We have developed and validated a novel LC-MS/MS method for simultaneously analyzing amino acids, biogenic amines, and their acetylated and methylated derivatives in plants. This method involves a one-step extraction of 2-5 mg of lyophilized plant material followed by fractionation of different biogenic amine forms, and exploits an efficient combination of hydrophilic interaction liquid chromatography (HILIC), reversed phase (RP) chromatography with pre-column derivatization, and tandem mass spectrometry (MS). This approach enables high-throughput processing of plant samples, significantly reducing the time needed for analysis and its cost. We also present a new synthetic route for deuterium-labeled polyamines. The LC-MS/MS method was rigorously validated by quantifying levels of nitrogen-related metabolites in seedlings of seven plant species, including Arabidopsis, maize, and barley, all of which are commonly used model organisms in plant science research. Our results revealed substantial variations in the abundance of these metabolites between species, developmental stages, and growth conditions, particularly for the acetylated and methylated derivatives and the various polyamine fractions. However, the biological relevance of these plant metabolites is currently unclear. Overall, this work contributes significantly to plant science by providing a powerful analytical tool and setting the stage for future investigations into the functions of these nitrogen-related metabolites in plants.

• Klíčová slova
• Acetylated amino acids, LC-MS/MS, acetylated biogenic amines, amino acids, biogenic amines, methylated amino acids, plant metabolism,
• MeSH
• Arabidopsis metabolismus   růst a vývoj   MeSH
• chromatografie kapalinová MeSH
• dusík * metabolismus   MeSH
• ječmen (rod) metabolismus   růst a vývoj   MeSH
• kapalinová chromatografie-hmotnostní spektrometrie MeSH
• kukuřice setá metabolismus   růst a vývoj   MeSH
• polyaminy metabolismus   analýza   MeSH
• rostliny metabolismus   MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dusík * MeSH
• polyaminy MeSH

OBJECTIVES: Accurate 25-hydroxyvitamin D (25-(OH)D) assays are essential for defining vitamin D status and ensuring appropriate clinical decisions. Standardization efforts, including the Vitamin D Standardization Program (VDSP), aim to minimize assay variability. This study evaluates the measurement uncertainty (MU) of various 25-(OH)D assays and their ability to detect physiologically relevant changes over time. METHODS: Seventeen pooled and eight single-donor serum samples were analyzed using two LC-MS/MS methods and 13 immunoassays, each applied in two independent laboratories. Imprecision, bias, and MU were assessed relative to the University of Ghent's reference measurement procedure (RMP). Results were compared against analytical performance specifications (APS) from VDSP, JCTLM-TF-RMSI, and IFCC C-BM based on physiological 25-(OH)D variation. A graphical approach was introduced to visualize MU in relation to clinical relevance. RESULTS: LC-MS/MS methods consistently met all APS criteria. Several immunoassays also achieved acceptable MU, although significant bias or inter-laboratory variability was observed for some of them. Slightly more than half of the assays met the desirable Joint Committee for Traceability in Laboratory Medicine Task Force on Reference Measurement System Implementation (JCTLM TF-RMSI) MU threshold (≤10 %), while four exceeded the minimum acceptable limit (≤15 %). The IFCC C-BM physiological approach identified a similar subset of assays. The graphical representation effectively illustrated method reliability across the tested concentration range. CONCLUSIONS: Measurement uncertainty remains a major challenge for 25-(OH)D assays. The integration of MU-based APS and graphical visualization provides a comprehensive framework for evaluating assay performance. These findings highlight the importance of selecting assays capable of reliably detecting clinically meaningful changes in vitamin D status.

• Klíčová slova
• 25-hydroxyvitamin D, LC-MS/MS, analytical performance specifications, immunoassays, measurement uncertainty, standardization,
• MeSH
• chromatografie kapalinová metody   normy   MeSH
• imunoanalýza normy   metody   MeSH
• kapalinová chromatografie-hmotnostní spektrometrie MeSH
• lidé MeSH
• nejistota MeSH
• tandemová hmotnostní spektrometrie * metody   normy   MeSH
• vitamin D * krev   analogy a deriváty   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 25-hydroxyvitamin D MeSH Prohlížeč
• vitamin D * MeSH

The use of pharmaceuticals entails a significant risk of environmental contamination. Wastewater treatment plants (WWTPs) are considered to be the main contributors to contamination as they ineffectively eliminate these compounds from wastewater. Simultaneously, they produce solid waste, sludge, which often contains a variety of retained pollutants, including pharmaceuticals. Since sewage sludge is frequently applied to agricultural soil due to its rich nutrient content, pollutants are introduced into the environment in this way. Only a few studies have been carried out on the topic of the analysis of pharmaceuticals in sludge. Therefore, information on the occurrence of pharmaceuticals in sludge is limited. The present study employed quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis to establish a simple and reliable procedure for determining 16 pharmaceuticals (antibiotics, anticonvulsants, antidepressants and β-blockers) in sewage sludge. The method has been thoroughly validated, and parameters such as linear range, accuracy, precision, matrix effects and detection and quantification limits were assessed. Our method achieved low limits of quantification (0.5-9.0 µg kg-1) and satisfactory recoveries (51-101%). Forty sludge samples from different WWTPs across the Czech Republic were analysed. Fourteen compounds were detected and quantified in most samples, with antidepressants having the highest detection frequency and overall content. Sertraline, with a mean concentration of 521.0 µg kg-1, was notably prevalent alongside its metabolite norsertraline (mean concentration 204.9 µg kg-1). The antibiotic azithromycin was also found at higher levels (mean concentration 185.1 µg kg-1).

• Klíčová slova
• LC–MS/MS, Micropollutants, Pharmaceuticals, QuEChERS, Sewage sludge,
• MeSH
• chemické látky znečišťující vodu analýza   MeSH
• chromatografie kapalinová MeSH
• kapalinová chromatografie-hmotnostní spektrometrie MeSH
• léčivé přípravky analýza   MeSH
• monitorování životního prostředí metody   MeSH
• odpadní voda chemie   MeSH
• odpadní vody * chemie   MeSH
• tandemová hmotnostní spektrometrie * MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• chemické látky znečišťující vodu MeSH
• léčivé přípravky MeSH
• odpadní voda MeSH
• odpadní vody * MeSH

Arginine-specific cleavage is the primary method used to prepare lysine-rich histone proteins in bottom-up proteomics. As the Arg-C enzyme has demonstrated suboptimal specificity, cleavage at the carboxyl side of arginine residues is typically achieved through the chemical derivatization of lysines followed by trypsin digestion. Recent improvements in proteolytic enzymes are reflected in the introduction of Arg-C Ultra, a recombinant proteinase with a substantially improved digestion specificity. Here, using mammalian histone extract, we demonstrate that Arg-C Ultra facilitates histone preparation for LC-MS/MS. We show the performance of Arg-C Ultra in terms of digestion specificity, number of modified forms identified, and yield of quantitative information compared with Arg-C and trypsin digestion combined with chemical derivatization with trimethylacetic anhydride. Importantly, we show that chemical derivatization at the peptide level, i.e., after Arg-C Ultra digestion, is still necessary to improve the quantification of short histone peptidoforms as well as positional isomers.

• MeSH
• arginin metabolismus   chemie   MeSH
• chromatografie kapalinová metody   MeSH
• histony * chemie   metabolismus   izolace a purifikace   MeSH
• kapalinová chromatografie-hmotnostní spektrometrie MeSH
• lidé MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• trypsin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• arginin MeSH
• histony * MeSH
• trypsin MeSH

Methoxphenidine (MXP), a dissociative anaesthetic derivative, has garnered the attention of toxicologists for their increasing abuse and associated toxicity. Despite that there is limited forensic and clinical toxicology data on MXP, especially regarding its metabolism and enantiomers. To fill this gap, we developed, validated and applied achiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) and chiral supercritical fluid chromatography-MS (SFC-MS) methods to quantify MXP and its primary metabolite, O-desmethyl-methoxphenidine (dmMXP), in rat serum and brain samples collected after a single subcutaneous dosing of racemic MXP. Serum samples were extracted by protein precipitation with 0.1 % formic acid in acetonitrile, and salting-out-assisted liquid-liquid extraction was utilised for brain extraction. The samples were analysed by a reversed-phase LC-MS/MS method equipped with a Poroshell 120 phenyl-hexyl column, and the enantioselective SFC-MS method was equipped with an Alcyon Amylose-SA column. Both methods were fully validated according to the European Medicines Agency guidelines. The LC-MS/MS method had a total run time of 4.8 min with a linear response up to 400 ng/mL in serum and 2400 ng/g in brain, and the limits of quantification were 1.00 ng/mL and 6.00 ng/g for MXP and 1.00 ng/mL and 1.50 ng/g for dmMXP. The enantioselective SFC-MS method had a total run time of 15 min, showing linear ranges up to 1000 ng/mL for individual enantiomers in serum and 7200 ng/g in brain. The limits of quantification of (R,S)-MXP were 12.5 ng/mL and 30.0 ng/g, while those of (R,S)-dmMXP were 25.0 ng/mL and 60.0 ng/g. The achiral LC-MS/MS method enabled quantification of racemic MXP and dmMXP, while the chiral SFC-MS method was used only for MXP enantiomers, as its higher lower limit of quantification did not allow for enantioselective quantification of dmMXP. Serum MXP peaked at 1600 ng/mL (0.5 h) and decreased to 5.87 ng/mL at 24 h, while dmMXP peaked at 11.8 ng/mL (1 h) and was below the limit of quantification at 24 h. In brain, MXP peaked at 13200 ng/g (0.5 h) and decreased to 36.1 ng/g (24 h), while dmMXP reached 67.1 ng/g (1 h) and decreased to 1.63 ng/g (24 h). Moreover, it was found that the (S)-MXP concentrations in the brain appeared to be higher than the (R)-enantiomer concentrations. The validated methods allow for the generation of pharmacokinetic curves for MXP within a behavioural study and provide a valuable tool for forensic and clinical toxicology for the study of the dissociative anaesthetic MXP and its metabolite in rat serum and brain.

• Klíčová slova
• Chiral SFC-MS, Dissociative anaesthetics, Enantiomers, LC-MS/MS, Methoxphenidine, Quantification, Rat serum and brain,
• MeSH
• chromatografie kapalinová metody   MeSH
• kapalinová chromatografie-hmotnostní spektrometrie MeSH
• krysa rodu Rattus MeSH
• limita detekce MeSH
• lineární modely MeSH
• mozek - chemie * MeSH
• mozek * metabolismus   MeSH
• piperidiny * analýza   krev   farmakokinetika   metabolismus   chemie   MeSH
• potkani Sprague-Dawley MeSH
• reprodukovatelnost výsledků MeSH
• stereoizomerie MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 1-(1-(2-methoxyphenyl)-2-phenylethyl)piperidine MeSH Prohlížeč
• piperidiny * MeSH

The success of bottom-up proteomic analysis frequently depends on the efficient removal of contaminants from protein or peptide samples before LC-MS/MS. For a peptide clean-up workflow, single-pot solid-phase-enhanced peptide sample preparation on carboxylate-modified paramagnetic beads (termed SP2) was evaluated for sodium dodecyl sulfate or polyethylene glycol removal from Arabidopsis thaliana tryptic peptides. The robust and efficient 40-min SP2 protocol, tested for 10-ng, 250-ng, and 10-µg peptide samples, was proposed and benchmarked thoroughly against the ethyl acetate extraction protocol. The SP2 protocol on carboxylated magnetic beads proved to be the most robust approach, even for the simultaneous removal of massive sodium dodecyl sulfate (SDS) and polyethylene glycol (PEG) contaminations from AT peptide samples in respect of the LC-MS/MS data outperforming ethyl acetate extraction.

• Klíčová slova
• Arabidopsis thaliana, LC-MS/MS, SP2, detergent, ethyl acetate extraction, magnetic beads, peptide clean-up, polyethylene glycol, sodium dodecyl sulfate,
• MeSH
• chromatografie kapalinová metody   MeSH
• dodecylsíran sodný MeSH
• kapalinová chromatografie-hmotnostní spektrometrie * MeSH
• peptidy analýza   MeSH
• polyethylenglykoly * MeSH
• proteomika metody   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dodecylsíran sodný MeSH
• ethyl acetate MeSH Prohlížeč
• peptidy MeSH
• polyethylenglykoly * MeSH

This study introduces an innovative approach to the long-standing challenge of determining the animal origin of blood used in artworks - an issue of central relevance to art historians aiming to understand historical techniques, symbolic meanings, and the cultural traditions associated with artistic materials. Using LC-MS/MS analysis, species-specific peptide sequences of blood proteins were identified, allowing for the discrimination of seven animal species (cat, cow, dog, goose, hen, human, and pig). This analytical approach was successfully applied to a series of model samples containing blood, confirming both the proteinaceous composition and the animal origin of the blood. Furthermore, the method enabled the identification of pig blood in historical samples taken from Japanese and Chinese lacquer artefacts dating from the 18th and 19th centuries. The novelty of this work lies in a new strategy for data evaluation that facilitates the creation of custom peptide databases tailored to distinguish specific animal species. This approach overcomes a major limitation in proteomic studies - namely, the lack of complete sequence data for many animals - by incorporating homologous sequences from closely related species. The strategy demonstrates a high degree of effectiveness when implemented within a clearly defined group of animal species, such as those historically utilised in the production of blood-based artistic materials. This approach offers a novel pathway for the molecular identification of animal origin in cultural heritage contexts and establishes a robust foundation for future interdisciplinary investigations bridging art history, conservation science, and molecular biology.

• MeSH
• chromatografie kapalinová metody   MeSH
• druhová specificita MeSH
• kapalinová chromatografie-hmotnostní spektrometrie MeSH
• krevní proteiny * analýza   chemie   MeSH
• lidé MeSH
• prasata MeSH
• psi MeSH
• sekvence aminokyselin MeSH
• skot MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• psi MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• krevní proteiny * MeSH

Saxitoxins (STXs) are potent neurotoxins produced by marine dinoflagellates or freshwater cyanobacteria known to cause acute and eventually fatal human intoxications, which are classified as paralytic shellfish poisonings (PSPs). Rapid analysis of STXs in blood plasma can be used for a timely diagnosis and confirmation of PSPs. We developed a fast and simple method of STX extraction based on plasma sample acidification and precipitation by acetonitrile, followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Our approach provides the results ≤30 min, with a limit of detection of 2.8 ng/mL and a lower limit of quantification of 5.0 ng/mL. Within-run and between-run precision experiments showed good reproducibility with ≤15% values. Standard curves for calibration were linear with correlation coefficients ≥0.98 across the assay calibration range (5-200 ng/mL). In an interlaboratory analytical exercise, the method was found to be 100% accurate in determining the presence or absence of STX in human plasma specimens, with recovery values of 86-99%. This simple method for STX determination in animal or human plasma can quickly and reliably diagnose STX exposures and confirm suspected PSP cases to facilitate patient treatment or expedite necessary public health or security actions.

• MeSH
• chromatografie kapalinová MeSH
• kapalinová chromatografie-hmotnostní spektrometrie * MeSH
• krevní plazma MeSH
• lidé MeSH
• reprodukovatelnost výsledků MeSH
• saxitoxin * MeSH
• tandemová hmotnostní spektrometrie MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• saxitoxin * MeSH

Microflow liquid chromatography interfaced with mass spectrometry (μLC-MS/MS) is increasingly applied for high-throughput profiling of biological samples and has been proven to have an acceptable trade-off between sensitivity and reproducibility. However, lipidomics applications are scarce. We optimized a μLC-MS/MS system utilizing a 1 mm inner diameter × 100 mm column coupled to a triple quadrupole mass spectrometer to establish a sensitive, high-throughput, and robust single-shot lipidomics workflow. Compared to conventional lipidomics methods, we achieve a ∼4-fold increase in response, facilitating quantification of 351 lipid species from a single iPSC-derived cerebral organoid during a 15 min LC-MS analysis. Consecutively, we injected 303 samples over ∼75 h to prove the robustness and reproducibility of the microflow separation. As a proof of concept, μLC-MS/MS analysis of Alzheimer's disease patient-derived iPSC cerebral organoid reveals differential lipid metabolism depending on APOE phenotype (E3/3 vs E4/4). Microflow separation proves to be an environmentally friendly and cost-effective method as it reduces the consumption of harmful solvents. Also, the data demonstrate robust, in-depth, high-throughput performance to enable routine clinical or biomedical applications.

• MeSH
• apolipoproteiny E MeSH
• chromatografie kapalinová metody   MeSH
• fenotyp MeSH
• kapalinová chromatografie-hmotnostní spektrometrie * MeSH
• lidé MeSH
• lipidomika MeSH
• reprodukovatelnost výsledků MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• apolipoproteiny E MeSH

Aderamastat (FP-025) is a small molecule, selective matrix metalloproteinase (MMP)-12 inhibitor, under development for respiratory conditions which may include chronic inflammatory airway diseases and pulmonary fibrosis. To support evaluation of the pharmacokinetic parameters of Aderamastat in humans, we developed and validated a high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analytical method for the quantification of Aderamastat in human plasma. This assay was validated in compliance with the Food and Drug Administration (FDA) Good Laboratory Practice Regulations (GLP) and European Medicines Agency (EMA) guidelines. K2EDTA human plasma samples were spiked with internal standard, processed by liquid-liquid extraction, and analyzed using reversed-phase HPLC with Turbo Ion Spray® MS/MS detection. Separation was done using a chromatographic gradient on 5 µm C6-Phenyl 110 Å, 50*2 mm analytical column at a temperature of 35 °C. The LC-MS/MS bioanalytical method, developed by QPS Taiwan to determine the concentration of Aderamastat in K2EDTA human plasma, was successfully validated with respect to linearity, sensitivity, accuracy, precision, dilution, selectivity, hemolyzed plasma, lipemic plasma, batch size, recovery, matrix effect, and carry-over. These data indicate that the method for determination of Aderamastat concentrations in human K2EDTA plasma can be used in pharmacokinetics studies and subsequent clinical trials with Aderamastat. Authors declare that, this novel data is not published and not under consideration for publication by another journal than this journal. All data will be made available on request.

• Klíčová slova
• Aderamastat, Bioanalysis Assay, FP-025, LC-MS/MS, Matrix Metalloproteinase (MMP)-12 inhibitor, Validation,
• MeSH
• adamantan analogy a deriváty   krev   farmakokinetika   chemie   MeSH
• chromatografie kapalinová metody   MeSH
• EDTA * chemie   krev   farmakokinetika   MeSH
• kapalinová chromatografie-hmotnostní spektrometrie MeSH
• lidé MeSH
• limita detekce MeSH
• lineární modely MeSH
• reprodukovatelnost výsledků MeSH
• stabilita léku MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adamantan MeSH
• EDTA * MeSH