E. Neil Lewis

Near-infrared spectral imaging for quality assurance of pharmaceutical products: Analysis of tablets to assess powder blend homogeneity

The objective of this study was to evaluate near-infrared (NIR) spectroscopic imaging as a tool to assess a pharmaceutical quality assurance problem—blend uniformity in the final dosage product. A system based on array detector technology was used to rapidly collect high-contrast NIR images of furosemide tablets. By varying the mixing, 5 grades of experimental tablets containing the same amount of furosemide and microcrystalline cellulose were produced, ranging from well blended to unblended. For comparison, these tablets were also analyzed by traditional NIR spectroscopy, and both approaches were used to evaluate drug product homogeneity. NIR spectral imaging was capable of clearly differentiating between each grade of blending, both qualitatively and quantitatively. The spatial distribution of the components was based on the variation or contrast in pixel intensity, which is due to the NIR spectral contribution to each pixel. The chemical nature of each pixel could be identified by the localized spectrum associated with each pixel. Both univariate and partial least squares (PLS) images were evaluated. In the suboptimal blends, the regions of heterogeneity were obvious by visual inspection of the images. A quantitative measure of blending was determined by calculating the standard deviation of the distribution of pixel intensities in the PLS score images. The percent standard deviation increased progressively from 11% to 240% from well blended to unblended tablets. The NIR spectral imaging system provides a rapid approach for acquiring spatial and spectral information on pharmaceuticals. The technique has potential for a variety of applications in product quality assurance and could affect the control of manufacturing processes.

High-Fidelity Raman Imaging Spectrometry: A Rapid Method Using an Acousto-optic Tunable Filter

In this communication, we describe a technique for obtaining high-fidelity Raman images and Raman spectra. The instrumentation provides the ability to rapidly collect large-format images with the number of image pixels limited only by the number of detector elements in the silicon charge-coupled device (CCD). Wavelength selection is achieved with an acousto-optic tunable filter (AOTF), which maintains image fidelity while providing spectral selectivity. Under computer control the AOTF is capable of µs tuning speeds within the operating range of the filter (400–1900 nm). The AOTF is integrated with the CCD and holographic Raman filters to comprise an entirely solid-state Raman imager containing no moving parts. In operation, the AOTF is placed in front of the CCD and tuned over the desired spectral interval. The two-dimensional CCD detector is employed as a true imaging camera, providing a full multichannel advantage over competitive Raman imaging techniques. Images and spectra are presented of a mixture of dipalmitoylphosphatidylcholine (DPPC) and L-asparagine, which serves as a model system for the study of both lipid/peptide and lipid/protein interactions in intact biological materials. The Raman images are collected in only several seconds and indicate the efficacy of this rapid technique for discriminating between multiple components in complex matrices. Additionally, high-quality Raman spectra of the spatially resolved microscopic regions are easily obtained.

Near-Infrared Acousto-Optic Filtered Spectroscopic Microscopy: A Solid-State Approach to Chemical Imaging:

A new instrumental approach for performing spectroscopic imaging microscopy is described. The instrument integrates an acousto-optic tunable filter (AOTF) and charge-coupled-device (CCD) detector with an infinity-corrected microscope for operation in the visible and near-infrared (NIR) spectral regions. Images at moderate spectral resolution (2 nm) and high spatial resolution (1 μim) can be collected rapidly. Data are presented containing 128 × 128 pixels, although images with significantly larger formats can be collected in approximately the same time. In operation, the CCD is used as a true imaging detector, while wavelength selectivity is provided by using the AOTF and quartz tungsten halogen lamp to create a tunable source. The instrument is entirely solid state, containing no moving parts, and can be readily configured for both absorption and reflectance spectroscopies. We present visible absorption spectral images of human epithelial cells, as well as NIR vibrational absorption images of a hydrated phospholipid suspension, to demonstrate the potential of the technique in the study of biological materials. Extensions and future applications of this work are discussed.

Real-Time, Mid-Infrared Spectroscopic Imaging Microscopy Using Indium Antimonide Focal-Plane Array Detection

A different approach toward mid-infrared spectroscopic imaging microscopy is introduced in which instrumentation is designed about an InSb multichannel, focal-plane array detector and a variable-bandpass dielectric filter. The system may be configured for either macroscopic or microscopic applications, and high-fidelity, chemically specific images may be acquired in real time. With the dielectric filter used in this assembly, continuous tuning is provided for the infrared 4000–2320 cm−1 spectral region with spectral resolutions of approximately 35–18 cm−1 at the extremes of this wavelength interval. The functioning of the imaging microscope is demonstrated with samples including polystyrene microspheres, preparations of lipids and an amino acid embedded in KBr disks, and a tissue sample derived from a coronal slice of a monkey cerebellum.

Infrared Spectroscopic Imaging of the Biochemical Modifications Induced in the Cerebellum of the Niemann-Pick type C Mouse.

We have applied Fourier transform infrared (IR) spectroscopic imaging to the investigation of the neuropathologic effects of a genetic lipid storage disease, Niemann-Pick type C (NPC). Tissue sections both from the cerebella of a strain of BALB/c mice that demonstrated morphology and pathology of the human disease and from control animals were used. These samples were analyzed by standard histopathological procedures as well as this new IR imaging approach. The IR absorbance images exhibit contrast based on biochemical variations and allow for the identification of the cellular layers within the tissue samples. Furthermore, these images provide a qualitative description of the localized biochemical differences existing between the diseased and control tissue in the absence of histological staining. Statistical analyses of the IR spectra extracted from individual cell layers of the imaging data sets provide concise quantitative descriptions of these biochemical changes. The results indicate that lipid is depleted specifically in the white matter of the NPC mouse in comparison to the control samples. Minor differences were noted for the granular layers, but no significant differences were observed in the molecular layers of the cerebellar tissue. These changes are consistent with significant demyelination within the cerebellum of the NPC mouse.

Development of Near-Infrared Fourier Transform Raman Spectroscopy for the Study of Biologically Active Macromolecules

General advantages and potential limitations of Fourier transform (FT) Raman spectroscopy using Nd:YAG laser excitation at 1064 nm have been considered for both routine analysis and specific biophysical applications. Optical design and operating parameters which affect the quality and reproducibility of the data are discussed. Moderately high resolution spectra (0.25 cm−1) of liquids are obtained with relative ease, and the results are compared with dispersive spectra. Particular emphasis has been placed on applications to biological systems where intrinsic fluorescence has traditionally limited the use of dispersive Raman spectroscopy. As an example of a biophysical study, we demonstrate the utility of FT-Raman spectroscopy in elucidating the interactions of polyene antibiotics with model membrane lipid bilayers as a means of understanding novel drug/membrane interactions at the molecular level.

Magnesium-induced lipid bilayer microdomain reorganizations: implications for membrane fusion.

Interactions between dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS), combined both as binary lipid bilayer assemblies and separately, under the influence of divalent Mg2+, a membrane bilayer fusogenic agent, are reported. Infrared vibrational spectroscopic analyses of the lipid acyl chain methylene symmetric stretching modes indicate that aggregates of the two phospholipid components exist as domains heterogeneously distributed throughout the binary bilayer system. In the presence of Mg2+, DPPS maintains an ordered orthorhombic subcell gel phase structure through the phase transition temperature, while the DPPC component is only minimally perturbed with respect to the gel to liquid crystalline phase change. The addition of Mg2+ induces a reorganization of the lipid domains in which the gel phase acyl chain planes rearrange from a hexagonal configuration toward a triclinic, parallel chain subcell. Examination of the acyl chain methylene deformation modes at low temperatures allows a determination of DPPS microdomain sizes, which decrease upon the addition of DPPC-d62 in the absence of Mg2+. On adding Mg2+, a uniform DPPS domain size is observed in the binary mixtures. In either the presence or absence of Mg2+, DPPC-d62 aggregates remain in a configuration for which microdomain sizes are not spectroscopically measurable. Analysis of the acyl chain methylene deformation modes for DPPC-d62 in the binary system suggests that clusters of the deuterated lipids are distributed throughout the DPPS matrix. Light scattering and fluorescence measurements indicate that Mg2+ induces both the aggregation and the fusion of the lipid assemblies as a function of the ratio of DPPS to DPPC. The structural reorganizations of the lipid microdomains within the DPPS-DPPC bilayer are interpreted in the context of current concepts regarding lipid bilayer fusion.

Applications of Fourier Transform Infrared Imaging Microscopy in Neurotoxicity

The spatial distribution of components within complex materials strongly influences both their physical and chemical properties. Thus, analytical methods that provide information on both the localization and molecular characteristics of composite materials are invaluable in disciplines as diverse as the design and fabrication of advanced materials or the chemical and biochemical elucidation of cellular systems. Spectroscopic imaging is a particularly attractive method since it can provide simultaneous information on both the spatial and chemical properties of an intact system while preserving sample integrity. In applications to biological specimens, for example, spectral imaging is also suitable for noninvasive biophysical analyses and biomedical diagnoses performed in vivo. Therefore, spectral imaging allows both the researcher and clinician to rapidly visualize sample chemistry with minimum sample preparation and disruption. Spectral imaging extends the power of spectroscopic analysis by generating spatial information while retaining the analytical capability provided by traditional, nonimaging spectroscopies. For example, nuclear magnetic resonance techniques are applied for whole-body diagnostic imaging, whereas for investigation of the microscale, fluorescence m i ~ r o s c o p y ~ . ~ is an effective chemical state imaging. Chemical visualization methods integrating micros-

Aggregate structure, morphology and the effect of aggregation mechanisms on viscosity at elevated protein concentrations.

Non-native aggregation is a common issue in a number of degenerative diseases and during manufacturing of protein-based therapeutics. There is a growing interest to monitor protein stability at intermediate to high protein concentrations, which are required for therapeutic dosing of subcutaneous injections. An understanding of the impact of protein structural changes and interactions on the protein aggregation mechanisms and resulting aggregate size and morphology may lead to improved strategies to reduce aggregation and solution viscosity. This report investigates non-native aggregation of a model protein, α-chymotrypsinogen, under accelerated conditions at elevated protein concentrations. Far-UV circular dichroism and Raman scattering show structural changes during aggregation. Size exclusion chromatography and laser light scattering are used to monitor the progression of aggregate growth and monomer loss. Monomer loss is concomitant with increased β-sheet structures as monomers are added to aggregates, which illustrate a transition from a native monomeric state to an aggregate state. Aggregates grow predominantly through monomer-addition, resulting in a semi-flexible polymer morphology. Analysis of aggregation growth kinetics shows that pH strongly affects the characteristic timescales for nucleation (τn) and growth (τg), while the initial protein concentration has only minor effects on τn or τg. Low-shear viscosity measurements follow a common scaling relationship between average aggregate molecular weight (Mw(agg)) and concentration (σ), which is consistent with semi-dilute polymer-solution theory. The results establish a link between aggregate growth mechanisms, which couple Mw(agg) and σ, to increases in solution viscosity even at these intermediate protein concentrations (less than 3w/v %).

Infrared spectroscopic study of ethanol-induced changes in rat liver plasma membrane

Vibrational infrared spectroscopy, a noninvasive method for probing the structural and dynamic properties of biological membranes, is used to characterize the in vivo and in vitro perturbations of ethanol on various liver plasma membrane preparations derived from alcohol-treated rats. Spectral frequency shifts of the bilayer lipid chain methylene carbon-hydrogen symmetric stretching modes indicate that the adaptive response of the liver plasma membranes of the alcohol-treated animals results in an increase in membrane order on the vibrational time scale. Additional in vitro ethanol treatment of these membrane preparations leads to further significant increases in bilayer order. The observed membrane ordering effects are consistent with a bilayer model of partial interdigitation, or chain overlap, of the opposing membrane monolayers near the bilayer center.