Performance Liquid Chromatography-tandem Mass Spectrometry Research Articles

Biomarker identification and risk assessment of cardiovascular disease based on untargeted metabolomics and machine learning

Cardiovascular disease (CVD) is the leading cause of mortality, disability, and healthcare costs, with a significant impact on the elderly and contributing to premature deaths across various age groups, including those below age 70. Despite decades of transformative discoveries and clinical efforts, the challenges of diagnosis, prevention, and treatment of CVD persist on a massive scale. This study aimed to unravel potential CVD-associated biomarkers and establish a machine learning model for the risk assessment of CVD. Untargeted metabolic assay with ultra-high performance liquid chromatography-tandem mass spectrometry and routine clinical biochemistry test were undertaken on the fasting venous blood specimens from 57 subjects. Four relevant clinical traits and 164 CVD-associated metabolites were identified, especially those related to glycerophospholipid metabolism and biosynthesis of unsaturated fatty acids. The machine learning model achieved from an integrated biomarker panel of palmitic amide, oleic acid, 138-pos (the 138th detected metabolomic feature in positive ion mode), phosphatidylcholine, linoleic acid, age, direct bilirubin, and inorganic phosphate, was able to improve the accuracy of CVD risk assessment up to a high satisfactory value of 0.91. The findings indicate that disorders in the metabolic processes of biological membranes and energy are significantly associated with increased risk of vascular damage in CVD patients. With machine learning methods, the pivotal metabolites and clinical biomarkers offer a promising potential for the efficient risk assessment and diagnosis of CVD.

Proteomic Profiling of Endothelial Cell Secretomes After Exposure to Calciprotein Particles Reveals Downregulation of Basement Membrane Assembly and Increased Release of Soluble CD59

Calciprotein particles (CPPs) are essential circulating scavengers of excessive Ca2+ and PO43− ions, representing a vehicle that removes them from the human body and precludes extraskeletal calcification. Having been internalised by endothelial cells (ECs), CPPs induce their dysfunction, which is accompanied by a remarkable molecular reconfiguration, although little is known about this process’s extracellular signatures. Here, we applied ultra-high performance liquid chromatography-tandem mass spectrometry to perform a secretome-wide profiling of the cell culture supernatant from primary human coronary artery ECs (HCAECs) and internal thoracic artery ECs (HITAECs) treated with primary CPPs (CPP-P), secondary CPPs (CPP-S), magnesiprotein particles (MPPs), or Ca2+/Mg2+-free Dulbecco’s phosphate-buffered saline (DPBS) for 24 h. Incubation with CPP-P/CPP-S significantly altered the profiles of secreted proteins, delineating physiological and pathological endothelial secretomes. Neither pathway enrichment analysis nor the interrogation of protein–protein interactions detected extracellular matrix- and basement membrane-related molecular terms in the protein datasets from CPP-P/CPP-S-treated ECs. Both proteomic profiling and enzyme-linked immunosorbent assay identified an increased level of protectin (CD59) and reduced levels of osteonectin (SPARC), perlecan (HSPG2), and fibronectin (FN1) in the cell culture supernatant upon CPP-P/CPP-S treatment. Elevated soluble CD59 and decreased release of basement membrane components might be considered as potential signs of dysfunctional endothelium.

A novel quantitative method for the determination of 10B-carrier boronophenylalanine in rat plasma by UHPLC-MS/MS and comparison with ICP-MS

L-Boronophenylalanine (BPA), a widely used 10B carrier for clinical boron neutron capture therapy (BNCT), was quantified in rat plasma through a simple, effective and stable ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. Chromatographic separation was performed on an ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm) column with the mobile phase of 0.5 % formic acid aqueous solution and acetonitrile. For the detector, the m/z ion pairs used for quantification were 209.1→120.1 for BPA and 210.1→120.1 for internal standard in a positive mode by electrospray ionization (ESI) using multiple reaction monitoring (MRM). The method is specific and robust with rare affection by endogenous substances in the matrix. A good linear relationship was observed over 80–80000 ng/mL (r2 = 0.9993). The values of inter- and intra-day accuracy and precision were within the acceptance criteria of ±15 %. BPA was found to be stable under different experimental conditions. This developed method was successfully applied on a pharmacokinetic experiment on Sprague-Dawley rats (intravenous injection, 125 mg/kg) and a comparation between UHPLC-MS/MS and ICP-MS for BPA was carried out.

Coadministration of Cariprazine with a Moderate CYP3A4 Inhibitor in Patients with Schizophrenia: Implications for Dose Adjustment and Safety Monitoring.

Cariprazine is metabolised mainly by CYP3A4 and to a lesser extent by CYP2D6. This study aimed to evaluate the effects of erythromycin, a moderate cytochrome P450 (CYP)3A4 inhibitor, on the pharmacokinetics of cariprazine in male patients with schizophrenia, and to assess the influence of CYP2D6 phenotypes on cariprazine metabolism. Forty-two patients received oral doses of 1.5 mg cariprazine alone for 28 days (to reach steady state), followed by a co-administration of cariprazine 1.5 mg daily with erythromycin 500 mg twice daily (BID) and Enterol 250 mg BID for 21 days, followed by a 14-day post-treatment period. Blood samples were collected at predefined time points and analysed for cariprazine, its two active metabolites: desmethyl cariprazine (DCAR) and didesmethyl cariprazine (DDCAR), and erythromycin using validated high performance liquid chromatography-tandem mass spectrometry methods. CYP2D6 phenotypes were determined by genotyping. The pharmacokinetic parameters were calculated using non-compartmental analysis. Erythromycin increased the area under the curve (AUCτ) and peak concentration (Cmax) of Total cariprazine (cariprazine + DCAR + DDCAR) by about 40-50% but did not affect the time to peak concentration (Tmax). The CYP2D6 phenotypes had no substantial effect on the pharmacokinetics of cariprazine and its metabolites, either alone or in combination with erythromycin. Cariprazine was well tolerated and safe. The findings suggest that co-administration of cariprazine with moderate CYP3A4 inhibitors may require dose adjustment or monitoring; however, pharmacogenetic testing for CYP2D6 is not necessary for optimising cariprazine therapy. Trial registration number (EudraCT Number): 2018-003721-28. Date of registration: 21-SEP-2018.

HPLC-MS/MS based method for the determination of 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid in human plasma

The report presents first high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based method for the determination of plasma 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid (HPPTCA), an adduct of cysteine and active form of vitamin B6 pyridoxal 5′-phosphate. The assay employs 4-deoxypyridoxine (4-DPD) as an internal standard. Sample preparation procedure primarily involves acetonitrile (ACN) extraction of HPPTCA from plasma proteins, sample deproteinization by ultrafiltration, and dilution of ultrafiltrate with mobile phase prior to chromatographic analysis. The chromatographic separations of HPPTCA and 4-DPD are achieved within 6 min at 20 °C on X-Bridge Glycan BEH Amide (100 × 2.1 mm, 2.5 μm) column using gradient elution. The eluent consists of 0.1% formic acid in a mixture of solvent A (water and ACN (95:5, v: v)) and solvent B (water and ACN (5:95, v: v)) delivered at a flow rate of 0.3 mL/min. The assay linearity was observed within 0.25-10 µmol/L in plasma. The limit of quantification was found to be 0.25 µmol/L. The method was successfully applied to plasma samples delivered by apparently healthy donors showing that the HPLC-MS/MS assay is suitable for human plasma screening. The presence of HPPTCA was confirmed in eleven of fifteen study samples. The HPPTCA concentration ranged from 0.55 to 8.39 µmol/L.

Citrullination Accompanies the Development of Carotid Atherosclerotic Plaques.

Citrullination represents a post-translational modification primarily mediated by peptidylarginine deiminase (PADI) 2 and 4 and resulting in the conversion of positively charged peptidylarginine to neutrally charged peptidylcitrulline. Molecular consequences of citrullination include the generation of neoepitopes which provoke the production of autoantibodies implicated in the development of autoimmune diseases. As citrullination initiates, promotes, and is enhanced by aseptic inflammation which plays a pivotal role in atherosclerosis, we proposed that citrullination might accompany the development of atherosclerotic vascular disease. To investigate features and patterns of citrullination in atherosclerotic plaques. We collected carotid atherosclerotic plaques (n = 14) and adjacent arterial segments (n = 14) which were pairwise excised during the carotid endarterectomy. The tissues were examined employing proteomic profiling (ultra-high performance liquid chromatography-tandem mass spectrometry analysis), haematoxylin and eosin staining, Western blotting and immunofluorescence staining for peptidylcitrulline, PADI2, and PADI4, and gene expression analysis. To better explore the mechanisms of citrullination in the neointima, we have also stained excised plaques for the extracellular vesicle markers (CD9 and CD81) and assessed co-localisation of PADI2 (a citrullination marker) with CD81 (an extracellular vesicle marker). In order to study the systemic response to citrullination in an atherosclerotic vascular disease setting, we measured the level of anti-citrullinated protein antibodies in the serum of patients with ischaemic stroke and healthy volunteers. Proteomic profiling found 213 plaque-specific and 111 intact arteria-specific proteins, as well as 46 proteins and 13 proteins which have been respectively upregulated or downregulated in plaques as compared with the adjacent intact segments. Among the top 20 upregulated proteins were atherogenic apolipoprotein B-100, iron-associated protein haptoglobin, and matrix metal-loproteinase-9, together indicating the advanced stage of plaque progression. In comparison with the intact arterial segments, plaques demonstrated protein signatures of innate immune response and oxidative stress, suggesting aseptic inflammation as a driver of atherosclerotic vascular disease. Both peptidylcitrulline and PADI2 have been abundant in the neointima but negligible in tunica media; further, the levels of peptidylcitrulline, PADI2, and PADI4 were elevated in plaque lysates in comparison with those from adjacent arterial segments (p = 0.025, 0.025, and 0.010, respectively). Notably, PADI2 and peptidylcitrulline were co-localised with the cells in the neointima and a considerable proportion of PADI2 was co-localised with CD81-positive extracellular vesicles (p = 0.003). Albeit citrullinated histone H3 and myeloperoxidase showed higher signal in the neointima than in tunica media (p = 0.048 and 0.023, respectively), we did not observe any signs of neutrophil extracellular traps (e.g., unwound chromatin or co-localisation of citrullinated histone H3 with neutrophil elastase) in the plaque tissue. Serum anti-citrullinated protein antibodies were not elevated in patients with ischaemic stroke (p = 0.71), suggesting that vascular citrullination likely does not trigger a generalised immune response. The development of carotid atherosclerosis is associated with citrullination, although it represents a local rather than systemic phenomenon in this clinical scenario.

Metabolomic profiling of human semen in patients with oligospermia using high performance liquid chromatography-tandem mass spectrometry

Male infertility is one of the most common reproductive dysfunctions. Despite oligospermia being a cause of infertility, few studies have been conducted on it. This study aimed to investigate differences in semen metabolic patterns in patients with oligospermia and to identify potential biomarkers associated with oligospermia. Semen samples from oligospermia patients (20 cases) and healthy controls (20 cases) were detected by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), and 72 and 89 metabolites were identified as potential markers in positive and negative ion modes, respectively. In addition, the results identified multiple metabolic pathways in patients with oligospermia, such as glycine serine and threonine metabolism, Synthesis and degradation of ketone bodies, Valine, leucine, and isoleucine degradation. These results described unique metabolic characteristics of semen in patients with oligospermia and provided novel insights into the mechanism of the semen disorder.

Highly selective guanidine-linked covalent organic framework for efficient removal of perfluoroalkyl carboxylic acids from water samples

In this study, guanidine-linked covalent organic framework (TPDGCl) was synthesized via a solvothermal method. The presence of guanidine groups in its structure facilitates electrostatic interactions and provides hydrogen bonding sites, enabling the selective enrichment of perfluoroalkyl carboxylic acids (PFCAs) from environmental water samples. The material exhibits rapid kinetic mass transfer, which facilitates the efficient removal of samples containing PFCAs from aqueous solutions within 5 min. Furthermore, TPDGCl exhibits several advantageous properties, including high selectivity for PFCAs, a high adsorption capacity (2005 mg/g), excellent chemical stability, and remarkable recyclability (five cycles). Additionally, density functional theory (DFT) studies have demonstrated that the adsorption of PFCAs on TPDGCl is enhanced by ion exchange, hydrophobic interactions, hydrogen bonding interactions and ordered channel structures. Finally, TPDGCl was used as an adsorbent for dispersive solid-phase extraction (d-SPE), in combination with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), to develop an analytical method for the efficient extraction and sensitive detection of PFCAs. The developed method exhibits highly sensitivity, with detection and quantification limits of 0.64–0.85 ng/L and 2.6–3.4 ng/L, respectively. The recoveries of actual water samples ranged from 75.4% to 113.7%. Moreover, TPDGCl effectively reduces the levels of PFCAs in real water samples to less than 70 ng/L within only 1 h. These results indicate that TPDGCl is an effective adsorbent for removing PFCAs from environmental water samples.

Vinpocetine Ameliorates Neuronal Injury After Cold-Induced Traumatic Brain Injury in Mice.

Traumatic brain injury (TBI), also known as intracranial injury, is a common condition with the highest incidence rate among neurodegenerative disorders and poses a significant public health burden. Various methods are used in the treatment of TBI, but the effects of cold-induced traumatic brain injury have not been thoroughly studied. In this context, vinpocetine (VPN), derived from Vinca minor, exhibits notable anti-inflammatory and antioxidant properties. VPN is known for its neuroprotective role and is generally utilized for treating various neurodegenerative disorders. However, the function of VPN after cold-induced TBI needs to be studied in more detail. This study aims to investigate the neuroprotective effects of VPN at varying doses (5mg/kg or 10mg/kg) after cold-induced TBI. C57BL/6 mice were sacrificed 2 or 28days after cold-induced TBI. Results indicate that VPN administration significantly reduces brain infarct volume, brain swelling, blood-brain barrier disruption, and DNA fragmentation in a dose-dependent manner. Additionally, VPN enhances neuronal survival in the ipsilesional cortex. In the long term, VPN treatment (5mg/kg/day or 10mg/kg/day, initiated 48h post-TBI) improved locomotor activity, cell proliferation, neurogenesis, and decreased whole brain atrophy, specifically motor cortex atrophy. We performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to elucidate the underlying mechanisms to profile proteins and signaling pathways influenced by prolonged VPN treatment post-TBI. Notably, we found that 192 different proteins were significantly altered by VPN treatment, which is a matter of further investigation for the development of therapeutic targets. Our study has shown that VPN may have a neuroprotective role in cold-induced TBI.

Accurate determination of three new antihypertensive drugs illegally added to food using ultra-high performance liquid chromatography-tandem mass spectrometry

Effective strategies are required to address food safety issues related to the illegal addition of antihypertensive drugs to food and claims of antihypertensive function. In this study, a novel ultra-high performance liquid chromatography-triple-quadrupole mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous determination of three antihypertensive drugs (azilsartan, candesartan cilexetil, and lacidipine) in 12 food matrices (pressed candies, solid beverages, alternative teas, tea drinks, biscuits, jellies, mixed liquors, oral liquids, medicinal teas, tablets, hard capsules, and soft capsules). Initially, mass spectrometry parameters, such as the collision energies of the three antihypertensive drugs, were optimized. Subsequently, the response intensities and chromatographic separation conditions of the three drugs in different mobile phases were compared. In addition, to enhance the recoveries, various extraction solvents and purification methods, including solid-phase extraction (SPE) columns and the QuEChERS technique, were investigated. In the developed method, sample determination involved three steps. First, the sample was extracted using 0.2% (v/v) formic acid in acetonitrile and then filtered using high-speed centrifugation, in addition, the extracted solution of alternative tea and medicinal tea was purified using the QuEChERS technique. Second, the supernatant was diluted with water, and filtered through a 0.22 μm polytetrafluoroethylene (PTFE) membrane. Finally, the analytes were separated on an Agilent Eclipse Plus RRHD C18 column (50 mm×2.1 mm, 1.8 μm) using a 5 mmol/L ammonium formate aqueous solution and acetonitrile as the mobile phases under gradient elution conditions and then detected using UHPLC-MS/MS with positive electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. Quantitative analysis was performed using a matrix-matched external standard method. Methodological validation showed good linear relationships for all three antihypertensive drugs in the investigated concentration ranges, with correlation coefficients (r2) greater than 0.996. The limit of detection (LOD) and limit of quantification (LOQ) of lacidipine were 0.02 mg/kg and 0.04 mg/kg, respectively, whereas those of the other two drugs were 0.01 mg/kg and 0.02 mg/kg, respectively. In the 12 food matrices, the average recoveries of the drugs ranged from 86.6% to 107.5% with relative standard deviations (RSDs) of 1.1%-10.9% (n=6) at low, medium, and high spiked levels. Furthermore, this method was successfully applied to the analysis of real food samples. Overall, the newly developed method is simple, rapid, sensitive, accurate, and suitable for the qualitative and quantitative determination of antihypertensive drugs in different food matrices. This work could provide technical support for food safety agencies in implementing measures against the illegal addition of antihypertensive drugs to food.

Influences of lactic acid bacteria strains on the flavor profiles, metabolites and quality characteristics of red yeast rice produced by solid-state fermentation

Red yeast rice (RYR) as a traditional fermented food produced by inoculation of the Monascus genus into steamed rice through solid-state fermentation, has been used intensively for thousands of years in China. Herein, we investigated the flavor profiles, metabolites and quality characteristics of RYR produced by mix fermentation with lactic acid bacteria (LAB) using the headspace solid phase microextraction gas chromatography–mass spectrometry (HS–SPME–GCMS) approach integrated with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS). 56 volatile organic compounds (VOCs) and 1287 non-volatile metabolites were identified and classified into eight classes, the inoculation of Lactiplantibacillus plantarum (LP) and Limosilactobacillus fermentum (LF) increased VOCs and enhanced the flavor characteristics of RYR, with LP being the more significant. The KEGG pathway enrichment analysis revealed that most of the differential metabolites were enriched in amino acid metabolism when integrating volatile and non-volatile omics data, meanwhile, the addition of LAB promoted the metabolism of amino acid, alcohols, fatty acid and some flavonoids, in particular, amino acid metabolism was almost upregulated in the LP group. Therefore, this study highlights the tremendous potential of LAB in improving the flavor quality of RYR. This study provides novel insights into RYR fermentation processes and establishes a theoretical foundation for developing and applying new RYR products.

Optimization of skin sampling based on tape stripping for lipidome analysis by nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry

In this study, skin sampling by tape stripping for lipid analysis was optimized by examining the lipid profiles of the stratum corneum (SC), focusing on the composition and levels of ceramides (Cer), diacylglycerols (DG), and triacylglycerols (TG), using nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry. Significant variations in the number and composition of the identified lipids, particularly Cer and neutral lipid species, were observed across different skin locations, including the forearm, forehead, cheek, and neck. Analysis of the layer-to-layer lipid profiles of the seven consecutive layers revealed a gradual decrease in DG and TG levels from the outermost to the innermost layers, with certain Cer subclasses showing increases in the second to fourth layers and subsequent decreases. Comparative analysis of lipid profiles from adjacent spots demonstrated statistical consistency and persistent differences between spots. Pooling layers were evaluated as an alternative method for representing SC layers, and their efficiencies were assessed by varying the number of pooled layers. We found that pooling five consecutive layers was effective in terms of the number and levels of identified lipids. Additionally, investigations into the matrix effect and extraction efficiency upon pooling layers indicated that pooling up to five layers did not significantly affect ionization suppression or reduce extraction recovery.

Associations of Advanced Glycation End Products with Sleep Disorders in Chinese Adults.

Advanced glycation end products (AGEs), a group of food processing byproducts, have been implicated in the development of various diseases. However, the relationship between circulating AGEs and sleep disorders remains uncertain. This cross-sectional study elucidated the association of plasma AGEs with sleep disorders among 1732 Chinese adults who participated in the initial visit (2019-2020) of the Tongji-Shenzhen Cohort (TJSZC). Sleep behavior was assessed using self-reported questionnaires and precise accelerometers. Plasma levels of AGEs, including Nε-(Carboxymethyl)lysine (CML), Nε-(Carboxyethyl)lysine (CEL), and Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine (MG-H1), were quantified by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In logistic regression, per IQR increment in individual AGEs was associated with an increased odds ratio of short sleep duration (CML: 1.11 [1.00, 1.23]; CEL: 1.16, [1.04, 1.30]), poor sleep quality (CML: 1.33 [1.10, 1.60]; CEL: 1.53, [1.17, 2.00]; MG-H1: 1.61 [1.25, 2.07]), excessive daytime sleepiness (CML: 1.33 [1.11, 1.60]; MG-H1: 1.39 [1.09, 1.77]), and insomnia (CML: 1.29 [1.05, 1.59]). Furthermore, in weighted quantile sum regression and Bayesian kernel machine regression analyses, elevated overall exposure levels of plasma AGEs were associated with an increased risk of sleep disorders, including short sleep duration, poor sleep quality, excessive daytime sleepiness, and insomnia, with CML being identified as the leading contributor. Insufficient vegetable intake and higher dietary fat intake was associated with an increase in plasma CEL. These findings support a significant association between plasma AGEs and sleep disorders, indicating that AGEs may adversely influence sleep health and reducing the intake of AGEs may facilitate preventing and ameliorating sleep disorders.

Dispersive solid-phase extraction using ZIF-8 coupled to high performance liquid chromatography–tandem mass spectrometry for the detection of 80 pesticides in poultry egg and their products

AbstractPeople are consuming increasing amounts of poultry eggs and their products, and their safety is always a public concern. However, the complex composition of poultry eggs and their products, particularly the proteins and lipids, can easily cause matrix effects, requiring the use of sorbents to remove these components to avoid affecting the detection results. Zeolitic imidazolate framework-8 (ZIF-8) is a MOF material with high specific surface area, high porosity, and high stability, making it widely used as a sorbent in pesticide residue detection. In this study, we evaluated the performance of ZIF-8 compared to classic solid phase extraction (SPE) and Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) in the pretreatment step. The results showed that ZIF-8 could better reduce the matrix effect in poultry eggs and their products. After optimizing the extraction solvents, chromatographic and mass spectrometric conditions, and pretreatment processes, we established a method using ZIF-8 as a sorbent combined with high performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) for the detection of 80 pesticide residues in seven different types of poultry eggs and their products, yielding satisfactory results. All the target analytes showed good linearity, both with values of r2 > 0.996. The average recovery and coefficients of variation (CVs), expressed as relative standard deviations, ranges from 72.5% to 113.2% (chicken egg) (CV: 0.1%–12.9%), 72.2%–114.2% (salted duck egg) (CV: 0.1%–9.8%), 70.3%–105.6% (goose egg) (CV: 0.1%–12.0%), 70.9–120.4% (marinated egg) (RSD: 0.1%–14.8%), 70.7%–108.5% (duck egg) (CV: 0.1%–12.3%), 71.1%–105.0% (quail egg) (CV: 0.2%–5.6%), 70.7%–111.5% (century egg) (CV: 0.1%–13.4%). The values of limit of detection (LOD) and limit of quantification (LOQ) were, respectively, ranging from 0.15 to 0.85 μg kg−1 and 0.34–2.6 μg kg−1. When this method was applied to the detection of real samples, one chicken egg sample was found to contain 0.013 mg kg−1 of fipronil, and one marinated egg sample was found to contain 0.0087 mg kg−1 of thiamethoxam, indicating the necessity of stringent safety monitoring for poultry eggs and their products.

High-performance liquid chromatography-tandem mass spectrometry was used to measure 20 antidepressants in human serum.

Aim: This study used high performance liquid chromatography-tandem mass spectrometry to quantify the blood concentrations of 20 antidepressants, such as bupropion and fluoxetine, in human serum samples.Methods: After direct precipitation with a 1:9 protein precipitant of methanol and acetonitrile, serum samples were examined using high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS). The material was separated using a Poroshell 120 EC-C18 column (3.0mm×50mm, 2.7μm) and gradient elution. The mobile phases were phase A 0.01% formic acid aqueous solution (containing 2mmol/ml ammonium acetate) and phase B methanol solution. A 0.45ml/min flow rate was used to divide the sample and inject 5μl. Electrospray ionization source positive ion mode and multiple reaction monitoring modes were used for analysis. Measurement was quantified using an internal standard technique.Results: Accuracy ranged from 90.3 to 114.3%, intra-day precision from 100.1 to 112.3%, inter-day precision from 100.4 to 112.6%, extraction recoveries from 85.5 to 114.5% and matrix effect from 85.6 to 98.7%.Conclusion: This approach is fast, accurate, sensitive and repeatable. It can identify 20 antidepressants in blood simultaneously. This can be used to monitor blood drug levels and medication metabolism.

Efficient Elimination of Matrix Effects for the Rapid Screening of β-Agonist Residues in Porcine Urine by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS)

An efficient and innovative clean-up method was developed for the rapid screening of β-agonists residues in porcine urine by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). 1% Aqueous ammonium hydroxide was shown to be effective for the removal of matrix components as a wash step in solid phase extraction (SPE). A significant reduction in matrix effects was observed with values from 81% to 108%. Furthermore, under the optimized conditions, the recoveries of all analytes at 0.3, 0.6, and 3.0 μg/L ranged from 70.1% to 109.0% when employing the external standard method for quantitative analysis, rather than relying on matrix-matched standards or isotope-labelled internal standards to mitigate matrix effects. The limits of detection (LOD) and limits of quantification (LOQ) were 0.1 μg/L and 0.3 μg/L, respectively. This method has been successfully applied to screen β-agonists in porcine urine and is a promising tool for multi-residue analysis.

Bioaccessibility and antioxidant activity of fermented chrysanthemum rice wine on In vitro simulated digestion

The purpose of this study was to investigate the bioaccessibility of total phenolic (TPC), total flavonoid (TFC), chlorogenic acid and antioxidant activity in fermented chrysanthemum rice wine (FRW) under in vitro gastrointestinal digestion model compared with rice wine (RW). The fermented samples were digested and the bioactive components and antioxidant activity during gastric phase (GP) and intestinal phase (IP) were analyzed. The results showed that TPC, TFC, ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and oxygen radical absorbance capacity (ORAC) decreased during the gastrointestinal digestion stage. TFC was higher in FRW during in vitro gastrointestinal digestion stage and increased to a maximum of 0.483 mg/mL at IP stage. 2.2- azinobis-3- ethyl-benzothiazoline-6 - sulfonic acid (ABTS) reached its maximum value of 91.62% in the late stage of intestinal digestion. The hydroxyl radical scavenging activity (HRSA) increased to 77.94% in the IP-2 phase. In addition, a total of 158 (FRW) and 131 (RW) polyphenols, including flavonoids, isoflavones, phenols and coumarins, were detected and identified by Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) analysis. The biotransformation was carried out between the isomers of phenolic compounds in FRW, where 4,5-dicaffeoylquinic acid might be changed into neochlorogenic acid and chlorogenic acid. Correlation analysis showed that 46.15% of metabolites in FRW were positively correlated with the decrease of DPPH, FRAP and ORAC, while 53.85% were positively correlated with the increase of ABTS and HRSA. The results showed higher bioavailability and antioxidative activity in FRW after in vitro digestion.

Improved skeletal muscle mass and strength through Protamex-mediated hydrolysis of perilla seed cake: Elevated rosmarinic acid levels as a contributing factor

Perilla seed cake (PSC) is a byproduct of oil extraction from perilla seeds. It is rich in proteins and bioactive compounds. PSC was enzymatically hydrolyzed to form PSC hydrolysate (PSCH) to enhance the absorption of PSC, and their effects on muscle health in mice were compared. High performance liquid chromatography-tandem mass spectrometry analysis revealed that PSC contains several polyphenols, including rosmarinic acid (RA), caffeic acid, apigenin, and luteolin. The hydrolysate showed 1.44- and 7.04-fold increases in RA and caffeic acid contents, respectively, compared to those of PSC. The intake of PSC, PSCH, and RA significantly improved muscle mass and exercise performance in mice by upregulating protein synthesis, myogenic differentiation, oxidative muscle fiber formation, fatty acid oxidation, and mitochondrial biogenesis; however, PSCH had better promoting effects than PSC. In conclusion, PSCH improves muscle health through its bioactive compounds (particularly RA), indicating the potential of PSCH and RA in functional foods.

Plasma Endocannabinoids Are Independently Associated With the Metabolic Function of White Adipose Tissue

Abstract Context Little is known about the link between the endocannabinoid (EC) system and the in vivo metabolic function of white adipose tissue (WAT). Objective We aimed to evaluate whether ECs are linked to postprandial fatty acid metabolism and WAT metabolic function. Methods Men and women, with (IGT, n = 20) or without impaired glucose tolerance (NGT, n = 20) underwent meal testing with oral and intravenous stable isotope palmitate tracers and positron emission tomography with intravenous [11C]-palmitate and oral [18F]-fluoro-thia-heptadecanoic acid to determine systemic and organ-specific dietary fatty acid (DFA) and nonesterified fatty acid (NEFA) metabolism and partitioning. We determined fasting and postprandial plasma levels of EC by ultra-high performance liquid chromatography–tandem mass spectrometry. Results All ECs of the 2-monoacylglycerol (2-MAG) family displayed a progressive postprandial increase up to 360 minutes after meal intake that was more pronounced in women with IGT. N-acylethanolamine (NAE) levels decreased between fasting and 180 minutes, followed by a return to preprandial values at 360 minutes and were also increased in women with IGT. Postprandial area under the curve (AUC) of palmitate appearance rate was significantly and independently associated with postprandial AUC of anandamide (AEA; P = .0003) and total energy expenditure (P = .0009). DFA storage in abdominal subcutaneous adipose tissue was positively predicted by fasting 2-arachidonoylglycerol (2-AG; P < .04). Conclusion EC levels of the NAE family independently follow plasma NEFA metabolism, whereas 2-MAG closely follow the spillover of triglyceride-rich lipoprotein intravascular lipolytic products. Whether these associations are causal requires further investigation.

Gibberellin 2-oxidase 1(CsGA2ox1) involved gibberellin biosynthesis regulates sprouting time in camellia sinensis

BackgroundTea is an important cash crop and buds are its main product. To elucidate the molecular mechanism of the sprouting time of tea plants, ‘Yuchunzao’, which was an early sprouting tea cultivar, was studied. ‘Echa 1’, sprout one week later than ‘Yuchunzao’ in spring, was used as the control.ResultsA total of 26 hormonal compounds and its derivatives in tea plants were qualified by using Ultra Performance Liquid Chromatography-Tandem mass spectrometry (UPLC-MS/MS). The result showed that GA20, GA3 and ICA were significantly different in ‘Yuchunzao’ than in ‘Echa 1’, with GA20 and GA3 up-regulated and ICA down-regulated. Based on the Illumina platform, transcriptome analysis revealed a total of 5,395 differentially expressed genes (DEGs). A diterpenoid biosynthesis related gene, gibberellin 2-oxidase 1 (CsGA2ox1), was downregulated in ‘Yuchunzao’ compared to ‘Echa 1’. CsGA2ox1 regulate the transformation of GA different forms in plants. The relative expression of CsGA2ox1 showed an adverse trend with the content of GA20 and GA3. Our results suggest that down regulation of CsGA2ox1 resulted in the accumulation of GA3 and GA20, and then promoted sprout of ‘Yuchunzao’.ConclusionThis study provides theoretical basis of tea plants sprout and guides the tea breeding in practice.