Abstract
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 the total of its lactone and carboxylate forms in human plasma. Linear response in analyte standard peak area were observed over the concentration range 0.05-10 ng/ml using 100-microliters plasma samples. The instability of the drug in the biological matrix necessitated that the plasma fraction was obtained within 5 min after blood sampling by centrifugation, immediately followed by protein precipitation with cold methanol (-30 degrees C). Stability studies have indicated that topotecan is stable in these methanolic extracts for at least 4.5 months at -30 degrees C and 2 months at -70 degrees C. For the total determination of the lactone plus lactone ring-opened forms of the drug as topotecan, plasma samples were deproteinated with methanol and, subsequently, acidified with 7% (v/v) perchloric acid. Plasma samples for the measurement of total levels of the lactone and the ring-opened forms of topotecan were stable for at least 4.5 months when stored at -30 degrees C. After centrifugation, the supernatants 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 (TEMED) in distilled water pH 6.0 (25:10:65, v/v) as the mobile phase. Detection was performed fluorimetrically. Within-run and between-run precision was always less than 12.1% in the concentration range of interest (0.05-10.0 ng/ml). The limit of quantitation is 0.05 ng/ml. Accuracy measurements ranged between 87.6 and 113.5%.