High-resolution Accurate Mass Spectrometry Research Articles

Protective effects on ovalbumin-induced mouse asthma models and qualitative and quantitative analysis of multiple compounds in Gerberae Piloselloidis Herba.

Gerberae Piloselloidis Herba is widely used to treat cough and asthma in China. However, its effects on allergic asthma as related to its chemical compositions have not been fully elucidated, and there is a scarcity of methods to determine multi-component contents for quality control. In this study, protective effects of Gerberae Piloselloidis Herba on ovalbumin-induced asthma models were investigated, while qualitative and quantitative analyses of multiple constituents in Gerberae Piloselloidis Herba were conducted by using an ultrahigh-performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry and an ultrahigh-performance liquid chromatography-photodiode array detection. The results showed that Gerberae Piloselloidis Herba could significantly mitigate asthma symptoms, reduce eosinophils counts in the bronchoalveolar lavage fluid, as well as decrease IgE, IL-5, and IL-13 concentration, and inflammatory cellular infiltration in lung tissues. A total of 51 compounds were tentatively identified, in which the content of 10 representative compounds was determined in 24 batches of Gerberae Piloselloidis Herba by using an ultrahigh-performance liquid chromatography method with good linearity, precision, repeatability, accuracy, and stability. This research presents a comprehensive strategy combining biological activity evaluation with chemical profiling, providing a useful and comprehensive reference for further application and quality control of Gerberae Piloselloidis Herba.

Metabolomics based plasma biomarkers for diagnosis of oral squamous cell carcinoma and oral erosive lichen planus.

Backgrounds: To identify diagnostic biomarkers for differentiating oral squamous cell carcinoma (OSCC) from oral erosive lichen planus (OELP) and investigate potential biomarkers associated with malignant transformation.Methods: In this study, 72 patients with OSCC, 75 patients with OELP subjects were recruited. Their plasma samples were analyzed by ultra-high-performance liquid chromatography quadrupole-Orbitrap high-resolution accurate mass spectrometry, (UHPLC/Q-Orbitrap HRMS). Principal component analysis, orthogonal partial least square discrimination analysis, t-test analysis and false discovery rate were used to identify different metabolites in patients with OSCC and OELP. The metabolic pathway analysis was performed by MetaboAnalyst. To further screen and identify the biomarkers of OSCC and establish a diagnostic panel, binary logistic regression analysis and receiver operating characteristic analysis were used. The data were then combined with blood samples from healthy individuals for mass spectrometry analysis to obtain biomarkers related to malignant transformation.Results: A total of 20 kinds of endogenous metabolites were identified from plasma samples of OSCC patients and OELP patients. Metabolic pathway analysis showed that the biomarkers associated with OSCC were closely related to cholic acid metabolism and amino acid metabolism. Finally, a diagnostic panel composed of decanoylcarnitine, cysteine and cholic acid was established. This diagnostic panel had good diagnostic efficiency with the AUC=0.998. Other metabolites including uridine, taurine, glutamate, citric acid and LysoPC(18:1) were identified to be general biomarkers for malignant transformation of OELP.Conclusion: Biomarkers based on plasma metabolomics are of great significance for the prediction of malignant transformation of OELP and early diagnosis of OSCC.

Using High-Sensitivity Lipidomics To Assess Microscale Heterogeneity in Oceanic Sinking Particles and Single Phytoplankton Cells.

Sinking particulate organic matter (POM) is a primary component of the ocean's biological carbon pump that is responsible for carbon export from the surface to the deep sea. Lipids derived from plankton comprise a significant fraction of sinking POM. Our understanding of planktonic lipid biosynthesis and the subsequent degradation of lipids in sinking POM is based on the analysis of bulk samples that combine many millions of plankton cells or dozens of sinking particles, which averages out natural heterogeneity. We developed and applied a nanoflow high-performance liquid-chromatography electrospray-ionization high-resolution accurate-mass mass spectrometry lipidomic method to show that two types of sinking particles─marine snow and fecal pellets─collected in the western North Atlantic Ocean have distinct lipidomes, providing new insights into their sources and degradation that would not be apparent from bulk samples. We pressed the limit of this approach by examining individual diatom cells from a single culture, finding marked lipid heterogeneity, possibly indicative of fundamental mechanisms underlying cell division. These single-cell data confirm that even cultures of phytoplankton cells should be viewed as mixtures of physiologically distinct populations. Overall, this work reveals previously hidden lipidomic heterogeneity in natural POM and phytoplankton cells, which may provide critical new insights into microscale chemical and microbial processes that control the export of sinking POM.

Metabolomics and Transcriptomics Analysis on Metabolic Characteristics of Oral Lichen Planus.

ObjectiveTo explore metabolic biomarkers related to erosive and reticulated oral lichen planus (OLP) by non-targeted metabolomics methods and correlate metabolites with gene expression, and to investigate the pathological network pathways of OLP from the perspective of metabolism.MethodsA total of 153 individuals were enrolled in this study, including 50 patients with erosive oral lichen planus (EOLP), 51 patients with reticulated oral lichen planus (ROLP), and 52 healthy controls (HC). The ultra-high-performance liquid chromatography quadrupole-Orbitrap high-resolution accurate mass spectrometry (UHPLC/Q-Orbitrap HRMS) was used to analyze the metabolites of 40 EOLP, 40 ROLP, and 40 HC samples, and the differential metabolic biomarkers were screened and identified. The regulatory genes were further screened through the shared metabolites between EOLP and ROLP, and cross-correlated with the OLP-related differential genes in the network database. A “gene-metabolite” network was constructed after finding the key differential genes. Finally, the diagnostic efficiency of the biomarkers was verified in the validation set and a diagnostic model was constructed.ResultCompared with HC group, a total of 19 and 25 differential metabolites were identified in the EOLP group and the ROLP group, respectively. A total of 14 different metabolites were identified between EOLP and ROLP. Two diagnostic models were constructed based on these differential metabolites. There are 14 differential metabolites shared by EOLP and ROLP. The transcriptomics data showed 756 differentially expressed genes, and the final crossover network showed that 19 differential genes were associated with 12 metabolites. Enrichment analysis showed that alanine, aspartate and glutamate metabolism were closely associated with the pathogenesis of OLP.ConclusionThe metabolic change of different types of OLP were clarified. The potential gene perturbation of OLP was provided. This study provided a strong support for further exploration of the pathogenic mechanism of OLP.

Association of Coronary Artery Disease and Metabolic Syndrome: Usefulness of Serum Metabolomics Approach.

IntroductionIndividuals with metabolic syndrome (MetS) are at increasing risk of coronary artery disease (CAD). We investigated the common metabolic perturbations of CAD and MetS via serum metabolomics to provide insight into potential associations.MethodsNon-targeted serum metabolomics analyses were performed using ultra high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry (UHPLC-Q-Orbitrap HRMS) in samples from 492 participants (272 CAD vs. 121 healthy controls (HCs) as cohort 1, 55 MetS vs. 44 HCs as cohort 2). Cross-sectional data were obtained when the participants were recruited from the First Affiliated Hospital of Zhengzhou University. Multivariate statistics and Student’s t test were applied to obtain the significant metabolites [with variable importance in the projection (VIP) values >1.0 and p values <0.05] for CAD and MetS. Logistic regression was performed to investigate the association of identified metabolites with clinical cardiac risk factors, and the association of significant metabolic perturbations between CAD and MetS was visualized by Cytoscape software 3.6.1. Finally, the receiver operating characteristic (ROC) analysis was evaluated for the risk prediction values of common changed metabolites.ResultsThirty metabolites were identified for CAD, mainly including amino acids, lipid, fatty acids, pseudouridine, niacinamide; 26 metabolites were identified for MetS, mainly including amino acids, lipid, fatty acids, steroid hormone, and paraxanthine. The logistic regression results showed that all of the 30 metabolites for CAD, and 15 metabolites for MetS remained significant after adjustments of clinical risk factors. In the common metabolic signature association analysis between CAD and MetS, 11 serum metabolites were significant and common to CAD and MetS outcomes. Out of this, nine followed similar trends while two had differing directionalities. The nine common metabolites exhibiting same change trend improved risk prediction for CAD (86.4%) and MetS (90.9%) using the ROC analysis.ConclusionSerum metabolomics analysis might provide a new insight into the potential mechanisms underlying the common metabolic perturbations of CAD and MetS.

Identification of serum biomarkers and evaluation of metabolism disorders in patients with oral squamous cell carcinoma

Objective: To explore the changes in serum metabolic profile in patients with oral squamous cell carcinoma (OSCC) and to identify the diagnostic biomarkers in order to provide new ideas for the early diagnosis of OSCC. Methods: In total, 76 OSCC patients who were diagnosed at the Department of Oral and Maxillofacial Surgery and 70 healthy individuals who at the Department of Medical Center of The First Affiliated Hospital of Zhengzhou University from August 2019 to January 2020 were recruited in The study. According to the random number table method, all subjects were divided into a test group (n=96) and a verification group (n=50). Subjects in the test group consisted of 51 OSCC patients and 45 healthy subjects and subjects in the verification group included 25 OSCC patients and 25 healthy individuals. Serum samples and clinical data of each of the subjects were collected. The serum samples were analyzed by ultra-high-performance liquid chromatography quadrupole-Orbitrap high resolution accurate mass spectrometry. Principal component analysis, orthogonal partial least square discrimination analysis and t-test were used to profile the differential metabolites in the test group. Pathway analysis of differential metabolites was performed. In addition, binary logistic regression analysis and receiver operating characteristic analysis were used in order to establish the potential diagnostic panel. Results: Twenty-one endogenous differential metabolites were identified showing significant association with OSCC. Results of pathway analysis suggested that OSCC associated with lipid metabolism and amino acid metabolism (P<0.05). A novel diagnostic panel consisting of lysophosphatidylcholine (LysoPC) (16∶0/0∶0), LysoPC[18∶1(9z)/0∶0], taurine and D-glutamic acid was defined. The panel performed a high area under the receiver operating characteristic curve (0.998, 95%CI: 0.994-0.999, P<0.05). Conclusions: There were obvious lipid and amino acid metabolism disorders in OSCC patients. It was an effective method to establish a diagnostic model by metabolomics.

Effects of pH and Temperature on Water under Pressurized Conditions in the Extraction of Nutraceuticals from Chaga (Inonotus obliquus) Mushroom.

Currently, there is increased interest in finding appropriate food-grade green extraction systems capable of extracting these bioactive compounds from dietary mushrooms for applications in various food, pharmacological, or nutraceutical formulations. Herein, we evaluated a modified Swiss water process (SWP) method using alkaline and acidic pH at low and high temperature under pressurized conditions as a suitable green food grade solvent to obtained extracts enriched with myco-nutrients (dietary phenolics, total antioxidants (TAA), vitamins, and minerals) from Chaga. Ultra-high performance liquid chromatography coupled to high resolution accurate mass tandem mass spectrometry (UHPLC-HRAMS-MS/MS) was used to assess the phenolic compounds and vitamin levels in the extracts, while inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the mineral contents. Over 20 phenolic compounds were quantitatively evaluated in the extracts and the highest total phenolic content (TPC) and total antioxidant activity (TAA) was observed at pH 11.5 at 100 °C. The most abundant phenolic compounds present in Chaga extracts included phenolic acids such as protocatechuic acid 4-glucoside (0.7–1.08 µg/mL), syringic acid (0.62–1.18 µg/mL), and myricetin (0.68–1.3 µg/mL). Vitamins are being reported for the first time in Chaga. Not only, a strong correlation was found for TPC with TAA (r-0.8, <0.0001), but also, with individual phenolics (i.e., Salicylic acid), lipophilic antioxidant activity (LAA), and total antioxidant minerals (TAM). pH 2.5 at 100 °C treatment shows superior effects in extracting the B vitamins whereas pH 2.5 at 60 and 100 °C treatments were outstanding for extraction of total fat-soluble vitamins. Vitamin E content was the highest for the fat-soluble vitamins in the Chaga extract under acidic pH (2.5) and high temp. (100 °C) and ranges between 50 to 175 µg/100 g Chaga. Antioxidant minerals ranged from 85.94 µg/g (pH7 at 100 °C) to 113.86 µg/g DW (pH2.5 at 100 °C). High temperature 100 °C and a pH of 2.5 or 9.5. The treatment of pH 11.5 at 100 °C was the most useful for recovering phenolics and antioxidants from Chaga including several phenolic compounds reported for the first time in Chaga. SWP is being proposed herein for the first time as a novel, green food-grade solvent system for the extraction of myco-nutrients from Chaga and have potential applications as a suitable approach to extract nutrients from other matrices. Chaga extracts enriched with bioactive myconutrients and antioxidants may be suitable for further use or applications in the food and nutraceutical industries.

Plasmalogen Deficiency and Overactive Fatty Acid Elongation Biomarkers in Serum of Breast Cancer Patients Pre- and Post-Surgery-New Insights on Diagnosis, Risk Assessment, and Disease Mechanisms.

Simple SummaryBreast cancer (BC) is the most commonly diagnosed cancer in women. Mammography and ultrasonography are commonly used for BC screening; however, they are associated with problems such as inconvenience, radiation exposure, and dependence on the skill level of operators. To overcome this problem, we performed a comprehensive lipid metabolomic analysis of serum using high-resolution accurate mass spectrometry from two case-control studies that included non-BC, BC subjects pre-surgery and BC subjects one-month post-surgery to determine if the metabolic signatures of over-active fatty acid elongation and other lipid changes could be detected in BC vs. non-BC subjects. The ratios of the linoleic acid to the oleic acid which were evaluated in multiple lipid pools were lower in pre-surgery BC subjects, however, these ratios increased at post-surgery and were no longer different from non-BC subjects. On the other hand, the ethanolamine plasmalogen levels were lower in pre-surgery BC subjects and were not recovered by surgical removal. These do not appear to be caused by BC tumor activity and may be pre-existent and a possible risk factor for BC. In this study, we have identified several lipid metabolic systems that detect both BC risk and BC activity.The polyunsaturated fatty acid (PUFA) elongase, ELOVL5, is upregulated in breast cancer (BC) vs. adjacent normal tissue. We performed a comprehensive lipid metabolomic analysis of serum using high-resolution accurate mass spectrometry from two case-control studies that included non-BC, BC subjects pre-surgery, and BC subjects one-month post-surgery to determine if the metabolic signatures of over-active fatty acid elongation and other lipid changes could be detected in BC vs. non-BC subjects: study 1 (n = 48: non-BC, n = 69: pre-surgery BC); study 2 (blinded validation: n = 121: non-BC, n = 62: pre-surgery BC, n = 31: one month post-surgery). The ratio of the ELOVL5 precursor, linoleic acid (18:2) to a non-ELOVL5 precursor, oleic acid (18:1) was evaluated in multiple lipid pools (phosphatidylethanolamine (PtdEtn), phosphatidylcholine (PtdCho), lyso-PtdCho, and free fatty acids). This ratio was lower in pre-surgery BC subjects in all pools in both studies (p < 0.001). At one-month post-surgery, the 18:2/18:1 ratios increased vs. pre-surgery and were no longer different from non-BC subjects (p > 0.05 expect for lyso-PtdCho). In contrast to the elongation biomarkers, docosahexaenoic acid (22:6n-3) containing ethanolamine plasmalogen (EtnPls) species were observed to be further decreased in BC subjects one-month post-surgery vs. pre-surgery levels (p < 0.001). These results are consistent with the hypothesis that ELOVL5 is upregulated in BC tissue, which would result in the selective depletion of 18:2 vs. 18:1 containing lipid species. Surgical removal of the tumor removes the overactive ELOVL5 effect on serum lipids. In contrast, the low EtnPls levels do not appear to be caused by BC tumor activity and may be pre-existent and a possible risk factor for BC. These results indicate that it may be possible to screen for both breast cancer risk and breast cancer activity using a simple blood test.

Capillary Electrophoresis-High Resolution Mass Spectrometry for Measuring In Vivo Arginine Isotope Incorporation in Alzheimer's Disease Mouse Models.

Immune-based metabolic reprogramming of arginine utilization in the brain contributes to the neuronal pathology associated with Alzheimer's disease (AD). To enable our long-term goals of differentiation of AD mouse model genotypes, ages, and sexes based on activity of this pathway, we describe here the novel dosing (using uniformly labeled (13C615N4) arginine) and analysis methods using capillary electrophoresis high-resolution accurate-mass mass spectrometry for isotope tracing of metabolic products of arginine. We developed a pseudoprimed infusion-dosing regimen, using repeated injections, to achieve a steady state of uniformly labeled arginine in 135-195 min post bolus dose. Incorporation of stable isotope labeled carbon and nitrogen from uniformly labeled arginine into a host of downstream metabolites was measured in vivo in mice using serially sampled dried blood spots from the tail. In addition to the dried blood spot time course samples, total isotope incorporation into arginine-related metabolites was measured in the whole brain and plasma after 285 min. Preliminary demonstration of the technique identified differences isotope incorporation in arginine metabolites between male and female mice in a mouse-model of sporadic Alzheimer's disease (APOE4/huNOS2). The technique described herein will permit arginine pathway activity differentiation between mouse genotypes, ages, sexes, or drug treatments in order to elucidate the contribution of this pathway to Alzheimer's disease.

An Entire Process Optimization Strategy for Comprehensive In Vivo Metabolite Profiling of Prucalopride in Rats Based on Ultra-Performance Liquid Chromatography With Q-Exactive Hybrid Quadrupole-Orbitrap High-Resolution Mass Spectrometry.

Prucalopride was widely used for chronic constipation, which is difficult to be adequately relieved by laxatives in adult patients in clinic. Due to the difficulty in metabolite identification, metabolic process of prucalopride had not been investigated in vivo. In this study, an efficient strategy was proposed for comprehensive metabolite profiling of prucalopride after oral administration in rat plasma, urine, and feces samples. This strategy was composed of five steps. First, the samples at multiple time points after oral administration were collected to increase the representativeness of the samples. Second, different sample preparation methods were investigated to obtain superior extraction efficiency. Third, the raw data of test sample and blank sample were acquired using ultra-performance liquid chromatography with Q-Exactive hybrid quadrupole–orbitrap high-resolution accurate mass spectrometry under the positive and negative full-scan/dd MS2 mode. Fourth, combined mass defect filter with background subtraction model in soft of compound discovery, all peaks were constructed to filter potential metabolites after retention time alignment and ion filtration, which could remove large amounts of interference ions. Besides, it can predict potential biotransformation, promoting to understand how to metabolize the drug. This provides multiple possibilities and prevents us conjecturing the potential metabolites blindly. Finally, the verification procedure was implemented through exporting the structure and MS2 spectrum to the analytical tool of Mass Frontier. The proposed strategy significantly improved the targeted detection and identification for metabolites in vivo. A total of 47 metabolites were tentatively characterized in the plasma, urine, and feces samples after oral administration of prucalopride. This study could provide a valuable reference for systematic metabolite profile of drug in vivo.

A novel class of sulfur-containing aminolipids widespread in marine roseobacters

Marine roseobacter group bacteria are numerically abundant and ecologically important players in ocean ecosystems. These bacteria are capable of modifying their membrane lipid composition in response to environmental change. Remarkably, a variety of lipids are produced in these bacteria, including phosphorus-containing glycerophospholipids and several amino acid-containing aminolipids such as ornithine lipids and glutamine lipids. Here, we present the identification and characterization of a novel sulfur-containing aminolipid (SAL) in roseobacters. Using high resolution accurate mass spectrometry, a SAL was found in the lipid extract of Ruegeria pomeroyi DSS-3 and Phaeobacter inhibens DSM 17395. Using comparative genomics, transposon mutagenesis and targeted gene knockout, we identified a gene encoding a putative lyso-lipid acyltransferase, designated salA, which is essential for the biosynthesis of this SAL. Multiple sequence analysis and structural modeling suggest that SalA is a novel member of the lysophosphatidic acid acyltransferase (LPAAT) family, the prototype of which is the PlsC acyltransferase responsible for the biosynthesis of the phospholipid phosphatidic acid. SAL appears to play a key role in biofilm formation in roseobacters. salA is widely distributed in Tara Oceans metagenomes and actively expressed in Tara Oceans metatranscriptomes. Our results raise the importance of sulfur-containing membrane aminolipids in marine bacteria.

Metabolic profiling of deschloro-N-ethyl-ketamine and identification of new target metabolites in urine and hair using human liver microsomes and high-resolution accurate mass spectrometry.

The aim of this study was to identify new markers of deschloro-N-ethyl-ketamine (O-PCE), a ketamine analogue that has been involved in acute intoxications with severe outcomes including death and whose metabolism has never been studied before. In vitro study after 2-h incubation with pooled human liver microsomes (HLMs) cross-checked by the analysis of urine and hair from a 43-year-old O-PCE user (male) were performed by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Acquired data were processed by the Compound Discoverer® software, and a full metabolic profile of O-PCE was proposed. In total, 15 metabolites were identified, 10 were detected in vitro (HLMs) and confirmed in vivo (urine and/or hair), two were present only in HLMs, and the remaining three metabolites were identified only in biological specimens. While O-PCE was no longer detected in urine, nine metabolites were identified allowing to increase its detection window. In descending order of metabolites abundance, we suggest using 2-en-PCA-N-Glu (34%, first), M3 (16%, second), O-PCA-N-Glu (15.4%, third), OH-O-PCE (15%, fourth) and OH-PCE (11.9%, fifth) as target metabolites to increase the detection window of O-PCE in urine. In hair, nine metabolites were identified. OH-PCA was the major compound (78%) with a relevant metabolite to parent drug ratio (=6) showing its good integration into hair and making it the best marker for long-term monitoring of O-PCE exposure.

Application of Lipidomics for Assessing Tissue Lipid Profiles of Patients With Squamous Cell Carcinoma.

Background: Lipid metabolism disorders play a key role in the pathogenesis of squamous cell carcinoma (SqCC). Herein we used lipidomics to study the tissue lipid profiles of 40 patients with SqCC. Methods: Lipidomics, based on ultrahigh-performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry, was applied to identify altered lipid metabolites between tumor and adjacent noninvolved tissues (ANIT), and partial least squares-discriminant analysis model facilitated the identification of differentially abundant lipids. The area under the receiver operator characteristic curve and variable importance in projection scores of the aforementioned model were calculated to select lipid profiles. Metabolic pathway analyses were completed using Kyoto Encyclopedia of Genes and Genomes and MetaboAnalyst. Results: Differences in lipid profiles were found between tumor and ANIT, early- and advanced-stage SqCC, and positive and negative lymph node metastases. The lipid profile panel was composed of five lipids—PC(44:4), diacylglycerol(36:5), sphingomyelin(d18:1/20:0), phosphatidylinositol(46:7), and HexCer-AP(t8:0/32:2 + O)—and could effectively differentiate between tumor and ANIT. Further, pathway analyses revealed alterations in several lipid metabolism pathways, including glycerophospholipid metabolism, glycosylphosphatidylinositol anchor biosynthesis, linoleic acid metabolism, glycerolipid metabolism, and sphingolipid metabolism. Conclusion: Our data revealed several changes in the tissue lipid profiles of patients with SqCC; moreover, we identified a lipid profile panel that could effectually distinguish tumor tissues from ANIT. We believe that our results provide new insights into the biological behavior of lung SqCC.

Detection and Identification of Catalpol Metabolites in the Rat Plasma, Urine and Faeces Using Ultra-high Performance Liquid Chromatography-Q Exactive Hybrid Quadrupole-orbitrap High-resolution Accurate Mass Spectrometry

Detection and Identification of Catalpol Metabolites in the Rat Plasma, Urine and Faeces Using Ultra-high Performance Liquid Chromatography-Q Exactive Hybrid Quadrupole-orbitrap High-resolution Accurate Mass Spectrometry

Integrated UPLC-MS/MS and UHPLC-Q-orbitrap HRMS Analysis to Reveal Pharmacokinetics and Metabolism of Five Terpenoids from Alpiniae oxyphyllae Fructus in Rats.

Alpiniae oxyphyllae Fructus (AOF), a traditional Chinese medicine (TCM), is widely used in the treatment of urinary, gastrointestinal and neurologic diseases in China. Although terpenoids are the main active ingredients of AOF, there are few researches on their pharmacokinetics and metabolism. In this study, a sensitive, rapid, accurate and novel ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established to evaluate the pharmacokinetic behavior of five terpenoids (oxyphyllenodiol B, (4S*,5E,10R*)-7-oxo-tri-nor-eudesm-5-en-4β-ol, 7-epi-teucrenone, (+)- (4R,5S,7R)-13-hydroxynootkatone, (E)-labda-12,14-dien-15(16)-olide-17-oic acid) in rats after oral administration of AOF extracts. 27 metabolic metabolites of the five terpenoids were identified by ultra high performance liquid chromatography -Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry (UHPLC-Q-Orbitrap HRMS) based on precise mass and fragment ions. The established pharmacokinetic analysis method showed good linearity over a wide concentration range, and the lower quantitative limit (LLOQ) ranged from 0.97 to 4.25 ng/mL. Other validation parameters were within the acceptable range. In addition, 27 metabolites were identified in plasma, urine and feces samples, and the metabolic pathways of five terpenoids were mainly focused on glucoside conjugation, dehydration, desaturation and glycine conjugation. This is the first study on the pharmacokinetics and metabolism of five terpenoids in AOF, illuminating the disposal process of terpenoids in vivo. It was expected that the results of this study would provide some references for the apprehension of the action mechanism and the further pharmacological study of five terpenoids in AOF.

Quantitative and qualitative analysis of edible oils using HRAM MS with an atmospheric pressure chemical ionisation (APCI) source

Fatty acids represent major components of cell membranes, serve as energy sources, modulate gene transcription and cell signalling and act as cytokine precursors. It is increasingly apparent that dietary fatty acids influence these vital functions and affect human health. Consequently, analytical techniques are required to identify and quantify the suite of fatty acids present in food and human tissues. Advances in mass spectrometry (MS) offer new opportunities to profile and quantify fatty acids in biological samples. Our aim was to demonstrate the use of GC- atmospheric pressure chemical ionisation (APCI)-ion mobility spectrometry (IMS)-TOF-MS to provide highly specific and sensitive quantification of known fatty acids plus a comprehensive overview of all the eluted analytes. Ionisation was achieved using an APCI source. This new approach was demonstrated on a range of commercial edible oils. Compared to standard GC techniques using flame ionisation detection (FID) or a single quadrupole MS with electron ionisation, GC-APCI-IMS-TOF-MS greatly increased compound selectivity and specificity, leading to greatly enhanced confidence in fatty acid methyl esters (FAME) identification and quantification. Our approach also added the fingerprint of high-resolution accurate mass (HRAM) discovery data, with collision cross section (CCS) values, relating to many other analytes. This method can be readily applied to study food provenance, food fraud and to identify fatty acid related illnesses.

Cloning, Yeast Expression, and Characterization of a β-Amyrin C-28 Oxidase (CYP716A249) Involved in Triterpenoid Biosynthesis in Polygala tenuifolia.

Polygala tenuifolia Willd. is a traditional Chinese herbal medicine that is widely used in treating nervous system disorders. Triterpene saponins in P. tenuifolia (polygala saponins) have excellent biological activity. As a precursor for the synthesis of presenegin, oleanolic acid (OA) plays an important role in the biosynthesis of polygala saponins. However, the mechanism behind the biosynthesis of polygala saponins remains to be elucidated. In this study, we found that CYP716A249 (GenBank: ASB17946) oxidized the C-28 position of β-amyrin to produce OA. Using quantitative real-time PCR, we observed that CYP716A249 had the highest expression in the roots of 2-year-old P. tenuifolia, which provided a basis for the selection of samples for gene cloning. To identify the function of CYP716A249, the strain R-BE-20 was constructed by expressing β-amyrin synthase in yeast. Then, CYP716A249 was co-expressed with β-amyrin synthase to construct the strain R-BPE-20 by using the lithium acetate method. Finally, we detected β-amyrin and OA by ultra-HPLC-Q Exactive hybrid quadrupole-Orbitrap high-resolution accurate mass spectrometry and GC-MS. The results of this study provide insights into the biosynthesis pathway of polygala saponins.

Abstract PO-163: Pre-diagnostic phthalates and other endocrine disruptors in relation to endometrial cancer risk in the Multiethnic Cohort (MEC) Study

Abstract Introduction: Endometrial cancer develops under conditions of high circulating estrogen levels unopposed by progesterone. We hypothesized that exposure to endocrine disrupting chemicals (EDCs), including those that are used in plastics such as phthalates, will increase endometrial cancer risk and may contribute to racial/ethnic differences in risk. Methods: We conducted a case-control study nested in the Multiethnic Cohort (MEC) Study to measure EDCs in pre-diagnostic urine samples. We identified 139 postmenopausal endometrial cancer cases and 139 female controls matched on race/ethnicity, study site, birth year, fasting hours, urine type, date/time of urine collection and current menopausal hormone therapy use. Concentrations of 17 EDCs including urinary phthalates, bisphenol A, parabens and triclosan were measured by highly sensitive liquid chromatography high-resolution accurate-mass mass spectrometry in the UHCC Analytical Biochemistry Shared Resource using validated published protocols. Creatinine-adjusted urinary EDC excretion values were divided into tertiles based on the distribution in controls. Conditional logistic regression, with matched sets used as strata, was used to calculate odds ratios (OR) and 95% confidence intervals (CI) for the association between EDC levels and endometrial cancer risk accounting for body mass index (BMI), diabetes, and the Mediterranean Diet Score. Results: The median (Q25, Q75) time between urine collection and endometrial cancer diagnosis was 6.6 years (3.4, 9.4). Fifty-two case-control sets were Japanese American, 35 were White, 26 were Native Hawaiian, 17 were Latina and nine were African American. In analyses of the full population, the butyl phthalate metabolite monoisobutyl phthalate (MIBP) was suggestively associated with endometrial cancer risk (tertile 3 (T3) vs. T1, OR 2.07 [CI 0.96-4.47]). This association was stronger in non-whites (n=104 case-sets; T3 vs. T1, OR 3.11 [CI 1.17-8.27]), who are thought to be exposed to higher levels of selected EDCs. We also observed a non-significant positive association among non-whites for mono n-butyl phthalate (MBP; T3 vs. T1, OR 2.55 [CI 0.90-7.22]). Conclusion: To our knowledge this is the first study to examine the role of several EDCs in relation to endometrial cancer risk using pre-diagnostic samples. These data suggest that exposure to di-butyl phthalates, which were observed in vitro to be weakly estrogenic in mammalian estrogen screens (Jobling et al., 1995, Environ Health Perspect, 103: 582-7), may represent a risk factor for endometrial cancer among non-white women that is independent of BMI and diabetes status. Replication of these findings in other large cohort studies is warranted, especially in non-whites. Citation Format: Danja Sarink, Loïc Le Marchand, Iona Cheng, Anna H. Wu, Adrian A. Franke, Lynne R. Wilkens, Kami K. White, Herbert Yu, Melissa A. Merritt. Pre-diagnostic phthalates and other endocrine disruptors in relation to endometrial cancer risk in the Multiethnic Cohort (MEC) Study [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-163.

Detection and Identification of Catalpol Metabolites in the Rat Plasma, Urine and Faeces Using Ultra-high Performance Liquid Chromatography-Q Exactive Hybrid Quadrupole-orbitrap High-resolution Accurate Mass Spectrometry.

Catalpol, an iridoid glycoside, is one of the richest bioactive components present in Rehmannia glutinosa. More and more metabolites of drugs have exhibited various pharmacological effects, thus providing guidance for clinical application. However, few researches have paid attention to the metabolism of catalpol. This study aimed to establish a rapid and effective method to identify catalpol metabolites and evaluate the biotransformation pathways of catalpol in rats. In this study, catalpol metabolites in rat urine, plasma and faeces were analyzed by UHPLC-Q-Exactive MS for the characterization of the metabolism of catalpol. Based on high-resolution extracted ion chromatograms (HREICs) and parallel reaction monitoring mode (PRM), metabolites of catalpol were identified by comparing the diagnostic product ions (DPIs), chromatographic retention times, neutral loss fragments (NLFs) and accurate mass measurement with those of catalpol reference standard. A total of 29 catalpol metabolites were detected and identified in both negative and positive ion modes. Nine metabolic reactions, including deglycosylation, hydroxylation, dihydroxylation, hydrogenation, dehydrogenation, oxidation of methylene to ketone, glucuronidation, glycine conjugation and cysteine conjugation, were proposed. A rapid and effective method based on UHPLC-Q-Exactive MS was developed to mine the metabolism information of catalpol. Results of metabolites and biotransformation pathways of catalpol suggested that when orally administrated, catalpol was firstly metabolized into catalpol aglycone, after which phase I and phase II reactions occurred. However, hydrophilic chromatography-mass spectrometry is still needed to further find the polar metabolites of catalpol.

Characterization of chemical components in the Guanxinning injection by liquid chromatography-mass spectrometry.

Guanxinning injection (GXNI) is widely used in the treatments of cerebral thrombosis, cerebral hemorrhage, sequela, coronary disease, stenocardia, arrhythmia, and so on. For the herbal injections, more components should be characterized and quantified as much as possible to guarantee the drug safety. However, large numbers of the chemical constituents in the GXNI still remain unknown. In this study, ultrahigh performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry (UHPLC-Q Orbitrap HRMS), in combination of nuclear magnetic resonance (NMR), was used to identify the components in GXNI, which led to the identification of 194 compounds. With the aid of solvent partition, more phthalides, diterpenoid quinines, and salvianolic acids were tentatively identified, and minor compounds with the other structural types were also detected. The structural diversity of phthalides and diterpenoid quinones were revealed by the structural network, and six phthalides and 13 diterpenoid quinones were further detected in GXNI with the help of the characteristic fragmentation pattern and structural network. In addition, NMR also revealed the presence of a series of primary metabolites in the GXNI, which could be used as a complimentary approach for the rapid identification of the chemical components in the traditional Chinese medicines (TCM). However, the unknown NMR signals of GXNI needed to be further identified to guarantee the drug safety.