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.