Abstract:

A method for the determination of four alkaloids (phellodendrine, jatrorrhizine, palmatine, berberine) in cell culture fluids was developed by pipette tip solid phase microextraction (SPME) and high performance liquid chromatography (HPLC). A weak cation exchange monolithic stationary phase was prepared by in-situ polymerization in a pipette tip. Methacrylate acid, ethylene dimethacrylate and azodiisobutyronitrile were the functional monomer, the cross-linker and the initiator, respectively. The samples were cleaned-up using the microextraction tips. Phellodendrine, jatrorrhizine, palmatine and berberine were extracted from cell culture fluids. The analytes were separated on a reversed-phase Diamonsil C18 column (150 mm×4.6 mm, 5 μm) and eluted gradiently with acetonitrile and 0.05 mol/L potassium dihydrogen phosphate solution. The detection wavelength was 270 nm. The extraction performance was investigated by varying the elution solution types, the extraction and elution time and the molar ratios among monomers, cross-linkers and porogens. The analytes were quantified using external standard method. The established method was successfully applied for the pretreatment and determination of four alkaloids from Cortex Phellodendri in cell culture fluids. Under the optimal conditions, the limits of detection (S/N=3) for the analytes were 0.16-0.39 μg/L. The recoveries were in the range of 92.73%-97.91% and the relative standard deviations (RSDs) were ranged from 0.14% to 3.31% (n=6). The method is simple, sensitive and accurate. It can be used in the determination of alkaloids in cell culture fluids.

Key words: Cortex Phellodendri, alkaloid, high performance liquid chromatography (HPLC), monolithic columns, solid phase microextraction (SPME)