Development and validation of a reverse-phase HPLC with fluorescence detector method for simultaneous determination of CZ48 and its active metabolite camptothecin in mouse plasma

摘要

A simple and sensitive high-performance liquid chromatography (HPLC) assay for the analysis of CZ48, a potent anticancer candidate, and its active metabolite camptothecin (CPT) in mouse plasma was developed and validated. CZ44 was used as an internal standard (IS). The samples were injected onto a C18 Synergi Polar-RP column (4 mu m, 150 mm x 4.60 mm) maintained at 30 degrees C. The identification of peaks showed high specificity. Shimadzu RF-10AXL fluorescence detector was used at the excitation and emission of 380 and 418 nm, respectively. The mean recoveries were 81.41 +/- 0.035%, 86.00 +/- 0.053% and 82.21 +/- 0.020% for CZ48 and 76.01 +/- 0.028%,77.04 +/- 0.042% and 85.93 +/- 0.023% for CPT at three concentrations of 10, 100 and 900 ng/ml, respectively. The calibration curve was linear (r(2) = 0.9999) over CZ48 and CPT concentrations ranging from 5 to 1000 ng/ml and 10-1000 ng/ml (n = 6), respectively. The method had an accuracy of >95% and intra- and inter-day precision (RE%) of <1.2% and <2.2% for CZ48 and CPT, respectively, at three different concentrations (10, 100 and 900 ng/ml). The lower limit of quantification (LLOQ) using 0.1 ml mouse plasma was 10 ng/ml for CZ48 and 5 ng/ml for CPT. Stability studies showed that CZ48 and CPT were stable in mouse plasma after 4 h incubation at room temperature or after 1 month storage at -80 degrees C with three freeze/thaw cycles. The method reported is simple, reliable, precise and accurate and confirmed by the determination of plasma samples in the mice after oral administration of CZ48. (D 2008 Elsevier B.V. All rights reserved.