MICROSCOPY & IMAGING
PHARMACEUTICAL
Damon Strom explores comprehensive particle analysis with correlative Raman microscopy
PARTICLE ANALYSIS
R
aman microscopy provides exceptional chemical sensitivity that allows data acquisition from very small material volumes, such
as those encountered in microparticle analysis. Te combination of Raman with other technologies, including confocal white light microscopy and scanning electron microscopy (SEM), to acquire data from the same measurement position is known as correlative Raman microscopy. Investigations of particles are especially
important in pharmaceutical research as a precise understanding of properties and composition is necessary to refine production processes and determine efficacy. Analyses of many particles and the identification of different substances within an individual particle are routine challenges. Te following measurement examples demonstrate how correlative Raman microscopy can expedite these tasks.
RAMAN AND CONFOCAL WHITE LIGHT MICROSCOPY For informative evaluations of powder samples, a large number of particles must be analysed. ParticleScout, an automated microparticle analysis tool for WITec’s alpha300 microscope series, accelerates this process by combining Raman spectroscopy with confocal white light microscopy and advanced algorithms to find, classify and identify microparticles. Here, an analgesic (pain-killing) and antipyretic (anti-fever) powder sample was investigated with ParticleScout by first acquiring a confocal white light image (Fig. 1A). Image stitching ensured high resolution and focus stacking sharply
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defined the edges of each particle, which were used to create a mask of their locations. Criteria were selected that focused the analysis on particles with a Feret diameter of less than 100 µm and an area of 1 µm² or larger. Tis highlighted a total of 3,052 particles (Fig. 1B). A Raman spectrum was then automatically acquired from each particle and identified with the integrated TrueMatch Raman spectral database software (Fig. 1C). Te measurement revealed that the majority of the particles were the analgesic agents acetaminophen and ethenzamide. Caffeine was present as an effect enhancer, lactose as a carrier, and white pigment was also detected.
RAMAN IMAGING AND SCANNING ELECTRON MICROSCOPY Microparticles are often used as carrier systems for drugs and their morphology and composition can affect the bioavailability of the delivered active pharmaceutical ingredient (API). Raman Imaging and Scanning Electron (RISE) microscopy can explore these properties in great detail within a common vacuum chamber. Shuttling the sample between measurement positions enables the correlation of structural and chemical information.
Tis study of a particle from an anti- asthma inhaler used a WITec/Tescan RISE microscope with a 532nm excitation laser to acquire a Raman image (Fig. 2A) that visualises the four chemical components identified and color-coded by their Raman spectra (Fig. 2B). Te particle consists mainly of lactose in two different hydration states (blue and green). Te
C: Particles colour coded
corresponding to spectra. Acetaminophen (blue), ethenzamide (cyan), lactose (green), caffeine (orange), and white pigment (light grey)
A: Confocal white light image of analgesic and antipyretic powder
FIG. 1. Correlative Raman- confocal white light microscopy particle analysis of a powdered pharmaceutical sample.
B: Selected particles for analysis
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