In-tube solid-phase microextraction with molecularly imprinted polymer to determine interferon alpha 2a in plasma sample by high performance liquid chromatography.

A molecularly imprinted sol­gel polymer (MIP) based on protein (biopharmaceutical) template with a mild template removal condition using protease was synthetized and evaluated as stationary phase for in-tube solid phase microextraction (in-tube SPME) of the interferon alpha 2a from plasma samples,followed by high performance liquid chromatography analysis with fluorescence detection (HPLC-FD).The developed MIP exhibited high selectivity for the analyte in a complex matrix. The in-tube SPME variables such as draw/eject cycles, draw/eject volume, and desorption conditions were optimized to establish the equilibrium conditions in a short time. The MIP in-tube SPME/HPLC-FD method presented linear response over a dynamic range of 8­300 ng mL−1, with a correlation coefficient of 0.997. The inter-assay precision presented coefficient of variation lower than 9.2%, and accuracy values between 92% and 98%. The developed MIP performed as well as other selective interferon alpha 2a stationary phases (e.g.,immunosorbent and restricted access material), with the advantage that it is robust, easy to handle and cheap to synthesize, in a addition to requiring smaller sample volume (50 L). Based on the analytical validation results, the proposed method (MIP in-tube SPME/HPLC-FD) can be a useful tool to determine interferon alpha 2a in plasma samples from patients receiving therapeutic dosages.