Reversed-phase high-performance liquid chromatography method for the simultaneous quantitation of the lactone and carboxylate forms of the novel natural product anticancer agent 10-hydroxycamptothecin in biological fluids and tissues.

Camptothecins are indole alkaloids isolated from a Chinese tree, Camptotheca acuminata, and have a wide spectrum of anticancer activity in vitro and in vivo. A novel camptothecin congener 10-hydroxycamptothecin (HCPT) has been shown to be more active and less toxic than camptothecin, and the lactone HCPT is believed to be responsible for its anticancer activity. In the present study, a reversed-phase high-performance liquid chromatography, (HPLC) with fluorescence detection was developed and validated for the simultaneous analysis of HCPT for lactone from (I) and carboxylate form (II) in plasma, urine and feces and tissues. Biological samples were prepared by a liquid-liquid extraction method using ice-cold methanol-acetonitrile (1:1, v/v). This method was shown to be reproducible and reliable, with intra- and inter-day variations being less than 7%, and accuracy being 94.3%-102.7%. The limits of determination were 2 ng/ml, 2 ng/ml, 2 ng/g, and 10 ng/ml for HCPT forms I and II in rat plasma, urine, feces, and tissues, respectively. The assay was liner over the range 2-2000 ng/ml (r = 0.999, P < 0.001) with recoveries of greater than 90% for plasma and urine and approximately 70-80% for feces and tissue homogenates through the extraction procedure. This analytic procedure has been successfully applied to a pharmacokinetic study of HCPT in experimental animals and should be useful in the future human studies.