An improved HPLC assay for S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721) in plasma

An HPLC assay is presented for the detection and quantitation of the radioprotective drug S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721, ethiofos) present in plasma. Improved selectivity and a 40-fold increase in sensitivity have been demonstrated over the method previously reported by this laboratory. Using precolumn derivatization with fluorescamine and S-3-(4-aminobutylamino)propyl phosphorothioate (WR-80855, a homolog of WR-2721) as the internal standard, drug levels of 0.05 to 1000 micrograms/mL were determined with excellent precision (CV less than or equal to 5% over the concentration range). An isocratic mobile phase of acetonitrile/ethanol/water (16:7:77) modified with 0.01 M tetrabutylammonium phosphate eluted the drug and the internal standard from the C-18 reverse-phase column in 23 minutes and 26 minutes, respectively. Detector response was linear over the entire range. The assay uses 150 microL of plasma and requires a total chromatography time of about 50 minutes. The method was found suitable for pharmacokinetic studies in a preliminary experiment with a beagle dog in which no interferences due to plasma constituents or drug metabolites were observed.