Immediate stabilization of human blood for delayed quantification of endogenous thiols and disulfides

Publication date: Available online 8 February 2016
Source:Journal of Chromatography B
Author(s): Daniela Giustarini, Federico Galvagni, Maurizio Orlandini, Paolo Fanti, Ranieri Rossi
Endogenous thiols undergo rapid and reversible oxidation to disulfides when exposed to oxidants and are, therefore, suitable biomarkers of oxidative stress. However, accurate analysis of thiols in blood is frequently compromised by their artifactual oxidation during sample manipulation, which spuriously elevates the disulfide levels. Here, we describe a validated pre-analytical procedure that prevents both artifactual oxidation of thiols during sample manipulation and their oxidative decay for months in biosamples that are stored at −80°C. Addition of N-ethylmaleimide to blood samples from healthy donors was used to stabilize whole blood, red blood cells, platelets and plasma disulfides, whereas addition of citrate buffer followed by dilution of plasma with H2O was used to stabilize plasma thiols. The concentrations of thiols and disulfides were stable in all biosamples for at least 6 months when analyzed by UV/Vis HPLC at regular intervals. Only 3ml of blood were needed to perform the analyses of thiols and disulfides in the different blood fractions. This pre-analytical procedure is reliable for use in both animal and human prospective studies. Its ease of implementation makes the method suitable for application to multicenter studies where blood samples are collected by different sites and personnel and are shipped to specific specialized laboratories.



Discovery of active components in herbs using chromatographic separation coupled with online bioassay

Publication date: Available online 8 February 2016 Source:Journal of Chromatography B Author(s): Li De-qiang, Jing Zhao, Dong Wu, Li Shao-ping Discovery of bioactive compounds from complex mixtures is a challenge. In past decad…



Metabolomics approach to explore the effects of Kai-Xin-San on Alzheimer’s disease using UPLC/ESI-Q-TOF mass spectrometry

Publication date: Available online 8 February 2016
Source:Journal of Chromatography B
Author(s): Hang Chu, Aihua Zhang, Ying Han, Shengwen Lu, Ling Kong, Jinwei Han, Zhidong Liu, Hui Sun, Xijun Wang
Alzheimer’s disease (AD) is a multifactorial neurodegenerative disease that influences elderly populations, with no effective method for its treatment so far. To improve its diagnosis and treatment, changes of small molecule metabolite during AD should be elucidated. Kai-Xin-San (KXS) is an herbal formulae that has been widely used to treat mental disorders, especially amnesia and depression in China. Experimental AD was induced in rats by an intraperitoneal injection of D-galactose (D-gal) and administered intragastrically with aluminum chloride (AlCl3) simultaneously for 105 days. Morris water maze task as a behavior test was used for testing the effects of KXS on AD model and pathological changes to the brain were assessed by hematoxylin-eosin staining and immunohistochemistry. The levels of Bcl-2 and ChAT in hippocampus were evaluated by western-blot. Furthermore, metabolite profiling of AD was performed through ultra-performance liquid chromatography/electrospray ionization quadruple time-of- flight-high-definition mass spectrometry (UPLC/ESI-Q-TOF/HDMS) combined with pattern recognition approaches and pathway analysis. D-gal and AlCl3-treated caused a decline in spatial learning and memory, hippocampal histopathological abnormalities and increased Aβ1-40 levels in the brain cortex and hippocampus along with decreased Bcl-2 and ChAT expression in the hippocampus. KXS significantly improved the cognitive impairment induced by D-gal and AlCl3, attenuated hippocampal histopathological abnormalities, reduced Aβ1-40 levels and increased Bcl-2 and ChAT expression in the hippocampus. A total of 48 metabolites were considered as potential biomarkers of AD, and 36 metabolites may correlate with the regulation of KXS treatment on AD. Changes in AD metabolic profiling were close to normal states through regulating multiple perturbed pathways after KXS treatment. This study has revealed the potential biomarkers and metabolic networks of AD, illuminated the biochemistry mechanism of AD and the metabolic pathways influenced by KXS.



ROLE OF DIMETHYL FUMARATE IN OXIDATIVE STRESS OF MULTIPLE SCLEROSIS: A Review

Publication date: Available online 8 February 2016 Source:Journal of Chromatography B Author(s): Suneetha A., Raja Rajeswari K. Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS affecting both white and grey matt…



Quantitative determination of nine urinary metabolites of organophosphate flame retardants using solid phase extraction and ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS)

Publication date: 1 March 2016
Source:Journal of Chromatography B, Volume 1014
Author(s): Ivana Kosarac, Cariton Kubwabo, Warren G. Foster
Over the last few years, the use of organophosphate flame retardants (OPFRs) has been on the rise; however, there are knowledge gaps in both the human health effects of OPFRs and levels of human exposure. Currently, human biomonitoring data on the levels of OPFR metabolites in the Canadian population are still non-existent. Herein we describe a novel method to measure nine urinary OPFR metabolites using solid phase extraction and ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). The method detection limits were between 0.08 and 0.25ng/mL for target metabolites. The newly developed and validated method was applied to the analysis of 24 urine samples collected in 2010–12 from pregnant Canadian women. The most frequently detected OPFR metabolite in urine of study participants (detection frequency: 97%) was diphenyl phosphate (DPHP), with concentrations ranging between <0.13–25.2ng/mL, followed (75%) by the sum of two metabolites (DoCP: Di-o-cresyl phosphate and DpCP: Di-p- cresyl phosphate) of tricresyl phosphate, with concentrations between <0.13–4.38ng/mL. With the exception of desbutyl-tris-(2-butoxy-ethyl) phosphate which was not detected in any of the samples, all other OPFR metabolites measured were found among study participants with variable detection frequency, suggesting pregnant Canadian women may be exposed to OPFRs.



Rapid separation of polysaccharides using a novel spiral coil column by high-speed countercurrent chromatography

The separation of polysaccharides is time-consuming. We developed and optimized a type-J counter-current chromatography system with a novel tri-rotor spiral coil column for the rapid separation of polysaccharide. The optimal composition of an aqueous …



Capillary electrophoresis combined in-line with solid-phase extraction using magnetic particles as new adsorbents for the determination of drugs of abuse in human urine

A simple approach is presented based on the in-line coupling between magnetic particles-based SPE and CE. Silica-coated iron oxide particles functionalized with C18 were successfully synthesized and used as a reverse-phase sorbent for in-line SPE-CE. …



Capillary electrophoresis combined in-line with solid-phase extraction using magnetic particles as new adsorbents for the determination of drugs of abuse in human urine

A simple approach is presented based on the in-line coupling between magnetic particles-based SPE and CE. Silica-coated iron oxide particles functionalized with C18 were successfully synthesized and used as a reverse-phase sorbent for in-line SPE-CE. …



Systematically characterize the metabolites of echinacoside and acteoside from Cistanche tubulosa in rat plasma, bile, urine and feces based on UPLC-ESI-Q-TOF-MS

Echinacoside(ECH) and acteoside(ACT), as the most and major active components of Cistanche tubulosa were reported to possess cardioactive, neuroprotective, hepatocyte protective effects, as well as antibacterial, antioxidative effects. In recent years, more and more study involved in it focused on pharmacological activities. However, their metabolic profile in vivo have not been sufficiently investigated. This study proposed an approach for rapidly identifying the complicated and unpredictable metabolites of ECH and ACT in rat plasma, bile, urine and feces, and systematically and comprehensively revealing major metabolic pathways of them, based on the powerful ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF MS). Plasma, bile, urine and feces were collected from rats after a single 200 mg/kg oral dose of them. A total of 49 metabolites were detected in rat biological samples. Through analyzing metabolites in bile samples, it found that ECH and ACT were subjected to a marked hepatic first-pass effect in liver. This article is protected by copyright. All rights reserved.



Urinary metabolite profiling provides potential differentiation to explore the mechanisms of adjuvant-induced arthritis in rats

To explore the pathogenesis of rheumatoid arthritis (RA) from the perspective of metabolomics, gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) technology was used to observe changes in the metabolic profiles of urine output from rats with adjuvant-induced arthritis (AA). Spague-Dawley (SD) rats were randomly divided into control group (NC) and experimental group, with 8 in each. Rats in experimental group were induced by intracutaneous innoculation of 0.1mL Freund’s complete adjuvant (FCA) to right paws. On day 20 after immunization, the metabolic profiles between rat control and experimental groups were compared by combining GC-TOF/MS technology with multivariate statistical approaches, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal projections to latent structures-discriminant analysis (OPLS-DA). Nine potential biomarkers were identified, including 2,2-dimethylsuccinic acid, tartronic acid, dehydroshikimic acid, hippuric acid, adenine, phenaceturic acid, L-dopa, 1,4-dihydroxy-2-naphthoic acid and melibiose. The findings indicate that the rats with AA are disturbed in metabolism of purine, amino acid, fat and energy. This study also demonstrates that the dysfunction in a range of biosynthetic and catabolic pathways, which leads to increased oxygen free radicals and inflammation, could cause underlying pathogenesis of RA. This article is protected by copyright. All rights reserved.



Plasma metabonomics study on toxicity biomarker in rats treated with Euphorbia fischeriana based on LC-MS

Lang-du (LD) has been traditionally used to treat human diseases in China. Plasma metabolic profiling was applied in this study based on LC-MS to elucidate the toxicity in rats induced by injected ethanol extract of LD. LD injection was given intraperitoneal injection at the doses of 0.1, 0.05, 0.025 and 0 g·kg-1 body weight per day to rats. The blood biochemical levels of ALT, DBIL, CRE, BMG and LDL increased in LD-injected rats, and the levels of TP and ALB decreased in these groups. The metabolic profiles of the samples were analyzed by multivariate statistics analysis, including PCA, PLS-DA and OPLS-DA. The metabolic characters in rats injected with LD were perturbed in a dose-dependent manner. By OPLS-DA, 18 metabolites were served as the potential toxicity biomarkers. Moreover, LD treatment resulted in an increase in the p-Cresol, p-Cresol sulfate, LPE (18:0), LPE (16:0), LPC (16:0) and 12-HETE concentrations, and a decrease in hippuric acid, cholic acid and N-Acetyl-L-phenylalanine. These results suggested that chronic exposure to LD could cause a disturbance in lipids metabolism and amino acids metabolism, etc. Therefore, an analysis of the metabolic profiles can contribute to the better understanding of the adverse effects of LD. This article is protected by copyright. All rights reserved.



Analytical Strategies for the Determination of Norepinephrine Reuptake Inhibitors in Pharmaceutical Formulations and Biological Fluids

Volume 46, Issue 1, January 2016, pages 40-66<br/>10.1080/10408347.2014.948679<br/>Cafer Saka



Current Applications of Chromatographic Methods in the Study of Human Body Fluids for Diagnosing Disorders

Volume 46, Issue 1, January 2016, pages 1-14<br/>10.1080/10408347.2014.929487<br/>Jagoda Jóźwik



An NGS-Independent Strategy for Proteome-Wide Identification of Single Amino Acid Polymorphisms by Mass Spectrometry

Analytical ChemistryDOI: 10.1021/acs.analchem.5b04417



Sequential Collision- and Ozone-Induced Dissociation Enables Assignment of Relative Acyl Chain Position in Triacylglycerols

Analytical ChemistryDOI: 10.1021/acs.analchem.5b04001



Metabolomics study on the antitumor effect of marine natural compound flexibilide in HCT-116 colon cancer cell line

Publication date: 1 March 2016 Source:Journal of Chromatography B, Volume 1014 Author(s): Dan Gao, Yini Wang, Weiyi Xie, Ti Yang, Yuyang Jiang, Yuewei Guo, Jin Guan, Hongxia Liu A marine natural compound flexibilide iso…



Determination of azithromycin residue in pork using a molecularly imprinted monolithic microcolumn coupled to liquid chromatography with tandem mass spectrometry

Using spiramycin as a dummy template, a molecularly imprinted polymer monolithic micro-column with high selection to azithromycin was prepared in a micropipette tip. The imprinting factor of the monolithic micro-column prepared was ca. 2.67 and the mo…



Comparative analysis of Cu (I)-catalyzed alkyne-azide cycloaddition (CuAAC) and strain-promoted alkyne-azide cycloaddition (SPAAC) in O-GlcNAc proteomics

O-linked β-N-acetylglucosamine (O-GlcNAc) is emerging as an essential protein posttranslational modification in a range of organisms. It is involved in various cellular processes such as nutrient sensing, protein degradation, gene expression and is associated with many human diseases. Despite its importance, identifying O-GlcNAcylated proteins is a major challenge in proteomics. Here, using peracetylated N-azidoacetylglucosamine (Ac4GlcNAz) as a bioorthogonal chemical handle, we described a gel-based mass spectrometry method for the identification of proteins with O-GlcNAc modification in A549 cells. In addition, we made a labeling efficiency comparison between two modes of azide-alkyne biothogonal reactions in click chemistry: copper-catalyzed azide-alkyne cycloaddition (CuAAC) with Biotin-Diazo-Alkyne and stain-promoted azide-alkyne cycloaddition (SPAAC) with Biotin-DIBO-Alkyne. After conjugation with click chemistry in vitro and enrichment via streptavidin resin, proteins with O-GlcNAc modification were separated by SDS-PAGE and identified with mass spectrometry. Proteomics data analysis revealed that 229 putative O-GlcNAc modified proteins were identified with Biotin-Diazo-Alkyne conjugated sample and 188 proteins with Biotin-DIBO-Alkyne conjugated sample, among which 114 proteins were overlapping. Interestingly, 74 proteins identified from Biotin-Diazo-Alkyne conjugates and 46 verified proteins from Biotin-DIBO-Alkyne conjugates could be found in the O-GlcNAc modified proteins database dbOGAP (http://cbsb.lombardi.georgetown.edu/hulab/OGAP.html). These results suggested that CuAAC with Biotin-Diazo-Alkyne represented a more powerful method in proteomics with higher protein identification and better accuracy compared to SPAAC. The proteomics credibility was also confirmed by the molecular function and cell component gene ontology (GO). Together, the method we reported here combining metabolic labeling, click chemistry, affinity-based enrichment, SDS-PAGE separation and mass spectrometry, would be adaptable for other post-translationally modified proteins in proteomics.
This article is protected by copyright. All rights reserved



Rapid N-glycan release from glycoproteins using immobilized PNGase F microcolumns

Publication date: Available online 6 February 2016 Source:Journal of Chromatography B Author(s): Marton Szigeti, Judit Bondar, Douglas Gjerde, Zsolt Keresztessy, Akos Szekrenyes, Andras Guttman N-glycosylation profiling of …



Enantioseparation and determination of isofenphos-methyl enantiomers in wheat, corn, peanut and soil with Supercritical fluid chromatography/tandem mass spectrometric method

Publication date: Available online 6 February 2016
Source:Journal of Chromatography B
Author(s): Xixi Chen, Fengshou Dong, Jun Xu, Xingang Liu, Zenglong chen, Na liu, Yongquan Zheng
Supercritical fluid chromatography/tandem mass spectrometry (SFC-MS/MS) is an effective tool in separation science which uses the nontoxic CO2 fluid for better control of analyte retention. Also the technology of a postcolumn additive to complement MS/MS ensure the high-selectivity determination. In this paper, a green and sensitive analytical method was developed for the enantioselective separation and determination of isofenphos-methyl enantiomers in foodstuff and soil by SFC-MS/MS. The enantioseparation was performed within 3.50min using Chiralpak IA-3 column with CO2/isopropanol (90:10, v/v) as the mobile phase at 2.2mL/min flow rate. The postcolumn compensation technology provided with 0.1% formic acid/methanol greatly improved the ionization efficiency of mass spectrometry. Column temperature, auto back pressure regulator pressure, and flow rate of compensation solvent were optimized to 30°C, 2200psi, and 0.1mL/min, respectively. The QuEChERs method was adopted in this study, which mean recoveries of isofenphos-methyl enantiomers ranged from 75.7% to 111.4%, with relative standard deviations less than 11.3% at three concentration levels in all matrices. The limits of detection for both enantiomers varied from 0.02μg/kg to 0.15μg/kg, while the limit of quantification did not exceed 0.50μg/kg. The proposed method was then successfully applied to analyze authentic samples, confirming that it was a versatile strategy for the analysis of isofenphos-methyl enantiomers in food and environmental matrices.