Tandem Mass Spectrometry for Structural Identification of Sesquiterpene Alkaloids from the Stems of Dendrobium nobile Using LC-QToF.

Fungal species are an attractive resource for physiologically functional food and drug precursor. Fomes officinalis Ames, a medicinal fungus, is traditionally used as a folk medicine in traditional Chinese medicine prescription for the therapy of cough and asthma. The water-soluble substances in Chinese herbal medicines are likely to play an important physiological function. However, information on probing and identifying chemical components of the aqueous extract of Fomes officinalis Ames (AFO) remains unknown. This study was conducted to screen and characterise the chemical components of AFO. An effective and sensitive ultrahigh-performance liquid chromatography tandem quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS) method with the Full MS/PIL/dd-MS2 acquisition approach was applied for the profiling of chemical components in AFO. An HSS T3 column was used for component separation, and a strategy of simultaneous targeted and untargeted multicomponent characterisation was implemented. Multiple identification approaches were used, including accurate molecular mass and elemental composition matching, literature and database searching, and fragmentation rules elucidation. A total of 115 components, including 20 amino acids and derivatives, six nucleobases, nine nucleosides, 75 dipeptides, two tripeptides, and three other components, were tentatively identified. Among them, the targeted exploring method screened six nucleobases and nine nucleosides including modified nucleosides. To our best knowledge, this is the first time a report has been done on the presence of the 115 compounds in AFO. Profiling and characterisation compounds of AFO enriched its material basis, which would lay the foundation for improving potential medicinal and nutritional values and effecting comprehensive quality control of Fomes officinalis Ames.

Protein Molecular Markers for Herbal Natures of Six Traditional Chinese Medicinal Herbs

Evaluation of the impact of Polygonum capitatum, a traditional Chinese herbal medicine, on rat hepatic cytochrome P450 enzymes by using a cocktail of probe drugs

Pyrrolizidine alkaloids (PAs) are a class of natural toxins with hepatotoxicity, genotoxicity and carcinogenicity. They are endogenous and adulterated toxic components widely found in food and herbal products. In this study, a sensitive and efficient ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was used to detect the PAs in 386 kinds of Chinese herbal medicines recorded in the Chinese Pharmacopoeia (2020). The estimated daily intake (EDI) of 0.007 μg/kg body weight (bw)/day was adopted as the safety baseline. The margin of exposure (MOE) approach was applied to evaluate the chronic exposure risk for the genotoxic and carcinogenic potential of PAs. Results showed that PAs was detected in 271 out of 386 samples with a content of 0.1–25,567.4 μg/kg, and there were 20 samples with EDI values above the baseline, 0.007 μg/kg bw/day. Beyond that, the MOE values for 10 out of 271 positive samples were below 10,000. Considering the actual situation, Haber’s rule was used to assume two weeks exposure every year during lifetime, and still the MOE values for four out of 271 positive samples were under 10,000, indicating these products may have potential health risk. The developed method was successfully applied to detect the PAs-containing Chinese herbal medicines. This study provides convincing data that can support risk management actions in China and a meaningful reference for the rational and safe use of Chinese herbal medicines.

Discrimination and chemical phylogenetic study of seven species of Dendrobium using infrared spectroscopy combined with cluster analysis

Inhibitory effect of different Dendrobium species on LPS-induced inflammation in macrophages via suppression of MAPK pathways

Regulatory mechanisms of natural compounds from traditional Chinese herbal medicines on the microglial response in ischemic stroke

Many species of the Dendrobium genus are traditional Chinese herbal medicine and ornamental plants. Flower fragrance is one of the most important horticultural ornamental characters and plays a crucial role in the ecology, economy, and aesthetics of plants. However, the volatile constituents and key regulatory genes related to floral biosynthesis are poorly understood. In this experiment, the flowers from two species of Dendrobium with high-scent smells, Dendrobium moniliforme (L.) Sw. (D. moniliforme), and light-scent smells, Dendrobium nobile “H1” (D. “H1”), were selected. The aim of this study was to explore the key gene expression profiles of floral biosynthesis by combining volatile constituent determination and transcriptome analysis in two different Dendrobium species. Physiological determination results showed that 60 volatile compounds were identified in D. moniliforme and 52 volatile compounds were identified in D. ‘H1’ flowers in four flowering stages, and the full bloom stage was the most complicated stage because there were 41 and 33 volatile compounds, respectively. These compounds belong to terpenes, aromatics, fatty acids, nitrogenous compounds, ketones, alcohols, and alkanes, respectively. The components identified in the gynandrium and petals revealed that the petals were probably the most important sites affecting the release of volatiles. The relative content of terpene compounds was the highest, with 77.25% (D. moniliforme) and 50.38% (D. “H1”), respectively. Transcriptome analysis showed that differentially expressed genes (DEGs) were highly enriched in terpenoid backbone biosynthesis and that four linalool synthetase (LIS) genes were up-regulated in high-scent smell species. This study is helpful to explore the key genes of flower fragrance and provides a theoretical basis for further understanding of the regulatory molecular functions of floral synthesis and release, as well as for the cultivation of new aromatic species.

To establish a method for determination of 15 pesticides residue in edible fungi by multiplug filtration clean-up(m-PFC) pretreatment technique with ultra-high performance liquid chromatography-tandem triple quadruple mass spectrometry(UPLC-MS/MS). The interferences of edible fungus samples were removed by extracting with acetonitrile and filtration type extraction column, which were fat, carbohydrates, water-soluble vitamins. Samples were separated with column of Waters ACQUITY UPLC®HSS T3(2.1 mm×100 mm, 1.8 μm), and were scanned by multiple reaction monitoring mode(MRM). Samples were quantified with matrix matching standard curve external standard method. The recoveries of 15 target compounds at the spiked levels of 10, 20, 50 μg/kg were 82.5%-118.5%, and the relative standard deviations were between 6.1% and 23.1%(n=6). The detection limit of 15 target compounds was 1-3 μg/kg, and the limit of quantification was 3-10 μg/kg. This method improves the efficiency of pretreatment, has good stability and high sensitivity, and could be used for the detection of 15 pesticides in edible fungi.

Due to the variety, chemical composition and complex structure, the quality control of Bupleuri Radix (BR) is a challenging task. There are still many trace compounds in BR that are difficult to extract and detect. To develop an innovative method of trisiloxane surfactant vesicles ultrasonic extraction (TSVUE) combined with ultrahigh-performance liquid chromatography tandem mass spectrometry for the identification from Bupleurum chinense DC. (BC) to Bupleurum scorzonerifolium Willd (BS) based on metabolomics. Based on extraction effect for BR, five different types of surfactants vesicles were prepared and compared. Then, a single-factor test and a response surface methodology study were adopted to obtain the optimal conditions for the surfactant vesicles ultrasonic extraction method. Finally, a non-targeted metabolomics method with information dependent acquisition mode was performed to analyse differential metabolites in BC and BS. Sugar-based surfactant containing trisiloxane [N-3-propyl-methyltrisiloxane-N-glucoheptonamne (Si(3)N-GHA)] displayed higher extraction efficiency compared to other types of surfactants when it comes to being used in pretreatment methods. And a TSVUE method was established and optimised. In total, 131 constituents were identified in two BR herbs, of which 35 were unreported, and 11 were characterised as chemical markers. This method provides promising perspectives for rapidly identifying trace compounds in complex systems of traditional Chinese medicine (TCM), as well as for laying the foundation in the identification of similar herbs from the same species. Meanwhile, these findings serve as a promising application of trisiloxane surfactant vesicles in the extraction field of TCM.

Oxypeucedanin, a furanocoumarin extracted from many traditional Chinese herbal medicines, has a variety of pharmacological effects. However, the independent pharmacokinetic characteristics and bioavailability of this compound remains elusive. In this study, a rapid, sensitive, and selective method using ultra-high performance liquid chromatography–tandem mass spectrometry (UPLC/MS/MS) was developed for evaluating the intravenous and oral pharmacokinetics of oxypeucedanin. After intravenous administration of oxypeucedanin (2.5, 5, and 10 mg/kg), and intragastric administration of oxypeucedanin (20 mg/kg), blood samples were collected periodically from the tail vein. The plasma concentration-time curves were plotted, and the pharmacokinetic parameters were calculated using a non-compartmental model analysis. After intravenous administration of oxypeucedanin (single dosing at 2.5, 5, and 10 mg/kg) to rats, the pharmacokinetics fit the linear kinetics characteristics, which showed that some parameters including average elimination half-life (T1/2Z of 0.61~0.66 h), mean residence time (MRT of 0.62~0.80 h), apparent volume of distribution (VZ of 4.98~7.50 L/kg), and systemic clearance (CLZ of 5.64~8.55 L/kg/h) are dose-independent and the area under concentration-time curve (AUC) increased in a dose-proportional manner. Single oral administration of oxypeucedanin (20 mg/kg) showed poor and slow absorption with the mean time to reach the peak concentration (Tmax) of 3.38 h, MRT of 5.86 h, T1/2Z of 2.94 h, and a mean absolute bioavailability of 10.26% in rats. These results provide critical information for a better understanding of the pharmacological effect of oxypeucedanin, which will facilitate its research and development.

Chapter 13 - Butterflyfish gill mucus metabolome reflects diet preferences and gill parasite intensities

Characterization of alkaloids in Sophora flavescens Ait. by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

Ultrahigh-performance liquid chromatography tandem mass spectrometry with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF) derivatization was developed for simultaneous determination of 20 free amino acids in Chinese jujube date. The MS/MS conditions, choice of mobile phase, the extraction process, and matrix effects were studied with a view to a method of optimization. The limits of detection for measurement of the amino acids ranged from 0.8 to 600.0 μg L−1. The correlation coefficients (r2 ≥ 0.9947) indicated good correlation between the concentrations of amino acids and the peak areas for the CNBF-derivatives. Recoveries for samples spiked at 25.0, 50.0, and 100.0 mg kg−1 ranged from 66.3 to 106.6 % (except tryptophan spiked at 25.0 and 50.0 mg kg−1, which provided recoveries of 61.2 and 60.4 %, and tyrosine spiked at three different concentrations, which provided recoveries of 54.8–58.6 %), with relative standard deviation values of less than 9.1 %.

Buprenorphine (BUP) is a psychoactive pharmaceutical drug largely used to treat opiate addiction. Short-term therapeutic monitoring is supported by toxicological analysis of blood and urine samples, whereas long-term monitoring by means of hair analysis is rarely used. Aim of this work was to develop and validate a highly sensitive ultrahigh-performance liquid chromatography tandem mass spectrometry method to detect BUP and norbuprenorphine (NBUP) in head hair. Interindividual correlation between oral dosage of BUP and head hair concentration was investigated. Furthermore, an intra-individual study by means of segmental analysis was performed on subjects with variable maintenance dosage. Hair samples from a population of 79 patients in treatment for opiate addiction were analyzed. The validated ultrahigh-performance liquid chromatography tandem mass spectrometry protocol allowed to obtain limits of detection and quantification at 0.6 and 2.2 pg/mg for BUP and 5.0 and 17 pg/mg for NBUP, respectively. Validation criteria were satisfied, assuring selective analyte identification, high detection capability, and precise and accurate quantification. Significant positive correlation was found between constant oral BUP dosage (1-32 mg/d) and the summed up head hair concentrations of BUP and NBUP. Nevertheless, substantial interindividual variability limits the chance to predict the oral dosage taken by each subject from the measured concentrations in head hair. In contrast, strong correlation was observed in the results of intra-individual segmental analysis, which proved reliable to detect oral dosage variations during therapy. Remarkably, all hair samples yielded BUP concentrations higher than 10 pg/mg, even when the lowest dosage was administered. Thus, these results support the selection of 10 pg/mg as a cutoff value.