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1.
Anal Chim Acta ; 1178: 338551, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34482862

RESUMO

Single-cell analysis can allow for an in-depth understanding of diseases, diagnostics, and aid the development of therapeutics. However, single-cell analysis is challenging, as samples are both extremely limited in size and complex. But the concept is gaining promise, much due to novel sample preparation approaches and the ever-improving field of mass spectrometry. The mass spectrometer's output is often linked to the preceding compound separation step, typically being liquid chromatography (LC). In this review, we focus on LC's role in single-cell omics. Particle-packed nano LC columns (typically 50-100 µm inner diameter) have traditionally been the tool of choice for limited samples, and are also used for single cells. Several commercial products and systems are emerging with single cells in mind, featuring particle-packed columns or miniaturized pillar array systems. In addition, columns with inner diameters as narrow as 2 µm are being explored to maximize sensitivity. Hence, LC column down-scaling is a key focus in single-cell analysis. But narrow columns are associated with considerable technical challenges, while single cell analysis may be expected to become a "routine" service, requiring higher degrees of robustness and throughput. These challenges and expectations will increase the need and attention for the development (and even the reinvention) of alternative nano LC column formats. Therefore, monolith columns and even open tubular columns may finally find their "killer-application" in single cell analysis.


Assuntos
Análise de Célula Única , Cromatografia Líquida , Espectrometria de Massas
2.
Metabolites ; 11(8)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34436480

RESUMO

Lipid mediators, small molecules involved in regulating inflammation and its resolution, are a class of lipids of wide interest as their levels in blood and tissues may be used to monitor health and disease states or the effect of new treatments. These molecules are present at low levels in biological samples, and an enrichment step is often needed for their detection. We describe a rapid and selective method that uses new low-cost molecularly imprinted (MIP) and non-imprinted (NIP) polymeric sorbents for the extraction of lipid mediators from plasma and tissue samples. The extraction process was carried out in solid-phase extraction (SPE) cartridges, manually packed with the sorbents. After extraction, lipid mediators were quantified by liquid chromatography-tandem mass spectrometry (LC-MSMS). Various parameters affecting the extraction efficiency were evaluated to achieve optimal recovery and to reduce non-specific interactions. Preliminary tests showed that MIPs, designed using the prostaglandin biosynthetic precursor arachidonic acid, could effectively enrich prostaglandins and structurally related molecules. However, for other lipid mediators, MIP and NIP displayed comparable recoveries. Under optimized conditions, the recoveries of synthetic standards ranged from 62% to 100%. This new extraction method was applied to the determination of the lipid mediators concentration in human plasma and mouse tissues and compared to other methods based on commercially available cartridges. In general, the methods showed comparable performances. In terms of structural specificity, our newly synthesized materials accomplished better retention of prostaglandins (PGs), hydroxydocosahexaenoic acid (HDoHE), HEPE, hydroxyeicosatetraenoic acids (HETE), hydroxyeicosatrienoic acid (HETrE), and polyunsaturated fatty acid (PUFA) compounds, while the commercially available Strata-X showed a higher recovery for dihydroxyeicosatetraenoic acid (diHETrEs). In summary, our results suggest that this new material can be successfully implemented for the extraction of lipid mediators from biological samples.

3.
Anal Chem ; 93(7): 3576-3585, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33534551

RESUMO

Liver organoids are emerging tools for precision drug development and toxicity screening. We demonstrate that electromembrane extraction (EME) based on electrophoresis across an oil membrane is suited for segregating selected organoid-derived drug metabolites prior to mass spectrometry (MS)-based measurements. EME allowed drugs and drug metabolites to be separated from cell medium components (albumin, etc.) that could interfere with subsequent measurements. Multiwell EME (parallel-EME) holding 100 µL solutions allowed for simple and repeatable monitoring of heroin phase I metabolism kinetics. Organoid parallel-EME extracts were compatible with ultrahigh-performance liquid chromatography (UHPLC) used to separate the analytes prior to detection. Taken together, liver organoids are well-matched with EME followed by MS-based measurements.


Assuntos
Organoides , Preparações Farmacêuticas , Fígado , Espectrometria de Massas , Membranas Artificiais
4.
Sci Rep ; 11(1): 273, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431985

RESUMO

The eye lens is a unique organ as no cells can be replaced throughout life. This makes it decisive that the lens is protected against damaging UV-radiation. An ultraviolet (UV)-absorbing compound of unknown identity is present in the aqueous humor of geese (wild and domestic) and other birds flying at high altitudes. A goose aqueous humor extract, that was believed to contain the UV protective compound which was designated as "compound X", was fractionated and examined using a variety of spectroscopic techniques including LC-MS and high field one- and two dimensional-NMR methods. A series of compounds were identified but none of them appeared to be the UV protective "compound X". It may be that the level of the UV protective compound in goose aqueous humor is much less than the compounds identified in our investigation, or it may have been degraded by the isolation and chromatographic purification protocols used in our investigations.


Assuntos
Aves , Olho/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Animais , Humor Aquoso/metabolismo , Ácido Ascórbico/metabolismo , Aves/metabolismo , Olho/metabolismo , Voo Animal
5.
Artigo em Inglês | MEDLINE | ID: mdl-32971370

RESUMO

3', 5' - Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that is involved in many cellular functions and biological processes. In several cell types, cholera toxin will increase the level of cAMP, which mediates toxic effects on cells. In this context, we have developed a fast and simple method based on extraction with 5% trichloroacetic acid (TCA) and quantitation with liquid chromatography-mass tandem spectrometry (LC-MS/MS) for measuring cAMP in cells. A main feature of the LC-MS method was employing a reversed phase C18 column (2.1 mm × 50 mm, 1.6 µm particles) compatible with a 100% aqueous mobile phase, providing retention of the highly polar analyte. Isocratic separations allowed for fast subsequent injections. Negative mode electrospray ionization detection was performed with a triple quadrupole (QqQ)MS. cAMP was extracted from cell samples (~106 cells per well) and spiked with a labelled internal standard, using 200 µL of 5% TCA. The extraction solvent was fully compatible for direct injection onto the reversed phase column. After 10 min incubation, the supernatant was removed, and 10 µL of the supernatant was directly analysed by LC-MS. The method was characterized by the simplicity of the extraction, and the speed (3 min retention time of cAMP), sensitivity (250 pg/mL detection limit), and selectivity (separation from interferences e.g. isomeric compounds) of the LC-MS method, and could be used for quantitation of cAMP in the range 1-500 ng/mL cell extract.


Assuntos
Cromatografia de Fase Reversa/métodos , AMP Cíclico/análise , AMP Cíclico/metabolismo , Técnicas Citológicas/métodos , Espectrometria de Massas em Tandem/métodos , Brefeldina A , Toxina da Cólera , Células HT29 , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes
6.
Front Chem ; 7: 835, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31850321

RESUMO

Prior to mass spectrometry, on-line sample preparation can be beneficial to reduce manual steps, increase speed, and enable analysis of limited sample amounts. For example, bottom-up proteomics sample preparation and analysis can be accelerated by digesting proteins to peptides in an on-line enzyme reactor. We here focus on low-backpressure 100 µm inner diameter (ID) × 160 mm, 180 µm ID × 110 mm or 250 µm ID × 140 mm vinyl azlactone-co-ethylene dimethacrylate [poly(VDM-co-EDMA)] monoliths as supports for immobilizing of additional molecules (i.e., proteases or drugs), as the monolith was expected to have few unspecific interactions. For on-line protein digestion, monolith supports immobilized with trypsin enzyme were found to be suited, featuring the expected characteristics of the material, i.e., low backpressure and low carry-over. Serving as a functionalized sample loop, the monolith units were very simple to connect on-line with liquid chromatography. However, for on-line target deconvolution, the monolithic support immobilized with a Wnt pathway inhibitor was associated with numerous secondary interactions when exploring the possibility of selectively trapping target proteins by drug-target interactions. Our initial observations suggest that (poly(VDM-co-EDMA)) monoliths are promising for e.g., on-line bottom-up proteomics, but not a "fit-for-all" material. We also discuss issues related to the repeatability of monolith-preparations.

7.
Analyst ; 144(24): 7090-7104, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31728480

RESUMO

Nano liquid chromatography (nanoLC), with columns having an inner diameter (ID) of ≤100 µm, can provide enhanced sensitivity and enable analysis of limited samples. NanoLC has become an established tool in omics research, and is gaining ground in other applications as well. There are several variants and formats of nanoLC columns, including packed columns, monoliths, open tubular columns, and the pillar array format. Most applications are done with packed columns, while e.g. the monolith and open tubular columns are still less established as routine tools. The pillar array format is a new variant with excellent resolution and low backpressure, and has recently been commercialized and used for bio-applications. In this minireview, we summarize and discuss recent research on nanoLC column development and uses, focusing on literature between 2016 and medio 2019.

8.
J Proteome Res ; 18(5): 2012-2020, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30964684

RESUMO

Glioblastoma is the most common and malignant brain tumor, and current therapies confer only modest survival benefits. A major obstacle is our ability to monitor treatment effect on tumors. Current imaging modalities are ambiguous, and repeated biopsies are not encouraged. To scout for markers of treatment response, we used NMR spectroscopy to study the effects of a survivin inhibitor on the metabolome of primary glioblastoma cancer stem cells. Applying high resolution NMR spectroscopy (1H resonance frequency: 800.03 MHz) to just 3 million cells per sample, we achieved sensitive and high resolving determinations of, e.g., amino acids, nucleosides, and constituents of the citric acid cycle. For control samples that were cultured, prepared, and measured at varying dates, peak area relative standard deviations were 15-20%. Analyses of unfractionated lysates were performed for straightforward compound identification with COLMAR and HMDB databases. Principal component analysis revealed that citrate levels were clearly upregulated in nonresponsive cells, while lactate levels substantially decreased following treatment for both responsive and nonresponsive cells. Hence, lactate and citrate may be potential markers of successful drug uptake and poor response to survivin inhibitors, respectively. Our metabolomics approach provided alternative biomarker candidates compared to spectrometry-based proteomics, underlining benefits of complementary methodologies. These initial findings make a foundation for exploring in vivo MR spectroscopy (MRS) of brain tumors, as citrate and lactate are MRS-visible. In sum, NMR metabolomics is a tool for addressing glioblastoma.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Ácido Cítrico/metabolismo , Glioblastoma/tratamento farmacológico , Imidazóis/uso terapêutico , Ácido Láctico/metabolismo , Metaboloma , Naftoquinonas/uso terapêutico , Biomarcadores Farmacológicos/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Sobrevivência Celular/efeitos dos fármacos , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ciclo do Ácido Cítrico/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Espectroscopia de Ressonância Magnética , Terapia de Alvo Molecular/métodos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Cultura Primária de Células , Análise de Componente Principal , Survivina/antagonistas & inibidores , Survivina/genética , Survivina/metabolismo
9.
J Steroid Biochem Mol Biol ; 192: 105309, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30779932

RESUMO

Oxysterols can contribute to proliferation of breast cancer through activation of the Estrogen Receptors, and to metastasis through activation of the Liver X Receptors. Endogenous levels of both esterified and free sidechain-hydroxylated oxysterols were examined in breast cancer tumours from Estrogen Receptor positive and negative breast tumours, using a novel fast liquid chromatography tandem mass spectrometry method. Multiple aliquots of five milligram samples of 22 tumours were analysed for oxysterol content to assess intra- and inter-tumour variation. Derivatization was performed with Girard T reagent (with and without alkaline hydrolysis) and sample clean-up was performed using a robust automatic on-line column switching system ("AFFL"). Oxysterols were separated isocratically on a 2.1 mm inner diameter column packed with ACE SuperPhenylHexyl core shell particles using a mobile phase consisting of 0.1% formic acid in H2O/methanol/acetonitrile (57/10/33, v/v/v) followed by a wash out step (0.1% formic acid in methanol/acetonitrile, 50/50, v/v). The total analysis time, including sample clean-up and column reconditioning, was 8 min (80% time reduction compared to other on-line systems). Analysis revealed large intra-tumour variations of sidechain oxysterols, resulting in no significant differences in endogenous oxysterols levels between Estrogen Receptor positive and Estrogen Receptor negative breast cancers. However, a correlation between esterified and free 27-hydroxycholesterol was observed. The same correlation was not observed for 24S-hydroxycholesterol or 25-hydroxycholesterol. The oxysterol heterogeneity of tumour tissue is a critical factor when assessing the role of these lipids in cancer.


Assuntos
Neoplasias da Mama/metabolismo , Cromatografia Líquida/métodos , Oxisteróis/análise , Oxisteróis/química , Espectrometria de Massas em Tandem/métodos , Neoplasias da Mama/patologia , Feminino , Humanos , Hidroxicolesteróis/metabolismo
10.
Future Sci OA ; 5(1): FSO359, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30652024

RESUMO

Aim: For isolation of exosomes, differential ultracentrifugation and an isolation kit from a major vendor were compared. Materials & methods: 'Case study' exosomes isolated from patient-derived cells from glioblastoma multiforme and a breast cancer cell line were analyzed. Results: Transmission electron microscopy, dynamic light scattering, western blotting, and so forth, revealed comparable performance. Potential protein biomarkers for both diseases were also identified in the isolates using nanoLC-MS. Western blotting and nanoLC-MS also revealed negative exosome markers regarding both isolation approaches. Conclusion: The two isolation methods had an overall similar performance, but we hesitate to use the term 'exosome isolation' as impurities may be present with both isolation methods. NanoLC-MS can detect disease biomarkers in exosomes and is useful for critical assessment of exosome enrichment procedures.

11.
Sci Total Environ ; 655: 1420-1426, 2019 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-30577133

RESUMO

Dried blood spot (DBS) sampling has gained attention in several scientific areas because of the low sampling burden. The study aimed to develop a method for the determination of poly- and perfluoroalkyl substances (PFASs) in DBS using a standardized blood volume. The DBS method using a simple methanol extraction followed by online solid phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry quantification was validated. Only 30 µL of blood is required. Based on the measurements of DBS dispersed areas from known blood volumes (20-70 µL), the blood volume on a 3 mm diameter DBS subsample was calculated to be 3.3 µL (median, n = 708 measurements, 59 adults). Strong correlations of PFAS concentrations between finger prick DBSs and venous whole blood samples (n = 57) were found (rho 0.72-0.97, p < 0.0001). Also, Passing-Bablok regressions and Bland-Altman plots demonstrated good agreements of PFAS concentrations in finger prick DBSs and venous whole blood samples. This finding indicates that the DBS method was satisfactory, and allows straightforward analysis of PFASs in DBS without hematocrit correction. This DBS method is reliable for accurate determination of PFASs and has a high potential for use of self-collected DBS in large-scale biomonitoring studies as well as for archived DBS samples.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Monitoramento Ambiental/métodos , Poluentes Ambientais/análise , Fluorcarbonetos/análise , Adulto , Cromatografia Líquida de Alta Pressão/métodos , Teste em Amostras de Sangue Seco/instrumentação , Monitoramento Ambiental/instrumentação , Humanos , Pessoa de Meia-Idade , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos
12.
Anal Chem ; 90(23): 13860-13866, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30384595

RESUMO

An online microfluidics-mass spectrometry platform was developed for determining proteotypic peptides from in-solution digested samples. Accelerated and selective sample cleanup was achieved by integrating proteotypic epitope peptide immunoextraction with nano liquid chromatography-tandem mass spectrometry (online IE-nanoLC-MS/MS). Ten individually prepared 180 µm inner diameter capillaries with ethylene glycol dimethacrylate- co-vinyl azlactone (EDMA- co-VDM) monoliths were immobilized with anti-protein antibodies that are used in routine immunoassays of the intact small cell lung cancer biomarker ProGRP. The resulting AB columns provided linearity correlation coefficients of 0.96-0.99 for protein amounts and concentrations of 10 pg to 5 ng and 0.5-250 ng/mL, respectively. The columns/platform gave relative peak area RSDs below 15%. The IE-nanoLC-MS/MS platform provided a limit of detection (LOD) of 520 pg/mL of ProGRP in human serum. The approach was applicable for other matrixes and proteins, i.e., primary glioblastoma cells and endogenous αV integrin chain. Thus, EDMA- co-VDM monoliths immobilized with antibodies are suited for automated peptide capture in microfluidic formats.


Assuntos
Acrilatos/química , Anticorpos/química , Anticorpos/imunologia , Biomarcadores Tumorais/sangue , Neoplasias Pulmonares/química , Nanotecnologia , Fragmentos de Peptídeos/sangue , Carcinoma de Pequenas Células do Pulmão/química , Acrilatos/imunologia , Biomarcadores Tumorais/imunologia , Cromatografia Líquida , Humanos , Imunoensaio , Neoplasias Pulmonares/sangue , Técnicas Analíticas Microfluídicas , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes/sangue , Proteínas Recombinantes/imunologia , Carcinoma de Pequenas Células do Pulmão/sangue , Espectrometria de Massas em Tandem
13.
J Chromatogr A ; 1534: 195-200, 2018 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-29290401

RESUMO

Open tubular liquid chromatography columns with organic polymer layers can be powerful tools for high sensitivity measurements in e.g. proteomics. However, these narrow columns are challenging to characterize. A two-electrode system, often used for bioimpendance measurements, was used to study poly(styrene-co-divinylbenzene = PS-DVB) polymer layered open tubular (PLOT) liquid chromatography columns with 10 µm inner diameters. The system performed electrical resistance measurements (ERM) for assessing layer thickness and porosity. Layer determination results were comparable (but more precise) to that obtained with scanning electron microscopy (SEM). Porosity examinations with ERM casted doubt on the presence/availability of pores in the layers investigated.


Assuntos
Cromatografia Líquida/métodos , Eletrodos , Microscopia Eletrônica de Varredura , Poliestirenos/química , Porosidade , Proteômica
14.
J Chromatogr A ; 1518: 104-110, 2017 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-28882340

RESUMO

An open tubular (OT) sample preparation/separation platform was developed. A multi-channel polymer layer open tubular (mPLOT) solid phase extraction (SPE) column was prepared by wall-coating the 126 channels (8µm inner diameter (ID) each) of a crystal fiber capillary with an organic polymer, namely poly(styrene-co-octadecene-co-divinylbenzene) (PS-OD-DVB). The mPLOT SPE was coupled on-line with a 10µm×2m poly(styrene-co-divinylbenzene) (PS-DVB) OT liquid chromatography column with nanospray mass spectrometry (OTLC-MS). Compared to using monolithic/particle-packed SPEs, mPLOT-SPE-OTLC allowed both fast loading and sufficient refocusing on the OT analytical column of small model compounds (sulfonamides≈300Da). Using automatic filtration/filter back-flushing (AFFL) plumbing, the mPLOT SPE column gave a constant and low back-pressure ≈35bar at 0.5µL/min. Surprisingly large sample volumes (10µL) were possible to be injected using a 12cm mPLOT.


Assuntos
Técnicas de Química Analítica/instrumentação , Cromatografia Líquida , Espectrometria de Massas , Extração em Fase Sólida/instrumentação , Poliestirenos/química , Polivinil/química
15.
Front Chem ; 5: 62, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28894734

RESUMO

A rugged and high throughput capillary column (cLC) LC-MS switching platform using large volume injection and on-line automatic filtration and filter back-flush (AFFL) solid phase extraction (SPE) for analysis of environmental water samples with minimal sample preparation is presented. Although narrow columns and on-line sample preparation are used in the platform, high ruggedness is achieved e.g., injection of 100 non-filtrated water samples did not result in a pressure rise/clogging of the SPE/capillary columns (inner diameter 300 µm). In addition, satisfactory retention time stability and chromatographic resolution were also features of the system. The potential of the platform for environmental water samples was demonstrated with various pharmaceutical products, which had detection limits (LOD) in the 0.05-12.5 ng/L range. Between-day and within-day repeatability of selected analytes were <20% RSD.

16.
Anal Chem ; 89(17): 8667-8673, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28783436

RESUMO

For counterterrorism purposes, a selective nano liquid chromatography-mass spectrometry (nanoLC-MS) platform was developed for detecting the highly lethal protein ricin from castor bean extract. Manual sample preparation steps were omitted by implementing a trypsin/Lys-C enzyme-immobilized multichannel reactor (MCR) consisting of 126 channels (8 µm inner diameter in all channels) that performed online digestion of proteins (5 min reaction time, instead of 4-16 h in previous in-solution methods). Reduction and alkylation steps were not required. The MCR allowed identification of ricin by signature peptides in all targeted mode injections performed, with a complete absence of carry-over in blank injections. The MCRs (interior volume ≈ 1 µL) have very low backpressure, allowing for trivial online coupling with commercial nanoLC-MS systems. The open tubular nature of the MCRs allowed for repeatable within/between-reactor preparation and performance.


Assuntos
Terrorismo Químico/prevenção & controle , Cromatografia Líquida/métodos , Ricina/análise , Espectrometria de Massas em Tandem/métodos , Reatores Biológicos , Semente de Rícino/química , Enzimas Imobilizadas/química , Metaloendopeptidases/química , Ricina/química , Ricina/isolamento & purificação , Tripsina/química
17.
Chem Phys Lipids ; 207(Pt B): 87-91, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28583433

RESUMO

Capillary electrophoresis (CE) can provide high separation efficiency with very simple instrumentation, but has yet to be explored regarding oxysterols/cholesterol. Cholesterol and 25-hydroxycholesterol (both are 4-ene-3-ketosteroids) were quantitatively transformed into hydrazones using Girard P reagent after enzymatic oxidation by cholesterol oxidase. Separation was achieved using non-aqueous capillary electrophoresis with UV detection at 280nm; the "charge-tagging" Girard P reagent ensured both charge and chromophore (which are requirements for CE-UV). Excess reagent was also separated from the two analytes, eliminating the need for removal prior to the analysis. The compounds were separated in less than 5min with excellent separation efficiency, using separation electrolytes fully compatible with mass spectrometry. The CE-UV method was used to optimize steps for charge-tagging, revealing that the procedure is affected by the analyte/reagent ratio and reaction time, but also the analyte structure.


Assuntos
Betaína/análogos & derivados , Colesterol/química , Colesterol/isolamento & purificação , Hidroxicolesteróis/química , Hidroxicolesteróis/isolamento & purificação , Betaína/química , Colesterol/metabolismo , Colesterol Oxidase/química , Colesterol Oxidase/metabolismo , Eletroforese Capilar , Hidroxicolesteróis/metabolismo , Conformação Molecular , Espectrofotometria Ultravioleta
18.
J Chromatogr A ; 1498: 111-119, 2017 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-28385266

RESUMO

Self-preparation of nano liquid chromatography (nLC) columns has advantages regarding cost and flexibility. For targeted proteomics, we evaluated several approaches for particle-packing nLC columns and manufacturing fritless silica-based monolithic trap columns (50µm inner diameter). Our preferred approach for nLC column preparation was to magnetically stir Accucore core shell particles (C18 stationary phase) in ACN/water (80/20, v/v) suspensions during pressure-driven filling of polymer-fritted standard fused silica capillaries. The columns were ready for use about one hour after preparation had begun. They had comparable peak capacities (peptides) to commercial columns, and satisfactory within/between-column retention time repeatability, suited for targeted proteomics. Packing with commercial capillary housings/nanospray emitters did not improve performance compared to packing with in-house fritted stock fused silica capillary tubing. For trap columns, several recipes for narrow bore silica-based monolithic columns were evaluated, and we found the recipe by Zou et al. (2005) to be reproducible. Compared to the standard C18 trap column for Accucore nLC columns, monolith trap columns (C8 stationary phase) significantly reduced peak widths. The readily prepared in-house columns were used for targeted detection of the enzyme CYP27A1 in cancer cells, which is associated with proliferation and metastasis of breast cancer.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Nanopartículas/química , Peptídeos/análise , Proteômica/métodos , Dióxido de Silício/química , Linhagem Celular Tumoral , Colestanotriol 26-Mono-Oxigenase/análise , Colestanotriol 26-Mono-Oxigenase/isolamento & purificação , Humanos , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Peptídeos/isolamento & purificação , Pressão
19.
Anal Chim Acta ; 957: 10-19, 2017 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-28107829

RESUMO

A rapid, sensitive and reliable method was developed for the determination of a broad range of poly- and perfluoroalkyl substances (PFASs) in various blood matrices (serum, plasma, and whole blood), and uses only 50 µL of sample material. The method consists of a rapid protein precipitation by methanol followed by high throughput online solid phase extraction (SPE), ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), and negative electrospray ionization detection. The method was developed for simultaneous determination of twenty-five PFASs, including polyfluoroalkyl phosphate esters (PAPs; 6:2, 8:2, 6:2/6:2, and 8:2/8:2), perfluoroalkyl phosphonates (PFPAs; C6, C8, and C10), perfluoroalkyl sulfonates (PFSAs; C4, C6, C7, C8, and C10), perfluoroalkyl carboxylates (PFCAs; C5C14), and perfluoroalkyl sulfonamides (FOSAs; C8, N-methyl, and N-ethyl). High linearity of matrix-matched calibration standards (correlation coefficients, R = 0.99-0.999) were obtained in the range of 0.006-45 ng mL-1 blood. Excellent sensitivity was achieved with method detection limits (MDLs) between 0.0018 and 0.09 ng mL-1, depending on the compound and matrix. The method was validated for serum, plasma, and whole blood (n = 5 + 5) at six levels in the range 0.0180-30 ng mL-1. The accuracy (n = 5) was on average 102± 12%. The intermediate precision (n = 10) ranged from 2 to 40% with an average between-batch of analyses difference of 10± 10%. Two human serum samples from a former interlaboratory comparison were analyzed and the differences between the applied method and the consensus values were below ≤22% (n = 5). The method was also successfully applied to samples of human plasma and whole blood with coefficients of variation in the range 0.8-15.2% (n = 5).


Assuntos
Cromatografia Líquida de Alta Pressão , Fluorcarbonetos/análise , Organofosfonatos/análise , Extração em Fase Sólida , Espectrometria de Massas em Tandem , Ésteres , Fluorcarbonetos/sangue , Humanos , Organofosfonatos/sangue , Fosfatos , Plasma/química , Soro/química
20.
J Clin Invest ; 127(2): 709-719, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-28112678

RESUMO

BACKGROUND: Sex, emotion, and reproduction are fundamental and tightly entwined aspects of human behavior. At a population level in humans, both the desire for sexual stimulation and the desire to bond with a partner are important precursors to reproduction. However, the relationships between these processes are incompletely understood. The limbic brain system has key roles in sexual and emotional behaviors, and is a likely candidate system for the integration of behavior with the hormonal reproductive axis. We investigated the effects of kisspeptin, a recently identified key reproductive hormone, on limbic brain activity and behavior. METHODS: Using a combination of functional neuroimaging and hormonal and psychometric analyses, we compared the effects of kisspeptin versus vehicle administration in 29 healthy heterosexual young men. RESULTS: We demonstrated that kisspeptin administration enhanced limbic brain activity specifically in response to sexual and couple-bonding stimuli. Furthermore, kisspeptin's enhancement of limbic brain structures correlated with psychometric measures of reward, drive, mood, and sexual aversion, providing functional significance. In addition, kisspeptin administration attenuated negative mood. CONCLUSIONS: Collectively, our data provide evidence of an undescribed role for kisspeptin in integrating sexual and emotional brain processing with reproduction in humans. These results have important implications for our understanding of reproductive biology and are highly relevant to the current pharmacological development of kisspeptin as a potential therapeutic agent for patients with common disorders of reproductive function. FUNDING: National Institute for Health Research (NIHR), Wellcome Trust (Ref 080268), and the Medical Research Council (MRC).


Assuntos
Emoções/efeitos dos fármacos , Kisspeptinas/administração & dosagem , Sistema Límbico/diagnóstico por imagem , Sistema Límbico/fisiologia , Comportamento Sexual/efeitos dos fármacos , Adulto , Método Duplo-Cego , Humanos , Masculino
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