The advantages of using mixtures of organic solvents for the separation of labeled oligosaccharides on the amide stationary phase under hydrophilic interaction liquid chromatography conditions are presented. The effect of the type of buffer as well as solvent or their mixtures on retention of uracil, saccharide labeling reagents (2-aminobenzoic acid, 2-aminobenzamide, ethyl 4-aminobenzoate, procainamide), and corresponding labeled saccharides were evaluated. The successful isocratic separation of labeled isomeric trisaccharides (maltotriose, panose, and isomaltotriose) was achieved in the mobile phase consisting of a 90% (v/v) mixture of organic solvents (methanol/acetonitrile 60:40) and 10% (v/v) 30 mM ammonium formate, pH 3.3. Changing the volume ratio between methanol/acetonitrile from 60:40 to 50:50 (v/v) allowed to obtain the separation of di-, tri-, and tetrasaccharides labeled by ethyl 4-aminobenzoate in less than 10.5 min.
- Klíčová slova
- HILIC, Mass spectrometry, Oligosaccharide, Saccharide/glycan labeling, Selectivity,
- MeSH
- acetonitrily chemie MeSH
- amidy chemie MeSH
- chemické techniky analytické přístrojové vybavení metody MeSH
- chromatografie kapalinová * MeSH
- formiáty chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- isomerie MeSH
- oligosacharidy izolace a purifikace MeSH
- ortoaminobenzoáty chemie MeSH
- rozpouštědla chemie MeSH
- sacharidy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetonitrile MeSH Prohlížeč
- acetonitrily MeSH
- amidy MeSH
- anthranilamide MeSH Prohlížeč
- anthranilic acid MeSH Prohlížeč
- formiáty MeSH
- formic acid MeSH Prohlížeč
- oligosacharidy MeSH
- ortoaminobenzoáty MeSH
- rozpouštědla MeSH
- sacharidy MeSH
An aberrant immune response developed early in life may trigger inflammatory bowel disease (IBD) and food allergies (e.g., celiac disease). Fecal levels of immune markers categorize an inflammatory response (e.g., food allergy, autoimmune) paralleled with the initial microbial colonization. The immunoaffinity assays are routinely applied to quantify circulating immune protein markers in blood/serum. However, a reliable, multiplex assay to quantify fecal levels of immune proteins is unavailable. We developed mass spectrometry assays to simultaneously quantify fecal calprotectin, myeloperoxidase, eosinophil-derived neurotoxin, eosinophil cationic protein, alpha-1-antitrypsin 1, and adaptive immunity effectors in 134 neonatal stool swabs. We optimized extraction and proteolytic protocol and validated the multiplex assay in terms of linearity of response (> 100; typically 0.04 to 14.77 µg/mg of total protein), coefficient of determination (R2; > 0.99), the limit of detection (LOD; 0.003 to 0.04 µg/mg of total protein), the limit of quantification (LOQ; 0.009 to 0.122 µg/mg of total protein) and robustness. The median CV of intra- and interday precision was 9.8% and 14.1%, respectively. We quantified breast milk-derived IGHA2 to differentiate meconium from feces samples and to detect the first food intake. An early life profiling of immune markers reflects disrupted intestinal homeostasis, and it is perhaps suitable for pre-symptomatic interception of IBD and food allergies.
- MeSH
- biologické markery metabolismus MeSH
- chemické techniky analytické metody MeSH
- feces chemie MeSH
- hmotnostní spektrometrie metody MeSH
- idiopatické střevní záněty etiologie metabolismus MeSH
- lidé MeSH
- novorozenec MeSH
- odběr biologického vzorku metody MeSH
- potravinová alergie etiologie metabolismus MeSH
- zánět diagnóza mikrobiologie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- biologické markery MeSH
A combination of two chromatographic and two enzymatic methods was used for the analysis of molecular species of lipids from Gram-positive bacteria of the genus Kocuria. Gram-positive bacteria contain a majority of branched fatty acids (FAs), especially iso- and/or anteiso-FAs. Two strains K. rhizophila were cultivated at three different temperatures (20, 28, and 37°C) and the majority phospholipid, i.e., the mixture of molecular species of phosphatidylglycerols (PGs) was separated by means of hydrophilic interaction liquid chromatography (HILIC). After enzymatic hydrolysis of PGs by phospholipase C and derivatization of the free OH group, the sn-1,2-diacyl-3-acetyl triacylglycerols (AcTAGs) were separated by reversed phase HPLC. Molecular species such as i-15:0/i-15:0/2:0, ai-15:0/ai-15:0/2:0, and 15:0/15:0/2:0 (straight chains) were identified by liquid chromatography-positive electrospray ionization mass spectrometry. The tandem mass spectra of both standards and natural compounds containing iso, anteiso and straight chain FAs with the same carbons were identical. Therefore, for identification of the ratio of two regioisomers, i.e. i-15:0/ai-15:0/2:0 vs. ai-15:0/i-15:0/2:0, they were cleavage by pancreatic lipase. The mixture of free fatty acids (FFAs) and 2-monoacylglycerols (2-MAGs) was obtained. After their separation by TLC and esterification and/or transesterification, the fatty acid methyl esters were quantified by GC-MS and thus the ratio of regioisomers was determined. It has been shown that the ratio of PG (containing as majority i-15: 0 / i-15: 0, i-15: 0 / ai-15: 0 and / or ai-15: 0 / i-15: 0 and ai-15: 0 / ai-15: 0 molecular species) significantly affected the membrane flow of bacterial cells cultured at different temperatures.
- Klíčová slova
- Gram-positive bacteria, RP-HPLC/MS-ESI(+), branched fatty acids, diacylglycerols, enzymatic hydrolysis, phosphatidylglycerols,
- MeSH
- chemické techniky analytické metody MeSH
- chromatografie kapalinová * MeSH
- diglyceridy chemie izolace a purifikace MeSH
- fosfolipidy chemie MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací * MeSH
- hydrofobní a hydrofilní interakce MeSH
- mastné kyseliny chemie MeSH
- Micrococcaceae chemie MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- diglyceridy MeSH
- fosfolipidy MeSH
- mastné kyseliny MeSH
About eight years ago, a new automation approach and flow technique called "Lab-In-Syringe" was proposed. It was derived from previous flow techniques, all based on handling reagent and sample solutions in a flow manifold. To date Lab-In-Syringe has evidently gained the interest of researchers in many countries, with new modifications, operation modes, and technical improvements still popping up. It has proven to be a versatile tool for the automation of sample preparation, particularly, liquid-phase microextraction approaches. This article aims to assist newcomers to this technique in system planning and setup by overviewing the different options for configurations, limitations, and feasible operations. This includes syringe orientation, in-syringe stirring modes, in-syringe detection, additional inlets, and addable features. The authors give also a chronological overview of technical milestones and a critical explanation on the potentials and shortcomings of this technique, calculations of characteristics, and tips and tricks on method development. Moreover, a comprehensive overview of the different operation modes of Lab-In-Syringe automated sample pretreatment is given focusing on the technical aspects and challenges of the related operations. We further deal with possibilities on how to fabricate required or useful system components, in particular by 3D printing technology, with over 20 different elements exemplarily shown. Finally, a short discussion on shortcomings and required improvements is given.
- Klíčová slova
- 3D printing of instrument elements, Lab-In-Syringe, automation of sample pretreatment, potentials and troubles, system setup and operation modes, tips and tricks in method development,
- MeSH
- chemické techniky analytické přístrojové vybavení metody normy MeSH
- injekční stříkačky * MeSH
- laboratorní automatizace * MeSH
- limita detekce MeSH
- reprodukovatelnost výsledků MeSH
- Publikační typ
- časopisecké články MeSH
A series of chiral indole phytoalexins with potential anticancer and antimicrobial activity were enantioseparated in supercritical fluid chromatography. Two polysaccharide-based chiral stationary phases composed of tris-(3,5-dimethylphenylcarbamate) derivatives of amylose or cellulose coated on 2.5 μm silica particles were successfully used. The influences of the polysaccharide backbone, co-solvent type and co-solvent content in the mobile phase on retention, enantioselectivity and enantioresolution of indole phytoalexins were investigated. Fast baseline separations were achieved for 26 from 27 tested compounds. Amylose-based chiral stationary phase provided higher number of baseline resolutions of the indole phytoalexins than the cellulose-based one. However, certain complementary enantioresolution results towards the studied compounds were observed between the investigated columns. The relationship between structure of the indole phytoalexins and their chromatographic behavior in supercritical fluid chromatography was discussed.
- Klíčová slova
- Amylose, Cellulose, Chiral separation, Chiral stationary phase, Indole phytoalexins, Supercritical fluid chromatography,
- MeSH
- amylosa chemie MeSH
- celulosa chemie MeSH
- chemické techniky analytické metody MeSH
- fytoalexiny MeSH
- indoly izolace a purifikace MeSH
- oxid křemičitý chemie MeSH
- polysacharidy chemie MeSH
- rozpouštědla chemie MeSH
- seskviterpeny izolace a purifikace MeSH
- stereoizomerie MeSH
- superkritická fluidní chromatografie * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- amylosa MeSH
- celulosa MeSH
- fytoalexiny MeSH
- indoly MeSH
- oxid křemičitý MeSH
- polysacharidy MeSH
- rozpouštědla MeSH
- seskviterpeny MeSH
Plasma protein-drug binding assays are routinely performed during the early stages of drug discovery and development, which creates demand for an automated high-throughput screening assay to increase laboratory efficiency. A comprehensive comparison of the four methods typically used for determining the binding parameters is presented in this study with respect to the above demand. Capillary electrophoresis-frontal analysis, isothermal titration calorimetry, circular dichroism and equilibrium dialysis were used to study the affinity of human serum albumin for diclofenac and lidocaine. These model drugs were chosen due to their different physico-chemical properties and different binding sites on the albumin molecule, also resulting in different binding strength. The binding parameters estimated under the conditions as similar as possible were comparable among all these approaches as well as to the literature values. Besides this, the comparison of the results and especially other considerations demonstrated the benefits and drawbacks of the selected methods, with capillary electrophoresis-frontal analysis being the best candidate for such studies.
- Klíčová slova
- Capillary electrophoresis-frontal analysis, Circular dichroism, Equilibrium dialysis, Isothermal titration calorimetry, Plasma protein-drug interaction,
- MeSH
- chemické techniky analytické metody MeSH
- krevní proteiny chemie metabolismus MeSH
- léčivé přípravky chemie metabolismus MeSH
- lidé MeSH
- reprodukovatelnost výsledků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- krevní proteiny MeSH
- léčivé přípravky MeSH
In this work we present a simple and cost-effective approach for the determination of selenium species in algae and yeast biomass, based on a combination of thin-layer chromatography (TLC) with diode laser thermal vaporization inductively coupled plasma mass spectrometry (DLTV ICP MS). Extraction of freeze-dried biomass was performed in 4M methanesulphonic acid and the selenium species were vaporized from cellulose TLC plates employing a continuous-wave infrared diode laser with power up to 4 W using a simple laboratory-built apparatus. Selenomethionine and selenocysteine were quantified with limits of detection 3 μg L-1 in a Se-enriched microalgae Chlorella vulgaris and yeast certified reference material SELM-1. Results delivered by TLC-DLTV ICP MS were consistent with those obtained by a routine coupling of high-performance liquid chromatography (HPLC) to ICP MS. In addition, the TLC approach is capable of analyzing extract containing even undiluted crude hydrolysates that could damage HPLC columns.
- Klíčová slova
- Chlorella vulgaris algae, Diode laser thermal vaporization (DLTV), Inductively coupled plasma mass spectrometry (ICP MS), Liquid chromatography (LC), Selenium speciation (selenomethionine, selenocysteine), Thin-layer chromatography (TLC),
- MeSH
- chemické techniky analytické ekonomika metody MeSH
- Chlorella vulgaris chemie MeSH
- chromatografie na tenké vrstvě * MeSH
- hmotnostní spektrometrie * přístrojové vybavení MeSH
- lasery polovodičové MeSH
- Saccharomyces cerevisiae chemie MeSH
- selenocystein analýza MeSH
- selenomethionin analýza MeSH
- spektrální analýza MeSH
- volatilizace MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- selenocystein MeSH
- selenomethionin MeSH
DNA extraction from soil samples is a critical step for molecular biology analyses. The present study compared the efficiency of two DNA isolation methods from non-polluted and polluted soils with or without the presence of a plant. Both applied methods used chemical and physical lyses, but method 1 had an additional physical disruption. The main difference between these two methods was the humic acid purification technique as it was carried out during cell lysis for method 1 and after cell lysis for method 2. Samples were assessed on the basis of their yield and DNA purity as well as their bacterial quantity and diversity. Based on our results, method 1 proved to be more effective at removing protein and RNA, whereas method 2 proved to be more effective at removing humic acids. Although no differences were obtained in terms of the DNA yield, both the bacterial quantity and community structure were affected by the method used. Method 1 allowed for the recovery of more information than method 2, and polluted soil was more sensitive to the DNA extraction procedure. We recommend carefully selecting the DNA extraction method, especially when soil is disturbed.
- MeSH
- Bacteria klasifikace genetika izolace a purifikace metabolismus MeSH
- chemické techniky analytické metody MeSH
- DNA bakterií genetika izolace a purifikace MeSH
- huminové látky MeSH
- látky znečišťující půdu analýza metabolismus MeSH
- polymerázová řetězová reakce MeSH
- půda chemie MeSH
- půdní mikrobiologie MeSH
- znečištění životního prostředí MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- Názvy látek
- DNA bakterií MeSH
- huminové látky MeSH
- látky znečišťující půdu MeSH
- půda MeSH
Analysis of the glycosylation of proteins is a challenge that requires orthogonal methods to achieve separation of the diverse glycoforms. A combination of reversed phase chromatography with tandem mass spectrometry (RP-LC-MS/MS) is one of the most powerful tools for glycopeptide analysis. In this work, we developed and compared RP-LC and hydrophilic interaction liquid chromatography (HILIC) in nanoscale on a chip combined with MS/MS in order to separate glycoforms of two peptides obtained from the tryptic digest of hemopexin. We observed reduction of the retention time with decreasing polarity of glycans attached to the same peptide backbone in HILIC. The opposite effect was observed for RP-LC. The presence of sialic acids prolonged the retention of glycopeptides in both chromatographic modes. The nanoHILIC method provided higher selectivity based on the composition of glycan, compared to nanoRP-LC but a lower sensitivity. The nanoHILIC method was able to partially separate linkage isomers of fucose (core and outer arm) on bi-antennary glycoform of SWPAVGDCSSALR glycopeptide, which is beneficial in the elucidation of the structure of the fucosylated glycoforms.
- Klíčová slova
- Glycoproteomics, Hemopexin, Hydrophilic interaction liquid chromatography, LC–MS/MS, Reversed phase chromatography,
- MeSH
- chemické techniky analytické metody normy MeSH
- chromatografie kapalinová * MeSH
- chromatografie s reverzní fází * MeSH
- glykopeptidy analýza MeSH
- hemopexin analýza MeSH
- hydrofobní a hydrofilní interakce MeSH
- polysacharidy chemie MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- glykopeptidy MeSH
- hemopexin MeSH
- polysacharidy MeSH
The aim of this article is to study the modification of an inner capillary wall with sol-gel coating (pure silica sol-gel or silica sol-gel containing porphyrin-brucine conjugate) and determine its influence on the separation process using capillary electrophoresis/electrochromatography method. After modification of the inner capillary surface the separation of analytes was performed using two different phosphate buffers (pH 2.5 and 9.0) and finally the changes in electrophoretic mobilities of various samples were calculated. To confirm that the modification of the inner capillary surface was successful, the parts of the inner surfaces of capillaries were observed using scanning electron microscopy. The analytes used as testing samples were oligopeptides, nucleosides, nucleobases and finally nucleotides.
- Klíčová slova
- Capillary electrochromatography (CEC), Nucleo-compounds, Oligopeptides, Open-tubular capillary electrochromatography (OT-CEC), Porphyrin, Scanning electron microscopy (SEM), Sol–gel methods,
- MeSH
- chemické techniky analytické přístrojové vybavení metody MeSH
- kapilární elektrochromatografie * MeSH
- mikroskopie elektronová rastrovací MeSH
- nukleosidy izolace a purifikace MeSH
- nukleotidy izolace a purifikace MeSH
- oligopeptidy izolace a purifikace MeSH
- změna skupenství MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- nukleosidy MeSH
- nukleotidy MeSH
- oligopeptidy MeSH
