Ultra-high Performance Liquid Chromatography-quadrupole Time-of-flight Mass Spectrometry Research Articles

Mass Spectral Character of Fentanyl Analogues.

Objective To provide the reference for the identification of unknown fentanyl analogues by studying the characteristic ions and main fragmentation pathways of fentanyl analogues in the modes of collision induced dissociation （CID） and electron ionization （EI）. Methods Nine fentanyl analogues （2, 2'-difluorofentanyl, acetyl fentanyl, fentanyl, butyl fentanyl, valeryl fentanyl, acryloyl fentanyl, furan fentanyl, 4-fluorine isobutyl fentanyl, carfentanyl） were selected and analyzed with ultra-high performance liquid chromatography-quadrupole time-of-flight-mass spectrometry （UHPLC-QTOF-MS） and gas chromatography-mass spectrometry （GC-MS）. The mass spectrum obtained was analyzed. The CID and EI fragmentation routes of fentanyl analogues were speculated. Results The CID and EI fragmentation pathways were highly similar. In the CID mode, characteristic ions were formed by the carbon-nitrogen bond cleavage between the piperidine ring and the N-phenyl-amide moiety, within the piperidine ring, and between the phenethyl and piperidine ring. While in the EI mode, dissociation of the piperidine ring, as well as cleavage between the piperidine ring and the phenethyl were the main fragmentation pathways. Conclusion This study summarizes the main fragmentation pathways and characteristic ions of fentanyl analogues in the CID and EI modes, which is useful for forensic laboratories to identify and structural analyze fentanyl type new psychoactive substance in practical work.

Study on chemical constituents of herbal formula Er Miao Wan and GC–MS based metabolomics approach to evaluate its therapeutic effects on hyperuricemic rats

Hyperuricemia strongly correlates with an increased risk of the development of gout, and cardiovascular and kidney diseases, etc. Er Miao Wan (EMW) is a classical traditional Chinese medicine (TCM) formula extensively used for the treatment of hyperuricemia and gout. However, the global components and action mechanism of the formula are still unknown. Here, the chemical constituents of EMW extract were identified by ultra-high performance liquid chromatography quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS) and gas chromatography–mass spectrometry (GC–MS). A total of 24 alkaloids, 15 organic acids, 4 terpenoids, 3 lactones, 3 glycosides, 46 volatile constituents and 3 other compounds were tentatively identified from the EMW extract. Additionally, based on the hyperuricemic rat model induced by long-term high-fructose feed, a GC–MS based metabolomics approach was conducted to holistically assess the mechanism of EMW. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were applied for screening differential metabolites. A total of 21 metabolites that markedly changed in hyperuricemic rats were identified. Further univariate analysis showed that 9 differential metabolites among them were profoundly reversed by EMW intervention. Metabolic pathway analysis revealed that the variations of these metabolites were mainly associated with glycerolipid metabolism, amino acid metabolism, primary bile acid metabolism, taurine and hypotaurine metabolism and purine metabolism. It was inferred that EMW possibly induced its anti-hyperuricemic effect through restoring multiple disturbed pathways to the normal state. This study could assist with elucidating the potential mechanisms of EMW.

The earliest excipient products of Traditional Chinese Medicine: Identification and analysis of samples from wooden lacquer box unearthed from Haihunhou tomb in the Western Han Dynasty

The processing of traditional Chinese medicine is an integral part of Chinese medical heritage. The recent discovery of the earliest processed drugs may provide strong evidence for the origins of traditional Chinese medicine processing. The remains were found in wooden lacquer boxes, unearthed from Haihunhou tomb in Nanchang, Jiangxi Province. By analyzing the appearance of the unearthed samples, it was found that the outer layers of the samples were composed of an auxiliary material and the inside was composed of plant material. As can be seen from the three-dimensional image taken using nuclear magnetic resonance screening, the sample had a hollow irregular rod structure. The outer layers were composed of a substance with weak signal strength, and the inside was similar to the fiber structure of plants with high water contents. Microscopic analysis showed that periderm, secondary xylem, and broad wood rays could be seen in the transverse section of the plants inside the samples. It can be further speculated that the plants inside the samples were derived from the roots or stems of dicotyledonous herbs. Additionally, the putative roots or stems of the dicotyledonous herbs had secondary structures and contained one to three clustered, red-brown cells. No starch granules, calcium oxalate crystals, or stone cells were observed in the transverse section. According to the microscopic database of the National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, it is speculated that the plants inside the samples were derived from the roots of Rehmannia species. A comparative study of the microscopic characteristics of wild Rehmannia chingii roots and the unearthed samples was carried out. It was found that both displayed periderm, broad secondary phloem, wood rays, similar vessel morphologies, and red-brown cells. The unearthed samples and R. chingii roots were analyzed by electrospray ionization mass spectrometry (ESI-MS) in negative ion mode. The results showed that the mass spectrum peak of m / z 623 in the unearthed samples agreed with that in the roots of R. chingii , and was putatively identified as acteoside or forsythiaside. Moreover, the peak at m / z 179 in the unearthed samples was identified as caffeic acid based on the molecular weight using high resolution tandem mass spectrometry (MS/MS) data, and was usually identified as a hydrolysate of acteoside or forsythiaside. There were about two to three outer layers of excipient on the samples. They were distributed unevenly, thicker at the top and thinner at the bottom. The plants and excipient layers were observed by microscope, and starch grains were found in the cross section and powder of the excipient layers, while no starch grains were found in the cross section or powder of the plant material inside. However, the reaction between the plant tissues and iodine-potassium iodide test solution produced the characteristic blue color. This phenomenon was related to the gelatinization of starch granules. Under a polarized light microscope, a large number of crystals were seen in the excipient layers. An aqueous extract of the excipient layers was analyzed by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS), and an ion fragment peak at m / z 341.11 was detected, indicating a disaccharide. When compared to the reference substances glucose, sucrose, fructose, and maltose, the retention times and mass spectra were very similar to that of sucrose. According to ancient literary and historical records, the tomb owner suffered from severe rheumatism before his death, which is consistent with the efficacy of R. chingii . It is further speculated that the samples in the wooden lacquer boxes are the remains of processed ancient traditional Chinese medicine products. The origin of the excipients in the processed medicines might be related to improving the taste, which could provide a scientific basis for further understanding the processing and application of ancient traditional Chinese medicines.

Structural characterisation and discrimination of the aerial parts of Paris polyphylla var. yunnanensis and Paris polyphylla var. chinensis by UHPLC-QTOF-MS coupled with multivariate data analysis.

As sources of Rhizoma Paridis are facing shortages, utilising the aerial parts of Paris polyphylla has emerged as a promising additional source. However, the components in the aerial parts still need to be explored, and it is difficult to distinguish the aerial parts of P. polyphylla Smith var. yunnanensis (PPY) and P. polyphylla var. chinensis (PPC), two varieties of P. polyphylla. This study aimed to establish a comprehensive platform to characterise steroid saponins from the aerial parts of PPY and PPC and to discriminate these two varieties. A dereplication approach and ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) analysis were used for the characterisation of steroidal saponins in the aerial parts of PPY and PPC. Multivariate statistical analysis was performed to differentiate these two varieties and screen discriminant variables. In addition, a genetic algorithm-optimised for support vector machines (GA-SVM) model was developed to predict P. polyphylla samples. The distribution of steroidal saponins in PPY and PPC was visualised by a heatmap. A total of 102 compounds were characterised from the aerial parts of PPY and PPC by dereplication. A clear separation of PPY and PPC was achieved, and 35 saponins were screened as marker compounds. The established GA-SVM model showed excellent prediction performance with a prediction accuracy of 100%. Many steroid saponins that have been reported in Rhizoma Paridis also exist in the aerial parts of P. polyphylla. Samples from the aerial parts of PPY and PPC could be discriminated using our platform.

Comprehensive analysis of the metabolomic characteristics on the health lesions induced by chronic arsenic exposure: A metabolomics study.

Early detection of the health lesions induced by chronic arsenic exposure (HLICAE) are crucial to prevent permanent arsenic-induced damage. If HLICAE can be identified in time, appropriate preventive and therapeutic measures may be provided without various avoidable lesions. The present study aims to assess the probability of HLICAE early recognition with metabolomics. Applying a case-control study, 94 participants with HLICAE (cases) and other 94 subjects without HLICAE (controls) were matched with gender and age (±1 year), coming from a previous chronic arsenic exposure cohort. Serum metabolomic profiles were assessed by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) and analyzed with univariate and multivariate statistics. A total of 210 and 364 features were detected in positive and negative ion modes (ESI+/ESI-), respectively. The altered metabolic pathways included lipid and amino acid metabolisms. 28 metabolomics-based biomarkers were significantly associated with HLICAE and provided areas under the curve (AUC, 95% confidence interval) of 0.898 (0.836, 0.960) and 0.908 (0.855, 0.960) in the discovery phase, 78.6% and 86.4% of positive predictive values in the validation phase, in distinguishing HLICAE from controls in ESI+/ESI-, respectively. This study provides novel insights on mechanisms of health effects probably induced by chronic arsenic exposure, and these biomarkers may be applied in HLICAE early detection.

Plasma metabolites associated with colorectal cancer: A discovery-replication strategy.

Colorectal cancer is known to arise from multiple tumorigenic pathways; however, the underlying mechanisms remain not completely understood. Metabolomics is becoming an increasingly popular tool in assessing biological processes. Previous metabolomics research focusing on colorectal cancer is limited by sample size and did not replicate findings in independent study populations to verify robustness of reported findings. Here, we performed a ultrahigh performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (UHPLC‐QTOF‐MS) screening on EDTA plasma from 268 colorectal cancer patients and 353 controls using independent discovery and replication sets from two European cohorts (ColoCare Study: n = 180 patients/n = 153 controls; the Colorectal Cancer Study of Austria (CORSA) n = 88 patients/n = 200 controls), aiming to identify circulating plasma metabolites associated with colorectal cancer and to improve knowledge regarding colorectal cancer etiology. Multiple logistic regression models were used to test the association between disease state and metabolic features. Statistically significant associated features in the discovery set were taken forward and tested in the replication set to assure robustness of our findings. All models were adjusted for sex, age, BMI and smoking status and corrected for multiple testing using False Discovery Rate. Demographic and clinical data were abstracted from questionnaires and medical records.

UHPLC-QTOF/MS-based metabolomics investigation for the protective mechanism of Danshen in Alzheimer's disease cell model induced by Aβ1-42.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder with neither definitive pathogenesis nor effective therapy so far. Danshen, the dried root and rhizome of Salvia miltiorrhiza Bunge, is used extensively in Alzheimer's disease treatment to ameliorate the symptoms, but the underlying mechanism remains to be clarified. To investigate potential biomarkers for AD and elucidate the protective mechanism of Danshen on AD cell model. An ultra high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-QTOF/MS)-based approach combined with partial least squares discriminant analysis (PLS-DA) has been developed to discriminate the metabolic modifications between human brain microvascular endothelial cell (hBMEC) and AD cell model induced by amyloid-β protein (Aβ1-42). To further elucidate the pathophysiology of AD, related metabolic pathways have been studied. Thirty-three distinct potential biomarkers were screened out and considered as potential biomarkers corresponding to AD, which were mostly improved and partially restored back to normalcy in Danshen pre-protection group. It was found that AD was closely related to disturbed arginine and proline metabolism, glutathione metabolism, alanine aspartate and glutamate metabolism, histidine metabolism, pantothenate and CoA biosynthesis, phenylalanine tyrosine and tryptophan biosynthesis, citrate cycle and glycerophospholipid metabolism, and the protective mechanism of Danshen in AD cell model may be related to partially regulating the perturbed pathways. These outcomes provide valuable evidences for therapeutic mechanism investigation of Danshen in AD treatment, and such an approach could be transferred to unravel the mechanism of other traditional Chinese medicine (TCM) and diseases.

Discovery and characterisation of lycorine-type alkaloids in Lycoris spp. (Amaryllidaceae) using UHPLC-QTOF-MS.

Lycorine, one of the most common alkaloids in Lycoris spp., is believed to possess pharmacological activity. To discover and identify lycorine-type alkaloids in the crude extracts of bulbs from six Lycoris spp. by ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) detection. A qualitative analytical method with a data mining strategy was utilised. Based on the fragmentation patterns of standards investigated in positive tandem mass spectrometry (MS/MS) mode, the fragmentation rules of lycorine-type alkaloids were summarised. These types of alkaloids were additionally classified as different subtypes based on structural features and MS/MS fragmentation patterns, and the diagnostic ions for characterisation of different subtypes of alkaloids were designated. Thirty-seven lycorine type alkaloids, including 16 previously undescribed compounds, were efficiently screened out and tentatively identified from the crude extracts of six Lycoris spp. Lycoris sprengri may be a preferable species for studying or extracting lycorine-type alkaloids because of elevated relative concentrations and highest diversity of alkaloids. The UHPLC-QTOF-MS and MS/MS data-mining strategy proved useful for the detection and tentative identification of lycorine-type alkaloids in bulbs of Lycoris spp. and could be extended to other Amaryllidaceae genera. The consequent profiling of the lycorine-type alkaloids will be useful in the quality control of raw materials of Lycoris species and the exploration of superior species.

Neuroprotective Effect of Dichloromethane Extraction From Piper nigrum L. and Piper longum L. on Permanent Focal Cerebral Ischemia Injury in Rats

Piper nigrum L. and Piper longum L. consist a classic formula in traditional Chinese Hui medicine and are widely used in treatment of stroke. To examine the therapeutic effect of neuron injury after apoplexy, we used a permanent middle cerebral artery occlusion model in rats to investigate the effects of dichloromethane fraction (DF) of Piper nigrum L. and Piper longum L. After subjecting the rats to permanent middle cerebral artery occlusion, DF (100 and 200 mg/kg) were administered for 14 days. Neurological deficits and the degree of cerebral tissue injury was detected by 2,3,5-Triphenyltetrazolium Chloride Staining Hematoxylin and eosin staining and Nissl staining. Postsynaptic density protein 95 (PSD-95), synapsin-I (syn-I), and α-synuclein (α-syn) were stained by immunohistochemistry. PSD-95, Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMK II), phosphorylated CaMK II (p-CaMK II), CaM, N-methyl D-aspartate receptor subtype 2B (NR2B) expression were detected by Western blot. Meanwhile, phytochemical profile of DF was determined through ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). DF alleviated neurological deficits and markedly prevented ischemia-induced cellular damage. Immunohistochemical micrographs revealed that PSD-95 and syn-I proteins increased, and α-syn presented reduced expression in brain samples from the sham group. Western blot analyses revealed that the model group exhibited a noticeable reduction in PSD-95, p-CaMK II, CaM, and NR2B. The DF-treated model group exhibited increased PSD-95, p-CaMK II, CaM, and NR2B. UPLC-Q-TOF/MS analysis revealed eight main components of DF, of which piperine accounted for the largest proportion.

Analysis of Active Metabolites of Sophora flavescens for Indoleamine 2,3-dioxygenase and Monoamine Oxidases using Ultra-Performance Liquid Chromatography-Quadrupole time-of-Flight Mass Spectrometry

As part of ongoing research on natural products derived from medicinal plants for enzyme inhibition, known dibenzoyl derivatives (1–3, 11 and 20), pterocarpans (4, 15 and 19), flavanones (5, 7, 10, 12–14, 18, 21–24, 26, 27, 29, 31–33, 35, 36, and 38–46), flavones (6, 16, 28, 30 and 37), isoflavones (8 and 17), furocoumarins (9), and chalcones (25 and 34) have been tentatively identified within fractions of Sophora flavescens roots (SFR) using the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTof-MS) technique. The extract and column fractions inhibited indoleamine 2,3-dioxygenase (IDO) and monoamine oxidases (MAOs) differently depending on the metabolite groups. The majority of rich fractions were shown to have residual activities of 49–59% at 10 μg/mL (IDO) and 11.7–34.9% at 50 μg/mL (MAOs) or below. In the total ion current (TIC) chromatogram, significant markers for the metabolites of the bioactive-guided fractions were identified; pterocarpans (4, 15 and 19), flavanones (5, 10, 12–14, 18, 21–23, 26, 29 31–33, 35, 36, and 38–46), isoflavones (8 and 17), furocoumarins (9), dibenzoyl derivatives (11 and 20), flavones (16, 28, 30 and 37), and chalcones (25 and 34) were evaluated among forty-six analyzed metabolites. Possible bioactive markers could be deduced using a data library and previous references, and information regarding spectroscopic characterization and optimal target metabolites was obtained.

Non-target metabonomic method provided new insights on the therapeutical mechanism of Gancao Fuzi decoction on rheumatoid arthritis rats

Gancao Fuzi decoction (GFD) is a classic Chinese medicine formula for the treatment of rheumatic and rheumatoid arthritis. The main active components of GFD are alkaloids, flavonoids and saponins. This study aimed to clarify the pharmacodynamic effects of the active components in GFD and investigate the mechanism of them treating rheumatoid arthritis rats by the method of metabonomics. Seven groups were studied, named as the normal group (NG), the model group (MG), the Gancao Fuzi decoction treatment group (GFDe), the alkaloids group (ALK), the compatibility of alkaloids with flavonoids group (AF), the compatibility of alkaloids with saponins group (AS) and the compatibility of alkaloids with flavonoids and saponins group (AFS), respectively. Firstly, the anti-inflammatory and analgesic effects of these groups were studied. Besides, urinary metabonomics based on ultra high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was employed for delineation of metabolic alterations in the rats. Based on our results, it is concluded that AFS showed better anti-inflammatory and analgesic activities in GFD. Urinary metabonomic study and multivariate statistical analyses were used to investigate the mechanism of different groups. 26 potential biomarkers have been identified. By the analysis of heat map combined with score plot, the AFS group was the closest group to the NG group after treatment in GFD. The changes of urinary endogenous metabolites showed that AFS exhibited better effect on regulating the taurine and hypotaurine metabolism, phenylalanine metabolism, TCA cycle, tryptophan metabolism, fatty acid metabolism, vitamin B6 metabolism, arginine and proline metabolism and purine metabolism pathways. The pharmacodynamics results showed that three components of flavonoids, saponins and alkaloids in GFD played an overall efficacy. Metabonomics studies showed that the compatibility of three components in GFD achieved the therapeutic effect by regulating the perturbations of multiple metabolic pathways.

Metabolomics analysis and rapid identification of changes in chemical ingredients in crude and processed Astragali Radix by UPLC-QTOF-MS combined with novel informatics UNIFI platform

Astragali Radix, the root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge., is widely used as a tonic decoction pieces in the clinic of traditional Chinese medicine (TCM). Astragali Radix has various processed products with varying pharmacological actions. There is no modern scientific evidence to explain the differences in pharmacological activities and related mechanisms. In the present study, we explore the changes in chemical components in Astragali Radix after processing, by ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) combined with novel informatics UNIFI platform and multivariate statistical analysis. Our results showed that the crude and various processed products could be clearly separated in PCA scores plot and 15 significant markers could be used to distinguish crude and various processed products by OPLS-DA in UNIFI platform. In conclusion, the present study provided a basis of chemical components for revealing connotation of different processing techniques on Astragali Radix.

Untargeted metabolomics profiles delineate metabolic alterations in mouse plasma during lung carcinoma development using UPLC-QTOF/MS in MSE mode.

In this work, an untargeted metabolomic method based on ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) in MSE (E represents collision energy) mode was exploited to determine the dynamic metabolic alterations in the plasma of male C57BL/6 mice during the onset and development of lung carcinoma. Plasma samples were collected from control and model mice (male C57BL/6 mice experimentally inoculated with the Lewis lung carcinoma cells) at 7 and 14 days post-inoculation (DPI). As a result, 15 dysregulated metabolites, including cholesterol sulphate, tiglylcarnitine, 1-palmitoylglycerophosphoinositol, 2-stearoylglycerophosphoinositol, stearoylcarnitine, PC(20:2(11Z,14Z)/16:0), PC(22:4(7Z,10Z,13Z,16Z)/14:0), PC(22:5(7Z,10Z,13Z,16Z,19Z)/14:0), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/16:0), 12,20-Dioxo-leukotriene B4, sphingosine 1-phosphate(d19:1-P), sphingomyelin(d18:0/16:1(9Z)), lysoPC(16:0), lysoPC(18:0) and lysoPC(20:4(5Z,8Z,11Z,14Z)), were identified in the plasma of model mice with xenografts at both 7 and 14 DPI. All the altered metabolites associated with the onset and development of lung carcinoma were involved in the metabolism of glycerophospholipid, fatty acid, sphingolipid and arachidonic acid. The feasible utility of these endogenous biomarkers as potential diagnostic indicators was validated through receiver operating characteristic curve analysis. Collectively, these findings provide a systematic view of metabolic changes linked to the onset and development of lung carcinoma.

Identification of metabolites of Helicid in vivo using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

Helicid is an active natural aromatic phenolic glycoside ingredient originating from a well-known traditional Chinese herbal medicine and has the significant effects of sedative hypnosis, anti-inflammatory analgesia and antidepressant. In this study, we analyzed the potential metabolites of Helicid in rats by multiple mass defect filter and dynamic background subtraction in ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). Moreover, we used a novel data processing method, 'key product ions', to rapidly detect and identify metabolites as an assistant tool. MetabolitePilot™ 2.0 software and PeakView™ 2.2 software were used for analyzing metabolites. Twenty metabolites of Helicid (including 15 phase I metabolites and five phase II metabolites) were detected by comparison with the blank samples. The biotransformation route of Helicid was identified as demethylation, oxidation, dehydroxylation, hydrogenation, decarbonylation, glucuronide conjugation and methylation. This is the first study simultaneously detecting and identifying Helicid metabolism in rats employing UHPLC-Q-TOF-MS technology. This experiment not only proposed a method for rapidly detecting and identifying metabolites, but also provided useful information for further study of the pharmacology and mechanism of Helicid in vivo. Furthermore, it provided an effective method for the analysis of other aromatic phenolic glycosides metabolic components in vivo.

Detection of novel metabolite for roxadustat doping by global metabolomics.

Roxadustat (FG-4592, Rox) is a stabilizer for hypoxia-inducible transcription factors (HIFs), which induce production of the erythroid growth factor erythropoietin, and has been listed by the World Anti-Doping Agency as a prohibited substance for athletes since 2011. Although the detection technologies for Rox and its glucuronide-conjugated metabolite (Rox-Gluc) have been developed exploiting triple quadrupole mass spectrometry (MS/MS), the production of metabolites from Rox in the human body remains to be clarified. Here, we established a protocol for the detection of unknown metabolites in plasma and urine samples from Rox-doping mice by global metabolomics using an ultra high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS). We identified methylated Rox (Rox-Methyl), a novel metabolite, and Rox-Gluc in mouse urine by principal component analysis and orthogonal partial least squares discriminant analysis based on detected features by UHPLC-QTOF/MS analysis. The estimated pharmacokinetic parameters of Rox-Methyl and Rox-Gluc in mouse plasma showed similar profiles to that of Rox and both compounds showed similar biological activities. Of note, Rox-Methyl showed shorter half-life than Rox-Gluc in vivo, implying the easy escape from anti-doping screen. These results demonstrate that the global metabolomics method introduced in this study will contribute to the identification and detection of HIF-analog doping.

A network pharmacology-integrated metabolomics strategy for clarifying the difference between effective compounds of raw and processed Farfarae flos by ultra high-performance liquid chromatography-quadrupole-time of flight mass spectrometry

This study aimed to clarify the difference between the effective compounds of raw and processed Farfarae flos using a network pharmacology-integrated metabolomics strategy. First, metabolomics data were obtained by ultra high-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF/MS). Then, metabolomics analysis was developed to screen for the influential compounds that were different between raw and processed Farfarae flos. Finally, a network pharmacology approach was applied to verify the activity of the screened compounds. As a result, 4 compounds (chlorogenic acid, caffeic acid, rutin and isoquercitrin) were successfully screened, identified, quantified and verified as the most influential effective compounds. They may synergistically inhibit the p38, JNK and ERK-mediated pathways, which would induce the inhibition of the expression of the IFA virus. The results revealed that the proposed network pharmacology-integrated metabolomics strategy was a powerful tool for discovering the effective compounds that were responsible for the difference between raw and processed Chinese herbs.

Qualitative and quantitative analysis of lipo-alkaloids and fatty acids in Aconitum carmichaelii using LC-MS and GC-MS.

Lipo-alkaloid (LA) is a kind of C19 -norditerpenoid alkaloid in Aconitum species, which usually contains an aconitane skeleton and one or two fatty acid residues. To qualify and quantify the fatty acids and lipo-alkaloids in Aconitum carmichaelii. An ultra-high performance liquid chromatography-triple quadrupole-mass spectrometry (UHPLC-QQQ-MS) method was established to quantify LAs, while the free fatty acids were identified by gas chromatography-mass spectrometry (GC-MS) and ultra-high performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (UHPLC-Q-TOF-MS). Six major LAs (1-6) containing linoleic, palmitic, and oleic acid residues as side chains were quantified. Eighteen fatty acids were determined by GC-MS, and 15 were detected as the side chains of LAs. The LAs containing these 15 fatty acid residues accounted for about a third of the total identified LAs. Moreover, the contents of linoleic, palmitic, and oleic acids were highest. In addition, 12 oxygenated fatty acids were also identified by UHPLC-Q-TOF-MS for the first time. The positive correlation between free fatty acids and LAs in A. carmichaelii indicated that the types and contents of LAs were influenced by free fatty acids.

Nuciferine and paeoniflorin can be quality markers of Tangzhiqing tablet, a Chinese traditional patent medicine, based on the qualitative, quantitative and dose-exposure-response analysis

BackgroundDiabetes is a chronic disease associated with significant morbidity and mortality. Tangzhiqing tablet (TZQ), a Chinese traditional patent medicine, has been in phase 2 clinical trial for the treatment of diabetes mellitus. However, the current quality evaluation of TZQ still remains rather obscure. PurposeThe promising quality markers (Q-markers) of TZQ will be sought for its quality assessment and process control based on the qualitative, quantitative and dose-exposure-response analysis. MethodsThe fingerprint analysis of TZQ was carried out through ultra high performance liquid chromatography- quadrupole time-of-flight/ mass spectrometry (UPLC-Q-TOF/MS) assay. Multicomponent quantitative analysis was implemented to the main ingredients of nuciferine, paeoniflorin, salvianolic acid B, hyperoside and rutin in TZQ by means of LC analysis. The dose-exposure-response relationship of TZQ was revealed by a placebo-controlled, 5-way crossover study in healthy Chinese subjects. The potential Q-markers in plasma were determined by a liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) method. The therapeutic effect of TZQ was expressed as the glucose-lowering profile. The exposure-response relevance was developed between the concentration of Q-markers and the glucose-lowering effect of TZQ. Results46 compounds were identified or tentatively characterized from TZQ. The contents of paeoniflorin, nuciferine, salvianolic acid B, hyperoside and rutin in TZQ were 6.40, 1.75, 1.70, 0.004, and 0.006 mg, respectively. However, salvianolic acid B, hyperoside and rutin could hardly be detected in human plasma under the current LC-MS/MS condition. The exposures of nuciferine and paeoniflorin (AUC0-3, Cmax) were dose-proportionality in human at the studied dosage ranges. The glucose-lowering effect appeared to increase proportionally with increasing TZQ dose in healthy volunteers. A clockwise hysteresis was displayed between the exposure of nuciferine and paeoniflorin and the glucose-lowering effect of TZQ. ConclusionNuciferine and paeoniflorin were identified as the promising Q-markers of TZQ based on the fingerprint qualitative analysis, multicomponent quantitative analysis and dose-exposure-response analysis. The two Q-markers are meaningful to ensure the quality assessment and process control of TZQ.

Detection of novel metabolite for Roxadustat doping by global metabolomics.

Roxadustat (FG-4592, Rox) is a stabilizer for hypoxia-inducible transcription factors (HIFs), which induce production of the erythroid growth factor erythropoietin, and has been listed by the World Anti-Doping Agency as a prohibited substance for athletes since 2011. Although the detection technologies for Rox and its glucuronide-conjugated metabolite (Rox-Gluc) have been developed exploiting triple quadrupole mass spectrometry (MS/MS), the production of metabolites from Rox in the human body remains to be clarified. Here, we established a protocol for the detection of unknown metabolites in plasma and urine samples from Rox-doping mice by global metabolomics using an ultra high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS). We identified methylated Rox (Rox-Methyl), a novel metabolite, and Rox-Gluc in mouse urine by principal component analysis and orthogonal partial least squares discriminant analysis based on detected features by UHPLC-QTOF/MS analysis. The estimated pharmacokinetic parameters of Rox-Methyl and Rox-Gluc in mouse plasma showed similar profiles to that of Rox and both compounds showed similar biological activities. Of note, Rox-Methyl showed shorter half-life than Rox-Gluc in vivo, implying the easy escape from anti-doping screen. These results demonstrate that the global metabolomics method introduced in this study will contribute to the identification and detection of HIF-analog doping.

Metabolomics Study of Cultivated Bulbus Fritillariae Cirrhosae at Different Growth Stages using UHPLC-QTOF-MS Coupled with Multivariate Data Analysis.

Bulbus fritillariae cirrhosae (known as Chuān bèi mǔ in China, BFC) contain fritillaria steroidal alkaloids as the bioactive ingredients and are widely used as traditional Chinese medicine for the treatment of cough and phlegm. Due to limited wild resources, the cultivated species are becoming predominantly used in Chinese traditional medicine markets. To assess the impact of different growth stages on the alkaloids of cultivated BFC and establish a reference for quality control and guidance for appropriate harvesting practices. The ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) metabolomic strategy was applied to determine potential chemical markers for the discrimination and quality control of cultivated BFC in different growth stages. The molecular feature extraction and multivariate statistical analysis were applied to alkaloid extraction and full metabolomic profiling of cultivated BFC for classification and marker compound characterisation. This approach allowed the establishment of a fast and efficient comparative multivariate analysis of the metabolite composition of 42 samples covering growth of cultivated BFC ranging in age from one to seven years old. Four alkaloid compounds were identified in cultivated BFC based on accurate mass, retention time, and MS/MS fragments. These compounds may be used as potential chemical markers for the classification and discrimination of cultivated BFC samples indifferent growth stages. The proposed analytical method in combination with multivariate statistical analysis comprised a useful and powerful strategy to explore the chemical ingredients and transforming mechanisms of cultivated BFC and for quality evaluation and control. Copyright © 2018 John Wiley & Sons, Ltd.