Characterisation of hederacoside C metabolites using ultrahigh-performance liquid chromatography quadrupole Orbitrap mass spectrometry based on automatic fragment ion search.

INTRODUCTION: Hederacoside C (HDC) is a bioactive natural triterpenoid saponins constituent originating from traditional Chinese medicines, playing an important role in the treatment of acute respiratory infections and chronic inflammatory bronchitis. Meanwhile, it is recognised by Korea as a botanical drug. OBJECTIVES: In order to develop an integrated template approach to analysing screening and identification of the metabolites of traditional Chinese medicines. This study will provide available information for further pharmaceutical studies of HDC and other triterpene saponins. METHODOLOGY: An analysis strategy based on ultrahigh-performance liquid chromatography quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) technique combined with automatic fragment ion search (FISh) was firstly exploited for the characterisation metabolites of HDC in vivo and in vitro. Accurate full mass scan combined with an on-line FISh annotations approach was developed to rapidly identify all the potential metabolites of HDC. Furthermore, FISh accurately located the structure of the target compound in a large number of mass spectral data. RESULTS: A total of 34 metabolites were detected and tentatively identified by analysing comprehensive biological samples. The results clearly demonstrated that HDC underwent general metabolic reactions including dealkylation, reduction, oxidation, desaturation, dehydration, cysteine conjugation, GSH conjugation, taurine conjugation, and glycine conjugation to produce 26 phase I and eight phase II metabolites. CONCLUSION: In the present study, UHPLC-Q-Exactive Orbitrap MS technique combined with FISh provided a rapid and efficient platform to characterise metabolites of HDC in vivo and in vitro. The proposed method could develop an integrated template approach to screen and identify the constituents and metabolites of traditional Chinese medicines.

Safety assessment and antioxidant evaluation of betulin by LC-MS combined with free radical assays.

Betulin, as a new type of natural food preservative, is widely used in various kinds of meat products. However, its detailed mechanism of action and metabolism have not been clarified. In this study, for further gain insight of the mechanism of betulin as a preservative, an efficient method has been applied for measuring the antioxidant capacity of betulin, based on the absorbance of the DPPH• and ABTS• radical cation. When the concentration of betulin was more than 2.0 mg/mL, the scavenging rate of ABTS and DPPH radical reached over 90%, which was equivalent to the antioxidant capacity of Trolox. It is indicated that betulin has significant DPPH and ABTS free radical scavenging ability. This should be one of the important mechanisms for betulin as a preservative. A sensitive method using UHPLC-Q-TOF-MS/MS was established to determine the metabolite profile in vivo and in vitro of betulin. 32 phase I and 2 phase II metabolites were structurally characterized. This study will provide theoretical support for the safety and effectiveness of betulin in the field of preservatives and provide theoretical basis for the further study of betulin and the other natural preservatives. This research also contributes to the development of the food industry.

A high-throughput metabolomics approach for the comprehensive differentiation of four Pulsatilla Adans herbs combined with a nontargeted bidirectional screen for rapid identification of triterpenoid saponins.

Pulsatilla Adans (PSA) herbs (Ranunculaceae) have been widely used in traditional medicine in China and other countries. However, the authentication and quality control of PSA herbs have always been a challenging task due to their similar morphological characteristics and the diversity of the multiple components that exist in the complicated matrix. Herein, a novel integrated strategy combining UHPLC/Q-Orbitrap-MS techniques with chemometrics analysis is proposed for the discrimination of PSA materials. We developed a comprehensive method integrating a nontargeted bidirectionally screened (NTBDS) MS data set and a targeted extraction peak area analysis for the characterization of triterpenoid saponins of PSA from different species. After that, partial least-squares discriminant analysis (PLS-DA) was performed on the obtained MS data set and the parameter variable importance for the projection (VIP) value and P value were employed to screen the valuable MS features to discriminate PSA from different species. In addition, the receiver operating characteristic (ROC) curve is used to verify the reliability of MS features. Finally, heatmap visualization was employed to clarify the distribution of the identified triterpenoid saponins, and four medicinal species of PSA were successfully differentiated. Additionally, 34 constituents were reported in PSAs for the first time, 81 triterpenoid saponins were identified as differential components, and 12 chemical ingredients were characterized as potential chemical markers to differentiate the four officinal PSA herbs. This is the first time that the differences in different PSA herbs have been observed systematically at the chemical level. The results suggested that using the identified characteristic components as chemical markers to identify different PSA herbs was effective and viable. This method provides promising perspectives in the analysis and identification of the ingredients of Chinese herbal medicines, and the identification of similar herbs from the same species.

Study on the metabolites of betulinic acid in vivo and in vitro by ultra high performance liquid chromatography with time-of-flight mass spectrometry.

Betulinic acid is a triterpenoid organic acid with remarkable antitumor properties and is naturally present in many fruits, condiments and traditional Chinese medicines. Currently, a strategy was developed for the identification of metabolites following the in vivo and in vitro biotransformation of Betulinic acid with rat intestinal bacteria utilizing ultra high performance liquid chromatography with time-of-flight mass spectrometry with polymeric solid-phase extraction. As a result, 46 metabolites were structurally characterized. The results demonstrated that Betulinic acid is universally metabolized in vivo and in vitro, and Betulinic acid could undergo general metabolic reactions, including oxidation, methylation, desaturation, loss of O and loss of CH2 . Additionally, the main metabolic pathways in vivo and in vitro were determined by calculating the relative content of each metabolite. This is the first study of Betulinic acid metabolism in vivo, whose results provide novel and useful data for better understanding of the safety and efficacy of Betulinic acid.