While the use of food additives is common manufacturing practice, the levels used in food have to be compliant with the prescribed legislation. For fast control of present levels of food additives in products, ultra-high performance liquid chromatography coupled to tandem mass spectrometry with a triple quadrupole linear ion trap (QTRAP) mass analyser was applied to develop a method for the simultaneous determination of 41 frequently added food additives and flavourings, including 16 water-soluble colourants, 14 illegal dyes, 7 sweeteners, 2 preservatives, and 2 purine alkaloids. The method was validated using energy drink, chilli powder, condiment, and jelly sweets as food sample matrices. The average recovery values were in the range of 70‒120%, and the relative standard deviations were less than 10% for the majority of the analytes. The validated method was applied for the analysis of 134 samples from the Czech market.

• Klíčová slova
• Food colourants, Sudan dyes, UPLC-MS/MS, adulteration, multimethod,
• MeSH
• analýza potravin * metody   MeSH
• chuťové esence analýza   MeSH
• kapalinová chromatografie-hmotnostní spektrometrie MeSH
• kontaminace potravin * analýza   MeSH
• lidé MeSH
• limita detekce MeSH
• nápoje * analýza   MeSH
• potravinářské přísady * analýza   MeSH
• reprodukovatelnost výsledků MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• validační studie MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• chuťové esence MeSH
• potravinářské přísady * MeSH

The use of contaminated raw materials can lead to the transfer of mycotoxins into the final product, including beer. This study describes the use of the commercially available immunoaffinity column 11+Myco MS-PREP® and UPLC-MS/MS for the determination of mycotoxins in pale lager-type beers brewed in Czech Republic and other European countries. The additional aim of the work was to develop, optimize and validate this analytical method. Validation parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), precision and accuracy were tested. The calibration curves were linear with correlation coefficients (R2 > 0.99) for all mycotoxins under investigation. The LOD ranged from 0.1 to 50 ng/L and LOQ from 0.4 to 167 ng/L. Recoveries of the selected analytes ranged from 72.2 to 101.1%, and the relative standard deviation under conditions repeatability (RSDr) did not exceed 16.3% for any mycotoxin. The validated procedure was successfully applied for the analysis of mycotoxins in a total of 89 beers from the retail network. The results were also processed using advanced chemometric techniques and compared with similar published studies. The toxicological impact was taken into account.

• Klíčová slova
• 11+Myco MS-PREP®, Beer, Dietary exposure, Immunoaffinity columns, Mycotoxins, UPLC-MS/MS,
• MeSH
• chromatografie kapalinová metody   MeSH
• dietární expozice analýza   MeSH
• mykotoxiny * analýza   MeSH
• pivo analýza   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mykotoxiny * MeSH

Alkaloids known as secondary metabolites are grouped by typical structural characteristics into large families such as pyrrolizidine alkaloids (PAs) comprising more than 350 individual heterocyclic compounds. The PAs present a serious health risk to human and livestock; hence there is a need for methods that allow these dangerous plant toxins to be determined. In this study, a fast, reliable and sensitive approach is proposed to identify and quantify PAs in feed samples. PAs including monocrotaline, senkirkine, senecionine, seneciphylline and retrorsine were determined by ultra-performance liquid chromatography coupled with tandem mass spectrometry. Sample preparation was based on a modified QuEChERS approach. The mean recovery, precision, matrix effects and limits of quantification were assessed for three matrices within the method validation. The presented method was used to inspect 41 various feed samples, where the presence of PAs was expected. Roughages and feed for rabbits contained the highest levels of PAs, in general.

• Klíčová slova
• Feeds, Plant toxins, Pyrrolizidine alkaloids, QuEChERS, UPLC–MS/MS,
• MeSH
• chromatografie kapalinová metody   MeSH
• pyrrolizidinové alkaloidy chemie   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• pyrrolizidinové alkaloidy MeSH

Milk thistle [Silybum marianum (L.) Gaertn.] achieved a significant increase in interest over the past few years from local and foreign pharmaceutical corporations. The silymarin complex of constituents extracted from milk thistle achenes provides compelling health benefits primarily thanks to antioxidant activities and hepatoprotective effects. However, consuming mycotoxin-contaminated plant material can cause immunosuppression and hepatotoxic problems. The aim of this study was to develop and validate a method for the determination of mycotoxin content in milk thistle. Fusarium toxins as T-2 and HT-2 toxins in grown milk thistle harvested from a breeding station in the Czech Republic during 2020-2021 were studied. The analysis of T-2 and HT-2 toxins was performed by UPLC-MS/MS after immunoaffinity columns EASI-EXTRACT® T-2 & HT-2 clean up. All analysed samples of milk thistle were contaminated with T-2 toxin and HT-2 toxin. The content of T-2 toxin in the samples from 2020 was in the range of 122.7-290.2 µg/kg and HT-2 toxin 157.0-319.0 µg/kg. In 2021, the content of T-2 toxin was in the range of 28.8-69.9 µg/kg and HT-2 toxin was 24.2-75.4 µg/kg. The results show that the climatic conditions of the year of harvesting have a highly statistically significant effect on the content of T-2 and HT-2 toxins in milk thistle.

• Klíčová slova
• HT-2 toxin, T-2 toxin, UPLC-MS/MS, immunoaffinity column, milk thistle, validation method,
• MeSH
• antioxidancia MeSH
• biologické přípravky * MeSH
• chromatografie kapalinová MeSH
• flavonoidy MeSH
• mykotoxiny * MeSH
• ostropestřec mariánský MeSH
• semena rostlinná MeSH
• silymarin * MeSH
• šlechtění rostlin MeSH
• T-2 toxin * analogy a deriváty   MeSH
• tandemová hmotnostní spektrometrie MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antioxidancia MeSH
• biologické přípravky * MeSH
• flavonoidy MeSH
• HT-2 toxin MeSH Prohlížeč
• mykotoxiny * MeSH
• silymarin * MeSH
• T-2 toxin * MeSH

Kalaharia uncinata (Schinz) Moldenke, is a tropical erect bushy shrub or subshrub of the Lamiaceae family. It is an endemic plant species of Southern Africa, widely used in the pharmacopoeia against upper respiratory tract infections. A previously conducted ethnobotanical survey revealed that it is believed to contain bioactive substances. However, no relevant phytochemical information was available. This study aimed to perform a phytochemical characterization of K. uncinata and also to discuss the potential bioactivity of the identified phytochemical constituents based on documented data. Ultra-performance liquid chromatography with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) was used for profiling and identification of the main phytochemical constituents from leaf extracts (MeOH 90 %, DCM, AcOEt, BuOH, hexane and residue) of K.uncinata. Twenty-four constituents, representing mainly flavonoids (14), followed by phenylethanoid glycosides (7), phenolic acids (2), and an iridoid glycoside (1) were tentatively identified. Most of the identified compounds are documented to have antiviral and anti-inflammatory properties, which could possibly be the rationale behind the use of K. uncinata against upper respiratory tract infections.

• Klíčová slova
• Kalaharia uncinata, Lamiaceae, UPLC-ESI-QTOF-MS, phytochemical profiling, upper respiratory tract infections,
• MeSH
• fytonutrienty chemie   MeSH
• glykosidy * MeSH
• hluchavkovité * MeSH
• tradiční lékařství MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Konžská demokratická republika MeSH
• Názvy látek
• fytonutrienty MeSH
• glykosidy * MeSH

A rapid, high-throughput method employing ultra-performance liquid chromatography with tandem quadrupole mass spectrometry (UPLC-MS/MS) was developed and optimized for simultaneous quantification and confirmation of 64 pesticide residues and their toxic metabolites in fruit extracts prepared by a buffered QuEChERS procedure. The total time required for UPLC-MS/MS analysis was 8 min plus 2 min for re-equilibration to the initial UPLC conditions. Performance characteristics were determined for apple extracts spiked at 10 microg kg(-1). The repeatability of measurements expressed as relative standard deviations was in the range 1.5-13% at this level for most analytes. Thanks to very low limits of quantification (<10 microg kg(-1)for the majority of pesticides), an optimized method allows for the reliable control of not only common maximum residue limits (MRLs) set by European Union regulation for various pesticides/fruit combinations, but also of a uniform MRL of 10 microg kg(-1)endorsed for baby food.

• MeSH
• časové faktory MeSH
• Evropská unie MeSH
• kalibrace MeSH
• kojenec MeSH
• lidé MeSH
• Malus chemie   MeSH
• maximální přípustná koncentrace MeSH
• potrava pro kojence normy   MeSH
• referenční standardy MeSH
• reprodukovatelnost výsledků MeSH
• rezidua pesticidů analýza   MeSH
• řízení kvality MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• validační studie MeSH
• Názvy látek
• rezidua pesticidů 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

BACKGROUND: In vivo solid-phase microextraction (SPME) is a minimally invasive, non-exhaustive sample-preparation technique that facilitates the direct isolation of low molecular weight compounds from biological matrices in living systems. This technique is especially useful for the analysis of phytocannabinoids (PCs) in plant material, both for forensic purposes and for monitoring the PC content in growing Cannabis spp. plants. In contrast to traditional extraction techniques, in vivo SPME enables continuous tracking of the changes in the level of PCs during plant growth without the need for plant material collection. In this study, in vivo SPME utilizing biocompatible C18 probes and liquid-chromatography coupled to quadrupole time-of flight mass spectrometry (LC-Q-TOF-MS) is proposed as a novel strategy for the extraction and analysis of the acidic forms of five PCs in growing medicinal cannabis plants. RESULTS: The SPME method was optimized by testing various parameters, including the extraction phase (coating), extraction and desorption times, and the extraction temperature. The proposed method was validated with satisfactory analytical performance regarding linearity (10-3000 ng/mL), limits of quantification, and precision (relative standard deviations below 5.5 %). The proposed method was then successfully applied for the isolation of five acidic forms of PCs, which are main components of growing medicinal cannabis plants. As a proof-of-concept, SPME probes were statically inserted into the inflorescences of two varieties of Cannabis spp. plants (i.e., CBD-dominant and Δ9-THC-dominant) cultivated under controlled conditions for 30 min extraction of tetrahydrocannabinolic acid (Δ9-THCA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabiviarinic acid (CBVA), and tetrahydrocannabivarinic acid (THCVA). SIGNIFICANCE AND NOVELTY: The results confirmed that the developed SPME-LC-Q-TOF-MS method is a precise and efficient tool that enables direct and rapid isolation and analysis of PCs under in vivo conditions. The proposed methodology is highly appealing option for monitoring the metabolic pathways and compositions of multiple PCs in medicinal cannabis at different stages of plant growth.

• Klíčová slova
• Biocompatible probes, Cannabis spp., In vivo sampling, Phytocannabinoids, Solid-phase microextraction,
• MeSH
• Cannabis * chemie   MeSH
• kanabinoidy * analýza   MeSH
• kapalinová chromatografie-hmotnostní spektrometrie * metody   MeSH
• mikroextrakce na pevné fázi * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kanabinoidy * MeSH

We have recently demonstrated the ability of a C18 stationary phase with a positively charged surface (PCS-C18) to provide superior chromatographic separation of peptides using mobile phase acidified with a mere 0.01 % formic acid, significantly improving MS sensitivity. Here, we examined three columns packed with different PCS-C18 phases using the MS-favorable mobile phase acidified with low formic acid concentrations to establish the impact of separation performance and better MS sensitivity on peptide identifications. The surface charge interaction was evaluated using the retention of nitrate. The highest interaction was observed for the AdvanceBio Peptide Plus column. A surface charge-dependent shift in the retention time of peptides was confirmed with a change in formic acid concentration in the mobile phase. The separation performance of the columns with MS-favorable mobile phase acidified with low concentrations of formic acid was evaluated using well-characterized peptides. The loading capacity was assessed using a basic peptide with three lysine residues. Good chromatographic peak shapes and high loading capacity were observed for the Acquity Premier CSH C18 column, even when using a mobile phase acidified with 0.01 % formic acid. The extent of improvement in peptide identification when using reduced formic acid concentration was evaluated by analyzing the tryptic digest of trastuzumab and tryptic digest of whole bacteria cell lysate. Each column provided improved MS signal intensity and peptide identification when using the mobile phase with 0.01 % formic acid. The ability of the Acquity Premier CSH C18 column to provide better separation of peptides, even with a reduced formic acid concentration in the mobile phase, boosted MS signal intensity by 65 % and increased the number of identified peptides from the bacterial sample by 19 %. Our study confirms that significant improvement in the proteomic outputs can be achieved without additional costs only by tailoring the chemistry of the stationary phase to the composition of the mobile phase. Our results can help researchers understand the retention mechanism of peptides on the PCS-C18 stationary phases using low-ionic strength mobile phases and, more importantly, select the best-suited stationary phases for their LC-MS proteomic applications.

• Klíčová slova
• Formic acid, Peptide separation, Proteomics, Reversed-phase liquid chromatography, Stationary phases,
• MeSH
• chromatografie kapalinová metody   MeSH
• formiáty * chemie   MeSH
• kapalinová chromatografie-hmotnostní spektrometrie MeSH
• peptidy chemie   analýza   izolace a purifikace   MeSH
• proteomika * metody   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• trastuzumab chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• formiáty * MeSH
• formic acid MeSH Prohlížeč
• peptidy MeSH
• trastuzumab MeSH

Ecdysteroids represent a large class of polyhydroxylated steroids which, due to their anabolic properties, are marketed as dietary supplements. Some ecdysteroids also act as important hormones in arthropods, where they regulate molting, development, and reproduction and many of these insects are miniature organisms that contain submicroliter levels of circulating biofluids. Analysis of ecdysteroids is further complicated by their very low abundance, large fluctuations during development, and difficult access to a pooled sample, which is important for quantitative measurements. In this work, we propose a new method that overcomes the described difficulties and allows validated quantification of four ecdysteroids in minimal amounts of biological material. After methanolic extraction, detectability of the ecdysteroids is increased 16- to 20-fold by conversion to their 14,15-anhydrooximes. These are further purified by pipette tip solid-phase extraction on a three-layer sorbent and subjected to HPLC-MS/MS analysis. Full validation was achieved using hemolymph from larvae of the firebug Pyrrhocoris apterus as a blank matrix and by the determination of ecdysteroids in a single Drosophila larva. The lower limit of quantifications for the four target ecdysteroids (20-hydroxyecdysone, ecdysone, makisterone A, and 2-deoxyecdysone) were 0.01; 0.1; 0.05; and 0.025 pg·ml-1 (20; 200; 100; 50 fmol ml-1), respectively, with very good accuracy, precision (expressed as relative standard deviation <15%) and recoveries (96%-119.9%). The application potential of the new method was demonstrated by quantification of ecdysteroids in various biological materials including human serum.

• Klíčová slova
• arthropods, dietary supplementation, ecdysteroid hormones, human body fluid, quantification, submilligram sample amount, ultratrace HPLC-MS analysis,
• MeSH
• ekdysteroidy * analýza   krev   chemie   MeSH
• hemolymfa chemie   metabolismus   MeSH
• kapalinová chromatografie-hmotnostní spektrometrie MeSH
• larva MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• validační studie MeSH
• Názvy látek
• ekdysteroidy * MeSH