Simple HPLC Method for the Determination of Caspofungin in Human Plasma

Abstract

Antifungal caspofungin (CPFG) was approved for the treatment of febrile neutropenia (FN) as the empiric treatment. However, the relationship between pharmacokinetic properties of CPFG and its clinical effects in patients with FN has not been fully established yet. Thus, in the present study, we tried to establish the simple and quantitative HPLC method to measure CPFG in human plasma with liquid-liquid extraction. CPFG in human plasma was extracted by liquid-liquid extraction and was separated by 5C18 column with mobile phase containing 20 mM phosphate buffer (pH 2.5) and acetonitrile (65:35). CPFG and p-hydroxybenzoate ethyl ester, used as an internal standard (IS), were detected by a fluorescence detector (Ex: 224 nm, Em: 304 nm) and by UV-VIS (254 nm), respectively. CPFG and IS were detected with retention times of 17.0 and 9.5 min, respectively, which were separated from matrix compounds. The calibration curves were linear from 1.0 to 20 μg/mL (R2>0.99). The limit of detection, the limit of quantification and the lower limit of quantification were 0.53 μg/mL, 0.89 μg/mL and 1.0 μg/mL, respectively. The validation study revealed that the intra- and inter-day accuracy and precision were within the acceptable range and that CPFG was fairly stable after freezing and thawing, reconstitution, in autosampler and in stock solution at ambient temperature up to 8-24 h. These results suggest that our established method to measure CPFG in human plasma would be applicable to measure plasma CPFG in patients with FN in order to establish the evidence for appropriate drug therapy.