Xianzhe Shi

Effect of traditional Chinese medicine berberine on type 2 diabetes based on comprehensive metabonomics

A comprehensive metabonomic method, in combination with fingerprint analysis and target analysis, was performed to reveal potential mechanisms of berberine action in the treatment of patients with type 2 diabetes and dyslipidemia. Serum samples of 60 patients before and after treatment with either berberine or placebo were collected. Ultra-performance liquid chromatography and quadrupole time-of-flight tandem mass spectrometry (UPLC Q-TOF MS) coupled with pattern recognition analysis were used to identify changes in global serum metabolites. Compared with placebo, patients before and after berberine treatment could be separated into distinct clusters as displayed by the orthogonal signal correction filtered partial least-squares discriminant analysis (OSC-PLS-DA) score plot, which indicated changes in circulating metabolites after berberine treatment. Among them, free fatty acids changed markedly. These were further quantified by UPLC combined with single quadrupole mass spectrometry (UPLC SQ MS). There was a highly significant decrease in the concentrations of 13 fatty acids following berberine administration. 10 fatty acids also differed statistically from placebo. These results suggest that berberine might play a pivotal role in the treatment of type 2 diabetes through down-regulating the high level of free fatty acids and that comprehensive metabonomic measurements are potentially very useful for studying the mechanisms of action of traditional Chinese medicines.

Analysis of catecholamines and their metabolites in adrenal gland by liquid chromatography tandem mass spectrometry.

Two simple, rapid and specific analytical methods for 13 catecholamines and their metabolites have been developed based on liquid chromatography tandem mass spectrometry in a multiple reaction monitoring mode. Tyrosine, dopamine, dihydroxyphenylalanine, epinephrine, norepinephrine, 3-methoxytyramine, normetanephrine, metanephrine and isoproterenol (internal standard) were separated on a Kromasil Cyano analytical column by a mobile phase consisting of 60% (v/v) acetonitrile and 40% (v/v) water adjusted with formic acid to pH 3.0, and detected by positive ionization electrospray tandem mass spectrometry. While vanillymandelic acid, 3,4-dihydroxymandelic acid, homovanillic acid, 3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxyphenylglycol and 5-hydroxy-2-indolecarboxylic acid (internal standard) were separated on a reversed-phase Shim-Pak VP-ODS column with the mobile phase of 60% (v/v) acetonitrile, and 40% (v/v) water adjusted with formic acid to pH 4.5 and detected in the negative ionization electrospray tandem mass spectrometry. The influence of various parameters such as column type and mobile phase composition on separation and sensitivity were investigated. The limits of detection were in the range of 0.5-20ngmL(-1). The mean recoveries determined from three different concentrations of each analyte were above 85.4%. The precision of the method calculated as relative standard deviation was lower than 5.3%. Deduced from the results of real sample analysis, adrenal gland synthesizes and stores the catecholamine hormones norepinephrine and epinephrine.

Facile synthesis of boronate-decorated polyethyleneimine-grafted hybrid magnetic nanoparticles for the highly selective enrichment of modified nucleosides and ribosylated metabolites.

Ribosylated metabolites, especially modified nucleosides, have been extensively evaluated as cancer-related biomarkers. Boronate adsorbents are considered to be promising materials for extracting them from complex matrices. However, the enrichment of ribosylated metabolites in low abundance is still a challenge due to the limited capacity and selectivity of the existing boronate adsorbents. In this study, a novel type of magnetic nanoparticles named Fe3O4@SiO2@PEI-FPBA was synthesized by grafting polyethyleneimine (PEI) onto the surface of Fe3O4@SiO2 before modification by boronate groups. The high density of the amino groups on the PEI chains supplied a large number of binding sites for boronate groups. Thus, the adsorption capacity (1.34 ± 0.024 mg/g) of the nanoparticles, which is 6- to 7-fold higher than that of analogous materials, was greatly improved. The unreacted secondary amines and tertiary amines of the PEI enhanced the aqueous solubility of the nanoparticles, which could efficiently reduce nonspecific adsorption. The nanoparticles were able to capture 1,2 cis-diol nucleosides from 1000-fold interferences. Moreover, the flexible chains of PEI were favorable for effective enrichment and quick equilibration (<2 min). Finally, 60 ribose conjugates were enriched from human urine using the nanoparticles. Among them, 43 were identified to be nucleosides and other ribosylated metabolites. Nine low abundance modified nucleosides were detected for the first time. In conclusion, Fe3O4@SiO2@PEI-FPBA is an attractive candidate material for the highly selective enrichment of 1,2-cis-diol compounds.

Urinary metabonomic study of lung cancer by a fully automatic hyphenated hydrophilic interaction/RPLC-MS system

Lung cancer is one of the most common and lethal cancers in the world. In this study, a home-devised hydrophilic interaction chromatography/RPLC-MS (HILIC/RPLC-MS) system was developed to study the urinary metabonomics of lung cancer patients. This system combined the orthogonal selectivity of HILIC and RPLC and could chromatographically reveal more comprehensive information of the urinary metabolites. Within a total analysis time of 50 min, we detected 577 polar metabolite ions on the first HILIC column and 261 apolar ones on the second RPLC column. In addition, an orthogonal signal correction partial least-squares discriminant analysis model was constructed to characterize differences between health and lung cancer cases. Eleven potential biomarkers, ten from HILIC column and one from the second RP column, were identified and all of these biomarkers were found upregulated in lung cancer patients. Overall, the results indicated that the developed HILIC/RPLC-MS system is a promising tool for metabonomic studies in revealing more information of highly complex samples.

A novel stop-flow two-dimensional liquid chromatography-mass spectrometry method for lipid analysis.

A novel on-line two dimensional liquid chromatography (2D LC) based on stop-flow mode coupled with electrospray ionization mass spectrometry (ESI-MS) method was established to separate lipids in human plasma. Hydrophilic interaction liquid chromatography (HILIC) in the first dimension and reversed-phase liquid chromatography (RP LC) in the second dimension were used to separate the lipids into six fractions based on their polar head groups and further into peaks based on aliphatic chains, respectively. A new stop-flow interface with a trap column and an extra make-up flow was designed to construct this system and trap the components eluted from the first dimension. Moreover, the same length of analytical columns and similar flow rates were used in the first and second dimensions. Therefore, the new stop-flow 2D LC system can avoid the sensitivity decrease caused by the dilution effect, which is the shortcoming of comprehensive 2D LC. Three hundred and seventy-two lipids were identified from plasma extract using this 2D LC coupled with ESI-MS in positive mode, and 88 more lipids were detected than one-dimensional RP LC analysis. Peak capacity of this stop-flow 2D LC was 415, which is similar to that of comprehensive 2D LC. The linearity, repeatability and sensitivity of this method were satisfactory, which demonstrated that this method was also suitable for quantitative analysis. All these results indicated that this on-line 2D LC method is powerful for qualitative and quantitative analysis of complex lipids.

Analysis of acetylcholine, choline and butyrobetaine in human liver tissues by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

The strong polar quaternary ammoniums, acetylcholine (ACh), choline (Ch) and butyrobetaine (BB, (3-carboxypropyl)trimethylammonium), are believed playing important roles in liver metabolism. These metabolites are at low levels and are weakly retained on reversed-phase liquid chromatographic (RP-LC) columns. Several hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) methods have been reported to analyze these compounds from different samples. However, no application to human liver tissues has been published. In this study, HILIC-MS/MS method was developed to simultaneously determine these three metabolites in human liver tissues. They were simply extracted from tissue, separated on a HILIC column, and detected by tandem MS in the mode of multiple reaction monitoring (MRM). Further studies on the recovery and repeatability based on real samples indicated the method was accurate and reliable. This method was successfully applied to measure the levels of ACh, Ch and BB in 61 human liver tissue samples including normal, hepatocellular carcinoma (HCC) and matched non-cancerous liver tissues. By comparison of Ch and ACh contents in 29 HCC with their matched non-cancerous liver tissues, it was found that ACh content increased in 11/29 HCC cases and decreased in 13/29 cases. Furthermore, the ACh/Ch ratio increased in 16/29 HCC cases, while it decreased in 8/29 cases. These results strongly indicated that there exist different patterns of ACh content in cancer tissues among HCC patients, thus highlighting the understanding of ACh and its relevant signal pathways in hepatic carcinogenesis and HCC progression.

Preparation and evaluation of a novel hybrid monolithic column based on pentafluorobenzyl imidazolium bromide ionic liquid.

To develop a novel hybrid monolithic column based on pentafluorobenzyl imidazolium bromide ionic liquid, a new ionic liquid monomer was synthesized from 1-vinylimidazole and pentafluorobenzyl bromide. By employing a facile one-step copolymerization of polyhedral-oligomeric-silsesquioxane-type (POSS) cross-linking agent and the home-made ionic liquid monomer, the hybrid monolithic columns were in situ fabricated in fused-silica capillary. The morphology of monolithic column was characterized by scanning electron microscope (SEM) and the chemical composition was confirmed by Fourier-transform infrared spectroscopy (FT-IR) and elemental analysis. Excellent mechanical stability and slight swelling propensity were exhibited which was ascribed to the rigid hybrid monolithic skeleton. Reproducibility results of run-to-run, column-to-column, batch-to-batch and day-to-day were investigated and the RSDs were less than 0.46%, 1.84%, 3.96% and 3.17%, respectively. The mixed-mode retention mechanism with hydrophobic interaction, π-π stacking, ion-exchange, electrostatic interaction and dipole-dipole interaction was explored systematically using analytes with different structure types. Satisfied separation capability and column efficiency were achieved for the analysis of small molecular compounds such as alkylbenzenes, polycyclic aromatic hydrocarbons, nucleosides and halogenated compounds.

Effect of Bisphenol A on Rat Metabolic Profiling Studied by Using Capillary Electrophoresis Time-of-Flight Mass Spectrometry

Bisphenol A (BPA), a chemical widely used in the manufacture of polycarbonate plastics, has raised considerable concern in recent decades because of its hormone-like properties. Whether BPA exposure is a health risk remains controversial in many countries. A metabolomics study based on capillary electrophoresis time-of-flight mass spectrometry (CE-TOF/MS) was performed to study the urine metabolic profiles of Sprague-Dawley rats fed with four dose levels of BPA (0, 1, 10, and 100 μg/kg body weight) for 45 days. Multivariate pattern recognition directly reflected the metabolic perturbations caused by BPA. On the basis of univariate analysis, 42 metabolites including amino acids, polyamines, nucleosides, organic acids, carbohydrates, pterins, polyphenols, and sugar phosphates were found as the most significantly differential metabolites. The marked perturbations were related with valine, leucine and isoleucine biosynthesis, D-glutamine and D-glutamate metabolism, etc. Significant alterations of neurotransmitters (glutamate, gamma-aminobutyric acid, and noradrenaline) and neurotransmitter-related metabolites (tyrosine, histamine, valine, and taurine) suggested that the toxic effects of small-dose BPA (below 50 mg/kg/day) may contribute to its interactions with the neuromediating system. Our study demonstrated that metabolomics may offer more specific insights into the molecular changes underlying the physiological effects of BPA.

A fully automated system with on-line micro solid-phase extraction combined with capillary liquid chromatography-tandem mass spectrometry for high throughput analysis of microcystins and nodularin-R in tap water and lake water.

Microcystins and nodularins are cyclic peptide hepatotoxins and tumour promoters from cyanobacteria. The present study describes the development, validation and practical application of a fully automated analytical method based on on-line micro solid-phase extraction-capillary liquid chromatography-tandem mass spectrometry for the simultaneous determination of seven microcystins and nodularin-R in tap water and lake water. Aliquots of just 100 μL of water samples are sufficient for the detection and quantification of all eight toxins. Selected reaction monitoring was used to obtain the highest sensitivity. Good linear calibrations were obtained for microcystins (50-2000ng/L) and nodularin-R (25-1000 ng/L) in spiked tap water and lake water samples. Excellent interday and intraday repeatability were achieved for eight toxins with relative standard deviation less than 15.7% in three different concentrations. Acceptable recoveries were achieved in the three concentrations with both tap water matrix and lake water matrix and no significant matrix effect was found in tap water and lake water except for microcystin-RR. The limits of detection (signal to noise ratio=3) of toxins were lower than 56.6 ng/L which is far below the 1 μg/L defined by the World Health Organization provisional guideline for microcystin-LR. Finally, this method was successfully applied to lake water samples from Tai lake and proved to be useful for water quality monitoring.

Recent development of ionic liquid stationary phases for liquid chromatography.

Based on their particular physicochemical characteristics, ionic liquids have been widely applied in many fields of analytical chemistry. Many types of ionic liquids were immobilized on a support like silica or monolith as stationary phases for liquid chromatography. Moreover, different approaches were developed to bond covalently ionic liquids onto the supporting materials. The obtained ionic liquid stationary phases show multi-mode mechanism including hydrophobic, hydrophilic, hydrogen bond, anion exchange, π-π, and dipole-dipole interactions. Therefore, they could be used in different chromatographic modes including ion-exchange, RPLC, NPLC and HILIC to separate various classes of compounds. This review mainly summarizes the immobilized patterns and types of ionic liquid stationary phases, their retention mechanisms and applications in the recent five years.