DEVELOPMENT, VALIDATION, AND TRANSFER OF GENERIC ANALYTICAL METHODS FOR FIGHTING AGAINST COUNTERFEIT MEDICINES

This thesis is part of the fight against poor quality medicines and especially their spread in developing countries. Its main goal is to contribute to the protection of public health in Rwanda and neighboring countries given the danger that this type of medicines represents to the health of the population. This contribution focuses on the development of reliable and inexpensive analytical methods that can be used in Rwanda. The technique of high performance liquid chromatography has been selected driven by the availability of LC equipment at the local level.Two therapeutic classes have been selected, namely antiretrovirals and antimalarials. They are among the medicines whose pathologies are classified in the TOP 10 causes of death and disability in Rwanda, so very widely used and therefore potentially targeted by counterfeiters. Two approaches of methods development have been applied. A first more complex but rich in analytical information (retention times, UV-Vis spectra, light absorption and intensity of peaks, sensitivity to parameter variations, etc.) that required modeling and statistical processing based on the experimental designs whose results make possible to determine the design spaces in which the analytical parameters can vary without altering the quality of the expected results. Another simple approach was followed for the development of simple methods in isocratic mode from other methods that must be adjusted to obtain good results on the separation of the targeted analytes. This has been applied for antimalarials. One part of the methods was validated using the total error strategy that includes the accuracy profile as a decision tool for compliance.Moreover, in support of HPLC, other orthogonal analytical techniques were used to confirm the cases of counterfeit samples. These are mainly the Raman spectroscopy, mass spectroscopy, and nuclear magnetic resonance.Thus in the Article 1, a general screening method for simultaneous analysis of 15 ARVs and 4 major excipients was optimized on a C18 column; 100 x 4.6 mm, 3.5 μm (dp); a generic screening method for the analysis of ARVs in liquid dosage forms; and another method of analysis for solid dosage forms have been developed and optimized. Subsequently, a formulation of emtricitabine (FTC), tenofovir disoproxil fumarate (TDF) and efavirenz (EFV) tablets was selected and fully validated under the analytical conditions of the method for solid dosage forms. Thanks to these methods, it is easy to detect counterfeits involving one or more of the studied analytes, but also other types of non-compliance such as overdoses or underdosages can be detected.In the Article 2, two general screening methods for simultaneous analysis of 8 antimalarial medicines used in Rwanda and 4 major excipients were developed and optimized on a C18 column, 250 x 4.6 mm, 5 μm (dp) at the analysis times of 100 and 65 minutes per injection, and we preferred the shorter of the two methods (the one at 65 minutes) which was then geometrically transferred to the columns of 150 mm, 100 mm, and 50 mm of length in order to obtain shorter analysis times: 41.5 minutes on the 150 mm column, 23.3 minutes on the 100 mm column, and 11 minutes on the 50 mm long column.In the Article 3, it is about simple development of methods through the adaptation or adjustment of other methods on less complex samples containing few analytes. In addition to this approach, another strategy based on sequentially followed experiments according to the obtained results has been applied, and we have developed three simple and rapid isocratic methods for the analysis of antimalarial medicines in less than 10 minutes per injection, or even less than 5 minutes.The Articles 4 and 5 deal with the applications of already developed methods. The Article 4 is about an application of the method for analysis of artemether/lumefantrine in various generic forms commonly marketed in Benin, the Democratic Republic of Congo and Rwanda in tablets forms. The first part of analysis was done in Benin using conventional techniques based on thin layer chromatography, disintegration and uniformity of mass tests. Subsequently, a sample was suspected to be counterfeit due to lack of characteristic spots of both analytes and abnormal disintegration time of more than 3 hours without effect. This sample was analyzed with other artemether/lumefantrine tablets collected in the DRC and Rwanda, and HPLC test results revealed that the Beninese sample was a fake medicine containing neither artemether nor lumefantrine! This counterfeit has been confirmed by Raman spectroscopy. As for Article 5, we had a case of samples of quinine tablets whose HPLC results revealed the absence of quinine as the declared active ingredient, but the substitution with another compound which was later elucidated with advanced spectroscopic techniques namely the Raman, NMR and mass spectrometry. The substituting molecule was surprisingly the metronidazole !