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Herbal medicines (HMs) usually consist of complex matrices of phytochemical compounds responsible for their efficacy. Supercritical fluid chromatography (SFC) and ultra-high performance SFC (UHPSFC) are efficient techniques that can be advantageously used for the analysis of complex HMs. Here, the comprehensive UHPSFC methods were established for separation of similar flavonoids, chemical profiling and differentiation, and quality assessment of Epimedium species (EPs). An efficient separation of 12 polar flavonoids was enabled in UHPSFC with an important additive, oxalic acid. Then, 51 flavonoids were characterized, and 28 potential markers enabling the differentiation of E. wushanense from other four species were discovered by UHPSFC-QTOF/MS combined pattern recognition multivariate statistical analysis. Additionally, the UHPSFC-PDA was developed and validated for simultaneous quantification of 7 predominant flavonoids in five EPs, and remarkable variation in their contents was observed. This integrated UHPSFC approach has potential applications in systematic analysis and quality evaluation of HMs containing hydrophilic flavonoids.

Radix Dipsaci (RD), the dried root of Dipsacus asper, is used in traditional Chinese medicine as a remedy for bone fractures, traumatic hematoma, threatened abortion, and uterine bleeding. A novel ultra high-performance liquid chromatography coupled with quadrupole-time-of-flight tanderm mass spectrometry (UHPLC-Q-TOF/MS) approach was performed to rapidly characterize the chemical constituents of RD. Consequently, 21 compounds, including 12 iridoid glycosides (IGs), 4 furofuran lignans (FLs), and 5 phenolic acids (PAs) were discovered and identified from RD. Among these compounds, 3 IGs were previously unreported. Furthermore, a rapid and reliable UHPLC-DAD-based method was developed. The linearity (R2 > 0.999), repeatability (RSDs

Epimedium herb is a well-known traditional Chinese medicine (TCM) that is used for treating kidney-yang deficiency, impotence and rheumatism, and flavonoids are the main active ingredients. The leaves and rhizomes of Epimedium herb are two separate kinds of medicinal materials with different functional indications and clinical applications. This study aimed to comprehensively analyze the chemical components of different parts of the herb from three Epimedium species (Epimedium sagittatum, E. pubescens and E. myrianthum) by using ultra high-performance liquid chromatography coupled with photo-diode array and quadrupole time-of-flight mass spectrometry (UHPLC-PDA-Q-TOF/MS) and multivariate statistical analysis to clarify the differences. Firstly, the workflow of UHPLC-Q-TOF/MS combined with UNIFI informatics was developed for characterizing the chemical compounds in different parts of Epimedium herb. Based on the exact mass information, the fragmentation characteristics and the retention times of compounds, all chromatographic peaks (74 chemical components) were identified. Secondly, 21 potential chemical markers for differentiating different parts of Epimedium herb were selected through PCA and PLS-DA analysis. The characteristic components in the leaves included flavonoids with Anhydroicaritin (type A, C-4' linked methoxy) as the backbone, and the characteristic components in the stems and rhizomes were Magnoline and flavonoids with Demethylanhydroicaritin (type B, C-4' linked hydroxyl) as the backbone. Thirdly, the UHPLC-PDA combined with heatmap visualization was employed to clarify the distribution of chemical components with high content in different parts of Epimedium herb. The results showed clear differences in the contents of chemical components in leaves, stems and rhizomes. The levels of flavonoids with Anhydroicaritin backbone were high in the leaves, and levels of flavonoids with Demethylanhydroicaritin backbone were high in the rhizomes. The levels of Magnoline in stems and rhizomes were higher than that in leaves. The contents of most of the compounds in stems remained low. The leaves and the other two parts (stems and rhizomes) can be distinguished by qualitative and semi-quantitative analysis of Magnoline and Epimedoside A (type B backbone). These results indicated that the different plant parts of Epimedium herb can be quickly and accurately distinguished by this method, establishing a foundation for the application of Epimedium herb.

In this work, a simple and rapid high-performance liquid chromatography coupled with charged aerosol detector (HPLC-CAD) method was first developed for the quantitation of toosendanin, the major constituent of the dried fruit of Melia toosendan Sieb. Et Zucc. Samples were well separated on an Agilent ZOBAX SB C18 column (4.6 mm × 250 mm, 5 μm) by isocratic elution using 33 % acetonitrile and 67 % water containing 0.1 % formic acid (v/v) at the flow rate of 1.0 mL min−1. The nitrogen inlet pressure of the charged aerosol detector (CAD) was 35 psi, and the nebulizer chamber temperature was 35 °C. The established method was well validated. Satisfactory linearity was achieved (r 2 > 0.9997) in a relatively wide concentration range (5–500 μg mL−1). The intra- and inter-day precisions, repeatability, and stability of the method were good with relative standard deviations (RSDs) of 1.05, 2.23, 2.39, and 2.03 %, respectively. The method also showed excellent accuracy with recovery rates of 97.42–101.87 %. Particularly, CAD showed much better sensitivity (LOQ 4 μg mL−1) than evaporative light scattering detector (LOQ 100 μg mL−1) for toosendanin’s determination. The established method was further applied in the quantitation of toosendanin in 39 batches of raw and stir-fried toosendan fructus. The HPLC-CAD method was rapid and accurate, and could be used for the routine analysis and quality control of toosendan fructus and its preparations.

Eleven novel furostanol saponins, named ophiofurospisides C–E, G–N (1–3, 5–12), one new spirostanol saponin, named ophiopogonin R (13), were isolated from the fresh tubers of Ophiopogon japonicus. Their structures were determined on the basis of spectroscopic techniques (1D and 2D NMR) and HRESIMS. The isolated furostanol saponins possessed two sugar chains located at C-3 and C-26, respectively. Six furostanol saponins (1, 5–9) with disaccharide moiety linked at position C-26 of the aglycone were rare in the plant kingdom.

Spirostanol saponins are important active components of some herb medicines, and their isolation and purification are crucial for the research and development of traditional Chinese medicines. We aimed to compare the separation of spirostanol saponins by ultra-high performance supercritical fluid chromatography (UHPSFC) and ultra-high performance liquid chromatography (UHPLC). Four groups of spirostanol saponins were separated respectively by UHPSFC and UHPLC. After optimization, UHPSFC was performed with a HSS C18 SB column or a Diol column and with methanol as the co-solvent. A BEH C18 column and mobile phase containing water (with 0.1% formic acid) and acetonitrile were used in UHPLC. We found that UHPSFC could be performed automatically and quickly. It is effective in separating the spirostanol saponins which share the same aglycone and vary in sugar chains, and is very sensitive to the number and the position of hydroxyl groups in aglycones. However, the resolution of spirostanol saponins with different aglycones and the same sugar moiety by UHPSFC was not ideal and could be resolved by UHPLC instead. UHPLC is good at differentiating the variation in aglycones, and is influenced by double bonds in aglycones. Therefore, UHPLC and UHPSFC are complementary in separating spirostanol saponins. Considering the naturally produced spirostanol saponins in herb medicines are different both in aglycones and in sugar chains, a better separation can be achieved by combination of UHPLC and UHPSFC. UHPSFC is a powerful technique for improving the resolution when UHPLC cannot resolve a mixture of spirostanol saponins and vice versa.

An ultra high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry approach coupled with multivariate statistical analysis was established and applied to rapidly distinguish the chemical differences between fibrous root and rhizome of Anemarrhena asphodeloides. The datasets of tR-m/z pairs, ion intensity and sample code were processed by principal component analysis and orthogonal partial least squares discriminant analysis. Chemical markers could be identified based on their exact mass data, fragmentation characteristics, and retention times. And the new compounds among chemical markers could be isolated rapidly guided by the ultra high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry and their definitive structures would be further elucidated by NMR spectra. Using this approach, twenty-four markers were identified on line including nine new saponins and five new steroidal saponins of them were obtained in pure form. The study validated this proposed approach as a suitable method for identification of the chemical differences between various medicinal parts in order to expand medicinal parts and increase the utilization rate of resources.