Labeling analysis of three neuropeptides（Met-enkephalin, Leu-enkephalin, and neurotensin） with 7-methoxycoumarin-3-carboxylic acid N-succinimidyl ester（MCSE） was investigated. The experimental conditions for labeling reaction and HPLC analysis of the neuropeptide derivatives were optimized. It was found that the labeling reatction could be achieved at 37 ℃ for one hour in H3BO3-NaCl-Na2B4O7 buffer（pH=8.3） with a labeling ratio of about 80%. The derivative mixture from the three peptides can be separated in a 40 min linear gradient from 60% to 20% of solvent A（100 mmol/L sodium acetate, pH=5.0） and from 40% to 80% of solvent B[60%（volume fraction） acetonitrile in water]. The resulting labeled products were also confirmed by MALDI-TOF-MS analysis. Compared with the native neuropeptides, the resulting labeled products have a longer absorption and fluorescence wavelengths, which is beneficial to the effective elimination of background fluorescence interference from biological matrix.
A monolithic molecularly imprinted polymer(monolithic MIP) for trimethoprim(TMP) was prepared by in situ polymerization method as the HPLC stationary phase. Methacrylic acid(MAA) and ethylene glycol dimethacrylate(EDMA) were used as functional monomer and cross-linker, respectively. The obtained MIP monoliths were characterized by high performance liquid chromatography and scanning electron microscope. The SEM analysis result clearly shows that many macropores and flow-through pores were embedded in the network skeleton of the TMP-imprinted monolith, which allowed the mobile phase to flow through the monolithic MIPs with a very low flow resistance. When the flow rate was high to 9.0 mL/min, the backpressure was only 11.1 MPa. Moreover, the imprinting factor was up to 10.3, which demonstrates that the obtained monolithic MIP showed a strong affinity and selectivity to the template molecule.
