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 × 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. Copyright © 2010 John Wiley & Sons, Ltd.