Hui Ouyang

A novel dereplication strategy for the identification of two new trace compounds in the extract of Gastrodia elata using UHPLC/Q-TOF-MS/MS

An ultra performance liquid chromatography (UHPLC) coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS/MS) was used in the structural determination of natural compounds in Gastrodia elata. A total of 64 compounds were identified or tentatively characterized. The strategy used for characterization was comparing their retention time and fragmentation behaviors with those of the reference standards, or investigating their accurate mass measurements and characteristic fragmentation patterns followed by low-energy collision dissociation tandem mass spectrometry (CID-MS/MS). Phenolic conjugates mainly underwent consecutive losses of gastrodin residues and combined losses of H2O and CO2 from their citric acid units under negative MS/MS conditions. According to these rules, we have successfully characterized fifteen potential novel compounds. To confirm the reliability of this strategy, two targeted unknown trace parishins were obtained from G. elata by LC/MS-guided isolation. Based on the analysis of data from NMR spectroscopy and other techniques, the two unknown parishins were identified as 2-[4-O-(β-d-glucopyranosyl)benzyl]-3-methyl-citrate (parishin J) and 1,2-di-[4-O-(β-d-glucopyranosyl)benzyl]-3-methyl-citrate (parishin K), respectively. The fully established structures were consistent with the MS-oriented structural elucidation. This study expanded our knowledge on parishins in Gastrodia species, and the proposed strategy was proven efficient and reliable in the discovery of new minor compounds from herbal extracts.

Ionic liquid-based ultrasound-assisted extraction and aqueous two-phase system for analysis of caffeoylquinic acids from Flos Lonicerae Japonicae

In this work, an ionic liquid-based ultrasonic-assisted extraction (ILUAE) method was developed to extract caffeoylquinic acids (CQAs) from Flos Lonicerae Japonicae (FLJ). ILUAE parameters were optimized by response surface methodology, including IL concentration, ultrasonic time, and liquid-solid ratio. Optimized ILUAE approach gained the highest extraction yields of 28.53, 18.21, 3.84mg/g for 3-O-caffeoylquinic acid (C1), 3,5-di-O-caffeoylquinic acid (C2), 3,4-di-O-caffeoylquinic acid (C3), respectively. C1-C3 are the three most abundant CQAs compounds in FLJ. The method showed comparable extraction yield and shorter extraction time compared with conventional extraction techniques. Subsequently, an aqueous two-phase system (ATPS) was applied in extraction solutions. Two trace CQAs, 5-O-caffeoylquinic acid (C4) and 4,5-di-O-caffeoylquinic acid (C5), were significantly enriched with signal to noise values increasing from less than 10 to higher than 1475. The results indicated that ILUAE and ATPS are efficient and environmentally-friendly sample extraction and enrichment techniques for CQAs from herbal medicines.

The rapid discovery and identification of physalins in the calyx of Physalis alkekengi L.var.franchetii (Mast.) Makino using ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry together with a novel three-step data mining strategy.

Physalins, uniquely discovered from genus physalis, showed significant bioactivities in many aspects. It is therefore very important for the exploration of natural resources rich of physalins. However, there is no efficient approach for rapid discovery and identification of this class of compounds due to their structural complexity. To address the issue, the fragmentation pathways and correspondingly fragmentation rules of physalins in negative MS/MS mode were thoroughly investigated in this study using seven physalin standards. As a result, diagnostic ions for the rapid screening of physalins and classification of different types of physalins were determined based on their MS/MS fragmentation patterns. On top of that, an integrated approach using UHPLC-QTOF-MS/MS together with a novel three-step data mining strategy was developed for the systematic analysis of physalins in complex samples. Consequently, 46 physalins including 20 novel ones were efficiently discovered and identified from the crude extracts of Ph. alkekengi calyx. The present study laid a foundation for future study of different parts of Ph. alkekengi and other physalis species with regard to rapid discovery of novel physalins. In addition, this study provided a base for establishing a quality control method of the raw materials of Ph. alkekengi according to the profile of physalins.

A robust platform based on ultra-high performance liquid chromatography Quadrupole time of flight tandem mass spectrometry with a two-step data mining strategy in the investigation, classification, and identification of chlorogenic acids in Ainsliaea fragrans Champ

The chlorogenic acids are the major bioactive constituents of the whole plant of Ainsliaea fragrans Champ. (Xingxiang Tuerfeng). These compounds are usually present as isomeric forms in Xingxiang Tuerfeng. Therefore, an efficient approach should be developed for the rapid discovery and identification of chlorogenic acids isomers through the fragmentation pathway and rules. In this study, the collision induced dissociation tandem mass spectrometry (CID-MS/MS) fragmentation routes of chlorogenic acids were systematically investigated by UHPLC-QTOF-MS/MS in the negative ion mode using eight chlorogenic acids standards. As a result, diagnostic product ions for rapid discovery and classification of chlorogenic acids isomers were determined according to their MS/MS fragmentation patterns and intensity analysis. Based on these findings, a novel two-step data mining strategy was established. The first key step was to screen different kinds of substitution and the skeleton of the quinic acid using the characteristic product ions and neutral losses. The second key step was to screen and classify different types of chlorogenic acids using their diagnostic product ions. It was apply to the rapid investigation, classification, and identification of chlorogenic acids. And the same carbon skeletons from a complex extract of Ainsliaea fragrans Champ. were effectively identified. 88 constituents, including 14 chlorogenic acids types, were rapidly discovered and identified, and in particular, 12 types of chlorogenic acids, including p-CoQC, FQA, BQC, CQA-Glu, CFQA, p-Co-CQC, di-p-CoQC, BCQA, di-CQA-Glu, PCQA, tri-QCA, and P-di-CQA, were first discovered in Ainsliaea fragrans Champ. In conclusion, UHPLC-QTOF-MS/MS method together with a systematic two-step data mining strategy was established as a feasible, effective, and rational technique for analyzing chlorogenic acids. Additionally, this study laid a foundation for the study of the active substances and quality control of Ainsliaea fragrans Champ.

Synthesis and biological evaluation of Hederacolchiside A1 derivatives as anticancer agents

Modification of Hederacolchiside A1 (HA1) on 28-COOH gave a series of novel triterpenoid saponin compounds containing ester or amide group. Comparing with natural product HA1, several derivatives showed decreased toxicity in the mice acute toxicity trial and increased the anticancer activity in vitro. Especially compound 1 exhibited the strongest antiproliferative activities against human cancer cell lines tested (IC50=1.1-4.6μM) and potent tumor inhibition rate in vivo (46.8%).

Systematic identification of shikonins and shikonofurans in medicinal Zicao species using ultra-high performance liquid chromatography quadrupole time of flight tandem mass spectrometry combined with a data mining strategy.

Shikonin, shikonofuran and their derivatives are the main bioactive components of Zicao, a traditional Chinese herbal medicine prepared with the dried roots of Lithospermum erythrorhizon, Arnebia euchroma or Arnebia guttata. However, approaches on the systematic discovery and identification of shikonins and shikonofurans, especially unknown ones, are still not available. To address this issue, the gas-phase CID-fragmentation routes for the shikonins and shikonofurans were established by using ESI-QTOF-MS in the negative ion mode and low-energy collision induced dissociation tandem mass spectrometry (CID-MS/MS) in this study using seventeen standards. As a result, diagnostic product ions for rapid discovery and classification of shikonins and shikonofurans were determined. In addition, various mobile phase compositions and UHPLC elution programs were evaluated to achieve optimal separation efficiency and detection response of these types of analytes. Based on these findings, an integral approach using UHPLC-ESI-QTOF-MS and CID-MS/MS analyses together with a novel two steps data mining strategy was developed for systematic analysis of shikonins and shikonofurans in complex samples. Consequently, 58 compounds including 32 novel ones were efficiently discovered and identified from the crude extract of Zicao. Moreover, comparative analyses of the 58 chemical components in three Zicao species including Lithospermum erythrorhizon, Arnebia euchroma and Arnebia guttata samples were conducted using the established analytical method, which can be instructive for future utilization of Zicao and its related medicinal products.

A rapid and sensitive LC-MS/MS method for the determination of Pulsatilla saponin D in rat plasma and its application in a rat pharmacokinetic and bioavailability study

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of Pulsatilla saponin D, a potential antitumor constituent isolated from Pulsatilla chinensis in rat plasma. Rat plasma samples were pretreated by protein precipitation with methanol. The method validation was performed in accordance with US Food and Drug Administration guidelines and the results met the acceptance criteria. The method was successfully applied to assess the pharmacokinetics and oral bioavailability of Pulsatilla saponin D in rats.

Application of a liquid chromatography-tandem mass spectrometry method to the pharmacokinetics, tissue distribution and excretion in the study of anemoside B4, a novel antiviral agent candidate, in rats

A simple, sensitive and reliable LC-MS/MS method was developed and validated for the quantification of anemoside B4, a potential antiviral constituent isolated from Pulsatilla chinensis in rat plasma, tissue, bile, urine and feces. All biological samples were prepared by protein precipitation method, and ginsenoside-Rg1 was chosen as the internal standard (IS). The analyte and IS were separated using a C18 column (2.1 × 50 mm, 1.8 μm) and a mobile phase consisting of 0.1% formic acid in water (v/v) and acetonitrile running at a flow rate of 0.2 mL/min for 5 min. The multiple reaction monitoring transitions were monitored at m/z 1219.5-749.5 for anemoside B4 and 845.4-637.4 for ginsenoside-Rg1 in electrospray ionization negative mode. The calibration curve was linear in the range of 10-2000 ng/mL for all biological matrices with a lower limit of quantification of 10 ng/mL. The validated method was successfully applied to a pharmacokinetics, tissue distribution and excretion study. These preclinical data will be beneficial for further development of anemoside B4 in future studies.

Structural characterization and discrimination of Chimonanthus nitens Oliv. leaf from different geographical origins based on multiple chromatographic analysis combined with chemometric methods

HIGHLIGHTSUHPLC‐QTOF‐MS/MS method was applied for comprehensive profiling of characterization constituents in CNOL by high‐resolution diagnostic product ions/neutral loss filtering offered.An HPLC‐PDA method was developed to fingerprint analysis and nine maker compounds were quantified in 21 batches of CNOL samples.The quantitative data of 21 batches from different geographical origins were statistically analyzed. ABSTRACT The Chimonanthus nitens Oliv. leaf (CNOL). is a widely used traditional Chinese medicine (TCM) used for treating colds and influenza. In the present study, a comprehensive strategy integrating multiple chromatographic analysis and chemometric methods was firstly proposed for structural characterization and discrimination of CNOL from different geographical origins. It consists of three steps: Firstly, the ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry (UHPLC‐QTOF‐MS/MS) method was applied for comprehensive profiling of characterization constituents in CNOL by high‐resolution diagnostic product ions/neutral loss filtering, and a total of 40 constituents were identified. Secondly, chemical fingerprints were established by HPLC coupled with photodiode array detector (PDA), and similarity analyses were calculated based on nineteen common characteristic peaks. Subsequently, the nine major constituents, including coumarins, flavonoids, and phenolic acids were quantified, and the quantitative data further analyzed by principal component analysis (PCA) and orthogonal partial least‐squares discriminant analysis (OPLS‐DA). Thirdly, a hot map visualization was conducted for clarifying the distribution of major compounds among different geographical origins. Also, nine constituents detected could be used as chemical markers for discrimination of CNOL from different provinces. Collectively, these results indicated that our proposed platform was a powerful tool for chemical profiling and discrimination of herbs with multiple botanical origins, providing promising perspectives in tracking the formulation process of TCMs products.

Metabolism and bioactivation of the tricyclic antidepressant amitriptyline in human liver microsomes and human urine.

AIM Amitriptyline is a widely used tricyclic antidepressant, but the metabolic studies were conducted almost 20 years ago using high-performance liquid chromatography coupled with ultraviolet detector or radiolabeled methods. RESULTS First, multiple ion monitoring (MIM)- enhanced product ion (EPI) scan was used to obtain the diagnostic ions or neutral losses in human liver microsome incubations with amitriptyline. Subsequently, predicted multiple reaction monitoring (MRM)-EPI scan was used to identify the metabolites in human urine with the diagnostic ions or neutral losses. Finally, product ion filtering and neutral loss filtering were used as the data mining tools to screen metabolites. Consequently, a total of 28 metabolites were identified in human urine after an oral administration using LC-MS/MS. CONCLUSION An integrated workflow using LC-MS/MS was developed to comprehensively profile the metabolites of amitriptyline in human urine, in which five N-acetyl-l-cysteine conjugates were characterized as tentative biomarkers for idiosyncratic toxicity.