High-performance liquid chromatography methods for the separation and quantitation of spironolactone and its degradation products in aqueous formulations and of its metabolites in rat serum.

HPLC assays have been developed for the determination of spironolactone and its degradation products 7 alpha-thiospirolactone and canrenone in aqueous solutions of beta-cyclodextrins and for the determination of spironolactone and its metabolites 7 alpha-thiospirolactone, 7 alpha-thiomethylspirolactone, 6 beta-hydroxy-7 alpha-thiomethylspirolactone and canrenone in rat serum samples. Both methods were well suited for their respective applications, i.e., studying the stability of spironolactone in liquid formulations of beta-cyclodextrins and the oral absorption of spironolactone in rats. The HPLC method developed for spironolactone and its metabolites in rat serum requires very small volumes of serum making it possible to take several blood samples over a period of time.

In-line moisture measurement during granulation with a four-wavelength near infrared sensor: an evaluation of particle size and binder effects.

Factors affecting in-line near infrared (NIR) moisture measurement with a four-wavelength sensor were evaluated (choice of binder used in granulation liquid and the increase in particle size). An entire NIR spectrum is not necessary for the measurement of water, and often the use of only a few NIR wavelengths around the water band enables reliable and high-speed detection of moisture. Glass ballotini and microcrystalline cellulose (MCC) were used as model test materials. The binders studied were poly[1-(2-oxo-1-pyrrolidinyl)ethylene] (PVP) and gelatin. Full off-line NIR spectra of test materials at different levels of binder solutions were measured. The major spectral features for both the binders were bands around 1700 nm (first overtones CH related stretches) and 2200 nm (combination bands). Gelatin also had an NH band around 1500 nm (first overtones of NH stretches) and combination bands at about 2050 nm. Particle size effects were observed as an increase in spectra baseline. All these factors should be considered when choosing NIR wavelengths used for detection of water with a fixed wavelength set-up. A robust calibration model enables the development of in-process control of wet granulation processes.

Visualization of fluid-bed granulation with self-organizing maps.

The degree of the instrumentation of pharmaceutical unit operations has increased. This instrumentation provides information of the state of the process and can be used for both process control and research. However, on-line process data is usually multidimensional, and is difficult to study with traditional trends and scatter plots. The Self-Organizing Map (SOM) is a recognized tool for dimension reduction and process state monitoring. The basics of the SOM and the application to on-line data collected from a fluid-bed granulation process are presented. As a batch process, granulation traversed through a number of process states, which was visualized with SOM as a two-dimensional map. In addition, it is demonstrated how the differences between granulation batches can be studied. The results suggest that SOM together with new in-line process analytical solutions support the in-process control of the pharmaceutical unit operations. Further, a novel research tool for understanding the phenomena during processing is achieved.

Water-soluble beta-cyclodextrins in paediatric oral solutions of spironolactone: preclinical evaluation of spironolactone bioavailability from solutions of beta-cyclodextrin derivatives in rats.

Water-soluble derivatives of beta-cyclodextrin have been considered for solubilization of spironolactone in the formulation of a safe liquid preparation for premature infants. The oral absorption of spironolactone was studied in rats to evaluate the need to adjust spironolactone dosage in prospective clinical studies. Spironolactone was administered in solutions of sulphobutyl ether beta-cyclodextrin (SBE7) or dimethyl-beta-cyclodextrin (DM-beta-CyD) and also as spironolactone-containing powder papers (reference preparation). Spironolactone in SBE7 solution was administered intravenously to assess the extent of intestinal absorption from the different formulations. Spironolactone and the metabolites 7alpha-thiospirolactone, 7alpha-thiomethylspirolactone and canrenone were determined in rat serum after intravenous administration of spironolactone. Half-lives for spironolactone, 7alpha-thiomethylspirolactone and canrenone were 0.72 +/- 0.17, 1.5 +/- 0.3 and 2.2 +/- 0.3 h, respectively. Although, according to Cmax values, 7alpha-thiomethylspirolactone was the major serum metabolite in rats, higher AUC (area under the serum concentration-time curve) values were obtained for canrenone. After oral administration of spironolactone the bioavailabilities evaluated from the AUC values of 7alpha-thiomethylspirolactone were 27.5 +/- 9.3%, 81.3 +/- 28.8% and 82.8 +/- 28.6% for powder papers, DM-beta-CyD and SBE7 solutions, respectively. The oral absorption of spironolactone by rats was better after administration of spironolactone in SBE7 and DM-beta-CyD solutions than after administration as powder papers. Both cyclodextrin formulations enhanced spironolactone bioavailability to a similar extent despite some deacetylation of spironolactone in the presence of SBE7. A reduction of spironolactone dosage would be recommended during clinical studies with premature infants. These results indicate that SBE7 could be a safe and suitable excipient for the solubilization of spironolactone in paediatric formulations.

Intestinal safety of water-soluble beta-cyclodextrins in paediatric oral solutions of spironolactone: effects on human intestinal epithelial Caco-2 cells.

Effects of water-soluble beta-cyclodextrins (beta CDs) on intestinal epithelial integrity were investigated, to establish the safe use of these beta CDs as solubilizers of spironolactone in paediatric enteral solutions. Mannitol permeability and transepithelial resistance (TER) of human intestinal epithelial Caco-2 cell monolayers during exposure to dimethyl-beta-cyclodextrin (DM beta CD), hydroxypropyl-beta-cyclodextrin (HP beta CD) and sulphobutyl ether beta-cyclodextrin (SBE beta CD) were followed. Staining methods were used to discern cells with damaged membranes and to study the integrity of cytoskeletal actin and tight junctions. Cytotoxicity of the beta CDs was tested by effects on intracellular dehydrogenase activity. Exposure to HP beta CD and SBE beta CD solutions had only minor effects on the integrity of Caco-2 cell monolayers. In contrast, DM beta CD clearly increased the epithelial permeability for the hydrophilic marker [14C]mannitol across Caco-2 monolayers, decreased TER and showed a dose-dependent cytotoxicity. According to staining, DM beta CD increased the permeability of the apical cell membrane without discernable effects on cytoskeletal actin. HP beta CD and SBE beta CD appear to be safe additives for use in enteral spironolactone preparations with respect to their acute local effects on epithelial integrity.

Next generation fluidized bed granulator automation.

A system for fluidized bed granulator automation with in-line multichannel near infrared (NIR) moisture measurement and a unique air flow rate measurement design was assembled, and the information gained was investigated. The multivariate process data collected was analyzed using principal component analysis (PCA). The test materials (theophylline and microcrystalline cellulose) were granulated and the calibration behavior of the multichannel NIR set-up was evaluated against full Fourier Transform (FT) NIR spectra. Accurate and reliable process air flow rate measurement proved critical in controlling the granulation process. The process data describing the state of the process was projected in two dimensions, and the information from various trend charts was outlined simultaneously. The absorbence of test material at correction wavelengths (NIR region) and the nature of material-water interactions affected the detected in-line NIR water signal. This resulted in different calibration models for the test materials. Development of process analytical methods together with new data visualization algorithms creates new tools for in-process control of the fluidized bed granulation.

Microscale granulation in a fluid bed powder processor using electrostatic atomisation.

The aim of this study was to use the electrostatic atomisation in miniaturised fluid bed granulation process and define the effect of process parameters. The process parameters included in the study were granulation liquid flow rate, atomisation voltage and binder concentration in the granulation liquid. Altogether 22 batches were granulated in Multichamber Microscale Fluid bed powder Processor (MMFP). Granule size distributions were measured with both sieves and image analyses. With these process conditions, the atomisation liquid flow rate had a strong positive correlation with the granule size. Increasing the atomisation voltage increased the granule size, which is contradictory with the expectations. The effect of the binder concentration remained unclear. Although it is challenging to model the fluid bed granulation process in micro-scale, multivariate methods such as principal component analysis (PCA) are helpful in studying the most important phenomena.

Optimization of moist heat sterilization.

The use of different combinations of sterilization time and temperature in a pilot scale autoclave, GEV 612 AR-2 (Getinge Ab, Sweden), in optimizing the sterilization process was studied. All three programs used had the same sterilization efficacy (F0 = 15 minutes) but different sterilization temperatures (116, 121, and 126 degrees C) and total process times (98, 57, and 44 minutes). The heat distribution during the sterilization phase was, in all cases, very uniform, the greatest difference being 0.5 degrees C. Also the F0 values differed only by +/- 0.5 minutes from each other. The F0 value increases linearly with all programs until the beginning of the cooling phase. The main effect of different sterilization temperatures on the cumulative F0 curves is an increase in the slope of the curves with increasing sterilization temperature. First order temperature change constants were determined both for the heating phase and the cooling phase. The numeric values of the rate constants for the heating and the cooling phases were 0.20 +/- 0.03 and 0.046 +/- 0.005 min-1, respectively. It is concluded that the pilot autoclave used in this study controls the sterilization process very accurately. The observed variations between F0 values at different positions in the autoclave chamber are acceptable. On the basis of this study an accurately engineered and controlled autoclave is required in process optimization. It also is possible to use higher sterilization temperatures than usually suggested in pharmacopeias and thus to shorten the process time.

Content-based image retrieval: a new promising technique in powder technology.

The aim of the present study was to introduce a new technique for analyzing powders by examining the content information of images of pharmaceutical powder systems. Texture features of images of microcrystalline cellulose were compared by using a content-based image retrieval system (CBIR), QBIC (Query-by-Image-Content). The rank order and image similarities were compared to particle sizes and appearances of different mixtures. The image order of the similarity values was in close agreement with the appearance and particle size of the mixtures. When the image of pure Avicel PH 101 was used as a query image, the most similar images were always from images of mixtures with a large number of particles with smaller particle mean sizes. When images of pure Avicel PH 200 were used as a query image, the closest matches of image similarity were from images of mixtures with a larger amount of larger particles. The results show that the CBIR system extracts applicable content information on images of powders, but the texture features used were not totally adequate for analysis of the powders used. In general, content-based image retrieval seems to be a promising approach to efficiently use the vast image information that is available from pharmaceutical powders. Nevertheless, to achieve an efficient CBIR tool for powder technology requires development of substantial algorithms for feature extraction.

Use of the near-infrared reflectance method for measurement of moisture content during granulation.

The purpose of this study was to demonstrate the use of a near-infrared (NIR) method for in-process control of a placebo formulation. An NIR setup with a multichannel detector was applied in the measurement of water during fluidized bed granulation. The effects of two critical granulation parameters were studied using the central composite design. The present NIR setup with three wavelengths proved applicable for in-line moisture measurement. The 1990 nm signal was used for measurement of water and the 1745 and 2145 nm signals were used to correct the change in spectra baseline during granulation. Variations in inlet air conditions proved to be critical factors, explaining differences in the granule size distributions. Differences in granule moistening and drying rates resulting from varying inlet air conditions could be measured with the NIR setup. The moisture content of granules at the end of the spraying phase explained part of the differences in granule size distributions. The moisture content of granules at the end of the drying phase affected the tableting behavior of granules. The results suggested that direct measurement of granule moisture content facilitates the in-process control of the granulation.

Comparison of different rubber stoppers; the effect of sterilization on the number of particles released.

The effect of sterilization on the number of particles released from five different types of rubber stoppers, as well as on their surface roughness and elemental composition before and after sterilization is described. The stoppers were immersed in 200 ml of 0.9% sodium chloride solution in conical flasks. The number of particles released into the sodium chloride solution was measured by Coulter Counter. The surface roughness and the elemental composition of the stoppers were determined by SEM/EDX. All measurements were made both before and after sterilization at 121 degrees C to F0 15 mins. The number of particles released from a stopper during sterilization varies considerably between different stoppers and even between different batches of the same stopper. The only non-siliconized stopper in this study performed well. The absence of surface siliconization may have contributed to this performance. The scanning electron micrographs revealed well the differences in the surface roughness of the stoppers. The sterilization generally increases the surface roughness of the samples. The x-ray microanalysis revealed that the elemental composition of the stoppers may vary not only between different types of stoppers but also between different batches of the same stopper.

The use of different time/temperature combinations in the optimization of sterilization of Ringers/glucose infusion solution.

The effect of sterilization temperature and sterilization efficacy (F0) on the amount of 5-hydroxymethyl-2-furaldehyde (5-HMF) and glucose, number of particles generated and color of Ringers/glucose infusion solution is described. The infusion solution was a 5% glucose solution containing Ringer-type electrolytes. The solutions were autoclaved in a pilot scale autoclave using different temperature/time combinations to produce the target F0 values of 10, 15, 20 and 25 minutes. The amounts of 5-HMF and glucose were determined by HPLC. The use of the shortest possible sterilization cycle to yield the target F0 resulted in the lowest 5-HMF concentrations in the Ringers/glucose infusion solutions studied. The spectrochromatograms of the sterilized solutions showed that glucose is degraded into various degradation products in the presence of Ringer-type electrolytes. It was also demonstrated by SEM/EDX that a significant part of the particles result from the leaching of the silicon oil used in the manufacture of rubber stoppers for LVP solutions. The number of particles generated in different solutions and the particle size distributions were determined with a Coulter-Counter. The particle size distributions show no clear correlation between sterilization process conditions and the number of particles. The results show that it is possible to minimize the amount of 5-HMF generated even when the same F0 is used, by choosing the highest sterilization temperature possible. This also leads in practice to the shorter sterilization processes, saving both process time and energy.