VALIDATED GRADIENT STABILITY INDICATING RP-HPLC METHOD FOR THE SIMULTANEOUS QUANTIFICATION OF 11 RELATED SUBSTANCES IN THE COMBINED DOSAGE FORMS OF LAMIVUDINE AND TENOFOVIR DISOPEOXIL FUMARATE

Objective: Development of a stability-indicating reverse phase liquid chromatographic (RP-HPLC) method for the simultaneous quantification of 11 impurities in the combined dosage forms of lamivudine and tenofovir disoproxil fumarate drug substances.Methods: Efficient chromatographic separation of all analytes was achieved on a Waters X-terra RP18 column (150 x 4.6 mm, 3.5 mm) using mobile phase A (ammonium acetate buffer, pH adjusted to 5.0±0.05 with dilute orthophosphoric acid) and mobile phase B (mixture of methanol and ammonium acetate buffer in the ratio of 20:80) with the flow rate of 1.0 ml/min in gradient elution mode at 260 nm.Results: The method was validated in terms of the limit of detection, limit of quantification, linearity, accuracy, precision and robustness according to the international conference on harmonisation (ICH Q2R1). Regression analysis showed that the correlation coefficient (r2) is greater than 0.997 for individual active drug substances as well as their related substances. The method has proven very accurate (94.6 % to 108.2 % with % RSD not more than 4.9), highly precise (% RSD of the Intra-day and the inter-day study was not more than 8.9) and robust enough to deliver accurate results, when the chromatographic conditions were altered intentionally. Forced degradation studies were conducted in acidic, basic, thermal, photolytic, humid and peroxide stress conditions, where all the degradation peaks were monitored. Highest degradation of lamivudine was observed under oxidative stress condition and tenofovir was more susceptible to degradation under acidic and alkaline conditions.Conclusion: The present method is able to separate all the related compounds with each other and with the main drug substances with the resolution more than 2.0. The test solution was found to be stable in diluent up to 24 h. The mass balance of forced degradation of formulations, close to 99 %, made this method as a stability indicating method

A critical review on modelling formalisms and simulation tools in computational biosystems

Integration of different kinds of biological processes is an ultimate goal for whole-cell modelling. We briefly review modelling formalisms that have been used in Systems Biology and identify the criteria that must be addressed by an integrating framework capable of modelling, analysing and simulating different biological networks. Aware that no formalism can fit all purposes we realize Petri nets as a suitable model for Metabolic Engineering and take a deeper perspective on the role of this formalism as an integrating framework for regulatory and metabolic networks.Research supported by PhD grant SFRH/BD/35215/2007 from the Fundacao para a Ciencia e a Tecnologia (FCT) and the MIT-Portugal program

Modelling and Analysing Genetic Networks: From Boolean Networks to Petri Nets

Abstract. In order to understand complex genetic regulatory networks researchers require automated formal modelling techniques that provide appropriate analysis tools. In this paper we propose a new qualitative model for genetic regulatory networks based on Petri nets and detail a process for automatically constructing these models using logic mini-mization. We take as our starting point the Boolean network approach in which regulatory entities are viewed abstractly as binary switches. The idea is to extract terms representing a Boolean network using logic minimization and to then directly translate these terms into appropri-ate Petri net control structures. The resulting compact Petri net model addresses a number of shortcomings associated with Boolean networks and is particularly suited to analysis using the wide range of Petri net tools. We demonstrate our approach by presenting a detailed case study in which the genetic regulatory network underlying the nutritional stress response in Escherichia coli is modelled and analysed.