Date of Award

1984

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Pharmaceutical Sciences

First Advisor

Bruce K. Birmingham

Abstract

Studies of doxorubicin pharmacokinetics have been bedeviled by the problems 0£ low assay specificity and the possible degradation of doxorubicin and its metabolites during extraction. The purpose of this investigation was to provide a sensitive, selective, efficient and reproducible assay method for doxorubicin and its metabolites. A C-18 reversed phase HPLC method was chosen to analyze the drug concentrations and the Sep-pak cartridges were used for sample preparation. The Sep-pak cartridge retained doxorubicin and its metabolites while interfering compounds (e.g., protein, cellular components) in the biological samples were eluted. Doxorubicin and its metabolites were then eluted with an acid-methanol mixture and concentrated in a water bath of 40° c. While plasma samples required no prior treatment before extraction, tissue samples were homogenized and released from binding to nuclear components by silver nitrate. lhe superiority of the Sep-pak method in sample preparation was established by comparing the ease of the efficiency, accuracy, processing time and operation with the conventional organic extraction method. The application of the assay method was tested in the plasma samples of human and rats, and tissue samples from rats. The results all showed a small variation and good agreement with the literature data. Pharmacokinetic profiles of these plasma samples were analyzed by AUTOAN and showed good correlation with those of literature and with each other. Plasma and kidney samples of the very young (2 months old) and the very old (2 years old) rats were analyzed but failed to observe any significant effect of age on doxorubicin pharmacokinetics. During the development of the assay method, it was necessary to perform a study of doxorubicin stability to ascertain the best conditions for drug analysis. Doxorubicin showed to be more stable in acidic medium and the effects of pH have been quantified. Its stability in solution could also be influenced by the buffering agents used. The study of doxorubicin stability in plasma revealed that frozen plasma samples remained stable for 1 month and the thawing/freezing of these samples should be avoided. Binding data obtained from the ultrafiltration method were unable to analyze due to high degree of binding to the Diaflo membranes. This membrane binding property of doxorubicin not only caused an inconsistency among repeated experiments but also failed to provide an observation of the fraction bound However, an ultracentrifugation method was performed and revealed that 0.7 fraction of doxorubicin was bound to 4 % albumin solution. This study clearly demonstrated that the coupling of Sep-pak method and the reversed phase HPLC system provided an efficient, sensitive, reproducible and accurate method for the pharmacokinetic studies of doxorubicin. This new method was also much easier to use than the organic extraction method. The stability studies indicated the suitable storage conditions for both plasma samples and doxorubicin solution during analysis. The binding data of 0.7 fraction bound of total doxorubicin was provided for future pharmacokinetic studies.

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