Absorbed Bioactive Compounds Replicate Guanxin II-Induced Endothelium-Associated in/ex vivo Vasodilation.

OBJECTIVE: To develop an interference-free and rapid method to elucidate Guanxin II (GX II)'s representative vasodilator absorbed bioactive compounds (ABCs) among enormous phytochemicals. METHODS: The contents of ferulic acid, tanshinol, and hydroxysafflor yellow A (FTA) in GX II/rat serum after the oral administration of GX II (30 g/kg) were detected using ultra-performance liquid chromatography-mass spectrometry. Totally 18 rats were randomly assigned to the control group (0.9% normal saline), GX II (30 g/kg) and FTA (5, 28 and 77 mg/kg) by random number table method. Diastolic coronary flow velocity-time integral (VTI), i.e., coronary flow or coronary flow-mediated dilation (CFMD), and endothelium-intact vascular tension of isolated aortic rings were measured. After 12 h of exposure to blank medium or 0.5 mmol/L H2O2, endothelial cells (ECs) were treated with post-dose GX II of supernatant from deproteinized serum (PGSDS, 300 µL PGSDS per 1 mL of culture medium) or FTA (237, 1539, and 1510 mg/mL) for 10 min as control, H2O2, PGSDS and FTA groups. Nitric oxide (NO), vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), superoxide dismutase (SOD), malondialdehyde (MDA) and phosphorylated phosphoinositide 3 kinase (p-PI3K), phosphorylated protein kinase B (p-AKT), phosphorylated endothelial nitric oxide synthase (p-eNOS) were analyzed. PGSDS was developed as a GX II proxy of ex vivo herbal crude extracts. RESULTS: PGSDS effectively eliminates false responses caused by crude GX II preparations. When doses equaled the contents in GX II/its post-dose serum, FTA accounted for 98.17% of GX II -added CFMD and 92.99% of PGSDS-reduced vascular tension. In ECs, FTA/PGSDS was found to have significant antioxidant (lower MDA and higher SOD, P<0.01) and endothelial function-protective (lower VEGF, ET-1, P<0.01) effects. The increases in aortic relaxation, endothelial NO levels and phosphorylated PI3K/Akt/eNOS protein induced by FTA/PGSDS were markedly abolished by NG-nitro-L-arginine methyl ester (L-NA, eNOS inhibitor) and wortmannin (PI3K/AKT inhibitor), respectively, indicating an endothelium-dependent vasodilation via the PI3K/AKT-eNOS pathway (P<0.01). CONCLUSION: This study provides a strategy for rapidly and precisely elucidating GX II's representative in/ex vivo cardioprotective absorbed bioactive compounds (ABCs)-FTA, suggesting its potential in advancing precision ethnomedicine.

Exploring Central Regulatory Effect of Chaishao Liujuntang on Chronic Atrophic Gastritis Rats with Liver Depression and Spleen Deficiency Syndrome Based on Metabolomics / 中国实验方剂学杂志

ObjectiveBased on ultra performance liquid chromatography-mass spectrometry（UPLC-MS） and non-targeted metabolomics technology to discuss the central regulatory effect of Chaishao Liujuntang on chronic atrophic gastritis（CAG） rats with liver-depression and spleen-deficiency， and to look for the correlation between cerebral cortex， hypothalamus and metabolic status of gastric tissues. MethodA CAG rat model with liver-depression and spleen-deficiency was established by chemical induction， hunger and satiety disorders， chronic restraint and tail clamping stimulation， lasting for 16 weeks. Twenty-eight Wistar rats were randomly divided into a blank group of 8 rats and a model group of 20 rats. After the completion of modeling， 4 rats in the model group were taken to observe the pathological changes of gastric mucosa. The remaining model rats were randomly divided into a model group of 8 rats and a Chaishao Liujuntang group of 8 rats. Chaishao Liujuntang group rats were given 5.1 g·kg-1 by gavage， and the remaining rats were given equal volume sterilized water by gavage for 4 weeks. Macroscopic characteristics， behavioral indicators and histopathological changes of the gastric mucosa of rats in each group were observed and compared. UPLC-MS non-targeted metabolomics was used to explore the metabolic regulation effect of Chaishao Liujuntang on the cerebral cortex， hypothalamus and stomach tissues of CAG rats with liver-depression and spleen-deficiency. Pearson correlation coefficient method was used to analyze the correlation between different tissue metabolites. ResultCompared with the model group， the macroscopic characteristics of rats in Chaishao Liujuntang group were improved， such as hair color， mental state and stool properties， and the number of times of crossing and standing in the open field experiment was significantly increased， and the static time of forced swimming was significantly reduced（P<0.01）， and the gastric mucosa atrophy was reduced. The metabolic data from the cerebral cortex of rats in each group identified a total of 3 common potential biomarkers， but not enriched in pathways， 26 common potential biomarkers were identified in the hypothalamus， and the key metabolic pathways involved were mainly enriched in purine metabolism， glycerol phospholipid metabolism， D-glutamine and D-glutamic acid metabolism. Seventeen common potential biomarkers were identified in the stomach， and the key metabolic pathways involved were mainly enriched in thiamine metabolism， valine， leucine and isoleucine biosynthesis， and taurine and taurine metabolism. Correlation analysis of metabolites in different tissues revealed that multiple amino acids and their derivatives mediated metabolic connections between the cerebral cortex， hypothalamus and stomach of rats. ConclusionThe metabolic disorders in the cerebral cortex， hypothalamus and stomach of CAG rats with liver-depression and spleen-deficiency have their own characteristics， mainly manifested by changes in the content of glycerol phospholipids， fatty acids and bile acid metabolites. Moreover， Chaishao Liujuntang may play a central regulatory role in CAG rats with liver-depression and spleen-deficiency by correcting the metabolic disorders of amino acids.

Identification of potential glioma drug resistance target proteins based on ultra-performance liquid chromatography-mass spectrometry differential proteomics.

In this study, to screen for candidate markers of temozolomide (TMZ) resistance in glioblastoma, we artificially established TMZ drug-resistant glioblastoma (GBM) cell lines, U251-TMZ and U87-TMZ. In the U251-TMZ and U87-TMZ cell lines, we screened and analyzed differentially expressed proteins using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) differential proteomics. Compared with the U251 and U87 control cell lines, 95 differential proteins were screened in the U251-TMZ and U87-TMZ cell lines, of which 28 proteins were upregulated and 67 proteins were down-regulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the co-upregulated proteins showed that most of the differentially expressed proteins were located in the cytoplasm and were significantly upregulated in the biological processes related to vesicular transport in the intimal system and inflammatory response mediated by myeloid leukocytes. Seven candidates were identified as potential GBM markers of TMZ resistance. Combined with existing research findings, our study supports that UAP1L1 and BCKDK are promising potential markers of TMZ resistance in GBM. This is important for further understanding the molecular mechanisms that drive the development and enhancement of TMZ resistance.

Validation and interlaboratory comparison of anticoagulant rodenticide analysis in animal livers using ultra-performance liquid chromatography-mass spectrometry.

Anticoagulant rodenticides (ARs) are used to control rodent populations. Poisoning of non-target species can occur by accidental consumption of commercial formulations used for rodent control. A robust method for determining ARs in animal tissues is important for animal postmortem diagnostic and forensic purposes. We evaluated an ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) method to quantify 8 ARs (brodifacoum, bromadiolone, chlorophacinone, coumachlor, dicoumarol, difethialone, diphacinone, warfarin) in a wide range of animal (bovine, canine, chicken, equine, porcine) liver samples, including incurred samples. We further evaluated UPLC-MS in 2 interlaboratory comparison (ILC) studies; one an ILC exercise (ICE), the other a proficiency test (PT). The limits of detection of UPLC-MS were 0.3-3.1 ng/g, and the limits of quantification were 0.8-9.4 ng/g. The recoveries obtained using UPLC-MS were 90-115%, and relative SDs were 1.2-13% for each of the 8 ARs for the 50, 500, and 2,000 ng/g spiked liver samples. The overall accuracy from the laboratories participating in the 2 ILC studies (4 and 11 laboratories for ICE and PT studies, respectively) were 86-118%, with relative repeatability SDs of 3.7-11%, relative reproducibility SDs of 7.8-31.2%, and Horwitz ratio values of 0.5-1.5. Via the ILC studies, we verified the accuracy of UPLC-MS for AR analysis in liver matrices and demonstrated that ILC can be utilized to evaluate performance characteristics of analytical methods.

Assessment of Purity, Functionality, Stability, and Lipid Composition of Cyclofos-nAChR-Detergent Complexes from Torpedo californica Using Lipid Matrix and Macroscopic Electrophysiology.

The main objective of the present study was to find detergents that can maintain the functionality and stability of the Torpedo californica nicotinic acetylcholine receptor (Tc-nAChR). We examined the functionality, stability, and purity analysis of affinity-purified Tc-nAChR solubilized in detergents from the Cyclofos (CF) family [cyclofoscholine 4 (CF-4), cyclofoscholine 6 (CF-6), and cyclofloscholine 7 (CF-7)]. The functionality of the CF-Tc-nAChR-detergent complex (DC) was evaluated using the Two Electrode Voltage Clamp (TEVC) method. To assess stability, we used the florescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) methodology. We also performed a lipidomic analysis using Ultra-Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI-MS/MS) to evaluate the lipid composition of the CF-Tc-nAChR-DCs. The CF-4-Tc-nAChR-DC displayed a robust macroscopic current (- 200 ± 60 nA); however, the CF-6-Tc-nAChR-DC and CF-7-Tc-nAChR-DC displayed significant reductions in the macroscopic currents. The CF-6-Tc-nAChR and CF-4-Tc-nAChR displayed higher fractional florescence recovery. Addition of cholesterol produced a mild enhancement of the mobile fraction on the CF-6-Tc-nAChR. The lipidomic analysis revealed that the CF-7-Tc-nAChR-DC displayed substantial delipidation, consistent with the lack of stability and functional response of this complex. Although the CF-6-nAChR-DC complex retained the largest amount of lipids, it showed a loss of six lipid species [SM(d16:1/18:0); PC(18:2/14:1); PC(14:0/18:1); PC(16:0/18:1); PC(20:5/20:4), and PC(20:4/20:5)] that are present in the CF-4-nAChR-DC. Overall, the CF-4-nAChR displayed robust functionality, significant stability, and the best purity among the three CF detergents; therefore, CF-4 is a suitable candidate to prepare Tc-nAChR crystals for structural studies.

Metabolomic Diversity of Human Milk Cells over the Course of Lactation-A Preliminary Study.

Human milk (HM) is a complex biofluid containing a wide cell variety including epithelial cells and leukocytes. However, the cellular compositions and their phenotypic properties over the course of lactation are poorly understood. The aim of this preliminary study was to characterize the cellular metabolome of HM over the course of lactation. Cells were isolated via centrifugation and the cellular fraction was characterized via cytomorphology and immunocytochemical staining. Cell metabolites were extracted and analyzed using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QqTOF-MS) in the positive and negative electrospray ionization modes. Immunocytochemical analysis revealed a high variability of the number of detected cells with relative median abundances of 98% of glandular epithelial cells, 1% of leukocytes, and 1% of keratinocytes. Significant correlations between the milk postnatal age with percentage of epithelial cells and leukocytes, and with total cell count were observed. Results from the Hierarchical Cluster Analysis of immunocytochemical profiles were very similar to those observed in the analysis of the metabolomic profiles. In addition, metabolic pathway analysis showed alterations in seven metabolic pathways correlating with postnatal age. This work paves the way for future investigations on changes in the metabolomic fraction of the cellular compartment of HM.

Steroid profiling for the diagnosis of congenital adrenal hyperplasia by microbore ultra-performance liquid chromatography-tandem mass spectrometry.

BACKGROUND: A rapid and accurate measurement approach for 17α-hydroxyprogesterone (17-OHP) and related steroids in amount/volume-limited clinic samples is of importance for precise newborn diagnosis of congenital adrenal hyperplasia (CAH) and its subtypes in clinic. METHODS: Sixteen steroids (17-OHP, androstenedione, cortisol, tetrahydro-11-deoxycortisol, pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, 21-deoxycortisol, 11-deoxycortisol, dehydroepiandrosterone, testosterone, aldosterone, 17α-hydroxypregnenolone, dihydrotestosterone and 18-hydroxycorticosterone) were included in the panel of high-throughput microbore ultra-performance liquid chromatography-tandem mass spectrometry. Samples were collected from 126 normal subjects and 65 patients including different subtypes of CAH. RESULTS: The method was validated with satisfactory analytical performance in linearity, repeatability, recovery and limit of detection. Reference intervals for 16 steroids were established by quantifying the level of steroids detected in normal infants. The applicability of the method was tested by differentiating steroid metabolic characteristics between normal infants and infants with CAH, as well as between infants with different CAH subtypes. The relevance of 17-OHP, 21-deoxycortisol, and 17-OHP/11-deoxycortisol for 21-hydroxylase deficiency screening was demonstrated. The level of 11-deoxycorticosterone, 11-deoxycortisol, progesterone and androstenedione can be used for the diagnosis of different rare subtypes of CAH. CONCLUSION: This study provides a strategy for highly efficient steroid analysis of amount/volume-limited clinic samples and holds great potential for clinical diagnosis of CAH.

Targeted lipidomics coupled with machine learning for authenticating the provenance of chicken eggs.

Free-range eggs are ethically desirable but as with all high-value commercial products, the establishment of provenance can be problematic. Here, we compared a simple one-step isopropanol method to a two-step methyl-tert-butyl ether method for extracting lipid species in chicken egg yolks before liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The isopropanol method extracted 937 lipid species from 20 major lipid subclasses with high reproducibility (CV < 30 %). Machine learning techniques could differentiate conventional cage, barn, and free-range eggs using an external test dataset with an accuracy of 0.94, 0.82, and 0.82, respectively. Lipid species that differentiated cage eggs were predominantly phosphocholines and phosphoethanolamines whilst the free-range egg lipidomes were dominated by acylglycerides with up to three fatty acids. The lipid profiles were found to be characteristic of the cage, barns, and free-range eggs. The lipidomic analysis together with the statistical modeling approach thus provides an efficient tool for verifying the provenance of conventional chicken eggs.

Evaluation of uncertainty in determination of cetrimonium bromide residue in polysaccharide vaccines by ultra performance liquid chromatography-mass spectrometry / 中国生物制品学杂志

@#Objective To determine the cetrimonium bromide(CTAB)residue in polysaccharide vaccines using ultra performance liquid chromatography-mass spectrometry(UPLC/MS-MS),and analyze and evaluate the uncertainty of the determination results.Methods By establishing a mathematical model,the sources and values of uncertainty introduced in the measurement process were analyzed,the uncertainty components of each influencing factor were calculated,and the standard uncertainty and expanded uncertainty were synthesized to form an uncertainty report.Results At 95% confidence interval,the expanded uncertainty was 0. 002 8 mg/kg. The determination result of CTAB residue in polysaccharide vaccine was reported as(1. 000 6 ± 0. 002 8)mg/kg(k = 2,confidence interval p = 95%).Conclusion The main factors affecting the accuracy of determination results are the preparation of standard solution and the introduction of recovery rate,which should be focused on and controlled in the experiment process to make the detection results more reliable.

Untargeted metabolism approach reveals difference of varieties of bud and relation among characteristics of grafting seedlings in Camellia oleifera.

Camellia oleifera is one of the essential wood oil trees in the world. C.oleifera was propagated by nurse seedling grafting. Since the scion of C.oleifera had a significant regulated effect on the properties of rootstock after grafting and impacted on the growth of the grafted seedlings, it was necessary to understand the characteristics of buds among varieties to cultivate high-quality grafted seedlings. The metabolome was thought to be a powerful tool for understanding connecting phenotype-genotype interactions, which has an important impact on plant growth and development. In this study, UPLC-MS was used to determine the metabolites of the apical buds of CL3, CL4, CL40, and CL53 spring shoots after 30 days of sprout and to measure the growth characteristics of roots and stems after grafting. Metabolomics analysis revealed 554 kinds of metabolites were significant differences among four varieties, and 29 metabolic pathways were identified to have significant changes (p< 0.05), including carboxylic acids and derivatives, fatty Acyls, organooxygen compounds, and prenol lipids metabolites. The metabolites appeared in all varieties, including phenethyl rutinoside in glycosyl compounds and hovenidulcioside A1 in terpene glycosides. Metabolite-metabolite correlations in varieties revealed more complex patterns in relation to bud and enabled the recognition of key metabolites (e.g., Glutamate, (±)Catechin, GA52, ABA, and cs-Zeatin) affecting grafting and growth ability. Each variety has a unique metabolite type and correlation network relationship. Differentiated metabolites showed different growth trends for development after grafting. Many metabolites regulate the growth of scions in buds before grafting, which plays a crucial role in the growth of seedlings after grafting. It not only regulates the growth of roots but also affects the development of this stem. Finally, those results were associated with the genetic background of each cultivar, showing that metabolites could be potentially used as indicators for the genetic background, indicating that metabolites could potentially be used as indicators for seedling growth characteristics. Together, this study will enrich the theoretical basis of seedling growth and lay a foundation for further research on the molecular regulation mechanism interaction between rootstock and scion, rootstock growth, and the development of grafted seedlings after grafting.

Nanoscale Iron trioxide catalyzes the synthesis of auxins analogs in artificial humic acids to enhance rice growth.

Humic acids (HAs), kinds of valuable active carbon, are critical for improving soil fertility. However, the majority of soils are poor in HAs, arousing the development of artificial HAs. In this study, two iron-based catalysts (nanoscale iron trioxide (nFe2O3) and FeCl3) were used to catalyze the hydrothermal humification of waste corn straw. With the help of ultra-performance liquid chromatography-mass spectrometry, we proposed the specific humification process with the action of catalysis for the first time, which is of great significance for the design, synthesis and application of artificial HAs in the future. Moreover, the growth-promoting effect and mechanisms of the artificial HAs were determined by rice planting in a greenhouse. Results showed that compared to no catalyst treatment, the FeCl3 and nFe2O3 catalysts increased the decomposition rate of macromolecular biomass by 39 and 14 %, respectively, increasing the yield of artificial HAs. During the humification process, nFe2O3 catalysts benefit the formation of many aromatic structure monomers including furfural and hydroxycaproic acids. These monomers were condensed into growth hormone analogs such as vanillin and methionine sulfoxide and were further built in the artificial HAs. Therefore, the artificial HAs from nFe2O3 catalytic treatment promoted the rice growth the best, showing that the resultant germination rate, root activity, and photosynthetic rate of rice increased by 50, 167, and 72 %, respectively; moreover, the uptake and accumulation of water and nutrient by roots as well as the contents of soluble protein and sugar of rice are also significantly increased. This could be ascribed to the upregulated expression of functional genes including OsRHL1, OsZPT5-07, OsSHR2 and OsDCL. Considering both the economic and environmental benefits, we suggested that the artificial HAs, especially that produced with the action of nFe2O3 catalysis, are promising in alleviating environmental stress from waste biomass and sustainably improving agricultural production.

Ultra-Performance Liquid Chromatography-Mass Spectrometry-Based Untargeted Metabolomics Reveals the Key Potential Biomarkers for Castor Meal-Induced Enteritis in Juvenile Hybrid Grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂).

The intensification of aquaculture to help kerb global food security issues has led to the quest for more economical new protein-rich ingredients for the feed-based aquaculture since fishmeal (FM, the ingredient with the finest protein and lipid profile) is losing its acceptability due to high cost and demand. Although very high in protein, castor meal (CM), a by-product after oil-extraction, is disposed-off due to the high presence of toxins. Concurrently, the agro-industrial wastes' consistent production and disposal are of utmost concern; however, having better nutritional profiles of these wastes can lead to their adoption. This study was conducted to identify potential biomarkers of CM-induced enteritis in juvenile hybrid-grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂) using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) alongside their growth and distal intestinal (DI) health evaluation. A total of 360 fish (initial weight = 9.13 ± 0.01g) were randomly assigned into three groups, namely, fish-meal (FM) (control), 4% CM (CM4), and 20% CM (CM20). After the 56-days feeding-trial, the DI tissues of FM, CM4, and CM20 groups were collected for metabolomics analysis. Principal components analysis and partial least-squares discriminant-analysis (PLS-DA, used to differentiate the CM20 and CM4, from the FM group with satisfactory explanation and predictive ability) were used to analyze the UPLC-MS data. The results revealed a significant improvement in the growth, DI immune responses and digestive enzyme activities, and DI histological examinations in the CM4 group than the others. Nonetheless, CM20 replacement caused DI physiological damage and enteritis in grouper as shown by AB-PAS staining and scanning electron microscopy examinations, respectively. The most influential metabolites in DI contents identified as the potential biomarkers in the positive and negative modes using the metabolomics UPLC-MS profiles were 28 which included five organoheterocyclic compounds, seven lipids, and lipid-like molecules, seven organic oxygen compounds, two benzenoids, five organic acids and derivatives, one phenylpropanoids and polyketides, and one from nucleosides, nucleotides, and analogues superclass. The present study identified a broad array of DI tissue metabolites that differed between FM and CM diets, which provides a valuable reference for further managing fish intestinal health issues. A replacement level of 4% is recommended based on the growth and immunity of fish.

[Nonspecific adsorption evaluation and general minimization strategy in peptide analysis based on ultra-performance liquid chromatography-mass spectrometry].

Proteomics technology is being increasingly used in the development of novel therapeutic peptides and protein drugs, and also in the intensive search for clinical biomacromolecule diagnostic biomarkers. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) is a rapid method to analyze peptides and proteins in low abundance. However, the nonspecific adsorption properties of peptides may lead to the loss or interference of the analytes throughout the analytical process. This unique nonspecific adsorption property is the main reason for the false negative and false positive results obtained through quantification, as well as for the poor precision, accuracy, linear range, and sensitivity, all of which impose significant challenges in the development of analytical methods. Accordingly, a general strategy was established to evaluate and reduce the negative impact of the nonspecific adsorption of peptides on UPLC-MS analysis. In this study, bovine serum albumin (BSA) was used as a model protein to explore the correlation between the physicochemical properties of 50 peptides obtained by the enzymatic digestion of BSA, as well as the degree of nonspecific adsorption. First, these peptides were classified into four categories according to their response and the degree of adsorption in the pretreatment containers and LC system. Next, the factors influencing the adsorption of 12 Class Ⅱ peptides, which were highly responsive and susceptible to adsorption, were systematically studied in terms of several aspects, including: (1) time-dependent adsorption on centrifuge tubes of three kinds (Protein-LoBind polypropylene tube and two types of polypropylene tubes); (2) time-dependent adsorption on sample vials of three kinds (Protein-LoBind polypropylene vial, polypropylene vial, and glass vial); (3) carryovers on chromatographic columns with six different stationary phases (Polar C18, Cortecs C18+, PFP, BEH C18, CSH C18, and BEH C8); (4) carryovers at different chromatographic gradients (2%B-30%B, 2%B-40%B, 2%B-50%B, and 2%B-60%B within 3 min), flow rates (0.2, 0.3, and 0.4 mL/min), and column temperatures (30, 40, 50, and 60 ℃); and (5) carryovers using different washing needle solutions (0.2% formic acid in 10% acetonitrile and 0.2% formic acid in 90% acetonitrile). The results showed that parameters such as the HPLC index and amino acid length of peptides were significantly correlated with their degree of adsorption (p<0.05), However, the above parameters can only explain the adsorption degree of 30% of the peptides. The use of the modified polypropylene material resulted in higher recovery (recovery rate>80% within 24 h) of the peptide solution during storage or pretreatment. During protein/peptide pretreatment and storage, good overall recoveries (recovery rate>80% within 24 h) were obtained using centrifuge tubes and sample vials made of the modified polypropylene material. Analysis and optimization of the LC conditions revealed that the use of the C8 chromatographic column, a high flow rate (0.4 mL/min), slow gradient (2%B-50%B within 3 min), and strong needle solution (0.2% formic acid in 90% acetonitrile) could minimize the carryover. However, the effect of the column temperature on the carryover varied considerably from peptide to peptide, and hence, requires further analysis for specific peptides. The combined optimization of the above experimental conditions resulted in minimal (approximately 1/150) or no adsorption of the Class Ⅱ peptides that were susceptible to adsorption in the analytical process. In this study, a workflow was designed to standardize the procedures for evaluating and reducing peptide adsorption. Detailed data were collected to elucidate the key risk factors and corresponding general mechanism of nonspecific adsorption throughout the analysis. Thus, this study serves as a reference for the development of analytical methods for peptides and proteins with different physicochemical properties. In future work, the risk factors should be assessed during the development and validation of protein-based macromolecular analysis methods. In conclusion, it is important to implement adequate and appropriate measures to ensure risk elimination or minimization.

Lipidome Alterations following Mild Traumatic Brain Injury in the Rat.

Traumatic brain injury (TBI) poses a major health challenge, with tens of millions of new cases reported globally every year. Brain damage resulting from TBI can vary significantly due to factors including injury severity, injury mechanism and exposure to repeated injury events. Therefore, there is need for robust blood biomarkers. Serum from Sprague Dawley rats was collected at several timepoints within 24 h of mild single or repeat closed head impacts. Serum samples were analyzed via ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) in positive and negative ion modes. Known lipid species were identified through matching to in-house tandem MS databases. Lipid biomarkers have a unique potential to serve as objective molecular measures of injury response as they may be liberated to circulation more readily than larger protein markers. Machine learning and feature selection approaches were used to construct lipid panels capable of distinguishing serum from injured and uninjured rats. The best multivariate lipid panels had over 90% cross-validated sensitivity, selectivity, and accuracy. These mapped onto sphingolipid signaling, autophagy, necroptosis and glycerophospholipid metabolism pathways, with Benjamini adjusted p-values less than 0.05. The novel lipid biomarker candidates identified provide insight into the metabolic pathways altered within 24 h of mild TBI.

Simultaneous determination of aflatoxins, fumonisin B1, T-2 and cyclopiazonic acid in agri-products by immunomagnetic solid-phase extraction coupled with UHPLC-MS/MS.

In the present study, a rapid and sensitive determination method of seven mycotoxins was developed using immunomagnetic solid-phase extraction (IMPSE) coupled with UHPLC-MS/MS. Monoclonal antibodies were conjugated with CNBr superparamagnetic beads, and the major parameters affecting the IMPSE efficiency were systematically investigated. Under the optimized conditions, the mycotoxins were purified with the IMSPE procedure within 15 min and simultaneously quantified by UHPLC-MS/MS. Good linearities of the analytic method were established with the determination coefficients (R2) ranging from 0.9952 to 0.9997. The limit of quantifications were 0.04 µg kg-1 âˆ¼ 0.16 µg kg-1, which fully satisfied the regulatory maximum levels. The recoveries were 84.5-112.7% with intra-day and inter-day precision less than 15.2%. Finally, the proposed IMSPE-UHPLC-MS/MS was successfully utilized to analyze seven mycotoxins in peanut, maize, and wheat, providing a simple and robust extraction and enrichment procedure of hydrophilic and zwitterionic analytes from complex matrix.

Study of Differential Serum Metabolites in Patients with Adenomatous Polyps of Colon and Yang-Deficiency Constitution Based on Ultra-Performance Liquid Chromatography-Mass Spectrometry.

OBJECTIVE: To study the differences between the serum metabolites in patients with adenomatous polyps of the colon and yang-deficiency constitution and those without colon polyps and with balanced constitution, and look for biomarkers that can be used to distinguish between the two groups. METHODS: General patient information was gathered, and Chinese medicine constitution were collected in 940 patients who underwent electronic colonoscopy. A total of 119 patients with adenomatous polyps of the colon and yang-deficiency constitution were included in the experimental group, and 150 patients without colon polyps and with balanced constitution were included in the control group. Metabolomics analysis was performed on the fasting venous blood obtained from each patient in both groups. Principal component analysis and orthogonal partial least squares discriminant analysis were performed on the detection results, potential biomarkers were screened, metabolic pathway changes were determined, and the metabolic processes involved were discussed. RESULTS: A total of 59 differential biomarkers between the experimental group and the control group were identified. The differential metabolites were found mainly in the glycerophospholipid metabolism pathway, and the bile acid 3-oxo-4,6-choladienoic acid was the biomarker that distinguished the experimental group from the control group. CONCLUSION: With the help of metabolomics analysis, the differential metabolites in patients with adenomatous polyps of the colon and yang-deficiency constitution and those in patients without colon polyps and with balanced constitution could be identified. The biomarker 3-oxo-4,6-choladienoic acid may have potential diagnostic value in patients with adenomatous polyp of the colon and yang-deficiency constitution. (Trial Registration No. NCT02986308).

Study of Differential Serum Metabolites in Patients with Adenomatous Polyps of Colon and Yang-Deficiency Constitution Based on Ultra-Performance Liquid Chromatography-Mass Spectrometry / 中国结合医学杂志

OBJECTIVE@#To study the differences between the serum metabolites in patients with adenomatous polyps of the colon and yang-deficiency constitution and those without colon polyps and with balanced constitution, and look for biomarkers that can be used to distinguish between the two groups.@*METHODS@#General patient information was gathered, and Chinese medicine constitution were collected in 940 patients who underwent electronic colonoscopy. A total of 119 patients with adenomatous polyps of the colon and yang-deficiency constitution were included in the experimental group, and 150 patients without colon polyps and with balanced constitution were included in the control group. Metabolomics analysis was performed on the fasting venous blood obtained from each patient in both groups. Principal component analysis and orthogonal partial least squares discriminant analysis were performed on the detection results, potential biomarkers were screened, metabolic pathway changes were determined, and the metabolic processes involved were discussed.@*RESULTS@#A total of 59 differential biomarkers between the experimental group and the control group were identified. The differential metabolites were found mainly in the glycerophospholipid metabolism pathway, and the bile acid 3-oxo-4,6-choladienoic acid was the biomarker that distinguished the experimental group from the control group.@*CONCLUSION@#With the help of metabolomics analysis, the differential metabolites in patients with adenomatous polyps of the colon and yang-deficiency constitution and those in patients without colon polyps and with balanced constitution could be identified. The biomarker 3-oxo-4,6-choladienoic acid may have potential diagnostic value in patients with adenomatous polyp of the colon and yang-deficiency constitution. (Trial Registration No. NCT02986308).

Root Metabolite Differences in Two Maize Varieties Under Lead (Pb) Stress.

To assess root metabolic differences of maize varieties in their response to lead (Pb) stress, the lead-tolerant variety Huidan No. 4 and the lead-sensitive variety Ludan No. 8 were tested under Pb-free and Pb-stressed conditions. Changes in metabolites were measured using ultra-performance liquid chromatography-mass spectrometry. Pb stress changed the levels of the amino acids proline, glutamine, lysine, and arginine in both varieties, whereas glutamate and phenylalanine levels changed only in Huidan No. 4. Pb stress altered cystine, valine, methionine, and tryptophan levels only in Ludan No. 8. Therefore, the synthesis and decomposition of amino acids may affect the response of maize to Pb stress. The degree of change in differential metabolites for Huidan No. 4 was greater than that for Ludan No. 8. In cell wall subcellular components, increases in superoxide dismutase (SOD), peroxidases (PODs), and Pb concentrations were greater in Huidan No. 4 than in Ludan No. 8. Therefore, the greater Pb tolerance of Huidan No. 4 could be due to better sequestration of Pb in cell walls and more effective removal of reactive oxygen species (ROS) from the plant. The levels of certain metabolites only increased in Ludan No. 8, indicating that Pb-sensitive varieties may use different metabolic pathways to cope with Pb stress. Both varieties showed increased levels of some metabolites related to antioxidant protection and osmotic regulation. This study provides an understanding of maize Pb tolerance mechanisms and a basis for further development of tools for use in maize breeding.

Alterations in Pattern Baldness According to Sex: Hair Metabolomics Approach.

Pattern baldness has been associated with the male hormone, dihydrotestosterone. In this study, we tried to determine how the overall metabolic pathways of pattern baldness differ in patients and in normal controls. Our study aimed to identify alterations in hair metabolomic profiles in order to identify possible markers of pattern baldness according to sex. Untargeted metabolomics profiling in pattern baldness patients and control subjects was conducted using ultra-performance liquid chromatography-mass spectrometry. To identify significantly altered metabolic pathways, partial least squares discriminant analysis was performed. Our analysis indicated differences in steroid biosynthesis pathway in both males and females. However, there was a remarkable difference in the androgen metabolic pathway in males, and the estrogen metabolic and arachidonic acid pathways in females. For the first time, we were able to confirm the metabolic pathway in pattern baldness patients using hair samples. Our finding improves understanding of pattern baldness and highlights the need to link pattern baldness and sex-related differences.

Quantification of phenolic compounds in vegetable oils by mixed-mode solid-phase extraction isotope chemical labeling coupled with UHPLC-MS/MS.

In the present work, a rapid, accurate and cost-effective method has been developed for the simultaneous quantification of phenolic compounds in oil using mixed-mode solid-phase extraction (SPE) coupled with chemical labeling UHPLC-MS/MS. Mix-mode SPE weak cation cartridges were selected to enrich and purify phenolic compounds in oil, and hydroxyl moiety was dansylation as stable-isotope internal standard. The major parameters that affected the extraction and chemical labeling efficiency were investigated, and the method was fully validated. The limit of quantifications and the limit of detections were 0.002 µg kg-1 ~ 0.10 µg kg-1 and 0.006 µg kg-1 ~ 0.30 µg kg-1, respectively. The recoveries were 61.2% ~ 129.3% with intra-day and inter-day precision less than 12%. The results for 38 rapeseed oils revealed that 14 phenolic compounds, including canolol, phenolic acids, phenolic alcohols, tyrosol and vanillin from trace levels to relatively high content.