consistent with the previous presumption that form A may be high water-content crystals. This study demonstrated that this XAFS spectroscopy method would be a potent alternative technique for evaluating APIs. ® © 2020 American Pharmacists Association . Published by Elsevier Inc. All rights reserved.
Keywords: Polymorph Absorption spectroscopy Phase transition X-ray powder diffraction Crystal structure
Introduction Elucidation of the phase transition of active pharmaceutical ingredients (APIs) is crucial for manufacturing the solid drug formulation because the solubility, dissolution rate and stability of the API differ according to its phase, resulting in deterioration of the formulation quality. The phase transitions of an API occur not only at the manufacturing stage but after oral administration, and may affect the efficacy of the API. For example, most stable form II crystals of the macrolide antibiotic clarithromycin (CAM) transit to gel,1 metastable CAM hydrochloride (CAM-HCl) hydrate salt form A crystals, and finally stable CAM-HCl hydrate salt form B crystals in sequence under acidic conditions.2 This ultimately results in changes in the dissolution properties after oral administration. The transition of solid API to gel has also been reported to retard the disintegration of CAM tablets.3,4 Characterization of the phases of APIs has been performed by using various analytical methods. Powder X-ray diffraction (PXRD) and thermal analyses are used most widely for
Abbreviations used: API, active pharmaceutical ingredient; CAM, clarithromycin; XAFS, X-ray absorption fine structure; XANES, X-ray absorption near-edge structure. * Corresponding author. E-mail address: [email protected] (S. Noguchi).
characterizing the polymorphs of APIs and their transitions. Crystals and the amorphous state of APIs also can be identified using spectroscopic methods such as infrared spectroscopy, Raman spectroscopy and solid-state nuclear magnetic resonance methods. As an extension to these established characterization methods, we focused our research on X-ray absorption fine structure (XAFS) spectroscopy.5 X-ray absorption spectra show sharp rises, called edges, at the energy of the core orbitals of elements. The features near or on the edges, around the energy from the edge to þ50 eV of the X-ray absorption spectra are called X-ray absorption near edge structure (XANES). The XANES shows unique features depending on the valences, coordination states and chemical bonds of the X-ray absorbing atoms. Therefore, the chemical environment of the X-ray absorbing atoms of a compound can be identified by comparing its XANES spectrum to those of standards whose structures are known. The information that XAFS spectroscopy reveals about the chemical state and environment of particular elements in the sample cannot be determined from analytical methods that are traditionally used in the pharmaceutical science field. XAFS spectroscopy is very useful for evaluating APIs that contain elements other than the light elements such as C, H, O, and N, because the APIs in pharmaceutics are usually coexistent with a wide variety of excipients, most of which consist of the light elements. Another merit of XAFS spectroscopy is that it is applicable to samples of not only solids but liquids, semi-solids, dispersions, and even gases.
https://doi.org/10.1016/j.xphs.2020.03.015 0022-3549/© 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
2
M. Ito et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-5
XAFS spectroscopy has been used to evaluate the valences and local structures of mainly metal ions,6,7 and its application to APIs has not yet been reported. In this study, XAFS spectroscopy was applied to analyze the metastable form A crystals of CAM-HCl hydrate salt. The crystal structure of form A has not been determined because of its low stability and the difficulty in preparing crystals suitable for Xray single crystal structure analysis. The chemical environment of the chloride ion in the CAM form A crystal was investigated by measuring its chlorine-K edge XAFS spectrum and comparing it to those of API crystals containing covalently bound chloride elements, HCl salt crystals of APIs containing tertiary amine moieties as in CAM or a secondary amine moiety and HCl aqueous solution. Materials and methods Materials CAM form II crystals were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All other reagents used were of the highest grade available from commercial sources (see Fig. 1).
Preparation of CAM-HCl hydrate form A and form B crystals Metastable form A crystals of CAM-HCl hydrate were prepared according to a method described previously with modifications.2 Briefly, 1.8 g of CAM form II crystals and 25 mL of 0.1 M hydrochloric acid solution were mixed with a mortar and pestle to induce the transition to a viscous gel and then to a suspension of glossy needle-shaped form A crystals. The form A crystals were sedimented by centrifugation at 7000 g for 5 min at 4 C. The supernatant was discarded and the sedimented form A crystals were gently suspended in 2 mL of ice-cold distilled water and centrifuged again. This washing process was repeated until the pH of the wash water became 3.4 or higher. The washed form A crystals were kept from drying and stored at 4 C. Stable form B crystals of CAM-HCl hydrate were prepared by incubating the wet form A crystals at 37 C for 4 h or more. The prepared form B crystals were dried at 37 C overnight and stored at room temperature. Form A crystals transitioned to an amorphous state when they were rapidly dried.
Fig. 1. Chemical structures of APIs containing chlorine used in this study.
M. Ito et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-5
3
Power X-Ray diffraction (PXRD) analyses
XAFS analyses
PXRD measurements were performed using a Mini Flex 600 Xray diffractometer and RINT 2100 (Rigaku Ltd., Tokyo, Japan) with Cu-Ka radiation at room temperature. The PXRD profiles were compared with those calculated from the crystal structures deposited to the Cambridge Structural Database to identify their crystal forms (Fig. 2a). All the chloride ions in the hydrochloride salt crystals formed hydrogen bonds with the protonated amine moieties (Supplementary Figure S1).
XAFS measurements were carried out at the BL6N1 beamline of the Aichi Synchrotron Radiation Center (Aichi, Japan). Dried crystalline powders of the APIs except for CAM-HCl form A crystals were placed on conductive double-sided adhesive carbon tape. Wet CAM form A crystals and a 0.1 M HCl solution were enclosed in polyethylene bags. These samples were set in the XAFS chamber and the atmosphere in the chamber was replaced with helium gas. The XAFS measurement was performed at 25 C. The X-ray energies
Fig. 2. PXRD profiles and XAFS spectra. (a) PXRD profiles of API crystals with covalently bound chlorine atoms (green lines) and API HCl salt crystals (blue lines). (b) XAFS spectra of API HCl salt crystals (colored as in (a)), and a HCl solution (red line). The absorption spectra are normalized so that the rises associated with the chlorine K-edges are 1. (c) Plot of normalized absorption values against E0. API crystals with covalently bound chlorine atoms are shown with green triangles, API HCl salt crystals with blue circles, and a 0.1 M HCl aqueous solution with a red square. (d) Plot of normalized absorption values against peak-top energies.
4
M. Ito et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-5
the highest peaks in the XANES spectra of HCl salt crystals were so broad. When the normalized absorption values at E0 were plotted against E0 or the peak-top energies of the highest peaks, the plots of the HCl salt crystals were clustered in the region with the lowest normalized absorption values and highest energy values apart from those of the APIs with covalently bound chlorine atoms (Fig. 2c and d). These plots might be useful for identifying by XAFS spectroscopy whether the chlorine atoms in the samples are ions or are covalently bound. To quantitatively compare the XANES spectra of API HCl salt crystals in detail, coefficients of determination (R2) of normalized absorption values were calculated using the XANES regions from 2823.0 to 2860.0 eV, where aspects of each spectrum were comparatively prominent (Table 1). All of the R2 values were higher than 0.47, and some could be grouped based on their higher R2 values. The R2 values among the HCl salt crystals of amitriptyline, imipramine, and tripelennamine were 0.974e0.989. The chloride ions in these crystals form one hydrogen bond with a protonated tertiary amine and also form van der Waals contacts or possibly hydrogen bonds with the hydrogen atoms of alkyl or aromatic groups.9 The higher R2 values may be ascribed to these common structural aspects around the chloride ions. Another example that supports the higher R2 values being related to structural similarities is found in the case of the CAM form B and cyproheptadine HCl crystals. The R2 value for the comparison of the XANES spectra of the CAM form B and cyproheptadine crystal was 0.943 while those between the CAM form B and others ranged from 0.477 to 0.918. In addition to the formation of hydrogen bonds with protonated tertiary amines, the chloride ions in the CAM form B and cyproheptadine HCl crystals formed hydrogen bonds with two and three water molecules, respectively, while no hydrogen bond involving a water molecule was formed in any other investigated crystals. The XANES spectra of diltiazem, promethazine, ranitidine and venlafaxine HCl crystals shared similarities with R2 values larger than approximately 0.97. Although no common interactions relating the chloride ions in these crystals were found other than the hydrogen bonds with the protonated tertiary amines, structural disorders were found in the promethazine and ranitidine HCl crystals. The tertiary amine moiety hydrogen bonded to the chloride ion in the promethazine HCl crystal was disordered, and the sp2-type secondary amines hydrogen bonded to the chloride ions in the ranitidine HCl crystal was disordered. When structures are disordered, the XANES spectra would be the average of the XANES of each disordered structure, and these spectra might coincidentally show higher R2 values similar to what was observed for these cases.
2.5
Amoxapine
CAM HCl form A 0.1M HClformB solution CAM CAM HCl form B HCl solution Amoxapine
Normalized absorption
2.0
1.5
CAM-form A
1.0
0.5
0.0 2810
2820
2830
2840
2850
2860
2870
2880
Energy (eV) Fig. 3. Superposition of the XANES spectra of a CAM form A crystal (blue thick line), form B crystal (blue thin line), and HCl aqueous solution (red thick chain-line). XANES spectrum of an amoxapine crystal (green thin chain-line) is also shown for comparison.
of the spectra ranged from 2800 to 2900 eV and included the Kedge energy of chlorine (2823.8 eV). The X-ray energy was calibrated by setting the first peak in the sulfur K-edge XAFS spectrum of the potassium sulfate powders to be 2481.70 eV. The XAFS spectra were recorded in the total-electron-yield mode for the dry crystalline powder samples and in the fluorescence mode for the samples enclosed in the insulating polyethylene bags. The XAFS spectra data were processed with ATHENA.8 The energies of the CleK edges, E0, in each spectrum were defined as the energies where second derivatives of the edge spectra were zero.
Results and discussion Shapes of the CleK XANES spectra were unique depending on the samples (Fig. 2b). The highest peaks near the edges were much broader in the cases of the API HCl salts compared with those of APIs with covalently bound chlorine atoms. The interactions that chloride ions form in crystals are ionic or hydrogen bonds, and the thermal vibration of chloride ions and atoms interacting with the chloride ions would make their bond length and bond angles vary from those of covalent bonds, leading the energy levels of the orbitals related to the chloride ions to vary. This might explain why
Table 1 Coefficients of determination between XANES spectra (2823e2860 eV) of drugs containing chloride ions.
1. CAM HCl form A 2. CAM HCl form B 3. Amitriptyline HCl 4. Imipramine HCl 5. Tripelennamine HCl 6. Diltiazem HCl 7. Promethazine HCl 8. Ranitidine HCl 9. Venlafaxine HCl 10. Procaine HCl 11. Cyproheptadine HCl 12. Propranolol HCl 13. Tetracaine HCl 14. 0.1 M HCl solution
CSD ID
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
e WOCFOJ YOVZEO IMIPRC FIMNIY CEYHUJ01 EAPTZC02 TADZAZ02 WOBMUV01 PROCHC01 CYPHEP01 PROPDD XISVOK01 e
0.786 0.827 0.768 0.780 0.741 0.747 0.745 0.774 0.843 0.629 0.873 0.801 0.977
0.799 0.787 0.759 0.763 0.831 0.724 0.725 0.805 0.943 0.918 0.477 0.689
0.974 0.977 0.933 0.931 0.914 0.926 0.933 0.757 0.912 0.708 0.750
0.989 0.940 0.942 0.915 0.911 0.918 0.774 0.889 0.623 0.666
0.959 0.953 0.947 0.943 0.944 0.743 0.897 0.679 0.688
0.986 0.989 0.985 0.960 0.752 0.915 0.702 0.647
0.974 0.962 0.964 0.820 0.941 0.655 0.644
0.990 0.962 0.701 0.894 0.743 0.659
0.968 0.684 0.906 0.791 0.697
0.730 0.858 0.330 0.512
0.947 0.785 0.768
0.685 0.788
0.826
14.
M. Ito et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-5
Although the relationship between the R2 values and the geometry of the bond distances and angles of the hydrogen bond between the chloride ions and protonated tertiary amines (distance between Cl and H atoms, between Cl and N atoms, and the angles formed by the Cl, H and N atoms of the Cl･･･HeN hydrogen bond) was investigated, no relationship was identified. This suggests that the geometry of the hydrogen bond with tertiary amines might not be dominant factors that determine the shapes of the XANES spectra of the API HCl salt crystals investigated in this study. The XANES spectra of the CAM form A showed lower similarity to those of all the API HCl salt crystals including the CAM form B (R2 ¼ 0.629e0.827), but showed considerably high similarity, R2 ¼ 0.977, to the XANES spectra of a 0.1 M hydrochloride aqueous solution (see Fig. 3). This indicates that the chemical environment of the chloride ion in the form A crystal is much more similar to that in HCl aqueous solution, where the chloride ion would predominantly interact with water molecules and be fully hydrated. Full hydration may be possible for the chloride ions in the form A crystals because the form A crystals are estimated to contain 10 or more water molecules per one CAM molecule and the volume ratio occupied by the water molecules in the form A crystal had a high value of 34.7%.2 Anions in salt crystals do not always interact with cations but with water molecules and are stabilized by hydration. In the case of the CAM citrate hydrate crystal, negatively charged citrate ions interacted not with the protonated tertiary amine of CAM but with the water molecules.3 In form A crystals, there may be a large space filled with water molecules, and most of the chloride ions would be in that space in a hydrated state rather than be hydrogen bonded to the protonated tertiary amine of CAM as observed in the form B crystal structure. The formation of the hydrogen bond between the chloride ion and protonated tertiary amine of CAM may be necessary to form the form B crystals and its absence in the form A crystal may be the reason why rapid drying of form A crystals induces the transition not to form B crystals, but to an amorphous state. Conclusion XAFS spectroscopy revealed for the first time that the chloride ion in the metastable CAM form A crystals is fully hydrated rather
5
than bound to the protonated tertiary amine of CAM with hydrogen bonds. XAFS spectroscopy would be a potent method for evaluating the APIs in pharmaceutics.
Acknowledgments The synchrotron radiation experiments were performed at the BL6N1 beam line of the Aichi Synchrotron Radiation Center, Aichi Science & Technology Foundation, Aichi, Japan (approval numbers 201705011 and 201706005). This work was supported by the Japan Society for the Promotion of Science KAKENHI Grant Nos. 17K08467 (to SN) and 18K14641 (to HS), and grants from the Takeda Science Foundation, from the Japan Prize Foundation, and from the Uehara Memorial Foundation (to HS).
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.xphs.2020.03.015.
References 1. Fujiki S, Iwao Y, Kobayashi M, Miyagishima A, Itai S. Stabilization mechanism of clarithromycin tablets under gastric pH condition. Chem Pharm Bull. 2011;59: 553-558. 2. Noguchi S, Takiyama K, Fujiki S, Iwao Y, Miura K, Itai S. Polymorphic transformation of antibiotic clarithromycin under acidic condition. J Pharm Sci. 2014;103:580-586. 3. Inukai K, Takiyama K, Noguchi S, Iwao Y, Itai S. Effect of gel formation on the dissolution behavior of clarithromycin tablets. Int J Pharm. 2017;521:33-39. 4. Inukai K, Noguchi S, Kimura S, Itai S, Iwao Y. Stabilization mechanism of roxithromycin tablets under gastric pH Conditions. J Pharm Sci. 2018;107:2514-2518. 5. Gaur A, Shrivastava BD, Nigam HL. X-ray absorption fine structure (XAFS) spectroscopyea review. Proc Indian Natl Sci Acad B Biol Sci. 2013;79:921-966. 6. Jaworski NJ, Kozack CV, Tereniak SJ, et al. Operando spectroscopic and kinetic characterization of aerobic allylic CeH acetoxylation catalyzed by Pd(OAc)2/4,5diazafluoren-9-one. J Am Chem Soc. 2019;26:10462-10474. 7. Arisawa M. Development of metal nanoparticle catalysis toward drug discovery. Chem Pharm Bull. 2019;67:733-771. 8. Ravel B, Newville M. ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT. J Synchrotron Radiat. 2005;12:537-541. €y CB, Evans TA, Seddon KR, P I. The CeH$$$ Cl hydrogen bond: does 9. Aakero alinko it exist? New J Chem. 1999;23:145-152.
Contents lists available at ScienceDirect
Journal of Pharmaceutical Sciences journal homepage: www.jpharmsci.org
Note
Chlorine K-edge X-ray absorption near-edge structure analysis of clarithromycin hydrochloride metastable crystal Masataka Ito, Rika Shiba, Hironori Suzuki, Shuji Noguchi* Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi-City, Chiba 274-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 January 2020 Accepted 18 March 2020
X-ray absorption fine structure (XAFS) spectra, especially X-ray absorption near-edge structure (XANES), show unique features depending on the chemical states and structures in the vicinity of the X-ray absorbing elements. As such, they can be used to identify the chemical environment of elements. XAFS spectroscopy was applied to investigate the chemical environment of chloride ions in a metastable form A crystal of the antibiotic clarithromycin hydrochloride hydrate. The XANES of the form A crystals showed low similarity to that of the active pharmaceutical ingredient (API) hydrochloride salt crystals, but showed a high similarity to that of a hydrochloride aqueous solution. This indicated that the chloride ion in the form A crystals predominantly interacted with water molecules and was fully hydrated, which was consistent with the previous presumption that form A may be high water-content crystals. This study demonstrated that this XAFS spectroscopy method would be a potent alternative technique for evaluating APIs. ® © 2020 American Pharmacists Association . Published by Elsevier Inc. All rights reserved.
Keywords: Polymorph Absorption spectroscopy Phase transition X-ray powder diffraction Crystal structure
Introduction Elucidation of the phase transition of active pharmaceutical ingredients (APIs) is crucial for manufacturing the solid drug formulation because the solubility, dissolution rate and stability of the API differ according to its phase, resulting in deterioration of the formulation quality. The phase transitions of an API occur not only at the manufacturing stage but after oral administration, and may affect the efficacy of the API. For example, most stable form II crystals of the macrolide antibiotic clarithromycin (CAM) transit to gel,1 metastable CAM hydrochloride (CAM-HCl) hydrate salt form A crystals, and finally stable CAM-HCl hydrate salt form B crystals in sequence under acidic conditions.2 This ultimately results in changes in the dissolution properties after oral administration. The transition of solid API to gel has also been reported to retard the disintegration of CAM tablets.3,4 Characterization of the phases of APIs has been performed by using various analytical methods. Powder X-ray diffraction (PXRD) and thermal analyses are used most widely for
Abbreviations used: API, active pharmaceutical ingredient; CAM, clarithromycin; XAFS, X-ray absorption fine structure; XANES, X-ray absorption near-edge structure. * Corresponding author. E-mail address: [email protected] (S. Noguchi).
characterizing the polymorphs of APIs and their transitions. Crystals and the amorphous state of APIs also can be identified using spectroscopic methods such as infrared spectroscopy, Raman spectroscopy and solid-state nuclear magnetic resonance methods. As an extension to these established characterization methods, we focused our research on X-ray absorption fine structure (XAFS) spectroscopy.5 X-ray absorption spectra show sharp rises, called edges, at the energy of the core orbitals of elements. The features near or on the edges, around the energy from the edge to þ50 eV of the X-ray absorption spectra are called X-ray absorption near edge structure (XANES). The XANES shows unique features depending on the valences, coordination states and chemical bonds of the X-ray absorbing atoms. Therefore, the chemical environment of the X-ray absorbing atoms of a compound can be identified by comparing its XANES spectrum to those of standards whose structures are known. The information that XAFS spectroscopy reveals about the chemical state and environment of particular elements in the sample cannot be determined from analytical methods that are traditionally used in the pharmaceutical science field. XAFS spectroscopy is very useful for evaluating APIs that contain elements other than the light elements such as C, H, O, and N, because the APIs in pharmaceutics are usually coexistent with a wide variety of excipients, most of which consist of the light elements. Another merit of XAFS spectroscopy is that it is applicable to samples of not only solids but liquids, semi-solids, dispersions, and even gases.
https://doi.org/10.1016/j.xphs.2020.03.015 0022-3549/© 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
2
M. Ito et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-5
XAFS spectroscopy has been used to evaluate the valences and local structures of mainly metal ions,6,7 and its application to APIs has not yet been reported. In this study, XAFS spectroscopy was applied to analyze the metastable form A crystals of CAM-HCl hydrate salt. The crystal structure of form A has not been determined because of its low stability and the difficulty in preparing crystals suitable for Xray single crystal structure analysis. The chemical environment of the chloride ion in the CAM form A crystal was investigated by measuring its chlorine-K edge XAFS spectrum and comparing it to those of API crystals containing covalently bound chloride elements, HCl salt crystals of APIs containing tertiary amine moieties as in CAM or a secondary amine moiety and HCl aqueous solution. Materials and methods Materials CAM form II crystals were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All other reagents used were of the highest grade available from commercial sources (see Fig. 1).
Preparation of CAM-HCl hydrate form A and form B crystals Metastable form A crystals of CAM-HCl hydrate were prepared according to a method described previously with modifications.2 Briefly, 1.8 g of CAM form II crystals and 25 mL of 0.1 M hydrochloric acid solution were mixed with a mortar and pestle to induce the transition to a viscous gel and then to a suspension of glossy needle-shaped form A crystals. The form A crystals were sedimented by centrifugation at 7000 g for 5 min at 4 C. The supernatant was discarded and the sedimented form A crystals were gently suspended in 2 mL of ice-cold distilled water and centrifuged again. This washing process was repeated until the pH of the wash water became 3.4 or higher. The washed form A crystals were kept from drying and stored at 4 C. Stable form B crystals of CAM-HCl hydrate were prepared by incubating the wet form A crystals at 37 C for 4 h or more. The prepared form B crystals were dried at 37 C overnight and stored at room temperature. Form A crystals transitioned to an amorphous state when they were rapidly dried.
Fig. 1. Chemical structures of APIs containing chlorine used in this study.
M. Ito et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-5
3
Power X-Ray diffraction (PXRD) analyses
XAFS analyses
PXRD measurements were performed using a Mini Flex 600 Xray diffractometer and RINT 2100 (Rigaku Ltd., Tokyo, Japan) with Cu-Ka radiation at room temperature. The PXRD profiles were compared with those calculated from the crystal structures deposited to the Cambridge Structural Database to identify their crystal forms (Fig. 2a). All the chloride ions in the hydrochloride salt crystals formed hydrogen bonds with the protonated amine moieties (Supplementary Figure S1).
XAFS measurements were carried out at the BL6N1 beamline of the Aichi Synchrotron Radiation Center (Aichi, Japan). Dried crystalline powders of the APIs except for CAM-HCl form A crystals were placed on conductive double-sided adhesive carbon tape. Wet CAM form A crystals and a 0.1 M HCl solution were enclosed in polyethylene bags. These samples were set in the XAFS chamber and the atmosphere in the chamber was replaced with helium gas. The XAFS measurement was performed at 25 C. The X-ray energies
Fig. 2. PXRD profiles and XAFS spectra. (a) PXRD profiles of API crystals with covalently bound chlorine atoms (green lines) and API HCl salt crystals (blue lines). (b) XAFS spectra of API HCl salt crystals (colored as in (a)), and a HCl solution (red line). The absorption spectra are normalized so that the rises associated with the chlorine K-edges are 1. (c) Plot of normalized absorption values against E0. API crystals with covalently bound chlorine atoms are shown with green triangles, API HCl salt crystals with blue circles, and a 0.1 M HCl aqueous solution with a red square. (d) Plot of normalized absorption values against peak-top energies.
4
M. Ito et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-5
the highest peaks in the XANES spectra of HCl salt crystals were so broad. When the normalized absorption values at E0 were plotted against E0 or the peak-top energies of the highest peaks, the plots of the HCl salt crystals were clustered in the region with the lowest normalized absorption values and highest energy values apart from those of the APIs with covalently bound chlorine atoms (Fig. 2c and d). These plots might be useful for identifying by XAFS spectroscopy whether the chlorine atoms in the samples are ions or are covalently bound. To quantitatively compare the XANES spectra of API HCl salt crystals in detail, coefficients of determination (R2) of normalized absorption values were calculated using the XANES regions from 2823.0 to 2860.0 eV, where aspects of each spectrum were comparatively prominent (Table 1). All of the R2 values were higher than 0.47, and some could be grouped based on their higher R2 values. The R2 values among the HCl salt crystals of amitriptyline, imipramine, and tripelennamine were 0.974e0.989. The chloride ions in these crystals form one hydrogen bond with a protonated tertiary amine and also form van der Waals contacts or possibly hydrogen bonds with the hydrogen atoms of alkyl or aromatic groups.9 The higher R2 values may be ascribed to these common structural aspects around the chloride ions. Another example that supports the higher R2 values being related to structural similarities is found in the case of the CAM form B and cyproheptadine HCl crystals. The R2 value for the comparison of the XANES spectra of the CAM form B and cyproheptadine crystal was 0.943 while those between the CAM form B and others ranged from 0.477 to 0.918. In addition to the formation of hydrogen bonds with protonated tertiary amines, the chloride ions in the CAM form B and cyproheptadine HCl crystals formed hydrogen bonds with two and three water molecules, respectively, while no hydrogen bond involving a water molecule was formed in any other investigated crystals. The XANES spectra of diltiazem, promethazine, ranitidine and venlafaxine HCl crystals shared similarities with R2 values larger than approximately 0.97. Although no common interactions relating the chloride ions in these crystals were found other than the hydrogen bonds with the protonated tertiary amines, structural disorders were found in the promethazine and ranitidine HCl crystals. The tertiary amine moiety hydrogen bonded to the chloride ion in the promethazine HCl crystal was disordered, and the sp2-type secondary amines hydrogen bonded to the chloride ions in the ranitidine HCl crystal was disordered. When structures are disordered, the XANES spectra would be the average of the XANES of each disordered structure, and these spectra might coincidentally show higher R2 values similar to what was observed for these cases.
2.5
Amoxapine
CAM HCl form A 0.1M HClformB solution CAM CAM HCl form B HCl solution Amoxapine
Normalized absorption
2.0
1.5
CAM-form A
1.0
0.5
0.0 2810
2820
2830
2840
2850
2860
2870
2880
Energy (eV) Fig. 3. Superposition of the XANES spectra of a CAM form A crystal (blue thick line), form B crystal (blue thin line), and HCl aqueous solution (red thick chain-line). XANES spectrum of an amoxapine crystal (green thin chain-line) is also shown for comparison.
of the spectra ranged from 2800 to 2900 eV and included the Kedge energy of chlorine (2823.8 eV). The X-ray energy was calibrated by setting the first peak in the sulfur K-edge XAFS spectrum of the potassium sulfate powders to be 2481.70 eV. The XAFS spectra were recorded in the total-electron-yield mode for the dry crystalline powder samples and in the fluorescence mode for the samples enclosed in the insulating polyethylene bags. The XAFS spectra data were processed with ATHENA.8 The energies of the CleK edges, E0, in each spectrum were defined as the energies where second derivatives of the edge spectra were zero.
Results and discussion Shapes of the CleK XANES spectra were unique depending on the samples (Fig. 2b). The highest peaks near the edges were much broader in the cases of the API HCl salts compared with those of APIs with covalently bound chlorine atoms. The interactions that chloride ions form in crystals are ionic or hydrogen bonds, and the thermal vibration of chloride ions and atoms interacting with the chloride ions would make their bond length and bond angles vary from those of covalent bonds, leading the energy levels of the orbitals related to the chloride ions to vary. This might explain why
Table 1 Coefficients of determination between XANES spectra (2823e2860 eV) of drugs containing chloride ions.
1. CAM HCl form A 2. CAM HCl form B 3. Amitriptyline HCl 4. Imipramine HCl 5. Tripelennamine HCl 6. Diltiazem HCl 7. Promethazine HCl 8. Ranitidine HCl 9. Venlafaxine HCl 10. Procaine HCl 11. Cyproheptadine HCl 12. Propranolol HCl 13. Tetracaine HCl 14. 0.1 M HCl solution
CSD ID
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
e WOCFOJ YOVZEO IMIPRC FIMNIY CEYHUJ01 EAPTZC02 TADZAZ02 WOBMUV01 PROCHC01 CYPHEP01 PROPDD XISVOK01 e
0.786 0.827 0.768 0.780 0.741 0.747 0.745 0.774 0.843 0.629 0.873 0.801 0.977
0.799 0.787 0.759 0.763 0.831 0.724 0.725 0.805 0.943 0.918 0.477 0.689
0.974 0.977 0.933 0.931 0.914 0.926 0.933 0.757 0.912 0.708 0.750
0.989 0.940 0.942 0.915 0.911 0.918 0.774 0.889 0.623 0.666
0.959 0.953 0.947 0.943 0.944 0.743 0.897 0.679 0.688
0.986 0.989 0.985 0.960 0.752 0.915 0.702 0.647
0.974 0.962 0.964 0.820 0.941 0.655 0.644
0.990 0.962 0.701 0.894 0.743 0.659
0.968 0.684 0.906 0.791 0.697
0.730 0.858 0.330 0.512
0.947 0.785 0.768
0.685 0.788
0.826
14.
M. Ito et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-5
Although the relationship between the R2 values and the geometry of the bond distances and angles of the hydrogen bond between the chloride ions and protonated tertiary amines (distance between Cl and H atoms, between Cl and N atoms, and the angles formed by the Cl, H and N atoms of the Cl･･･HeN hydrogen bond) was investigated, no relationship was identified. This suggests that the geometry of the hydrogen bond with tertiary amines might not be dominant factors that determine the shapes of the XANES spectra of the API HCl salt crystals investigated in this study. The XANES spectra of the CAM form A showed lower similarity to those of all the API HCl salt crystals including the CAM form B (R2 ¼ 0.629e0.827), but showed considerably high similarity, R2 ¼ 0.977, to the XANES spectra of a 0.1 M hydrochloride aqueous solution (see Fig. 3). This indicates that the chemical environment of the chloride ion in the form A crystal is much more similar to that in HCl aqueous solution, where the chloride ion would predominantly interact with water molecules and be fully hydrated. Full hydration may be possible for the chloride ions in the form A crystals because the form A crystals are estimated to contain 10 or more water molecules per one CAM molecule and the volume ratio occupied by the water molecules in the form A crystal had a high value of 34.7%.2 Anions in salt crystals do not always interact with cations but with water molecules and are stabilized by hydration. In the case of the CAM citrate hydrate crystal, negatively charged citrate ions interacted not with the protonated tertiary amine of CAM but with the water molecules.3 In form A crystals, there may be a large space filled with water molecules, and most of the chloride ions would be in that space in a hydrated state rather than be hydrogen bonded to the protonated tertiary amine of CAM as observed in the form B crystal structure. The formation of the hydrogen bond between the chloride ion and protonated tertiary amine of CAM may be necessary to form the form B crystals and its absence in the form A crystal may be the reason why rapid drying of form A crystals induces the transition not to form B crystals, but to an amorphous state. Conclusion XAFS spectroscopy revealed for the first time that the chloride ion in the metastable CAM form A crystals is fully hydrated rather
5
than bound to the protonated tertiary amine of CAM with hydrogen bonds. XAFS spectroscopy would be a potent method for evaluating the APIs in pharmaceutics.
Acknowledgments The synchrotron radiation experiments were performed at the BL6N1 beam line of the Aichi Synchrotron Radiation Center, Aichi Science & Technology Foundation, Aichi, Japan (approval numbers 201705011 and 201706005). This work was supported by the Japan Society for the Promotion of Science KAKENHI Grant Nos. 17K08467 (to SN) and 18K14641 (to HS), and grants from the Takeda Science Foundation, from the Japan Prize Foundation, and from the Uehara Memorial Foundation (to HS).
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.xphs.2020.03.015.
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