Determination of LY295501 in human plasma by reverse phase HPLC with UV detection

A high performance liquid chromatographic (HPLC) assay was developed and validated for the quantitative determination of LY295501 in human plasma. A structural analog, LY186641, was selected as the internal standard. The samples were processed by protein precipitation with acetonitrile followed by concentration of the supernatants and reconstitution. Chromatographic resolution of LY295501 from endogenous plasma components was accomplished with a Waters Novapak C18 HPLC column (3.9 x 150 mm, d(p) 4 mm). Detection was by absorbance at 260 nm. The linear dynamic range was from 5 to 400 microg ml(-1) of human plasma using a 0.25 ml aliquot. The inter-day precision (%RSD) and accuracy (%RE) in plasma ranged from 2.4 to 4.7, and -4.9 to 1.4, respectively. This assay is both simple and rapid, and has been used to successfully analyze over 1500 samples from human clinical trials.