Deborah Huck is Product Technical Specialist – Morphological Imaging Systems, Malvern Instruments.

Deborah Huck

The way in which a suspension nasal spray product interacts with the body depends not only on the droplet size of the delivered droplet, but also on the particle size of the suspended active pharmaceutical ingredient (API). These dependencies are clearly recognised in the FDA draft guidance document '

Bioequivalance (BE) and bioavailability (BA) studies for nasal sprays and nasal aerosols for local action

  which also recommends appropriate analytical techniques to quantify these two parameters. 

For suspensions, the guidance states that 'drug particle size may be important for rate of dissolution and availability to sites of action within the nose'. For 

 bioequivalence studies, it recommends measurement of the API particle size distribution within the product formulation prior to actuation, and in the spray following actuation in order to characterise the influence of the device on de-agglomeration. In highlighting the presence of insoluble suspending agents/excipients as a complicating factor in this measurement, the guidance recommends the use of light microscopy, or alternative related techniques. However, it can be difficult to differentiate these excipients from the API using only light microscopy.  

With respect to droplet size and particle size distribution, the regulatory guidance states, that this "

is an important property influencing the nasal deposition of aerosols and sprays

". Droplets in the sub-10 µm region may be drawn into the lung rather than remaining in the nasal cavity, while excessively large droplets may be deposited primarily in the front of the nose and not at the intended site of deposition. Most prescription nasal sprays achieve the maximum therapeutic effect when the droplets deposit beyond the nasal valve in the posterior two thirds of the nasal cavity. Laser diffraction is recommended for droplet size measurement.  

Continuing high interest in nasal drug delivery intensifies the need for fast and detailed nasal spray characterisation that meets the demands of the regulators. In the study described here, laser diffraction is used to measure droplet size and to investigate the dynamics of spray formation in a commercial device. The API particle size distribution of the formulation, before and after dispersion, is measured using an automated imaging system with Raman spectroscopy capabilities. Much faster than manual light microscopy, automated imaging techniques provide statistically relevant analysis of size, shape and, in this case composition for precise API characterisation. 

Investigating the dynamics of spray formation

Laser diffraction is an ensemble particle sizing technique that measures size distributions in the range 0.1 to 3000 µm, comfortably spanning the area of interest for nasal sprays. Fast and non-destructive, laser diffraction is suitable for measuring both dry and wet samples, with instruments designed for spray analysis measuring at rates of up to 10 kHz. There are now several suppliers of laser diffraction equipment. Such systems capture the evolution of droplet size in real-time, throughout the duration of a spray event, providing detailed information on which to base decisions about a formulation or device.

 shows laser diffraction (Spraytec, Malvern Instruments) droplet size data for a commercial nasal spray product actuated at different velocities. Investigating the impact of actuation profile on droplet size is essential since in-use operation will vary from patient to patient. Here, tests were carried out at actuation velocities of 40, 70 and 100 mm/s. Samples were measured at a distance of 30 mm from the laser beam at a frequency of one measurement every 400 µs. Measuring over a period of 400 ms the spray event is tracked through to completion. The results are presented in the form of a particle size profile — a plot of Dv50 (the particle size below which 50% of the droplet population lies) with time. 

Figure 1: Particle size profiles and averaged particle size distributions (from the stable phase), for a commercial nasal spray product actuated at 40, 70 and 100 mm/s.  

Nasal spray events can be divided into three phases: formation, fully developed/stable, and dissipation. In the formation phase, flow through the spray pump nozzle is relatively low, droplet size is large, and the output of the nasal spray product is not yet stable. Flow is also low during dissipation at the end of the spray event when the metering chamber is empty. The FDA therefore recommends that data from the fully developed phase is used for statistically valid comparisons of the performance of the product under different conditions. 

The results show that as actuation velocity increases, the duration of the fully developed phase decreases; the dose is delivered more rapidly. In terms of atomisation behaviour, actuation at 70 and 100 mm/s produces similar results, generating a stable Dv50 of 35.4 µm and 32.2 µm, respectively. In contrast, the slowest actuation profile (40 mm/s), as well as producing a longer fully developed phase, atomises droplets to a much larger diameter, a Dv50 of 72.1 µm. This means that the performance of this product will be markedly different if the actuation profile applied by the patient is below a certain velocity, some way between 40 and 70 mm/s. The regulatory guidance states that pumps should be tested at velocities that are typical of hand actuation by patients, so care should be taken to select the appropriate settings for automated actuation during laser diffraction studies.  

Figure 2: Example particle images of API (left) and excipient (right) from a commercially available corticosteroid nasal spray formulation. The individual Circular Equivalent Diameter is indicated below each image (images are not to same scale).  

Automated particle imaging for API measurement 

Reliable measurement of the API of a suspension nasal spray formulation relies on the successful differentiation of API particles from any other suspended solids present, such as insoluble excipients for example. Traditionally, manual microscopy is employed for this application, with the operator visually discriminating between particles on the basis of their appearance. However, this approach is both operator dependent and extremely labour intensive, and the results tend to be relatively subjective with low statistical significance, pragmatism limiting the number of particles that can be measured. 

Figure 3: Overlay of the API CE diameter distribution before and after spraying.  

Using automated particle image analysis, tens of thousands of particles can be characterised in minutes. Individual images of every detected particle are captured, and analysed to generate statistically relevant descriptors of size, shape and transparency. For nasal sprays the formulation is simply dispersed on to a microscope slide prior to measurement. 

 shows example images, captured using automated image analysis, of an API and insoluble excipient present in the nasal spray formulation previously studied, as measured prior to actuation of the device. 

Figure 4: Size classification of the API particles before and after spraying.  

In this case, the active and excipient particles are quite different in terms of their morphological characteristics. Automatic classification of the particle images on the basis of a relevant shape parameter therefore enables identification of the API population and the gathering of data for this discrete group. 

show particle size distribution data, specifically for the API, measured before and after spraying. A slight shift in the distribution towards a finer particle size suggests that some shear-induced de-agglomeration occurs within the device during pump actuation. 

Figure 5: The Raman spectra of two individual particles, showing the ability of Raman todifferentiate API and excipients particles. Reference spectra for the API and excipient are shown in the green boxes.  

Although automated image analysis offers significant practical advantage relative to manual microscopy, it shares the limitation of being unable to discriminate between API and excipient particles that are visually identical. The addition of a Raman microprobe and spectrometer to an automated imaging system (Morphologi G3-ID, Malvern Instruments) overcomes this problem, enabling the chemical identification of API particles that are morphologically similar to insoluble excipients. Following acquisition of the images of individual particles, via routine automated imaging, the Raman spectra of selected particles can be measured and correlated with reference spectra to chemically differentiate the population of interest (

). By using the imaging data in this way to target the acquisition of Raman spectra — so called Morphologically Directed Raman spectroscopy — the time for measurements can be significantly reduced compared with standard Raman mapping methods. In addition, the measurement set up is simplified compared with standard methods, as the position of the particles is automatically determined prior to analysis, removing any operator subjectivity. 

To illustrate the benefits of this approach, the nasal spray product previously investigated was analysed. Following dispersion of the sample on to a slide, chemical identification data were gathered for around 9000 particles from a single scan area. 

 shows scatter-plots of the correlation scores for individual particles, referenced to the Raman spectra for the API and excipient. These scatter-plots chemically differentiate populations of API and excipient particles. Of the approximately 9000 particles analysed, around 450 were classified as API, this is consistent with the stated API : excipient ratio of the formulation which is 1:20 by weight. 

Figure 6: Scatterplots for Raman correlation scores of API and excipient particles and examples of associated particle images for the two chemical classes defined by the yellow regions.  

On the basis of this chemical classification it is possible to measure the API particle size distribution (

). Additionally, it is possible to compare the ingredient-specific particle size and shape distributions for the API and excipient (

). These data suggest that, in fact, the bulk of the excipient population can be excluded from chemical analysis on the basis of shape alone, by automatic classification using the shape parameter of elongation. Particles with an elongation greater than 0.4 are not API, and so do not need to be chemically identified using Raman. This finding opens up a route to improved measurement times. 

Figure 7: Particle size distribution of API in a nasal spray based upon Raman chemical classification.  

For this product, applying a morphological filter reduces the volume of spectral data which has to be generated, and would ease the analytical burden. In this particular example, classification on the basis of elongation would mean that only around 3000 of the original 9000 particles (approx.) would need to be chemically identified, a time saving of 66%.  

Figure 8: Comparison of the particle shape distributions for API and excipient in a nasal spray based upon chemical classification. Shaded area shows particles which can be excluded as being API based upon their elongation alone.  

Using rapid automated imaging in this way, as a pre-selection tool for chemical analysis of selected particles, is an efficient and powerful way of streamlining measurement towards routine quality control. It also allows for a more objective and statistically sound measurement to compare innovator and generic nasal spray products as required by the FDA draft BE guidance. In addition, of course, the detailed information provided by these techniques allows the insightful investigation of nasal spray products, and indeed other pharmaceuticals, in support of product development goals. 

Laser diffraction and automated imaging combined with Raman spectroscopy are powerful, complementary techniques for nasal spray product characterisation. Laser diffraction measurements relate to the formulation in its entirety and support the development of formulation and device towards optimal dispersion. Laser diffraction studies are also required for routine quality control and to determine

 bioequivalence. In contrast, automated image analysis in combination with Raman spectroscopy enables particle size analysis of the API alone, an important variable with respect to bioavailiabilty. Both techniques are fast and efficient, with automated imaging offering substantial advantage — quicker measurement, better reproducibility — compared with manual microscopy. Together they promote the efficient commercialisation of new nasal spray products in accordance with the regulatory guidances. 

is Product Group Manager – Micrometrics, Malvern Instruments. 

is Product Technical Specialist – Morphological Imaging Systems, Malvern Instruments. 

is Sales Specialist – Analytical Imaging Systems, Malvern Instruments. 

is Product Technical Specialist – Diffraction, Malvern Instruments. 

1. FDA Guidance — Bioequivalance (BE) and bioavailability (BA) studies for nasal sprays and nasal aerosols for local action, April 2003. 

 the authors discuss in more detail the value of laser diffraction in helping formulators achieve optimal nasal spray product performance by enabling the capture of nasal droplet size data in real time. 

Articles

The way in which a suspension nasal spray product interacts with the body depends not only on the droplet size of the delivered droplet, but also on the particle size of the suspended API.