High-performance Liquid Chromatography With Mass Spectrometry Research Articles

Greenness of dispersive microextraction using molecularly imprinted polymers

Abstract Molecularly imprinted polymers (MIPs) as materials with determined levels of selectivity and specificity for designated analytes have recently gained much attention in various application fields. However, with the growing adoption of green analytical chemistry (GAC) principles, it is essential to investigate the greenness of MIP synthesis and its subsequent application in sample preparation, as well as to evaluate the “green” nature of the developed analytical methodologies, such as dispersive solid-phase microextraction (DSPME). Accordingly, the main objective of this research was to evaluate the greenness of MIP-based glycidyl methacrylate synthesis and MIP use as a DSPME sorbent prior to high-performance liquid chromatography with mass spectrometry (HPLC-MS). The green perspective of MIP-DSPME prior to HPLC-MS was investigated using various analytical metric tools such as the Analytical Eco-Scale, the Green Analytical Procedure Index, and Analytical GREEnness (AGREE). Since these analytical tools are not fully implementable for the assessment of the greenness of the MIP synthesis, some alternative approaches were used to optimize the synthesis parameters to make the MIP DSPME sorbent as close as possible to the GAC principles. The calculated AGREE score (0.62) and 91 points in the Analytical Eco-Scale for the proposed DSPME technique using MIP indicated a high level of greenness.

Si-Ni-San Reduces Hepatic Lipid Deposition in Rats with Metabolic Associated Fatty Liver Disease by AMPK/SIRT1 Pathway.

Metabolic associated fatty liver disease (MAFLD) is a chronic disease characterized by excessive lipid deposition in the liver without alcohol or other clear liver-damaging factors. AMP-activated protein kinase (AMPK)/silencing information regulator (SIRT)1 signaling pathway plays an important role in MAFLD development. Si-Ni-San (SNS), a traditional Chinese medicine, has shown reducing hepatic lipid deposition in MAFLD rats, however, the underlying mechanisms of SNS are barely understood. The aim of this research was to investigate the mechanisms of SNS in reducing hepatic lipid deposition in MAFLD rats by regulating AMPK/SIRT1 signaling pathways. The components of SNS were determined by high performance liquid chromatography with mass spectrometry (HPLC-MS) analysis. MAFLD rats were induced by high-fat and high-cholesterol diet (HFHCD), and treated by SNS. SNS-containing serum and Compound C (AMPK inhibitor) were used to treat palmitic acid (PA)-induced HepG2 cells. To elucidate the potential mechanism, lipid synthesis-related proteins (SREBP-1c and FAS), fatty acid oxidation (PPARα and CPT-1), and AMPK/SIRT1 signaling pathway (p-AMPK and SIRT1) were assessed by Western blot. SNS improved serum lipid levels, liver function and reduced hepatic lipid deposition in MAFLD rats. SNS-containing serum reduced lipid deposition in PA-induced HepG2 cells. SNS could up-regulate protein expressions of PPARα, CPT-1, p-AMPK and SIRT1, and down-regulate protein expressions of SREBP-1c and FAS. Similar effects of SNS-containing serum were observed in PA-induced HepG2 cells. Meanwhile, Compound C weakened the therapeutic effects of SNS-containing serum on lipid deposition. SNS could reduce hepatic lipid deposition by inhibiting lipid synthesis and promoting fatty acid oxidation, which might be related with activating the AMPK/SIRT1 signaling pathway. This study could provide a theoretical basis for the clinical use of SNS to treat MAFLD.

The study of metabolites of new promising strains of bacterial‐antagonists of the genus <i>Bacillus</i> to increase the effectiveness of fungicidal biological products based on them

Aim. To study the metabolomic profile of bacterial strains of the genus Bacillus promising for the development of biofungicides using thin layer chromatography (TLC), bioautography and high performance liquid chromatography with mass spectrometry (HPLC‐MS).Materials and Methods. The objects of study are strains of bacteria of the genus Bacillus. Exometabolites were isolated from the liquid culture and their metabolomic profile was analyzed by TLC, bioautography, and HPLC‐MS.Results. By the method of bioautography with a test culture of the fungus F. oxysporum var. orthoceras BZR F6 metabolites of bacterial strains of the genus Bacillus surfactin, iturin and fengycin were identified. Quantitative analysis using HPLC‐MS analysis allows us to state that the B. velenzensis BZR 336g and B. amyloliquefaciens BZR 277 strains produce more surfactin than the others. An increased content of ituric lipopeptides was found in strains B. velezensis BZR 517 and B. velenzensis BZR 336g. According to the ability to produce fengycins, strains of B. velezensis BZR 517 and B. velenzensis BZR 336g are ahead of other strains.Conclusion. Studies using two analytical methods reveal that the strains produce all three antifungal lipopeptides. This is important, since metabolites are able not only to suppress phytopathogenic fungi, but also to enhance the antifungal effect due to synergism. The results obtained allow us to state the possibility of using all four strains as producers of effective biofungicides.

Effects of ultrasound on the formation of oxidative pigments in a model red wine solution containing glutathione

Xanthylium cations and its ethyl ester are important pigments derived from (+)-catechin and glyoxylic acid in wine. In this paper, ultrasound treatment was applied in model wine containing glutathione to study its effects on the formation of xanthylium cation pigments triggered by free radicals from the ultrasound cavitation. The changes in color parameters, concentration of pigment compounds and free radicals were investigated during wine storage by the means of ultraviolet visible (UV–vis) spectrophotometer, colorimeter, high performance liquid chromatography with mass spectrometry (HPLC-MS) and electron paramagnetic resonance (EPR), respectively. Results indicate that longer ultrasonic treatment in conjunction with glutathione can significantly enhance the formation of xanthylium cation pigments and accelerate the red wine color evolution, and no extra (+)-catechin was involved, which might be attributed to the stronger 1-hydroxyethyl radicals intensity induced by ultrasound irradiation. In summary, ultrasound treatment can accelerate the oxidative coloration of wine with GSH during ageing, which would contribute to well understand the changing mechanism of wine color properties modified by ultrasound.

Highly enhanced degradation of organic pollutants in hematite/sulfite/photo system

Sulfate radical (SO4−)-based advanced oxidization processes (SR-AOPs) are most commonly explored by activation of peroxydisulfate (PDS), peroxymonosulfate (PMS) or sulfite by various transition-metal ions with or without UV irradiation. Here sulfite activation in the presence of hematite (α-Fe2O3) under UV (hematite/sulfite/photo) in the presence of O2 was successfully developed. The performance of this system towards organic pollutant degradation was evaluated, and the reactive species involved were identified. The results demonstrated that roxarsone (ROX), an aromatic organoarsenic pollutant, could be effectively degraded by this system and the degradation efficiency was greatly affected by initial pH, hematite dosage, and sulfite concentration. Replacement of Na2SO3 with CaSO3 greatly improved the degradation efficiency, and the same amount of sulfite source applied with multiple-dosing mode was more beneficial to pollutant removal than the single-dosing mode. The quenching experiments, electron spin resonance (ESR) spectra, and high-performance liquid chromatography with mass spectrometry (HPLC-MS) analysis of the reaction products showed that SO4− radicals mainly contributed to the highly efficient ROX degradation. This system also had high degradation efficiencies towards other recalcitrant organic pollutants, such as atrazine, bisphenol A, rhodamine, and methylene blue and could use sunlight instead of artificial UV irradiation. This work provides a practical and cost-effective method for degrading recalcitrant organics in wastewaters, and also exhibits a beneficial treatment strategy to replace Na2SO3 with CaSO3 to enhance the decay of organics, and thus advances the application of this oxidation system.

It's Only a Part of the Story: Analytical Investigation of the Inks and Dyes Used in the Privilegium Maius.

The Privilegium maius is one of the most famous and spectacular forgeries in medieval Europe. It is a set of charters made in the 14th century upon commitment by Duke Rudolf IV, a member of the Habsburg family, to elevate the rank and the prestige of his family. These five charters, now kept at the Österreichisches Staatsarchiv in Vienna, have been subjected to a thorough interdisciplinary study in order to shed light on its controversial story. The charters are composed of pergamenaceous documents bound to wax seals with coloured textile threads. The present contribution concerns the characterisation of the inks used for writing and of the dyes used to colour to the threads: Are they compatible with the presumed age of the charters? Though showing only a part of the whole story of the charters, dyes analysis could contribute in assessing their complex history from manufacturing to nowadays. The dyes were characterised with non-invasive in situ measurements by means of fibre optic (FORS) and with micro-invasive measurements by means of Surface Enhanced Raman Spectroscopy (SERS) and High-Performance Liquid Chromatography with Mass Spectrometry (HPLC-MS) analysis. The results showed that the threads of four of the charters (three dyed with madder, one with orchil) were apparently coloured at different dyeing stages, then re-dyed in the 19–20th century.

Inflammatory responses relate to distinct bronchoalveolar lavage lipidome in community-acquired pneumonia patients: a pilot study

BackgroundCommunity-acquired pneumonia (CAP) is a leading cause of morbidity and mortality worldwide. Antibiotics are losing their effectiveness due to the emerging infectious diseases, the scarcity of novel antibiotics, and the contributions of antibiotic misuse and overuse to resistance. Characterization of the lipidomic response to pneumonia and exploring the “lipidomic phenotype” can provide new insight into the underlying mechanisms of pathogenesis and potential avenues for diagnostic and therapeutic treatments.MethodsLipid profiles of bronchoalveolar lavage fluid (BALF) samples were generated through untargeted lipidomic profiling analysis using high-performance liquid chromatography with mass spectrometry (HPLC-MS). Principal component analysis (PCA) was applied to identify possible sources of variations among samples. Partitioning clustering analysis (k-means) was employed to evaluate the existence of distinct lipidomic clusters.ResultsPCA showed that BALF lipidomes differed significantly between CAP (n = 52) and controls (n = 68, including 35 healthy volunteers and 33 patients with non-infectious lung diseases); while no clear separation was found between severe CAP and non-severe CAP cases. Lactosylceramides were the most prominently elevated lipid constituent in CAP. Clustering analysis revealed three separate lipid profiles; subjects in each cluster exhibited significant differences in disease severity, incidence of hypoxemia, percentages of phagocytes in BALF, and serum concentrations of albumin and total cholesterol (all p < 0.05). In addition, SM (d34:1) was negatively related to macrophage (adjusted r = − 0.462, p < 0.0001) and PE (18:1p/20:4) was positively correlated with polymorphonuclear neutrophil (PMN) percentages of BALF (adjusted r = 0.541, p < 0.0001). The 30-day mortality did not differ amongst three clusters (p < 0.05).ConclusionsOur data suggest that specific lower airway lipid composition is related to different intensities of host inflammatory responses, and may contribute to functionally relevant shifts in disease pathogenesis in CAP individuals. These findings argue for the need to tailor therapy based on specific lipid profiles and related inflammatory status.Trial registrationClinicalTrials.gov (NCT03093220). Registered on 28 March 2017 (retrospectively registered).

The effects of hemorrhage on the pharmacokinetics of tranexamic acid in a swine model.

The early use of tranexamic acid (TXA) is strongly advocated in patients who are likely to require massive transfusion to decrease mortality. This study determines the influence of hemorrhage on the pharmacokinetics of TXA in a porcine model. The investigation was a prospective experimental study in Yucatan minipigs. First, in vitro plasma-cell partitioning of TXA was evaluated by inoculating whole blood with known aliquots, centrifuging, and measuring the supernatant with high-performance liquid chromatography with mass spectrometry (HPLC-MS). Then, using in vivo modeling, normovolemic and hypovolemic (35% reduction in blood volume) swine (n = 4 per group) received 1 g of intravenous TXA and had blood sampled at 14 time points over 4 hours to determine baseline clearance via HPLC-MS. Additional swine (n = 4) were hemorrhaged 35% of their blood volume, and TXA was administered as a 15 mg/kg infusion over 10 minutes followed by infusion of 1.875 mg/kg per hour to simulate massive hemorrhage scenario. During the first hour of TXA administration, one total blood volume was hemorrhaged and simultaneously replaced with TXA free blood. Serial blood samples and the hemorrhaged blood were analyzed by HPLC-MS to determine the percentage of dose lost via hemorrhage. Clearance of TXA was diminished in the hypovolemic group compared with the normovolemic group (115 ± 4 vs 70 ± 7 mL/min). Percentage of dose lost via hemorrhage averaged 25%. The lowest measured plasma level during the exchange transfusion was 34 μg/mL. Mean 25% of the present 2017 Joint Trauma System Clinical Practice Guideline dosing of TXA can be lost to hemorrhage if a blood volume is transfused within an hour of initiating therapy. In the case of TXA, which has limited distribution and is administered during active hemorrhage and massive blood transfusions, replacement strategies should be developed and tested to find simple methods of adjusting the current dosing guidelines to maintain therapeutic plasma concentrations. Therapeutic, level II.

CYP3A4 activity and haloperidol effects in alcohol addicts.

Haloperidol is one of the most commonly used typical antipsychotics [2]. It has a powerful antipsychotic activity blocking mesolimbic postsynaptic dopamine receptors. Unwanted adverse effects accompany the use of haloperidol. Therefore alcohol abusers' attitudes towards haloperidol are ambiguous and often negative, which sometimes limits it's use in patients with addictive disorders [3]. Cytosolic carbonyl reductase reduces haloperidol to reduced form, which has 10-20% of the activity of the parent molecule. It is further metabolized by CYP3A4 to a tetrahydropyridine and then conjugated by glucuronidation and sulphation. Reduced haloperidol is back-oxidized to haloperidol by CYP3A4 and CYP2D6. Haloperidol is N-dealkylated by CYP3A4 and CYP2D6 to 4-chlorophenyl-4-hydroxypiperidine and p-fluorobenzoyl propionic acid. The correlation between CYP2D6 and CYP3A4 activity and the rate of biotransformation of haloperidol was demonstrated in a number of studies on patients with schizophrenia [1, 2, 5]. At the same time other studies deny or disaffirm this correlation [4]. To estimate the correlation between CYP3A4 isoenzyme activity and the efficacy and safety of haloperidol in patients with alcohol abuse during the exacerbation of the addiction. The study involved 15 men, alcohol abusers, in exacerbation of their addiction, who were hospitalized in Moscow Research and Practical Centre for Narcology of the Departament of Public Health. All 15 patients received haloperidol in tablets and injections. Determination of CYP3A4 activity was performed using high performance liquid chromatography with mass spectrometry (HPLC/ MS) by determination of endogenous substrate of this isoenzyme and its metabolite in urine - the ratio: cortisol/6-beta-hydroxycortisol. We used international psychometric scales to assess efficacy of haloperidol (the scale of determining the severity of addiction of The National Research Center on Addictions of the Ministry of Health Of Russia, Hamilton Anxiety Research Scale (HARS)). The safety of haloperidol was estimated by the UKU Side-Effect Rating Scale. Scales express the clinical picture of the abuse. The higher the score, the more pronounced the addiction is. Calculating the differences in scores of the scales allowed for clinical assessment of haloperidol effects. The larger the difference in scores was, the more pronounced were the changes in clinical picture of abuse, and the higher the efficacy of therapy was assumed. Statistical analysis of the results of the study was performed by non-parametric statistics by the program STATISTICA v10.0 («StatSoft Inc.», USA). The normality of sample distribution was estimated by Shapiro-Wilk's W-test, and the homogeneity of dispersion, that was estimated by Fisher's T-test (in case of comparison of two samples). The differences were evaluated as significant in case of p < 0,05 (statistic power >80%). To determine the correlation between the quantitative characteristics Spearman rank R coefficient was calculated. The value of correlation coefficient r from 0,3 to 0,7 (p < 0,05) indicated positive moderate, but significant correlation between the characteristics, r>0,7 (p < 0,05) - strong and significant correlation, negative value of r indicated inverse correlation. Data analysis demonstrated a correlation between the activity of isoenzyme CYP3A4 and the scores of pathological addiction (r1 = -0,36), HARS (r2 = -0,45), UKU Side-Effect Rating Scale (r3 = -0.15) in the entire group (p < 0.05). In a group of patients, who received the higher doses of haloperidol (more than 7.5 mg per day in tablets or 5 mg per day in injections), the following results were received in the same groups of data: r1 = -0.68, r2 = -0.71, r3 = -0.76 (p < 0.05). The results demonstrate the correlation between CYP3A4 activities and the efficacy and safety of haloperidol in alcohol abusers during the exacerbation of the addiction. The inverse correlation indicates that the higher the activity of CYP3A4 is, the lower the efficacy of haloperidol is. Also it can be assumed that the presence of strong correlation between the activity of CYP3A4 and the efficacy of haloperidol in group of patients, who received higher doses of haloperidol, may indicate that CYP3A4 is involved in haloperidol metabolism when it is used at higher doses. It should be noted that in this research the activity of CYP3A4 was determined using high performance liquid chromatography with mass spectrometry (HPLC/MS) by determination the ratio of cortisol/6-beta-hydroxycortisol for the fist time. To increase the level of our confidence in the results further studies with a larger number of people are necessary.

Study of the transformation of two salicylates used in personal care products in chlorinated water

Disinfection of swimming pool water is essential to inactivate pathogenic microorganisms. However chlorine based disinfectants, the most commonly used, are known to lead to the formation of disinfection by-products (DBPs), some of which have been associated with adverse health effects. Precursors of DBPs include the organic matter present in the water used to fill the swimming pool, human body fluids and personal care products (PCPs) used by swimmers and bathers. The increased use, in the last years, of PCPs lead to an increased concern about the fate of PCPs in swimming pool waters and potential health risks of formed DBPs. In this study, the chemical transformations of two salicylates, benzyl salicylate (BzS) and phenyl salicylate (PS), incorporated in several PCPs, in chlorinated water were investigated. High-performance liquid chromatography (HPLC) with UV-diode-array detection (HPLC-UV-DAD) was used to follow the reaction kinetics and HPLC with mass spectrometry (HPLC-MS) was used to tentatively identify the major transformation by-products. Under the experimental conditions used in this work both salicylates reacted with chlorine following pseudo-first order kinetics: rate constant k = (0.0038 ± 0.0002) min−1 and half-life t1/2 = (182 ± 10) min for BzS and rate constant k = (0.0088 ± 0.0005) min−1 and half-life t1/2 = (79 ± 4) min for PS (mean ± standard deviation). The reactions of the two salicylates in chlorinated water led to the formation of DBPs that were tentatively identified as mono- and dichloro- substituted compounds. Most probably they result from an electrophilic substitution of one or two hydrogen atoms in the phenolic ring of both salicylates by one or two chlorine atoms.

Purification and characterization of the biosurfactant rhamnolipid

Biosurfactant rhamnolipid is a metabolic intermediate produced by microorganisms under a certain condition. There are the polar hydrophilic group and the non-polar hydrophobic group in rhamnolipid molecule which always exhibits high surface or interfacial activity. A reliable separation and purification method as well as component identification technique is essential for success of production process. The rhamnolipid was produced by aerobic fermentation using Pseudomonas aeruginosa CCTCC AB93066 in this study. It was separated from the culture by acid precipitation and purified by column chromatography until two groups of monorhamnolipid and dirhamnolipid were obtained. High performance liquid chromatography with mass spectrometry (HPLC-MS) examination showed that either the monorhamnolipid or the dirhamnolipid contained three major species. They were RhaC10C10, RhaC10C12-H2, RhaC10C12 for monorhamnolipid and Rha2C10 C10, Rha2C10 C12-H2, Rha2 C10 C12 for dirhamnolipid. The results of the study suggested that Pseudomonas aeruginosa CCTCC AB93066 is a good strain for rhamnolipid production. Acid precipitation-column chromatography technique is good for purification of rhamnolipid. Meanwhile, HPLC-MS is a reliable method for identifying components of rhamnolipid with high sensitivity and accuracy.

Study on Active Compounds of &lt;i&gt;Apocynum venetum&lt;/i&gt; L

Although Apocynum leaves have been used for the treatment of cardiovascular diseases, the study on active compounds of Apocynum venetum L. is little. In this paper,high performance liquid chromatography with mass spectrometry (HPLC-MS) were used for the active ingredient in qualitative analysis of Apocynum leaves, based on the information of the mass spectrum molecular ion peak [M-and some characteristic fragment ions, and combined with UV spectral characteristics we initially speculated that Apocynum leaves water extract isolated to 10 components.

Use of the Local False Discovery Rate for Identification of Metabolic Biomarkers in Rat Urine Following Genkwa Flos-Induced Hepatotoxicity

Metabolomics is concerned with characterizing the large number of metabolites present in a biological system using nuclear magnetic resonance (NMR) and HPLC/MS (high-performance liquid chromatography with mass spectrometry). Multivariate analysis is one of the most important tools for metabolic biomarker identification in metabolomic studies. However, analyzing the large-scale data sets acquired during metabolic fingerprinting is a major challenge. As a posterior probability that the features of interest are not affected, the local false discovery rate (LFDR) is a good interpretable measure. However, it is rarely used to when interrogating metabolic data to identify biomarkers. In this study, we employed the LFDR method to analyze HPLC/MS data acquired from a metabolomic study of metabolic changes in rat urine during hepatotoxicity induced by Genkwa flos (GF) treatment. The LFDR approach was successfully used to identify important rat urine metabolites altered by GF-stimulated hepatotoxicity. Compared with principle component analysis (PCA), LFDR is an interpretable measure and discovers more important metabolites in an HPLC/MS-based metabolomic study.

Isolation and Identification of Strawberry Phenolics with Antioxidant and Human Cancer Cell Antiproliferative Properties

Studies suggest that consumption of berry fruits, including strawberries ( Fragaria x ananassa Duch.), may have beneficial effects against oxidative stress mediated diseases such as cancer. Berries contain multiple phenolic compounds, which are thought to contribute to their biological properties. Comprehensive profiling of phenolics from strawberries was previously reported using high-performance liquid chromatography with mass spectrometry (HPLC-MS) detection. The current study reports the isolation and structural characterization of 10 phenolic compounds from strawberry extracts using a combination of Amberlite XAD16-resin and C18 columns, HPLC-UV, and nuclear magnetic resonance (NMR) spectroscopy methods. The phenolics were cyanidin-3-glucoside ( 1), pelargonidin (2), pelargonidin-3-glucoside (3), pelargonidin-3-rutinoside (4), kaempferol (5), quercetin (6), kaempferol-3-(6'-coumaroyl)glucoside) (7), 3,4,5-trihydroxyphenyl-acrylic acid (8), glucose ester of ( E)- p-coumaric acid (9), and ellagic acid . Strawberry crude extracts and purified compounds 1- 10 were evaluated for antioxidant and human cancer cell antiproliferative activities by the Trolox equivalent antioxidant capacity (TEAC) and luminescent ATP cell viability assays, respectively. Among the pure compounds, the anthocyanins 1 (7156 microM Trolox/mg), 2 (4922 microM Trolox/mg), and 4 (5514 microM Trolox/mg) were the most potent antioxidants. Crude extracts (250 microg/mL) and pure compounds (100 microg/mL) inhibited the growth of human oral (CAL-27, KB), colon (HT29, HCT-116), and prostate (LNCaP, DU145) cancer cells with different sensitivities observed between cell lines. This study adds to the growing body of data supporting the bioactivities of berry fruit phenolics and their potential impact on human health.

新しい質量分析法による複合糖質の構造解析

Mass spectrometric analyses of glycoconjugates are most commonly performed by fast atom bombardment (FAB) or liquid matrix-assisted secondary ion mass spectrometry (SIMS). However, more recent ionization methods, such as electrospray (ESI) and matrix-assisted laser desorption (MALD), have made mass spectrometric analyses applicable to even larger, thermally labile biomolecules. ESI often produces multiply-charged ions, and a molecular weight is obtained by the deconvolution of those multiply-charged ions. On the other hand, MALD may produce intact molecular ions up to half a million daltons. Combination of high performance liquid chromatography with mass spectrometry (HPLC-MS) is often a strategy of choice for biological samples in which contamination of other chemical species is usually unavoidable. Tandem mass spectrometry (MS/MS) with collision-induced dissociation (CID) often provides structural information for unknown sample molecules. In particular, charge-remote fragmentation applying a high-energy collision sometimes gives detailed information about the locations of unsaturation and branchings of metal-cationated molecules.