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 250X4 mm LiChrospher 100, RP-18 (5 pm) in conjunction with a 4X4 mm LiChrospher 100, RP-18 guard column (5 mu m). 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 mu M for calphostin C in 50 mu l of plasma. Intra- and inter-assay variabilities were less than 6% in plasma. The lowest detection limit of calphostin C in 50 mu l plasma was 0.02 mu M at a signal-to-noise ratio of similar to 3. The availability of this assay will now permit detailed pharmacodynamic and pharmacokinetic studies of calphostin C in vivo. (C) 1999 Elsevier Science B.V. All rights reserved.