Ultra-high-performance Liquid Chromatography Tandem Quadrupole Time-of-flight Mass Spectrometry Research Articles

A strategy for intelligent chemical profiling-guided precise quantitation of multi-components in traditional Chinese medicine formulae-QiangHuoShengShi decoction

Due to the tremendous clinical value, more and more Traditional Chinese Medicines (TCMs) and their formulae are attracted by world's attention. QiangHuoShengShi (QHSS) decoction is one of classic TCM formulae, which is clinically used for treating various rheumatic diseases. However, the phytochemical constituents of QHSS have rarely been reported. A simple, intelligent, and comprehensive strategy was developed to characterize the phytochemical-fingerprint and quantify the chemical-markers for precise quality evaluation of QHSS. Firstly, a new deep-learning assisted mass defect filter (MDF) method was built for rapid and accurate classification of mass spectrum (MS) ions acquired by ultra-high performance liquid chromatography quadrupole time of flight tandem mass spectrometry (UHPLC-Q-TOF/MS). Subsequently, herb species-specific chemical-category and characteristic identification were used for further characterization of multi-components. As the result, seven major types of compounds in QHSS were intelligently differentiated and 183 phytochemical compounds were tentatively identified. Finally, a sensitive scheduled multiple reaction monitoring (sMRM) detection method was applied to precisely quantify 37 target analytes in QHSS decoction. This integrated strategy would provide an alternative method for chemical-material basis study of more herbal medicine or natural products.

An Integrative Pharmacology-Based Strategy to Uncover the Mechanism of Xiong-Pi-Fang in Treating Coronary Heart Disease with Depression.

Objectives: This study aimed to explore the mechanism of Xiong-Pi-Fang (XPF) in the treatment of coronary heart disease (CHD) with depression by an integrative strategy combining serum pharmacochemistry, network pharmacology analysis, and experimental validation. Methods: An ultrahigh performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS) method was constructed to identify compounds in rat serum after oral administration of XPF, and a component-target network was established using Cytoscape, between the targets of XPF ingredients and CHD with depression. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed to deduce the mechanism of XPF in treating CHD with depression. Finally, in a chronic unpredictable mild stress (CUMS)-and isoproterenol (ISO)-induced rat model, TUNEL was used to detect the apoptosis index of the myocardium and hippocampus, ELISA and western blot were used to detect the predicted hub targets, namely AngII, 5-HT, cAMP, PKA, CREB, BDNF, Bcl-2, Bax, Cyt-c, and caspase-3. Results: We identified 51 compounds in rat serum after oral administration of XPF, which mainly included phenolic acids, saponins, and flavonoids. Network pharmacology analysis revealed that XPF may regulate targets, such as ACE2, HTR1A, HTR2A, AKT1, PKIA, CREB1, BDNF, BCL2, BAX, CASP3, cAMP signaling pathway, and cell apoptosis process in the treatment of CHD with depression. ELISA analysis showed that XPF decreased Ang-II content in the circulation and central nervous system, inhibited 5-HT levels in peripheral circulation, and increased 5-HT content in the central nervous system and cAMP content in the myocardia and hippocampus. Meanwhile, western blot analysis indicated that XPF could upregulate the expression levels of PKA, CREB, and BDNF both in the myocardia and hippocampus. TUNEL staining indicated that the apoptosis index of myocardial and hippocampal cells increased in CUMS-and ISO-induced CHD in rats under depression, and XPF could increase the expression of Bcl-2, inhibit the expression of Bax, Cyt-c, and caspase-3, and rectify the injury of the hippocampus and myocardium, which exerted antidepressant and antimyocardial ischemia effects. Conclusion: Our study proposed an integrated strategy, combining serum pharmacochemistry and network pharmacology to investigate the mechanisms of XPF in treating CHD with depression. The mechanism of XPF in treating CHD with depression may be related to the activation of the cAMP signaling pathway and the inhibition of the apoptosis.

Discovery of potential biomarkers in acute kidney injury by ultra-high-performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS).

The LC-MS/MS-based non-targeted metabolomics method was used to differentially screen serum and urine metabolites of acute kidney injury (AKI) patients and healthy people, to explore potential biomarkers of AKI and analyze related pathways, and explain the potential mechanism and biological significance of AKI. The serum and urine samples from 30 AKI patients and 20 healthy people were selected to conduct a non-targeted metabolomics study by ultra-high-performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS). The differential metabolites between the two groups were searched by the human metabolome (HMDB) database ( https://hmdb.ca/ ) and the related pathways of these potential biomarkers were identified by searching the Kyoto encyclopedia of genes and genomes (KEGG) database ( https://www.kegg.jp/ ). The total metabolic pathways were analyzed by the MS Peaks to Pathways module of MetaboAnalyst ( https://www.metaboanalyst.ca/ ). Multivariate data analysis found that serum and urine metabolism in AKI patients was significantly different from healthy people. We found three metabolites in urine (2-S-glutathionyl glutathione acetate, 5-L-Glutamyl-taurine, and L-Phosphoarginine) contributing to the separation of AKI patients from healthy people, and major metabolic pathways associated with these potential biomarkers including cytochrome P450 metabolism, arginine, and proline metabolism. 2-S-glutathionyl glutathione acetate, 5-L-Glutamyl-taurine, and L-Phosphoarginine were associated with AKI patients, which could be selected as potential biomarkers to predicate AKI disease.

Target and Suspect HRMS Metabolomics for the Determination of Functional Ingredients in 13 Varieties of Olive Leaves and Drupes from Greece.

The huge interest in the health-related properties of foods to improve health has brought about the development of sensitive analytical methods for the characterization of natural products with functional ingredients. Greek olive leaves and drupes constitute a valuable source of biophenols with functional properties. A novel ultra-high-performance liquid chromatography–quadrupole time of flight tandem mass spectrometry (UHPLC-QTOF-MS) analytical method was developed to identify biophenols through target and suspect screening in Greek olive leaves and drupes of the varieties: Koroneiki, Throumbolia, Konservolia, Koutsourelia, Kalamon, Petrolia, Amigdalolia, Megaritiki, Mastoeidis, Agouromanakolia, Agrilia, Adramitiani and Kolovi. The method’s performance was evaluated using the target compounds: oleuropein, tyrosol and hydroxytyrosol. The analytes demonstrated satisfactory recovery efficiency for both leaves (85.9–90.5%) and drupes (89.7–92.5%). Limits of detection (LODs) were relatively low over the range 0.038 (oleuropein)–0.046 (hydroxytyrosol) and 0.037 (oleuropein)–0.048 (hydroxytyrosol) for leaves and drupes, respectively For leaves, the precision limit ranged between 4.7% and 5.8% for intra-day and between 5.8% and 6.5% for inter-day experiments, and for drupes, it ranged between 3.8% and 5.2% for intra-day and between 5.1 and 6.2% for inter-day experiments, establishing the good precision of the method. The regression coefficient (r2) was above 0.99 in all cases. Furthermore, the preparation of herbal tea from olive leaves is suggested after investigating the optimum infusion time of dried leaves in boiling water. Overall, 10 target and 36 suspect compounds were determined in leaves, while seven targets and thirty-three suspects were identified in drupes, respectively.

Authentication of Greek PDO Kalamata Table Olives: A Novel Non-Target High Resolution Mass Spectrometric Approach.

Food science continually requires the development of novel analytical methods to prevent fraudulent actions and guarantee food authenticity. Greek table olives, one of the most emblematic and valuable Greek national products, are often subjected to economically motivated fraud. In this work, a novel ultra-high-performance liquid chromatography–quadrupole time of flight tandem mass spectrometry (UHPLC-QTOF-MS) analytical method was developed to detect the mislabeling of Greek PDO Kalamata table olives, and thereby establish their authenticity. A non-targeted screening workflow was applied, coupled to advanced chemometric techniques such as Principal Component Analysis (PCA) and Partial Least Square Discriminant Analysis (PLS-DA) in order to fingerprint and accurately discriminate PDO Greek Kalamata olives from Kalamata (or Kalamon) type olives from Egypt and Chile. The method performance was evaluated using a target set of phenolic compounds and several validation parameters were calculated. Overall, 65 table olive samples from Greece, Egypt, and Chile were analyzed and processed for the model development and its accuracy was validated. The robustness of the chemometric model was tested using 11 Greek Kalamon olive samples that were produced during the following crop year, 2018, and they were successfully classified as Greek Kalamon olives from Kalamata. Twenty-six characteristic authenticity markers were indicated to be responsible for the discrimination of Kalamon olives of different geographical origins.

Dynamic urinary metabolomics analysis based on UHPLC-Q-TOF/MS to investigate the potential biomarkers of blood stasis syndrome and the effects of Danggui Sini decoction

Blood stasis syndrome (BSS) is one of the common syndromes in traditional Chinese medicine (TCM). It involves abnormal blood circulation, which can progress to produce many severe diseases. Danggui Sini decoction (DSD) is a classical TCM prescription frequently used to treat BSS by decreasing blood stasis and improving blood circulation. However, understanding of the therapeutic mechanism of DSD during the development of BSS is still limited, as the development of BSS is a slow dynamic process. Therefore, a dynamic urinary metabolomics analysis based on ultra-high-performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry (UHPLC-Q-TOF/MS) combined with multivariate statistical analysis was used to explore the distinctive metabolic patterns of BSS development and the efficacy of DSD. The dynamic changes of endogenous metabolites over time revealed the progression of BSS and allowed the overall efficacy of DSD in rats with BSS to be evaluated. The effects of the DSD compatibilities were also explored. A total of 21 metabolites were identified during the development of BSS. They are involved in the metabolic pathways of tryptophan metabolism, phenylalanine metabolism, riboflavin metabolism, nicotinate and nicotinamide metabolism, pentose and glucuronate interconversions, histidine metabolism, steroid hormone biosynthesis, and starch and sucrose metabolism. A receiver operating characteristic (ROC) curve analysis showed that 10 metabolites with an area under the curve (AUC) value >0.9, which can be used as potential biomarkers for the diagnosis of BSS. In conclusion, a dynamic urinary metabolomics approach was applied to identify potential biomarkers of the development of BSS and to clarify the therapeutic mechanism of DSD in BSS. The results could provide a theoretical basis for further research on the therapeutic mechanism of DSD.

In Vitro and In Vivo Metabolomic Profiling after Infection with Virulent Newcastle Disease Virus.

Newcastle disease (ND) is an acute, febrile, highly contagious disease caused by the virulent Newcastle disease virus (vNDV). The disease causes serious economic losses to the poultry industry. However, the metabolic changes caused by vNDV infection remain unclear. The objective of this study was to determine the metabolomic profiling after infection with vNDV. DF-1 cells infected with the vNDV strain Herts/33 and the lungs from Herts/33-infected specific pathogen-free (SPF) chickens were analyzed via ultra-high-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) in combination with multivariate statistical analysis. A total of 305 metabolites were found to have changed significantly after Herts/33 infection, and most of them belong to the amino acid and nucleotide metabolic pathway. It is suggested that the increased pools of amino acids and nucleotides may benefit viral protein synthesis and genome amplification to promote NDV infection. Similar results were also confirmed in vivo. Identification of these metabolites will provide information to further understand the mechanism of vNDV replication and pathogenesis.

Rapid Screening of Forskolin-Type Diterpenoids of Blumea aromatica DC Using Ultra-High-Performance Liquid Chromatography Tandem Quadrupole Time-of-Flight Mass Spectrometry Based on the Mass Defect Filtering Approach.

The discovery of new active compounds of natural products tends to be increasingly more challenging due to chemical complexity and unpredictable matrices. Forskolin is an active natural labdane-type diterpenoid ingredient widely used worldwide for the treatment of glaucoma, heart failure, hypertension, diabetes, and asthma, and is expected to be a promising anticancer, anti-inflammation, and anti-HIV agent. In recent years, demand for forskolin in the medicine market has increased dramatically. However, natural forskolin originates exclusively from traditional Indian herb medicine Coleus forskohlii (Willd.) Briq. In a previous study, we isolated a series of diterpenoids including an 8,13-epoxy-14ene labdane carbon skeleton from Blumea aromatica DC. In order to identify alternative plant resources, a novel and effective strategy was proposed for the screening of potential forskolin-type diterpenoids (FSKD) compounds obtained from B. aromatica, using the mass defect filtering (MDF) strategy via ultra-high-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) approach. Within a narrow, well-defined mass defect range, the strategy developed could significantly improve the detection efficiency of selected FSKD compounds by filtering out certain major or moderate interference compounds. Additionally, the MS/MS cleavage behavior and the characteristic diagnostic ions of the FSKD compounds were proposed to be used in aiding structural identification of the filtration compounds. As a result, a total of 38 FSKD of B. aromatica were filtered out and tentatively identified. To the best of our knowledge, it was the first time that these forskolin-type diterpenoids were identified in B. aromatica, which significantly expands our understanding of the chemical constituents of Blumea species, and allows B. aromatica to be used as a potential alternative plant resource that contains these forskolin-type active compounds. The strategy proposed was proven efficient and reliable for the discovery of novel compounds of herbal extracts.

Non-targeted Metabolomic Analysis Based on Ultra-High-Performance Liquid Chromatography Quadrupole Time-of-Flight Tandem Mass Spectrometry Reveals the Effects of Grafting on Non-volatile Metabolites in Fresh Tea Leaves ( Camellia sinensis L.).

To investigate the effects of grafting on non-volatile metabolites in tea, non-targeted metabolomic analyses of fresh leaves were performed on the basis of ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF/MS). One non-grafted YingHong No. 9 and four grafted tea [grafting scion YingHong No. 9 on four different rootstocks, BaiMao No. 2 (BM2), BaiYeDanCong (BY), HeiYeShuiXian (HY), and WuLingHong (WLH)] were chosen as materials. In total, 32 differential metabolites were identified, including phenolic acids, flavan-3-ols, dimeric catechins, flavonol and flavonol/flavone glycosides, etc. Partial least squares discrimination analysis and hierarchical cluster analysis showed various effects of different rootstocks on metabolites. Thereinto, rootstocks of WLH and BY showed extremely outstanding performance in up- and downregulating these metabolites, respectively. Differential metabolites were enriched into three crucial pathways, including biosynthesis of phenylpropanoids, flavonoid biosynthesis, and flavone and flavonol biosynthesis, which might influence the quality of tea. This study provides a theoretical basis for grafting-related variations of non-volatile metabolites in fresh tea leaves.

Extraction of Extracellular Matrix in Static and Dynamic Candida Biofilms Using Cation Exchange Resin and Untargeted Analysis of Matrix Metabolites by Ultra-High-Performance Liquid Chromatography-Tandem Quadrupole Time-of-Flight Mass Spectrometry (UPLC-Q-TOF-MS).

Fungal infections caused by Candida albicans poses a great threat to human health. The ability of biofilm formation is believed to be associated with resistance-related Candida infections. Currently, knowledge on extracellular matrix (EM) of C. albicans biofilm is limited. In this study, we introduced ion exchange resin, i.e., cation exchange resin (CER) and anion exchange resin (AER), in EM extraction of C. albicans biofilm as well as several non-albicans Candida (NAC) biofilms under static and dynamic states in combination with vortexing and ultrasonication (VU). The metabolites extracted from the dynamic C. albicans biofilm matrix using the CER-VU and VU were identified with ultra-high-performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) via untargeted filtration. Compared with other physical and chemical extraction methods, CER-VU was demonstrated to be an ideal approach with high-yield acquisitions of EM constituents including proteins, triglycerides and carbohydrates and low-level damages on fungal cell viability and integrity. The untargeted MS analysis further showed the high efficacy of CER-VU, as a large quantity of metabolites (217 versus 198) was matched comprising a great number of lipids, carbohydrates, amino acids, nucleic acids and their derivatives together with a high involvement of signaling pathways compared with the VU alone. However, combining the results from both the CER-VU and VU methods could generate more metabolites. In summary, the EM analysis of the dynamic C. albicans biofilm expands our understanding upon a comprehensive depiction of matrix components and provides another effective approach for EM extraction.

Identification of novel serum biomarker for the detection of acute myeloid leukemia based on liquid chromatography-mass spectrometry

Acute myeloid leukemia (AML) is a life-threatening hematological malignancy. Traditional diagnosis of AML depends on invasive bone marrow biopsies. To recognize the metabolic characteristics related with AML and search for early non-invasive biomarkers of AML, in this work we applied ultra-high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOFMS)-based metabolomoc method to profile serum metabolites from 55 de novo AML patients and 45 age- and gender-matched healthy subjects and to screen and validate AML biomarkers. We observed AML-related metabolic differences mainly involved in alanine, aspartate and glutamate metabolism; d-Glutamine and d-glutamate metabolism; tryptophan metabolism; taurine and hypotaurine metabolism; and phenylalanine metabolism as well as fatty acid metabolism. A serum metabolite biomarker panel consisting of glutamic acid, kynurenine and oleic acid was defined and validated based on binary logistic regression analysis and receiver operating characteristic curves (ROC) analysis, yielding an area under the ROC curve (AUC) of 0.981 with 0.975 sensitivity and 0.933 specificity in the discovery set and an AUC of 0.973 with 0.933 sensitivity and 0.933 specificity in the validation set. This work demonstrated the UHPLC- MS-based metabolomics as a low invasive potential tool for the detection of AML, and this composite serum metabolite panel exhibited good diagnostic performance for AML in this case-control study and deserved further validation in a large-scale clinical trial. The identified metabolic pathways were also potentially worthy of further studying the pathogenesis of AML.

Oxidation pattern of curdlan with TEMPO-mediated system

In this study, the TEMPO-mediated (TEMPO/NaBr/NaClO) oxidation pattern of curdlan was investigated through comprehensively structural analysis of the corresponding oxidized products. During the structural analysis, infrared spectroscopy (IR), nuclear magnetic resonance (NMR) spectroscopy, gel permeation chromatography tandem multiple angle laser scattering (GPC-MALS) and ultra-high performance liquid chromatography tandem quadrupole time of flight mass spectrometry (UHPLC-Q/TOF-MS) were applied. As a result, the homogenous β1-3 polyglucuronic acids (MW, 49.8, 29.8 and 7.0 kDa) were obtained with proper amount of oxidant (5.36 mmol NaClO) at various temperatures (4, 25, 50 °C), respectively. Compared to the oxidation of 1–4 linked glucan (starch and cellulose) with TEMPO-mediated system at same reaction conditions, higher degree of specific oxidation and less degradation were observed in that of 1–3 linked curdlan. The glycosylation at position 3 could stabilize the sugar ring, which inactivates the non-specific oxidation related hydroxyl groups on the sugar ring. Thus, the TEMPO-mediated system has higher selectivity to oxidize the primary hydroxyl groups of 1–3 linked curdlan and form polyglucuronic acid than those observed in the oxidation of starch and cellulose. In addition, same as those observed in previous work about starch, higher the temperature was used in the oxidation with TEMPO system, higher the activity of oxidant (NaClO solution) was, more non-specific oxidation occurred, and more the degradation were observed.

Differentiation of Furanocoumarin Isomers with Ratio of Relative Abundance of Characteristic Fragment Ions and Application in Angelicae dahuricae Radix

C-5-substituted and C-8-substituted furanocoumarin isomers, two important kinds of furanocoumarin, are widely documented as the main active constituents in Angelicae dahuricae radix. Due to the similar polarity and mass fragmentation pathways of such isomers, it is difficult to distinguish them using mass spectrometric methods. To address this issue, we developed a strategy employing combined full scan and product ion scan modes on an ultra high performance liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS) platform to differentiate four pairs of furanocoumarin isomer, viz. xanthotoxin and bergapten, imperatorin and isoimperatorin, psoralen and isopsoralen, and impinellin and isoimpinellin. A novel method using the ratio of relative abundance (RRA) of characteristic fragment ions was established to distinguish C-5-substituted and C-8-substituted furanocoumarin isomers, using the formula R = M/N, where M and N represent the ratios of relative abundance of characteristic fragment ions of a pair of furanocoumarin isomers. For R value greater than 1, compound M is substituted at C-5, whereas for R value less than 1, compound M is substituted at C-8. This method with good repeatability was applied to identify five pairs of isomeric furanocoumarins in Angelicae dahuricae radix. This is the first method to distinguish C-5-substituted and C-8-substituted furanocoumarin isomers, and can be used in complex matrix.

Identification and Quantification Analysis on the Chemical Constituents from Traditional Mongolian Medicine Flos Scabiosae Using UHPLC-DAD-Q-TOF-MS Combined with UHPLC-QqQ-MS.

In this study, a systematic method was established for the qualitative and quantitative analysis of the major constituents in Flos Scabiosae (FS). First, Ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was developed for the identification of the multi-constituents in FS. A total of 48 compounds (9 phenolic acids, 24 flavonoids, 8 iridoids and 7 others) were unambiguously or tentatively identified, including 25 compounds (flavonoids, phenolic acids) identified in FS for the first time. Second, ultra-high performance liquid chromatography combined with triple quadrupole mass spectrometry (UHPLC-MS-MS) was developed for the quantitative analysis of 10 phenolic compounds. Ten compounds, either with high contents or strong bioactivities, were chosen as markers. This analytical method was validated through intra- and inter-day precision, repeatability and stability, with respective relative standard deviations <4.43, <8.64, <4.60 and <3.65%, respectively. The limits of detection and quantification were <1.09 and <16.96 ng/mL, respectively. The overall recoveries ranged from 96.47 to 103.94% .Then the validated method was applied to determine 10 batches of FS. The results indicated that the new method can be applied for the qualitative and quantitative analysis of FS.

The Study of Senkyunolide I Degradation Products by Ultra-High Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry

Ligusticum chuanxiongHort. (Umbelliferae) has been prescribed widely to treat cardiovascular diseases in China for centuries. Senkyunolide I is a major bioactive component inL. chuanxiongthat shows pharmacological activity against migraines and oxidative damage. Here, we identified the degradation products of senkyunolide I under different conditions (temperature, light alkaline) by ultra-high-performance liquid chromatography–quadrupole time-of-flight–tandem mass spectrometry (UPLC-QTOF-MS) analyses. Senkyunolide I was separated on an ACQUITY UPLC HSS T3 C18 (100 mm × 2.1 mm i.d., 1.8 μm) column using a binary eluent under gradient conditions. Analytes were detected by ESI/QTOF–MS/MS in positive ion mode to obtain MS and MS/MS spectra together with extracted molecular weights. Upon comparison with reference data, we concluded that senkyunolide I undergoes a ring-opening reaction under alkaline conditions (pH &gt;10.0), and dual-key addition or hydration on 3,4-unsaturated bonds and branched terminal hydroxyl groups under high-temperature and aerobic conditions. With auxiliary application1H-NMR, we determined that partial isomerization of senkyunolide I takes place under bright light. In the method detailed here, the advantages of separation and identification of a complex system of small and medium-sized molecules was exploited using UPLC–QTOF–MS, not only to avoid complicated separation and purification, but also to isolate and identify trace amounts of degradation products that were previously difficult to identify.