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

Abstract

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 μm, 150 mm × 4.60 mm) maintained at 30 °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 °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.