Performance Liquid Chromatography-tandem Mass Research Articles

Exploring the Structure and Substance Metabolism of a Medicago sativa L. Stem Base.

The stem base of alfalfa is a critical part for its overwintering, regeneration, and yield. To better understand the specificity and importance of the stem base, we analyzed the structure, metabolic substances, and transcriptome of the stem base using anatomical techniques, ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), and RNA sequencing (RNA-seq), and compared it with stems and roots. The anatomical structure shows that the ratio of xylem to phloem changes at the base of the stem. A total of 801 compounds involved in 91 metabolic pathways were identified from the broadly targeted metabolome. Transcriptome analysis revealed 4974 differentially expressed genes (DEGs) at the stem base compared to the stem, and 5503 DEGs compared to the root. Comprehensive analyses of differentially accumulated compounds (DACs) and DEGs, in the stem base vs. stem, identified 10 valuable pathways, including plant hormone signal transduction, zeatin biosynthesis, α-Linolenic acid metabolism, histidine metabolism, carbon metabolism, carbon fixation in photosynthetic organisms, pentose phosphate pathway, galactose metabolism, and fructose and mannose metabolism. The pathways of plant hormone signal transduction and carbon metabolism were also identified by comparing the stem base with the roots. Taken together, the stem base of alfalfa is the transition region between the stem and root in morphology; in terms of material metabolism, its growth, development, and function are regulated through hormones and sugars.

PRM1201 effectively inhibits colorectal cancer metastasis via shaping gut microbiota and short- chain fatty acids

BackgroundPRM1201 is a traditional medicine with beneficial effects against colorectal cancer (CRC) metastasis. However, the underlying mechanism of this action remains to be determined. HypothesisRemodeling microbiota and short-chain fatty acids (SCFAs) metabolism might be a potential mechanism to explain the anti-metastatic action of PRM1201, as this gut-microbiota dependent effect involves downregulation of histone deacetylation and EMT. MethodsTo investigate this possibility, clinical specimens were sequenced and the correlation between the anti-metastatic efficacy of PRM1201 and the restoration of SCFA-producing bacteria was studied. To obtain solid causal evidence, a mouse metastasis model was established to detect the influence of PRM1201 on cancer metastasis. Specifically, 16S amplicon sequencing, ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) analysis, and bacterial manipulation were used to examine the gut microbiota-driven anti-metastatic action of PRM1201. ResultsClinical data showed that PRM1201 increased both the number of SCFA-producing bacteria and generation of SCFAs in the feces of CRC patients. A positive correlation between the anti-metastatic efficacy of PRM1201 and the restoration of SCFAs observed. The animal experiments demonstrated that PRM1201 effectively blocked CRC metastasis in a dose-dependent manner. PRM1201 treatment modulated the composition of gut microbiota, and promoted the proliferation of beneficial SCFAs producers such as Akkermansia, Lachnospiraceae_NK4A136_group and Blautia, while simultaneously reducing the abundance of pathogenic bacteria like Escherichia-Shigella. In addition, PRM1201 led to augmentation of SCFAs content. Further results indicated that the anti-cancer metastatic mechanism of PRM1201 was linked to inhibition of histone deacetylation and suppression of epithelial-to-mesenchymal transition (EMT) in metastatic lesions. Microbiota depletion treatment and fecal microbiota transplantation (FMT) underscored the microbiota-dependent nature of this phenomenon. Moreover, this anti-colorectal cancer metastatic effect and mechanism of total SCFAs and single SCFA were also confirmed. ConclusionIn summary, PRM1201 exerts its anti-metastatic effects by modulating SCFA-producing bacteria and enhancing the production of SCFAs. Furthermore, the prebiotic-like actions of PRM1201, along with the PRM1201-treated bacteria, function as inhibitors of histone deacetylases (DHACs) thereby effectively suppressing EMT events.

Simultaneous measurement of COVID-19 treatment drugs (nirmatrelvir and ritonavir) in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study

PAXLOVID™ (Co-packaging of Nirmatrelvir with Ritonavir) has been approved for the treatment of Coronavirus Disease 2019 (COVID-19). The goal of the experiment was to create an accurate and straightforward analytical method using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to simultaneously quantify nirmatrelvir and ritonavir in rat plasma, and to investigate the pharmacokinetic profiles of these drugs in rats. After protein precipitation using acetonitrile, nirmatrelvir, ritonavir, and the internal standard (IS) lopinavir were separated using ultra performance liquid chromatography (UPLC). This separation was achieved with a mobile phase composed of acetonitrile and an aqueous solution of 0.1% formic acid, using a reversed-phase column with a binary gradient elution. Using multiple reaction monitoring (MRM) technology, the analytes were detected in the positive electrospray ionization mode. Favorable linearity was observed in the calibration range of 2.0–10000 ng/mL for nirmatrelvir and 1.0–5000 ng/mL for ritonavir, respectively, within plasma samples. The lower limits of quantification (LLOQ) attained were 2.0 ng/mL for nirmatrelvir and 1.0 ng/mL for ritonavir, respectively. Both drugs demonstrated inter-day and intra-day precision below 15%, with accuracies ranging from −7.6% to 13.2%. Analytes were extracted with recoveries higher than 90.7% and without significant matrix effects. Likewise, the stability was found to meet the requirements of the analytical method under different conditions. This UPLC-MS/MS method, characterized by enabling accurate and precise quantification of nirmatrelvir and ritonavir in plasma, was effectively utilized for in vivo pharmacokinetic studies in rats.

Determination of cinnamaldehyde, thymol and eugenol in essential oils by LC–MS/MS and antibacterial activity of them against bacteria

Plant essential oils contain many secondary metabolites, some of which can effectively inhibit the growth of pathogenic microorganisms, so it is a very promising antibacterial agent. In this study, a qualitative and quantitative method based on high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was developed for the simultaneous determination of three bioactive substances, cinnamaldehyde (CNM), thymol (THY), and eugenol (EUG), in the essential oils of plants. Necessary tests for linearity, limit of quantification, recovery, carryover contamination and precision of the method were carried out. Then, the antibacterial activity of 3 bioactive compounds against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was evaluated by minimal inhibitory concentration and the synergistic antimicrobial effect. The results indicated that CNM, THY and EUG had good antibacterial activity. According to the results of fractional inhibitory concentration index (FICI), it is considered that CNM + THY and CNM + THY + EUG has obvious synergistic inhibitory effect on E. coli, and CNM + THY and CNM + EUG has obvious synergistic inhibitory effect on S. aureus. Finally, we analyzed the effect of the bioactive compounds on trace elements in bacteria and found significant changes in magnesium, calcium, copper and iron.

Using monoclonal antibody to establish a sensitive icELISA for the determination of tangeretin in citrus and herbal samples

Tangeretin, a polymethoxyflavone abundant in citrus fruits, is acclaimed for its diverse biological benefits to human health and is a significant component of traditional Chinese medicine. This study reports the development of an indirect competitive enzyme-linked immunosorbent assay (icELISA) with monoclonal antibody for quantifying tangeretin in various citrus and herbal medicines. The optimized icELISA demonstrated a half-maximal inhibitory concentration (IC50) of 5.57 ng/mL, encompassing a dynamic range of 1.54–25.38 ng/mL, and showcased average recoveries ranging from 81.65 % to 105.75 % in citrus peel and pulp samples. Extensive analysis of six citrus cultivar samples and four herbal samples was conducted using the developed assay, with results corroborated by Ultra–Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC–MS/MS). These findings confirm the assay’s efficacy and reliability for tangeretin detection, offering a practical method for evaluating this bioactive compound in food.

A multi-technique based analytical platform for characterization and identification of a newly emerged steroid 6β-chlorotestosterone and its in-vivo epimeric metabolites

Doping athletes tend to use new and non-approved drugs in an attempt to circumvent the dope test. Recently, a package of unknown drug was obtained from black market claiming to contain steroid-like compound, which could not be readily identified due to the lack of commercially available reference standards. In order to characterize and identify the chemical structure of the unknown compound, ultra performance liquid chromatography high resolution mass spectrometry (UPLC-HRMS), high performance liquid chromatography tandem quadrupole mass spectrometry (HPLC-MS/MS), gas chromatography tandem quadrupole mass spectrometry (GC–MS/MS) and nuclear magnetic resonance (NMR) instruments were applied in this work. Based on the combined data, the unknown compound was identified as 6β-chlorotestosterone. The aim of this study was to find urinary metabolic biomarkers of 6β-chlorotestosterone for clinical research and doping control analysis. Ten in-vivo metabolites including four phase I metabolites and six phase II metabolites were characterized and potentially identified. The detailed structure of the phase I metabolite was successfully obtained by GC–MS/MS analysis of trimethylsilylation (TMS) released upon dissolution, overcoming the absence of useful fragment ions to demonstrate the structure of sulfate metabolites. Two strategies including metabolic mechanisms and retention time variation of steroids were introduced to determine and distinguish 3α/β- and 5α/β- epimeric metabolites. A comprehensive evaluation was conducted on time of appearance and detection of each metabolite. The sulfate metabolite 6β-chloro-5β-androstan-17-one-3β-O-sulfate (S1) could be detected up to 12 days after oral administration, which was considered to be the long term biomarker for 6β-chlorotestosterone abuse in clinical research and doping control analysis.

Amino acid metabolomics and machine learning for assessment of post-hepatectomy liver regeneration

ObjectiveAmino acid (AA) metabolism plays a vital role in liver regeneration. However, its measuring utility for post-hepatectomy liver regeneration under different conditions remains unclear. We aimed to combine machine learning (ML) models with AA metabolomics to assess liver regeneration in health and non-alcoholic steatohepatitis (NASH).MethodsThe liver index (liver weight/body weight) was calculated following 70% hepatectomy in healthy and NASH mice. The serum levels of 39 amino acids were measured using ultra-high performance liquid chromatography–tandem mass spectrometry analysis. We used orthogonal partial least squares discriminant analysis to determine differential AAs and disturbed metabolic pathways during liver regeneration. The SHapley Additive exPlanations algorithm was performed to identify potential AA signatures, and five ML models including least absolute shrinkage and selection operator, random forest, K-nearest neighbor (KNN), support vector regression, and extreme gradient boosting were utilized to assess the liver index.ResultsEleven and twenty-two differential AAs were identified in the healthy and NASH groups, respectively. Among these metabolites, arginine and proline metabolism were commonly disturbed metabolic pathways related to liver regeneration in both groups. Five AA signatures were identified, including hydroxylysine, L-serine, 3-methylhistidine, L-tyrosine, and homocitrulline in healthy group, and L-arginine, 2-aminobutyric acid, sarcosine, beta-alanine, and L-cysteine in NASH group. The KNN model demonstrated the best evaluation performance with mean absolute error, root mean square error, and coefficient of determination values of 0.0037, 0.0047, 0.79 and 0.0028, 0.0034, 0.71 for the healthy and NASH groups, respectively.ConclusionThe KNN model based on five AA signatures performed best, which suggests that it may be a valuable tool for assessing post-hepatectomy liver regeneration in health and NASH.

A simple and sensitive ultra-high performance liquid chromatography tandem mass spectrometry method for the quantitative analysis of VX-548 in monkey plasma: Method validation and application to pharmacokinetic study.

VX-548 is an orally active and highly selective NaV 1.8 inhibitor that is undergoing development for the treatment of acute pain. The aim of this study was to develop a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the measurement of VX-548 in monkey plasma. VX-548 was extracted from the plasma using acetonitrile-mediated protein precipitation. The quantitative analysis was performed on a Thermo Vantage TSQ mass spectrometer with ibrutinib as an internal standard. Chromatography was performed on a Waters ACQUITY UPLC BEH C18 column with 0.1% aqueous formic acid and acetonitrile as mobile phase. The precursor-to-product ion transitions were m/z 474.2 > 165.0 and m/z 441.2 > 138.1 for VX-548 and internal standard, respectively. This developed method was successfully validated in the concentration range of 1-1000 ng/mL. The calibration curve showed excellent linearity with a correlation coefficient of >0.999. The precision expressed as relative standard deviation (RSD) was <8.4%, whereas the accuracy denoted as relative error (RE) ranged from -5.0% to 9.1%. The mean recovery was >84%. VX-548 was stable in monkey plasma after storage under certain conditions. The validated method was successfully applied to the pharmacokinetic study of VX-548 in monkey plasma after single oral (2 mg/kg) and intravenous (1 mg/kg) administrations.

Comprehensive analysis of the dynamic changes of volatile and non-volatile metabolites in boletus edulis during processing by HS-SPME-GC–MS and UPLC-MS/MS analysis

In order to investigate the dynamic changes of flavor compounds, Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS) combined with Headspace Solid Phase Microextraction Gas Chromatography Mass Spectrometry (HS-SPME-GC–MS) was used to detect the metabolites in different drying processes. A total of 80 volatile compounds and 1319 non-volatile compounds were identified. The trend in the changes of C-8 compounds and sulfur-containing compounds were generally consistent with the trend of key enzyme activities. 479 differential metabolites were identified and revealed that metabolic profiles of compounds in Boletus edulis were altered with increased organic acids and derivatives and lipids and lipid-like molecules. Fatty acids and amino acids were transformed into volatile compounds under the action of enzymes, which played a significant role in the formation of the distinctive flavor of Boletus edulis. Our study provided a theoretical support for fully comprehending the formation mechanism of flavor from Boletus edulis during drying processes.

Human dementia with Lewy bodies brain shows widespread urea elevations

IntroductionSeveral recent studies have uncovered the presence of widespread urea elevations in multiple neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease dementia (PDD), vascular dementia (VaD), and Huntington's disease (HD). However, it is currently unknown whether dementia with Lewy bodies also shows these alterations in urea. This study aimed to investigate if and where urea is perturbed in the DLB brain. MethodsTissues from ten brain regions were obtained from 20 diagnosed cases of DLB and 19 controls. Urea concentrations were measured using ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). Case–control differences were assessed by nonparametric Mann-Whitney U tests, and s-values, E-values, effect sizes, and risk ratios were determined for each brain region. The results were compared to those previously obtained for AD, PDD, VaD, and HD. ResultsAs with other previously investigated dementia diseases, DLB shows widespread urea elevations, affecting all ten regions investigated in the current study; the degree of these elevations is lower than that seen in AD or PDD, similar to that seen in HD, and higher than that observed in VaD. The highest urea fold-change was observed in the pons and the lowest in the primary visual cortex. ConclusionUrea elevations appear to be a shared alterations across at least five neurodegenerative diseases, despite their many differences in clinical and neuropathological presentation. The cause and effects of this perturbation should be the focus of future studies, for its possible contributions to the pathology of these conditions.

Quantitative investigation of drug-drug interaction between bergenin and vilazodone in rats through UPLC-MS/MS assay

In this study, we firstly established and verified a method by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for the analysis of vilazodone and its metabolite M10 in rat plasma, then this method was used to explore the pharmacokinetics of vilazodone and M10 present or absence of 80 mg/kg bergenin in rats. Protein precipitation with acetonitrile was used to prepare the samples in this research. The mobile phase for liquid chromatography was consisted of 0.1% formic acid aqueous solution and acetonitrile. Brexpiprazole was used as the internal standard (IS), and the multiple reaction monitoring (MRM) mode was used for detection. The verification items required by the US Food and Drug Administration (FDA) guidelines such as selectivity, sensitivity, linearity, stability, recovery and matrix effect of this method were all met the standards. Besides, rats were used to explore the drug-drug interaction between vilazodone and bergenin, which were divided into two groups, and separately gavaged with the same-volume of carboxymethyl cellulose sodium (CMC-Na) solution and 80 mg/kg bergenin, respectively. The results showed that bergenin significantly affected the metabolism of vilazodone. It suggested that there was a potential drug-drug interaction between bergenin and vilazodone in rats. In clinical application, we should pay attention to the dose of vilazodone when in combination with bergenin.

Abstract PO5-13-03: Early Detection of Breast Cancer using Targeted Plasma Metabolomic Profiling

Abstract Background: Breast cancer (BC) is the second leading cause of cancer death among women. Accordingly, early diagnosis is key to the successful treatment, management, and care of BC. Recent studies confirmed that plasma metabolites could be reliable cancer biomarkers, allowing for the development of a minimally invasive routine blood test that can be used for screening, as well as for monitoring disease evolution in patients. The exact metabolic pathways involved in early BC development remain unclear. Metabolomic profiling of women with BC may help to identify new biomarkers to predict breast cancer long before symptoms appear. The purpose of this study was to validate a plasma metabolomic biomarker panel for an improved risk assessment for early detection BC in 241 patients, and to understand the potential role and the relationship between BC subtypes and hormone receptor status. Methods: Our study included a total of 185 plasma samples from women with biopsy-confirmed BC and 56 plasma samples from healthy controls. A targeted, quantitative mass spectrometry (MS)-based metabolomics approach was used to analyze 138 metabolites in plasma samples using a combination of direct injection (DI) MS and reverse-phase high performance liquid chromatography (HPLC) tandem mass spectrometry (MS/MS). The sample set was split into a discovery set and validation set. Metabolite concentration data, clinical data, and hormones receptor status were used to determine optimal biomarker sets. The same biomarkers and regression models were used and assessed on the validation models. The area under the receiver operator characteristic curves (AUROC), sensitivities and specificities at selected cut off points were calculated for each subgroup. Results: A large proportion of BC patients were at an early stage, with 98 at stage I (53.0 %), 70 at stage II (37.8%), and 17 at stage III (9.2%). The BC patients were of all subtypes: 138 luminal A (76,2%), 23 luminal B (12.7%), 5 hormone receptor-negative/HER2-positive (2.8%), and 15 triples negative (8.3%). A feature selection was performed on the training set using Partial Least-Squares Discriminant Analysis (PLS-DA), and the top performing metabolites were identified as the most important in discriminating BC from healthy subjects. The impact of age was also investigated. Features with &amp;gt;80% missing values were removed. To predict BC, the best signature comprised 9 variables implicated in fatty acid metabolism, amino acid metabolism, polyamine biosynthesis and related signaling pathways We further developed and validated a logistic regression with AUROC &amp;gt; 0.9 using these metabolites and other clinical data for detecting different stages and subtypes of BC. Conclusion: This study identified and validated a simple, high-performing, metabolite-based test for the early detection of BC. After confirmation in other independent cohort studies, our findings could provide the foundations for the development of a blood-based routine test for women at the highest risk for BC that is cost-effective, accurate and reliable. In addition, this approach could be used to complement other modalities especially for BC patients with dense breasts. Citation Format: Jean-Francois Haince, Lun Zhang, Rashid Ahmed Bux, Paramjit S. Tappia, Bram Ramjiawan, David Wishart, Andrew Maksymiuk. Early Detection of Breast Cancer using Targeted Plasma Metabolomic Profiling [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-13-03.

ARGININE METABOLISM AND ARTERIAL STIFFNESS IN HIGHLANDERS WITH HIGH ALTITUDE POLYCYTHEMIA

Objective: High altitude polycythemia (HAPC) is a health condition associated with hypoxemia and erythrocytosis. Patients with chronic hypoxemia often exhibit arterial stiffness. In the advanced stages of HAPC, patients can present changes in the pulmonary circulation, but limited information exists regarding arterial stiffness. Our study aims to investigate the relationship between HAPC and arterial stiffness, and explore the underlying mechanisms. Design and method: At an altitude of 3500m, we conducted a study examining arterial stiffness and vascular endothelial function in two groups of hypertensive highlanders with and without HAPC, each consisting of 30 participants. Plasma samples were measured using Ultra Performance Liquid Chromatography Tandem Mass Spectroscopy. Results: Hypertensive highlanders with HAPC exhibited notable vascular dysfunction, as indicated by significantly impaired flow-mediated dilation (FMD) (3.97±1.21% vs. 6.82±1.46%, P &lt; 0.001), and higher brachial-ankle pulse wave velocity (BaPWV) (1556±239 cm/s vs. 1890±459 cm/s, P &lt; 0.001). Metabolomics analysis revealed alterations in arginine/proline metabolism and the arginine biosynthesis pathway, involving L-arginine, L-ornithine, creatine and 4-guanidinobutanoic acid. The area under the curve (AUC) values for 4 metabolites and L-arginine/L-ornithine ratio were 0.753, 0.782, 0.720, 0.717 and 0.819, respectively. Pearson correlation analysis showed that L-arginine, L-ornithine, and L-arginine/L-ornithine ratio were associated with BaPWV (r=-0.4486, P=0.00032; r=0.3400, P=0.0078; r=0.3921, P=0.0019). L-arginine, L-ornithine, 4-guanidinobutanoic acid, creatine, and L-arginine/L-ornithine ratio were correlated with FMD. Multiple linear regression analysis revealed that L-arginine and L-arginine/L-ornithine ratio were independently correlated with BaPWV (beta= -0.370, t=-3.114, P=0.003; beta=-0.268, t=-2.211, P=0.032) and FMD (beta=0.434, t=3.114, P=0.003; beta= 0.457, t=3.445, P=0.001), but L-ornithine was not independently related to BaPWV and FMD. Conclusions: Our study indicates that HAPC may accelerate the progression of arterial stiffness in hypertensive patients. In fact, L-arginine takes an important role in the forming of NO in vascular endothelium. So The disorder of arginine metabolism may serve as the underlying metabolic mechanism.

Evaluation of new method for detection of creatine and guanidinoacetate by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)

Evaluation of new method for detection of creatine and guanidinoacetate by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)

A novel enrichment and sensitive method for rapid determination of 4 sulfonamide antibiotics residues in fish

Sulfonamide antibiotics with superior performance are widely used in aquaculture. However, the residues of sulfonamide antibiotics in aquatic products may pose a threat to human health. In this work, a novel type of magnetic molecularly imprinted polymer (mMIP) was synthesized and coupled with ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) for the selective separation and determination of 4 sulfonamide antibiotics residues in tilapia. The structure and adsorption properties of the polymers were characterized and evaluated. The key mMIP variables were investigated to determine the optimal magnetic solid phase extraction conditions. Results showed that the mMIPs had saturated adsorption capacities of 137.4, 78.7, 65.2, and 58.4 mg/g for trimethoprim, sulfadiazine, sulfamethazine, and sulfamethoxazole, respectively. Under optimized conditions, the linear range of the method for the detection of the 4 sulfonamides was 0.1–100 ng mL−1 (R > 0.991), the detection limit was 0.015 μg kg−1 (S/N = 3), the recovery rate was 78.6–101%, and the relative standard deviation (RSD%) was 4.2–7.8%. This new rapid and ultra-sensitive pretreatment method was successfully applied to the monitoring of trace sulfonamide antibiotics residues in fish samples.

Elucidating hormone transduction and the protein response of soybean roots to drought stress based on ultra performance liquid chromatography–tandem mass spectrometry and four-dimensional data-independent acquisition

Drought stress can affect the growth and development of soybean, an important cultivated crop. As the main water-absorbing organ, it is particularly important to study the drought response mechanism of roots. Plant hormones are associated with almost all basic biological processes and are used as candidate targets for improving plant stress resistance by regulating cell signal transduction related to stress resistance. In this study, ultra performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) and four-dimensional data-independent (4D-DIA) techniques were used to study the root hormone changes (5 %, 10 %, 15 % and 20 % drought stress level) and proteomic responses (15 % drought stress level) of different drought-tolerant varieties (drought-resistant HN44 and drought-sensitive HN65) under drought stress. The root activity, CAT activity and soluble sugar content of HN44 were basically higher than those of HN65 under different drought stress levels and days. In the two varieties, some plant hormones, such as abscisic acid (ABA) and salicylic acid (SA), accumulated with an increase in drought degree, and the accumulation of these hormones can give plants a certain degree of drought resistance. Most of the plant hormones peaked at 5–15 % drought stress level, and the overall content of jasmonic acid (JA) and cytokinin (CKs) decreased. Differences in the expression of key enzymes involved in hormone signal transduction, such as GH3, TCH4, and PR1, affect a variety of processes and lead to differences in drought resistance between the two varieties. In addition, HN44 had more upregulated proteins in important antioxidant pathways such as glutathione metabolism, phenylpropanoid biosynthesis, and isoflavone biosynthesis. Important drought-responsive proteins, such as PAL, 4CL, and VR, have also been characterized in HN44. In general, the drought-resistant variety was substantially better than the drought-sensitive variety in terms of hormone signaling and antioxidant capacity. These candidate proteins can be used as potential targets for improving drought resistance.

Establishment of Reference Value of 20 Amino Acids for Toddlers by High Performance Liquid Chromatography Tandem Mass Spectrometry

BACKGROUND: Amino acids are one of the essential metabolites, especially the 20 amino acids that are preserved as the building blocks of protein. Alterations in amino acid concentrations are related to disease such as inborn error of metabolism, cancer, as well as nutritional status. Hence, it is necessary to define reference values of 20 plasma-free amino acids for Indonesian toddlers and to establish a robust measurement technique using chromatography with tandem mass spectrometry (MS).METHODS: This study was a cross-sectional preliminary study to establish reference values. The sample was prepared by mixing plasma with 20% sulfosalicylic acid. Plasma-free amino acids were measured with high-performance liquid chromatography (HPLC) non-derivatization technique using column XTerra for chromatographic separation coupled with tandem MS. Amino acids reference values were taken from 101 healthy Indonesian toddlers aged 1-3 years old. Since amino acids data were not Gaussian distributed, the lower and upper of the reference value was established from the 5th percentile and the 95th percentile, respectively.RESULTS: Analysis for 20 amino acids was validated. The accuracy ranged from 90.53-105.39% and the precision ranged from 0.06-3.80%. The limit of detection range was 1-2 nmol/mL, and the limit of quantification range was 2-4 nmol/mL. The result was linear, with R2 higher than 0.998. There was no significant difference between boys and girls for all amino acids except for glycine.CONCLUSION: HPLC with tandem MS method can be used to evaluate amino acids in clinical practice. The reference values obtained are specific for aged 1-3 years old from urban areas in Indonesia. The study suggests that for each population, the reference values for amino acids should be established.KEYWORDS: amino acids, high-performance liquid chromatography, tandem mass spectrometry, reference values, Indonesia

Aqueous preparation of arginyl-fructosyl-glucose (a maltose-arginine AC) and determination of Amadori compounds (ACs) in red ginseng by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)

Amadori compounds (ACs) are key Maillard intermediates in various foods after thermal processing, and are also important non-saponin components in red ginseng. Currently, due to the difficulty in obtaining AC standards, the determination of multiple ACs is limited and far from optimal. In this study, an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated. A green synthetic method was developed for arginyl-fructosyl-glucose (AFG), the major AC in red ginseng with potential health benefits. The UPLC-MS/MS method was then applied in identification and quantification of ACs in red ginseng samples, which showed for the first time that 12 other ACs also exist in red ginseng in addition to AFG and arginyl-fructose (total 98.88 % of all ACs). Contents of AFG and arginyl-fructose in whole red ginseng were 36.23 and 10.80 mg/g dry weight, respectively. Raw ginseng can be steamed and then dried whole to obtain whole red ginseng, or sliced before drying to obtain sliced red ginseng. Slicing before drying was found to reduce ACs content. Results of the present study will help to reveal the biological functions of red ginseng and related products associated with ACs and promote the standardization of red ginseng manufacture.

Development of a UPLC-MS/MS-based method for simultaneous determination of advanced glycation end products and heterocyclic amines in stewed meat products

A novel analytical strategy was proposed to simultaneously quantify two advanced glycation end products (AGEs) including Nε-(Carboxymethyl)lysine (CML), Nε-(Carboxyethyl)lysine (CEL) and eight heterocyclic amines (HAs) including IQ, MeIQ, MeIQx, 4,8-DiMeIQx, 7,8-DiMeIQx, PhIP, Harman, and Norharman. The procedure was based on a two-step extraction, solid phase extraction (SPE) purification followed by ultra performance liquid chromatography tandem mass spectrometry. The established method showed a good linearity (R2 ≥ 0.9950), rapid processing time (8 min per sample), satisfactory recoveries (matrix spiked recoveries range from 72.2% to 119.6%) and precision (intra-day and inter-day RSDs were <19.3%). The limit of quantification (LOQ) and limit of detection (LOD) resulted to be between 0.05–15 ng/g and 0.2–50 ng/g, respectively. The validated technique was further applied to determine HAs and AGEs in eight stewed meat product samples consumed in Shanghai, with the amount of HAs and AGEs ranging from 2.851 to 18.289 ng/g and 118.158–543.493 ng/g, respectively.

Coprococcus eutactus screened from healthy adolescent attenuates chronic restraint stress-induced depression-like changes in adolescent mice: Potential roles in the microbiome and neurotransmitter modulation

The onset of depression commonly occurs in adolescence; therefore, depressive prevention and intervention are pivotal during this period. It is becoming evident that neurotransmitter imbalance and gut microbiota dysbiosis are prominent causes of depression. However, the underlying links and mechanisms remain poorly understood. In this study, with 16S ribosomal RNA gene sequencing, genus Coprococcus markedly differentiated between the healthy and unmedicated depressive adolescents. Based on this, transplantation of Coprococcus eutactus (C.e.) was found to dramatically ameliorate the chronic restraint stress (CRS) induced depression-like changes and prevent synaptic loss and glial-stimulated neuroinflammation in mice. The Ultra-high performance liquid chromatography tandem mass spectrometry analysis (UHPLC-MS/MS) further showed that neurotoxic neurotransmitters in kynurenine pathway (KP) such as 3-hydroxykynurenine (3−HK) and 3-hydroxyanthranilic acid (3-HAA) decreased in mouse brains, mechanistically deciphering the transfer of the tryptophan metabolic pathway to serotonin metabolic signaling in the brain after C.e. treatment, which was also verified in the colon. Molecularly, blockage of KP activities mediated by C.e. was ascribed to the restraint of the limit-step enzymes responsible for kynurenine, 3-HK, and quinolinic acid generation. In the colon, C.e. treatment significantly recovered goblet cells and mucus secretion in CRS mice which may ascribe to the rebalance of the disordered gut microbiota, especially Akkermansia, Roseburia, Rikenella, Blautia, and Alloprevotella. Taken together, the current study reveals for the first time the beneficial effects and potential mechanisms of C.e. in ameliorating CRS-induced depression, unraveling the direct links between C.e. treatment and neurotransmitter rebalance, which may provide efficacious therapeutic avenues for adolescent depressive intervention.