Stability of new potential ACE inhibitor in the aqueous solutions of different pH.

Angiotensin-converting enzyme (ACE) inhibitors are a group of active substances binding to an active site of ACE. Many authors who studied the structure activity relationship suggested the structural elements needed for a potent ACE inhibitor. While many authors studied the activity of ACE inhibitor substances only a few structure stability studies have been presented. In this paper the stability properties of molecule xPRIL were studied by determination of degradation path and rate of degradation in aqueous solutions with different pH (2.0, 6.8 and 12.0) and temperatures (40, 60 and 80 degrees C). The degradation of molecule through two main degradation paths was identified and confirmed by liquid chromatography and mass spectroscopy (LC-MS). Stability properties of xPRIL were determined in a stability study evaluated by high-performance liquid chromatography (HPLC). The first order kinetics of degradation reaction of xPRIL and Arrhenius equations for each pH were determined at observed conditions. xPRIL showed the highest stability at pH 2 solution. The degradation kinetics of xPRIL was compared to the degradation kinetics of enalapril maleate (EM) and perindopril (PER) in bio relevant solutions with pH 2.0 and 6.8. In addition to the stability study of xPRIL the forced degradation study of all three molecules at rigorous conditions was conducted. From the obtained results the structural element having the highest influence on stability properties of the studied molecules was identified. The fragmentation paths of xPRIL, its cyclization degradation product and its hydrolysis degradation product were identified and confirmed by MS/MS method.

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.

Use of microcalorimetry in determination of stability of enalapril maleate and enalapril maleate tablet formulations.

The stability properties of enalapril maleate (EM) and of different tablet formulations including EM were studied by isothermal microcalorimetry and by high performance liquid chromatography (HPLC). It was shown that water content of the sample and elevated temperature have a high impact on stability properties of the substance itself and of the formulations including this substance. The degradation is more extensive at higher water content and at elevated temperature. The type of the tablet formulation (5 or 20mg EM tablet formulation) also has an impact: the 5 EM tablet formulation is the less stable one. The heat output of individual tablet formulations was used to evaluate the enthalpy changes and to calculate the difference in the amount of degraded EM between various samples. These results agreed satisfactorily with those obtained by HPLC. Isothermal microcalorimetry proved to be a fast and predictive method that could be used in preformulation studies to accelerate the pharmaceutical development and shorten the time before launching the product to the market.

Determination of main low molecular weight antioxidants in urinary bladder wall using HPLC with electrochemical detector.

The aim of the present work was to develop validated HPLC method using electrochemical detector for simultaneous detection of low molecular weight antioxidants (LMWA) in urinary bladder. Furthermore, the method was applied to study the distribution of LMWA in urinary bladder wall. The ascorbic acid (AA), glutathione in reduced (GSH) and oxidized (GSSG) form and uric acid (UA) were resolved by isocratic elution from C18 reversed-phase column. The bladder tissue sample preparation involved extraction with meta-phosphoric acid solution for LMWA stabilization. The AA, GSH and UA tissue peak was identified by different approaches. The obtained method validation parameters were in acceptable range: intra-day precision (<4.4%), intra-day accuracy (<8.4%), inter-day precision (<9.4%) and inter-day accuracy (<15.6%). Additionally, the method provided good linearity (r2>0.99) and recoveries (98.9-112.6%). The distribution of LMWA in urinary bladder was determined by measuring their concentration in bladder wall layers: urothelium, lamina propria, muscularis and serosa. The validated method was able to quantify the reduced form of all three LMWA in all four bladder wall layers. The LMWA concentrations were decreasing from urothelium to serosa except of UA. The developed HPLC method with electrochemical detection of LMWA is simple, fast and can be used for simultaneous quantification of LMWA in tissues, which contain low concentrations of antioxidants.

Stability of ascorbyl palmitate in topical microemulsions.

Ascorbyl palmitate and sodium ascorbyl phosphate are derivatives of ascorbic acid, which differ in stability and hydro-lipophilic properties. They are widely used in cosmetic and pharmaceutical preparations. In the present work the stability of both derivatives was studied in microemulsions for topical use as carrier systems. The microemulsions were of both o/w and w/o types and composed of the same ingredients. The stability of the less stable derivative ascorbyl palmitate was tested under different conditions to evaluate the influence of initial concentration, location in microemulsion, dissolved oxygen and storage conditions. High concentrations of ascorbyl palmitate reduced the extent of its degradation. The location of ascorbyl palmitate in the microemulsion and oxygen dissolved in the system together significantly influence the stability of the compound. Light accelerated the degradation of ascorbyl palmitate. In contrast, sodium ascorbyl phosphate was stable in both types of microemulsions. Sodium ascorbyl phosphate is shown to be convenient as an active ingredient in topical preparations. In the case of ascorbyl palmitate, long-term stability in selected microemulsions was not adequate. To formulate an optimal carrier system for this ingredient other factors influencing the stability have to be considered.

Optimization of HPLC method for stability testing of bacitracin.

A stability indicating HPLC assay for bacitracin has been developed and validated. The assay is based on a gradient elution, reversed phase column and UV diode array detection. On the basis of our previous analytical work several additional systematic HPLC tests for optimization of analytical method were performed. In order to achieve the highest selectivity of HPLC method, tests were conducted with extremely complex samples -- zinc bacitracin feed grade as food additive for animals. The influence of pH of mobile phase and type of columns on chromatographic separation of active (A, B(1) and B(2)) and inactive (F) polypeptide components of bacitracin were investigated in detail. It was found also that the peak B(1) comprises three and the peak F two subunits -- probably isomers. The obtained analytical procedure proved to be very selective and effective for the simultaneous determination of active polypeptide A, B(1) and B(2), impurities, known and unknown degradation products and ballast material.

Are calculated log P values for some guanine derivatives by different computer programs reliable?

The log P values of n-octanol/water for some guanine derivatives, acyclovir, deoxyaciclovir and their acetyl congeners, were calculated by some commercially available computer programs for log P calculation. These values were compared with those obtained by the conventional shake-flask method. It was established that the calculations of log P values for examined guanine derivatives by these computation programs do not give reliable results.

Bioavailability of piroxicam: oral and rectal multiple application in humans.

The aim of the work is to evaluate the bioequivalence of piroxicam administered orally and rectally in 20 mg dose every 24 hours. The corresponding "in vivo" study was undertaken and plasma samples were collected during the ninth dosing interval. HPLC method was used for piroxicam plasma concentrations determination. AUC and C were calculated and the obtained data were statistically analyzed. Analog-hybrid simulation was used to confirm additionally the similarity between the discussed formulations. No significant differences were observed using paired t-test and two-way analysis of variance while the methods of Hauck and Westlake, looking strictly, gave nonbioequivalence. Simulated response of one compartment model is suitable for "in vivo" data in both cases. Measured and simulated average steady state concentrations are equal and in complete accordance with those given in literature. Finally it can be concluded that oral and rectal application are bioequivalent in the sense of expected clinical effects.