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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, info@witec.de
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, info@witec.de
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, info@witec.de
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, info@witec.de
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).