Development and validation of a high-throughput HPLC-MS/MS method for the simultaneous determination of exatecan and its cathepsin B-sensitive prodrug in rat plasma†

Abstract

Exatecan is a potent topoisomerase I inhibitor. However, its clinical application is limited by its high toxicity and poor tumor targeting. In this study, a cathepsin B-sensitive fatty acid prodrug (C12-VC-PAB-Exa) with high albumin affinity was introduced and prepared into albumin nanoparticles with the aim of improving the safety of exatecan in blood circulation and accumulation at tumor sites. To assess the pharmacokinetic behavior of exatecan and its prodrug, a reliable, selective, and high-throughput HPLC-MS/MS method for the simultaneous determination of exatecan and C12-VC-PAB-Exa in rat plasma was established and fully validated. The samples were prepared using one-step protein precipitation, and then separated on a ZORBAX SB-C18 column (2.1 × 50 mm, 3.5 μm) using the gradient elution procedure, and detected using a triple–quadrupole tandem mass spectrometer with a positive electrospray ionization interface in multiple reaction monitoring mode. Exatecan and C12-VC-PAB-Exa have excellent linear correlations over the concentration range of 0.5–2000 ng mL⁻¹ and 5–20 000 ng mL⁻¹ (r > 0.995), with lower limits of quantification of 0.5 ng mL⁻¹ and 5 ng mL⁻¹, respectively. The intra- and inter-day precision and accuracy were within ±15%. The validated method was successfully applied to the pharmacokinetic study of exatecan solution and C12-VC-PAB-Exa nanoparticles in Sprague-Dawley rats for the first time. The results of the pharmacokinetics demonstrated that the nano-delivery system of the prodrug is a promising strategy to improve the physicochemical properties of exatecan. The analytical method could provide technical support for the subsequent in vivo studies of exatecan and its derivatives.