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There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression products with a high potential for abuse in horseracing. As these substances are not covered by most routine anti-doping analytical approaches, they represent an entire class of compounds that are not readily detectable. The nucleotide sequence for each oligonucleotide is highly specific, which makes targeted analysis for these agents problematic. Accordingly, we have developed a non-targeted approach to detect the presence of specific product ions that are not naturally present in ribonucleic acids. Briefly, serum samples were extracted using solid-phase extraction with a mixed-mode cartridge following the disruption of protein interactions to isolate the oligonucleotides. Following the elution and concentration steps, chromatographic separation was achieved utilizing reversed-phase liquid chromatography. Following an introduction to a Thermo Q Exactive HF mass spectrometer using electrospray ionization, analytes were detected utilizing a combination of full-scan, parallel reaction monitoring and all ion fragmentation scan modes. The limits of detection were determined along with the accuracy, precision, stability, recovery, and matrix effects using a representative 13mer oligonucleotide. Following method optimization using the 13mer oligonucleotide, the method was applied to successfully detect the presence of specific product ions in three unique oligonucleotide sequences targeting equine-specific transcripts.

Fluoroquinolones (FQs) are broad-spectrum antibiotics widely used to treat animal and human infections. The use of FQs in these activities has increased the presence of antibiotics in wastewater and food, triggering antimicrobial resistance, which has severe consequences for human health. The detection of antibiotics residues in water and food samples has attracted much attention. Herein, we report the development of a highly sensitive online solid-phase extraction methodology based on a selective molecularly imprinted polymer (MIP) and fluorescent detection (HPLC-FLD) for the determination of FQs in water at low ng L−1 level concentration. Under the optimal conditions, good linearity was obtained ranging from 0.7 to 666 ng L−1 for 7 FQs, achieving limits of detection (LOD) in the low ng L−1 level and excellent precision. Recoveries ranged between 54 and 118% (RSD < 17%) for all the FQs tested. The method was applied to determining FQs in river water. These results demonstrated that the developed method is highly sensitive and selective., Depto. de Química Analítica, Fac. de Ciencias Químicas, TRUE, pub

The objective of this research is to describe the impact of different advanced oxidation processes used for the removal of sulfamethoxazole on wastewater quality. Ozone, UV, a combination of ozone and UV, and plasma discharge were employed. Concentrations of sulfamethoxazole were analysed by LC-MS/MS. Chemical and microbiological analyses and ecotoxicological tests were conducted to investigate the quality of treated wastewater. The results of this study show that the most effective technique for the removal of sulfamethoxazole is O3, followed by plasma discharge, O3 + UV and, finally, UV. A significant effect of tested advanced oxidation processes on the chemical composition of wastewater was not observed; however, the number of microorganisms was reduced. In the case of ecotoxicological tests with Lemna minor and crustacean Daphnia magna, a significant negative effect was only observed when plasma was applied.

The objective of this research is to describe the impact of different advanced oxidation processes used for the removal of sulfamethoxazole on wastewater quality. Ozone, UV, a combination of ozone and UV, and plasma discharge were employed. Concentrations of sulfamethoxazole were analysed by LC-MS/MS. Chemical and microbiological analyses and ecotoxicological tests were conducted to investigate the quality of treated wastewater. The results of this study show that the most effective technique for the removal of sulfamethoxazole is O3, followed by plasma discharge, O3 + UV and, finally, UV. A significant effect of tested advanced oxidation processes on the chemical composition of wastewater was not observed; however, the number of microorganisms was reduced. In the case of ecotoxicological tests with Lemna minor and crustacean Daphnia magna, a significant negative effect was only observed when plasma was applied.

The objective of this research is to describe the impact of different advanced oxidation processes used for the removal of sulfamethoxazole on wastewater quality. Ozone, UV, a combination of ozone and UV, and plasma discharge were employed. Concentrations of sulfamethoxazole were analysed by LC-MS/MS. Chemical and microbiological analyses and ecotoxicological tests were conducted to investigate the quality of treated wastewater. The results of this study show that the most effective technique for the removal of sulfamethoxazole is O3, followed by plasma discharge, O3 + UV and, finally, UV. A significant effect of tested advanced oxidation processes on the chemical composition of wastewater was not observed; however, the number of microorganisms was reduced. In the case of ecotoxicological tests with Lemna minor and crustacean Daphnia magna, a significant negative effect was only observed when plasma was applied.

FLAG® tag (DYKDDDDK) is a small epitope peptide employed for the purification of recombinant proteins such as immunoglobulins, cytokines, and gene regulatory proteins. It provides superior purity and recoveries of fused target proteins when compared to the commonly used His-tag. Nevertheless, the immunoaffinity-based adsorbents required for their isolation are far more expensive than the ligand-based affinity resin used in combination with the His-tag. In order to overcome this limitation we report herein the development of molecularly imprinted polymers (MIPs) selective to the FLAG® tag. The polymers were prepared by the epitope imprinting approach using a four amino acids peptide, DYKD, including part of the FLAG® sequence as template molecule. Different kinds of magnetic polymers were synthesised in aqueous and organic media also using different sizes of magnetite core nanoparticles. The synthesised polymers were used as solid phase extraction materials with excellent recoveries and high specificity for both peptides. The magnetic properties of the polymers confer a new, effective, simple, and fast method in the purification using FLAG® tag., Ministerio de Economía, Comercio y Empresa (España), Depto. de Química Analítica, Fac. de Ciencias Químicas, TRUE, pub

This paper proposes a simple ion chromatographic approach to determine trace amounts of perchlorate in river water samples. Determination of the trace perchlorate by ion chromatography typically faces two challenges: interference by matrix ions such as chloride, nitrate, and sulfate in the samples and insufficient detection sensitivity. In the present study, online pretreatment of the samples with an OnGuard II Ba/Ag/H disposable sample pretreatment cartridge prevented the sulfate peak tailing from overlapping with the perchlorate peak on the chromatogram. In addition, the matrix removal enabled as large as 10 mL of sample to be loaded into a high exchange capacity anion concentrator, significantly improving perchlorate's detection sensitivity. The proposed approach achieved a detection limit (S/N = 3) of 0.046 µg L-1 without using a costly mass spectrometer and successfully determined sub µg L-1 levels of perchlorate in river water.

The objective of this research is to describe the impact of different advanced oxidation processes used for the removal of sulfamethoxazole on wastewater quality. Ozone, UV, a combination of ozone and UV, and plasma discharge were employed. Concentrations of sulfamethoxazole were analysed by LC-MS/MS. Chemical and microbiological analyses and ecotoxicological tests were conducted to investigate the quality of treated wastewater. The results of this study show that the most effective technique for the removal of sulfamethoxazole is O3, followed by plasma discharge, O3 + UV and, finally, UV. A significant effect of tested advanced oxidation processes on the chemical composition of wastewater was not observed; however, the number of microorganisms was reduced. In the case of ecotoxicological tests with Lemna minor and crustacean Daphnia magna, a significant negative effect was only observed when plasma was applied.

The objective of this research is to describe the impact of different advanced oxidation processes used for the removal of sulfamethoxazole on wastewater quality. Ozone, UV, a combination of ozone and UV, and plasma discharge were employed. Concentrations of sulfamethoxazole were analysed by LC-MS/MS. Chemical and microbiological analyses and ecotoxicological tests were conducted to investigate the quality of treated wastewater. The results of this study show that the most effective technique for the removal of sulfamethoxazole is O3, followed by plasma discharge, O3 + UV and, finally, UV. A significant effect of tested advanced oxidation processes on the chemical composition of wastewater was not observed; however, the number of microorganisms was reduced. In the case of ecotoxicological tests with Lemna minor and crustacean Daphnia magna, a significant negative effect was only observed when plasma was applied.

The objective of this research is to describe the impact of different advanced oxidation processes used for the removal of sulfamethoxazole on wastewater quality. Ozone, UV, a combination of ozone and UV, and plasma discharge were employed. Concentrations of sulfamethoxazole were analysed by LC-MS/MS. Chemical and microbiological analyses and ecotoxicological tests were conducted to investigate the quality of treated wastewater. The results of this study show that the most effective technique for the removal of sulfamethoxazole is O3, followed by plasma discharge, O3 + UV and, finally, UV. A significant effect of tested advanced oxidation processes on the chemical composition of wastewater was not observed; however, the number of microorganisms was reduced. In the case of ecotoxicological tests with Lemna minor and crustacean Daphnia magna, a significant negative effect was only observed when plasma was applied.

This paper proposes a simple ion chromatographic approach to determine trace amounts of perchlorate in river water samples. Determination of the trace perchlorate by ion chromatography typically faces two challenges: interference by matrix ions such as chloride, nitrate, and sulfate in the samples and insufficient detection sensitivity. In the present study, online pretreatment of the samples with an OnGuard II Ba/Ag/H disposable sample pretreatment cartridge prevented the sulfate peak tailing from overlapping with the perchlorate peak on the chromatogram. In addition, the matrix removal enabled as large as 10 mL of sample to be loaded into a high exchange capacity anion concentrator, significantly improving perchlorate's detection sensitivity. The proposed approach achieved a detection limit (S/N = 3) of 0.046 µg L-1 without using a costly mass spectrometer and successfully determined sub µg L-1 levels of perchlorate in river water.

The objective of this research is to describe the impact of different advanced oxidation processes used for the removal of sulfamethoxazole on wastewater quality. Ozone, UV, a combination of ozone and UV, and plasma discharge were employed. Concentrations of sulfamethoxazole were analysed by LC-MS/MS. Chemical and microbiological analyses and ecotoxicological tests were conducted to investigate the quality of treated wastewater. The results of this study show that the most effective technique for the removal of sulfamethoxazole is O3, followed by plasma discharge, O3 + UV and, finally, UV. A significant effect of tested advanced oxidation processes on the chemical composition of wastewater was not observed; however, the number of microorganisms was reduced. In the case of ecotoxicological tests with Lemna minor and crustacean Daphnia magna, a significant negative effect was only observed when plasma was applied.

Our current knowledge of the chemical composition of ocean dissolved organic matter (DOM) is limited, mainly because of its extreme molecular diversity, low concentration of individual compounds and the elevated ionic strength of ocean waters. As a result, many analytical methods require a previous extraction step. The efficiency and selectivity of the extraction method defines the representativeness of the extracted DOM fraction. Nowadays, the most widespread procedure for concentrating DOM is solid phase extraction (SPE) using styrene divinyl benzene polymer cartridges (PPL). Here, we investigate the effect of SPE-PPL on DOM elemental and optical properties to assess the efficiency and selectivity of this extraction method on water samples from the main intermediate and deep water masses of Arctic, Mediterranean and Antarctic origin present in the Cape Vert Frontal Zone (CVFZ, NW Africa). Furthermore, North and South Atlantic Central waters converge in this area and coastal DOM is injected by the giant upwelling filament of Cape Blanc. On one side, the colored fraction of DOM (CDOM) presented extraction efficiencies comparable to that of the bulk dissolved organic carbon (DOC), but decreased significantly with increasing wavelength, suggesting an affinity of PPL cartridges for low molecular weight organic compounds. While the protein-like fluorescent fraction of DOM (FDOM) was also extracted with the same efficiency than DOC, the extraction efficiency of the humic-like fraction was comparatively much higher. On the other side, dissolved organic nitrogen (DON) extraction efficiencies were about half that of DOC. These contrasting extraction efficiencies of the different DOM pools indicated that the extracts were enriched in N-poor, low molecular weight and recalcitrant DOM, therefore showing less variability than the corresponding bulk DOM. Furthermore, DOC, DON, CDOM and FDOM extracted were not homogeneous through the water column but displayed certain significant

UV-benzotriazoles have been identified as water micropollutants that cause serious problems for human health and the environment. Their low concentration in water bodies complicates their detection by direct water analysis, slowing the corrective actions to avoid bioaccumulation. In this regard, the use of graphene-based materials with a high affinity for non-polar molecules has been demonstrated to be a potential tool for the optimal separation and concentration of this type of molecules in solid phase extraction (SPE) processes. This work evaluates the potential of novel reduced graphene oxide aerogels (rGO) as extractants of mixtures of three UV-benzotriazoles in water at low concentrations. These rGO aerogels incorporate graphenic domains into a tough structure of polymeric chains by adding graphene oxide during the synthesis of resorcinol-formaldehyde gels. Aerogels with a different content and ordering of graphenic domains were obtained and characterized using Raman, XRD, SEM and nitrogen adsorption isotherms (−196 °C). The rGO aerogels that performed better as solid phase extractants were those containing 60% rGO. Aerogels with lower rGO contents (40%) required a high-temperature (2000 °C) treatment to render competitive results. The SPE methodology using selected rGO aerogels was optimized by varying the elution solvent, elution time and volume. The best performances, i.e., recoveries of 80–100% and enrichment factors of 12.5–50, were accomplished when using 0.8 mL of tetrahydrofuran (THF) as an elution solvent. As a result, a fast (10 min) and simple extraction method of UV-benzotriazoles in water was attained, achieving a detection limit of 1 ng mL−1. Selected aerogels were finally tested for the SPE of spiked samples of river waters, showing a similar performance to that observed with synthetic mixtures.

The adequate quantification of endocannabinoids and related N-acylethanolamines can be complex due to their low endogenous levels, structural diversity, and metabolism. Therefore, advanced analytical approaches, involving LC–MS, are required to quantify these molecules in plasma, tissues, and other matrices. It has been shown that endocannabinoid congeners synthesized from n-3 poly-unsaturated fatty acids (n-3 PUFAs), such as docosahexaenoylethanolamide (DHEA) and eicosapentaenoylethanolamide (EPEA), have interesting immunomodulatory and tumor-inhibiting properties. Recent work has shown that DHEA and EPEA can be further enzymatically metabolized by cyclo-oxygenase 2 (COX-2), forming oxygenated metabolites. Here, an LC–MS-based method for the quantification of the n-3 PUFA-derived endocannabinoid congeners DHEA and EPEA is described, which is also suited to measure a wider spectrum of endocannabinoids. The chapter contains a step-by-step protocol for the analysis of (n-3) endocannabinoids in plasma, including sample collection and solid phase extraction, LC–MS analysis, and data processing. In addition, protocol modifications are provided to allow quantification of n-3 PUFA-derived endocannabinoids and their COX-2 metabolites in tissues and cell culture media. Finally, conditions that alter endocannabinoid concentrations are briefly discussed.

This paper describes the synthesis of novel molecularly imprinted magnetic nano-beads for the selective extraction (MISPE) of zearalenone mycotoxin in river and tap waters and further analysis by high-performance liquid chromatography (HPLC) with fluorescence detection (FLD). A semi-covalent imprinting approach was achieved for the synthesis of the molecularly imprinted polymers (MIP). The nanoparticles were prepared by covering the starting Fe3O4 material with a first layer of tetraethyl orthosilicate (TEOS) and then with a second layer using cyclododecyl 2-hydroxy-4-(3-triethoxysilylpropylcarbamoyloxy) benzoate. The last was used with a dual role, template and functional monomer after the extraction of the template molecule. The material was characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopies (FT-IR). The solid phase extraction was optimized in all the steps: loading, washing and elution. The optimal conditions allowed the determination of zearalenone in trace levels of 12.5, 25 and 50 µg L−1 without significant differences between the fortified and found level concentrations., MICIN, Depto. de Química Analítica, Fac. de Ciencias Químicas, TRUE, pub

Fluoroquinolones (FQs) are broad-spectrum antibiotics widely used to treat animal and human infections. The use of FQs in these activities has increased the presence of antibiotics in wastewater and food, triggering antimicrobial resistance, which has severe consequences for human health. The detection of antibiotics residues in water and food samples has attracted much attention. Herein, we report the development of a highly sensitive online solid-phase extraction methodology based on a selective molecularly imprinted polymer (MIP) and fluorescent detection (HPLC-FLD) for the determination of FQs in water at low ng L−1 level concentration. Under the optimal conditions, good linearity was obtained ranging from 0.7 to 666 ng L−1 for 7 FQs, achieving limits of detection (LOD) in the low ng L−1 level and excellent precision. Recoveries ranged between 54 and 118% (RSD < 17%) for all the FQs tested. The method was applied to determining FQs in river water. These results demonstrated that the developed method is highly sensitive and selective., MINECO, Depto. de Química Analítica, Fac. de Ciencias Químicas, TRUE, pub

Последњих година примећен је све већи број акутних тровања неуролептицима, како намерних услед злоупотребе, тако и случајних због њихове уске терапијске ширине., Lately, an increasing number of acute neuroleptic poisonings has been observed, both intentional due to abuse and accidental due to their narrow therapeutic range.

Analysis of biological samples is affected by interfering substances with chemical properties similar to those of the target analytes, such as drugs. Biological samples such as whole blood, plasma, serum, urine and saliva must be properly processed for separation, purification, enrichment and chemical modification to meet the requirements of the analytical instruments. This causes the sample preparation stage to be of undeniable importance in the analysis of such samples through methods such as microextraction techniques. The scope of this review will cover a comprehensive summary of available literature data on microextraction techniques playing a key role for analytical purposes, methods of their implementation in common biological samples, and finally, the most recent examples of application of microextraction techniques in preconcentration of analytes from urine, blood and saliva samples. The objectives and merits of each microextration technique are carefully described in detail with respect to the nature of the biological samples. This review presents the most recent and innovative work published on microextraction application in common biological samples, mostly focused on original studies reported from 2017 to date. The main sections of this review comprise an introduction to the microextraction techniques supported by recent application studies involving quantitative and qualitative results and summaries of the most significant, recently published applications of microextracion methods in biological samples. This article considers recent applications of several microextraction techniques in the field of sample preparation for biological samples including urine, blood and saliva, with consideration for extraction techniques, sample preparation and instrumental detection systems., QC 20220316

Our previously reported, first validated, UPLC-MS/MS-based simultaneous analysis of five human milk B-vitamins revealed severe matrix effects. High levels of endogenous lactose fouled the electrospray ionization source affecting the analysis. We evaluated solid-phase extraction (SPE), liquid-solid extraction (LSE), protein precipitation (PPT), and liquid chromatography effluent diversion for lactose-removal. SPE failed to separate lactose from vitamins; LSE using 2-propanol reduced lactose and vitamin recoveries. PPT-solvent, milk volume, and reconstitution solvent influenced flavin adenine dinucleotide, pyridoxal and nicotinamide recoveries. Using an optimized LC-gradient enabled chromatographic separation of lactose from vitamins and its removal using a post-column switch-valve. Only 40 µL milk was subjected to methanol-PPT and non-polar matrix removal by methyl tert-butyl ether. B-vitamin recoveries were established (81.9-118.6%; CV ≤ 11.9%; precision: 4.9-13.7%) with greatly reduced matrix effects, and improved process efficiency, and recovery.

Nucleotides fulfill many essential functions in plants. Compared to non-plant systems, these hydrophilic metabolites have not been adequately investigated in plants, especially the less abundant nucleotide species such as deoxyribonucleotides and modified or damaged nucleotides. Until recently, this was mainly due to a lack of adequate methods for in-depth analysis of nucleotides and nucleosides in plants. In this review, we focus on the current state-of-the-art of nucleotide analysis in plants with liquid chromatography coupled to mass spectrometry and describe recent major advances. Tissue disruption, quenching, liquid–liquid and solid-phase extraction, chromatographic strategies, and peculiarities of nucleotides and nucleosides in mass spectrometry are covered. We describe how the different steps of the analytical workflow influence each other, highlight the specific challenges of nucleotide analysis, and outline promising future developments. The metabolite matrix of plants is particularly complex. Therefore, it is likely that nucleotide analysis methods that work for plants can be applied to other organisms as well. Although this review focuses on plants, we also discuss advances in nucleotide analysis from non-plant systems to provide an overview of the analytical techniques available for this challenging class of metabolites.

Humans are exposed to a broad range of organic chemicals. Although targeted gas chromatography mass spectrometry techniques are used to quantify a limited number of persistent organic pollutants and trace organic contaminants in biological samples, nontargeted, high-resolution mass spectrometry (HRMS) methods assess the human exposome more extensively. We present a QuEChERS extraction for targeted and nontargeted analysis of trace organic contaminants using HRMS and compare this method to a traditional, cartridge-based solid-phase extraction (SPE). Following validation using reference and spiked serum samples, the method was applied to plasma samples (n = 75) from the Prospective investigation of Obesity, Energy, and Metabolism (POEM) study. We quantified 44 analytes using targeted analysis and 6247 peaks were detected using the nontargeted approach. Over 90% of targeted analytes were at least 90% recovered using the QuEChERS method in spiked serum samples. In nontargeted analysis, 84% of the peaks were above the method detection limit with area counts up to 3.0 × 105 times greater using the QuEChERS method. Of the targeted compounds, 88% were also identified in the nontargeted analysis. We categorized the 4212 chemicals assigned an identity in using EPA's CompTox Dashboard and 1076 chemicals were found in at least one list. The category with the highest number of chemicals was "androgen or estrogen receptor activity." The findings demonstrate that a QuEChERS technique is suitable for both targeted and nontargeted analysis of trace organic contaminants in biological samples.

Bacillus subtilis EA-CB0015 was isolated from the phyllosphere of a banana plant and tested for its potential to produce bioactive compounds against Mycosphaerella fijiensis. Using a dual plate culture technique the cell-free supernatant of B. subtilis EA-CB0015 produced inhibition values of 89 +/- 1%. The active compounds were purified by solid-phase extraction and HPLC, and their primary structures determined using mass spectrometry and amino acid analysis. A new fengycin isoform, fengycin C, with the amino acid sequence Glu-Orn-Tyr-Thr-Glu-Val-Pro-Gln-Thr-Ile was isolated. The peptidic moiety differs from fengycin B at position 9 and from fengycin A at positions 6 and 9. The beta-hydroxy fatty acyl chain is connected to the N-terminal of the decapeptide and can be saturated or unsaturated, ranging from 14 to 18 carbons. The C-terminal residue of the peptidic moiety is linked to the tyrosine residue at position 3, forming the branching point of the acyl peptide and the eight-membered cyclic lactone.

A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated in human plasma for the simultaneous determination of aripiprazole (ARI) and its metabolite dehydro-aripiprazole (DARI); olanzapine (OLA), risperidone (RIS), paliperidone (PAL), quetiapine (QUE), clozapine (CLO) and caffeine (CAF). CAF is included to the method because it can have an influence on drug metabolism due to competitive inhibition. The above mentioned compounds and their isotope-labeled internal standards were extracted from 200 µL human plasma samples by both, effective phospholipids-eliminating three-step microelution-solid-phase extraction (µ-SPE) and protein precipitation (PPT) for comparison. A combination of formic acid (0.2%)-acetonitrile (pH 3.0; 65:35, v/v) was used as mobile phase and the chromatogram was run under gradient conditions at a flow rate of 0.6 mL/min. Run time lasted 6 min, followed by a re-equilibration time of 3 min. All analytes were monitored by mass spectrometric detection operating in multiple reaction monitoring mode and the method was validated covering the corresponding therapeutic ranges: 0.18-120 ng/mL for ARI, 0.25-80 ng/mL for DARI, 1.00-100 ng/mL for OLA, 0.70-60 ng/mL for RIS, 0.20-30 ng/mL for PAL, 0.50-160 ng/mL for QUE, 0.50-1000 ng/mL for CLO, and finally 1200-3700 ng/mL for CAF. The method was validated based on the recommendations of regulatory agencies through tests of precision, accuracy, extraction recovery, identity confirmation, trueness, matrix effect, process efficiency, stability, selectivity, linearity and carry-over effect fulfilling the guideline requirements. Our µ-SPE method results in the elimination of more than 99% of early eluting and more than 92% of late-eluting phospholipids compared to PPT. Additionally, the method was successfully applied for quantifying ARI and OLA plasma concentrations from healthy volunteers.

Nekódující RNA, mezi něž patří i miRNA, jsou předmětem výzkumu s cílem jejich použití jako biomarkerů pro odlišení fyziologického od patologického stavu lidského organismu. To vedlo k potřebě jejich účinné izolace z komplexních biologických vzorků za účelem jejich následné detekce. Tato diplomová práce se zabývá izolací miRNA pomocí TiO2 mikrosfér z biologických vzorků představujících buňky a sérum. Hlavní úkol spočíval v zavedení možnosti izolace miRNA z lidského séra pomocí TiO2 mikrosfér. K tomu bylo nutné provést několik optimalizačních kroků v již známém protokolu s cílem minimalizovat působení RNas při zachování účinnosti izolace, které je běžně dosahováno např. u buněčného lyzátu. Samotné vyšetření séra je oproti cytologickému vyšetření např. biopsii mnohem méně invazivní, což umožňuje nejen diagnostiku v počátku onemocnění, ale také možnost průběžného sledování stavu pacienta. Dále byla zkoumána kompatibilita optimalizovaného izolačního postupu s detekcí pomocí polymerázové řetězové reakce, která se pro detekci miRNA využívá z důvodu vysoké citlivosti. Metoda s vysokou citlivostí je zapotřebí kvůli přirozeně nízké koncentraci miRNA v biologických vzorcích. Tato metoda izolace miRNA pomocí TiO2 mikrosfér a následné detekce pomocí polymerázové řetězové reakce by mohla potencionálně sloužit pro sledování změn v expresi miRNA v sérech pacientů. Na základě změn exprese miRNA by docházelo k včasnému odhalení rakovinného onemocnění bez nutnosti odběru tkáně., The non-coding RNAs, including microRNAs, are researched with the aim to use them as biomarker to distinguish physiological and pathological conditions of the human organism. Because of this, there is a greater demand for efficiency of their isolation from complex biological samples for a purpose of their subsequent detection. This diploma thesis deals with the isolation of miRNAs from biological samples such as cells and serum with the use of TiO2 microspheres. The main task was to implement a method with the option of miRNAs isolation from human serum. For that, it was necessary to optimize steps of an already established protocol to minimize the effect of RNases, while maintaining the isolation efficiency, commonly achieved, for example with a cell lysate. The collection process for serum samples is much less invasive in comparison to cytological samples (biopsy). This provides, not only the possibility of an early diagnosis at the beginning stage of the disease but also the chance to monitor the pacient´s conditions continuously. Next, the compatibility of the optimized isolation procedure with the polymerase chain reaction was studied. This method is used for miRNAs detection because of its sensitivity. A method with hight sensitivity is necessary to use due to the naturally low concentration of miRNAs in biological samples. This miRNAs isolation method using TiO2 microspheres and subsequent detection by polymerase chain reaction has a great potential for the observation of the changes in miRNAs expression in patient´s serum. With accurate and reliable data about the changes in miRNAs expression, the cancer disease would be detect in time without the need for tissue collection., Fakulta chemicko-technologická, 1. Prezentace výsledků diplomové práce. 2. Diskuze k posudkům vedoucího a oponenta diplomové práce. 3. Studentka zodpověděla všechny dotazy a připomínky k DP., Dokončená práce s úspěšnou obhajobou

This study presents a novel mixed-mode weak cation-exchange (WCX) material. This material was prepared by means of the functionalization of a mesoporous divinylbenzene (DVB) resin with maleic acid (maleic acid-DVB), which yielded a high carboxylic moiety content resulting in WCX interactions as well as suitable specific surface area for reversed-phase interactions. After the optimization of the solid-phase extraction (SPE) protocol to enhance the selectivity of the sorbent, this material was evaluated as a novel WCX sorbent in the SPE of a group of drugs from environmental water samples. The method is based on SPE followed by liquid chromatography (LC) coupled to high resolution mass spectrometry (HRMS) with an Orbitrap analyzer, and was validated and applied for the determination of basic drugs in river, effluent and influent wastewater samples. Maleic acid-DVB sorbent yielded suitable recovery rates (57% to 89%) and an acceptable matrix effect ( < 32%) thanks to the effective washing step included when these environmental waters were loaded through the novel resin. The method was applied to different environmental water samples and some basic drugs were suitably quantified in these environmental samples. (c) 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )

This work describes a method for the simultaneous determination of organic acids in fungi fermentation broths. The sample preparation technique and the chromatographic conditions were optimized to enable the monitoring of the fermentation process. The determination of the citric and oxalic acid was done with a high resolution liquid chromatograph adapted to a detector with diode arrangement, an IC-Pak Ion-Exclusion 7μm (7.8 x 300 mm) column and a mobile phase of 0.001N H2SO4 with isocratic elusion. The validation parameters show efficiency, adequate linearity, and standard deviation values (%RSD) from 0.018% and 4.650%. the quantification limits (LDC) were 50.76 mg/L for citric acid and 20.18 mg/L for oxalic acid and the detection limits (LDD) were 0.6 mg/L for both acids. The method was applied in the analysis of organic acids in fermentation broths of Aspergillus niger., Este trabajo describe un método para la determinación simultanea de ácidos orgánicos en caldos de fermentación de hongos. La técnica de preparación de la muestra y las condiciones cromatográficas fueron optimizadas para permitir el seguimiento del proceso de fermentación. La determinación del ácido cítrico y oxálico se realizó empleando un cromatógrafo liquido de alta resolución acoplado a un detector con arreglo de diodos, una columna IC-Pak Ion-Exclusion 7μm (7.8 x 300 mm) y fase móvil de 0.001N H2SO4 con elución isocrática. Los parámetros de validación exhiben eficiencia, adecuada linealidad y valores de desviación estándar (%RSD) entre 0.018% y 4.650%. Los límites de cuantificación (LDC) fueron de 50.76 mg/L para el ácido cítrico y 20.18 mg/L para el ácido oxálico y los límites de detección (LDD) fue de 0.6 mg/L para ambos ácidos. El método fue aplicado en el análisis de ácidos orgánicos en caldos de fermentación de Aspergillus niger.

Работа посвящена твердофазному концентрированию различных типов фенольных соединений из водных экстрактов лекарственного растительного сырья семейств Зверобойные (Hypericum perforatum L.) и Яснотковые (Thymus serpyllum L., Salvia officinalis L.) с использованием различных сорбционных материалов для их дальнейшей идентификации. В качестве твердофазных сорбционных материалов использовали известные в аналитической практике октадецилсиликагель (Strata C18-E), сополимер стирола и дивинилбензола, химически модифицированный N-винилпирролидоном (Strata X), сополимер дивинилбензола и N-винилпирролидона, проявляющий гидрофильно-липофильную двойственность (Oasis HLB), а также непористый графитированный углерод (Supelclean ENVI-Carb). Для каждого из сорбентов получены основные сорбционные характеристики по отношению к целевым соединениям, а также их десорбционные параметры. Использование полимерных материалов Strata X и Oasis HLB позволяет извлекать из водных экстрактов растений целевые соединения при пяти-, а в случае с гидрофильно-липофильным сорбентом при 32- и 20-кратном концентрировании. Октадецилсиликагель показывает полноту десорбции флавоноидов (R ≥ 90 %), однако по отношению к фенолкарбоновым кислотам его использование нецелесообразно ввиду низких значений степеней извлечения аналитов (R ≤ 40 %). Достаточно универсальными сорбционными свойствами обладают гидрофильно-липофильные материалы (Oasis HLB), обеспечивающие высокие значения коэффициентов концентрирования при приемлемых значениях степеней извлечения фенолкарбоновых кислот (R ≤ 96 %) и флавоноидов (R ≤ 91 %). Сорбция фитокомпонентов непористым углеродным сорбентом Supelclean ENVI-Carb дает достаточно высокие характеристики, но процесс десорбции данных соединений затруднителен и требует дальнейшего изучения. Использование различных типов сорбентов показывает, что минорные компоненты, которые не детектируются в обычных условиях их хроматографического определения, могут быть сконцентрированы ТФЭ для дальнейшей их ид, The current work is devoted to the solid-phase concentration of phenolic compounds from the aqueous extracts of medicinal plant raw materials using the various sorbents for their further identification. Plants of the Hypericaceae (Hypericum perforatum L.) and Lamiaceae (Thymus serpyllum L., Salvia officinalis L.) families were selected as samples in this effort. For each of the sorbents, the main sorption characteristics («breakthrough volumes» and dynamic sorbents capacities) in relation to the target compounds, as well as their desorption parameters (concentration factor and recoveries) were obtained. Strata X and Oasis HLB polymeric materials allowed extracting the target compounds from the water extracts of plants at five-, and in the case of hydrophilic-lipophilic sorbent at 32- and 20-fold concentration. Strata C18-Е showed complete desorption of flavonoids (R ≥ 90%), but in relation to the phenolic acids, its use was impractical due to the low values of recovery analytes (R ≤ 40%). The Oasis HLB sorbent had universal sorption properties, which provided high concentration coefficients at acceptable values of recoveries of phenolic acids (R ≤ 96%) and flavonoids (R ≤ 91%). The sorption of phytocomponents with the Supelclean ENVI-Carb sorbent gave quite high characteristics, but the process of desorption of these compounds was difficult and requires further study. The use of the various types of sorbents showed that minor components that were not detected under the usual conditions of their chromatographic determination could be concentrated by SPE for their further identification, which would provide an extension of the range of definable compounds in various medicinal plants.

In the brain, approximately 90% of oxylipins are esterified to lipids. However, the significance of this esterification process is not known. In the present study, we (1) validated an aminopropyl solid phase extraction (SPE) method for separating esterified lipids using 100 and 500 mg columns and (2) applied the method to quantify the distribution of esterified oxylipins within phospholipids (PL) and neutral lipids (NL) (i.e. triacylglycerol and cholesteryl ester) in rats subjected to head-focused microwave fixation (controls) or CO2 -induced hypercapnia/ischemia. We hypothesized that oxylipin esterification into these lipid pools will be altered following CO2 -induced hypercapnia/ischemia. Lipids were extracted from control (n = 8) and CO2 -asphyxiated (n = 8) rat brains and separated on aminopropyl cartridges to yield PL and NL. The separated lipid fractions were hydrolyzed, purified with hydrophobic-lipophilic-balanced SPE columns, and analyzed with ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry. Method validation showed that the 500 mg (vs 100 mg) aminopropyl columns yielded acceptable separation and recovery of esterified fatty acid epoxides but not other oxylipins. Two epoxides of arachidonic acid (ARA) were significantly increased, and three epoxides of docosahexaenoic acid (DHA) were significantly decreased in brain NL of CO2 -asphyxiated rats compared to controls subjected to head-focused microwave fixation. PL-bound fatty acid epoxides were highly variable and did not differ significantly between the groups. This study demonstrates that hypercapnia/ischemia alters the concentration of ARA and DHA epoxides within NL, reflecting an active turnover process regulating brain fatty acid epoxide concentrations.

The aim of this study was to optimize the extraction conditions of Microcystin-LR (MC-LR), Microcystin-RR (MC-RR), Microcystin-YR (MC-YR) and Cylindrospermopsin (CYN) simultaneously from mussels by using response surface methodology (RSM) and to validate the method by a dual solid phase extraction (SPE) system combined with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The optimal parameters were: 90% MeOH (% v/v) for the extraction, a solvent/sample ratio of 75 and 15% MeOH in the extract before loading onto SPE. Mussels were spiked at 10; 37.5 and 75 ng g−1 fresh weight (f.w) of the 4 toxins, showing linear ranges of 0.5–75 ng g−1 f.w; low values for the limits of detection (0.01–0.39 ng g−1 f.w.) and quantification (0.23–0.40 ng g−1 f.w.); acceptable recoveries (70.37–114.03%) and relative standard deviation (%RSDIP) values (2.61–13.73%). The method was successfully applied to edible mussels exposed to cyanobacterial extracts under laboratory conditions, and it could allow the monitoring of these cyanotoxins in environmental mussel samples.

Being performance enhancing hormones, endogenous anabolic androgenic steroids (EAAS) are banned from most competitive sports by the World Anti-doping Agency (WADA). In anti-doping control laboratories, routine assays are mainly performed on urine samples of athletes in and out of competitions. Serum constitutes a promising alternative to urine as it is less subjected to manipulation or contamination that may influence the method sensitivity. The simultaneous determination of EAAS including conjugated metabolites using LC-MS is very challenging due to their contradicting chemical behaviors at the ionization interface of the mass spectrometer. This may prejudice their detection or limit the method sensitivity. Herein, we have addressed these challenges and developed a new method for the simultaneous determination of unconjugated, sulphate- and glucuronide-conjugated EAAS (Androsterone, Etiocholanolone, testosterone, epitestosterone, dihydrotestosterone, dehydroepiandrosterone, androstenedione and 17a-hydroxyprogesterone) in human serum using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The use of mass spectrometric detection in full scan mode facilitated the study of the most versatile adducts for detection and quantitation. A solid phase extraction method was developed for the sample preparation prior to analysis. The method limits of quantitation ranged from 0.006 to 7.904 ng/mL and the recoveries ranged from 70.2% to 96.5%. The method calibration was performed in untreated serum representing realistic matrix composition with correlation coeffecients ranged from 0.9859 to 0.9988. Finally, the serum-levels of the investigated steroids were determined in 4 male and 1 female human subjects to provide estimates of baseline levels based on individual values.

The aim of this study was to optimize the extraction conditions of Microcystin-LR (MC-LR), Microcystin-RR (MC-RR), Microcystin-YR (MC-YR) and Cylindrospermopsin (CYN) simultaneously from mussels by using response surface methodology (RSM) and to validate the method by a dual solid phase extraction (SPE) system combined with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The optimal parameters were: 90% MeOH (% v/v) for the extraction, a solvent/sample ratio of 75 and 15% MeOH in the extract before loading onto SPE. Mussels were spiked at 10; 37.5 and 75 ng g−1 fresh weight (f.w) of the 4 toxins, showing linear ranges of 0.5–75 ng g−1 f.w; low values for the limits of detection (0.01–0.39 ng g−1 f.w.) and quantification (0.23–0.40 ng g−1 f.w.); acceptable recoveries (70.37–114.03%) and relative standard deviation (%RSDIP) values (2.61–13.73%). The method was successfully applied to edible mussels exposed to cyanobacterial extracts under laboratory conditions, and it could allow the monitoring of these cyanotoxins in environmental mussel samples.

This work describes a method for the simultaneous determination of organic acids in fungi fermentation broths. The sample preparation technique and the chromatographic conditions were optimized to enable the monitoring of the fermentation process. The determination of the citric and oxalic acid was done with a high resolution liquid chromatograph adapted to a detector with diode arrangement, an IC-Pak Ion-Exclusion 7µm (7.8 x 300 mm) column and a mobile phase of 0.001N H2SO4 with isocratic elusion. The validation parameters show efficiency, adequate linearity, and standard deviation values (%RSD) from 0.018% and 4.650%. the quantification limits (LDC) were 50.76 mg/L for citric acid and 20.18 mg/L for oxalic acid and the detection limits (LDD) were 0.6 mg/L for both acids. The method was applied in the analysis of organic acids in fermentation broths of Aspergillus niger, Este trabajo describe un método para la determinación simultanea de ácidos orgánicos en caldos de fermentación de hongos. La técnica de preparación de la muestra y las condiciones cromatográficas fueron optimizadas para permitir el seguimiento del proceso de fermentación. La determinación del ácido cítrico y oxálico se realizó empleando un cromatógrafo liquido de alta resolución acoplado a un detector con arreglo de diodos, una columna IC-Pak Ion-Exclusion 7µm (7.8 x 300 mm) y fase móvil de 0.001N H2SO4 con elución isocrática. Los parámetros de validación exhiben eficiencia, adecuada linealidad y valores de desviación estándar (%RSD) entre 0.018% y 4.650%. Los límites de cuantificación (LDC) fueron de 50.76 mg/L para el ácido cítrico y 20.18 mg/L para el ácido oxálico y los límites de detección (LDD) fue de 0.6 mg/L para ambos ácidos. El método fue aplicado en el análisis de ácidos orgánicos en caldos de fermentación de Aspergillus niger

This work describes a method for the simultaneous determination of organic acids in fungi fermentation broths. The sample preparation technique and the chromatographic conditions were optimized to enable the monitoring of the fermentation process. The determination of the citric and oxalic acid was done with a high resolution liquid chromatograph adapted to a detector with diode arrangement, an IC-Pak Ion-Exclusion 7µm (7.8 x 300 mm) column and a mobile phase of 0.001N H2SO4 with isocratic elusion. The validation parameters show efficiency, adequate linearity, and standard deviation values (%RSD) from 0.018% and 4.650%. the quantification limits (LDC) were 50.76 mg/L for citric acid and 20.18 mg/L for oxalic acid and the detection limits (LDD) were 0.6 mg/L for both acids. The method was applied in the analysis of organic acids in fermentation broths of Aspergillus niger, Este trabajo describe un método para la determinación simultanea de ácidos orgánicos en caldos de fermentación de hongos. La técnica de preparación de la muestra y las condiciones cromatográficas fueron optimizadas para permitir el seguimiento del proceso de fermentación. La determinación del ácido cítrico y oxálico se realizó empleando un cromatógrafo liquido de alta resolución acoplado a un detector con arreglo de diodos, una columna IC-Pak Ion-Exclusion 7µm (7.8 x 300 mm) y fase móvil de 0.001N H2SO4 con elución isocrática. Los parámetros de validación exhiben eficiencia, adecuada linealidad y valores de desviación estándar (%RSD) entre 0.018% y 4.650%. Los límites de cuantificación (LDC) fueron de 50.76 mg/L para el ácido cítrico y 20.18 mg/L para el ácido oxálico y los límites de detección (LDD) fue de 0.6 mg/L para ambos ácidos. El método fue aplicado en el análisis de ácidos orgánicos en caldos de fermentación de Aspergillus niger

Endothelial cell (EC) metabolism is thought to be one of the driving forces for angiogenesis. Here we report the identification of the hexosamine D-mannosamine (ManN) as an EC mitogen and survival factor for bovine and human microvascular EC, with an additivity with VEGF. ManN inhibits glycosylation in ECs and induces significant changes in N-glycan and O-glycan profiles. We further demonstrate that ManN and two N-glycosylation inhibitors stimulate EC proliferation via both JNK activation and the unfolded protein response caused by ER stress. ManN results in enhanced angiogenesis in a mouse skin injury model. ManN also promotes angiogenesis in a mouse hindlimb ischemia model, with accelerated limb blood flow recovery compared to controls. In addition, intraocular injection of ManN induces retinal neovascularization. Therefore, activation of stress pathways following inhibition of protein glycosylation can promote EC proliferation and angiogenesis and may represent a therapeutic strategy for treatment of ischemic disorders.

In the brain, approximately 90% of oxylipins are esterified to lipids. However, the significance of this esterification process is not known. In the present study, we (1) validated an aminopropyl solid phase extraction (SPE) method for separating esterified lipids using 100 and 500 mg columns and (2) applied the method to quantify the distribution of esterified oxylipins within phospholipids (PL) and neutral lipids (NL) (i.e. triacylglycerol and cholesteryl ester) in rats subjected to head-focused microwave fixation (controls) or CO2 -induced hypercapnia/ischemia. We hypothesized that oxylipin esterification into these lipid pools will be altered following CO2 -induced hypercapnia/ischemia. Lipids were extracted from control (n = 8) and CO2 -asphyxiated (n = 8) rat brains and separated on aminopropyl cartridges to yield PL and NL. The separated lipid fractions were hydrolyzed, purified with hydrophobic-lipophilic-balanced SPE columns, and analyzed with ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry. Method validation showed that the 500 mg (vs 100 mg) aminopropyl columns yielded acceptable separation and recovery of esterified fatty acid epoxides but not other oxylipins. Two epoxides of arachidonic acid (ARA) were significantly increased, and three epoxides of docosahexaenoic acid (DHA) were significantly decreased in brain NL of CO2 -asphyxiated rats compared to controls subjected to head-focused microwave fixation. PL-bound fatty acid epoxides were highly variable and did not differ significantly between the groups. This study demonstrates that hypercapnia/ischemia alters the concentration of ARA and DHA epoxides within NL, reflecting an active turnover process regulating brain fatty acid epoxide concentrations.

Harmful cyanobacterial blooms compromise human and environmental health, mainly due to the cyanotoxins they often produce. Microcystins (MCs) are the most commonly measured group of cyanotoxins and are hepatotoxic, neurotoxic, and cytotoxic. Due to MCs ability to covalently bind to proteins, quantification in complex matrices is difficult. To analyze bound and unbound MCs, analytical methods were optimized for analysis in sediment and clam tissues. A clean up step was incorporated to remove lipids, improving percent yield. This method was then applied to sediment and clam samples collected from the Sacramento-San Joaquin River Delta (Delta) in the spring and fall of 2017. Water samples were also tested for intracellular and extracellular MCs. These analyses were used to quantify the partitioning of MCs among sediment, clams, and water, and to examine whether MCs persist during non-summer months. Toxin analysis revealed that multiple sediment samples collected in the Delta were positive for MCs, with a majority of the positive samples from sites in the San Joaquin River, even while water samples from the same location were below detection limit. These data highlight the importance of analyzing MCs in complex matrices to accurately evaluate environmental risk.

The aim of this study was to optimize the extraction conditions of Microcystin-LR (MC-LR), Microcystin-RR (MC-RR), Microcystin-YR (MC-YR) and Cylindrospermopsin (CYN) simultaneously from mussels by using response surface methodology (RSM) and to validate the method by a dual solid phase extraction (SPE) system combined with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The optimal parameters were: 90% MeOH (% v/v) for the extraction, a solvent/sample ratio of 75 and 15% MeOH in the extract before loading onto SPE. Mussels were spiked at 10; 37.5 and 75 ng g−1 fresh weight (f.w) of the 4 toxins, showing linear ranges of 0.5–75 ng g−1 f.w; low values for the limits of detection (0.01–0.39 ng g−1 f.w.) and quantification (0.23–0.40 ng g−1 f.w.); acceptable recoveries (70.37–114.03%) and relative standard deviation (%RSDIP) values (2.61–13.73%). The method was successfully applied to edible mussels exposed to cyanobacterial extracts under laboratory conditions, and it could allow the monitoring of these cyanotoxins in environmental mussel samples.

This work describes a method for the simultaneous determination of organic acids in fungi fermentation broths. The sample preparation technique and the chromatographic conditions were optimized to enable the monitoring of the fermentation process. The determination of the citric and oxalic acid was done with a high resolution liquid chromatograph adapted to a detector with diode arrangement, an IC-Pak Ion-Exclusion 7µm (7.8 x 300 mm) column and a mobile phase of 0.001N H2SO4 with isocratic elusion. The validation parameters show efficiency, adequate linearity, and standard deviation values (%RSD) from 0.018% and 4.650%. the quantification limits (LDC) were 50.76 mg/L for citric acid and 20.18 mg/L for oxalic acid and the detection limits (LDD) were 0.6 mg/L for both acids. The method was applied in the analysis of organic acids in fermentation broths of Aspergillus niger, Este trabajo describe un método para la determinación simultanea de ácidos orgánicos en caldos de fermentación de hongos. La técnica de preparación de la muestra y las condiciones cromatográficas fueron optimizadas para permitir el seguimiento del proceso de fermentación. La determinación del ácido cítrico y oxálico se realizó empleando un cromatógrafo liquido de alta resolución acoplado a un detector con arreglo de diodos, una columna IC-Pak Ion-Exclusion 7µm (7.8 x 300 mm) y fase móvil de 0.001N H2SO4 con elución isocrática. Los parámetros de validación exhiben eficiencia, adecuada linealidad y valores de desviación estándar (%RSD) entre 0.018% y 4.650%. Los límites de cuantificación (LDC) fueron de 50.76 mg/L para el ácido cítrico y 20.18 mg/L para el ácido oxálico y los límites de detección (LDD) fue de 0.6 mg/L para ambos ácidos. El método fue aplicado en el análisis de ácidos orgánicos en caldos de fermentación de Aspergillus niger

Certain estrogen metabolites have been implicated in the pathophysiology of breast cancer. Moreover, the estrogen metabolite profiles of healthy women and those with (a high risk of) breast cancer differ significantly. The development of an analytical method to determine the relative levels of all the estrogen biotransformation products has been described in van der Berg et al. [1]. An improvement on previously developed methods was the ability to also detect molecules such as sulphate and glucuronide conjugates as well as progesterone, estradiol precursors, and metabolites from the 16-hydroxylation metabolic pathway of estrogens simultaneously with all other estrogen metabolites. The data presented here describe the optimisation of a solid phase extraction method with different fractionation steps for LC-MS/MS analysis of 27 estrogen-related metabolites from small urine volumes. Conditions that were optimised include the elution and washing solvent concentration, the urine, loading, washing, and elution volumes, as well as pH. All raw data used to construct the bar graphs presented in this article are included in the supplementary data file. The data indicated that fractionation was necessary in order to elute estrogen metabolites with different chemical properties at different eluate compositions. Only one of the fractions (containing the less water-soluble metabolites) underwent derivatisation before LC-MS/MS analysis

Certain estrogen metabolites have been implicated in the pathophysiology of breast cancer. Moreover, the estrogen metabolite profiles of healthy women and those with (a high risk of) breast cancer differ significantly. The development of an analytical method to determine the relative levels of all the estrogen biotransformation products has been described in van der Berg et al. [1]. An improvement on previously developed methods was the ability to also detect molecules such as sulphate and glucuronide conjugates as well as progesterone, estradiol precursors, and metabolites from the 16-hydroxylation metabolic pathway of estrogens simultaneously with all other estrogen metabolites. The data presented here describe the optimisation of a solid phase extraction method with different fractionation steps for LC-MS/MS analysis of 27 estrogen-related metabolites from small urine volumes. Conditions that were optimised include the elution and washing solvent concentration, the urine, loading, washing, and elution volumes, as well as pH. All raw data used to construct the bar graphs presented in this article are included in the supplementary data file. The data indicated that fractionation was necessary in order to elute estrogen metabolites with different chemical properties at different eluate compositions. Only one of the fractions (containing the less water-soluble metabolites) underwent derivatisation before LC-MS/MS analysis

The aim of this study was to optimize the extraction conditions of Microcystin-LR (MC-LR), Microcystin-RR (MC-RR), Microcystin-YR (MC-YR) and Cylindrospermopsin (CYN) simultaneously from mussels by using response surface methodology (RSM) and to validate the method by a dual solid phase extraction (SPE) system combined with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The optimal parameters were: 90% MeOH (% v/v) for the extraction, a solvent/sample ratio of 75 and 15% MeOH in the extract before loading onto SPE. Mussels were spiked at 10; 37.5 and 75 ng g−1 fresh weight (f.w) of the 4 toxins, showing linear ranges of 0.5–75 ng g−1 f.w; low values for the limits of detection (0.01–0.39 ng g−1 f.w.) and quantification (0.23–0.40 ng g−1 f.w.); acceptable recoveries (70.37–114.03%) and relative standard deviation (%RSDIP) values (2.61–13.73%). The method was successfully applied to edible mussels exposed to cyanobacterial extracts under laboratory conditions, and it could allow the monitoring of these cyanotoxins in environmental mussel samples.

In the brain, approximately 90% of oxylipins are esterified to lipids. However, the significance of this esterification process is not known. In the present study, we (1) validated an aminopropyl solid phase extraction (SPE) method for separating esterified lipids using 100 and 500 mg columns and (2) applied the method to quantify the distribution of esterified oxylipins within phospholipids (PL) and neutral lipids (NL) (i.e. triacylglycerol and cholesteryl ester) in rats subjected to head-focused microwave fixation (controls) or CO2 -induced hypercapnia/ischemia. We hypothesized that oxylipin esterification into these lipid pools will be altered following CO2 -induced hypercapnia/ischemia. Lipids were extracted from control (n = 8) and CO2 -asphyxiated (n = 8) rat brains and separated on aminopropyl cartridges to yield PL and NL. The separated lipid fractions were hydrolyzed, purified with hydrophobic-lipophilic-balanced SPE columns, and analyzed with ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry. Method validation showed that the 500 mg (vs 100 mg) aminopropyl columns yielded acceptable separation and recovery of esterified fatty acid epoxides but not other oxylipins. Two epoxides of arachidonic acid (ARA) were significantly increased, and three epoxides of docosahexaenoic acid (DHA) were significantly decreased in brain NL of CO2 -asphyxiated rats compared to controls subjected to head-focused microwave fixation. PL-bound fatty acid epoxides were highly variable and did not differ significantly between the groups. This study demonstrates that hypercapnia/ischemia alters the concentration of ARA and DHA epoxides within NL, reflecting an active turnover process regulating brain fatty acid epoxide concentrations.

The aim of this study was to optimize the extraction conditions of Microcystin-LR (MC-LR), Microcystin-RR (MC-RR), Microcystin-YR (MC-YR) and Cylindrospermopsin (CYN) simultaneously from mussels by using response surface methodology (RSM) and to validate the method by a dual solid phase extraction (SPE) system combined with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The optimal parameters were: 90% MeOH (% v/v) for the extraction, a solvent/sample ratio of 75 and 15% MeOH in the extract before loading onto SPE. Mussels were spiked at 10; 37.5 and 75 ng g−1 fresh weight (f.w) of the 4 toxins, showing linear ranges of 0.5–75 ng g−1 f.w; low values for the limits of detection (0.01–0.39 ng g−1 f.w.) and quantification (0.23–0.40 ng g−1 f.w.); acceptable recoveries (70.37–114.03%) and relative standard deviation (%RSDIP) values (2.61–13.73%). The method was successfully applied to edible mussels exposed to cyanobacterial extracts under laboratory conditions, and it could allow the monitoring of these cyanotoxins in environmental mussel samples.

This work describes a method for the simultaneous determination of organic acids in fungi fermentation broths. The sample preparation technique and the chromatographic conditions were optimized to enable the monitoring of the fermentation process. The determination of the citric and oxalic acid was done with a high resolution liquid chromatograph adapted to a detector with diode arrangement, an IC-Pak Ion-Exclusion 7µm (7.8 x 300 mm) column and a mobile phase of 0.001N H2SO4 with isocratic elusion. The validation parameters show efficiency, adequate linearity, and standard deviation values (%RSD) from 0.018% and 4.650%. the quantification limits (LDC) were 50.76 mg/L for citric acid and 20.18 mg/L for oxalic acid and the detection limits (LDD) were 0.6 mg/L for both acids. The method was applied in the analysis of organic acids in fermentation broths of Aspergillus niger, Este trabajo describe un método para la determinación simultanea de ácidos orgánicos en caldos de fermentación de hongos. La técnica de preparación de la muestra y las condiciones cromatográficas fueron optimizadas para permitir el seguimiento del proceso de fermentación. La determinación del ácido cítrico y oxálico se realizó empleando un cromatógrafo liquido de alta resolución acoplado a un detector con arreglo de diodos, una columna IC-Pak Ion-Exclusion 7µm (7.8 x 300 mm) y fase móvil de 0.001N H2SO4 con elución isocrática. Los parámetros de validación exhiben eficiencia, adecuada linealidad y valores de desviación estándar (%RSD) entre 0.018% y 4.650%. Los límites de cuantificación (LDC) fueron de 50.76 mg/L para el ácido cítrico y 20.18 mg/L para el ácido oxálico y los límites de detección (LDD) fue de 0.6 mg/L para ambos ácidos. El método fue aplicado en el análisis de ácidos orgánicos en caldos de fermentación de Aspergillus niger

This paper describes a procedure for the isolation of 20 organic micro-pollutants among pesticides, drugs, recreational drugs, flame retardants from river sediments. After a solid-liquid extraction with a methanol:water (50:50, v/v) solution, the supernatant was diluted with water and cleaned up by stir-disc solid-phase extraction (SPE). The disc was made of buckypaper, a self-supporting entangled assembly of carbon nanotubes, which was used as a highly porous, two-sided, sorbent membrane. In the preliminary activation step, the membrane was oxidised for 2-hours with nitric acid to extend its extraction capability also to more polar compounds. All extracts were analysed by ultra-high-performance liquid chromatography-tandem mass spectrometry. A comparative investigation with commercial Strata-X Polymeric Reversed Phase SPE cartridges proved the effectiveness of the lab-made device. On the average, the analytes were recovered with yields around 69% (low spike level) and 80% (medium and high spike level), while only a couple of analytes exhibited values less than 50%. The relative standard deviation was always less than 20%. Limits of detections were in the range 0.02–9.9 ng g−1. The validated method was then applied for the analysis of sediment samples from different sites of the River Turia basin in the area of Valencia (Spain), finding tris(2-chloroisopropyl)-phosphate in all sediments at a level ranging from 6.9 to 387.9 ng g−1. Other compounds, such as pesticides and pharmaceuticals were more sporadically in these samples.

The developing fetus represents a highly sensitive period of exposure to endocrine disrupting compounds (EDCs). However, risk assessment of EDCs is hampered by the lack of data on direct in utero exposure. In this study, we developed a robust analytical methodology for the identification of a wide range of known and unknown EDCs in full-term amniotic fluid (AF). First, a method for extraction and fractionation of a broad range of polar and nonpolar EDCs was developed and validated. Maximal recoveries of reference compounds and minimal interference from the matrix were achieved with a combination of solid phase extraction and dispersive liquid/liquid extraction. Bioassay analysis using cell-based reporter gene assays revealed estrogenic, androgenic, and dioxin-like activity in AF extract corresponding to 1.4 nmol EEQ/L, 76.6 pmol DHT-EQ/L, and 10.1 pmol TEQ/L, respectively. Targeted analysis revealed 13 xenobiotics, phytoestrogens, and endogenous hormones in the AF extract that partly contributed to the bioassay activity. Separation of the complex mixture of chemicals in the AF extract with reversed-phase chromatographic fractionation and subsequent bioassay analysis revealed activity in fractions over a wide range of polarity, indicating diverse (unidentified) substances with potential ED activity. The method developed here represents the first methodological step in an effect-directed analysis approach to identify unknown biologically active compounds in the fetal environment.

Drugs used to enhance human performance in sport competitions are prohibited by the world anti-doping association (WADA). Biological samples from athletes are continuously tested for adverse analytical findings regarding the identity and/or quantity of the banned substances. The current thesis deals with the development of new analytical methods to determine the concentrations of certain drugs used by athletes and even by regular users for therapeutic purposes. The developed methods aim to analyze the contents of these drugs in the biological matrices; plasma, serum and saliva to provide a successful approach towards either doping detection or therapeutic monitoring. β-adrenergic blockers such as propranolol and metoprolol are used in sports to relief stress and as therapeutic agents in the treatment of hypertension. Both drugs are in chiral forms and available only as racemic mixtures. The different pharmacology of each enantiomer necessitates the monitoring of each enantiomer by stereoselective analytical technique such as chiral liquid chromatography for separation and mass spectrometry for selective detection. The Endogenous anabolic androgenic steroids (EAAS) on the other hand are only notoriously used in sports to increase muscle mass and strength. A method utilizing high-resolution mass spectrometry (HRMS) coupled to ultra-high performance liquid chromatography (UHPLC) was developed for the simultaneous determination of EAAS and their conjugated metabolites to provide a better insight into the steroidal module of the athlete biological passport (ABP). Moreover, the steroidal profile was assessed in serum using the proposed method after the administration of Growth hormone injection as an approach toward the implementation of a new endocrinological module based on steroids biomarkers to hormone doping. Biological samples contain many components that may interfere with the analytical measurements. Therefore, sample preparation methods were developed using soli, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.