A simple, specific, and sensitive high-performance liquid chromatographic (HPLC) assay utilizing ultraviolet (UV) detection for the determination of
bisnafide in human plasma was developed, validated, and applied to plasma samples from patients undergoing
cancer therapy. Plasma samples, containing an internal standard, XE842, were first deproteinized with 2.0 ml
acetonitrile, and subsequently, 1.0 ml and pH 9
boric acid-
potassium chloride-
sodium hydroxide buffer (0.1 M) was added. To this mixture, 9.0 ml of
ethyl ether was added then vortex mixed. Following centrifugation, the
ether layer was back-extracted into 250 microliters of 0.1 M
phosphoric acid, then removed by vacuum aspiration. A portion of the remaining
acid layer was directly injected onto the HPLC.
Bisnafide was quantified using a Shiseido
Capcell Pak C8 HPLC column and ultraviolet detection (274 nm). The lower limit of quantification was 10 ng ml-1 using 1.0 ml plasma. The intraday precision (RSD) ranged from 2.7 to 8.6% over a concentration range of 10-1000 ng ml-1. The interday precision (RSD) ranged from 5.6 to 11.5%. Overall mean accuracy was +/- 5.2%. The
drug was stable in frozen heparinized human plasma stored at -20 degrees C for at least 1 year and stable throughout at least two freeze-thaw cycles. This method was successfully utilized for quantifying plasma concentrations needed to study the clinical pharmacokinetics of
bisnafide in patients undergoing
cancer therapy.