Determination of atrasentan by high performance liquid chromatography with fluorescence detection in human plasma

Atrasentan is an endothelin antagonist selective for the ET(A) receptor in development at Abbott Laboratories for the treatment of cardiovascular disease and cell proliferation disorders. A simple and sensitive chromatographic method for the determination of atrasentan in human plasma has been developed and validated. The analytical method involves acidification of the plasma samples with 0.3 N HCl prior to extraction with 1:1 (v:v) hexane/tert-butylmethylether. The organic extract was evaporated to dryness, reconstituted with 20:80 (v:v) acetonitrile/0.05 M K(2)HPO(4) and washed with 75:25 (v:v) hexane/tert-butylmethylether. The organic layer was discarded and the aqueous layer was injected into the HPLC. Atrasentan and internal standard (ABT-790) were separated from interference using a 250 x 4.6 mm, 5 microm, 120 A Phenomenex Spherisorb C(8) analytical column with a 50 x 4.6 mm, Alltech Absorbosphere 5 microm CN guard cartridge using a mobile phase consisting of 25:15:5:55 (v:v:v:v) acetonitrile/isopropanol/methanol/0.05 M K(2)HPO(4), pH 7.0, at a flow rate of 1.0 mL/min. Fluorescence detection was achieved using lambda(ex) 278 nm and lambda(em) 322 nm. For a 1.0 mL plasma sample volume, the limit of quantitation was approximately 200 pg/mL. The method was linear from 0.2 to 1300 ng/mL (r(2) = 0.9986). Inter- and intra-day assay RSD (n = 6) were less than 10%. Mean accuracy determinations showed the quality control samples to range between 94 and 99% of the theoretical concentration.