Triacylglycerols (TAGs) containing positional isomers of hypogeic (Hy), palmitoleic (Po), and palmitvaccenic (Pv) acids from three microorganisms (top-fermenting brewer's yeast Saccharomyces cerevisiae, green alga Coccomyxa elongata, and arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis) were analyzed. Dozens of regioisomers and enantiomers of TAGs containing one, two or three hexadecenoic acids have been identified by means of reversed phase chromatography/mass spectrometry (RP-HPLC/MS). The regioisomers of TAGs containing two palmitic acids and any hexadecenoic acid were separated. Analysis of regioisomers of TAGs having one Pv residue showed that asymmetric molecular species such as PvPP or PPPv were dominant in Rhizophagus. TAGs were also analyzed on a chiral phase column and nine molecular species of TAGs containing two palmitic and any of three hexadecenoic acids were separated and identified. In the case of TAGs containing one palmitic and two hexadecenoic acids, the separation was successful only if the hexadecenoic acids were identical. Separation of TAGs containing three hexadecenoic acids was successful only if all three hexadecenoic acids were identical. Regardless of the type of TAG, it was found that TAGs in the AM fungus and containing palmitvaccenic acid bound at the sn-1 position of the glycerol backbone were dominant, suggesting similarity in the biosynthesis of the different TAGs. The covalent adduct chemical ionization method was used for identification of TAGs as adduct with (1-methyleneimino)-1-ethenyl ion, which reacted with double bond of the unsaturated fatty acid. Tandem MS thus makes it possible to identify TAGs containing various hexadecenoic acids.
A development of robust and rapid method with simple sample preparation for the analysis of steroids of C18-, C19-, C21- families is of interest of many research groups. Here we present a novel LC-MS/MS method for the simultaneous quantification of 32 steroid hormones in human plasma. Twenty-two of them were analyzed directly without the need for derivatization, while ten were derivatized with 2-fluoro-1-methylpyridinium p-toluenesulfonate. The steroids were separated on a C18 column with a gradient elution consisting of methanol and water with the addition of 0.1% formic acid. The mass spectrometer was operated in positive ESI mode. Validation demonstrated that the method was applicable for the quantitative analysis of two C18- steroids (estrone, estradiol), nineteen C19- steroids (testosterone, epitestosterone, dihydrotestosterone, 11-ketodihydrotestosterone, 11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketotestosterone, dehydroepiandrosterone, 7α-hydroxydehydroepiandrosterone, 7β-hydroxydehydroepiandrosterone, 7-ketodehydroepiandrosterone, androsterone, epiandrosterone, androstenedione, androstenediol, 5α-androstane-3α,17β-diol, 5α-androstane-3β,17β-diol, 5β-androstane-3α,17β-diol, 5β-androstane-3β,17β-diol), and eleven C21- steroids (cortisol, 21-deoxycortisol, 11-deoxycortisol, cortisone, corticosterone, 11-deoxycorticosterone, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, 5α-dihydroprogesterone). The lower limits of quantification are appropriate for analyses in both physiological and various pathophysiological conditions. The accuracy, intra- and inter-day precision values as well as stability tests were in accordance with FDA Guidelines. The method will be a useful tool in investigating the mechanisms of steroid-related diseases and will serve as a steppingstone for the development of other methods for steroid analyses in various biological matrices such as prostate tissue, cerebrospinal fluid, urine, seminal fluid, and saliva.
- MeSH
- androgeny MeSH
- androstendion * MeSH
- chromatografie kapalinová metody MeSH
- estron MeSH
- lidé MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Blood is a complex biological matrix providing valuable information on nutritional, metabolic, and immune status. The detection of blood biomarkers requires sensitive analytical methods because analytes are at very low concentrations. Peripheral blood monocytes play a crucial role in inflammatory processes, and the metabolites released by monocytes during these processes might serve as important signalling molecules and biomarkers of particular physiological states. Headspace solid-phase microextraction (HS-SPME) combined with two different mass spectrometric platforms, two-dimensional (2D) gas chromatography coupled to time-of-flight mass spectrometry (2D-GC/TOF-MS) and one-dimensional gas chromatography coupled to Orbitrap mass spectrometry (GC/Orbitrap-MS), were applied for the investigation of volatile organic compounds (VOCs) produced by human peripheral blood monocytes. An optimized method was subsequently applied for the characterization of changes in VOCs induced by lipopolysaccharides (LPS) and zymosan (ZYM) stimulation. Overall, the 2D-GC/TOF-MS and the 1D-GC/Orbitrap-MS analyses each yielded about 4000 and 400 peaks per sample, respectively. In total, 91 VOCs belonging to eight different chemical classes were identified. The samples were collected in two fractions, conditioned media for monitoring extracellularly secreted molecules and cell pellet samples to determine the intracellular composition of VOCs. Alcohols, ketones, and hydrocarbons were the main chemical classes of the metabolic profile identified in cell fractions. Aldehydes, acids and cyclic compounds were characteristic of the conditioned media fraction. Here we demonstrate that HS-SPME-2D-GC/TOF-MS is more suitable for the identification of specific VOC profiles produced by human monocytes than 1D-GC/Orbitrap-MS. We define the signature of VOCs occurring early after monocyte activation and characterise the signalling compounds released by immune cells into media.
- MeSH
- lidé MeSH
- mikroextrakce na pevné fázi MeSH
- monocyty metabolismus MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí metody MeSH
- reprodukovatelnost výsledků MeSH
- těkavé organické sloučeniny * analýza izolace a purifikace metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
In this study, a GC-MS method was developed for the quantification of saccharides in complex mixtures such as bio-oils and bio-oil aqueous phases produced by ablative pyrolysis of lignocellulosic biomass. The samples were first treated using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and the trimethylsilylated (more volatile) derivatives were analyzed by GC-MS. The method offers advantages of great separation capability and simultaneous identification of unknown peaks by comparison of the mass spectra and retention indices with extensive libraries available online. However, even with these tools at hand, the identification of several saccharide-resembling compounds can be challenging especially in such highly complex samples as pyrolysis bio-oils. For this reason, we devised a novel procedure, which eliminates certain saccharides depending on their specific chemical properties before subjecting the samples to the GC-MS analysis. The procedure was based on the combination of aniline treatment (elimination of reducing aldoses), and hydrolysis (elimination of anhydrosugars, glycosides, disaccharides and oligosaccharides). Based on the differences in chromatograms before and after the procedure, the unknown compounds were assigned into groups based on their susceptibility to each treatment. The combination of all methods above has allowed more accurate identification and quantification of saccharides, some of which were not as of today found in bio-oils.
This study presents a novel sample preparation method for the determination of both specific and non-specific pesticide metabolites in human urine samples. The method combines a deconjugation step with QuEChERS-based method and solid-phase extraction. In total, 15 pesticide metabolites (diethyl phosphate; diethyl thiophosphate; dimethyl phosphate; diethyl thiophosphate; 2,4-dichlorophenoxyacetic acid; 3-phenoxybenzoic acid; 4-fluoro-3-phenoxybenzoic acid; coumaphos; diethyl dithiophosphate; malathion dicarboxylic acid; p-nitrophenol; cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid; 3,5,6-trichloro-2-pyridinol; N,N-diethyl-3-methylbenzamid and 2-isopropyl-4-methyl-6-hydroxypyrimidine) were separated using liquid chromatography coupled to a mass spectrometer and isotope dilution method for quantitation. The method was validated using recovery tests with recoveries generally ranging from 80 to 120%. Additionally, 20 urine samples collected from South African children were analysed using the presented method. The median levels of pesticide metabolites found in the urine samples ranged from not detected (N,N-diethyl-3-methylbenzamid) to 22.36 µg/g creatinine (dimethyl phosphate). The novel method developed in this study is sensitive, selective, robust and reproducible while also conserving the amount of sample, chemicals, material and time required. Due to the low limits of detection obtained for individual pesticide metabolites, the method is capable of quantifying trace levels of pesticide metabolites in urine, which thus makes it an ideal tool for biomonitoring studies.
- MeSH
- chromatografie kapalinová metody MeSH
- dítě MeSH
- hmotnostní spektrometrie metody MeSH
- lidé MeSH
- limita detekce MeSH
- mladiství MeSH
- pesticidy moč MeSH
- reprodukovatelnost výsledků MeSH
- vystavení vlivu životního prostředí analýza MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Jihoafrická republika MeSH
A simple, sensitive HPLC-MS/MS method was developed and validated for the determination of lidocaine in skin and plasma of rats. The methods were established and validated assessing lower limit of quantitation (LLOQ), linearity, intra and inter-day precision and accuracy, selectivity, recovery and matrix effect. Chromatography was done on a Gemini column embedded with C18 stationary phase (50 mm × 2.0 mm, 5 µm particle size), using a gradient with mobile phases consisting of 0.1% HCOOH in bidistilled water and 0.1% HCOOH in acetonitrile. The mass spectrometer worked with electrospray ionization in positive ion mode and selected reaction monitoring, using target ions m/z 235.10 for lidocaine and m/z 245.10 for lidocaine-d10, used as internal standard. RESULTS: The linearity of the method was in the ranges of lidocaine concentrations 10.0-200.0 ng/mL for skin homogenate (accuracy 94.1-105.5%; R2 ≥ 0.998) and 0.025-2 ng/mL for plasma (accuracy 96.2-104.8%; R2 ≥ 0.996). The intra- and inter-day precision and accuracy determined on three quality control samples (20, 75 and 170 ng/mL for skin and 0.075, 0.4 and 1.5 ng/mL for plasma) were ≤4.2% and 103.8-108.2% for skin and ≤12.4% and 95.5-101.4% for plasma. The LLOQ was 10 ng/mL in skin homogenate and 0.025 ng/mL in plasma. The applicability of the method was demonstrated by measuring lidocaine in skin and plasma after exposure to medicated patches containing 5% lidocaine.
- MeSH
- krysa rodu rattus MeSH
- kůže chemie MeSH
- lidokain aplikace a dávkování analýza farmakokinetika MeSH
- limita detekce MeSH
- lineární modely MeSH
- reprodukovatelnost výsledků MeSH
- stabilita léku MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- transdermální náplast * MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The composition of a sample solvent has a crucial impact on separations in hydrophilic interaction liquid chromatography (HILIC). In this short communication, we studied the effect of an organic modifier in the sample solvent on the solubility of different tryptic glycopeptides of hemopexin and haptoglobin proteins. The results showed that the solubility of glycopeptides in solvents with a high acetonitrile content depends on the type of attached N-glycan. We observed lower solubility in larger glycans attached to the same peptide backbone, and we demonstrated that glycopeptides containing sialic acids precipitate more readily than those without sialic acid. Therefore, the sample solvent composition in HILIC must be carefully optimized for accurate quantitative data collection and for adequate separation.
- MeSH
- acetonitrily chemie MeSH
- glykopeptidy analýza chemie izolace a purifikace MeSH
- hydrofobní a hydrofilní interakce MeSH
- kyselina N-acetylneuraminová chemie MeSH
- polysacharidy chemie MeSH
- rozpouštědla chemie MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
In this study, the ionic profile and pH of exhaled breath condensate (EBC) in a group of patients with acid and weakly acid reflux and no-reflux controls were compared. A portable sampler was used for non-invasive EBC collection from five exhalations. The ionic profile (anions, cations, organic acids) and pH of the collected EBC samples were measured by capillary electrophoresis with contactless conductivity detection and a pH microelectrode, respectively. Several ions were elevated in the patient groups. Sodium cation was elevated in weakly acid reflux (significance level p < 0.01) and acid reflux (p < 0.05) compared to no-reflux controls. Butyrate and propionate were elevated in both acid reflux and weakly acid reflux compared to no-reflux controls (butyrate: p < 0.01, propionate: p < 0.05). The median values of pH (after de-aeration with N2) were also significantly higher (p < 0.01) in groups with acid reflux and weakly acid reflux than in the control group with no reflux. The ionic analysis and simultaneous pH measurement offer a simple, cheap, fast, and non-invasive approach in gastroesophageal reflux disease diagnostics.
- MeSH
- butyráty analýza MeSH
- dechové testy metody MeSH
- dospělí MeSH
- elektroforéza kapilární metody MeSH
- gastroezofageální reflux diagnóza metabolismus MeSH
- ionty analýza metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- lidé středního věku MeSH
- lidé MeSH
- propionáty analýza MeSH
- senioři MeSH
- sodík analýza MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Deoxynivalenol (DON) is one of the most common mycotoxins produced by field fungi (especially Fusarium). Contamination of livestock feed is a significant risk factor, especially for pigs that are highly susceptible to the toxic effects of deoxynivalenol. In this study, validated ultra-high performance liquid chromatography (U-HPLC) combined with a HR-Orbitrap-MS analysis method is described for the identification and quantitative determination of the mycotoxin compounds (DON and deepoxy-deoxynivalenol (DOM-1)) in pig colostrum (milk) and serum. Pre-treatment of the samples involved a deproteinisation step with methanol followed by a purification step by solid phase extraction (HLB cartridges). The chromatographic separation was performed on a C18 column with 1.7 μm-particle size using a water-methanol mobile phase. Detection of analytes was achieved on the tandem hybrid mass spectrometer Q Exactive, with a heated electrospray ionisation probe measured in positive mode (H-ESI+). For the confirmation of identification, a mass spectrometer was utilized in the full scan mode with resolving power (PR) = 140,000 (FWHM) and for quantification analysis, it was utilized in the parallel reaction monitoring mode (PRM). The method has been fully validated according to the requirements of Commission Decision 2002/657/EC for confirmatory analyses, plus the addition of a mass accuracy (MA) parameter. For the confirmation of the presence of these analytes in pig colostrum and serum, matching of the retention time with mass accuracy for the precursor ion from MS and product ions from MS/MS was used. A deuterium isotopically labelled internal standard and a matrix-matched calibration curve were employed for quantification. The linear range of quantification was 0.5-20 μg L-1 and the correlation coefficient (R2) was >0.999 for all calibrations. The limit of detection for DON and DOM-1 in colostrum was 0.48 μg L-1 and 0.54 μg L-1, respectively, and in serum 0.24 μg L-1 and 0.36 μg L-1, respectively. The limit of quantification for DON and DOM-1 in colostrum was 0.80 μg L-1 and 0.89 μg L-1, respectively, and in serum 0.39 μg L-1 and 0.60 μg L-1, respectively. The method was successfully evaluated using the obtained samples of pig colostrum and serum.
- MeSH
- chromatografie kapalinová metody MeSH
- kolostrum chemie MeSH
- kontaminace potravin analýza MeSH
- krmivo pro zvířata MeSH
- limita detekce MeSH
- lineární modely MeSH
- prasata MeSH
- reprodukovatelnost výsledků MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- těhotenství MeSH
- trichotheceny analýza MeSH
- zvířata MeSH
- Check Tag
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Lipid oxidation is one of the most important processes occurring in living cells and has been investigated through stable end-products. Currently, new insights into many physiological and pathophysiological processes provide a measurement of the first products of oxidation, e.g., oxidized glycerophosphatidylcholines (oxGPCs). Here, we evaluate the capacity of untargeted global metabolomics to measure oxGPCs in serum samples. This evaluation covered analytical reproducibility and data quality as well as the ability to capture metabolic alterations in diverse conditions. The analytical evaluation was performed based on the quality control samples, while the comparative analysis was based on the model of the development of type 2 diabetes mellitus (T2DM). The novelty of this approach arises not only from the measurement of oxGPCs instead of lipid peroxide-derived aldehydes but also from the stratification of the patients according to body mass index (BMI). Such a scenario was dictated by the fact that, despite the well-known relationship between obesity and T2DM development, there are lean individuals suffering from T2DM as well as obese people with normal glucose homeostasis. Our results provided evidence to support the ability of nontargeted metabolomics to measure oxGPCs. Comparative analysis of measured oxGPCs revealed differences in the level of oxGPCs either between different stages of disease development (insulin resistance, prediabetes) or BMI groups (normal weight, overweight, obese). The obtained results provided new insights into the metabolic processes leading to the development of T2DM and opened new paths in the investigation of the impact of body mass in T2DM progress.
- MeSH
- chromatografie kapalinová MeSH
- diabetes mellitus 2. typu krev metabolismus MeSH
- dospělí MeSH
- glycerylfosforylcholin krev chemie MeSH
- lidé středního věku MeSH
- lidé MeSH
- metabolom fyziologie MeSH
- metabolomika metody MeSH
- oxidace-redukce MeSH
- tandemová hmotnostní spektrometrie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
