Development and validation of a RP-HPLC method with fluorescence detection for simultaneous determination of 10-methoxycamptothecin and its metabolite 10-hydroxycamptothecin in rat plasma.

Both 10-methoxycamptothecin (MCPT) and 10-hydroxycamptothecin (HCPT) are the natural bioactive derivatives of camptothecin (CPT) isolated from Camptotheca acuminata, and have been confirmed to possess high anti-cancer properties. In the present study, HCPT was identified as the major metabolite of MCPT in rat plasma through HPLC/photodiode array detection (PDA) and LC-MS/MS analysis. A sensitive and reliable RP-HPLC method with fluorescence detection was developed and validated for the simultaneous analysis of MCPT and HCPT in rat plasma. The parental CPT was used as an internal standard (IS). A piecewise linear function was used over lower and higher concentrations, respectively. The calibration curves were linear (r² > 0.999) over concentrations from 1.25 to 20 ng/mL and 20 to 320 ng/mL for both MCPT and HCPT. The method had an accuracy of 92.24-113.90%, and the intra- and inter-day precision (RSD%) were 10.05% or less for MCPT and HCPT. The stability data showed no significant degradation occurred under the experimental conditions. The mean recoveries at concentrations of 2.5, 40 and 160 ng/mL were 95.09±3.94%, 98.67±1.40% and 95.65±2.15% for MCPT and 84.06±4.39%, 84.85±3.10% and 81.03±4.44% for HCPT, respectively. The lower limit of quantification (LLOQ) using 0.1 mL of plasma was 1.25 ng/mL for both MCPT and HCPT. This method was successfully applied to the pharmacokinetic study of MCPT and its metabolite HCPT in rat plasma after intravenous administration.