Development and application of novel approaches to quantitate therapeutic drugs in human blood

The research described in this thesis utilised novel extraction phases and analytical approaches in order to develop new methods for the separation and determination of important pharmaceuticals in complex biological matrices. The project employed bioanalytical chemistry to solve real-world biological and clinical problems. The first project involved the development and validation of an analytical protocol for the extraction, separation and determination of rifampicin and efavirenz in HIV-positive patients. The protocol was developed to support the hypothesis that ‘concomitant treatment with rifampicin-containing anti-tuberculosis (TB) therapy and the anti- HIV therapy, efavirenz, results in a decreased rifampicin half life’. The emphasis in this work was the determination of both drugs simultaneously in one sample in a simple, rapid and cost effective assay. This was achieved using solid phase extraction and LC-UV analysis. The main challenge was developing a low cost assay to detect the drugs within a clinically relevant concentration range using standard chromatography equipment in order to make it applicable to resource-limited settings, in particular in areas where there are high incidences of HIV infections, such as sub-Saharan Africa, Eastern Europe and Asia. Successful development of a LC-UV method preceded by SPE obtained satisfactory results fit for this purpose and was employed to measure drug levels in HIV-positive patients. Using a Zorbax SB-Phenyl reverse-phase analytical column, good separation and detection of the drugs was attained within a 10 minute run time. Intra- and inter-assay precision RSD values were found to be less than 15% at the concentrations examined (0.1-20 μg/mL). The LOQ was found to be 0.1 μg/mL for each agent and the assay was found to generate a linear response up to 20 μg/mL. Drug levels were measurable and significant differences in circulating rifampicin levels were found among the patients. Having completed the above, a second analytical protocol was developed to support evaluation of an emerging hypothesis that ‘cutting a scopolamine transdermal patch in half will reduce the side effects associated with the medication while still having effective protection from motion sickness’. The validated assay utilised solid-phase extraction coupled to LC-MS to sensitively and accurately estimate the serum drug concentrations evident in subjects who participated in a pilot study undertaken as part of this project. The main challenge met here was developing a fast, isocratic method capable of determining the extremely low concentrations of scopolamine in serum post patch and half patch application. As the circulating level of scopolamine, especially when only half a patch is applied, is very low, enormous challenges had to be overcome to couple extraction and analysis methods and push them to their very limits to reliably and reproducibly measure the levels of drug encountered. This was accomplished by coupling SPE and LC-MS methods to detect the drug at pg/mL levels in blood. Using an Agilent Zorbax SB-Phenyl column, intra- and inter-assay precision RSD values were found to be less than 15% at the concentrations examined (10-200 pg/mL). The assay LOQ was 10 pg/mL with a linear response up to 200 pg/mL. Successful determination of scopolamine levels in circulating blood levels of patients administered the transdermal patch, both full and cut in half was achieved. It was found cutting the patch significantly impacted on the anticipated pharmacokinetics of the drug