Time Stability Studies Research Articles

Impact of gaseous interferents on palladium expansion for hydrogen optical sensing: A time stability study

Continuous monitoring of hydrogen (H2) concentration is critical for safer use, which can be done using optical sensors. Palladium (Pd) is the most commonly used transducer material for this monitoring. This material absorbs H2 leading to an isotropic expansion. This process is reversible but is affected by the interaction with interferents, and the lifetime of Pd thin films is a recurring issue. Fiber Bragg Grating (FBG) sensors are used to follow the strain induced by H2 on Pd thin films. In this work, it is studied the stability of Pd-coated FBGs, protected with a thin Polytetrafluoroethylene (PTFE) layer, 10 years after their deposition to assess their viability to be used as H2 sensors for long periods of time. It was found that Pd coatings that were PTFE-protected after deposition had a longer lifetime than unprotected films, with the same sensitivities that they had immediately after their deposition, namely 23 and 10 pm/vol% for the sensors with 150 and 100 nm of Pd, respectively, and a saturation point around 2 kPa. Furthermore, the Pd expansion was analyzed in the presence of H2, nitrogen (N2), carbon dioxide (CO2), methane (CH4) and water vapor (H2O), finding that H2O is the main interferent. Finally, an exhaustive test for 90 h is also done to analyze the long-term stability of Pd films in dry and humid environments, with only the protected sensor maintaining the long-term response. As a result, this study emphasizes the importance of using protective polymeric layers in Pd films to achieve the five-year lifetime required for a real H2 monitoring application.

Screening techniques for monitoring the sub-visible particle formation of free fatty acids in biopharmaceuticals

Free fatty acid (FFA) particles that originate from the enzymatic hydrolysis of polysorbate (PS) via co-purified host cell proteins generally appear abruptly in drug products during real-time (long-term) storage. Efforts were taken to understand the kinetics of FFA particle formation, aiming for a mitigation strategy. However, it is rather challenging particularly in the sub-visible particle (SVP) range, due to either the insufficient sensitivity of the analytical techniques used or the interference of the formulation matrices of proteinaceous drug products. In this study, we examined the feasibility of Raman microscopy, backgrounded membrane imaging (BMI) and total holographic characterization (THC) on the detection of FFA sub-visible particles (SVPs). The results indicate that THC is the most sensitive technique to track their occurrence during the course of PS hydrolysis. Moreover, with this technique we are able to distinguish different stages of FFA particle formation in the medium. In addition, a real time stability study of a biopharmaceutical was analyzed, demonstrating the viability of THC to monitor SVPs in a real sample and correlate it to the visible particles (VPs) occurrence.

Implication of Data Obtained from Real Time Stability Studies of Pharmaceutical Preparations

The major reason for carrying out stability studies of a drug product is to establish the shelf life of a drug during the period of storage so as to guarantee its quality, effectiveness and safety. Real time stability studies data from 2018-2022 were obtained from selected pharmaceutical industries located within North Central, Nigeria. The analysis of variance (ANOVA) of the data obtained for the real time stability studies was carried out using IBM SPSS Statistics Version 23. The results obtained showed that both chemical and microbiological parameters checked were within the pre-set specifications despite the changes in the results as the storage increases due to environmental factors such as: temperature, humidity, light, exposure to oxygen and container –closure system used for the packaging of the drug product. Pathogenic bacteria such as: Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Pseudomonas aeruginosa recorded 0.00 cfu/ml and the values obtained for total viable aerobic mesophilic bacteria plate count and fungi were within the pre-set specifications while Staphylococcus aureus and Candida albicans were sensitive to Methylated Spirit. There was a significant difference in the data analysed (P<0.05). The implication of this study is that the drug product may not be stable if the shelf life increases beyond what was stated for them. Hence, these results provided clues to how the quality of the product changes with time under different environmental factors (temperature, relative humidity, exposure to oxygen and light) and the interaction between the drug product and container-closure system used. This study suggests the need for pharmaceutical industries to continuously carry out stability studies of drug products in order to know the changes that may likely occur during the storage and to establish the shelf life of the drug product.

Formulation, optimization and evaluation of fast dissolving tablets of Lamotrigine using natural disintegrant agent

Objective: The present investigative work is aimed for formulating, optimizing and evaluating the Fast Dissolving Tablets (FDT) of Lamotrigine in improving the disintegration time and dissolution rate with Locust bean gum as natural disintegrant agent in combination with Sodium Starch Glycolate (SSG). Methods: Employed 32 full factorial design for optimization of Lamotrigine blend formulations. Concentrations of Locust bean gum (X1) and concentration of SSG (X2) are two factor independent variables. Experiments are conducted to identify their effect on three dependent variables are disintegration time (Y1), drug release in 5 min (Y2), and drug release in 30 min (Y3). Drug interactions between Lamotrigine and Locust bean gum were investigated with FTIR, DSC, XRD compatibility studies. Direct compression method is used for tablet manufacturing. Results: Pre and post compression parameters of Lamotrigine FDT’s are evaluated and found within acceptable limits. Among multiple formulations, LF8 formulation has demonstrated lower disintegration time in 40 ± 3.81sec and wetting time in 38 ± 0.31sec, maximum drug release is 97.88% in 30min. In short time stability studies, no major transformations are identified in physical and chemical properties of Lamotrigine FDT’s. In-vivo studies of Lamotrigine fast dissolving tablets showed faster absorption rate.

Decomposition of Vitamins in Multivitamin Syrups of Different Brands under Various Storage Conditions

Stability of multivitamins containing drug products especially the syrups remains one of the most important factor which restrains from getting the desired therapeutic effects as its ingredients decompose quickly as compared to other Active Pharmaceutical Ingredients (APIs), therefore, stability assessment is necessary to check the effect of climatic conditions on syrups’ decomposition. The study is aimed to assess the level of decomposition of vitamins in different syrups by placing them in stability chambers with different storage conditions and then comparison of assay contents of ingredients in different syrups. Different brands were selected for the estimation of assay values of vitamins/ingredients when stored at Real Time and Accelerated stability conditions as recommended by ICH for the countries categorized in climatic zone IV-A. The samples then analyzed by different analytical techniques such as HPLC, UV- Spectrophotometry and redox titration method at 3rd and 6th month timepoints. The stability study conducted showed that nicotinamide had assay values ranging from 30% to 95%, Pyridoxine 79% to 95%, Thiamine 22 % to 66%, Riboflavin 35% to 58% and Ascorbic acid 21% to 71% at real time (300C) storage condition. The study revealed that nicotinamide had assay values ranging from 26% to 89%, Pyridoxine 71% to 94%, Thiamine 9 to 35%, Riboflavin 24 to 55%, and Ascorbic acid 10 to 55% at accelerated (400C) storage condition. The most stable ingredient was Pyridoxine which showed assay values ranging from 74% to 95% and the most stable sample was Sample no A1.
 It is evident from the data presented that the degradation is more frequent when the product were stored under accelerated conditions i.e. 400C±2/75%±5RH when compare with the results of stability studies conducted under real time stability studies’ conditions i.e. 300C±2 C/65%±5RH. It is concluded that such products must be stored below 25oC. The manufacturers of such products should mention ‘Store Below 25 C0’ on the label and outer carton of the product.

Bhallatakadi Ghrita: Development and evaluation with reference to Murcchana and Shata-Dhauta process

BackgroundAyurveda is primarily based upon use of herbs either singly or in combination (polyherbal). The cow ghee (clarified butterfat) is considered as a precious base for preparing medicines in Ayurveda. Processing of ghee with plant ingredients is renowned for enhancing their therapeutic efficacy. ObjectiveIn present research work, the attempt was made to develop cow ghee based Polyherbal Bhallatakadi Ghrita formulations and evaluate them with reference to ‘Murcchana’ and ‘Shata-Dhauta’ process. Materials and methodsThe research plants were identified, procured, authenticated and processed. The extracts of plant materials were prepared and used for development of Polyherbal Bhallatakadi Ghrita (PHBG), Polyherbal Bhallatakadi Murcchita Ghrita and Polyherbal Bhallatakadi Shata-Dhauta Ghrita formulations as per Ayurvedic procedures. The prepared ghrita formulations were subjected to organoleptic (colour, odour, taste, appearance and touch), physicochemical (pH, viscosity, moisture content, specific gravity, refractive index, acid value, saponification value, iodine value, peroxide value, Rechert Meissl value and Polenske value) evaluation, in-vitro antioxidant and GC-MS analysis. The accelerated and real time stability studies were carried out to determine shelf life of ghrita formulations. ResultsThe results of evaluations indicate that, developed PHBG formulations retained the organoleptic and physicochemical characteristics of ghee. The shelf life of formulations was found to be in the range of 1.6 to 3.3 years at accelerated and 2.2 to 3.8 years at real time stability conditions. All ghrita formulations exhibited antioxidant activity in dose dependent manner. ConclusionThe standardization or evaluation of Polyherbal Bhallatakadi Ghrita formulations was found to be crucial for the establishment of a steady biological, chemical or simply a quality assurance profile of the drugs.

FORMULATION AND EVALUATION OF MINOXIDIL GEL USING ACRYLAMIDE/SODIUM ACRYLOYLDIMETHYL TAURATE COPOLYMER FOR ALOPECIA AREATA

Alopecia areata is a common, chronic inflammatory disease, characterized by patchy hair loss on the scalp, affecting about 2.1% of world population. Presently, minoxidil has been used for treatment of alopecia as topical lotion, but associated with many drawbacks like systemic side effects and low contact time with skin. Therefore, in the present work, minoxidil gel was prepared using a novel copolymer, Sepineo P 600 to overcome these drawbacks. The prepared gel was characterized for pH, drug content, viscosity, spreadability, skin adhesivity, occlusivity, in vitro drug release, ex vivo skin permeation, stability and finally for skin corrosivity. The drug content of the finalized gel was found to be 99.80 ± 0.82%. The formulation showed good spreadability, occlusivity, adhesiveness and viscosity. In vitro release studies showed that the drug release from prepared gel followed matrix release pattern as compared to lotion. Mathematical modelling of the drug release data suggested Higuchi release model. The formulated minoxidil gel was found to be non-corrosive and stable when subjected to accelerate as well as real time stability studies. Overall, the minoxidil gel formulation was suitable for skin application and can be an effective dosage form for the treatment of Alopecia areata.

Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS)

A method for the simultaneous determination of warfarin and its active metabolites (warfarin alcohols) in dried blood spots samples was developed and validated. The procedure consisted of the collection of blood sample spots on Whatman 903 filter paper and the liquid extraction of the analytes by a 3:1v/v methanol/acetonitrile mixture from 6mm diameter disks punched out from dried blood spots. Extracted samples were analyzed by ultra-high performance liquid chromatography coupled to electrospray positive ionization and tandem mass spectrometry. Chromatographic separation was carried out in isocratic condition at 25°C on a Poroshell 120 EC-C18 reversed phase column with a mobile phase consisting of 65% water containing 0.1% (v/v) formic acid and 35% acetonitrile containing 0.1% (v/v) formic acid at a flow rate of 0.5mLmin−1.The method did not show any detectable interference or matrix effect. The limits of detection were 0.007, 0.006 and 0.004ng/mL for RR/SS-warfarin alcohols, RS/SR-warfarin alcohols and warfarin, respectively. Warfarin and warfarin alcohols recovery from dried blood spots sample was almost quantitative (range 96–103%). The intra- and inter-day precision (expressed as relative standard deviation) was always below 10%. Time stability studies highlighted that the concentrations of warfarin and both diastereoisomers of warfarin alcohols in a pooled blood sample were stable for at least 1month at −20°C and after 4cycles of thaw–freeze. Moreover, warfarin, RR/SS- and RS/SR-warfarin alcohols concentrations in the dried blood spots were stable for at least 7days at 25°C.Hematocrit value had a minor influence on the analytical result compared to blood drop volume. The method was successfully applied to the analysis of 15 blood samples from patients undergoing warfarin therapy and demonstrated its suitability as an alternative for warfarin and warfarin alcohols measurement in plasma. Good correlations between concentrations in plasma and dried blood spots were demonstrated for all three analytes, with correlation coefficients r≥0.95. Warfarin concentration in dried blood spot correlated well with warfarin dosage (r=0.70, p<0.01) but not with international normalized ratio (r=0.47, p=0.0784) when all the enrolled patients (n=15) were considered. However, this correlation increased (r=0.80, p=0.0302) if a single patient was monitored over time.These preliminary results highlighted that the developed method can provide useful information for the therapeutic drug monitoring of warfarin and its active metabolites, although further studies in a larger clinical trial are needed.

Spectroscopic investigations of novel pharmaceuticals: Stability and resonant interaction with laser beam

This paper presents data about photophysics of two novel thio-hydantoins that exhibit promising pharmaceutical properties in multidrug resistance control. Time stability studies are necessary to establish the proper use of these compounds in different applications. As for their administration as drugs, it is imperative to know their shelf life, as well as storage conditions.At the same time, laser induced modified properties of the two new compounds are valuable to further investigate their specific interactions with other materials, including biological targets.The two new thio-hydantoins under generic names SZ-2 and SZ-7 were prepared as solutions in dimethyl sulfoxide at different concentrations, as well as in deionised water. For the stability assay they were kept in various light/temperature conditions up to 60days. The stability was estimates based on UV–vis absorption measurements.The samples in bulk shape were exposed different time intervals to laser radiation emitted at 266nm as the fourth harmonic of a Nd:YAG laser. The resonant interaction of the studied compounds with laser beams was analysed through spectroscopic methods UV–vis and FTIR absorption, as well as laser induced fluorescence spectroscopy.As for stability assay, only solutions kept in dark at 4°C have preserved the absorption characteristics, considering the cumulated measuring errors, less than one week.The vibrational changes that occur in their FTIR and modified fluorescence spectra upon laser beam exposure are also discussed.A result of the experimental analysis is that modifications are induced in molecular structures of the investigated compounds by resonant interaction with laser radiation. This fact evidences that the molecules are photoreactive and their characteristics might be shaped through controlled laser radiation exposure using appropriate protocols. This conclusion opens many opportunities both in the biomedical field, but also in other industrial activities involving the use of hydantoins.

High throughput peptide mapping method for analysis of site specific monoclonal antibody oxidation

Oxidation of therapeutic monoclonal antibodies (mAbs) often occurs on surface exposed methionine and tryptophan residues during their production in cell culture, purification, and storage, and can potentially impact the binding to their targets. Characterization of site specific oxidation is critical for antibody quality control. Antibody oxidation is commonly determined by peptide mapping/LC–MS methods, which normally require a long (up to 24h) digestion step. The prolonged sample preparation procedure could result in oxidation artifacts of susceptible methionine and tryptophan residues. In this paper, we developed a rapid and simple UV based peptide mapping method that incorporates an 8-min trypsin in-solution digestion protocol for analysis of oxidation. This method is able to determine oxidation levels at specific residues of a mAb based on the peptide UV traces within <1h, from either TBHP treated or UV light stressed samples. This is the simplest and fastest method reported thus far for site specific oxidation analysis, and can be applied for routine or high throughput analysis of mAb oxidation during various stability and degradation studies. By using the UV trace, the method allows more accurate measurement than mass spectrometry and can be potentially implemented as a release assay. It has been successfully used to monitor antibody oxidation in real time stability studies.

Time stability studies of electrorheological response of dispersions with different types of charge carriers

The behavior of hydrous and anhydrous fillers of various types in the electric field is investigated. Rheological and dielectric properties of dispersions containing 20 wt% of the filler in the insulating oil are determined. The comparative results of the experimental determination of the change in shear stress and current density over time at the impact of the electric field of different intensity are presented.

Real time stability and viability prediction of the anticancer BCG after lyophilization

To test if trehalose could be a better cryoprotectant for BCG than the usually used lactose and predict viability of BCG during shelf-life, BCG was suspended into three stabilizer systems containing 15% w/v trehalose, trehalose–gelatin mixture (in ratio, 30:1 w/w) or lactose. Each formula was lyophilized and several quality parameters were tested before and after lyophilization including sterility, safety, viability, morphology and moisture content. Samples of lyophilized formulae were tested for their reconstitution time and others were charged to stability chambers at 5 °C for the performance of real time study. Shelf-life of each formula was estimated and correlation between moisture content and loss in viability was established at each time interval of the real time stability study. Sterility, safety and morphology were retained after lyophilization. Just after lyophilization, minute diminish in viability was observed in the presence of each stabilizer (0.02–0.05%). There was no significant difference in reconstitution time of the three lyophilized formulae. Lactose BCG had the highest shelf-life among the used cryoprotectants during the real time stability studies. Also, moisture content was highly correlated to viability with correlation coefficient ranged between 0.97 and 0.99 and so, the former could be used for prediction of viability throughout the vaccine shelf-life.

HPLC-UV/FD Methods for Scopoletin and Asiatic acid: Development, Validation and Application in WHO Recommended Stability Testing of Herbal Drug Products

WHO recommended accelerated stability study was conducted on commercially available herbal products containing Shankhpushpi along with Bacopa monnieri (A) or Centella asiatica (B) at a temperature of 40 ± 2°C and 75 ± 5% RH for six months. Control sample of each product was stored at 4°C. Stability samples were withdrawn after 1, 3 and 6 months. Each control and stability sample was analysed for the content of scopoletin, asiatic acid and bacoside A by HPLC methods because such methods prove more selective, sensitive, efficient, reproducible, and accurate than spectroscopic and chemical methods. These methods also have advantages of sample handing, cost effectiveness and versatility in analyzing chemically diverse compounds over other methods like GSC and GLC. New methods developed and validated for scopoletin and asiatic acid were proved sufficiently precise, accurate and robust for analysis of scopoletin (1-500 ng/ml) and asiatic acid (10-1000 μg/ml). Bacoside A was not detected in any of the control samples of products A, indicating that B. monnieri is either absent in A or the content of Bacoside A is too low to be detectable. Content of scopoletin and asiatic acid was found to vary widely among different batches as well as products (scopoletin 165.78-206.15 ng/ml in A and 2.61-28.78 ng/ml in B, and asiatic acid 30.14-44.92 μg/ml in B), which indicate a probable variability in therapeutic efficacy of the products. The content of markers decreased significantly after 6 months of storage under accelerated conditions, which implies that therapeutic efficacy of the product may also decrease substantially with storage. These findings further suggest real time stability studies as per the WHO and ICH guidelines involving marker’s quantification and evaluation of therapeutic effects through appropriate in vitro/in vivo methods to establish actual shelf life of the products.

A fast, stability-indicating, and validated liquid chromatography method for the purity control of lercanidipine hydrochloride in tablet dosage form.

A robust, sensitive, and stability-indicating rapid resolution liquid chromatography method for the simultaneous determination of process impurities and degradation products of lercanidipine hydrochloride in pharmaceutical dosage form was developed and validated. The chromatographic separation was performed on the Zorbax SB C18 [(50 × 4.6) mm] 1.8 μm column, using gradient elution of a potassium dihydrogen phosphate buffer (pH 3.5, 0.01 M) and acetonitrile. The flow rate was 1.0 ml/min and UV detection was performed at 220 nm. The method was further evaluated for its stability-indicating capability by hydrolytic, oxidative, thermal, thermal with moisture, and photolytic degradation studies. All acceptance criteria of the International Conference on Harmonization guidelines for validation were covered in the method validation. This method can be used for purity control during manufacture and real time stability studies. A shorter run time of 10 minutes and good solution stability for at least 48 hours allowed the quantification of more than 50 samples per day with comparatively lower costs than existing methods.

Development of a Reference Standard of Escherichia coli DNA for Residual DNA Determination in China

This collaborative study developed the first national Escherichia coli (E. coli) DNA reference standard for standardizing quantitative residual DNA assay methods, fluorescence dye (PicoGreen) and quantitative PCR (q-PCR), which were widely employed to measure residual DNA contents of prokaryotic-derived recombinant products. High purity of E. coli strain BL21 was extracted by the cetyl triethyl ammonium bromide (CTAB)/phenol chloroform method, analyzed by UV-visible spectrophotometry and electrophoresis, diluted with tris-EDTA (TE) buffer and manually dispensed. Then, with a cooperative calibration among six laboratories, including five manufacturers and one national control laboratory, the concentration of E. coli DNA standard solution was determined as 96.2 μg/mL (95% C.I: 95.5–96.9 μg/mL, CV 3.4%). The candidate showed excellent stability both from accelerated degradation study and real time stability study. The applicability study showed that the E. coli DNA reference could reach the sensitivity of 0.781 ng/mL and 1 fg/μL, respectively, in fluorescent dye and q-PCR assay, and also had good linearity and precision. The consistency of the reference could meet the requirements of the national reference standard. As a conclusion, the candidate material was suitable to serve as a China national standard for E. coli residual DNA determination. The successful establishment of the E. coli DNA standard will facilitate the standardization of quantitative methods for testing residual host cell DNA.

Time Stability Studies of Quinazoline Derivative Designed to Fight Drug Resistance Acquired by Bacteria

The 3-[2-(dimethylamino)ethyl]-6-nitroquinazolin-4(3H)-one (BG1188) may modify the antibiotic susceptibility of human Gram-negative bacteria which present multidrug resistant phenotype. This application requires to know the time stability of BG1188 solutions. This paper reports for the first time about the time stability of the BG1188 solutions measured by optical absorption; the solvents were ultrapure de-ionized water and dimethyl sulfoxide (DMSO). In ultrapure water, BG1188 concentration varied between 2×10-3M (stock solution) and 10-6M, at pH = 6.7. In DMSO, the concentration was 10-3M (recommended by higher activity on bacteria). Solutions in ultrapure water were kept in dark at 4°C and measured at room temperature (22°C). The ultraviolet-visible (UV-Vis) absorption spectra have shown that only monomer forms were present in solutions. One of the obtained results is that the time evolution of the absorption spectra at 10-3M in ultrapure water shows a reproducibility within the measuring errors (±1.045%) for time intervals up to 1032 hours (more than 40 days) after preparation, showing a high stability of BG1188 solutions; generally, samples are stable within the experimental errors at concentrations higher than 10-5M, but the stability time interval may be variable: from 119 days at 10-4M to 34 days at 10-5M. As for solutions in the DMSO, the absorption spectra show that the samples exhibit modifications immediately after preparation, in terms of peak intensities and shapes, so that the BG1188 solutions in DMSO may be considered as unstable.

Spectrophotometric study of time stability and acid-base characteristics of chelerythrine and dihydrochelerythrine

Abstract Time stability, acid-base and UV-VIS spectral properties of dihydrochelerythrine (DHCHE) were studied spectrophotometrically in water:methanol and water:ethanol media. DHCHE is stable in strongly acid milieu (pH HCl, HNO3, H2SO4, H3PO4 and their mixtures. Remarkable shifts of formation parts of absorbance-pH (A-pH) curves to the alkaline medium were observed depending on the type and concentration of inert electrolyte (most remarkable for HNO3 and HCl). The corresponding equilibrium constants pKR+ of the transition reaction between charged iminium Q+ and uncharged QOH (pseudo-base, 6-hydroxy-dihydro derivative) forms of chelerythine were calculated using a numerical interpretation of A-pH curves by a SQUAD-G computer program which ranged from 8.51–9.31. The highest changes of ΔpK R+ (0.75 and 0.53) were observed for H3PO4 and H2SO4, respectively. The priority effect of ionic species and ionic strength was confirmed in the presence of additions of NaCl and KCl. The strength of interaction of CHE with biomacromolecular compounds (i.e., peptides, proteins, nucleic acids etc.) may be affected because of the observed influence of both cation and anion of the inert electrolyte on acid-base behavior.

Extremely long time stability study of selected antibiotic standards

A study was conducted to investigate the quality of reference substances which were produced long before the introduction of advanced analysis and purification techniques. The quality and antimicrobial activity of WHO and EP oxytetracycline, doxycycline, colistin and spiramycin reference standards were assessed. The oldest substance was stored for 54 years in a freezer. Assay and purity tests were conducted according to Ph. Eur. 6.0. Additionally, antibacterial activity was tested with the microbiological method according to Ph. Eur. 4.0 (agar diffusion method). The results of the study show that several of the tested substances remained stable for over 40 years and one for over 50 years of storage. In most cases, the determined potency is close to the declared one, regardless of the method used (HPLC or microbiological). Composition analysis of multi-substance antibiotics (colistin and spiramycin) showed important differences compared with new reference substances. Results also indicate that no excessive degradation occurred during the entire storage period and impurity levels have not changed significantly.

A radial basis function method for the shallow water equations on a sphere

The paper derives the first known numerical shallow water model on the sphere using radial basis function (RBF) spatial discretization, a novel numerical methodology that does not require any grid or mesh. In order to perform a study with regard to its spatial and temporal errors, two nonlinear test cases with known analytical solutions are considered. The first is a global steady-state flow with a compactly supported velocity field, while the second is an unsteady flow where features in the flow must be kept intact without dispersion. This behaviour is achieved by introducing forcing terms in the shallow water equations. Error and time stability studies are performed, both as the number of nodes are uniformly increased and the shape parameter of the RBF is varied, especially in the flat basis function limit. Results show that the RBF method is spectral, giving exceptionally high accuracy for low number of basis functions while being able to take unusually large time steps. In order to put it in the context of other commonly used global spectral methods on a sphere, comparisons are given with respect to spherical harmonics, double Fourier series and spectral element methods.

Lipase-catalyzed reactions in vesicles as an approach to vesicle self-reproduction.

As a continuation of our endeavor to find conditions under which bounded aggregate structures are able to self-reproduce, we have investigated the reactivity of lipase, both in free solution and vesicle-entrapped, against mixed oleic acid/oleate/ethyl oleate vesicles. Three types of vesicles have been prepared and characterized: (A) oleic acid/oleate vesicles; (B) oleic acid/oleate/ethyl oleate vesicles; and (C) lipase containing oleic acid/oleate vesicles. Long time stability studies by quasi elastic light scattering show that whereas (B) and (C) vesicles remain stable with a diameter of 110-130 nm and monodisperse for over a period of one month, vesicles (A) separated from an initial single population of 105 nm diameter into two populations, having respectively 70 nm diameter (more than 95% of the particles) and 180-210 nm diameter (less than 5% of the total population). In the case of vesicles (C), it could be shown that the enzyme remains localized inside the vesicles and it does not protrude into the water bulk phase. The enzymatic hydrolysis of ethyl oleate (which is water-insoluble) incorporated in the B-vesicles was studied under two configurations: (I) by adding lipase externally to the B-vesicles; (II) by mixing vesicles (B) and vesicles (C). In both cases, the reaction progressed to 100% hydrolysis. In the first case, the reaction was attended by an increase of the number of vesicles, and since this hydrolysis reaction takes place within the boundary of the parent vesicles, the criteria of autopoietic self-reproduction of vesicles are satisfied. In the case (II) instead, no increase of the population number of particles could be detected. The possible reasons for this difference are discussed.