Heat Conduction Microcalorimetry Research Articles

The use of microcalorimetry and HPLC for the determination of degradation kinetics and thermodynamic parameters of Perindopril Erbumine in aqueous solutions

Perindopril Erbumine (PER) is one of the newly used angiotensin-converting enzyme inhibitors (ACE inhibitors) and is used for the treatment of patients with hypertension and symptomatic heart failure. It has two main degradation pathways, i.e. the degradation by hydrolysis and the degradation by cyclization. An isothermal heat conduction microcalorimetry (MC) and high pressure liquid chromatography (HPLC) were used for the characterization of aqueous solutions of PER and its stability properties. The rates of heat evolved during degradation of perindopril were measured by MC as a function of temperature and pH and from these data rate constant and change in enthalpy of the reactions were determined. With the HPLC method the concentration of perindopril and its degradation products were measured as a function of time in aqueous solutions of different pH that were stored at different temperatures. We demonstrated that reactions of degradation of perindopril at observed conditions follow the first order kinetics. The Arrhenius equation for each pH was determined. At pH 6.8 only one degradation pathway is present, i.e. the degradation by hydrolysis. Degradation constants for this pathway calculated from MC data are in good agreement with those obtained from HPLC. MC as a non-specific technique was shown to be useful in studies of PER when one reaction was present in the sample and also when more chemical and physical processes were simultaneously running.

Phase Equilibrium System of LuCl3−CdCl2−HCl(7.47 %)−H2O at 298.15 K and Standard Molar Enthalpies of Formation of New Solid-Phase Compounds

The equilibrium solubility of the quaternary system LuCl3−CdCl2−HCl−H2O was determined at 298.15 K, and the corresponding equilibrium diagram was constructed in this paper. The quaternary system is complicated with four equilibrium solid phases, CdCl2·H2O, Cd3LuCl9·14H2O (3:1 type), CdLuCl5·9H2O (1:1 type), and LuCl3·6H2O, of which the new compounds Cd3LuCl9·14H2O and CdLuCl5·9H2O were found to be congruently soluble in the system. The two new compounds obtained were identified and characterized by the methods of X-ray diffraction, thermogravimetry, and differential thermogravimetry. The standard molar enthalpies of solution of Cd3LuCl9·14H2O and CdLuCl5·9H2O in deionized water were measured to be 28.79 ± 0.22 kJ·mol−1 and −15.48 ± 0.30 kJ·mol−1, respectively, by heat conduction microcalorimetry. Their standard molar enthalpies of formation were calculated to be −6427.5 ± 1.3 kJ·mol−1 and −4133.7 ± 0.7 kJ·mol−1, respectively.

Study on Phase Equilibrium in the Quaternary System CsCl−LuCl3−HCl (10.06 %)−H2O at 298.15 ± 0.1 K and New Solid-Phase Compounds

The equilibrium solubility of the quaternary system CsCl−LuCl3−HCl−H2O was determined at 298.15 K, and the corresponding equilibrium diagram was constructed. The quaternary system is complicated with five equilibrium solid phases, CsCl, Cs4LuCl7·5H2O (4:1 type), Cs9Lu5Cl24·29H2O (9:5 type), Cs2LuCl5·2H2O (2:1 type), and LuCl3·6H2O, of which the new compounds Cs4LuCl7·5H2O and Cs9Lu5Cl24·29H2O were found to be congruently soluble and the new compound Cs2LuCl5·2H2O was found to be incongruently soluble in the system. The three new compounds obtained were identified and characterized by the methods of X-ray diffraction, thermogravimetry, and differential thermogravimetry. The standard molar enthalpy of solution of Cs4LuCl7·5H2O, Cs9Lu5Cl24·29H2O, and Cs2LuCl5·2H2O in deionized water was measured to be (15.6 ± 0.3) kJ·mol-1, (−78.8 ± 0.6) kJ·mol-1, and (−62.5 ± 0.5) kJ·mol-1, respectively, by heat conduction microcalorimetry. Their standard molar enthalpies of formation were calculated to be (−4313.0 ± 2.1) kJ·mol-1, (−17871.6 ± 5.3) kJ·mol-1, and (−2526.5 ± 1.2) kJ·mol-1, respectively.

Solid lipid nanodispersions containing mixed lipid core and a polar heterolipid: Characterization

This paper describes the characterization of solid lipid nanodispersions (SLN) prepared with a 1:1 mixture of theobroma oil and goat fat as the main lipid matrix and Phospholipon 90G ® (P90G) as a stabilizer heterolipid, using polysorbate 80 as the mobile surfactant, with a view to applying the SLN in drug delivery. The 1:1 lipid mixture and P90G constituting the lipid matrix was first homogeneously prepared by fusion. Thereafter, the SLN were formulated with a gradient of polysorbate 80 and constant lipid matrix concentration by melt-high pressure homogenisation. The SLN were characterized by time-resolved particle size analysis, zeta potential and osmotic pressure measurements, differential scanning calorimetry (DSC) and wide angle X-ray diffraction (WAXD). Transmission electron microscopy (TEM) and isothermal heat conduction microcalorimetry (IMC) which monitors the in situ crystallization were also carried out on the SLN containing P90G and 1.0 % w/w of polysorbate 80. The results obtained in these studies were compared with SLN prepared with theobroma oil with and without phospholipid. Particle size analysis of SLN indicated reduction in size with increase in concentration of mobile surfactant and was in the lower nanometer range after 3 months except SLN prepared without P90G or polysorbate 80. The lipid nanoparticles had negative potentials after 3 months. WAXD and DSC studies revealed low crystalline SLN after 3 months of storage except in WAXD of SLN formulated with 1.0 % w/w polysorbate 80. TEM micrograph of the SLN containing 1.0 % w/w polysorbate 80 revealed discrete particles whose sizes were in consonance with the static light scattering measurement. In situ crystallization studies in IMC revealed delayed crystallization of the SLN with 1.0 % w/w polysorbate 80. Results indicate lipid mixtures produced SLN with lower crystallinity and higher particle sizes compared with SLN prepared with theobroma oil alone with or without P90G, and would lead to higher drug incorporation efficiency when used in formulation of actives. Mixtures of theobroma oil and goat fat would be suitable for the preparation of nanostructured lipid carriers. SLN of theobroma oil containing phospholipid could prove to be a good ocular or parenteral drug delivery system considering the low particle size, particle size stability and in vivo tolerability of the component lipids. SLN prepared with lipid admixture, which had higher increase in d 90% on storage are suitable for preparation of topical and transdermal products.

Investigation of surface-modified solid lipid nanocontainers formulated with a heterolipid-templated homolipid

There is increasing interest in the search for improved drug delivery systems with greater versatility. Consequently, many drug delivery systems have been studied. In this study, surface-modified lipid nanocontainers were formulated with a homolipid from Capra hircus (goat fat) templated with a heterolipid (Phospholipon 90G ®) which was also the surface modifier. The solid lipid nanocontainers (SLN) were formulated by hot high pressure homogenisation using increasing concentrations of polysorbate 80 as the mobile surfactant. Prior to SLN preparation, the templated homolipid was formulated by fusion to obtain a homogeneous lipid matrix, which was characterized using differential scanning calorimetry (DSC), polarized light microscopy (PLM) and wide angle X-ray diffraction (WAXD) to obtain its thermal and crystal characteristics. Isothermal heat conduction microcalorimetry (IMC) and freeze-fracture transmission electron microscopy (FFTEM) studies were carried out on the templated homolipid and SLN containing 1.0% (w/w) of polysorbate 80 to study their in situ crystallization kinetics and morphology, respectively. The formulated SLN were also subjected to time-resolved DSC, WAXD and particle size analyses for one month. The thermal and crystal characteristics were compared with those of the bulk lipid matrix (templated homolipid). Result of the particle size analysis indicated that the particles size remained roughly within the lower nanometer range after one month. FFTEM micrograph of the lipid matrices revealed lamellar sheets for Phospholipon 90G ® and layered triglyceride structures for the homolipid and Phospholipon 90G ®-templated homolipid. FFTEM micrograph of SLN revealed anisometric structures. PLM of the templated homolipid did not show, but goat fat (homolipid) alone showed slight growth in crystals with time. WAXD and DSC studies revealed minor increase in crystallinity of the new lipid matrix after one month and DSC also detected templation of homolipid by the heterolipid noted by the disappearance of the lower melting peak of the homolipid. However, for the SLN, WAXD results showed low crystalline particles while DSC only showed a very little endothermic process after one month of storage at 20°C. The implication of this finding is that progression of the SLN to highly ordered particles over time would not occur. This will be favourable for any incorporated drug as drug expulsion, due to increase in crystallinity, will not occur. Result obtained from analysis of the isothermal crystallization exotherms indicated that the templated homolipid and SLN1 containing 1.0% polysorbate 80 possess similar nucleation mechanisms and growth dimensions different from the pure homolipid. The SLN containing 0.5 and 1.0% polysorbate 80 possessed good properties and could prove to be good delivery systems for drugs for parenteral or ocular administration. The result of this study also shows a method of improving natural lipids for use in particulate drug delivery systems.

Further characterization of theobroma oil–beeswax admixtures as lipid matrices for improved drug delivery systems

There is an increasing interest in lipid based drug delivery systems due to factors such as better characterization of lipidic excipients and formulation versatility and the choice of different drug delivery systems. It is important to know the thermal characteristics, crystal habit, texture, and appearance of a new lipid matrix when determining its suitability for use in certain pharmaceutical application. It is line with this that this research was embarked upon to characterize mixtures of beeswax and theobroma oil with a view to applying their admixtures in drug delivery systems such as solid lipid nanoparticles and nanostructured lipid carriers. Admixtures of theobroma oil and beeswax were prepared to contain 25% w/w, 50% w/w, and 75% w/w of theobroma oil. The admixtures were analyzed by differential scanning calorimetry (DSC), small angle X-ray diffraction (SAXD), wide angle X-ray diffraction (WAXD), and isothermal heat conduction microcalorimetry (IMC). The melting behavior and microstructures of the lipid admixtures were monitored by polarized light microscopy (PLM). Transmission electron microscopy (TEM) was used to study the internal structures of the lipid bases. DSC traces indicated that the higher melting peaks were roughly constant for the different admixtures, but lower melting peaks significantly increased ( p < 0.05). The admixture containing 25% w/w of theobroma oil possessed highest crystallinity index of 95.6%. WAXD studies indicated different reflections for the different lipid matrices. However, new interferences were detected for all the lipid matrix admixtures between 2 θ = 22.0° and 2 θ = 25.0°. The lipid matrices containing 50% w/w and 25% w/w of theobroma oil showed absence of the weak reflection characteristic of pure theobroma oil, while there was disappearance of the strong intensity reflection of beeswax in all the lipid matrix admixtures at all stages of the study. PLM micrographs revealed differences with regard to the thermal and optical behaviors depending on the composition of the matrix. The lipid matrix consisting of 75% w/w of theobroma oil showed a spherulite texture after 4 weeks of isothermal storage. Crystallization exotherms of lipid matrices containing 50% w/w and 25% w/w of theobroma oil showed change in modification after 30 min with the latter having a greater time-dependent crystallization. Generally, low non-integral Avrami exponents and growth rate constants were obtained for all the lipid matrices, with the admixture containing 25% w/w theobroma oil having the lowest Avrami exponent and growth rate constant. Based on the results obtained, admixtures containing 50% w/w and 75% w/w of theobroma oil could be applied in the formulation of solid lipid nanoparticles and nanostructured lipid carriers as these lipid matrices possessed crystal characteristics that favour such drug delivery systems.

Effect of partial substitution of nickel for copper on LaNi5 activation

The first cycles of hydrogen absorption-desorption in LaNi5-H2 and LaNi4.5Cu0.5-H2 systems were studied using Tian-Calvet differential heat-conduction microcalorimetry, volumetric measurements, and powder X-ray diffraction. The diffraction profiles were analyzed, and the microstructure characteristics of the LaNi5 and LaNi4.5Cu0.5 systems at different stages of activation were determined. The pressure-composition isotherms were plotted, and the enthalpies of phase transitions α→β and β→α were calculated. The effect of substitution on the change in the thermodynamic parameters and microstructure characteristics of the hydride-forming intermetallic compounds during activation was shown.

A critical study of novel physically structured lipid matrices composed of a homolipid from Capra hircus and theobroma oil

There is increasing interest in drug formulation using lipids. In this study, some physically structured lipid matrices were formulated and characterized for drug delivery applications. Lipid matrices containing a novel homolipid from Capra hircus (goat fat) and theobroma oil, at 25, 50 and 75% (w/w) concentration of the homolipid were formulated by fusion. The lipid matrices were subjected to some characterization procedures such as differential scanning calorimetry (DSC) to ascertain their supramolecular properties, small angle X-ray diffraction (SAXD), wide angle X-ray diffraction (WAXD), polarized light microscopy (PLM) and isothermal heat conduction microcalorimetry (IMC). The internal structures of some selected lipid matrices were also studied by freeze-fracture transmission electron microscopy (FFTEM). DSC results obtained indicated that goat fat has a pre-transition at 15.9 ± 0.2 °C (after 1 week) and melts completely with two detectable melting peaks at 33.0 ± 0.2 and 49.9 ± 0.1 °C, and total enthalpy of 99.9 ± 2.5 mJ/mg determined after 6 weeks of preparation. The melting enthalpy of goat fat changed after 3 weeks but remained constant after 6 weeks while the melting enthalpy of the lipid matrix containing 50% (w/w) goat fat changed after 3 and 6 weeks. An increase in lower melting peak was observed in the lipid matrix containing 25% (w/w) goat fat after 6 weeks. WAXD and SAXD of the physically structured lipid matrices showed reflections of the different pure lipids but new interferences were detected in WAXD mostly between 2 θ = 17.5° and 2 θ = 27.5°. PLM observation revealed the presence of Maltese crosses for the homolipid at 37 °C, which disappeared upon heating at 51.0 °C. PLM of the structured lipid matrix containing 25% (w/w) goat fat showed distinct crystal growth after 4 weeks among the admixtures. However, IMC studies did not reveal any change in recrystallization behaviour in this lipid matrix within 24 h. Analysis of the crystallization exotherms indicated that the lipid matrix containing 50% (w/w) goat fat showed unique crystallization kinetics and possessed the lowest Avrami exponent, while goat fat alone showed slight change within the first 45 min of isothermal crystallization. Physically structured lipid matrix containing 75% (w/w) goat fat possessed the lowest growth rate constant.

Thermal analysis of the crystallization and melting behavior of lipid matrices and lipid nanoparticles containing high amounts of lecithin

Lipid nanoparticles (LNP) based on triglycerides containing high amounts of the amphiphilic lipid lecithin have been proposed as a promising alternative drug delivery system with regard to drug loading capacity. Aim of the present study is to evaluate the influence of lecithin within the lipid matrix (LM) on the crystallization behavior by thermoanalysis and wide angle X-ray diffraction (WAXD). The crystallinity of LM and LNP is mainly determined by the triglyceride content. However, lecithin influences the crystallization behavior significantly. WAXD shows an accelerated polymorphic transition of the LM to the β-modification upon storage with increasing lecithin content. Both, the melting point and the crystallization temperature are not affected by the lecithin concentration and are comparable to recrystallized triglyceride bulk. However, the crystallinity indices (CI) of LM show a general decrease by 10% suggesting an incomplete crystallization. For the formation of LNP at least 10% lecithin is necessary and all systems are present in the stable β-modification. In comparison to the undispersed LM, the crystallization temperature of LNP is significantly decreased by about 20 °C whereas the melting point is reduced by about 5 °C only. Melting enthalpy is comparable to the untreated triglyceride bulk and elevated in comparison to the undispersed LM. Isothermal heat-conduction microcalorimetry (IMC) enables the determination of crystallization kinetics after fitting of the heat flow volume according to the Avrami equation.

Use of isothermal heat conduction microcalorimetry, X-ray diffraction, and optical microscopy for characterisation of crystals grown in steroid combination-containing transdermal drug delivery systems

The combined application of the steroids estradiol (E2) hemihydrate and norethindrone acetate (NEA) is desirable for hormone replacement therapy. Transdermal drug delivery systems (TDDS) enable a controlled delivery of these drugs to the skin. However, in order to attain high skin permeation rates the concentration of the dissolved drugs in the TDDSs has to be high. This often results in supersaturated systems with a high crystallisation tendency. The combination of NEA and E2-hemihydrate in the acrylic matrix of patches yields crystals that are different from single drug systems. A new crystal phase showing additional X-ray powder diffraction peaks and a new feather-like crystal shape appeared. The crystal formation was considerably accelerated and enhanced by increasing E2 contents in the patches. The new crystal phase seems to be kinetically favoured compared with crystals appearing from pure E2-hemihydrate or NEA. A crystallisation enthalpy of −7.9±0.95 kJ/mol in the matrix containing a 1:3 mixture of E2-hemihydrate and NEA was determined by isothermal microcalorimetry. The crystallisation rate increased with higher drug concentrations. In addition, the influence of patch pre-treatment at 80°C prior to storage on crystallisation was investigated. This treatment enabled a slight reduction of the crystallisation in the TDDSs. Microcalorimetry enabled the classification of various additives according to their influence on the crystallisation process.

Kinetic studies of the degradation of an aminopenicillin antibiotic (amoxicillin trihydrate) in aqueous solution using heat conduction microcalorimetry

Recent developments in isothermal microcalorimetry allow the direct determination of kinetic and thermodynamic parameters for slow reactions from studies conducted at appropriate temperatures and under designated environmental control. The degradation kinetics of amoxicillin trihydrate has been investigated as a function of pH (1-10) and temperature (303.15-318.15 K) at 0.5 M ionic strength using heat conduction microcalorimetry. Equations were developed incorporating calorimetric accessible data, rate constants and change in enthalpy, which showed that the degradation of amoxicillin trihydrate in aqueous solution followed pseudo-first-order kinetics under our experimental conditions. The enthalpy of degradation reaction was found to be exothermic in nature. The values of the rate constant k for individual steps were determined from the values of the overall rate constants at different pH. Energy of activation of overall reaction as a function of pH and for individual rate constants was determined. The log k-pH profiles indicated specific-acid and specific-base catalysis and there were inflection points near pH 3 and pH 7 corresponding to the pKa1 and pKa2 values. Quantitatively, there was good correlation between calorimetric determined half-life (t1/2) and the literature value in the acidic region determined by other methods at 310.15 K. The presence of a beta-lactam ring and of an alpha-amino group in the C-6 side chain played a critical role in the degradation of amoxicillin trihydrate and the zwitterionic form of the drug was found to be more stable.

Determination of the activation energies of and aggregate rates for exothermic physico-chemical changes in UHMWPE by isothermal heat-conduction microcalorimetry (IHCMC)

Exothermic heat flow rates ( Q=μW=μJ/s), as a function of elapsed time, were measured by isothermal heat-conduction microcalorimetry (IHCMC) in order to study the aggregate rate of physico-chemical change in specimens of unsterilized and sterilized ultra-high-molecular-weight polyethylene (UHMWPE). Standard protocols for performing the IHCMC tests were developed and are described. Use of the standard protocols yielded the desired results—data that were not significantly different among either replicate sets of unsterilized specimens or as a function of which calorimeter test well was used. Heat flow rates measured in air at 20°C, 25°C, 35°C, and 45°C yielded estimates of activation energies of 47, 11, and 41 kJ/mol for unsterilized, γ-radiation sterilized, and ethylene oxide gas (EtO) sterilized polymer, respectively. These results support the ideas that (a) initial exothermic degradation takes place much more easily in the radiation-sterilized material, due to direct oxidation of readily available free radicals, and (b) the much slower degradation process in EtO-sterilized UHMWPE is not appreciably different than in unsterilized polymer. Comparison with other activation energy data suggests that the rate-limiting process in EtO- or un-sterilized polymer is oxygen diffusion into the polymer. For shelf storage in air, for periods up to 8 months, the mean exothermic heat flow in air, at 25°C ( Q m) [determined from the Q values averaged over the time period between 15 and 20 h after test start], from UHMWPE γ-radiation sterilized in air was significantly higher than for unsterilized material (2.91±0.11 vs. 0.73±0.11 μW). The higher rate can be attributed to oxidation of radiation-induced free radicals in the polymer near its surface. For the γ-irradiated polymer, the decline in Q m with shelf storage time suggests that, eventually, degradation might become oxygen diffusion limited in this case also. However, in vivo, surface wear of an UHMWPE articular component may continue to expose unoxidized free radicals, keeping the exothermic reaction rate high and, possibly, continuing to produce an oxidized UHMWPE surface prone to wear.

Use of isothermal heat-conduction microcalorimetry (IHCMC) for the evaluation of synthetic biomaterials.

Isothermal heat-conduction microcalorimetry (IHCMC) allows measurement of extremely small rates of heat flow-on the order of 0.1 microwatt. This provides, for example, the ability to directly observe-and quantitate in a few days-rates of degradation as low as 1% per year at body temperature, in solid material samples of a few grams. Also, one method of IHCMC data analysis allows direct determination of the reaction-rate constant at the temperature of interest, thereby avoiding possible errors due to rate mechanism changes with temperature, an issue that needs to be considered when the Arrhenius method is used. IHCMC can also be used to measure transient phenomena, such as heat of adsorption, and initial metabolic responses of cellular entities to biomaterials. The purposes of this review article are to (a) explain the basic principles, attractive features, limitations, and methods of IHCMC; (b) describe biomaterials applications to date--including studies of the stability of ultra-high-molecular-weight polyethylene and implant-grade calcium sulfate, setting reactions of dental adhesives, and macrophage response to biomaterial particles; (c) provide a discussion of issues and concerns that should be addressed in order to maximize the utility of IHCMC in biomaterials studies; and (d) suggest a number of possible future biomaterials applications for this technique.

Application of heat conduction calorimetry to high explosives

This paper describes some thermal analysis experiments conducted on high explosive samples. These employ differential scanning calorimetry to monitor thermal effects at elevated temperatures (around 200 °C) and heat conduction calorimetry to record thermal effects at much lower temperatures (below 100 °C). The work shows that, due to the generally high thermal stability of many high explosive compositions, heat generation rates are very low, if detectable at all, at normal storage temperatures, even when using a very sensitive instrument. The sensitivity and reproducibility of this technique has been investigated in detail by Wilker et al. [S. Wilker, U. Ticmanis, G. Pantel, Detailed investigation of sensitivity and reproducibility of heat flow calorimetry, in: Proceedings of the 11th Symposium on Chemical Problems Connected with the Stability of Explosives, Sweden, 1998] and shown to be capable of recording heat generation rates of less than a microwatt. This allows continuous measurement of decomposition processes in nitrate ester based propellants at temperatures as low as 40 °C. However, the measurement of very low levels of heat generation is difficult, time consuming and therefore expensive. If the assumption is made that the life limiting process is invariably the slow decomposition of the energetic component, this will frequently lead to very long service lifetime predictions. A number of possible complications are identified. Firstly, due to its low detection threshold, a heat conduction calorimeter may detect other reactions which will not lead to failure, but which may still dominate the heat flow signal. Secondly, the true failure process may generate little energy and be overlooked. In view of these considerations, at present it seems unwise to rely on heat conduction microcalorimetry as the only tool for the assessment of the life of high explosive energetic systems. Based on examples of life terminating processes in high explosives during storage and use, it is clear that decomposition of the energetic material is not invariably the cause of system failure. It is also by no means the only reaction that may take place in, and be observed by, a heat conduction calorimeter.

Solid-state reactions from isothermal heat conduction microcalorimetry: Theoretical approach and evaluation via simulated data

Several recent publications from this laboratory have reported developments in the capacity to calculate thermodynamic and kinetic parameters, such as rate constant, enthalpy, order of reaction, from isothermal micro-calorimetric data. To date these developments have all been associated with the calculation of the desired parameters from solution phase reactions. This paper furthers these developments to a theoretical consideration of solid-state reactions and the calculation of the values for the rate coefficient, k, the fitting parameters m and n, the total number of joules released over the lifetime of reaction, Q, and hence either the specific enthalpy or the molar enthalpy of reaction, H.

Kinetics and thermodynamics of heat conduction microcalorimetry

Heat conduction microcalorimetry offers an important technique in the context of physical organic chemistry for the study of chemical reactions in solution. The thermodynamic basis of the technique is discussed. Model reaction schemes are used in an examination of the dependence on time of the rate of heat production. The impact of rate constants and enthalpies of reaction on the recorded dependence is reviewed for first- and second-order reactions. Copyright © 2002 John Wiley & Sons, Ltd.

Quantification of low levels (

During the processing of pharmaceutical solids (e.g. milling, spray drying, tablet compaction, wet granulation and lyophilisation), various degrees of disorder in the form of crystal defects and/or amorphous regions may be generated. Even relatively low levels of amorphous material (<10%) may have a detrimental impact on the stability, manufacturability and dissolution characteristics of the formulated drug product. In this paper an isothermal heat conduction microcalorimetry and dynamic vapour sorption technique have been evaluated for the quantification of low levels (<10%) of amorphous material within a crystalline active. Both techniques were able to detect a 0.5% amorphous content, and in each case the limit of detection may be further lowered by increasing the sample size. The impact of micronisation on the crystallinity of a batch of active was evaluated using the two methods. The isothermal microcalorimetry and dynamic vapour sorption data showed excellent agreement (±0.2% amorphous content) and indicated that the amount of amorphous material generated is extremely sensitive to small changes in the operating conditions of the microniser. The techniques described in this paper have been developed at a very early stage of the actives development program such that the impact of small quantities of amorphous material on the quality attributes of the formulation can be fully assessed. The methods can be applied to any active, the only criteria is that the amorphous material will recrystallise on exposure to moisture or solvent vapours, and no hydrates or solvates are formed.

The application of heat conduction microcalorimetry to study the metabolism and pharmaceutical modulation of cultured mammalian cells

The heat produced by animal cells in culture can be used as the primary indicator of the kinetics of their metabolism because the scalar flux of it is a function of the metabolic flux. The validity of the relationship between heat and metabolism was demonstrated theoretically through the concept of thermal advancement and in experiments by the use of continuous cultures. This validation permitted the application of heat flux as a probe of the metabolic state of cells in culture. It consisted of an on-line heat conduction microcalorimeter that measures the instantaneous heat flow and dividing the smoothed signal with one obtained simultaneously using a dielectric spectrometer that records the change in capacitance as an estimate of the amount of viable biomass. In this mini-review, it is shown with Chinese hamster ovary cells (CHO320) genetically engineered to produce interferon-γ (IFN-γ) that heat flux is an early signal of deteriorating metabolism in cultures that produce considerable amounts of toxic lactate under fully aerobic conditions. The early detection favours the use of heat flux as the control variable in fed-batch cultures. This is a particularly useful finding in the context of the pharmaceutical industry because it will help to ensure the high fidelity of the cytokines, antibodies and vaccines produced in large-scale cultures. The monotonic relationship between the fluxes for heat and metabolism means that the enthalpy balance method can be employed to test the validity of the growth reaction for cells in culture. This showed that the crucial ratio between the substrates, glucose and glutamine, in the culture medium was incorrect at 5.5:1 instead of about 3:1, depending on the phase of the culture. Together with other changes to the medium composition, an improved formulation was made that ensured faster cell growth and greater specific rate (flux) of IFN-γ constitutive secretion while decreasing glucose utilisation and, most importantly, halving the excretion of lactate, that is toxic to the cells and harmful to the fidelity of their secondary products. Indirect calorimetry (oxygen uptake rate, OUR) is often favoured over the direct technique, but the former only measures aerobic metabolism. The environmental conditions in cultures favours lactate production even under fully aerobic conditions. Developments in measuring OUR mean that the stationary liquid phase balance can be used successfully to make the calorimetric:respirometric (CR) ratio a valuable tool in optimising cell culture to grow cells that synthesise the maximum amounts of the high fidelity secondary products. Besides the value of heat flux in improving the cultures of animal cells producing heterologous products, three different techniques are examined that should be valuable in the testing the many compounds that are produced on a speculative basis as potential drugs. They are: (i) a thin-film thermopile transducer as an immunosensor; (ii) infra-red imaging of cells cultured in multi-well microtitre plates and (iii) integrated circuit (IC) calorimetry for small samples and low detection limit. One or more of these methods could well find favour with industry in the near future.

An outline of new calculation methods for the determination of both thermodynamic and kinetic parameters from isothermal heat conduction microcalorimetry

An outline of equations allowing calculation, from calorimetric data, of both thermodynamic and kinetic parameters for reactions which proceed to completion is given. In addition equilibrium constants are calculable for reactions which proceed to an equilibrium position. Advantages of the methods for solid state kinetic and stability studies are briefly discussed.

Calorimetric investigations of the lipase-catalysed hydrolysis of acylated acyclonucleosides

Thermal power accompanying the hydrolysis of mono- and bi-acylated acyclonucleosides:(R,S)-1-N-(1-O-acetyl-3-hydroxypropoxymethyl)-thymine (ThL),1-N-(1,3-di-O-acetylpropoxymethyl)-thymine (ThAc), and1-N-(1,3-di-O-acetylpropoxymethyl)-5-fluorouracil (FAc), catalysed by lipase from Candida Cylindracea (Rugosa) was investigated by isothermal heat conduction microcalorimetry. Changes of the thermal power in time and the total heat effects were determined in long-lasting measurements. Hydrolysis of these compounds was examined also by TLC.