application note Lise-Meitner-Straße 6, D-89081 Ulm, Germany Tel. +49 (0) 731 140 700, Fax. +49 (0) 731 140 70200 www.witec.de, [email protected] Investigations of Pharmaceutical Drug Delivery Systems with Topographic Confocal Raman Imaging Knowledge about the morphology and chemical composition of heterogeneous materials is crucial for the pharmaceutical development of new material properties for highly specified drug delivery systems. However, several properties are difficult to study with conventional characterization techniques due to the inability of these methods to chemically differentiate materials with sufficient spatial resolution, without damage or staining. The combination of confocal Raman Imaging with TrueSurface Microscopy provide unique topographic as well as chemical information to nondestructively and non-invasively characterize a sample three-dimensionally, underneath and at the surface. It also facilitates accurate chemical characterization of even rough and inclined samples. This contributes to a detailed qualitative description of a pharmaceutical drug system and effectively supports pharmaceutical research and development. The application note gives an overview of several pharmaceutical dosage forms and drug systems investigated by topographic confocal Raman imaging. Confocal Raman Imaging A Raman spectrum shows the energy shift of the excitation light (laser) as a result of inelastic scattering by the molecules in a sample. The excitation light excites or annihilates vibrations of the chemical bonds within the molecules which results in an energy shift of the photon scattered from this molecule. Different chemical species consist of different atoms and bonds, so each molecule can be easily identified by its unique Raman spectrum. As only molecular vibrations are excited (or annihilated), Raman spectroscopy is a nondestructive technique. In Raman imaging the Raman spectra are collected with a highthroughput confocal microscope/Raman spectrometercombination. A high-sensitivity CCD camera connected to a powerful computer and software system is used to detect the Raman signal. With specialized software tools the imaging capabilities can be expanded even further. For example, it is possible to generate images by integrating over selected spectral areas, determining the peak width, peak position or by even more sophisticated procedures such as the fitting of complete spectra or cluster analysis. Raman Principle TrueSurface® Microscopy The key element of this novel imaging mode is a topographic sensor that works using the principle of chromatic aberration. With this non-contact, purely optical profilometer technique it is possible to trace a sample's topography and follow it in a subsequent Raman measurement, thus remaining in focus throughout. For profilometry a white light point-source is focused onto the sample with a hyperchromatic lens assembly: A lens system with a good point mapping capability, but a strong linear chromatic error. Every color has therefore a different focal distance. The light reflected from the sample is collected with the lens and focused through a pinhole into a spectrometer. As only one color is in focus at the sample surface, only this light can pass through the confocal pinhole. The detected wavelength is therefore related to the surface topography. Scanning the sample in the XY plane reveals a topographic map of the sample. This map can then be followed in a subsequent Raman image so that the Raman laser is always kept in focus with the sample surface (or at any distance below the surface). The results are images revealing chemical and/or optical properties at the surface of the sample, even if the surface is rough or inclined. Integrated TrueSurface® Microscopy TrueSurface® Principle Above (without TrueSurface®): When imaging rough surfaces in confocal imaging mode only parts of the sample are in focus. TrueSurface®: Each color corresponds to a certain focal Below (with TrueSurface®): Following the topography in distance. confocal imaging mode enables the surface to always stay in focus. application note Lise-Meitner-Straße 6, D-89081 Ulm, Germany Tel. +49 (0) 731 140 700, Fax. +49 (0) 731 140 70200 www.witec.de, [email protected] Methods The experiments were performed using a WITec alpha500R microscope for large-area confocal Raman imaging and a WITec alpha300 R+ microscope for automated confocal Raman imaging. The microscopes were equipped with TrueSurface® Microscopy for topographic profiling. For the investigations, an excitation wavelengths of 532 nm was used. The TrueSurface® topographic confocal Raman imaging extension was directly integrated into the objective turret of the microscope. Due to the non-destructive measurement techniques it was not required to stain, fix or dissect the samples. The data were acquired, evaluated and processed with the WITec Project FOUR Software. (b) (a) (c) drug 1 drug 2 excipient Example TrueSurface® Microscopy measurement. A height profile of a pharmaceutical tablet was scanned with TrueSurface®. The topographic variation was 300 µm (a). The sample’s topography was then followed in confocal Raman imaging mode. The confocal Raman image was superimposed on the height profile resulting in a topographic confocal Raman image. The drugs are labeled red and blue, while the excipient is shown in green (b). Corresponding Raman spectra with the same color code (c). application note Lise-Meitner-Straße 6, D-89081 Ulm, Germany Tel. +49 (0) 731 140 700, Fax. +49 (0) 731 140 70200 www.witec.de, [email protected] Lyophilisate (c) (d) (a) (b) Investigation of the protein conformation of a highly structured lyophilisate section. (a) Lyophilisate image visualizing the highly structured surface. (b) Surface topography profile of a lyophilisate section acquired with TrueSurface® Microscopy. (c) Plain confocal Raman image of the investigated area. (d) Overlay of the topography profile with the corresponding confocal Raman image. The resulting three dimensional, colorcoded image indicates the native protein conformation in blue and the denatured protein conformation in red. Scale bars: 400 µm. Tablet Cross Section (a) 100 µm (b) 100 µm Cross sections of tablets (two components) fabricated with different milling techniques. The topography profile was acquired with TrueSurface® Microscopy and overlaid with the confocal Raman Image. (a) Both components unground. (b) Co-ground components. application note Lise-Meitner-Straße 6, D-89081 Ulm, Germany Tel. +49 (0) 731 140 700, Fax. +49 (0) 731 140 70200 www.witec.de, [email protected] Membrane Layer Coating Exterior Extrudate Surface (a) (a) (b) (b) 200 µm Confocal Raman Image of a membrane layer coating. The sample’s topography was first investigated by TrueSurface® Microscopy. Then this topographic information was used to follow the sample’s surface structure for confocal Raman imaging. The color-coded Raman image shows the membrane in red and the lipid layer coating in blue. (a) Membrane with inconsistent coating (b) consistent lipid layer coating on top of the membrane. Component distribution analysis of the exterior extrudate surface. (a) Photograph of the extrudate with dimension scale (b) Overlay of the topography profile acquired with TrueSurface® and the colorcoded confocal Raman image revealing the component distribution on the exterior surface. The drug is shown in blue and the lipid matrix in green. Images pages 3 + 4 courtesy of Dr. Maike Windbergs, Department of Biopharmaceutics and Pharmaceutical Technology, Saarland University, Germany. Further reading: B. Kann, M. Windbergs, Chemical imaging of drug delivery systems with structured surfaces-a combined analytical approach of confocal Raman microscopy and optical profilometry. The AAPS journal 15, 505-510 (2013).
© Copyright 2024