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 microm) in conjunction with a 4x4 mm LiChrospher 100, RP-18 guard column (5 microm). 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 microM for calphostin C in 50 microl of plasma. Intra- and inter-assay variabilities were less than 6% in plasma. The lowest detection limit of calphostin C in 50 microl plasma was 0.02 microM at a signal-to-noise ratio of approximately 3. The availability of this assay will now permit detailed pharmacodynamic and pharmacokinetic studies of calphostin C in vivo.
Chen
C L
CL
Department of Pharmaceutical Sciences, Hughes Institute, Roseville, MN 55113, USA.
Chen
H
H
Zhu
D M
DM
Uckun
F M
FM
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000097:blood* / Q000493:pharmacokinetics / Q000627:therapeutic use; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000097:blood / Q000493:pharmacokinetics / Q000627:therapeutic use; D005260:Female; D051379:Mice; D009281:Naphthalenes / Q000097:blood* / Q000493:pharmacokinetics / Q000627:therapeutic use; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013057:Spectrum Analysis
D000903:Antibiotics, Antineoplastic; D004791:Enzyme Inhibitors; D009281:Naphthalenes; D011493:Protein Kinase C; C058819:calphostin C