Quantitative high-performance liquid chromatography-based detection method for calphostin C,a naturally occurring perylenequinone with potent antileukemic activity

Calphostin C is a potent inhibitor of protein kinase C and can induce Ca²⁺-dependent apoptosis in human ALL cells. Further development of calphostin C will require detailed pharmacodynamic studies in preclinical animal models. Therefore, we established a sensitive and accurate high-performance liquid chromatography (HPLC)-based quantitative detection method for the measurement of calphostin C levels in plasma. Extraction of calphostin C from plasma was performed by precipitation of plasma protein using acetonitrile and an aliquot of extracted supernatant was injected onto a Hewlett-Packard HPLC system constituting a 250×4 mm LiChrospher 100, RP-18 (5 μm) in conjunction with a 4×4 mm LiChrospher 100, RP-18 guard column (5 μm). The eluted compounds were detected by diode array detection set at a wavelength of 479 nm. Acetonitrile–water containing 0.1% trifluoroacetic acid and 0.1% triethylamine (70:30, v/v) was used as the mobile phase. The average extraction recovery from plasma was 97.3%. Good linearity (r>0.999) was observed throughout the concentration range of 0.05–40 μM for calphostin C in 50 μl of plasma. Intra- and inter-assay variabilities were less than 6% in plasma. The lowest detection limit of calphostin C in 50 μl plasma was 0.02 μM at a signal-to-noise ratio of ∼3. The availability of this assay will now permit detailed pharmacodynamic and pharmacokinetic studies of calphostin C in vivo.