Untargeted metabolite profiling using high-resolution mass spectrometry coupled with liquid chromatography (LC-HRMS), followed by data analysis with the Compound Discoverer 2.0™ software, was used to study the metabolism of imatinib in humans with chronic myeloid leukemia. Plasma samples from control (drug-free) and patient (treated with imatinib) groups were analyzed in full-scan mode and the unknown ions occurring only in the patient group were then, as potential imatinib metabolites, subjected to multi-stage fragmentation in order to elucidate their structure. The application of an untargeted approach, as described in this study, enabled the detection of 24 novel structurally unexpected metabolites. Several sulphur-containing compounds, probably originating after the reaction of reactive intermediates of imatinib with endogenous glutathione, were found and annotated as cysteine and cystine adducts. In the proposed mechanism, the cysteine adducts were formed after the rearrangement of piperazine moiety to imidazoline. On the contrary, in vivo S-N exchange occurred in the case of the cystine adducts. In addition, N-O exchange was observed in the collision cell in the course of the fragmentation of the cystine adducts. The presence of sulphur in the cysteine and cystine conjugates was proved by means of ultra-high resolution measurements using Orbitrap Elite. The detection of metabolites derived from glutathione might improve knowledge about the disposition of imatinib towards bioactivation and help to improve understanding of the mechanism of its hepatotoxicity or nephrotoxicity in humans.

• Klíčová slova
• Compound Discoverer™, Glutathione, Imatinib, LC-HRMS, Metabolization, Untargeted metabolite profiling,
• MeSH
• chromatografie kapalinová MeSH
• cystein metabolismus   MeSH
• cystin metabolismus   MeSH
• imatinib mesylát krev   metabolismus   moč   MeSH
• inhibitory proteinkinas krev   metabolismus   moč   MeSH
• lidé MeSH
• protinádorové látky krev   metabolismus   moč   MeSH
• síra krev   metabolismus   moč   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cystein MeSH
• cystin MeSH
• imatinib mesylát MeSH
• inhibitory proteinkinas MeSH
• protinádorové látky MeSH
• síra MeSH

Nontargeted screening (NTS) utilizing liquid chromatography electrospray ionization high-resolution mass spectrometry (LC/ESI/HRMS) is increasingly used to identify environmental contaminants. Major differences in the ionization efficiency of compounds in ESI/HRMS result in widely varying responses and complicate quantitative analysis. Despite an increasing number of methods for quantification without authentic standards in NTS, the approaches are evaluated on limited and diverse data sets with varying chemical coverage collected on different instruments, complicating an unbiased comparison. In this interlaboratory comparison, organized by the NORMAN Network, we evaluated the accuracy and performance variability of five quantification approaches across 41 NTS methods from 37 laboratories. Three approaches are based on surrogate standard quantification (parent-transformation product, structurally similar or close eluting) and two on predicted ionization efficiencies (RandFor-IE and MLR-IE). Shortly, HPLC grade water, tap water, and surface water spiked with 45 compounds at 2 concentration levels were analyzed together with 41 calibrants at 6 known concentrations by the laboratories using in-house NTS workflows. The accuracy of the approaches was evaluated by comparing the estimated and spiked concentrations across quantification approaches, instrumentation, and laboratories. The RandFor-IE approach performed best with a reported mean prediction error of 15× and over 83% of compounds quantified within 10× error. Despite different instrumentation and workflows, the performance was stable across laboratories and did not depend on the complexity of water matrices.

• Publikační typ
• časopisecké články MeSH

Gene-directed enzyme/prodrug therapy represents one of the experimental treatment approaches. The system based on conversion of nontoxic prodrug 5-fluorocytosine to chemotherapeutic 5-fluorouracil by cytosine deaminase or fusion cytosine deaminase::uracil phosphoribosyl transferase belongs to the most frequently used. The detailed analysis of 5-fluorocytosine, 5-fluorouracil and its metabolites enables to understand various responses of tumour cells to treatment as well as mechanisms of resistance. A fast, sensitive and accurate methods based on liquid chromatography with high-resolution mass spectrometry (LC-HRMS) for the identification and quantification of 5-fluorocytosine, 5-fluorouracil and its major metabolites were developed. Two different hybrid high-resolution mass spectrometers sufficient for study of metabolic pathways were used. The LC-ESI IT-TOF MS method was successfully used for identification of 5-fluorocytosine, 5-fluorouracil and its metabolites in complex biological matrices (mesenchymal stromal cells and tumour cells media) and for confirmation of the metabolic conversion of 5-fluorocytosine even in chemoresistant tumour cells media samples. For quantification, the LC-HESI QExactive MS method was developed and validated. The developed method demonstrated a very good linear range for 5-fluorocytosine from 1 ng/mL to 1000 ng/mL and for its major metabolites from 5 ng/mL to 1000 ng/mL. The limits of detection and limits of quantification ranged from 1.1 to 26 ng/mL and from 3.6 to 87 ng/mL, respectively. Both developed methods confirmed the ability of gene-directed enzyme prodrug therapy to metabolically convert 5-fluorocytosine to 5-fluorouracil and its major metabolites in real samples of tumour cell media and mesenchymal stromal cells.

• Klíčová slova
• 5-fluorouracil, Cancer therapy, Chemotherapeutics, High resolution mass spectrometry, Metabolites,
• MeSH
• chromatografie kapalinová MeSH
• cytosindeaminasa MeSH
• flucytosin * MeSH
• fluoruracil MeSH
• hmotnostní spektrometrie MeSH
• prekurzory léčiv * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytosindeaminasa MeSH
• flucytosin * MeSH
• fluoruracil MeSH
• prekurzory léčiv * MeSH

Global climate changes cause water scarcity in many regions, and the sustainable use of recycled water appears crucial, especially in agriculture. However, potentially hazardous compounds such as pharmaceuticals can enter the food chain and pose severe risks. This paper aims to study the presence of selected pharmaceutical active compounds (PhACs) and their metabolites in crops grown in aeroponic conditions and evaluate the potential of PhAC plant uptake. A solvent extraction with an acidified mixture of acetonitrile and water followed by LC-HRMS was developed and validated for quantifying nine pharmaceuticals and their nine metabolites in three plants. We aimed for a robust method with a wide linear range because an extensive concentration range in different matrices was expected. The developed method proved rapid and reliable determination of selected pharmaceuticals in plants in the wide concentration range of 10 to 20,000 ng g-1 and limit of detection range 0.4 to 9.0 ng g-1. The developed method was used to study the uptake and translocation of pharmaceuticals and their metabolites in plant tissues from an aeroponic experiment at three different pH levels. Carbamazepine accumulated more in the leaves of spinach than in arugula. On the other hand, sulfamethoxazole and clindamycin evinced higher accumulation in roots than in leaves, comparable in both plants. The expected effect of pH on plants' uptake was not significant.

• Klíčová slova
• Extraction, Pharmaceutical, Plant uptake, Soil pollution,
• MeSH
• koncentrace vodíkových iontů MeSH
• léčivé přípravky MeSH
• voda MeSH
• zemědělské plodiny * MeSH
• zemědělství * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• léčivé přípravky MeSH
• voda MeSH

In Italy a particularly valuable chestnut is "Marrone di Roccadaspide", a protected geographical indication (PGI) product, deriving from a Castanea sativa cultivar, typical of Salerno province in Campania region. As chestnut industrial processes yield a large amount of shell by-products, in this study the possibility to retrain this waste food as potential source of bioactives was investigated. The ability of "Marrone di Roccadaspide" shell MeOH extract to modulate the pro-inflammatory transcriptional factor NF-κB after LPS stimulation, along with the antioxidant activity by a cell-based in vitro test, were evaluated. To correlate the NF-κB inhibition (67.67% at 5 μg/mL) and the strong antioxidant activity to the chemical composition, an analytical approach based on LC-ESI/LTQOrbitrap/MS/MSn along with NMR characterization of isolated compounds was developed. The identification of hydrolysable and condensed tannins, along with flavonoids, phenol glucosides, ellagic acid derivatives, and triterpenoids was accomplished. The most representative compounds were quantitatively analyzed by LC-ESI/QTrap/MS/MS, showing bartogenic acid as the compound occurring in the highest amount (103.08 mg/100 g shells). With the aim to explore the possibility to employ chestnut shells as suitable source of bioactives for the preparation of functional ingredients, the chemical composition and the antioxidant activity of "eco-friendly" extracts (EtOH and EtOH:H2O 7:3) was finally evaluated, showing a high superimposability of the EtOH:H2O (7:3) extract to the MeOH extract.

• Klíčová slova
• Antioxidant activity, LC-HRMS/MS(n), Shells, Tannins, Triterpenes, “Marrone di Roccadaspide” PGI, “green” extracts,
• MeSH
• antioxidancia analýza   MeSH
• Fagaceae chemie   MeSH
• fenoly analýza   MeSH
• flavonoidy analýza   MeSH
• glukosidy analýza   MeSH
• hodnotící studie jako téma MeSH
• hydrolyzovatelné taniny analýza   MeSH
• kalibrace MeSH
• kyselina ellagová analýza   MeSH
• lidé MeSH
• NF-kappa B metabolismus   MeSH
• ořechy chemie   MeSH
• proantokyanidiny analýza   MeSH
• proliferace buněk MeSH
• racionalizace * MeSH
• reaktivní formy kyslíku MeSH
• rostlinné extrakty analýza   MeSH
• tandemová hmotnostní spektrometrie MeSH
• taniny analýza   MeSH
• triterpeny analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Itálie MeSH
• Názvy látek
• antioxidancia MeSH
• bartogenic acid MeSH Prohlížeč
• fenoly MeSH
• flavonoidy MeSH
• glukosidy MeSH
• hydrolyzovatelné taniny MeSH
• kyselina ellagová MeSH
• NF-kappa B MeSH
• proantokyanidiny MeSH
• reaktivní formy kyslíku MeSH
• rostlinné extrakty MeSH
• taniny MeSH
• triterpeny MeSH

A conventional evaluation methodology for drinking water pollution focuses on analysing hundreds of compounds, usually by liquid chromatography-tandem mass spectrometry. High-resolution mass spectrometry allows comprehensive evaluation of all detected signals (compounds) based on their elemental composition, intensity, and numbers. We combined target analysis of 192 emerging micropollutants with nontarget (NT) full-scan/MS/MS methods to describe the impact of treatment steps in detail and assess drinking water treatment efficiency without compound identification. The removal efficiency based on target analytes ranged from - 143 to 97%, depending on the treatment section, technologies, and season. The same effect calculated for all signals detected in raw water by the NT method ranged between 19 and 65%. Ozonation increased the removal of micropollutants from the raw water but simultaneously caused the formation of new compounds. Moreover, ozonation byproducts showed higher persistence than products formed during other types of treatment. We evaluated chlorinated and brominated organics detected by specific isotopic patterns within the developed workflow. These compounds indicated anthropogenic raw water pollution but also potential treatment byproducts. We could match some of these compounds with libraries available in the software. We can conclude that passive sampling combined with nontargeted analysis shows to be a promising approach for water treatment control, especially for long-term monitoring of changes in technology lines because passive sampling dramatically reduces the number of samples and provides time-weighted average information for 2 to 4 weeks.

• Klíčová slova
• Drinking water treatment, Log2FoldChange, Nontarget analysis, Organic micropollutants, Removal efficiency, Treatment effect,
• MeSH
• chemické látky znečišťující vodu * analýza   MeSH
• čištění vody * metody   MeSH
• monitorování životního prostředí metody   MeSH
• ozon * analýza   MeSH
• pitná voda * analýza   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chemické látky znečišťující vodu * MeSH
• ozon * MeSH
• pitná voda * MeSH

Every day we are exposed to a cocktail of anthropogenic compounds many of which are biologically active and capable of inducing negative effects. The simplest way to monitor contaminants in a population is via human biomonitoring (HBM), however conventional targeted approaches require foreknowledge of chemicals of concern, often have compound specific extractions and provide information only for those compounds. This study developed an extraction process for human biomarkers of interest (BoE) in urine that is less compound specific. Combining this with an ultra-high resolution mass spectrometer capable of operating in full scan, and a suspect and non-targeted analysis (SS/NTA) approach, this method provides a more holistic characterization of human exposure. Sample preparation development was based on enzymatically hydrolysed urine spiked with 34 native standards and extracted by solid-phase extraction (SPE). HRMS data was processed by MzMine2 and 80% of standards were identified in the final data matrix using typical NTA data processing procedures.

• Klíčová slova
• Biomonitoring, Exposome, HBM4EU, Non-targeted, Sample preparation, Xenobiotic,
• MeSH
• biologické markery MeSH
• biologický monitoring MeSH
• hmotnostní spektrometrie metody   MeSH
• lidé MeSH
• monitorování životního prostředí * metody   MeSH
• vystavení vlivu životního prostředí * analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biologické markery MeSH

Rilmenidine is an alpha 2 adrenoreceptor agonist used in the treatment of mild and moderate hypertension. In this study, a fast and accurate liquid chromatographic method with tandem mass spectrometric detection has been validated in order to assure quantification of rilmenidine in human serum. The fragmentation pathway of protonated rilmenidine was studied using high-resolution mass spectrometry (HRMS). This study compared selectivity, linearity, accuracy, precision, extraction efficiency, matrix effect and sensitivity using common liquid-liquid extraction (LLE) and solid-phase extraction (SPE) procedures. The limit of quantitation for both extraction techniques was 0.1 ng/ml. Several differences between the LLE and SPE have been observed in terms of linearity, accuracy, precision and matrix effect. Additionally, the advantages of SPE included less manual work load and increased recovery of rilmenidine in human serum to approximately 80% (LLE, 57%). The developed method involving SPE was found to be accurate (relative error (RE) < 5%), reproducible (relative standard deviation, RSD < 7%), robust and suitable for quantitative analysis of rilmenidine in serum samples obtained from patients under antihypertensive treatment.

• MeSH
• antihypertenziva krev   chemie   farmakokinetika   MeSH
• chromatografie kapalinová metody   MeSH
• extrakce na pevné fázi MeSH
• lidé MeSH
• metoda nejmenších čtverců MeSH
• oxazoly krev   chemie   farmakokinetika   MeSH
• reprodukovatelnost výsledků MeSH
• rilmenidin MeSH
• senzitivita a specificita MeSH
• stabilita léku MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• antihypertenziva MeSH
• oxazoly MeSH
• rilmenidin MeSH

Pancreatic lipase (PNLIP, EC 3.1.1.3) plays a pivotal role in the digestion of dietary lipids, a metabolic pathway directly related to obesity. One of the effective strategies in obesity treatment is the inhibition of PNLIP, which is possible to be achieved by specific phenolic compounds occurring in high abundance in some plants. In this study, a multidisciplinary approach is presented investigating the PNLIP inhibitory effect of 33 plants belonging in the Asteraceae botanical family. In the first stage of the study, a rapid and cost-efficient PNLIP assay in a 96-microwell plate format was developed and important parameters were optimized, e.g., the enzyme substrate. Upon PNLIP assay optimization, aqueous and dichloromethane Asteraceae plant extracts were tested and a cut-off inhibition level was set to further analyze only the samples with a significant inhibitory effect (inhibitory rate > 40%), using an ultra-high-performance liquid chromatography hybrid quadrupole time-of-flight mass spectrometry (UHPLC-q-TOF-MS) method. Specifically, a metabolomic suspect screening was performed and 69 phenolic compounds were tentatively identified, including phenolic acids, flavonoids, flavonoid-3-O-glycosides, and flavonoid-7-O-glycosides, amongst others. In the case of aqueous extracts, phytochemicals known for inducing PNLIP inhibitory effect, e.g., compounds containing galloyl molecules or caffeoylquinic acids, were monitored in Chrysanthemum morifolium, Grindella camporum and Hieracium pilosella extracts. All in all, the presented approach combines in vitro bioactivity measurements to high-end metabolomics to identify phenolic compounds with potential medicinal and/or dietary applications.

• Klíčová slova
• bioprospecting, enzyme assay, in vitro testing, metabolomics, obesity, phytochemicals, polyphenols, suspect screening, ultra-high-performance liquid chromatography hybrid quadrupole time-of-flight mass spectrometry,
• MeSH
• Asteraceae * chemie   MeSH
• chromatografie kapalinová MeSH
• fenoly analýza   MeSH
• flavonoidy chemie   MeSH
• fytonutrienty analýza   MeSH
• glykosidy MeSH
• hmotnostní spektrometrie MeSH
• lipasa MeSH
• lipidy MeSH
• methylenchlorid MeSH
• obezita MeSH
• rostlinné extrakty chemie   farmakologie   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fenoly MeSH
• flavonoidy MeSH
• fytonutrienty MeSH
• glykosidy MeSH
• lipasa MeSH
• lipidy MeSH
• methylenchlorid MeSH
• rostlinné extrakty MeSH

Within the Human Biomonitoring for Europe initiative (HBM4EU), a study to determine new biomarkers of exposure to pesticides and to assess exposure patterns was conducted. Human urine samples (N = 2,088) were collected from five European regions in two different seasons. The objective of the study was to identify pesticides and their metabolites in collected urine samples with a harmonized suspect screening approach based on liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) applied in five laboratories. A combined data processing workflow included comprehensive data reduction, correction of mass error and retention time (RT) drifts, isotopic pattern analysis, adduct and elemental composition annotation, finalized by a mining of the elemental compositions for possible annotations of pesticide metabolites. The obtained tentative annotations (n = 498) were used for acquiring representative data-dependent tandem mass spectra (MS2) and verified by spectral comparison to reference spectra generated from commercially available reference standards or produced through human liver S9 in vitro incubation experiments. 14 parent pesticides and 71 metabolites (including 16 glucuronide and 11 sulfate conjugates) were detected. Collectively these related to 46 unique pesticides. For the remaining tentative annotations either (i) no data-dependent MS2 spectra could be acquired, (ii) the spectral purity was too low for sufficient matching, or (iii) RTs indicated a wrong annotation, leaving potential for more pesticides and/or their metabolites being confirmed in further studies. Thus, the reported results are reflecting only a part of the possible pesticide exposure.

• Klíčová slova
• Exposomics, HBM4EU, Human biomonitoring, LC-HRMS, Pesticide metabolites, Pesticides, Suspect screening,
• Publikační typ
• časopisecké články MeSH