Triple Quadrupole-linear Ion Trap Mass Research Articles

Single-Base Methylome Analysis of Sweet Cherry (Prunus avium L.) on Dwarfing Rootstocks Reveals Epigenomic Differences Associated with Scion Dwarfing Conferred by Grafting

Plant grafting using dwarfing rootstocks is one of the important cultivation measures in the sweet cherry (Prunus avium) industry. In this work, we aimed to explore the effects of the dwarfing rootstock “Pd1” (Prunus tomentosa) on sweet cherry ‘Shuguang2’ scions by performing morphological observations using the paraffin slice technique, detecting GA (gibberellin) and IAA (auxin) contents using UPLC-QTRAP-MS (ultra-performance liquid chromatography coupled with a hybrid triple quadrupole-linear ion trap mass spectrometer), and implementing integration analyses of the epigenome and transcriptome using whole-genome bisulfite sequencing and transcriptome sequencing. Anatomical analysis indicated that the cell division ability of the SAM (shoot apical meristem) in dwarfing plants was reduced. Pd1 rootstock significantly decreased the levels of GAs and IAA in sweet cherry scions. Methylome analysis showed that the sweet cherry genome presented 15.2~18.6%, 59.88~61.55%, 28.09~33.78%, and 2.99~5.28% methylation at total C, CG, CHG, and CHH sites, respectively. Shoot tips from dwarfing plants exhibited a hypermethylated pattern mostly due to increased CHH methylation, while leaves exhibited a hypomethylated pattern. According to GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, DMGs (differentially methylated genes) and DEGs (differentially expressed genes) were enriched in hormone-related GO terms and KEGG pathways. Global correlation analysis between methylation and transcription revealed that mCpG in the gene body region enhanced gene expression and mCHH in the region near the TSS (transcription start site) was positively correlated with gene expression. Next, we found some hormone-related genes and TFs with significant changes in methylation and transcription, including SAURs, ARF, GA2ox, ABS1, bZIP, MYB, and NAC. This study presents a methylome map of the sweet cherry genome, revealed widespread DNA methylation alterations in scions caused by dwarfing rootstock, and obtained abundant genes with methylation and transcription alterations that are potentially involved in rootstock-induced growth changes in sweet cherry scions. Our findings can lay a good basis for further epigenetic studies on sweet cherry dwarfing and provide valuable new insight into understanding rootstock–scion interactions.

Metabolite identification of salvianolic acid A in rat using post collision-induced dissociation energy-resolved mass spectrometry

BackgroundAs one of the most famous natural products, salvianolic acid A (SAA) is undergoing clinical trials for the treatments of angina pectoris and coronary heart disorders. However, the in vivo metabolites of SAA have only been tentatively identified, leading to a barrier for precise therapeutical drug monitoring.MethodsUltra-high performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry (UPLC–Qtof-MS/MS) was firstly employed to acquire high-resolution MS1 and MS2 spectra for all metabolites. Through paying special attention onto the features of ester bond dissociation, metabolism sites were restricted at certain regions. To further determine the metabolism site, such as the monomethylated products (M23, M25, and M26), post collision-induced dissociation energy-resolved mass spectrometry (post-CID ER-MS) was proposed through programming progressive exciting energies to the second collision chamber of hybrid triple quadrupole-linear ion trap mass spectrometry (Qtrap-MS) device.ResultsAfter SAA oral administration, 29 metabolites (M1–M29), including five, thirteen, and sixteen ones in rat plasma, urine, and feces, respectively, were detected in rats. The metabolism route was initially determined by applying well-defined mass fragmentation pathways to those HR-m/z values of precursor and fragment ions. Metabolism site was limited to SAF- or DSS-unit based on the fragmentation patterns of ester functional group. Through matching the dissociation trajectories of concerned 1st-generation fragment ions with expected decomposition product anions using post-CID ER-MS strategy, M23 and M25 were unequivocally assigned as 3'-methyl-SAA and 3''-methyl-SAA, and M26 was identified as 2-methyl-SAA or 3-methyl-SAA. Hydrolysis, methylation, glucuronidation, sulfation, and oxidation were the primary metabolism channels being responsible for the metabolites' generation.ConclusionTogether, the metabolism regions and sites of SAA metabolites were sequentially identified based on the ester bond dissociation features and post-CID ER-MS strategy. Importantly, the present study provided a promising way to elevate the structural identification confidence of natural products and metabolites.Graphical abstract

Floating plastics as integrative samplers of organic contaminants of legacy and emerging concern from Western Mediterranean coastal areas

This study investigates the role of floating plastics as integrative samplers of organic contaminants. To this end, plastics items were collected in two Western Mediterranean coastal areas: the Mar Menor lagoon, and the last transect of Ebro river. Floating plastics were identified and characterized by attenuated total reflection Fourier-transform infrared spectrometry. Then, organic contaminants were extracted from plastic items by ultrasonic extraction with methanol, and the concentrations of 168 regulated and emerging contaminants were analysed. These compounds were analysed by stir bar sorptive extraction coupled to gas chromatography–mass spectrometry (GC–MS), except for bisphenol analogues, which were analysed with a ultraperformance liquid chromatography pump coupled to a triple quadrupole mass spectrometer (UHPLC-MS/MS), and pharmaceutical compounds, determined by UPLC coupled to hybrid triple quadrupole-linear ion trap mass spectrometer (UPLC-MS/MS). All the contaminants groups considered were detected in the samples, being particularly relevant the contribution of plastic additives. The most frequently detected contaminants were UV-filters, PAHs, pharmaceuticals and synthetic musks. Apart from plasticizers, the individual contaminants octocrylene, homosalate, galaxolide, salycilic acid and ketoprofen were frequently detected in plastics items. The results pointed out to urban and touristic activities as the main sources of pollution in the coastal areas investigated. The utility of floating plastics as integrative samplers for the detection of organic contaminants in aquatic ecosystems has been demonstrated.

Plasma metabolomic signatures of breast cancer.

Breast cancer is a common malignant tumor. A large number of medical evidence shows that breast cancer screening can improve the early diagnosis rate and reduce the mortality rate of breast cancer. In the present study, a wide range of targeted metabolomics profiling was conducted to investigate the plasma signatures of breast cancer. A total of 86 patients with benign breast abnormalities (L group) and 143 patients with breast cancer (E group) were recruited. We collected their plasma samples and clinical information. Metabolomic analysis, based on the coverage of a wide range of targeted metabolomics was conducted with ultraperformance liquid chromatography- triple quadrupole-linear ion trap mass spectrometer (UPLC-QTRAP-MS). We identified 716 metabolites through widely-targeted metabolomics. Serotonergic synapse was the main different metabolic pathway. The fold change of 14 metabolites was considered significantly different (fold change <0.67 or fold change >2; p < 0.05). By combining all the 14 metabolites, we achieved differentiation of L group vs. E group (AUC = 0.792, 95%Cl: 0.662-0.809). This study provided new insights into plasma biomarkers for differential diagnosis of benign abnormalities and breast cancer.

Eco-friendly simultaneous extraction of pectins and phenolics from passion fruit (Passiflora edulis Sims) peel: Process optimization, physicochemical properties, and antioxidant activity

The objective of this study was to simultaneously extract passion fruit (Passiflora edulis) peel pectins and phenolics using deep eutectic solvents, to evaluate their physicochemical properties and antioxidant activity. By taking L-proline: citric acid (Pro-CA) as the optimal solvent, the effect of extraction parameters on the yields of extracted passion fruit peel pectins (PFPP) and total phenolic content (TPC) was explored by response surfaces methodology (RSM). A maximum pectin yield (22.63%) and the highest TPC (9.68 mg GAE/g DW) were attained under 90 °C, extraction solvent pH = 2, extraction time of 120 min and L/S ratio of 20 mL/g. In addition, Pro-CA-extracted pectins (Pro-CA-PFPP) and HCl-extracted pectins (HCl-PFPP) were subjected to high performance gel permeation chromatography (HPGPC), Fourier transform infrared spectroscopy (FT-IR), thermogram analysis (TG/DTG) and rheological measurements. Results verified that the Mw and thermal stability of Pro-CA-PFPP were higher than those of HCl-PFPP. The PFPP solutions featured a non-Newtonian behavior, and compared with commercially pectin solution, PFPP solution exhibited a stronger antioxidant activity. Additionally, passion fruit peel extract (PFPE) exhibited stronger antioxidant effects than PFPP. The results of ultra-performance liquid chromatography hybrid triple quadrupole-linear ion trap mass spectrometry (UPLC-Qtrap-MS) and high performance liquid chromatography (HPLC) analysis showed that (−)-epigallocatechin, gallic acid, epicatechin, kaempferol-3-O-rutin and myricetin were the main phenolic compounds in PFPE and PFPP. Our results suggest that Pro-CA can be considered as an eco-friendly solvent for high-efficient extraction of high-value compounds from agricultural by-products.

Constituent Analysis and Quality Evaluation of Epimedii Folium from Different Species

Epimedii Folium (EF) is a commonly used traditional Chinese drug that includes many species. In Chinese Pharmacopoeia (2020 edition), EF includes Epimedium brevicornu Maxim, E. Sagittatum (Sieb. et Zucc) Maxim, E. pubescens Maxim, E. koreanum Nakai, and Epimedium Wushanense T.S. Ying. It has been reported that the active constituents in EF are different, resulting in the uneven quality of commercial medicinal materials. To explore the specific differences and make a comprehensive quality evaluation of EF, we established an analytical method to simultaneously detect 45 constituents including 23 flavonoids, 4 phenolic acids, 12 amino acids, 5 nucleosides, and 1 alkaloid in 5 species of EF, based on ultrafast performance liquid chromatography coupled with triple quadrupole-linear ion trap mass spectrometry (UFLC-QTRAP-MS/MS). In addition, orthogonal partial least squares discriminant analysis (OPLS-DA), analysis of variance (ANOVA), and grey correlation analysis (GRA) were used to discriminate and evaluate different species of samples. The results showed that EF could be divided into three categories and EBM was superior to the other four species. This study provides data basis for the comprehensive evaluation and a new perspective on the quality control of EF from different species.

LC-MS-based metabolite profiling of aqueous extract of Pergularia tomentosa L. and its anti-hyperglycemic effect.

In this study, to find scientific evidence for the traditional use of Pergularia tomentosa as an anti-diabetic remedy, the effects of its aqueous extract on streptozotocin-induced diabetes mellitus in rats were evaluated. Wistar rats were fasted overnight and diabetes mellitus was induced using streptozotocin (50 mg/kg body weight). The rats were randomly and equally divided into four groups (n=5): group I (normoglycaemic control), group II (diabetic rats), group III (diabetic rats treated with 200 mg/kg BW of an aqueous extract of P. tomentosa), group IV (normoglycemic rats treated with 200 mg/kg BW of an aqueous extract of P. tomentosa). Chemical profiling of the aqueous extract was carried out using liquid chromatography coupled with electrospray ionization and multiple-stage linear ion-trap and orbitrap high-resolution mass spectrometry (LC-ESI/LTQOrbitrap/MS/MS). In addition, the quantitative determination of the main cardenolides in the extract was carried out by an analytical approach based on LC coupled to tandem mass spectrometry with ESI source and hybrid triple quadrupole-linear ion trap mass analyzer (LC-ESI/QTrap/MS/MS). Aqueous extract of P. tomentosa showed a reasonable reduction in blood glucose level. Probably, the P. tomentosa effect on hyperglycemic and hyperlipidemic diabetic animals was associated with antioxidant properties, triglyceride levels, as well as liver enzymes. Meanwhile, LC-ESI/LTQOrbitrap/MS/MS analysis led to identification of double-linked cardenolides along with cardenolides and flavone glycosides as the main bioactive compounds. The extract decreased the glucose level and induced a beneficial effect on the lipid profile, probably due to the presence of cardenolide glycosides.

Development and Validation of a Highly Sensitive and Rapid LC-MS3 Strategy to Determine Oxcarbazepine and Its Active Metabolite in the Serum of Patients with Epilepsy and Its Application in Therapeutic Drug Monitoring

A sensitive and rapid bioanalytical method based on the LC-triple-stage fragmentation (LC-MS3) strategy on a hybrid triple quadrupole-linear ion trap mass spectrometer in combination with protein precipitation extraction for sample pretreatment has been developed and validated for the simultaneous determination of the antiepileptic drug oxcarbazepine (OXC) and its main active metabolite (MHD) in human serum. The separation was performed on a Waters XBridge BEH C18 column (2.5 µm, 2.1 × 50 mm) in isocratic elution with 0.1% formic acid in water and methanol (50:50, v:v) as the mobile phase. The run time for each sample was 2.0 min. The calibration curves ranging from 25 to 1600 ng/mL for OXC and from 0.5 to 32 μg/mL for MHD showed correlation coefficients (r) better than 0.99. All of the validation data, such as precision, accuracy and other parameters, fit the requirements of the current bioanalytical method validation guidelines. The LC-MS3 method for quantitation of OXC and MHD was compared with the LC-MRM based method. Passing–Bablok regression coefficients and Bland–Altman plots showed that the developed LC–MS3 method is a reliable method for quantitative analysis of OXC and MHD. The proposed LC-MS3 method was successfully applied to determine the serum concentrations of OXC and MHD to support a clinical study.

Quality Evaluation of Tetrastigmae Radix from Two Different Habitats Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Multivariate Statistical Analysis.

Tetrastigmae Radix, also known as Sanyeqing (SYQ) in Chinese, is an important traditional Chinese medicine with a long history. Tetrastigma hemsleyanum Diels et Gilg mainly grows in the south of the Yangtze River and is widely distributed. The content of bioactive constituents in SYQ varies greatly in different habitats, and there are obvious differences in the content of bioactive constituents between southwestern SYQ (WS) and southeastern SYQ (ES). To distinguish and evaluate the quality of ES and WS, an analytical method based on ultrafast performance liquid chromatography coupled with triple quadrupole-linear ion trap mass spectrometry (UFLC-QTRAP-MS/MS) was established for the simultaneous determination of 60 constituents including 25 flavonoids, 9 phenolic acids, 15 amino acids, and 11 nucleosides in 47 samples from ES and WS. In addition, orthogonal partial least squares discriminant analysis (OPLS-DA), t-test, and gray correlation analysis (GRA) were used to discriminate and evaluate the ES and WS samples based on the contents of 60 constituents. The results showed that there were significant differences in the bioactive constituents between ES and WS, and ES was superior to WS in terms of quality evaluation. This study not only provides basic information for differentiating ES and WS but also provides a new perspective for the comprehensive evaluation and quality control of SYQ from two different habitats.

Changes in Phospholipid Composition of the Human Cerebellum and Motor Cortex during Normal Ageing.

(1) Background: Changes in phospholipid (phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, i.e., PC, PE and PS) composition with age in the mitochondrial and microsomal membranes of the human cerebellum and motor cortex were examined and compared to previous analyses of the prefrontal cortex, hippocampus and entorhinal cortex. (2) Methods: Nano-electrospray ionization on a hybrid triple quadrupole–linear ion trap mass spectrometer was used to analyse the brain regions of subjects aged 18–104 years. (3) Results: With age, the cerebellum showed many changes in the major phospholipids (>10% of the phospholipid class). In both membrane types, these included increases in PE 18:0_22:6 and PS 18:0_22:6, decreases in PE 18:0_20:4 and PS 18:0_18:1 and an increase in PC 16:0_16:0 (microsomal membrane only). In addition, twenty-one minor phospholipids also changed. In the motor cortex, only ten minor phospholipids changed with age. With age, the acyl composition of the membranes in the cerebellum increased in docosahexaenoic acid (22:6) and decreased in the arachidonic (20:4) and adrenic (22:4) acids. A comparison of phospholipid changes in the cerebellum, motor cortex and other brain areas is provided. (4) Conclusions: The cerebellum is exceptional in the large number of major phospholipids that undergo changes (with consequential changes in acyl composition) with age, whereas the motor cortex is highly resistant to change.

Systematic Qualitative and Quantitative Analyses of Wenxin Granule via Ultra-High Performance Liquid Chromatography Coupled with Ion Mobility Quadrupole Time-of-Flight Mass Spectrometry and Triple Quadrupole-Linear Ion Trap Mass Spectrometry.

Wenxin granule (WXG) is a popular traditional Chinese medicine (TCM) preparation for the treatment of arrhythmia disease. Potent analytical technologies are needed to elucidate its chemical composition and assess the quality differences among multibatch samples. In this work, both a multicomponent characterization and quantitative assay of WXG were conducted using two liquid chromatography–mass spectrometry (LC-MS) approaches. An ultra-high performance liquid chromatography–ion mobility quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) approach combined with intelligent peak annotation workflows was developed to characterize the multicomponents of WXG. A hybrid scan approach enabling alternative data-independent and data-dependent acquisitions was established. We characterized 205 components, including 92 ginsenosides, 53 steroidal saponins, 14 alkaloids, and 46 others. Moreover, an optimized scheduled multiple reaction monitoring (sMRM) method was elaborated, targeting 24 compounds of WXG via ultra-high performance liquid chromatography–triple quadrupole linear ion trap mass spectrometry (UHPLC/QTrap-MS), which was validated based on its selectivity, precision, stability, repeatability, linearity, sensitivity, recovery, and matrix effect. By applying this method to 27 batches of WXG samples, the content variations of multiple markers from Notoginseng Radix et Rhizoma (21) and Codonopsis Radix (3) were depicted. Conclusively, we achieved the comprehensive multicomponent characterization and holistic quality assessment of WXG by targeting the non-volatile components.

Degradation of Ochratoxin A by a UV-Mutated Aspergillus niger Strain.

Ochratoxin A (OTA) is a mycotoxin that can contaminate a wide range of crops such as grains and grapes. In this study, a novel fungal mutant strain (FS-UV-21) with a high OTA degradation rate (74.5%) was obtained from Aspergillus niger irradiated with ultraviolet light (15 W for 20 min). The effect of pH, temperature, and inoculation concentration on the degradation of OTA by FS-UV-21 was investigated, and the results revealed that the detoxification effect was optimal (89.4%) at a pH of 8 and a temperature of 30 °C. Ultra-performance liquid chromatography-tandem mass spectrometry was used to characterize the degraded products of OTA, and the main degraded product was ochratoxin α. Triple quadrupole-linear ion trap-mass spectrometry combined with LightSight software was used to analyze the biotransformation pathway of OTA in FS-UV-21, to trace the degraded products, and to identify the main metabolite, P1 (C19H18ClNO6, m/z 404). After the FS-UV-21 strain was treated with OTA, the HepG2 cellular toxicity of the degradation products was significantly reduced. For the real sample, FS-UV-21 was used to remove OTA from wheat bran contaminated by mycotoxins through fermentation, resulting in the degradation of 59.8% of OTA in wheat bran. Therefore, FS-UV-21 can be applied to the degradation of OTA in agricultural products and food.

Target profiling of flavonol glycosides in the extract of Ginkgo biloba leaf and their pharmacokinetics in rat plasma by ultra‐high‐performance liquid chromatography with tandem mass spectrometry

The extract of Ginkgo biloba leaf is a popular herbal product or dietary supplement in the world to treat various diseases, and flavonol glycosides are considered as the main bioactive constituents. In this study, 37 flavonol glycosides were rapidly screened out by precursor ion scanning in positive ion mode with production ions at m/z 287.05, 303.05, and 317.06. Subsequently, a reliable and sensitive ultra-high-performance liquid chromatography coupled with triple quadrupole-linear ion trap mass spectrometry approach was established and validated to quantify the 20 prototype flavonol glycosides in rat plasma. Calibration curves showed good linearity (R2 ≥ 0.9894) over the corresponding concentration range. The precision, accuracy, extraction recovery, matrix effect, and stability were also satisfactory. The validated method was successfully applied to a pharmacokinetic study of prototype flavonol glycosides in rat after oral administration of the extract of G. biloba leaf. As a result, the Tmax of flavonol glycosides was short at 0.11-0.60 h. Quercetin-3-O-(2",6″-di-O-rhamnosyl)-glucoside, kaempferol-3-O-(2'',6''-di-O-rhamnosyl)-glucoside, quercetin-3-O-rutinoside, quercetin- 3-O-glucosyl-(1-2)-O-rhamnoside, and kaempferol-3-O-glucoside presented relatively high systemic exposure levels with AUC0-∞ >500 μg h/L and Cmax >100 μg/L. This study would provide the valuable information for further scientific research and clinical application of the extract of G. biloba leaf.

Determination of lipophilic marine biotoxins in shellfish foods by ultra performance liquid chromatography-hybrid triple quadrupole linear ion trap-mass spectrometry

An ultra performance liquid chromatography-hybrid triple quadrupole linear ion trap-mass spectrometry(UPLC-QqLIT-MS) was established for determination of lipophilic marine biotoxins in shellfish. And the 12 lipophilic marine biotoxins in shellfish were surveyed. The lipophilic marine biotoxins in homogenized shellfish were ultrasonically extracted by methanol in super-sonic instrument, and cleaned up by solid phase extraction of Strata-X column, and eluted with methanol(containing 0.3% ammonia water). The elution was diluted with water, and cleaned by 0.22 μm millipore filter. The filtrate was separated on a Waters ACQUITY UPLC BEH C_(18) column(150 mm×2.1 mm, 1.7 μm)by gradient elution in 12 minutes with acetronitrile-water(containing 0.01%(V/V) ammonia and 2 mmol/L ammonium formate) as mobile phase, and detected by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS), identified by electrospray ionization(ESI) in simultaneous scanning mode of positive and negative ions using multiple reaction monitoring, and quantified with external standards. Information dependent acquisition scan function(IDA) combined with enhanced production scan(EPI) was used to confirm the 12 lipophilic marine biotoxins. The calibration curves of 12 lipophilic marine biotoxins showed good linearity in the range of 0.5-50 μg/L with correlation coefficients were 0.9984-0.9999.The detection limits of the method were 0.15-0.29 μg/kg. The recoveries of three spiking levels ranged from 80.0% to 116.0%, and the relative standard deviation(RSD) were 0.6%-6.4%(n=7). The method for determination of 12 lipophilic marine biotoxins in shellfish by UPLC-QqLIT-MS was of operation convenience, less interference from impurities and good accuracy, which could meet the requirements for the determination of 12 lipophilic marine biotoxins residues in sea foods.

Simultaneous Determination of Iridoid Glycosides, Phenylpropanoid Glycosides, Organic Acids, Nucleosides and Amino Acids in Scrophulariae Radix Processed by Different Processing Methods by HPLC-QTRAP-MS/MS.

Scrophulariae Radix is one of the widely used traditional Chinese medicines. In this study, a high-performance liquid chromatography coupled with triple quadrupole-linear ion trap mass spectrometry method was established for the simultaneous determination of multiple bioactive constituents including four iridoid glycosides, two phenylpropanoid glycosides, six organic acids, 11 nucleosides and 16 amino acids in Scrophulariae Radix. The validated method was used to analyze nine Scrophulariae Radix samples processed by different processing methods. In addition, Grey relational analysis and DTOPSIS were used to evaluate the samples according to the content of 39 ayalytes. The results showed that the quality of Scrophulariae Radix processed by cutting into slices, sun drying and "sweating" methods were better. All the results proved that the developed method was available and could be used to evaluate the quality of Scrophulariae Radix.

Establishment of the tandem mass spectrometric fingerprints of taxane-based anticancer compounds.

Compounds in the taxane drug family are among the most successful and effective chemotherapeutic agents used in the treatment of solid tumors, such as breast, ovarian, and prostate cancers. The tandem mass spectrometric (MS/MS) fragmentation behavior of these compounds is described in detail, and a generalized MS/MS fingerprint is established for the first time. Five compounds, namely paclitaxel, docetaxel, cabazitaxel, cephalomannine, and baccatin III, were evaluated. A hybrid quadrupole orthogonal time-of-flight (Q-TOF) mass spectrometer was used to obtain accurate mass measurements, whereas MS/MS and second-generation MS/MS (MS3 ) analyses were performed using a triple quadrupole-linear ion trap mass spectrometer. Both instruments were equipped with an electrospray ionization source operated in the positive ion mode. All taxanes showed an abundant singly charged [M + H]+ species in the single-stage analysis with mass accuracies less than 3ppm. The evaluated compounds exhibited common fragmentation behavior in their MS/MS analysis, which allowed for the production of a universal fragmentation pattern. MS3 experiments confirmed the genesis of the various product ions proposed in the fragmentation pathway. In addition, diagnostic product ions were originated from a cleavage in the ester bond between the core diterpene ring structure and the side chain. Varying functional groups present in these compounds resulted in unique product ions that are specific to each structure. The established MS/MS fingerprints will be used in the near future for identification and for the development of multiple reaction monitoring liquid chromatography-MS/MS quantification methods.

Metabolite Profiling and Characterization of LW6, a Novel HIF-1α Inhibitor, as an Antitumor Drug Candidate in Mice.

A novel HIF (hypoxia-inducible factor)-1α inhibitor, the (aryloxyacetylamino)benzoic acid derivative LW6, is an anticancer agent that inhibits the accumulation of HIF-1α. The aim of this study was to characterize and determine the structures of the metabolites of LW6 in ICR mice. Metabolite identification was performed using a predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (pMRM-IDA-EPI) method in negative ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (QTRAP). A total of 12 metabolites were characterized based on their MS/MS spectra, and the retention times were compared with those of the parent compound. The metabolites were divided into five structural classes based on biotransformation reactions: amide hydrolysis, ester hydrolysis, mono-oxidation, glucuronidation, and a combination of these reactions. From this study, 2-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)acetic acid (APA, M7), the metabolite produced via amide hydrolysis, was found to be a major circulating metabolite of LW6 in mice. The results of this study can be used to improve the pharmacokinetic profile by lowering the clearance and increasing the exposure relative to LW6.

Exploration of biodegradation mechanism by AFB1-degrading strain Aspergillus niger FS10 and its metabolic feedback

Significant progress has been made in the biological degradation of aflatoxin B1 (AFB1), which is the most toxic and carcinogenic mycotoxin commonly found in food and feed products; however, research in this area has currently focused on the effective methods for detoxifying AFB1 and the mechanisms underlying the degradation process. The relationship between AFB1 biotransformation and the metabolic feedback by the AFB1-degrading strain has rarely been reported. This study investigated the biotransformation pathway of AFB1 in a highly efficient degrading strain, Aspergillus niger FS10, and the metabolic regulation of FS10 during AFB1 degradation. We analyzed the AFB1 metabolic degradation products and evaluated its toxicity based on its structures by triple quadrupole-linear ion trap-mass spectrometry (Q-Trap-MS) coupled with LightSight™ Software. Four metabolic degradation products were obtained: Product 1 (C17H14O6), Product 2 (C17H14O7), Product 3 (C16H12O5), and Product 4 (C27H31N3O13, AFB2-GOH). Two biotransformation pathways were proposed based on the structures of these degradation products. Metabolomics data revealed that FS10 exhibited different degrees of bioremediation under low-, medium- and high- dose AFB1. The results also suggested the significant involvement of glutathione, a metabolite, in the AFB1 biotransformation pathway and its formation of AFB2-GOH with AFB1. The four products could probably be less toxic than AFB1, as reflected by changes in the AFB1 toxicity site. Therefore, the AFB1-degrading strain FS10 exhibits great potential for application in the detoxification of AFB1 in food and feed products.

P0390THERAPEUTIC EFFECTS AND SERUM METABOLOMIC VARIATIONS IN FOCAL SEGMENTAL GLOMERULOSCLEROSIS MICE ON TREATMENT WITH HUMAN UMBILICAL MESENCHYMAL STEM CELLS

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) mediates the kidney podocyte dysfunction and leads to chronic renal diseases worldwide. Current treatment options are limited, owing to the poor understanding of the metabolic pathologic variations and regeneration of FSGS. Mesenchymal stem cells (MSC) offer the better insights to prevent renal injury and could promote the recovery of renal structure and function through complex mechanisms. Due to the safe, feasible, and therapeutic features, we first designed to investigate the role of human umbilical mesenchymal stem cells (HUMSCs) on FSGS mice and explored the potential serum metabolomic variations. Method Nephropathy was induced by adriamycin (ADR) in male Balb/C mice to study FSGS via intravenous administration. 2*106 HUMSCs are provided to treat FSGS model for 3 times. Renal function was measured by urine protein, serum urea nitrogen (BUN) and serum creatinine (SCr) in the study. Serum metabolic profiles from three groups of Balb/C mice were analysed using ultra- performance liquid chromatography coupled with a triple quadrupole-linear ion trap mass spectrometer and a novel mass spectrometry (UPLC-MS/MS) data collection technique. Results The concentrations of SCr and BUN were apparently decreased after HUMSCs injection for three times compared with the FSGS model and body weight from HUMSCs group was improved significantly. A total of 532 differential metabolites were identified between FSGS group and the normal control group. Bile acid dysregulation and valine/leucine/ isoleucine metabolism pathway, especially, N-acetylvaline and L-valine, were positively associated to ADR-induced FSGS. A panel of identified specific metabolites (pyrrole-2- carboxylic acid, urocanic acid and 6-hydroxynicotinic acid) as well as valine/leucine/isoleucine metabolism pathway rather than bile acid metabolism were related to the renal repair after HUMSCs treatment. Conclusion The administration of HUMSCs in FSGS induced ADR provides the promising protection owing to the improvement of metabolomics. Figure 2. The distribution of DiR-HUMSCs in vivo. Figure 3. Kidney pathology using periodic acid-Schiff (PAS) and Masson staining. Figure 4. Metabolome profiling analysis and Venn diagram for comparisons of numbers from each group. Figure 5. Metabolic pathway analysis results from FSGS + Saline and FSGS + HUMSCs groups. Figure S1. The quality control (QC) of serum metabolomics was designed and performed as follows. Figure S2. Metabolic pathway analysis results from FSGS + Saline and Normal + Saline groups. Figure S3. Metabolic pathway analysis results from FSGS + HUMSCs and Normal + Saline groups.

Identification of Potential Biomarkers for Early Prediction of Gestational Diabetes

Abstract Objectives Gestational Diabetes Mellitus (GDM) is present in up to 10% of pregnancies in the United States. The occurrence of GDM causes severe short- and long-term complications for the mother and offspring. baby pre- and post-partum. Identification of the metabolites and potential biomarkers involved in GDM could improve the prediction of its occurence. The integration of food data with metabolite results could provide innovative diet intervention strategies. The objective of this study is to identify metabolites that differed in the first and third trimesters of GDM versus Non-GDM pregnancies. Methods Participants were 68 OW/OB pregnant women enrolled in the Healthy Beginnings Trial and completed blood draws at first (10–16 weeks) and third trimester (28–35 weeks). Participants from the control and dietary intervention group who developed GDM (n = 34; GDM group) were matched on age, BMI, ethnicity, and treatment group with those who did not develop GDM (n = 34; Non-GDM group). Plasma samples were analyzed by ultra-high-performance liquid chromatography-hybrid triple-quadrupole linear ion trap mass spectrometry (UPLC-QTRAP) using three targeted metabolomics assays for primary metabolomics, aminomics and lipdomics. Dietary intake was estimated using 24 hour recalls in order to assess potential dietary differences between groups. Results A total of 243 metabolites were identified in the plasma samples. At first trimester, several complex lipids, including cholestryl esters and phospholipids, were higher in the GDM group (P &amp;lt; 0.05). Furthermore, the purine derivative hypoxanthine was also higher in GDM subjects (P &amp;lt; 0.05). At third trimester, multiple acylcarnitines, associated with utilization of fat for energy, were lower in the GDM group (P &amp;lt; 0.05). Conclusions Metabolite differences between GDM and Non-GDM groups in plasma samples collected during first trimester may predict the development of GDM. Further research is required to identify the roles these metabolite changes play in the development of this disease. Funding Sources NIH National Heart, Lung, and Blood Institute (NHLBI; HL114377), ARI #58,875, Cal Poly CAFES SURP.