Study On The Determination Of Risedronate By High Performance Capillary Electrophoresis

Risedronate (Rsd), a novel, orally administered pyridinyl bisphosphonatescommonly used in the treatment of osteoporosis and Paget’s disease without seriousadverse reactions as it inhibits osteoclast-mediated bone resorption and modulatesbone metabolism. Oral bioavailability is approximately0.6%at the therapeutic doseleading to only low ng/ml levels presenting in blood and other biological fluids. Dueto the high polarity of Rsd, the development of a determination method for theanalysis of Rsd presented major difficulties. The main objective of the current workwas to develop a validated capillary zone electrophoresis (CZE) technique with UVdetection for the analysis of Rsd in bulk drug, tablets and rat plasma throughscreening of the electrophoresis conditions. There are three main parts in this thesis.Firstly, the effects towards the crucial factors such as the buffer type,concentration and pH, applied voltage and injection time were investigated. Initiallyseparation and determination was obtained utilizing15mM citric acid-disodiumhydrogen phosphate buffer at pH6.0as a background electrolyte with a directdetection at262nm on a40cm effective length, uncoated fused-silica capillarycolumn (50μm i.d.×360μm o.d.). Samples were injected electrokinetically at20kVfor8s and the capillary temperature was at25oC. An applied voltage of20kV(positive polarity) led to an analysis time less than5min. This analysis method israpid, simple and cost effective.In this paper, a CZE-UV method was established for the determination of Rsd inbulk drug and in tablets. The oxidative stressed samples, with a10%(v/v) H2O2, Rsddegraded rapidly and showed approximately40.5%of degradation in6h. The effectsof the pH and concentration of citric acid-disodium hydrogen phosphate buffer on theresolution (Rs) between Rsd and its degradation product were investigated for furtheroptimization of capillary electrophoresis conditions. The optimized method wasvalidated on the basis of the ICH requirements. The optimized method demonstratedgood performance concerning selectivity, robustness, linearity (r~2=0.9997),sensitivity (limit of detection:3μg/ml), accuracy (95.32~97.43%) and precision (<2.26%). The stability indicating capability of the method was established by theenforced degradation studies combined with the peak purity assessment. Wecalculated the response ratio of Rsd using peak areas integrated at both210and262 nm. As a good alternative to the HPLC method, this approach has been successfullyemployed for the determination of the commercially available Rsd tablets.This paper achieved a further study in development and validation of CZE-UVmethod for the determination of Rsd in rat plasma. In this work, sample pretreatmentmethod consisted of protein precipitation with trichloroacetic acid (TCA) andco-precipitation with calcium at alkaline pH. The method was linear at a concentrationrange of300~2500ng/ml. Endogenous components interfered slightly with theexamination of Rsd in the plasma during the retention time under the present CEconditions. The limits of detection and quantitation at signal-to-noise ratios of3and10were100and300ng/ml, respectively. Precision, expressed as relative standarddeviation (%RSD), was always lower than8.52%. All extraction recoveries for Rsdwere greater than87.95%. The results obtained analyzing plasma samples from Wisterrats1h after single doses administration of a50mg Rsd aqueous solution weresatisfactory in terms of precision and selectivity.