Sub-100 µm Spatial Resolution Ambient Mass Spectrometry Imaging of Rodent Brain with Laser Ablation Atmospheric Pressure Photoionization (LAAPPI) and Laser Ablation Electrospray Ionization (LAESI).

In this study, we applied a new IR laser-beam-focusing technique to enable sub-100 µm spatial resolution in laser ablation atmospheric pressure photoionization (LAAPPI) and laser ablation electrospray ionization (LAESI) mass spectrometry imaging (MSI). After optimization of operational parameters, both LAAPPI- and LAESI-MSI with a spatial resolution of 70 µm produced high-quality MS images, which allowed accurate localization of metabolites and lipids in the mouse and rat brain. Negative and positive ion LAAPPI- and LAESI-MS detected many of the same metabolites and lipids in the brain. Many compounds were also detected either by LAAPPI- or LAESI-MS, indicating that LAAPPI and LAESI are more complementary than alternative methods.

Pectin and Mucin Enhance the Bioadhesion of Drug Loaded Nanofibrillated Cellulose Films.

PURPOSE: Bioadhesion is an important property of biological membranes, that can be utilized in pharmaceutical and biomedical applications. In this study, we have fabricated mucoadhesive drug releasing films with bio-based, non-toxic and biodegradable polymers that do not require chemical modifications. METHODS: Nanofibrillar cellulose and anionic type nanofibrillar cellulose were used as film forming materials with known mucoadhesive components mucin, pectin and chitosan as functional bioadhesion enhancers. Different polymer combinations were investigated to study the adhesiveness, solid state characteristics, film morphology, swelling, mechanical properties, drug release with the model compound metronidazole and in vitro cytotoxicity using TR146 cells to model buccal epithelium. RESULTS: SEM revealed lamellar structures within the films, which had a thickness ranging 40-240 µm depending on the film polymer composition. All bioadhesive components were non-toxic and showed high adhesiveness. Rapid drug release was observed, as 60-80% of the total amount of metronidazole was released in 30 min depending on the film formulation. CONCLUSIONS: The liquid molding used was a straightforward and simple method to produce drug releasing highly mucoadhesive films, which could be utilized in treating local oral diseases, such as periodontitis. All materials used were natural biodegradable polymers from renewable sources, which are generally regarded as safe.

Wood hemicelluloses as effective wall materials for spray-dried microcapsulation of polyunsaturated fatty acid-rich oils.

The most commonly-used and effective wall materials (WMs) for spray-dried microencapsulation of bioactive compounds are either costly, or derived from unsustainable sources, which lead to an increasing demand for alternatives derived from sustainable and natural sources, with low calories and low cost. Wood hemicelluloses obtained from by-products of forest industries appear to be attractive alternatives as they have been reported to have good emulsifying properties, low viscosity at high concentrations, high heat stability and low heat transfer. Here, we investigated the applicability of spruce galactoglucomannans (GGM) and birch glucuronoxylans (GX), to encapsulate flaxseed oil (FO, polyunsaturated fatty acid-rich plant based oil) by spray drying; and the results were compared to those of the highly effective WM, gum Arabic (GA). It was found that depending on solid ratios of WM:FO (1:1, 3:1 and 5:1), encapsulation efficiency of GGM was 88-96%, and GX was 63-98%. At the same encapsulation ratio, both GGM and GX had higher encapsulation efficiency than GA (49-92%) due to their ability to produce feed emulsions with a smaller oil droplet size and higher physical stability. In addition, the presence of phenolic residues in GGM and GX powders enabled them to have a greater ability to protect oil from oxidation during spray drying than GA. Physiochemical properties of encapsulated powders including thermal properties, morphology, molecular structure, particle size and water adsorption intake are also investigated. The study has explored a new value-added proposition for wood hemicelluloses which can be used as effective WMs in the production of microcapsules of polyunsaturated fatty acid-rich oils for healthy and functional products in food, pharmaceutical and cosmetic industries.

Rapid particle size measurement using 3D surface imaging.

The present study introduces a new three-dimensional (3D) surface image analysis technique in which white light illumination from different incident angles is used to create 3D surfaces with a photometric approach. The three-dimensional features of the surface images created are then used in the characterization of particle size distributions of granules. This surface image analysis method is compared to sieve analysis and a particle sizing method based on spatial filtering technique with nearly 30 granule batches. The aim is also to evaluate the technique in flowability screening of granular materials. Overall, the new 3D imaging approach allows a rapid analysis of large amounts of sample and gives valuable visual information on the granule surfaces in terms of surface roughness and particle shape.

Effect of colloidal silicon dioxide and moisture on powder flow properties: Predicting in-process performance using image-based analysis.

The effect of colloidal silicon dioxide (CSD) on powder flow properties of poor-flowing excipient lactose 200 M was investigated. Binary mixtures of different ratios of CSD as glidant were examined using a modern image-based flow measuring technique. Special attention was placed to subtle variations in powder flow from small changes in glidant concentration (0.025% w/w). Understanding the modes of interaction of particles and their effects on flowability using the method predicted the die filling performance during tablet manufacture. In addition, the importance of moisture content on powder flow properties was empirically underlined. A more efficient range of CSD was detected from 0.10 to 0.50% w/w in most of the tested conditions, which revealed a significant improvement in powder flow performance compared to higher amounts typically handled in the pharmaceutical industry.

Mass Spectrometry Imaging of Arabidopsis thaliana Leaves at the Single-Cell Level by Infrared Laser Ablation Atmospheric Pressure Photoionization (LAAPPI).

In this study, we show that infrared laser ablation atmospheric pressure photoionization mass spectrometry (LAAPPI-MS) imaging with 70 µm lateral resolution allows for the analysis of Arabidopsis thaliana (A. thaliana) leaf substructures ranging from single-cell trichomes and the interveinal leaf lamina to primary, secondary, and tertiary veins. The method also showed its potential for depth profiling analysis for the first time by mapping analytes at the different depths of the leaf and spatially resolving the topmost trichomes and cuticular wax layer from the underlying tissues. Negative ion LAAPPI-MS detected many different flavonol glycosides, fatty acids, fatty acid esters, galactolipids, and glycosphingolipids, whose distributions varied significantly between the different substructures of A. thaliana leaves. The results show that LAAPPI-MS provides a highly promising new tool to study the role of metabolites in plants.

Ultrasound-assisted powder-coating technique to improve content uniformity of low-dose solid dosage forms.

An ultrasound-assisted powder-coating technique was used to produce a homogeneous powder formulation of a low-dose active pharmaceutical ingredient (API). The powdered particles of microcrystalline cellulose (MCC; Avicel® PH-200) were coated with a 4% m/V aqueous solution of riboflavin sodium phosphate, producing a uniform drug layer on the particle surfaces. It was possible to regulate the amount of API in the treated powder. The thickness of the API layer on the surface of the MCC particles increased near linearly as the number of coating cycles increased, allowing a precise control of the drug content. The tablets (n = 950) prepared from the coated powder showed significantly improved weight and content uniformity in comparison with the reference tablets compressed from a physical binary powder mixture. This was due to the coated formulation remaining uniform during the entire tabletting process, whereas the physical mixture of the powders was subject to segregation. In conclusion, the ultrasound-assisted technique presented here is an effective tool for homogeneous drug coating of powders of irregular particle shape and broad particle size distribution, improving content uniformity of low-dose API in tablets, and consequently, ensuring the safe delivery of a potent active substance to patients.

Nano-coating of beta-galactosidase onto the surface of lactose by using an ultrasound-assisted technique.

We nano-coated powdered lactose particles with the enzyme beta-galactosidase using an ultrasound-assisted technique. Atomization of the enzyme solution did not change its activity. The amount of surface-attached beta-galactosidase was measured through its enzymatic reaction product D-galactose using a standardized method. A near-linear increase was obtained in the thickness of the enzyme coat as the treatment proceeded. Interestingly, lactose, which is a substrate for beta-galactosidase, did not undergo enzymatic degradation during processing and remained unchanged for at least 1 month. Stability of protein-coated lactose was due to the absence of water within the powder, as it was dry after the treatment procedure. In conclusion, we were able to attach the polypeptide to the core particles and determine precisely the coating efficiency of the surface-treated powder using a simple approach.

Image-based characterization of powder flow to predict the success of pharmaceutical minitablet manufacturing.

Powder flowability plays an important role in die filling during tablet manufacturing. The present study introduces a novel small-scale measuring technique for powder flow. Based on image analysis, the flow was defined depending on the variation of luminous intensity and the movement of powder inside the measurement cuvette. Using quantities around 100 mg it was possible to characterize a wide range of common pharmaceutical powders, especially in distinguishing subtle differences in flow caused by minor changes in samples characteristics. The method was compared with powder rheometry, which is widely used in the pharmaceutical literature, and showed a significant improvement in predicting the success of pharmaceutical minitablet manufacture (d = 5 mm). Tablet weight variation (RSD) was defined as the most efficient way to assess relevant powder flow behaviour in tablet production when using the novel device. The proposed method was distinguished from others by its ability to classify different grades of microcrystalline cellulose in the die-filling process. Subsequently, eight common pharmaceutical powders, both excipients and APIs, were properly ranked as a function of flowability based on their physical properties. The method showed a high repeatability, with a relative standard deviation not more than 10%.

Effective modification of particle surface properties using ultrasonic water mist.

The goal of the present study was to design a new technique to modify particle surface properties and, through that, to improve flowability of poorly flowing drug thiamine hydrochloride and pharmaceutical sugar lactose monohydrate of two different grades. The powdered particles were supplied by a vibratory feeder and exposed to an instantaneous effect of water mist generated from an ultrasound nebulizer. The processed and original powders were evaluated with respect to morphology (scanning electron microscopy, atomic force microscopy, and spatial filtering technique), flow, and solid state properties. It was found that rapid exposition of pharmaceutical materials by water mist resulted in the improvement of powder technical properties. The evident changes in flowability of coarser lactose were obviously due to smoothing of particle surface and decreasing in the level of fines with very slight increment in particle size. The changes in thiamine powder flow were mainly due to narrowing in particle size distribution where the tendency for better flow of finer lactose was related to surface and size modifications. The aqueous mist application did not cause any alteration of the crystal structures of the studied materials. The proposed water mist treatment technique appears to be a robust, rapid, and promising tool for the improvement of the technological properties of pharmaceutical powders.

Predicting tablet tensile strength with a model derived from the gravitation-based high-velocity compaction analysis data.

In the present study, a model was developed to estimate tablet tensile strength utilizing the gravitation-based high-velocity (G-HVC) method introduced earlier. Three different formulations consisting of microcrystalline cellulose (MCC), dicalcium phosphate dihydrate (DCP), hydroxypropyl methylcellulose (HPMC), theophylline and magnesium stearate were prepared. The formulations were granulated using fluid bed granulation and the granules were compacted with the G-HVC method and an eccentric tableting machine. Compaction energy values defined from G-HVC data predicted tensile strength of the tablets surprisingly well. It was also shown, that fluid bed granulation improved the compaction energy intake of the granules in comparison to respective physical mixtures. In addition, general mechanical properties and elastic recovery were also examined for all samples. In this study it was finally concluded, that the data obtained by the method was of practical relevance in pharmaceutical formulation development.

Analysis of steroids in urine by gas chromatography-capillary photoionization-tandem mass spectrometry.

A new heated capillary photoionization (CPI) ion source design was developed to photoionize analytes inside a transfer capillary between a gas chromatograph (GC) and a mass spectrometer (MS). The CPI setup included a wide, oval-shaped vacuum-ultraviolet (VUV) transparent magnesium fluoride (MgF2) window to maximize photoionization efficiency and thus sensitivity. The source contained a nitrogen housing around the ionization chamber inlet to avoid undesirable hydrolysis and oxidation reactions with ambient air and to maximize the proportion of formed molecular radical cations of analytes. The feasibility of the ion source was studied by analyzing 18 endogenous steroids in urine as their trimethylsilyl (TMS) derivatives with gas chromatography-tandem mass spectrometry (GC-MS/MS). The method was validated and applied to human urine samples. To our best knowledge, this is the first time that a capillary photoionization ion source has been applied for quantitative analysis of biological samples. The GC-CPI-MS/MS method showed good chromatographic resolution (peak half-widths between 3.1 to 5.3 s), acceptable linearity (coefficient of determination between 0.981 to 0.996), and repeatability (relative standard deviation (RSD%) between 5 to 18%). Limits of detection (LOD) were between 2 to 100 pg mL-1 and limits of quantitation (LOQ) were between 0.05 to 2 ng mL-1. In total, 15 steroids were quantified either as a free steroid or glucuronide conjugate from the urine of volunteers. The new CPI source design showed excellent sensitivity for analysis of steroids in complex biological samples.

Controlling granule size by granulation liquid feed pulsing.

The effects of inlet air humidity, granulation liquid feed rate and granulation liquid feed pulsing on the particle-size distribution of final granules were studied in a laboratory scale fluid-bed granulator using a central composite study design. The factors examined were modelled using three different particle-size measurement techniques (sieve analysis, laser light diffraction and the spatial filtering technique). Best goodness of fit (R2=0.94) and goodness of prediction (Q2=0.90) values were obtained using particle-size results of the spatial filtering technique. Increasing inlet air humidity and granulation liquid feed rate resulted in greater median granule size, as expected. When pulsing (interruption of granulation liquid feed in predetermined sequences) was used, the median granule size decreased clearly. This effect was strong, especially with high inlet air humidity and rapid liquid feed rate processes. Granulation liquid feed pulsing is an effective way to modify the particle size of final granules. Pulsing can be used as a controlling tool to compensate for excessive moisture content in the granulation process.

Effect of fluidisation activity on end-point detection of a fluid bed drying process.

The influence of inlet air humidity variations on fluid bed drying end-point detection was the primary focus here. Various drying end-point criteria based on temperature and humidity measurements were compared. Seasonally changing inlet air humidity affects the moisture content of the finished granules, as long as the drying process remains unchanged. However, a specific moisture content of the finished granules is commonly desired after fluid bed drying. When experimental batches of varying inlet air humidity were compared at the beginning of the drying phase, the temperature of the granules increased linearly as the humidity of the inlet air increased. This effect causes variation in moisture contents of the final granules of different batches when the fixed temperature of the mass is used as an end-point criterion. With varying inlet air humidity, the often used DeltaT temperature difference method resulted in more precise estimation of the drying end-point than the constant temperature criterion. In this study new insights were found into the correlation between moisture content and temperature of the fluidising mass. Fluidisation activity greatly affected detection of drying end-point. Use of the DeltaT criterion requires proper fluidisation throughout the process.

Particle size, moisture, and fluidization variations described by indirect in-line physical measurements of fluid bed granulation.

The aim of this study was to evaluate an instrumentation system for a bench scale fluid bed granulator to determine the parameters expressing the changing conditions during the spraying phase of a fluid bed process. The study focused mainly on four in-line measurements (dependent variables): fluidization parameter (calculated by inlet air flow rate and rotor speed), pressure difference over the upper filters, pressure difference over the granules (lower filter), and temperature of the fluidizing mass. In-line particle size measured by the spatial filtering technique was an essential predictor variable. Other physical process measurements of the automated granulation system, 25 direct and 12 derived parameters, were also utilized for multivariate modeling. The correlation and partial least squares analyses revealed significant relationships between various process parameters highlighting the particle size, moisture, and fluidization effect. Fluidization parameter and pressure difference over upper filters were found to correlate with in-line particle size and therefore could be used as estimates of particle size during granulation. The pressure difference over the granules and the temperature of the fluidizing mass expressed the moisture conditions of wet granulation. The instrumentation system evaluated here is an invaluable aid to gaining more control for fluid bed processing to obtain repeatable granules for further processing.

Comparison of melibiose and trehalose as stabilising excipients for spray-dried ß-galactosidase formulations.

Spray-dried protein formulations commonly require stabilising excipients to prevent protein degradation during processing and storage, and trehalose has been commonly used. The purpose of this work was to evaluate melibiose in spray-dried protein formulations in comparison to trehalose. The protein-activity-preserving efficacy, process behaviour and storage stability were studied. Spray drying of ß-galactosidase was carried out using different process temperature, drying air flow and feed liquid atomisation settings. Both melibiose and trehalose reduced protein activity loss during drying. A decrease in activities was observed when the process temperature exceeded a threshold temperature. During storage (30 days at 18% RH and 20 or 40 °C), the formulations dried below this threshold temperature showed no further activity loss, and the stabilising efficacy of the two disaccharides was equal. With higher process temperatures, the remaining protein activities after storage trended higher with melibiose formulations. All formulations remained amorphous. The powder yields of melibiose formulations were similar to trehalose. There was a difference in residual moisture contents, with melibiose formulations giving drier products. In conclusion, protein formulations with melibiose could be spray dried into amorphous powders that were physically stable, contained lower moisture contents and protected protein activity at least as well as trehalose formulations.

Novel description of a design space for fluidised bed granulation.

The physical measurements of a fluid bed granulator can be exploited in construction of an operating window, a design space, for process performance. The purpose of this study was to determine the influence of inlet air humidity changes on temperature in different parts of a granulator system, on fluidisation behaviour and on the particle size of the final granules. A humidifying setup was constructed on a bench-scale fluid bed granulator that enabled elevated humidity levels and sharp humidity changes of the inlet air. Ibuprofen granules were produced at the various inlet air humidity levels classified as low, intermediate and high. A novel fluidisation parameter was developed. The more improperly the particles were fluidising the smaller was the relationship of airflow rate and fan speed. Four different failure modes were identified and classified, based on the fluidisation parameter: over-fluidisation, risk of improper fluidisation, improper fluidisation and collapsed bed. It was possible to construct process trajectories for smooth fluidisation, which the optimal granulation process should follow.

A Simple Method for Improving the Spatial Resolution in Infrared Laser Ablation Mass Spectrometry Imaging.

In mass spectrometry imaging of tissues, the size of structures that can be distinguished is determined by the spatial resolution of the imaging technique. Here, the spatial resolution of IR laser ablation is markedly improved by increasing the distance between the laser and the focusing lens. As the distance between the laser and the lens is increased from 1 to 18 m, the ablation spot size decreases from 440 to 44 µm. This way, only the collimated center of the divergent laser beam is directed on the focusing lens, which results in better focusing of the beam. Part of the laser energy is lost at longer distance, but this is compensated by focusing of the radiation to a smaller area on the sample surface. The long distance can also be achieved by a set of mirrors, between which the radiation travels before it is directed to the focusing lens and the sample. This method for improving the spatial resolution can be utilized in mass spectrometry imaging of tissues by techniques that utilize IR laser ablation, such as laser ablation electrospray ionization, laser ablation atmospheric pressure photoionization, and matrix-assisted laser desorption electrospray ionization. Graphical Abstract ᅟ.

Spray-dried amorphous isomalt and melibiose, two potential protein-stabilizing excipients.

The possibility of producing amorphous isomalt and melibiose by spray drying was studied. The impact of process parameters on yield and solid-state stability was compared to sucrose and trehalose. All powders remained amorphous during 2-3 weeks. Processing was challenging due to powder stickiness. Low-temperature and low-humidity drying processes generally performed best. Most isomalt and sucrose powder was retrieved when using 60°C inlet temperature, 800L/h atomizing rate, 1.4ml/min feed rate, 15% concentration and 100% aspirator rate, giving 42-43°C outlet temperature. Isomalt was the most problematic, because it had the lowest Tg and became sticky very easily, therefore process parameters needed to be precisely balanced. There was more freedom in designing processes for melibiose but best yields were obtained with low-temperature (50°C inlet temperature, 800L/h atomizing rate, 4.9ml/min feed rate, 10% concentration and 100% aspirator, 39°C outlet temperature). Trehalose was different in that higher temperatures resulted in better yields. Yet, trehalose generally contained the highest moisture contents. The possibility to produce amorphous isomalt and melibiose at low-temperature process conditions makes them promising considering spray drying applications for heat-sensitive proteins. Melibiose is a better candidate than isomalt because of easier processability and superior solid-state stability.

Controlled Expansion of Supercritical Solution: A Robust Method to Produce Pure Drug Nanoparticles With Narrow Size-Distribution.

We introduce a robust, stable, and reproducible method to produce nanoparticles based on expansion of supercritical solutions using carbon dioxide as a solvent. The method, controlled expansion of supercritical solution (CESS), uses controlled mass transfer, flow, pressure reduction, and particle collection in dry ice. CESS offers control over the crystallization process as the pressure in the system is reduced according to a specific profile. Particle formation takes place before the exit nozzle, and condensation is the main mechanism for postnucleation particle growth. A 2-step gradient pressure reduction is used to prevent Mach disk formation and particle growth by coagulation. Controlled particle growth keeps the production process stable. With CESS, we produced piroxicam nanoparticles, 60 mg/h, featuring narrow size distribution (176 ± 53 nm).