Development and validation of a simple reversed-phase HPLC method for the determination of camptothecin in animal organs following administration in solid lipid nanoparticles

Abstract

A simple, sensitive and specific high-performance liquid chromatography (HPLC) assay for the quantification of camptothecin (CPT), a potent anticancer candidate, incorporated into solid lipid nanoparticles (SLN) in several rat organs (brain, heart, kidneys liver, lung, spleen) and serum was developed and validated. The sample pre-treatment involved organs homogenisation followed by CPT extraction. The samples were injected onto an analytical reversed-phase (RP) Mediterranea™ Sea18 column maintained at 30°C. The chromatographic separation was achieved by gradient elution consisting of triethyamine buffer pH 5.5 and acetonitrile at a flow rate of 1.2mL/min in 16min of run time and retention time of 9.8min (lactone). Fluorescence detection was used at the excitation and emission of 360 and 440nm, respectively. The calibration curves in the different organs, serum and in PB3 were linear (r2>0.9999) over CPT concentrations ranging from 1 to 200ng/mL or 0.5 to 200ng/mL (n=6), respectively. The method was shown to be specific, accurate (between 94.4±4.5% and 108.9±0.6%) and precise at the intra-day and inter-day levels as reflected by the coefficient of variation (CV<6.3%) at three different concentrations (10, 50 and 100ng/mL) in all matrices. Stability studies showed that CPT was stable in all matrices after 24h of incubation at room temperature (RT), after 24h in the autosampler or after three freeze/thaw cycles. The mean recoveries of CPT in suspension, loaded into SLN and in a physical mixture with SLN at three concentrations of 10, 50 and 200ng/mL were higher than 86.4%. The detection limit (DL) was ≤0.2ng/mL and the quantification limit (QL) was ≤0.5ng/mL. The method developed is reliable, precise and accurate and can be used in the determination of CPT amount in rat organ samples after i.v. administration of CPT in suspension, in physical mixture with SLN and incorporated in SLN.