A comprehensive profiling and identification of liquiritin metabolites in rats using ultra-high-performance liquid chromatography coupled with linear ion trap-orbitrap mass spectrometer.

Liquiritin (LQ), a main component of liquorice, exerts various biological activities. However, insufficient attentions have been paid to the metabolism study on this natural compound until now. Our present study was conducted to investigate the LQ metabolites in rats urine, faeces and plasma using UHPLC-LTQ-Orbitrap mass spectrometer in both positive and negative ion modes. Meanwhile, post-acquisition data-mining methods including high-resolution extracted ion chromatogram (HREIC), multiple mass defect filters (MMDFs), neutral loss fragments (NLFs) and diagnostic product ions (DPIs) were utilised to screen and identify LQ metabolites from HR-ESI-MS to ESI-MSn stage. As a result, a total of 49 metabolites were detected and characterised unambiguously or tentatively. These metabolites were presumed to generate through glucuronidation, sulfation, deglucosylation, dehydrogenation, methylation, hydrogenation, hydroxylation, ring cleavage and their composite reactions. Our results not only provided novel and useful data to better understand the biological activities of LQ, but also indicated that the proposed strategy was reliable for a rapid discovery and identification drug-related constituents in vivo.

Metabolism study of hesperetin and hesperidin in rats by UHPLC-LTQ-Orbitrap MS n.

Hesperidin (HPD) and hesperetin (HPT), as a kind of flavonone compounds, are abundant in citrus fruits with various pharmacological effects. HPD and HPT are not always consistent in some biological activities, even if they have the same skeletal structure. The aim of this study was to screen and identify HPT and HPD metabolites in rats using UHPLC-LTQ-Orbitrap MS n , compare the possible metabolism and provide the research basis for further understanding the similarities and differences in pharmacodynamics and pharmacokinetics of HPT and HPD. A total of 17 and 52 metabolites were identified in rats after oral administration of HPT or HPD, respectively. Three of HPT and HPD metabolites, glucuronidation, sulfation and diglucuronidation of HPT, were the same and could be the active components for the same pharmacodynamic action of them. We could find prototype in the urine sample of HPD group, but no prototypes in any samples of HPT group. There were hardly any general phase I metabolites of HPT, while 33 phase I metabolites of HPD could be identified. These data suggested that the poorer bioavailability of HPD compared with HPT.

Characterization of forsythoside A metabolites in rats by a combination of UHPLC-LTQ-Orbitrap mass spectrometer with multiple data processing techniques.

Forsythoside A (FTA), the main active constituent isolated from Fructus Forsythiae, has various biological functions including anti-oxidant, anti-viral and anti-microbial activities. However, while research on FTA has been mainly focused on the treatment of diseases on a material basis, FTA metabolites in vivo have not been comprehensively evaluated. Here, a rapid and sensitive method using a UHPLC-LTQ-Orbitrap mass spectrometer with multiple data processing techniques including high-resolution extracted ion chromatograms, multiple mass defect filters and diagnostic product ions was developed for the screening and identification of FTA metabolites in rats. As the result, a total of 43 metabolites were identified in biological samples including 42 metabolites in urine, 22 metabolites in plasma and 15 metabolites in feces. These results demonstrated that FTA underwent a series of in vivo metabolic reactions including methylation, dimethylation, sulfation, glucuronidation, diglucuronidation, cysteine conjugation and their composite reactions. The research enhanced our understanding of FTA metabolism and built a foundation for further toxicity and safety studies.

A Comprehensive Screening and Identification of Genistin Metabolites in Rats Based on Multiple Metabolite Templates Combined with UHPLC-HRMS Analysis.

Genistin, an isoflavone belonging to the phytoestrogen family, has been reported to possess various therapeutic effects. In the present study, the genistin metabolites in rats were investigated by UHPLC-LTQ-Orbitrap mass spectrometer in both positive and negative ion modes. Firstly, the data sets were obtained based on data-dependent acquisition method and then 10 metabolite templates were established based on the previous reports. Then diagnostic product ions (DPIs) and neutral loss fragments (NLFs) were proposed to efficiently screen and ascertain the major-to-trace genistin metabolites. Meanwhile, the calculated Clog P values were used to identify the positional isomers with different retention times. Consequently, a total of 64 metabolites, including prototype drug, were positively or putatively characterized. Among them, 40 metabolites were found according to the templates of genistin and genistein, which was the same as the previous research method. After using other metabolite templates, 24 metabolites were added. The results demonstrated that genistin mainly underwent methylation, hydrogenation, hydroxylation, glucosylation, glucuronidation, sulfonation, acetylation, ring-cleavage and their composite reactions in vivo biotransformation. In conclusion, the research not only revealed the genistein metabolites and metabolic pathways in vivo comprehensively, but also proposed a method based on multiple metabolite templates to screen and identify metabolites of other natural compounds.

Proteomic Signature of Endothelial Dysfunction Identified in the Serum of Acute Ischemic Stroke Patients by the iTRAQ-Based LC-MS Approach.

Acute ischemic stroke (AIS) is a devastating cerebrovascular disorder that leads to permanent physical and neurological disabilities in adults worldwide. Proteins associated with stroke pathogenesis may appear in the serum of AIS patients due to blood-brain barrier dysfunction, thus permitting the development of blood-based biomarkers for early diagnosis of stroke. These biomarkers could perhaps be an adjunct to the existing imaging modalities and aid in better management and therapeutic intervention during the course of the disease. For this exploratory study, a combination of multiplexed isobaric tagging using iTRAQ reagents and high resolution tandem mass spectrometry was used to identify differentially expressed proteins in serum samples from AIS patients. The quantitative proteomic analysis of serum from both AIS and control subjects revealed 389 high confidence protein identifications and their relative levels. Among them, 60 proteins showed a ≥1.5-fold change in the AIS subjects. We verified the altered serum levels of candidate proteins such as vWF, ADAMTS13, S100A7, and DLG4 through ELISA, and the results also corroborate with the experimental findings. vWF and ADAMTS13 are key players that regulate blood hemostasis, and their altered concentration may contribute to endothelial dysfunction. S100A7 is a novel candidate protein identified in this study that is also known to mediate inflammation, endothelial proliferation, and angiogenesis. The current study provided a potential and novel biomarker panel that may in turn provide diagnostic aid to the existing imaging modalities for the rapid diagnosis of ischemic stroke.

Rapid identification of polymethoxylated flavonoids in traditional Chinese medicines with a practical strategy of stepwise mass defect filtering coupled to diagnostic product ions analysis based on a hybrid LTQ-Orbitrap mass spectrometer.

INTRODUCTION: The methodology of stepwise mass defect filtering (MDF) approach coupled to diagnostic product ions (DPIs) analysis on a hybrid linear trap quadrupole (LTQ)/orbitrap mass spectrometer was the first to be established to screen and identify structural analogues from complex herbal extracts. OBJECTIVE: To develop an analytical methodology that could be adopted to screen and identify structural analogues in traditional Chinese medicines (TCMs) rapidly and accurately. METHODS: Taking polymethoxylated flavonoids (PMFs) in the leaves of Citrus reticulata Blanco as an example, high-resolution mass data were acquired by high-performance liquid chromatography (HPLC) coupled with a LTQ/orbitrap mass spectrometer. The stepwise MDF with multiple mass defect windows or mass windows enabled the original data to be analysed much faster and more accurately by reducing the potential interferences of matrix ions. Additionally, analysis of DPIs could provide a criterion to classify the target constituents detected into certain chemical families. RESULTS: In total, 81 PMFs, including 50 polymethoxyflavones and 31 polymethoxyflavanones or polymethoxychalcones, were screened and identified from the original data and preliminarily identified. CONCLUSION: The analytical methodology developed could be used as a rapid, effective technique to screen and identify compounds from TCM extracts and other organic matter mixtures with compounds that can also be classified into families based on the common carbon skeletons.

Rapid profiling and identification of puerarin metabolites in rat urine and plasma after oral administration by UHPLC-LTQ-Orbitrap mass spectrometer.

Puerarin, a bioactive natural C-glycoside isoflavonoid isolated from Pueraria lobata (Willd.) Ohwi, possesses many potential health benefits. However, the in vivo metabolic fates of puerarin have not been comprehensively clarified yet. In this study, an efficient strategy based on UHPLC-LTQ-Orbitrap mass spectrometer in both positive and negative ion modes was developed to profile and characterize puerarin metabolites in rat urine and plasma. Meanwhile, post-acquisition data-mining methods including high-resolution extracted ion chromatogram (HREIC) and multiple mass defect filtering (MMDF) were utilized to screen potential puerarin metabolites from HR-ESI-MS1 stage. The mass fragmentation behaviors of five reference standards, including puerarin, daidzin, daidzein, genistin, and genistein, were comprehensively studied for construction of diagnostic product ions (DPIs), which could be employed to implement rapid identification of puerarin metabolites. Finally, a total of 66 metabolites (prototype compound included) were tentatively or positively identified based on standard substances, chromatographic retention times, accurate mass measurement, and corresponding ClogP values. Our results demonstrated that puerarin underwent multiple in vivo metabolic reactions including methylation, hydroxylation, dehydroxylation, hydrogenation, deglycosylation, glycosylation, sulfonation, glucuronidation, and their complicated reactions. In conclusion, the newly discovered puerarin metabolites significantly expanded the understanding on its pharmacological effects and built the foundation for further toxicity and safety studies.

Rapid screening and identification of target constituents using full scan-parent ions list-dynamic exclusion acquisition coupled to diagnostic product ions analysis on a hybrid LTQ-Orbitrap mass spectrometer.

A highly sensitive and effective strategy for rapid screening and identification of target constituents has been developed using full scan-parent ions list-dynamic exclusion (FS-PIL-DE) acquisition coupled to diagnostic product ions (DPIs) analysis on a hybrid LTQ-Orbitrap mass spectrometer. The FS-PIL-DE was adopted as a survey scan to trigger the MS/MS acquisition of all the predictable constituents contained in traditional Chinese medicines. Additionally, DPIs analysis can provide a criterion to judge the target constituents detected into certain chemical families. Results from analyzing polymethoxylated flavonoids (PMFs) in the leaves of Citrus reticulata Blanco demonstrated that FS-PIL-DE was capable of targeting a greater number of constituents than FS, FS-PIL and FS-DE, thereby increasing the coverage of constituent screening. As a result, 135 PMFs including 81 polymethoxyflavones, 54 polymethoxyflavanones or polymethoxychalcones were identified preliminarily. And this was the first time to systematically report the presence of PMFs in the leaves of Citrus reticulata Blanco, especially for polymethoxylated flavanones and chalcones, most of which were new compounds. The results indicated that the developed FS-PIL-DE coupled to DPIs analysis methodology could be employed as a rapid, effective technique to screen and identify target constituents from TCMs extracts and other organic matter mixtures whose compounds contained can also be classified into families based on the common carbon skeletons.