HPLC-UV method for quantifying etoposide in plasma and tumor interstitial fluid by microdialysis: application to pharmacokinetic studies.

A simple and sensitive bioanalytical method was developed and validated for determination of etoposide in plasma and microdialysis samples of Walker-256 tumor-bearing rats. A microdialysis probe was implanted in the center of a subcutaneous tumor and Ringer's solution was used as perfusion medium. Chromatographic separation was conducted on a Shimadzu CLC-C8 column using a mobile phase consisting of water-acetonitrile (70:30; v/v) adjusted to pH 4.0 ± 0.1 with formic acid at a gradient flow rate of 1.0-0.6 mL/min, an injection volume of 30 μL and UV detection at 210 nm. Microdialysate samples were analyzed without processing and plasma samples (100 μL) were spiked with phenytoin as internal standard (IS) (1 µg/mL) followed by extraction with tert-butyl methyl ether. The organic layer was evaporated and reconstituted with 100 μL of mobile phase before injection. The methods for plasma and microdialysate were linear in the ranges of 25-10,000 ng/mL and of 10-1500 ng/mL, respectively. All the validation parameters such as intra- and inter-day precision and accuracy and stability were within the limits established by international guidelines. The present method was successfully applied in the investigation of etoposide pharmacokinetics in rat plasma and microdialysate tumor samples following a single 15 mg/kg intravenous dose.