Ingredient-specific particle sizing (ISPS) has historically been a challenge for the manufacturers of drugs delivered by nasal aerosols and sprays, as well as those who design their delivery systems. Measuring particle-size distribution of the active ingredients in formulated nasal sprays is critical to understanding the drug-dissolution rate and drug availability to active sites within the nasal cavity. Special requirements for reporting drug-particle size statistics have been established according to the US Food and Drug Administration (FDA) Guidance for Industry for Bioavailability and Bioequivalence Studies.¹ Assessing drug particles in the presence of insoluble suspending agents as well as any influence the delivery device may have on their agglomeration is of great interest to formulation developers.

click to enlarge Figure 1. Raman dispersive spectra of fluticasone propionate (A) and mometasone furoate (B) formulation components. The yellow areas define the spectral boundaries used in the Raman Chemical Imaging measurement. (Source: ChemImage Corporation)

While a number of established methods for particle sizing (e.g. laser-light scattering, optical microscopy, and cascade impaction) are available to drug developers, none of these methods can identify individual ingredients in the formulated suspensions, especially when spatial and chemical information is of critical value. Optical microscopy is used to measure drug-particle size in formulated suspensions.¹ Powerful software algorithms then derive information about size, shape, color, and morphology from optical images. Unfortunately, this approach to active pharmaceutical ingredient (API) recognition is subjective and difficult to validate, particularly when there are several insoluble components in the suspension. As the complexity of formulations and their delivery systems increases and new formulations are tested on a regular basis, the characterization of drug-particle size distribution within the formulation becomes even more imperative. This article describes a novel method for measuring the ISPS of one or more active ingredients in the presence of multiple insoluble excipients in a nasal formulation sample.

Raman spectroscopy, a laser-based vibrational spectroscopy technique, provides high specificity for determining the chemical composition of a target analyte. Wide-field Raman chemical imaging (RCI) is a hyperspectral imaging method based on Raman spectroscopy. RCI determines the chemical identity of individual components of a heterogeneous sample by combining Raman imaging with optical microscopy. Wide-field RCI is a more objective method for drug-particle size analysis than optical microscopy because the additional information about component identity ultimately minimizes human error. It delivers the size distribution of a given component in the matrix, spectroscopically identified and registered within each pixel of an image.² In addition, wide-field RCI enables drug developers to study aggregates and formulation stability in an automated, reproducible, and reliable manner from early development to scale up and batch processing of the final formulation. For nasal-spray suspensions, this information can potentially assess the bioavailability and efficacy of an API in a final pharmaceutical product based on particle-size distributions. This has uses for both innovators and generic manufacturers.³

Wide-field RCI provides unprecedented value for a variety of applications ranging from pharmaceutical research and development to materials science, anatomic pathology, forensics, and others.⁴ Liquid crystal-based optical tunable filter technology enables the transmission of a spatially resolved and user-defined wavelength of light. The Falcon II from ChemImage Corporation (Pittsburgh, Pa.) is a microscope-based wide-field RCI system that can collect spectroscopically resolved data in two or three dimensions.

click to enlarge Figure 2a. Concatenated bright-field/RCI overlay for both APIs in formulations. Figure 2b. Ingredient-specific particle sizing of fluticasone propionate and mometasone furoate in a fluticasone propionate formulation and mometasone furoate formulation actuated droplets (concatenated images): false colored Raman fusion image after global processing. Figure 2c. Corresponding Raman spectra from particles in the image representing two APIs: fluticasone propionate (green) and mometasone furoate (blue). Figure 2d. Fluticasone propionate particle size distribution histogram. Figure 2e. Mometasone furoate particle size distribution histogram. (Source: ChemImage Corporation)

With RCI, a full or partial spectrum is obtained for each pixel in the chemical image. These pixels represent fields of view, which can be stitched together to form a three-dimensional montage image possessing X (spatial), Y (spatial), and wavelength (spectroscopic) dimensions. The spectral differences of each chemical entity in the image can be isolated by its unique spectral profile and mapped onto an associated optical image. For a single ingredient of interest, spectral planes at the wavelengths exhibiting the highest Raman intensities can be used for identification and sizing purposes. For multiple ingredients of interest, advanced chemometric techniques are needed to isolate the unique Raman signals and size each analyte in the mixture. Unsupervised algorithms to monitor, control, and optimize ISPS are in great demand and have been recently reported.⁵

The fluticasone propionate and mometasone furoate formulations differ not only in active pharmaceutical ingredient, but also in the number of inactive ingredients. Figure 1 shows the composition of both formulations based on pure component spectra. Yellow highlighting represents the spectral regions appropriate for chemically imaging the actuated droplets of two formulations. In wide-field imaging experiments, Raman bands specific for fluticasone propionate and mometasone furoate are included. Discrimination of one drug from the other—in addition to each of the formulation’s ingredients—is feasible based on spectra in each pixel.

Drug-specific ISPS analysis can be best illustrated by presenting both drugs in a side-by-side view. Bright-field optical images were collected with a 50× microscope objective representing an area of approximately 0.16 mm². Raman chemical imaging of the samples was also performed, and the data was processed using ChemImage Xpert software. A specific frame representing each active ingredient was extracted from the RCI and processed to create the false-color overlay highlighting all drug particles (for both APIs) in the droplets sample as shown in Figure 2a. Drug-specific image deconvolution and grouping of pixels based on their similar spectral profile allow one to discriminate particles of fluticasone propionate (green) and mometasone furoate (blue) within the respective formulations (Figure 2b). The specificity of RCI allows analysts to distinguish an API from other ingredients and from one another based on Raman spectra that are shown for two randomly selected particles in the Raman chemical image on Figure 2c. Spectral unmixing allows for the creation of a drug-specific map of one ingredient versus another and sizes them separately (Figure 2d, 2e). As clearly shown here, fluticasone propionate and mometasone furoate formulations possess different drug-particle size distribution and can be compared directly by analyzing RCI of the concatenated, side-by-side image for ISPS.

Conclusion
RCI is a valuable tool to obtain ISPS for nasal-spray suspensions and inhalation products. Drug-particle size distribution information is obtained based on spectral and optical imaging registration. This technology is especially valuable for aqueous suspensions containing two or more insoluble active ingredients within a complex matrix. In such cases, optical microscopy alone cannot objectively and accurately report size statistics for each of the active ingredients. As shown with the example of mometasone furoate and fluticasone propionate nasal spray formulations, it is possible to obtain drug-specific size statistics and also compare drug-specific particle-size distribution for two formulations using RCI. Wide-field RCI enables drug developers and formulators to study aggregation, formulation stability, and much more.

References
1. Draft Guidance for Industry Bioavailability and Bioequivalence Studies for Nasal Aerosols and Nasal Sprays for Local Action. Food and Drug Administration Web site. Available at http://www.fda.gov/cder/guidance/5383DFT.pdf. Accessed on May 20, 2009.
2. Doub WH, Adams WP, Spencer JA, Buhse LF, Nelson MP, Treado PJ. Raman chemical imaging for ingredient-specific particle size characterization of aqueous suspension nasal spray formulations: a progress report. Pharm Res. 2007;24(5):934-45.
3. Critical Path Opportunities for Generic Drugs. Food and Drug Administration Web site. Available at http://www.fda.gov/oc/initiatives/criticalpath/reports/generic.html#sprays. Accessed on May 20, 2009.
4. Treado PJ,Nelson MP. Raman Imaging. Handbook of Raman Spectroscopy. Lewis IR and Edwards GM (eds). New York, 2001; p. 140-159.
5. Priore RJ, Olkhovyk O, Klueva O, Fuhrman M. Automation of ingredient-specific particle sizing employing Raman chemical imaging. Respiratory Drug Delivery 2009.

About the Authors
Dr. Oksana Olkhovyk received her PhD in physical chemistry from Kent State University. She also holds an MS with honors in chemical engineering from Lviv Polytechnic University (Ukraine). Dr. Olkhovyk joined ChemImage in 2006 as a senior scientist and develops chemical imaging instrumentation and applications.