Determination of GDC-0449, a small-molecule inhibitor of the Hedgehog signaling pathway, in human plasma by solid phase extraction-liquid chromatographic-tandem mass spectrometry

J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Mar 15;878(9-10):785-90. doi: 10.1016/j.jchromb.2010.01.039. Epub 2010 Feb 1.


To support clinical development, a solid phase extraction (SPE) liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method for the determination of GDC-0449 concentrations in human plasma has been developed and validated. Samples (200 microl) were extracted using an Oasis MCX 10 mg 96-well SPE plate and the resulting extracts were analyzed using reverse-phase chromatography coupled with a turbo-ionspray interface. The method was validated over calibration curve range 5-5000 ng/mL. Quadratic regression and 1/x(2) weighing were used. Within-run relative standard deviation (%RSD) was within 10.1% and accuracy ranged from 88.6% to 109.0% of nominal. Between-run %RSD was within 8.6% and accuracy ranged from 92.4% to 105.3% of nominal. Extraction recovery of GDC-0449 was between 88.3% and 91.2% as assessed using quality control sample concentrations. GDC-0449 was stable in plasma for 315 days when stored at -70 degrees C and stable in reconstituted sample extracts for 117 h when stored at room temperature. Quantitative matrix effect/ion suppression experiment was performed and no significant matrix ion suppression was observed. This assay allows for the determination of GDC-0449 plasma concentrations over a sufficient time period to determine pharmacokinetic parameters at relevant clinical doses.

Publication types

  • Evaluation Study

MeSH terms

  • Anilides / blood*
  • Anilides / pharmacokinetics
  • Chromatography, Liquid / methods*
  • Hedgehog Proteins / antagonists & inhibitors*
  • Hedgehog Proteins / metabolism*
  • Humans
  • Pyridines / blood*
  • Pyridines / pharmacokinetics
  • Signal Transduction*
  • Solid Phase Extraction / methods*
  • Tandem Mass Spectrometry / methods*


  • Anilides
  • Hedgehog Proteins
  • HhAntag691
  • Pyridines