Analysis of CP-122,288 at femto-molar levels from human plasma using solid-supported extraction and HPLC column switching

A sensitive and specific method for the analysis of CP-122,288 in plasma has been developed and validated using column-switching HPLC with fluorescence detection. Solid-phase extraction (SPE) cartridges (Chem-Elut) are used to extract analyte and internal standard from human plasma samples. The extracts are analysed by heart-cutting from one analytical HPLC column to a second column with fluorescence detection using excitation and emission wave-lengths of 225 and 350 nm respectively, after post column UV irradiation at 254 nm. The method has a lower limit of quantification (LLOQ) of 10 pgmL⁻¹ and is linear over a fifty-fold range, up to 500 pg mL⁻¹. An internal standard (CP-122,638) is used which is a close chemical analogue of the compound. This validated method has been used extensively for the analysis of CP-122,288 in plasma samples from clinical trials.