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Chemical Characterization of Pharmaceutical Samples by Confocal Raman Microscopy and Correlative Techniques

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Chemical Characterization of Pharmaceutical Samples by Confocal Raman Microscopy and Correlative Techniques APPLICATION NOTE Chemical Characterization of Pharmaceutical Samples by Confocal Raman Microscopy and Correlative Techniques WITec GmbH, Lise-Meitner-Str. 6, 89081 Ulm, Germany 89081 Ulm, 6, Lise-Meitner-Str. GmbH, WITec +49 (0) 731 140 70 200 fax +49 (0) 731 140 700, fon www.WITec.de [email protected], Chemical Characterization of Pharmaceutical Samples by Confocal Raman Microscopy and Correlative Techniques Introduction topographic 3D Raman images, depth-pro- These multi-spectrum files are then analy- files and correlative Raman-AFM images zed to display and image the distribution Development, production and quality the analyzing capabilities of CRM and of chemical sample properties. control in the pharmaceutical industry its correlative techniques will be shown. requires efficient and reliable control Furthermore an example of a statistical Atomic Force Microscopy mechanisms to ensure the safety and the- quantitative evaluation of a spectral Ra- Atomic Force Microscopy (AFM) provides rapeutic effect of the final products. These man dataset will be given. topographic high-resolution information products can vary widely in composition by recording the interaction forces bet- and application. Several of their properties Materials & Methods ween the surface and a sharp tip mounted can be difficult to study with conventi- on a cantilever. In addition, local material onal techniques due to the inability of Confocal Optical Microscopy properties such as adhesion or stiffness these methods to chemically differentiate The primary benefit of confocal microsco- can be investigated. materials with sufficient spatial resolution py is the suppression of light from outside Combining CRM with AFM the high spatial and without damage or staining. There- the focal plane. This is realized firstly by a and topographical resolution obtained fore analyzing methods that provide both point illumination in the focal plane and with an AFM can be directly linked to the comprehensive chemical characterization secondly by a pinhole in the conjugate molecular information provided by con- and the flexibility to adjust the method to plane of the detection beam path. focal Raman spectroscopy. the investigated specimen are preferred in Due to the point illumination and detecti- pharmaceutical research. on, only information from a single point is Topographic Confocal Raman Imaging Confocal Raman Microscopy (CRM) is a determined at a time. In order to generate Although confocal Raman imaging is con- well-established and widely-used spec- an image the sample is scanned point-by- sidered to be a non-destructive method troscopic method for the investigation of point and line-by-line. it is common to create a smooth and the chemical composition of a sample. In The advantages of confocal microscopy even sample surface in order to keep the pharmaceutical research, CRM can be used over conventional wide-field microscopy surface in the focal plane while measu- to probe the distribution of components are an improved resolution in the lateral ring in confocal Raman imaging mode. within formulations, to characterize the plane, a high axial discriminatory power, a However, this intervention can lead to the homogeneity of pharmaceutical samples, reduced background signal and the ability misinterpretation of the analytical results. to determine the state of drug substan- to collect optical sections in series from To avoid any sample preparation topogra- ces and excipients and to characterize different focal planes to generate depth phic confocal Raman imaging (TrueSurface contaminants and foreign particulates [1]. profiles or 3D images. Microscopy, WITec GmbH, Germany) can be The information obtained by CRM is also applied. It is especially useful for samples useful for drug substance design, for the Confocal Raman Microscopy with rough or inclined surfaces. The highly development of solid and liquid formula- By combining a research-grade confocal precise sensor scans the topography of the tions, as a tool for process analytics and optical microscope with a high-sensitivity sample. The topographic coordinates are for patent infringement and counterfeit Raman spectrometer, the resolution of the used to trace the sample‘s surface in con- analysis [2, 3, 4]. Being a non-destructive optical microscope can complement the focal Raman imaging mode to ensure that method it is possible to combine CRM with analytical power of Raman spectroscopy. the Raman laser is always kept in focus. other imaging techniques such as Atomic Thus it is not only possible to obtain Ra- The result is a confocal Raman image that Force Microscopy (AFM), Scanning Electron man spectra from extremely small sample can be overlaid on the topographic image Microscopy (SEM) or fluorescence analysis. volumes, but also to collect high-resolution and visualized three dimensionally. Raman images that show the distribution The Aim of the Study of chemical species and crystallographic Experimental Setup properties with sub-µm resolution. The A confocal Raman microscope (alpha300 Based on various samples the article will sample is scanned point-by-point and line- R, WITec GmbH) was used to study the demonstrate how CRM can be beneficially by-line, and at every image pixel a com- chemical composition of various pharma- applied in the pharmaceutical field. With plete Raman spectrum is acquired. This ceutical samples. The laser light is guided high-resolution, large-area Raman images, process is called hyperspectral imaging. to the microscope through an optical fiber. It can be focused to a diffraction-li- ve, back-illuminated CCD camera. was acquired, therefore the large image mited spot and therefore acts as a point is the result of an evaluation of 4,194,304 light source. This light is focused onto the Results & Discussion Raman spectra. The integration time per sample using a dichroic beam splitter that spectrum was 2 ms and the raw data file reflects the exciting laser beam, but is High-Resolution, Large-Area Confocal size is 12.5 GB. The consecutive zoom-in fully transparent for the frequency-shifted Raman Imaging, Volume Scans and 3D images (figure 1b and 1c) of the same data- Raman light. The sample is scanned with a Visualizations set illustrate the extremely high-resolution piezo-electric scanning table with capaciti- In the first example a pharmaceutical of the large-area scan shown in figure 1a. ve feedback correction for high resolution. emulsion was investigated. The active The Raman data were acquired, evalua- The Raman scattered light is collected pharmaceutical ingredient (API) was ted and processed with the Project FOUR with the same objective and is focused dissolved in water. In figure 1a a large-area, Software (WITec GmbH, Germany). In the into the core of another optical fiber that high-resolution Raman image of the emul- resulting color-coded images the water is connected to a spectrometer. The light is sion is shown. The image scan range is 180 and API-containing phase is presented in dispersed inside the spectrometer and the x 180 µm2 with 2048 x 2048 pixels. At each blue, whereas with green the oil-matrix is spectra are acquired with an ultra-sensiti- image pixel a complete Raman spectrum displayed. In addition to the distribution of a) b) c) Figure 1: a) Large-area, high-resolution confocal Raman image of a pharmaceutical emulsion. Scan range: 180 µm x 180 µm; 2048 x 2048 pixels = 4,194,304 Raman spectra; raw data file size: 12.5 GB. Blue: active pharmaceutical ingredient; green: oil; red: silicon impurities. b) and c) consecutive zoom-in images of the same dataset. the known materials, silicon-based impuri- Figure 2: Confocal 3D Raman ties could be visualized (red in the images). volume image. The green oil is Volume scans are a valuable tool for pro- partially removed in the image viding information about the dimensions to facilitate a better identifica- tion of the red impurities. Scan of objects or the distribution of a certain range: 25 x 25 x 20 µm3, 200 x compound throughout the sample. The 200 x 50 pixels = 2,000,000 generation of volume scans and 3D images Raman spectra, integration always requires large data sizes. In order time per spectrum: 10 ms, data to generate 3D images, confocal 2D Raman file size: 6 GB. images of parallel focal planes are acqui- red by scanning throughout the sample in the z-direction. The 2D images are then combined into a 3D image stack. To investigate the volume of the impurities of the emulsion from figure 1a in more detail, a 3D scan was performed. For that a volume of 25 x 25 x 20 µm3 was analyzed a) with 200 x 200 x 50 pixels. At each image pixel a complete Raman spectra with 10 ms integration time per spectrum was acquired. The image stack contains the information of a total of 2 million Raman spectra and is based on a raw data file size of 6 GB (figure 2). In the following an example emulsion was studied. Therefore carbon tetrachloride (CCl4) was mixed with an alkane, water and oil. A high-resolution 3D Raman image of the emulsion was acquired (figure 3). It shows the three-dimensional distribu- tion of the components and reveals CCl4 dissolved in the oil phase. b) Figure 3: Confocal, high-resolution Raman microscopy of an emulsion of CCl4, alkane, water and oil. a) Three-dimensional confocal color-coded Raman image of the emulsion; alkane: green, water: blue, CCl4 + oil: yellow. Image parameters: 200 x 200 x 20 pixels; scan area: 100 x 100 x 10 µm3; in- tegration time per spectrum: 0.06 s; excitation laser wavelength: 532 nm. b) corresponding Raman spectra. Topographic Raman Imaging the sample’s surface in focus over the green. In this way the generated 3D Raman A pharmaceutical tablet with height entire scanned area without any Raman image provides the full information about variations of 300 µm on the surface signal decrease.
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