RESUMO
By using a double loop technique based on a set of mechanical transmission components and a flow path system,a double function of injection and fraction collection was realized. On the basis of double loop technique,a novel type of online/offline interface for two-dimensional liquid chromatography was designed to construct an efficient separation system coupling two different separation modes with a higher peak capacity, and the functions of the interface were evaluated. By means of connecting the interface to an HPLC system, the multiple functions of injection, fraction collection and injection again were fulfilled for preparation and purity analysis of 4 kinds of aromatic compounds. As for combination of 2 sets of HPLC system,5 kinds of protein samples were separated preliminarily by strong cation exchange chromatography, and the components difficult to separate were collected and injected into reversed phase chromatographic system for further separation. Furthermore, the interface was applied to coupling two chromatographic systems in both strong cation exchange mode and microcolumn reversed-phase mode for the two-dimensional separation of bovine serum albumin enzymatic digest. When 1 mAU was set as the integral threshold,a total number of 292 peaks were identified. With the help of the online/offline interface, the preparation of microscale samples, fine separation of hardly separated samples and two-dimensional separation of complex samples were achieved flexibly. The result indicated that the system was a potent tool for the construction of two-dimensional chromatographic system and separation research.
RESUMO
Herein, a strategy is proposed for the simultaneous determination of primary coumarins in Peucedani Radix (Chinese name: Qianhu). The methodology consists of three consecutive steps: 1) Semi-preparative LC in combination with a home-made automated fraction collection module to fragment the universal metabolome standard into ten fractions (Frs. I-X); 2) LC-accurate MS/MS and quantitative H NMR spectroscopy conducted in parallel to acquire the qualitative and quantitative data of each fraction; 3) Robust identification and quantification of components by use of LC coupled to multiple reaction monitoring. In this final step, the most significant fractions (Frs. III-X) were pooled to serve as the pseudo-mixed standard solution. Meticulous online parameter optimization was performed to obtain the optimal parameters, including ion transitions and collision energies. Concerns were particularly paid onto pursuing the parameters being capable of monitoring -specific isomers, notably praeruptorin E 3'-isovaleryl-4'-angeloylkhellactone. The quantitative performance of the method was validated according to diverse assays. Eleven primary coumarins (-) were unambiguously identified and absolutely quantified, even though no external reference compound was used. Above all, the integrated strategy not only provides a feasible pipeline for the quality assessment of Peucedani Radix, but more importantly, shows the potential for authentic compound-free quantitative evaluation of traditional Chinese medicines.