A sensitive high performance liquid chromatographic (HPLC) assay has been developed and validated for the quantitation of the novel anticancer agent topotecan and topotecan as its lactone plus carboxylate forms in rat and dog plasma. Linear responses in analyte standard peak areas were observed over the concentration ranges 0.10-10 ng ml-1 using 100 microliters of rat plasma and 0.2-100 ng ml-1 using 100 microliters of dog plasma. Due to the instability of the drug in the biological matrix it was necessary to obtain the plasma fraction within 5 min after blood sampling by centrifugation, immediately followed by protein precipitation with cold methanol (-30 degrees C). For the determination of total drug levels (lactone plus lactone ring-opened form), plasma samples were deproteinated with methanol and subsequently acidified with 2% (v/v) perchloric acid. The samples were analysed by HPLC using a Zorbax SB-C18 Stable Bond column and methanol-0.1 M hexane-1-sulfonic acid in methanol-0.01 M N,N,N'N'-tetramethylethylenediamine in distilled water pH 6.0 (25:10:65, v/v/v) as the mobile phase. The detection was performed fluorimetrically. The analytical column was thermostated at 19-21 degrees C to obtain baseline resolution between an interfering endogenous compound in rat and dog plasma and topotecan. This endogenous peak was absent in human plasma. Variation of chromatography temperature appeared to be a very useful tool in the bioanalysis of topotecan. It allowed optimization of the separation between the endogenous compound and the analyte; different mechanisms of solute interactions are apparently involved in this reversed-phase ion-pair chromatographic system.
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