Ultrahigh-performance Liquid Chromatography-tandem Mass Spectrometry Research Articles

Pharmacokinetic evaluation of 24 representative components of Ling-Gui-Zhu-Gan decoction in acute myocardial infarction model rats via a validated ultrahigh-performance liquid chromatography-tandem mass spectrometry method.

Ling-Gui-Zhu-Gan decoction (LGZGD), one of the 100 herbal classic formulas, is clinically used to treat chronic heart failure with remarkable curative effect. However, LGZGD pharmacokinetic parameters in pathological model rats are poorly understood, in particular for special components. As physicochemical properties are specific to each representative component, no standard sample preparation is available for absolute quantification of representative components of LGZGD in rat plasma. A specific, sensitive and high-throughput ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS) method capturing 24 representative components was developed and applied to evaluate the pharmacokinetic parameters of LGZGD in acute myocardial infarction (AMI) rat plasma after intragastric administration (2.4, 4.8 and 9.6g/kg). Precipitation and extraction were selected and optimized for plasma preparation, and isopropanol precipitation could offer higher recovery and broader coverage. It was expected that AMI could cause less absorption and slower elimination of most of active components of LGZGD. Most of newly reported special components absorbed quickly and eliminated slowly. The average elimination half-life of the 24 representative components was 10.09 h, which is consistent with the dosage of LGZGD (twice daily). The specificity, linearity, precision and accuracy, recovery, matrix effect and stability were validated according to Food and Drug Administration guidance. The validation results demonstrated that the method could be applied to evaluate the pharmacokinetic parameters of LGZGD in AMI rats. The pharmacokinetic parameters showed substantial improvement in quality research of LGZGD, thereby laying the groundwork for preclinical and clinical trials in chronic heart failure clinical efficacy.

In vitro blood[sbnd]brain barrier permeability study of four main active ingredients from Alpiniae oxyphyllae fructus

The fruits of Alpinia oxyphylla Miq., a broadly utilized traditional Chinese medicine, have a number of effects on the central nervous system (CNS). The main active constituents of Alpiniae oxyphyllae fructus (AOF) were nootkatone, tectochrysin, chrysin and protocatechuic acid. An immortalized human brain microvascular endothelial cell (hCMEC/D3) and astrocyte (HA1800) coculture model was used to investigate the permeability of the blood-brain barrier (BBB). The validation of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) methods for the four compounds was conducted following industry guidelines. Calibration curves were generated with mean coefficients (R2) better than 0.99. The inter-day and intra-day precisions were less than 8.53% and 7.12%, respectively. The accuracies were lower than ± 11.57%, and recoveries were greater than 86.07%. The samples of the transport experiment were examined, and the apparent permeability coefficients (Papp) were calculated. The efflux ratios of the four compounds are all less than 2. The Papp values of protocatechuic acid, chrysin, nootkatone, tectochrysin were at the level of 10−5, 10−6, 10−6, and 10−7 cm/s, respectively. All four compounds crossed the BBB by passive diffusion, with protocatechuic acid having high permeability, and tectochrysin having poor permeability. This research indicated the permeability of protocatechuic acid, chrysin, nootkatone and tectochrysin through the BBB and offered a foundation for related research on AOF in the treatment of CNS illnesses.

Transfer and biotransformation of the COVID-19 prodrug molnupiravir and its metabolite β-D-N4-hydroxycytidine across the blood-placenta barrier

Molnupiravir is an orally bioavailable prodrug of the nucleoside analogue β-D-N4-hydroxycytidine (NHC) and is used to treat coronavirus disease 2019 (COVID-19). However, the pharmacokinetics and transplacental transfer of molnupiravir in pregnant women are still not well understood. In the present study, we investigated the hypothesis that molnupiravir and NHC cross the blood-placenta barrier into the fetus. A multisite microdialysis coupled with a validated ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC‒MS/MS) system was developed to monitor the dialysate levels of molnupiravir and NHC in maternal rat blood and conceptus (the collective term for the fetus, placenta, and amniotic fluid). Molnupiravir was administered intravenously (100mg/kg, i.v.) on gestational day 16. To investigate the mechanism of transport of molnupiravir across the blood-placenta barrier, we coadministered nitrobenzylthioinosine (NBMPR, 10mg/kg, i.v.) to inhibit equilibrative nucleoside transporter (ENT). We report that molnupiravir is rapidly metabolized to NHC and then rapidly transformed in the fetus, placenta, amniotic fluid, and maternal blood. Our pharmacokinetics analysis revealed that the area under the concentration curve (AUC) for the mother-to-fetus ratio (AUCfetus/AUCblood) of NHC was 0.29±0.11. Further, we demonstrated that the transport of NHC in the placenta may not be subject to modulation by the ENT. Our results show that NHC is the predominant bioactive metabolite of molnupiravir and rapidly crosses the blood-placenta barrier in pregnant rats. The NHC concentration in maternal blood and conceptus was above the average median inhibitory concentration (IC50) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), suggesting a therapeutic effect. These findings support the use of molnupiravir in pregnant patients infected with COVID. This study was supported in part by research grants from the National Science and Technology Council of Taiwan (NSTC 111-2113-M-A49-018 and NSTC 112-2321-B-A49-005).

Characterization and determination of casein glycomacropeptide in dairy products by UHPLC–MS/MS based on its characteristic peptide

The ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS) method was built to quantify the casein glycomacropeptide (CGMP) in bovine dairy products accurately based on targeted proteomics. Qualitative analysis of theoretical peptides was carried out using high-resolution mass spectrometry (HRMS) and protein software. Isotope-labeled characteristic peptides were acquired via the labeled amino acid condensation method to correct the matrix effects. Peptide MAIPPK was the representative characteristic peptide for distinguishing the CGMP from κ-casein through trypsin digestion. After optimizing the pre-treatment conditions, the final 8% oxidant concentration was selected and the 10% formic acid concentration with 2.5 h oxidation time. Moreover, the results of methodological verification showed that the recovery rate was 103.7%, meanwhile the precision of inter-day and intra-day was less than 5%. In conclusion, the research demonstrated the characteristic peptide MAIPPK could quantitatively applied to detect CGMP in dairy products.

High-Fat Diet Alters Acylcarnitine Metabolism of the Retina and Retinal Pigment Epithelium/Choroidal Tissues in Laser-Induced Choroidal Neovascularization Rat Models.

Choroidal neovascularization (CNV) is age-related macular degeneration's (AMD) main pathological change. High-fat diet (HFD) is associated with a form of CNV; however, the specific mechanism is unclear. Mitochondrial dysfunction, characterized by abnormal acylcarnitine, occurs during metabolic screening of serum or other body tissues in AMD. This study investigates HFD's role in retinal and retinal pigment epithelium (RPE)/choroidal acylcarnitine metabolism in CNV formation. Chow diet and HFD-BN rats are laser-treated to induce CNV. Acylcarnitine species are quantitatively characterized by ultrahigh-performance liquid chromatography-tandem mass spectrometry. Optical coherence tomography and fundus fluorescein angiography evaluate CNV severity. HFD promotes weight gain, dyslipidemia, and CNV formation. In CNV rats, few medium-chain fatty acids (MCFAs) acylcarnitine in the RPE/choroid are initially affected. When an HFD is administered to these, even MCFA acylcarnitine in the RPE/choroid is found to decline. However, in the retina, odd acylcarnitines are increased, revealing "an opposite" change within the RPE/choroid, accompanied by influencing glycolytic key enzymes. The HFD+CNV group incorporated fewer long-chain acylcarnitines, like C18:2, into the retina than controls. HFD hastens choroidal neovascularization. The study comprehensively documented acylcarnitine profiles in a CNV rat model. Acylcarnitine's odd-even and carbon-chain length properties may guidefuture therapeutics.

Association between prenatal exposure to perfluoroalkyl substance mixtures and intrauterine growth restriction risk: A large, nested case–control study in Guangxi, China

Intrauterine growth restriction (IUGR) is an abnormal fetal growth pattern that can lead to neonatal morbidity and mortality. IUGR may be affected by prenatal exposure to environmental pollutants, including perfluoroalkyl substances (PFASs). However, research linking PFAS exposure to IUGR is limited, with inconsistent results. We aimed to investigate the association between PFAS exposure and IUGR by using nested casecontrol study based on Guangxi Zhuang Birth Cohort (GZBC), in Guangxi, China. A total of 200 IUGR cases and 600 controls were enrolled in this study. The maternal serum concentrations of nine PFASs were measured using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLCMS). The associations single and mixed effects of prenatal PFAS exposure on IUGR risk were assessed using conditional logistic regression (single-exposure), Bayesian kernel machine regression (BKMR) and quantile g-computation (qgcomp) models. In the conditional logistic regression models, the log10-transformed concentrations of perfluoroheptanoic acid (PFHpA, adjusted OR: 4.41, 95% CI: 3.03–6.41), perfluorododecanoic acid (PFDoA, adjusted OR: 1.94, 95% CI: 1.14–3.32), and perfluorohexanesulfonate (PFHxS, adjusted OR: 1.83, 95% CI: 1.15–2.91) were positively associated with risk of IUGR. In the BKMR models, the combined effect of PFASs was positively associated with IUGR risk. In the qgcomp models, we also found an increased IUGR risk (OR=5.92, 95% CI: 2.33–15.06) when all nine PFASs increased by one tertile as a whole, and PFHpA (43.9%) contributed the largest positive weights. These findings suggested prenatal exposure to single and mixtures of PFASs may increase IUGR risk, with the effect being largely driven by the PFHpA concentration.

Determination of the Dissipation Dynamics and Terminal Residue of Bupirimate and Its Metabolites in Cucumber by QuEChERS-Based UPLC-MS/MS.

Bupirimate is widely used as a highly active systemic fungicide. However, the frequent and heavy use of bupirimate has led to pesticide residues in crops that threaten human health and food safety. At present, there is limited research on the detection of ethirimol, which is the metabolite of bupirimate. This study established an ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to simultaneously detect bupirimate and ethirimol residues based on QuEChERS pretreatment. The average recoveries of bupirimate and ethirimol in cucumber were between 95.2 and 98.7%, respectively, with relative standard deviations (RSDs) of 0.92-5.54% at fortified levels of 0.01, 0.1, and 5 mg L-1. The established method was used to determine the residues in field trials in 12 regions of China, and the final residues of bupirimate were all less than the maximum residue limit (MRL). Since the risk quotient (RQ) of bupirimate and ethirimol in cucumber was less than 1.3%, the dietary risk assessment indicated that bupirimate and ethirimol had a low long-term dietary risk to the general population in China. This study provides effective guidance on the proper use of bupirimate in cucumber fields and a reference for establishing the MRL of bupirimate in China.

Ultrahigh-performance liquid chromatography-tandem mass spectrometry quantitation of o-cresol in hydrolyzed human urine to assess toluene exposure.

Toluene is a volatile organic compound used in domestic and industrial applications. The main routes of workplace exposure to toluene are inhalation and dermal contact. As toluene exposure can cause severe nervous system damage, its quantification is crucial to prevent occupational illness. Toluene is metabolized mainly as hippuric acid, S-benzylmercapturic acid and epoxides. These are rapidly converted to o-/p-cresol, which is then excreted in the urine as conjugated glucuronides and sulfates. o-Cresol and its conjugates can be chemically hydrolyzed to form free o-cresol, which can then serve as a urinary biomarker of toluene exposure. Current analytical methods for quantifying o-cresol in hydrolyzed urine are, however, either weakened by interference, are not sensitive enough or require water-sensitive sample preparation. Development of a liquid chromatography-tandem mass spectrometry method for assessing exposure to toluene is thus required. Urine samples were acidified and heated to form free o-cresol and then derivatized with dansyl chloride and diluted. Extracts were separated by reverse-phase chromatography on a BEH phenyl column and then analyzed using a triple quadrupole instrument in selected reaction monitoring mode. The dansyl chloride derivatization step was optimized to produce the derivative within a reaction time of 3min. Hydrolysis efficiency in forming free o-cresol from conjugated metabolites was evaluated using o-cresol-β-d-glucuronidespiked human urine: complete hydrolysis occurred in 45 min. Dynamic range was 0.4 to 40 μM, and the method was useful for toluene monitoring in non-occupational (0.1μmol/mmol creatinine) as well as occupational (0.3μmol/mmol creatinine) exposure. The calculated limit of detection and limit of quantitation of the method were 0.06 and 0.21 μM, respectively. Intraday and interday precisions were 3.2% and 4.4%, respectively. Method accuracy was established as 99% using ClinChek® urine controls. An ultrahigh-performance liquid chromatography-tandem mass spectrometry method for analysis of o-cresol was developed for biological monitoring of toluene exposure in human urine. This is the method of choice used by occupational health and safety practitioners in the province of Québec, Canada.

Analysis of photocatalytic degradation of polyamide microplastics in metal salt solution by high resolution mass spectrometry

Microplastic pollution has become one of the most concerned focuses in the world. Among many treatment methods, photocatalysis is considered to be one of the most environmentally friendly methods. In this work, the photodegradation behavior of polyamide microplastics is studied by using polyamide 6 PA6) as model microplastics and FeCl3 as catalyst. It is hoped that the PA6 fiber can be effectively degraded by utilizing the strong oxidizing active species that can be produced after FeCl3 is irradiated in water. The results shows that PA6 fiber can be almost completely degraded after 10 days of irradiation in FeCl3 aqueous solution, indicating that it is promising to use this new method to solve the problem of PA6 type microplastics. In addition, the chain scission mechanism and degradation process of PA6 are analyzed in detail by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS), which provides a new insight for the study of polymer degradation mechanism.

Integrated Multiomics and Synergistic Functional Network Revealed the Mechanism in the Tolerance of Different Ecotypes of Salvia miltiorrhiza Bge. to Doxycycline Pollution.

Tetracycline pollution in soil irreversibly damages the biosafety of plants by inhibiting the mitochondrial function. Some traditional Chinese medicine (TCM) plants, such as Salvia miltiorrhiza Bunge, have a strong tolerance to mitochondrial damage. We comprehensively compared the doxycycline (DOX) tolerances of two ecotypes of S. miltiorrhiza in the Sichuan and Shandong provinces and found that the Sichuan ecotype had a lower yield reduction, more stable accumulation of medicinal ingredients, higher mitochondrial integrity, and a more robust antioxidant system. The synergetic response networks under DOX pollution of both ecotypes were constructed using RNA sequencing and ultrahigh-performance liquid chromatography-tandem mass spectrometry. The differentiation of the downstream pathways of aromatic amino acids (AAAs) produced variations in the DOX tolerance of S. miltiorrhiza in different regions. The Sichuan ecotype maintained redox homeostasis and xylem development by activating salvianolic acid and indole biosynthesis, while the Shandong ecotype balanced chemical and mechanical defenses by regulating the flavonoid biosynthesis. Rosmarinic acid, a downstream AAA molecule, maintains the mitochondrial homeostasis of plant seedlings under DOX pollution by targeting the ABCG28 transporter. We also highlight the significance of downstream AAA small molecules in guiding the development of bio-based environmental pollution remediation agents.

Integrative analysis of the gut microbiota and faecal and serum short-chain fatty acids and tryptophan metabolites in patients with cirrhosis and hepatic encephalopathy

ObjectiveThe purpose of this study was to describe the changes in the gut microbiome of patients with cirrhosis and hepatic encephalopathy (HE), as well as quantify the variations in short-chain fatty acid (SCFA) and tryptophan metabolite levels in serum and faeces.MethodsFresh faeces and serum were collected from 20 healthy volunteers (NC group), 30 cirrhosis patients (Cir group), and 30 HE patients (HE group). Then, 16S rRNA sequencing and metabolite measurements were performed using the faeces. Gas chromatography‒mass spectrometry and ultrahigh-performance liquid chromatography-tandem mass spectrometry were used to measure SCFA and tryptophan levels, respectively. The results were analysed by SIMCA16.0.2 software. Differences in species were identified using MetaStat and t tests. The correlations among the levels of gut microbes and metabolites and clinical parameters were determined using Spearman correlation analysis.ResultsPatients with cirrhosis and HE had lower microbial species richness and diversity in faeces than healthy volunteers; these patients also had altered β-diversity. Serum valeric acid levels were significantly higher in the HE group than in the Cir group. Serum SCFA levels did not differ between the Cir and NC groups. Serum melatonin and 5-HTOL levels were significantly higher in the HE group than in the Cir group. The Cir and NC groups had significant differences in the levels of eight serum tryptophan metabolites. Furthermore, the levels of faecal SCFAs did not differ between the HE and Cir groups. Faecal IAA-Ala levels were significantly lower in the HE group than in the Cir group. There were significant differences in the levels of 6 faecal SCFAs and 7 faecal tryptophan metabolites between the Cir and NC groups. Certain gut microbes were associated with serum and faecal metabolites, and some metabolites were associated with certain clinical parameters.ConclusionReduced microbial species richness and diversity were observed in patients with HE and cirrhosis. In both serum and faeces, the levels of different SCFAs and tryptophan metabolites showed varying patterns of change. In HE patients, the levels of some serum tryptophan metabolites, and not SCFAs, were correlated with liver function and systemic inflammation. Systemic inflammation in patients with cirrhosis was correlated with faecal acetic acid levels. In summary, this study identified metabolites important for HE and cirrhosis.

Development and validation of a UPLC-MS/MS method to investigate the plasma pharmacokinetics of a KCa 2.2/KCa 2.3 positive allosteric modulator in mice.

There is currently no treatment for spinocerebellar ataxias (SCAs), which are a group of genetic disorders that often cause a lack of coordination, difficulty walking, slurred speech, tremors, and eventually death. Activation of KCa 2.2/KCa 2.3 channels reportedly exerts beneficial effects in SCAs. Here, we report the development and validation of an analytical method for quantitating a recently-developed positive allosteric modulator of KCa 2.2/KCa 2.3 channels (compound 2q) in mouse plasma. Mouse plasma samples (10 μL) containing various concentrations of 2q were subjected to protein precipitation in the presence of a structurally similar internal standard (IS). Subsequently, the analytes were separated on a C18 UPLC column and detected by a tandem mass spectrometer. The method was validated using FDA guidelines. Finally, the validated assay was applied to the measurement of the plasma concentrations of 2q in plasma samples taken from mice after single intravenous doses of 2 mg/kg of 2q, and the pharmacokinetic parameters of 2q were determined. The calibration standards were linear (r2 ≥ 0.99) in the range of 1.56 - 200 nM of 2q with intra- and inter-run accuracy and precision values within the FDA guidelines. The lower limit of quantitation of the assay was 1.56 nM (0.258 pg on the column). The recoveries of 2q and IS from plasma were > 94%, with no appreciable matrix effect. The assay showed no significant carryover, and the plasma samples stored at -80o C or the processed samples stored in the autosampler at 10o C were stable for at least three weeks and 36 h, respectively. After intravenous injection, 2q showed a bi-exponential decline pattern in the mouse plasma, with a clearance of 30 mL/min/kg, a terminal volume of distribution of 1.93 mL/kg, and a terminal half-life of 45 min. The developed assay is suitable for preclinical pharmacokinetic-pharmacodynamic studies of 2q as a potential drug candidate for ataxias.

The role of metabolic perturbations in mediating the effects of ambient air pollution on lung cancer in the cancer prevention studies.

10541 Background: Exposure to ambient air pollution is an established risk factor for lung cancer. Investigation on the underlying mechanisms of air pollution carcinogenicity remains challenging due to the complex composition of air pollution and a lack of sensitive biomarkers to accurately measure exposure and corresponding response. To address the knowledge gaps, we applied high-resolution metabolomics to identify metabolic signatures of exogenous air pollution exposures and endogenous processes involved in lung carcinogenesis. Methods: A total of 683 matched lung cancer and control pairs within the established Cancer Prevention Study-II (CPS-II) Nutrition and CPS-3 cohorts were included in this study. The plasma metabolome was profiled with ultrahigh-performance liquid chromatography-tandem mass spectrometry. Assessment of exposure to six ambient air pollutants, including carbon monoxide (CO), nitrogen dioxide (NO2), particulate matter (PM10), fine particulate matter (PM2.5), sulfur dioxide (SO2), and ozone (O3), was conducted using spatiotemporally-resolved models based on residential address at blood draw and calculated based on yearly average levels. We conducted metabolome-wide association studies with multiple linear regression models to assess associations of ambient air pollution and lung cancer with a meet-in-the-middle approach. Models controlled for potential confounders and covariates including age at blood draw, sex, race, body mass index, alcohol consumption, smoking status, passive smoke exposure, vegetable and fruit intake, and educational level. Metabolites significant at the FDR < 0.2 level in the air pollution model were analyzed in the lung cancer model. High-dimensional mediation analysis was used as a secondary analysis to compare results from the meet-in-the-middle analysis. Results: Among 1,204 metabolic features extracted from the blood samples, seven features were significantly associated with air pollution exposure and lung cancer incidence at the FDR < 0.2 level in the meet-in-the-middle analysis. Six features were significant at the p < 0.01 level through high-dimensional mediation analysis. All confirmed metabolites are enriched within peptide, lipid, and amino acid pathways. Gamma-glutamylglutamine and gamma-glutamylmethionine were statistically significantly associated with CO, NO2, and PM10 exposure and lung cancer incidence in both analyses. Conclusions: This is the largest prospective metabolomics study examining biological perturbations associated with air pollution exposure and lung cancer incidence. Peptide metabolism may play an important role in mediating the association between air pollution and lung cancer. Findings from this study will lay the foundation for future studies to further understand the biological mechanisms underlying the carcinogenicity of air pollution.

Rapid microwave synthesis of MOF microrods: Dispersive SPE coupled with UHPLC–MS/MS to determine fluoroquinolones in honey

A novel sorbent Cu–S metal–organic framework (MOF) microrods was prepared for dispersive solid-phase extraction via microwave synthesis and used to determine 12 fluoroquinolones (FQs) in honey samples employing ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). The best extraction efficiency was achieved by optimizing sample pH, sorbent quantity, eluent type/volume, and extraction and elution time. The proposed MOF exhibits advantages such as rapid synthesis time (20 min) and outstanding adsorption ability toward zwitterionic FQs. These advantages can be attributed to multiple interactions, including hydrogen bonding, π–π interaction, and hydrophobic interaction. The limits of detection of analytes were 0.005–0.045 ng g−1. Acceptable recoveries (79.3%–95.6%) were obtained under the optimal conditions. Precision (relative standard deviation, RSD) was <9.2%. These results demonstrate the utility of our sample preparation method and the high capacity of Cu–S MOF microrods for rapid and selective extraction of FQs from honey samples.

Toward High-Throughput Analysis of Aroma Compounds Using Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry: Screening of Key Food Odorants in Various Foods.

Recent studies show the immense capacities of the unified quantitation of aroma and taste compounds using liquid chromatography-mass spectrometry (LC-MS). The goal of this study was to highlight the broad application of this unified method. Thus, a stable isotope dilution analysis quantification method of the most important key food odorants in various food categories by LC-MS was developed. Using the well-known derivatization agent 3-nitrophenylhydrazine for carbonyl derivatization and a newly developed approach for alcohol and thiol derivatization, a method for the quantitation of 20 key food odorants was established. Intraday precision was determined to be ≤26%, and interday precision was between 24 and 31%. Limits of quantitation were determined between 0.014 and 283 μg/kg. The work shows that a wide array of aroma compounds can be analyzed accurately by LC-MS.

Widely untargeted metabolomic profiling unearths metabolites and pathways involved in leaf senescence and N remobilization in spring-cultivated wheat under different N regimes.

Progression of leaf senescence consists of both degenerative and nutrient recycling processes in crops including wheat. However, the levels of metabolites in flag leaves in spring-cultivated wheat, as well as biosynthetic pathways involved under different nitrogen fertilization regimes, are largely unknown. Therefore, the present study employed a widely untargeted metabolomic profiling strategy to identify metabolites and biosynthetic pathways that could be used in a wheat improvement program aimed at manipulating the rate and onset of senescence by handling spring wheat (Dingxi 38) flag leaves sampled from no-, low-, and high-nitrogen (N) conditions (designated Groups 1, 2, and 3, respectively) across three sampling times: anthesis, grain filling, and end grain filling stages. Through ultrahigh-performance liquid chromatography-tandem mass spectrometry, a total of 826 metabolites comprising 107 flavonoids, 51 phenol lipids, 37 fatty acyls, 37 organooxygen compounds, 31 steroids and steroid derivatives, 18 phenols, and several unknown compounds were detected. Upon the application of the stringent screening criteria for differentially accumulated metabolites (DAMs), 28 and 23 metabolites were differentially accumulated in Group 1_vs_Group 2 and Group 1_vs_Group 3, respectively. From these, 1-O-Caffeoylglucose, Rhoifolin, Eurycomalactone;Ingenol, 4-Methoxyphenyl beta-D-glucopyranoside, and Baldrinal were detected as core conserved DAMs among the three groups with all accumulated higher in Group 1 than in the other two groups. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that tropane, piperidine, and pyridine alkaloid biosynthesis; acarbose and validamycin biosynthesis; lysine degradation; and biosynthesis of alkaloids derived from ornithine, lysine, and nicotinic acid pathways were the most significantly (p < 0.05) enriched in Group 1_vs_Group 2, while flavone and flavonol as well as anthocyanins biosynthetic pathways were the most significantly (p < 0.05) enriched in Group 1_vs_Group 3. The results from this study provide a foundation for the manipulation of the onset and rate of leaf senescence and N remobilization in wheat.

Immediate, sensitive and specific time-resolved fluorescent immunoassay strips based on immune competition for the detection of procymidone in vegetables

As an organochlorine pesticide, procymidone has become an increasing serious problem in vegetables in recent years. In order to solve a series of problems such as low sensitivity of the immunological methods, a time-resolved fluorescent immunoassay strip (TRFIAS) was developed with low limit of detection (LOD). Based on the principle of antigen-antibody immune competition, TRFIAS could detect procymidone in vegetables. After the targets being added, the samples were incubated at 37 °C for 10 min. TRFIAS was observed under a UV lamp of 365 nm. The results were processed by ImageJ software to complete the qualitative and quantitative analysis of TRFIAS. Under the optimal working conditions, a linear range of TRFIAS was 5–250 ng/mL with a LOD of 0.33 ng/mL and a visual limit of detection (VDL) of 50 ng/mL. And the TRFIAS had good specificity, precision and accuracy. Comparing the results of TRFIAS with those of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), the two assays were in good agreement. Therefore, TRFIAS has the advantages of high sensitivity, high specificity, simple operation and relatively short assay time. Meanwhile, it shows absolutely great advantages in both quantitative and qualitative analysis.

Development of QuEChERS coupled with UHPLC–MS/MS for simultaneous determination of eight neonicotinoid pesticides in breast milk

A speedy and hypersensitive method was built to detect eight neonicotinoid insecticides (neonics) in breast milk by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The breast milk was extracted with a mixture of acetonitrile and water and purified with primary secondary amine (PSA) and C18. The recovery of the method ranged from 74.3 to 105.9% with relative standard deviations (RSDs) of less than 10%, and the limit of detection ranged from 0.05 to 0.18ng/mL. Among 32 samples obtained from women 1month postpartum, thiamethoxam and imidacloprid were the most frequently detected neonics. Moreover, thiacloprid and imidaclothiz were not detected in any samples. The concentrations of neonics in breast milk ranged from 1.90 to 149.95ng/mL. Considering the toxic effects on mammals and even humans, infants who are exposed to neonics through ingestion of breast milk should receive extensive attention in future studies.

Simultaneous determination of nine phenolic compounds in imitation wild Dendrobium officinale samples using ultrahigh-performance liquid chromatography-tandem mass spectrometry.

Dendrobium officinale Kimura et Migo (D. officinale), one of the nine everlasting types of grass, has gained increasing attention owing to its important roles in alternative medicines and drug discovery. Due to its natural resources being in danger of being extinct, imitation wild planting is becoming increasingly common. To assess the product's quality completely, an efficient ultrahigh performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) method was established to simultaneously quantify nine phenolic compounds in D. officinale samples. The extraction parameters, including solvent, solvent concentration, solid-liquid ratio, and extraction time, were systematically optimized with the single-factor test. The results demonstrated that extraction with a 1:200 solid-to-liquid ratio of 80% methanol for 1.5 h was the most efficient condition for the extraction of flavonoids. Satisfactory retention times and resolution of the nine analytes were acquired on the Thermo Scientific Hypersil GOLD column with multiple reaction monitoring in negative ion scanning mode. The method was validated to demonstrate its selectivity, linearity, precision, accuracy, and robustness. Thus, the verified UHPLC-QQQ-MS/MS method was successfully applied to the quantification of phenolic components present in D. officinale samples. The results indicated that the quantity and composition of phenolic components in D. officinale from various provenances were significantly different. This work provides a theoretical foundation for the cultivation and assessment of wild D. officinale quality.

Renal Function Underpins the Cyclooxygenase-2: Asymmetric Dimethylarginine Axis in Mouse and Man

Through the production of prostacyclin, cyclooxygenase (COX)-2 protects the cardiorenal system. Asymmetric dimethylarginine (ADMA), is a biomarker of cardiovascular and renal disease. Here we determined the relationship between COX-2/prostacyclin, ADMA, and renal function in mouse and human models. We used plasma from COX-2 or prostacyclin synthase knockout mice and from a unique individual lacking COX-derived prostaglandins (PGs) because of a loss of function mutation in cytosolic phospholipase A2 (cPLA2), before and after receiving a cPLA2-replete transplanted donor kidney. ADMA, arginine, and citrulline were measured using ultra-high performance liquid-chromatographytandem mass spectrometry. ADMA and arginine were also measured by enzyme-linked immunosorbent assay (ELISA). Renal function was assessed by measuring cystatin C by ELISA. ADMA and prostacyclin release from organotypic kidney slices were also measured by ELISA. Loss of COX-2 or prostacyclin synthase in mice increased plasma levels of ADMA, citrulline, arginine, and cystatin C. ADMA, citrulline, and arginine positively correlated with cystatin C. Plasma ADMA, citrulline, and cystatin C, but not arginine, were elevated in samples from the patient lacking COX/prostacyclin capacity compared to levels in healthy volunteers. Renal function, ADMA, and citrulline were returned toward normal range when the patient received a genetically normal kidney, capable of COX/prostacyclin activity; and cystatin C positively correlated with ADMA and citrulline. Levels of ADMA and prostacyclin in conditioned media of kidney slices were not altered in tissue from COX-2 knockout mice compared to wildtype controls. In human and mouse models, where renal function is compromised because of loss of COX-2/PGI2 signaling, ADMA levels are increased.