Direct High-Resolution Imaging of Crystalline Components in

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Direct High-Resolution Imaging of Crystalline Components in Pharmaceutical Dosage Forms Using Low-Frequency Raman Spectroscopy Hiroshi Hisada, Motoki Inoue, Tatsuo Koide, James Carriere, Randy Heyler, and Toshiro Fukami Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.5b00329 • Publication Date (Web): 23 Oct 2015 Downloaded from http://pubs.acs.org on October 26, 2015

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Organic Process Research & Development

Direct High-Resolution Imaging of Crystalline Components in Pharmaceutical Dosage Forms Using Low-Frequency Raman Spectroscopy

Hiroshi Hisada1, Motoki Inoue1 Tatsuo Koide2, James Carriere3, Randy Heyler3, and Toshiro Fukami1*

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Department of Molecular Pharmaceutics, Meiji Pharmaceutical University, Kiyose, Tokyo

204-8588 Japan 2

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Division of Drugs, National Institute of Health Sciences, Setagaya, 158-8501 Tokyo Japan Ondax Inc. 850 E. Duarte Rd Monrovia, 91016 CA USA

*Corresponding author: Toshiro Fukami PhD Department of Molecular Pharmaceutics, Meiji Pharmaceutical University 2-522-1 Noshio, Kiyose, Tokyo 204-8588 Japan TEL & FAX: +81-42-495-8936 E-mail: [email protected]

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ABSTRACT Crystalline forms of active pharmaceutical ingredients need to be clearly understood and characterized by the pharmaceutical industry to ensure the correct dosage is produced. In this study, we evaluated the crystalline form of two different pharmaceutical cocrystals and a physical mixture consisting of caffeine and 4-hydroxy benzoic acid using a Raman microscopy system equipped with a measurement module to access the low-frequency region. We also demonstrated the differences between a low-frequency Raman spectroscopy image of a cocrystal and its physical mixture in a pharmaceutical dosage form. The measured pharmaceutical dosage forms were: a prepared pharmaceutical cocrystal, a physical mixture, and microcrystalline cellulose. The spectral patterns of the cocrystal and physical mixture were easily distinguished in the low-frequency region of the Raman spectrum. Based on the spectrum of the cocrystal and physical mixture, two different crystalline forms in the pharmaceutical dosage form were visualized using Raman microscopy. We concluded that low-frequency Raman spectroscopy is able to directly visualize the crystalline form of active pharmaceutical ingredients in pharmaceutical dosage forms without any pretreatment.

Keywords: Chemical imaging; Pharmaceutical cocrystals; Low-frequency Raman spectroscopy; pharmaceutical dosage form

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1. INTRODUCTION Crystalline forms of active pharmaceutical ingredients (API) affect physicochemical properties such as stability, solubility, dissolution rate, and bioavailability1,2. Cocrystallization of API is one of the promising techniques that can easily improve these properties without requiring chemical modifications3-5. Crystalline forms of the APIs are usually evaluated by powder X-ray diffraction and differential scanning calorimetry6,7. However, it is difficult to analyze specific particles of crystalline API that are dispersed over a pharmaceutical dosage form using these techniques8,9. Raman spectroscopy has been widely recognized as a useful analytical method and is an essential technique in the pharmaceutical industry7,10-14. In particular, Raman spectroscopy can provide a characteristic pattern that indicates the chemical structure of compounds in the conventional spectral region (200 - 1800 cm-1) 13. Recently, the low-frequency region (10 - 200 cm-1) of the Raman spectrum has been utilized to obtain information about intermolecular interactions in crystalline forms of API12,15. The European Medicine Agency proposed in its reflection paper that the integrity of the cocrystal during the entire manufacturing process should be experimentally confirmed16. In this study, we attempted to evaluate two types of crystalline components and visualize their distribution in the dosage form using Raman microscopy at low-frequencies in a non-contact and non-destructive manner. The Raman spectrum at low-frequencies has also been measured to evaluate the molecular state of the APIs containing microcrystalline cellulose (MCC) in the 4


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pharmaceutical dosage form, where MCC is a traditional pharmaceutical additive exhibiting fluorescence that interferes with the detection of conventional Raman scattering.

2. EXPERIMENTAL SECTION 2.1. Materials Caffeine (CAF) and 4-hydroxybenzoic acid (4HBA) were purchased from Sigma-Aldrich Co. (St Louis, MO). Microcrystalline cellulose (MCC) was kindly donated from Asahi Kasei Chemicals Corporation (Tokyo, JAPAN). All chemicals were reagent grade and used as received. A 2:1 molar ratio of 4HBA and CAF was mixed using an agate mortar (physical mixture; PM). Cocrystals of 4HBA and CAF were prepared using the solvent evaporation and slurry methods17.

The prepared cocrystal was confirmed by XRD (see the supporting

information (S1)). 7.5% (w/w) pharmaceutical cocrystal consisting of CAF and 4HBA (molar ratio at 2:1) and 2.5% (w/w) PM were mixed with 90% (w/w) microcrystalline cellulose (MCC) and compressed to prepare pharmaceutical dosage forms. In addition, 10% (w/w) cocrystal with 90% (w/w) MCC was substituted for the cocrystal to evaluate the dissociation during manufacturing.

2.2. Raman microscopy The Raman spectra of the prepared pharmaceutical dosage form were acquired with a Raman 5



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microscope (called a “Work StationTM” by Kaiser Optical Systems Inc, Ann Arbor, MI USA) with 785 nm excitation laser and an exposure time of 10 seconds for reference spectra. For measurement of the low-frequency region, a SureBlock™ TR-MICRO module (Ondax Inc, Monrovia, CA USA) with a 976 nm excitation laser was attached to the Raman microscope. The addition of this module enabled the collection of Raman signals to within ~5cm-1 from the laser line for both the Stokes and anti-Stokes (standard cutoff of Kaiser Work Station is ~150cm-1 at 785nm excitation). Exposure times of 10 seconds for reference spectra, and 2 seconds at every point for chemical imaging were used. Chemical imaging was performed by scanning the Raman microscope over a 2000 × 2000 µm area consisting of 2500 points (x : 50, y : 50 points, respectively). The spot size using a 50-times objective lens was approximately 16 µm in diameter. Raman images were generated by partial least squares (PLS) type 2 using chemical imaging software called ISysTM (Malvern Instruments Ltd. UK)18.

3. RESULTS AND DISCUSSION Conventional Raman spectra of CAF, 4HBF, PM and cocrystal in the region from 200 to 1800 cm-1 are shown in Figure 1. The spectrum of the PM exhibited very strong peaks at 554 cm-1, medium peaks at 1327, 1599 cm-1, and a similar spectrum was obtained from the cocrystal. These scattering peaks seemed to be superimposed from 4HBA and CAF and then overlapped throughout the measured region. By comparison, the Raman spectra of the PM 6


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and cocrystal in the low-frequency region were significantly different (Figure 2). The spectrum of the PM has weak peaks at 22 and 38, 92, 112, 140 cm-1, meanwhile, strong characteristic peaks appeared at 15 and 48 cm-1 in the spectrum of the cocrystal. This result indicated that the crystalline structure of a cocrystal was formed with a rearrangement of the original crystalline structures of 4HBA and CAF, thus causing the peaks of 4HBA around 90 to 140 cm-1 to disappear. Recently, Larkin et al. reported an attempt at assignment of the Raman peaks they observed in the low-frequency region12. We are currently considering the exact assignment of each peak measured in this study. The significant spectral differences between the PM and cocrystal observed in the low-frequency region reflect the different crystal structures of the compounds. To distinguish crystalline components in the pharmaceutical dosage form by chemical imaging, model tablets were prepared using MCC as a typical pharmaceutical additive. Baseline drift derived from the fluorescence induced by exposure of the excitation laser (785 nm) to various cellulose derivatives, made it difficult to measure the dosage forms containing MCC in the conventional region (data not shown). The fluorescence of MCC made it difficult to obtain chemical imaging, since the fluorescence dominated the spectrum of the APIs. The low-frequency region of the Raman spectra for the PM and cocrystal in the model tablets are shown in Figure 3. The results verify that Raman analysis of the low-frequency region can detect slight differences in crystal form. Figure 4 shows the chemical imaging of model 7



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tablets containing different crystal forms. In this study, cocrystal, PM, and crystalline cellulose are illustrated red, green and blue, respectively. As shown in Figure 4, the dispersed cocrystal and PM can be visualized in the model tablet, and the total area of cocrystal and PM closely correspond to the amount of each component. We therefore conclude that the crystalline components can be identified using low-frequency Raman microscopy under non-contact and non-destructive conditions. Chemical imaging of pharmaceutical cocrystals have also been demonstrated by using terahertz spectroscopic imaging19. Since they employed terahertz spectroscopy, the experiment was performed under vacuum conditions to avoid water absorption. Furthermore, in order to avoid absorption in the terahertz spectrum, polystyrene was used to simulate pharmaceutical additives. It has been reported that a spatial resolution of several tens of micrometers is required to properly observe the dispersion state in the pharmaceutical dosage form20,21. Our system has enough spatial resolution (ca. 16 µm) to visualize the dispersion state of different crystalline forms in MCC tablets and analyze the pharmaceutical dosage forms, while terahertz spectroscopy (ca. 600 µm) does not. Furthermore, our system can be measured under ambient atmosphere, since water adsorption can be ignored. In conclusion, low-frequency Raman microscopy is a valuable technique to confirm the crystalline or molecular state of pharmaceutical ingredients for pharmaceutical dosage forms. This technique could be instantly applied to evaluate other crystalline forms such as 8


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polymorphism, hydration and dehydration, and their transitions in pharmaceutical dosage forms during manufacturing and/or storage.

Acknowledgements This work was supported in part by JSPS KAKENHI, Grant-in-Aid for Scientific Research (C), Grant Number 26460045 (to T. F. and M. I.). Mr Akinori Okuyama is also acknowledged for their assistance in conducting the experiments.

Supporting Information The figure showing the powder XRD patterns indicating formation of the cocrystal is provided in the supplementary material.

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Figures 555

1327

Raman Intensity (Arb.Unit)

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740 1698 1654

643

483

CAF 444

1359 1609 1287

1264 1220 1160

4HBA

PM

Cocrystal

Raman Shift (cm-1) Figure 1 Conventional Raman spectra of CAF, 4HBA, PM, and cocrystal.

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CAF Raman Intensity (Arb.Unit)

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200

4HBA

38 140

22

PM

112 92

Cocrystal

100

0

-100

-200

Raman Shift (cm-1) Figure 2 Low-frequency region of the Raman spectra for CAF, 4HBA, PM, and cocrystal.

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Raman Intensity (Arb.Unit)


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Cocrystal

PM

MCC

200

100

0

-100

-200

Raman Shift (cm-1) Figure 3 Low-frequency of the Raman spectra for cocrystal, PM and MCC in the pharmaceutical dosage form.

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Figure 4 Chemical imaging of (a) ternary components of cocrystal / PM / MCC, and (b) two components of cocrystal / MCC in the pharmaceutical dosage form.

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Direct High-Resolution Imaging of Crystalline Components in

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