Development of Analytical Methods for Measurement of Drugs against Malaria in Plasma and Whole Blood

Abstract: The aim of this thesis was to develop analytical methods for measuring drugs in blood and/or plasma. Solid phase extraction was used for the enrichment and purification of the drugs from the sample matrix. Automatic extraction procedures using a solid phase extraction robot to reduce the workload of the analyst and to minimize variations in the extraction procedure were developed. To determine sample concentrations, liquid chromatography was used together with UV absorbance detection through out this work. Determination of Pyronaridine in whole blood utilised a weak cation exchanger to extract Pyronaridine from blood. From the beginning there were problems in separating Pyronaridine from the internal standard but by adding sodium perclorate as an ion-pairing agent, good separation was achieved. For the simultaneous quantification of the highly lipophilic Atovaquone and the strong basic drug Proguanil with metabolites, a novel mixed mode solid phase extraction column was used. It combines the properties of a carboxylic acid (CBA) column and a non-polar octyl-silica (C8) column to extract the compounds from plasma. Their different physiochemical properties also required a gradient separation on the liquid chromatography system. Stability is an important factor when developing new methods. A new approach was used to evaluate the stability of Amodiaquine in blood and plasma. This included the use of a stability marker, in this case Chloroquine, a stable compound which was added together with Amodiaquine when preparing the stability samples. When using the ratio of Amodiaquine and the stability marker, between-run variations and variations associated with the preparation of new stock solutions and calibration standards were eliminated.