An HPLC assay for the lipophilic camptothecin analog AR-67 carboxylate and lactone in human whole blood

AR-67 (7-t-butyldimethylsilyl-10-hydroxycamptothecin, DB-67) is a camptothecin analog currently in early stage clinical trials. The lactone moiety of camptothecins hydrolyzes readily in blood to yield the pharmacologically inactive carboxylate form. However the lactone form of third-generation lipophilic congeners, such as AR-67, is more stable, possibly due to partitioning into red cell membranes. This prompted us to develop a reverse-phase HPLC method with fluorescence detection (excitation 380 nm/emission 560 nm), which could quantitate the concentration of AR-67 lactone and carboxylate in whole blood. Samples were prepared by red cell lysis, protein precipitation with methanol and centrifugation to remove denatured materials. Recovery was estimated to be >85%. Analytes were eluted isocratically with 0.15 M ammonium acetate buffer containing 10 mM TBAP (pH 6.5) and acetonitrile (65:35, v/v) on a Nova-Pak C₁₈ column (4 μm; 3.9 x 150 mm). The assay was linear in the ranges 0.5-300 and 2.5-300 ng/mL for carboxylate and lactone, respectively. Accuracy and precision were acceptable. AR-67 forms were stable in whole blood and in methanolic supernatants. This assay has been successfully applied to measure AR-67 concentrations in whole blood of patients enrolled in a phase I study.