Development Of Two-dimensional Liquid Chromatography Tandem Mass Spectrometry Detection For The Analysis Of Constituents Of Zhizichi Decoction

Two-dimensional liquid chromatography is a new type of chromatographic separationtechnology developed in recent years, it can significantly improve the chromatographicpeak capacity and selectivity, and can solve the problem of separation analysis whichdifficult to resolve by a single chromatographic separation mode, it has been widely usedfor the analysis of complex samples. Zhizichi decoction is from the “treatise on febriledisease” wrote by Zhang zhong jing, and is made up of Gardenia jasminoides Ellis andSemen sojae praeparatum. It was applied to the treatment of menopausal depression,insomnia, neurasthenia and other diseases, and has broad prospects for development andutilization in clinic. Take the Zhizichi decoction as research object in this work, thetheoretical study and practical application of off-line two-dimensional liquidchromatography (2D-LC) were developed to analysis of Gardenia jasminoides Ellis, Semensojae praeparatum and the whole prescriptions. Combined with mass spectrometry methodto do the further research of constituents in Zhizichi decoction.This paper is divided into five chapters as follows：Chapter1briefly introduced the background and significance of the thesis, includingthe principle, evaluation index and classification of2D-LC, the applications and importanceof in separation and analysis of traditional Chinese medicines (TCMs) used in2D-LC.Chapter2established the off-line RPLC-HILIC two-dimensional liquidchromatography orthogonality of evaluation system. The2D-RPLC/HILIC system and2D-HILIC/RPLC system were established to estimate the orthogonality and peak capacityby selecting the same size column, respectively. The orthogonality of this two2D-LC system was69.92%and46.82%while the practical peak capacity was7303and4551,respectively. The result showed that the2D-RPLC/HILIC system is more prominent, andcould be selected as the analysis method of the single herbs and the whole prescription inZhizichi decoction.Chapter3developed the off-line2D-RPLC/HILIC system for the analysis of Gardeniajasminoides Ellis. A semi-preparative C18column and a analytical HILIC column wereperformed using RPLC mode and HILIC mode, respectively, in order to raise the peakcapacity and detection sensitivity in the second dimension. Totally896peaks wereseparated by this2D-LC system. At the same time combined with mass spectrometrymethod to identified and characterized the16constituents in Gardenia jasminoides Ellis.Chapter4developed the heart-cutting mode off-line2D-RPLC/HILIC system for theanalysis of Semen sojae praeparatum. In view of the instability quality problem of Semensojae praeparatum, we adopt a heart-cutting mode to separate different polaritycomponents. A Xaqua C18column and An XAmide column were performed using RPLCmode and HILIC mode, respectively. Totally136peaks were separated from polar fractionand198peaks were separated from medium-polar fraction by this2D-LC system,respectively. At the same time combined with mass spectrometry method to identified andcharacterized the11constituents in Semen sojae praeparatum.Chapter5developed the off-line2D-RPLC/HILIC system for the analysis of thewhole prescription in Zhizichi decoction. A XAqua C18column and An XAmide columnwere performed using RPLC mode and HILIC mode, respectively. Totally441peaks wereseparated by this2D-LC system. A comprehensive characterization of the constituents inZhizichi decoction through two-dimensional liquid chromatography and mass spectrometrymethod was developed. Compared with the2D-LC analysis results of Gardeniajasminoides Ellis and Semen sojae praeparatum, the difference and changes between thewhole prescription and single herbs in Zhizichi decoction were speculated preliminary.