High-performance liquid chromatographic assay for etoposide in human plasma

A sensitive, selective reversed phase-high performance liquid chromatographic (HPLC) assay that uses UV detection has been developed for etoposide (4'-demethylepipodophyllotoxin 9-[4,6-O-(R)-ethylidene-beta-D-glucopyranoside) 1. Parent drug is separated from its known metabolites, the cis-picrolactone 2, the hydroxy derivative 3, and the aglycone 4. After the addition of the internal standard (teniposide, 5) to 0.5 mL of plasma, 3 mL of chloroform is added and the sample is centrifuged. The lower organic layer is removed, evaporated in a stream of nitrogen, and reconstituted with 500 microL of mobile phase prior to injection. A phenyl mu Bondapak column (30 cm X 4 mm) and a mobile phase, consisting of 10 microM ammonium acetate (pH 5.5) in methanol:water:acetonitrile (50:45:5), were used to separate the compounds. The flow rate was 2 mL/min. Detection was achieved with a UV monitor set at 230 nm. The aglycone 4, etoposide (1), cis-picrolactone 2, and teniposide (5) had retention times of 3, 4, 4.8, and 9 min, respectively. The extraction efficiency of etoposide ranged from 88 to 94% with a coefficient of variation of 12% at 0.8 micrograms/mL and 4% at 28 micrograms/mL. This assay has an intraday and interday coefficient of variation of 6%, quantitates etoposide at concentrations as low as 0.4 micrograms/mL, and separates etoposide from its known metabolites. The procedure described represents an alternative to a previously published assay that also separated etoposide from its metabolites, but used electrochemical detection.