High‐throughput determination of atrasentan in human plasma by high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Atrasentan (A‐147627) is an endothelin antagonist receptor being developed at Abbott Laboratories for the treatment of prostate cancer. A quick and sensitive method for the determination of atrasentan in human plasma has been developed and validated using high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. A dual‐column, single mass spectrometer system is used to provide a reliable and routine means to increase sample throughput. The analytical method involves liquid–liquid extraction and internal standard (A‐166790). The plasma samples and internal standard are acidified with 0.3 m hydrochloric acid prior to being extracted into 1:1 (v[sol ]v) hexanes–methyl t ‐butyl ether. The organic extract was evaporated to dryness using heated nitrogen stream and reconstituted with mobile phase. Atrasentan and internal standard were separated with no interference in a Zorbax SB‐C 18 analytical column with 2.1 × 50 mm, 5 µm, and a Zorbax C 8 guard column using a mobile phase consisting of 50:50 (v:v) acetonitrile–0.05 m ammonium acetate, pH 4.5, at a flow rate of 0.30 mL[sol ]min to provide 4 min chromatograms. For a 250 µL plasma sample volume, the limit of quantitation was approximately 0.3 ng[sol ]mL. The calibration was linear from 0.30 to 98.0 ng[sol ]mL ( r 2 > 0.995). A significant advantage of the method is the ability to employ parallel HPLC separations with detection by a single MS[sol ]MS system to provide sensitivity and selectivity sufficient to achieve robust analytical results with a lower limit of quantitation of 0.30 ng[sol ]mL and high throughput. Copyright © 2005 John Wiley & Sons, Ltd.