Cormac Smyth is a product manager and Breda O’Driscoll is managing director at Ultrasonic Scientific Ltd, 1 Richview Office Park, Clonskeagh, Dublin 14, Ireland. Tel. +353 1 218 0600 Fax +353 1 218 0601 info@ultrasonic-scientific.com www.ultrasonic-scientific.com

Cormac Smyth

Pharmaceutical manufacturing processes involve a variety of chemical or biological materials. The quality control (QC) of materials and formulations for pharmaceutical use include, for example, the confirmation of a chemical purity, polymorphism and water content. Other important characteristics are physical and functionality-related properties, which can vary between different grades of the same chemical entity. Such characteristics include dissolution rate; physical state (dispersed/aggregates); thermal stability (thermal transition); and particle size. This is true, in particular, for biologically active macromolecules such as proteins, polysaccharides or synthesized polymers. The specific physical and functionality-related properties can be tested by different methods such as ultraviolet (UV)/visible spectroscopy, nuclear magnetic resonance (NMR), laser diffraction, calorimetry and dynamic rheology. All have their limitations and, therefore, the search for new, fast and effective methods for wide-range characterization of material (raw materials, intermediates, excipients and active ingredients) is a hot issue in drug production quality. 

High-resolution ultrasonic spectroscopy (HR-US) is a nondestructive technique with enormous potential for the analysis of materials and formulations used in the pharmaceutical industry. This technique is based on precision measurements of parameters of acoustical waves at high frequencies propagating through materials. Unlike current analytical methods, optical transparency is not required because ultrasonic waves propagate through opaque samples. These instruments require small sample volumes, down to 0.03 mL, and give excellent resolution. They can be used for analysing composition; aggregation; gelation; micelle formation; crystallization; dissolution; sedimentation; enzyme activity; conformational transitions in polymers; ligand binding; antigen-antibody interactions; and many other processes that play a key role for functional properties of material used in drug production. Capable of dealing with a wide range of samples and dynamic processes, HR-US generates product quality information in real process time. It allows fast analysis of formulation consistency; batch-to-batch variation, stability assessment and so forth. 			

Two independent parameters, ultrasonic attenuation and ultrasonic velocity, are measured in ultrasonic spectroscopy. Ultrasonic attenuation is determined by the energy losses in ultrasonic waves and can be expressed in terms of the high-frequency viscosity of the medium or its longitudinal loss modulus. This allows the analysis of kinetics of fast chemical reactions and the microstructure of materials including particle sizing, aggregation, gelation, crystallization, and other processes and characteristics. Ultrasonic velocity is determined by the density and elastic response of the sample to the oscillating pressure in the ultrasonic wave and thus, can be expressed in terms of compressibility or storage modulus. This parameter is extremely sensitive to the molecular organization, composition and intermolecular interactions in the analysed medium, and is responsible for the majority of applications of HR-US for analysis of chemical properties of materials. HR-US spectrometers, with their superior resolution, small sample volume and robust design, are the first commercial instruments that allow the user to enjoy the full potential of high-resolution ultrasonic analysis. Standard sample cells for the HR-US spectrometers have a 1 mL capacity. A range of cells is also available, which include: 					

flow-through cells that allow analysis in-flow

cells with small volume capacities (as low as 0.03 mL).

All measurements can be made at temperatures as low as –60 °C and as high as 120 °C.  

The paper describes some applications of HR-US for different types of material analysis in pharmaceutical production. Several examples are illustrated including the measurements of the particle size in drug emulsions with different polymer coating; monitoring the dissolution of drug substances; the characterization of thermal stability of polymers used in drug formulations; and the analysis of water content in different forms of the same drug compound. 

Control of particle size in the dispersed phase is a critical issue in pharmaceutical formulations. Particle characteristics can affect many different areas including inhalation delivery systems, tablet dissolution characteristics, formulation quality, and solubility or absorption. Batch-to-batch variations in particle size can lead to unpredictable variations in the life span and stability (shelf life/heat stability) of a product. Traditionally, the characterization of the particles in dispersion is made by optical methods such as light scattering. This means that the sample must be diluted to reach optical transparency and avoid multiple scattering effects. However, the application of HR-US measurements allows direct analysis of the particle size and their volume fraction in the dispersions (even concentrated samples, e.g., 40%), thus avoiding the problems associated with dilution (such as change in the particle size and aggregation rate). 							

In the current example, HR-US was used to analyse the effect of different polymer coatings on the sedimentation rate and the evolution of the particle size in drug suspension. The samples of drug suspensions were prepared using hydroxypropyl cellulose as a drug carrier system. The drug particles in Sample 1 were coated with polymer, which had a higher molecular weight (approximately twice) compared with the polymer used in the preparation of Sample 2. Each of the samples were shaken vigorously before the measurements and loaded into an ultrasonic cell. The sedimentation processes were monitored continuously over 30 min.  

Sedimentation is reflected in ultrasonic measurements as a change in velocity and attenuation with time. HR-US software deconvolutes these changes and provides the change in particle size and volume fraction during the sedimentation process. Evolution of particles' size and their volume fraction in two types of drug suspension is compared in Figure 1. Initial particle size (after loading the sample into the cell) in Sample 1 was 0.8 μm, which is 40% larger than the size in Sample 2. However, the particle size in Sample 2 increases faster with time as a result of the aggregation. Particle growth is accompanied by a decrease in volume fraction, which can be attributed to the sedimentation, by about 0.05% in Sample 1 and 0.3% in Sample 2 within 25 min. Both processes (sedimentation and aggregation) are more evident in Sample 2, indicating lower stability compared with Sample 1. One of the possible factors for the higher stability of Sample 1 can be a 'thicker' polymer layer on the drug particles in this sample, which protects the particles against aggregation. 

 Figure1. Evolution of changes in volume fraction of drug particles and particlesÃÂ´; size calculated from the ultrasonic velocity and attenuation data measured in two undiluted drug suspensions with different polymer coating at 25 Â°C. 			

The effect of dilution on the evolution of the drug particles' size was analysed by measuring the sedimentation profile (similar to those in Figure 1) in diluted suspensions. Figure 2 shows that average particle size decreases with dilution. This decrease can be explained by the effect of the particle concentration on aggregation. Stirring the suspension before the measurements are taken results in the breakage of the particle aggregates into smaller particles. In undisrupted suspension, the particles' growth in size is a result of an aggregation. However, because the aggregation rate decreases at lower drug concentration, the average particle size measured after the same time interval (i.e., 25 min) is smaller in undiluted suspension. Figure 2 also illustrates the effect of dilution on the particle size depends on the particle coating. In very diluted samples (e.g., 1:20) the drug particles are larger in Sample 1. This can be attributed to a 'thicker' layer formed by a higher molecular weight polymer on the original particles. The results show that an increase in the drug concentration overall enhances the aggregation and that this process is less pronounced in Sample 1 because of a more effective protection polymer layer on drug particles as previously discussed. Optimization of drug coating and the improvement of sample stability can be effectively controlled using high-resolution ultrasonic measurements. 

Figure2. Change in size of the drug particles with concentration for two different polymer coatings as determined by the HR-US measurements. The particle size was measured after 25 min of the sedimentation and giving as relative to one in undiluted sample 1.			

Control of water content in raw material and active compounds is one of the key issues in drug production. HR-US spectrometers allow analysis of water concentration using high-precision velocity measurements. The ultrasonic analysis is based on the fact that for many solutions and mixtures, the ultrasonic velocity is proportional to the concentration of solute component. When the contributions of the components to the ultrasonic velocity are known (e.g., after calibration) the concentration of the components can be precisely determined from the measured ultrasonic velocity. This approach was applied to measure water content in the crystalline and amorphous forms of the drug active compound.  			

Two types of drug compounds — amorphous and crystalline — were dissolved in methanol at 0.13 g/cm

 concentration and loaded into ultrasonic cells of the HR-US 102 spectrometer (Ultrasonic Scientific Ltd, Ireland) for the measurements of relative ultrasonic velocity (using methanol as a reference). The comparison of ultrasonic parameters of two drugs solutions (at the same nominal concentration of drug powder, mg/cm

) at 20 °C is given in Figure 3(a). The data demonstrates that samples can be easily distinguished by their ultrasonic profiles. The measured difference in ultrasonic velocity is several orders higher than the accuracy of the HR-US 102 spectrometer. Water content was calculated from the measured ultrasonic velocities and the calibration curve, and is shown in Figure 3(b). The total amount of water in the samples is between 1–1.5%. Water content is higher in the amorphous form of drug compounds compared with the crystalline version by approximately 0.5%. 

Figure 3. Water content in two forms of the same drug substance measured with an HR-US 102 spectrometer. The values of the ultrasonic velocity are given for 1 mg/mL drug suspension relative to methanol.			

An example of the practical use of HR-US is monitoring thermal transitions in polymers used in different drug formulations. Hydroxypropylmethylcellulose (HPMC) is used to encapsulate hard capsule products. This polymer dissolves slowly in cold water to form a viscous colloidal solution and upon heating becomes insoluble in water forming a thermally reversible gel. This use is petitioned as an alternative to gelatin (animal based) capsules. HPMC has many other uses as an emulsifier, thickening agent, stabilizer, gellant and suspending agent.  			

In this study, the HR-US 102 spectrometer was used for comparative analysis of the gelling transition in two samples of HPMC (HPMC 1 and HPMC 2) that differ in the hydroxypropyl content. The solutions of HPMC samples in water (2 w/v) were loaded into an ultrasonic cell and their thermal gelling profile was measured from 40–90°C at a heating rate of 0.5°C/min.

Figure 4. Monitoring of thermal transition in two types of hydroxypropylmethylcellulose. Evolution of ultrasonic velocity in polymer solution upon heating from 40-95 Â°C: (a) original data and (b) after the subtraction of baseline.			

Figure 4 shows the change in ultrasonic velocity with temperature in solutions of two different HPMC samples. The velocity profile at low temperature exhibits a linear decrease with increasing temperature and was used as a baseline. Subtracting a temperature dependent baseline shows the details of the gelling transitions in two samples. The decrease in velocity in this gel transition is caused by a decrease in the hydration level of the atomic groups of the HPMC molecules (the compressibility of water in the hydration shells is typically less than the compressibility of the bulk water, and, therefore, the transfer of hydration water into the bulk water reduces the ultrasonic velocity). The gelation temperature can be determined as the point of inflection on the transition curves in Figure 2. The data show that the gelation temperature in HPMC 1 (56°C) is lower than in HPMC 2 (72°C). The overall magnitude of change in ultrasonic velocity characterizes the degree of cross-linking and is an indicator of the gel strength. According to the ultrasonic data, HPMC 1 forms the strongest gel networks in comparison with the HPMC 2 sample.  

Thus, HR-US measurements allow analysis of the gelling profiles, detection of the gelling temperatures and characterization of cross-links in the gel.  

Dissolution rates of raw materials, active ingredients and excipients are important functional characteristics that determine drug efficiency. HR-US offers a direct and simple method for the analysis of the dissolution rate. In the following example, the dissolution rate of two different tablets in an acidic buffer, mimicking the gastric acid environment, was monitored using the HR-US 102 spectrometer.  			

Figure 5 presents the dissolution rate of two tablets determined from the ultrasonic velocity data. The gradual dissolving of solid drug components results in the growth of solute concentration, thus changing the ultrasonic velocity. The drugs dissolved completely after 2 h. It can be seen from Figure 5 that Tablet 1 initially dissolves at a much slower rate than Tablet 2. Half of Tablet 1 is dissolved in the first 12 min, whereas it takes about 35 min to dissolve 50% of Tablet 2. However, the dissolution rate of both tablets is found to be similar after 20 min. One of the advantages of the ultrasonic method for measuring the dissolution rate is that it allows measurements of practically any compound in various environments (e.g., different temperatures, pH and solvents).

Figure 5. The rate of dissolution of two tablets as monitored with high-resolution ultrasonic velocity measurements.			

The examples described show the high potential of HR-US for analysing a broad range of important properties of different raw materials, excipients and final formulations used in drug production. The arrival of the next generation of ultrasonic spectrometers and analysers will have a profound impact on the efficiency and profitability of pharmaceutical processes.			

Evgeny Kudryashov, Cormac Smyth, Breda O'Driscoll 

are senior scientist, product manager and managing director at Ultrasonic Scientific, Ireland.

 is head of the laboratory of Physical Chemistry of Biocolloids at the Department of Chemistry, University College Dublin, Ireland.

Articles

HR-US is a nondestructive technique with enormous potential for the analysis of materials and formulations used in the pharmaceutical industry.

Analysing Raw Materials and Formulations Using High Resolution Ultrasonic Spectroscopy

This article introduces the application of high-resolution ultrasonic spectroscopy (HR-US) for the analysis of emulsions and suspensions. The authors outline the principles of the technique and illustrate its application for analysis of the crystallization of lysozyme and the formation of a microemulsion.	

High-resolution ultrasonic spectroscopy (HR-US) is a novel approach for the analysis of materials that uses the important ability of ultrasound to probe systems non-invasively and report their constitution, microstructure and intermolecular interactions. The analytical power of ultrasound is well known through its application in medicine, for example, in the screening of unborn babies wherein the ability of the waves to pass through opaque media without causing damage is particularly important. 	

However, the use of HR-US in analysing a variety of other materials and solutions has only recently evolved to become an essential laboratory technique. This process is extremely sensitive to the molecular organization, is non-destructive, requires no markers, and can be used on non-transparent and concentrated samples, making it advantageous compared with traditional methods.	

Stable dispersions can be divided into two main groups: suspensions and emulsions. A suspension is a dispersion of fine particles of a solid within a liquid, the particles being the dispersed phase, whereas the suspending medium is the continuous phase. Apart from the mixing of an insoluble solid in a liquid, a suspension can also result from the aggregation of dissolved solids; crystallization is an example of such a process.		

Figure 1 HR-US principles of operation.  (Click to enlarge)				

An emulsion is a dispersion of one liquid in another immiscible liquid, usually in the presence of stabilizer molecules. Emulsions may be either oil droplets dispersed in water or water droplets dispersed in oil, wherein the droplet diameters are in the micron range. Fat in milk is a familiar example of an oil-in-water emulsion. The droplets are usually formed by high shear mechanical processes and stabilized against coalescence by electrostatic and/or steric barriers around the droplets provided by the stabilizers. Often, this colloidal dispersion is complex because the dispersed phase is partially solidified or because the continuous phase contains crystalline material (such as in ice cream). 	

Emulsions and suspensions have two things in common: first, they are thermodynamically unstable and second, because they are mostly optically opaque systems, they are difficult to study using optical analysis methods. Examples of pharmaceutical emulsions include lotions, liniments, creams and vitamin drops. Except for the medicinal ingredients, the base vehicles in creams, ointments and lotions are quite similar in formulation to cosmetic preparations. 	

It has been found that properly formulated pharmaceutical emulsions and dispersions provide more accurate control of the dosage. They will also furnish a method of combining many immiscible ingredients into a single, stable product, which improves the ease of application. The proper formulation and control of particle size and stability bring about the controlled release of the active ingredients. Emulsions are difficult to characterize and their behaviour is challenging to understand, however, their application is universal and they are widespread in nature. 	

There is a special class of emulsions: microemulsions. In contrast to 'normal' emulsions, they are thermodynamically stable, the droplet size in microemulsions is small (in the range 10–100 nm) and they are transparent. Microemulsions have attracted considerable interest during the years as potential drug delivery systems because of their unique properties, which include stability, ease of preparation and their ability to form spontaneously.

Analysis of microstructure and intermolecular interactions in emulsions and suspensions is a challenging task. Many traditional analytical techniques require dilution to adjust the transparency of emulsion to an acceptable range. This often affects the weak balance of intermolecular forces in emulsion and its structure. (Microemulsions suffer from similar dilution problems to emulsions, although in the case of a microemulsion formulated with a short-chain cosurfactant, dilution often leads to the total destruction of the system. Hence it is imperative that most microemulsions are measured in their concentrated state.) Another limitation is the effect of the measuring device on the structure of emulsions, which is common for rheological (viscometry and dynamic rheology) and other measurements.		

Figure 2 Monitoring lysozyme crystallization by ultrasonic spectroscopy. The inset shows the measurements during the first 5 hours of crystallization.  (Click to enlarge)	

Proteins are a class of macromolecules found universally in living creatures. Mutation of a protein or a combi-nation of proteins working together can cause diseases such as bovine spongiform encephalopathy (BSE). Subsequently, having a good understanding of proteins can help fight disease. To appreciate the function of a protein and how it performs this specific function, scientists need to understand its structure.  				

The method used to study the molecular structure of proteins is X-ray diffraction, in which the protein must be in a pure crystal form. However, because of the complex nature of proteins, and their sensitivity to their environmental conditions, it is difficult to produce good quality crystals. By running thousands of protein crystal screens that use different variables such as pH, salt concentration and temperature, scientists can narrow down the conditions that are needed to produce high quality crystals. However, this can be a long and arduous process taking months or years. The use of a real-time analytical method that can follow the crystallization reaction can speed up this process. 	

HR-US is a novel technique for non-destructive material analysis based on precision measurements of parameters of high-frequency sound waves propagating through analysed samples (Figure 1). These waves propagate through most materials, including opaque samples, and allow direct probing of intermolecular forces. HR ultrasonic spectrometers provide an unprecedented range of new analytical capabilities for research, product development, quality and process control in the biotech, pharmaceutical, food, chemical and petrochemical, and polymer industries. This rapid, inexpensive and non-destructive technique has previously established itself as a powerful tool for 				

conformational transitions in polymers and biopolymers

formation of micelles and critical micellization concentration measurements

High-resolution ultrasonic spectrometers can be used for characterization of emulsions and suspensions, and the evaluation of their stability and structure (particle size measurements). The instruments measure two parameters, ultrasonic attenuation and velocity, which are physically independent, allowing the probing of different levels of the organization of the samples to be characterized. 	

Attenuation is mainly determined by the scattering of ultrasonic waves in non-homogeneous samples (emulsions and dispersions) and fast chemical relaxation (in homogeneous mixtures). The density and the elasticity of the medium determine ultrasonic velocity. A major advantage of the ultrasonic technique is the ability to make measurements directly, in the original emulsion, without having to make dilutions. The following examples illustrate the application of the technology for analysis of the crystallization process and the formation of a microemulsion.	

Lysozyme protects the human body from bacterial infection by attacking bacterial cell walls, causing them to rupture. By varying the experimental conditions, different amounts and sizes of protein crystals can be produced. The amount of crystals as well as an assessment of their sizes and interactions is an important part of routine analysis in the pharmaceutical industry. HR-US spectrometers allow measurement as the crystal grows with time and temperature and assessment of the amount of crystals in a sample. Monitoring of crystal formation, in particular the kinetics of the reaction, is essential for the optimization of process control in the batch crystallization of pharmaceutical compounds. 		

Figure 3 Ultrasonic analysis of microstructural rearrangements and formation of microemulsion.  (Click to enlarge)				

Crystallization of lysozyme measured by HR-US is shown in Figure 2. In this measurement, 1 mL of a solution of lysozyme (40 mg/mL) in 0.1 M sodium acetate (pH 4.8) was placed into the ultrasonic cell. A precipitating agent was added to the sample and the kinetics of crystallization was followed. The spectrometer detects three stages in the crystallization process. During the first 3.4 h of the reaction, no significant change is detected. At the end of stage I the ultrasonic velocity and attenuation start to increase because of the formation of crystals. 	

This increase continues through stage II as the concentration and size of the crystals grow. The increase in ultrasonic velocity is caused by the increase in rigidity (decrease in compressibility) of the sample as a result of crystal formation. The rise in ultrasonic attenuation can be attributed to the scattering of the ultrasonic wave on the solid crystals that are formed. The scattering contribution is dependent on the ratio of crystal size and frequency (for example, the attenuation at the higher frequencies is more sensitive to the formation of small particles). Therefore, multi-frequency ultrasonic attenuation measurements allow analysis of the crystal structure and size. During stage II the ultrasonic velocity starts to decrease; in stage III the ultrasonic attenuation nearly levels off, indicating the end of crystal formation in the micron range size. However, the velocity continues to decrease, as a consequence of the sediment of a small percentage of large crystals from the system.	

This example describes the application of the technology for analysis of the formation of a microemulsion in a system consisting of pharmaceutically accepted components,

), n-propanol as a cosurfactant in a w/w ratio 6:1:1 and water.

 The measurements were performed at 20 Â°C using an HR-US spectrometer equipped with a standard 1 mL cell in the frequency range 2–20 MHz. 				

Figure 3 shows the typical dependence of ultrasonic velocity and attenuation on the concentration of water in the system (at 5 MHz). Molecular dissolving of water in the mixture at low concentrations (up to 2 wt%), which is accompanied by hydration of lipid and cosurfactant, is shown by the steady increase in ultrasonic velocity. Following this, a microstructural reorganization takes place, indicated by an additional increase in ultrasonic velocity in the range 2–6% water concentration. Formation of the microemulsion can be seen from approximately 8 wt% with ultrasonic attenuation quickly increasing, which is caused by the scattering of the ultrasonic wave on the particles. It should be noted that when compared with light scattering, good agreement above the break point was found whereas below the break point, the possible structuring of the system is not observable with the light scattering. This shows the potential of the technique in providing information previously unattainable by existing methods of analysis.	

1.	M.J. Lawrence and G.D. Rees, "Microemulsion-Based Media as Novel Drug Delivery Systems," 

2.	R. Aboofazeli and M.J. Lawrence, "Investigations into the Formation and Characterization of Phospholipid Microemulsions. I. Pseudo-Ternary Phase Diagrams of Systems Containing Water-Lecithin-Alcohol-Isopropyl Myristate,"

www.aapspharmsci.org/view.asp?art=ps020319

4.	K. Shinoda et al., "Lecithin-Based Microemulsions: Phase Behaviour and Microstructure," 

This article introduces the application of high-resolution ultrasonic spectroscopy (HR-US) for the analysis of emulsions and suspensions. The authors outline the principles of the technique and illustrate its application for analysis of the crystallization of lysozyme and the formation of a microemulsion.