High performance liquid chromatography with tandem laser induced fluorescence and mass spectrometry detection methods to monitor the metabolic profile of doxorubicin and its alteration with age

Abstract

In this thesis, a combination of high-performance liquid chromatography (HPLC), laser-induced fluorescence (LIF) and mass spectrometry (MS) is used to assess the metabolic profile of doxorubicin in vitro using the post-mitochondrial fraction (PMF) from the liver of Fischer 344 rats. Monitoring the metabolism of xenobiotics and drugs is important in the development, screening and assessment of new chemical compounds for use in therapies. This often requires the use of two or more techniques in order to collect the relevant data. This is the case for the analysis of metabolic products of the anticancer drug doxorubicin. An HPLC instrument with tandem LIF and MS detection was developed and then used to quantify and identify the metabolic products of doxorubicin in vitro. Using this instrumentation, the consumption of doxorubicin and the appearance of 7-deoxydoxorubicinone and 7-deoxydoxorubicinolone were monitored in rat liver post-mitochondrial fractions. This application demonstrates the potential of the tandem LIF-MS detection scheme in quantification and characterization of biotransformations of fluorescent xenobiotics of biomedical and environmental relevance. The HPLC-LIF-MS instrumentation and in vitro methods were then applied to investigate the changes in metabolism between young adult (10 months-old, 100% survival rate) and old (26 months-old, ~25% survival rate) Fischer 344 rats. Results suggest that with aging there is decrease in the rate of biotransformation of doxorubicin and that the timeframes needed to reach steady metabolite and doxorubicin levels are longer. On the other hand, the levels of metabolites and doxorubicin concentrations are not statistically different between the two age groups. In the future, the new methodologies presented here could be applied to investigate age-related changes in metabolism of drugs already in use, new compounds and xenobiotics with health relevance (e.g. pesticides or environmental pollutants).