Optimization of a Raman microscopy technique to efficiently detect amorphous-amorphous phase separation in freeze-dried protein formulations.

A confocal Raman microscopic technique was optimized to more efficiently detect amorphous-amorphous phase separation in freeze-dried protein formulations. A Renishaw Raman inVia confocal microscope was used to collect 100-200 µm line maps (2 µm step size) of freeze-dried protein-excipient formulations. At each point across the line map, the composition was evaluated from the intensity of the nonoverlapping peaks representative of each component. Collection aperture, scan time, and line map length significantly contributed to the phase-separation analysis, whereas different sample preparation methods did not affect the analysis. Using the optimized parameters (i.e., large aperture 5 s scan time, 200 µm line map), phase separation was successfully detected in binary polymer formulations and was comparable to the previously developed Raman method. However, the previous method required 2.5 h/sample, whereas the optimized method only requires 0.5 h/sample. Phase separation was detected in the following protein-excipient formulations: lysozyme-trehalose (1:1), lysozyme-isomaltose (1:1), ß-lactoglobulin-dextran (1:1), ß-lactoglobulin-dextran (1:3), and ß-lactoglobulin-trehalose (1:1). Phase separation was not detected in lysozyme-sucrose (1:1) and ß-lactoglobulin-sucrose (1:1) formulations. The optimized method successfully detected phase separation in several protein formulations, where phase separation was previously suspected, and promised to be a useful tool for detection of phase separation in amorphous therapeutic formulations.

Sensitivity-enhanced transmission Raman spectroscopy.

Transmission Raman sensitivity for a representative commercial pharmaceutical tablet was increased by a factor of 40 using optics that returned lost laser and Raman photons to the tablet surface. A new achromatic one-way mirror is introduced that uses the spatial coherence of laser light to nondestructively force laser photons through the reflective tablet coating. Transmission Raman mapping and spatially offset Raman spectroscopy (SORS) mapping were developed and used to better understand the sensitivity-enhancement technology. Fundamental limitations of the sensitivity-enhancement approach are described and used to guide the design of the optics. The sensitivity-enhancement optics are compatible with commercial transmission Raman instruments.

Transmission fourier transform Raman spectroscopy of pharmaceutical tablet cores.

Transmission Fourier transform (FT) Raman spectroscopy of pharmaceutical tablet cores is demonstrated using traditional, unmodified commercial instrumentation. The benefits of improved precision over backscattering Raman spectroscopy due to increased sample volume are demonstrated. Self-absorption effects on analyte band ratios and sample probe volume are apparent, however. A survey of near-infrared (NIR) absorption spectra in the FT-Raman spectral range (approximately 0 to 3500 wavenumber shift from 1064 nm, or 1064 to 1700 nm) of molecules with a wide range of NIR-active functional groups shows that although absorption at the laser wavelength (1064 nm) is relatively small, some regions of the Raman spectrum coincide with NIR absorbances of 0.5 per cm or greater. Fortunately, the pharmaceutically important regions of the Raman shift spectrum from 0 to 600 cm(-1) and from 1400 to 1900 cm(-1) exhibit low self-absorption for most organic materials. A statistical analysis of transmission FT-Raman noise in spectra collected from different regions of a pharmaceutical tablet provides insight into both spectral distortion and reduced sampling volume caused by self-absorption.

Control of out-of-focus light intensity in confocal raman microscopy using optical preprocessing.

Optical preprocessing, the manipulation of imaging-forming light prior to detection, is used in many forms of microscopy to enhance image information and interpretation. The rejection of out-of-focus light by a pinhole in confocal microscopy is an example of optical preprocessing, where spatial filtering is carried out in a plane conjugate to the object focal plane. The rejection efficiency afforded by this arrangement is, however, sometimes insufficient. Insufficient rejection of out-of-focus light intensity can lead to incorrect interpretations of confocal Raman depth and line maps. An alternative approach is to reject out-of-focus light by implementing spatial filtering in one of the several planes conjugate to the pupil plane. This paper shows that mapping enhanced by structured pupils (MESP) provides substantial additional rejection of out-of-focus intensity relative to traditional confocal microscopy, yielding a potential solution to the problem of misleading Raman maps. In addition, lossless MESP is proposed, wherein simple phase masks simultaneously direct in-focus and out-of-focus light intensity transmitted by confocal optics to different regions of a charge-coupled device (CCD) detector.

Monolithic Teflon membrane valves and pumps for harsh chemical and low-temperature use.

Microfluidic diaphragm valves and pumps capable of surviving conditions required for unmanned spaceflight applications have been developed. The Pasteur payload of the European ExoMars Rover is expected to experience temperatures ranging between -100 degrees C and +50 degrees C during its transit to Mars and on the Martian surface. As such, the Urey instrument package, which contains at its core a lab-on-a-chip capillary electrophoresis analysis system first demonstrated by Mathies et al., requires valving and pumping systems that are robust under these conditions before and after exposure to liquid samples, which are to be analyzed for chemical signatures of past or present living processes. The microfluidic system developed to meet this requirement uses membranes consisting of Teflon and Teflon AF as a deformable material in the valve seat region between etched Borofloat glass wafers. Pneumatic pressure and vacuum, delivered via off-chip solenoid valves, are used to actuate individual on-chip valves. Valve sealing properties of Teflon diaphragm valves, as well as pumping properties from collections of valves, are characterized. Secondary processing for embossing the membrane against the valve seats after fabrication is performed to optimize single valve sealing characteristics. A variety of different material solutions are found to produce robust devices. The optimal valve system utilizes a membrane of mechanically cut Teflon sandwiched between two thin spun films of Teflon AF-1600 as a composite "laminated" diaphragm. Pump rates up to 1600 nL s(-1) are achieved with pumps of this kind. These high pumping rates are possible because of the very fast response of the membranes to applied pressure, enabling extremely fast pump cycling with relatively small liquid volumes, compared to analogous diaphragm pumps. The developed technologies are robust over extremes of temperature cycling and are applicable in a wide range of chemical environments.

Rapid, nondestructive near-infrared assay for water in sealed dimethyl sulfoxide compound repository containers.

Water contamination in dimethyl sulfoxide (DMSO) compound libraries is a recognized problem in the pharmaceutical industry. We describe a nondestructive, near-infrared (NIR) assay for water contamination inside the sealed containers used for compound libraries. This assay does not require opening the containers. We also report our observation of the permeation of water from laboratory air through the sealed container and into the DMSO solution over a period of months, as well as the rate of water absorption by DMSO directly exposed to laboratory air.

Measurement of amphotericin B concentration by resonant Raman spectroscopy--a novel technique that may be useful for non-invasive monitoring.

We wished to determine whether Resonant Raman Spectroscopy (RRS) could be used to measure Amphotericin B (AmB) at therapeutic and subtherapeutic concentrations in a model system mimicking the anterior chamber of the eye. The goal was to develop a technique for non-invasive measurement of AmB levels in the aqueous humor (AH) of the eye. A krypton-ion laser source (406.7 nm) was used for excitation and Resonant Raman Spectra were captured with a confocal system in an anterior chamber (AC) model. These spectra were used to develop a correlation curve for prediction of AmB levels. Subsequently, one rabbit was evaluated with this system after 5 days of intravenous AmB administration (1 mg/kg/day) and AmB concentrations measured by RRS were compared to those measured by high-performance liquid chromatography (HPLC). AmB exhibited a unique spectral peak at 1557 cm(-1). Integrated area of this peak linearly correlated with AmB concentration in our model AC. When integrated peak area from multiple in vivo measurements in one animal at steady-state was plotted on this correlation curve, we were able to predict AmB levels. These closely approximated those measured by HPLC. These measurements were not significantly affected by photobleaching or depth profile at acquisition. RRS at 406.7 nm is a method that may be useful for non-invasive monitoring of intraocular AmB levels. This instrument can help physicians decide when repeat, invasive delivery of this drug is warranted based on measurement of actual drug levels in the AH. Also, there is the potential to measure the ocular concentrations of other pharmaceutical agents with similar instruments.