Fabrication of a molecularly imprinted monolithic column via the epitope approach for the selective capillary microextraction of neuropeptides in human plasma.

A novel molecularly imprinted monolithic (MIM) column was designed and fabricated using the epitope approach, and was used for the selective capillary microextraction (CME) of the neuropeptides neurotensin (NT) and neuromedin N (NmN). The MIMs were synthesized in a capillary by thermally initiated polymerization of the functional monomer (methacrylic acid (MAA)), in the presence of a dummy template (Pro-Tyr-Ile-Leu (PYIL)), a crosslinker and porogens. The resulting monoliths were characterized by scanning electron microscopy, pore size distribution measurement, and Fourier transform infrared spectroscopy. Different synthesis conditions of the MIM column were investigated. The parameters affecting the MIM-CME performance, including loading, washing and elution protocols, were optimized as well. The MIMs were used to enrich NT and NmN from human plasma prior to HPLC-UV analysis. The imprinted monolith showed an excellent maximum adsorption capacity of 245-711 mg mL-1 and selectivity (imprinting factor of 5.7-13.4) towards its target peptides. Low detection limits of 0.62 and 1.20 nM, and satisfactory recoveries (82.5-98.8%) were obtained for NT and NmN, respectively. The proposed MIM-CME/HPLC-UV method was found suitable to be used as an effective tool for the highly efficient and specific analysis of NT and NmN in human plasma samples.