UHPLC–ESI-MS/MS
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Three dimensions of chromatographic separation, using split-flow two-dimensional liquid chromatography (SF-2D-LC) with two parallel second dimensions, LC × 2LC, combined with quadruple parallel mass spectrometry (LC3MS4) is demonstrated for analysis of NIST SRM 1849a adult/infant formula. The first dimension, 1D, was a conventional non-aqueous reversed-phase (NARP) HPLC separation using two C18 columns in series, followed by detection using an ultraviolet (UV) detector, a fluorescence detector (FLD), with flow then split to a corona charged aerosol detector (CAD), and then dual parallel mass spectrometry (MS), conducted in atmospheric pressure photoionization (APPI) and electrospray ionization (ESI) modes. The first second dimension, 2D(1), UHPLC was conducted on a 50.0 mm C30 column using a NARP-UHPLC parallel gradient for separation of short-chain triacylglycerols (TAGs) from long-chain TAGs, with detection by UV and ESI-MS. The second dimension, 2D(2), UHPLC was conducted using a 100.0 mm C30 column with a NARP-UHPLC parallel gradient for improved separation of TAG isomers, with detection by UV, an evaporative light scattering detector, and high-resolution, accurate-mass (HRAM) ESI-MS. Transferred eluent dilution was used to refocus peaks and keep them sharp during elution in both 2Ds. The separation space in the 2D(2) was optimized using multi-cycle (aka, "constructive wraparound") elution, which employed flow rate programming. In the 1D, calibration lines for quantification of fat-soluble vitamins were constructed. A lipidomics approach to TAG identification and quantification by HRAM-ESI-MS was applied to the 2D(2). These experiments can be represented: LC1MS2 × (LC1MS1 + LC1MS1) = LC3MS4, or three-dimensional liquid chromatography with quadruple parallel mass spectrometry.
Seaweeds have been exploited as both food products and therapeutics to manage human ailments for centuries. This study investigated the metabolite profile of five seaweeds (Halimeda spp., Spyridia hypnoides (Bory de Saint-Vincent) Papenfuss, Valoniopsis pachynema (G. Martens) Børgesen, Gracilaria fergusonii J. Agardh and Amphiroa anceps (Lamarck) Decaisne using ultra-high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (UHPLC-ESI-MS/MS). Furthermore, these seaweeds were assessed for antioxidant and inhibitory effects against α-amylase, α-glucosidase, acetyl-cholinesterase (AChE), butyryl-cholinesterase (BChE) and tyrosinase. Valoniopsis pachynema and A. anceps yielded the highest flavonoid (4.30 ± 0.29 mg RE/g) and phenolic content (7.83 ± 0.08 mg RE/g), respectively. Additionally, A. anceps exhibited significant antioxidant properties with all assays and significantly depressed BChE (IC50 = 6.68 ± 0.83 mg/mL) and α-amylase activities (IC50 = 5.34 ± 0.14 mg/mL). Interestingly, the five seaweeds revealed potent inhibitory effects against tyrosinase activity. In conclusion, A. anceps might be considered as a key source of phytoantioxidants and a potential candidate to develop nutritional supplements. Besides, the five tested seaweeds warrant further study and may be exploited as promising natural sources for managing hyperpigmentation.
The ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) method was optimized and validated for the determination of oxylipins in human plasma using the targeted approach with selected reaction monitoring (SRM) in the negative-ion electrospray ionization (ESI) mode. Reversed phase UHPLC separation on an octadecylsilica column enabled the analysis of 63 oxylipins including numerous isomeric species within 12-min run time. The method was validated (calibration curve, linearity, limit of detection, limit of quantification, carry-over, precision, accuracy, recovery rate, and matrix effect) and applied to 40 human female plasma samples from breast cancer patients and age-matched healthy volunteers (control). Thirty-six oxylipins were detected in human plasma with concentrations above the limit of detection, and 21 of them were quantified with concentrations above the limit of quantitation. The concentrations determined in healthy controls are in a good agreement with previously reported data on human plasma. Quantitative data were statistically evaluated by multivariate data analysis (MDA) methods including principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA). S-plot and box plots showed that 13-HODE, 9-HODE, 13-HOTrE, 9-HOTrE, and 12-HHTrE were the most upregulated oxylipin species in plasma of breast cancer patients.
- MeSH
- analýza hlavních komponent MeSH
- chromatografie s reverzní fází metody MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- lidé MeSH
- limita detekce MeSH
- multivariační analýza MeSH
- nádory prsu krev MeSH
- oxylipiny krev MeSH
- reprodukovatelnost výsledků MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
Neurotransmitters, small molecules widely distributed in the central nervous system are essential in transmitting electrical signals across neurons via chemical communication. Dysregulation of these chemical signaling molecules is linked to numerous neurological diseases including tauopathies. In this study, a precise and reliable liquid chromatography method was established with tandem mass spectrometry detection for the simultaneous determination of aspartic acid, asparagine, glutamic acid, glutamine, γ-aminobutyric acid, N-acetyl-l-aspartic acid, pyroglutamic acid, acetylcholine and choline in human brain tissue. The method was successfully applied to the analysis of human brain tissues from three different tauopathies; corticobasal degeneration, progressive supranuclear palsy and parkinsonism-dementia complex of Guam. Neurotransmitters were analyzed on ultra-high performance chromatography (UHPLC) using an ethylene bridged hybrid amide column coupled with tandem mass spectrometry (MS/MS). Identification and quantification of neurotransmitters was carried out by ESI+ mass spectrometry detection. We optimized sample preparation to achieve simple and fast extraction of all nine analytes. Our method exhibited an excellent linearity for all analytes (all coefficients of determination >0.99), with inter-day and intra-day precision yielding relative standard deviations 3.2%-11.2% and an accuracy was in range of 92.6%-104.3%. The present study, using the above method, is the first to demonstrate significant alterations of brain neurotransmitters caused by pathological processes in the brain tissues of patient with three different tauopathies.
- MeSH
- chromatografie kapalinová metody MeSH
- hmotnostní spektrometrie metody MeSH
- lidé MeSH
- limita detekce MeSH
- lineární modely MeSH
- mozek - chemie fyziologie MeSH
- neurotransmiterové látky analýza metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- tauopatie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The potential of ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) and ultrahigh-performance supercritical fluid chromatography (UHPSFC) coupled to negative-ion electrospray ionization mass spectrometry (ESI-MS) for the analysis of 46 oxylipins and 2 fatty acid standards is compared in terms of their chromatographic resolution with the emphasis on distinguishing isobaric interferences and the method sensitivity. UHPLC provides the baseline separation of 24 isobaric oxylipins within 13min, while UHPSFC enables the separation of only 20 isobaric oxylipins within 8min. Moreover, the UHPLC/ESI-MS method provides an average improvement of sensitivity by 3.5-fold. A similar trend is observed in the analysis of human plasma samples, but lower ion suppression effects caused by lysophospholipids (LPL) are observed in case of UHPSFC/ESI-MS due to better separation of LPL. Both methods are fully applicable for the analysis of oxylipins, but UHPLC/ESI-MS method is preferred due to better separation and higher sensitivity, which results in the identification of 31 oxylipins in human plasma based on available standards and additional tentative 20 identifications based on accurate m/z values and the fragmentation behavior known from the literature.
- MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- lidé MeSH
- oxylipiny krev chemie MeSH
- rozpouštědla chemie MeSH
- superkritická fluidní chromatografie metody MeSH
- teplota MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Management of the therapy of life-threatening bacterial infection is extremely based on an optimal antibiotic treatment. Achieving the correct vancomycin dosage in blood and target tissues can be complicated in special situations, e.g., where large fluid sequestration and/or acute renal failure occur. A UHPLC-MS/MS method operating in electrospray (ESI) positive ion mode was applied for the determination of vancomycin in serum, urine and peritoneal/pleural effusion. Sample pretreatment was composed of dilution and simple protein precipitation where only a small volume (50μL) of serum, urine or peritoneal/pleural effusion was required. The separation of vancomycin was performed on a Meteoric Core C18 BIO column (100×4.6mm, 2.7μm) by gradient elution with 0.1% formic acid in water and acetonitrile. The total time of analysis was 4.5min. The method was found to be linear in the range of 2-60μM (or 0.5-10μM) for serum, 0.27-10μM (or 2-60μM) for peritoneal/pleural effusion and 25-300μM for urine, which was adequate for the determination of vancomycin in patient samples. The intra- and inter-day precision was below 8% RSD, and accuracy was from 89 to 104%. The UHPLC/MS-MS method offers a fast and reliable approach to determine vancomycin concentrations in three different human body fluid samples (serum, urine and peritoneal/pleural effusion) with a simple sample pretreatment that was the same for all selected specimens. This method should be applicable to large sample series in clinical (pharmacokinetic/pharmacodynamic) studies.
It is assumed that human exposure to phthalates may be associated with adverse health effects. The indicators of urinary phthalate metabolite concentrations in healthy adults are limited. In this study, the phthalate metabolites concentrations were detected from 24-h urine collection in non-obese Czech adults (n = 201). Each participant filled in an 80-item questionnaire (FANTOM-SQ 2013) regarding the outdoor and indoor sources of phthalates, the use of personal care products and food intake sources. The concentrations of 15 phthalates metabolites were analysed following enzymatic cleavage of the glucuronide using ultra-high-performance liquid chromatography-electrospray ionisation tandem mass spectrometry (UHPLC-ESI-MS/MS). The indicators of chronic or acute exposure phthalate-containing materials were identified. It is shown that higher fruit consumption was positively and significantly associated with a higher level of total 15 urinary phthalates biomarkers (p < 0.001). Regular meat consumption showed a negative significant association with total 15 phthalates metabolites (p < 0.01). The use of personal care products was significantly and positively correlated with monoethyl phthalate urine concentrations (p < 0.05). The analysis of the dietary behaviour and personal care products use in the Czech non-obese population showed it to be a predictable tool in the level of phthalates exposure when high fruit consumption and personal care products use are linked to higher phthalate metabolite contents in the urine. However, this topic deserves more research.
- MeSH
- biologické markery moč MeSH
- kontaminace potravin analýza MeSH
- kyseliny ftalové metabolismus moč MeSH
- lidé MeSH
- maso analýza MeSH
- ovoce chemie MeSH
- tandemová hmotnostní spektrometrie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zdraví dobrovolníci pro lékařské studie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
The triterpenoid plant hormones brassinosteroids (BRs) are believed to influence almost every aspect of plant growth and development. We have developed a sensitive mass spectrometry-based method for the simultaneous profiling of twenty-two naturally occurring brassinosteroids including biosynthetic precursors and the majority of biologically active metabolites. Using ultra-high performance liquid chromatographic (UHPLC) analysis, the run time was reduced up to three times (to 9 min) in comparison to standard HPLC BRs analyses, the retention time stability was improved to 0.1-0.2 % RSD and the injection accuracy was increased to 1.1-4.9 % RSD. The procedures for extraction and for two-step purification based on solid-phase extraction (SPE) were optimised in combination with subsequent UHPLC analysis coupled to electrospray ionisation tandem mass spectrometry (ESI-MS/MS) using Brassica flowers and Arabidopsis plant tissue extracts. In multiple reaction monitoring (MRM) mode, the average detection limit for BRs analysed was close to 7 pg, and the linear range covered up to 3 orders of magnitude. The low detection limits for this broad range of BR metabolites enabled as little as 50 mg of plant tissue to be used for quantitative analyses. The results of determinations exploiting internal standards showed that this approach provides a high level of practicality, reproducibility and recovery. The method we have established will enable researchers to gain a better understanding of the dynamics of the biosynthesis and metabolism of brassinosteroids and their modes of action in plant growth and development.
- MeSH
- Arabidopsis chemie MeSH
- Brassica napus chemie MeSH
- brassinosteroidy analýza MeSH
- extrakce na pevné fázi metody MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- limita detekce MeSH
- reprodukovatelnost výsledků MeSH
- rostlinné extrakty chemie MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
INTRODUCTION: The pathogenesis of preterm labor is fragmentarily explained. The most widely accepted theory points out to infection and inflammation as possible causes, which can be mediated by potentially different factors, including sphingolipid mediators. Sphingolipids are a class of lipids that have been shown as important mediators in various cell processes such as: proliferation, growth, apoptosis, stress response, necrosis and inflammation. The aim of the study was to assess plasma concentrations of selected sphingolipids in patients with preterm labor. MATERIAL AND METHODS: We used ultra-high performance liquid chromatography with triple mass spectrometry (UHPLC-ESI-MS/MS) to assess plasma concentrations of the 11 sphingolipids in patients presenting with symptoms of preterm labor (n=61) and threatened preterm labor (n=40). RESULTS: We observed a statistically significant increase (p-value<0.004) in plasma concentrations of C16-Cer in patients with preterm labor as compared to the control group. We also found C16-Cer to be the best predictor of preterm labor in the group of patients with symptoms occurring after 32 weeks of gestation. CONCLUSIONS: Our findings show a possible involvement of selected sphingolipids, especially C16-Cer, in the pathogenesis of preterm labor. Their role as predictors of preterm delivery needs to be validated in the future on larger group of patients.
- MeSH
- biologické markery krev MeSH
- ceramidy krev MeSH
- dospělí MeSH
- gestační stáří MeSH
- lidé MeSH
- novorozenec nedonošený MeSH
- předčasná porodní činnost krev diagnóza patofyziologie MeSH
- senzitivita a specificita MeSH
- studie případů a kontrol MeSH
- tandemová hmotnostní spektrometrie MeSH
- těhotenství MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) method using two 15cm sub-2μm particles octadecylsilica gel columns is developed with the goal to separate and unambiguously identify a large number of lipid species in biological samples. The identification is performed by the coupling with high-resolution tandem mass spectrometry (MS/MS) using quadrupole - time-of-flight (QTOF) instrument. Electrospray ionization (ESI) full scan and tandem mass spectra are measured in both polarity modes with the mass accuracy better than 5ppm, which provides a high confidence of lipid identification. Over 400 lipid species covering 14 polar and nonpolar lipid classes from 5 lipid categories are identified in total lipid extracts of human plasma, human urine and porcine brain. The general dependences of relative retention times on relative carbon number or relative double bond number are constructed and fit with the second degree polynomial regression. The regular retention patterns in homologous lipid series provide additional identification point for UHPLC/MS lipidomic analysis, which increases the confidence of lipid identification. The reprocessing of previously published data by our and other groups measured in the RP mode and ultrahigh-performance supercritical fluid chromatography on the silica column shows more generic applicability of the polynomial regression for the description of retention behavior and the prediction of retention times. The novelty of this work is the characterization of general trends in the retention behavior of lipids within logical series with constant fatty acyl length or double bond number, which may be used as an additional criterion to increase the confidence of lipid identification.
- MeSH
- časové faktory MeSH
- chromatografie s reverzní fází metody MeSH
- erytrocyty chemie MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- lidé MeSH
- lipidy analýza krev chemie moč MeSH
- mozek - chemie MeSH
- prasata MeSH
- superkritická fluidní chromatografie MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
