Optimization of pressurized liquid extraction using a multivariate chemometric approach and comparison of solid phase extraction cleanup steps for the determination of polycyclic aromatic hydrocarbons in mosses

A factorial design was used to optimize the extraction of polycyclic aromatic hydrocarbons (PAHs) from mosses, plants used as biomonitors of air pollution. The analytical procedure consists of pressurized liquid extraction (PLE) followed by solid-phase extraction (SPE) cleanup, in association with analysis by high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD). For method development, homogeneous samples were prepared with large quantities of the mosses Isothecium myosuroides Brid. and Hypnum cupressiforme Hedw., collected from a Spanish Nature Reserve. A factorial design was used to identify the optimal PLE operational conditions: 2 static cycles of 5 min at 80°C. The analytical procedure performed with PLE showed similar recoveries (∼70%) and total PAH concentrations (∼200 ng g-1) as found using Soxtec extraction, with the advantage of reducing solvent consumption by 3 (30 mL against 100 mL per sample), and taking a fifth of the time (24 samples extracted automatically in 8 h against 2 samples in 3.5 h). The performance of SPE normal phases (NH2, Florisil®, silica and activated aluminium) generally used for organic matrix cleanup was also compared. Florisil® appeared to be the most selective phase and ensured the highest PAH recoveries. The optimal analytical procedure was validated with a reference material and applied to moss samples from a remote Spanish site in order to determine spatial and inter-species variability

Reconstructing historical trends of polycyclic aromatic hydrocarbon deposition in a remote area of Spain using herbarium moss material

Herbarium mosses from 1879-1881, 1973-1975 and 2006-2007 were used to investigate the historical changes of atmospheric deposition of polycyclic aromatic hydrocarbons (PAHs) at a remote site located in Northern Spain. Natural abundance of nitrogen and carbon isotopes was also measured in order to assess the evolution of emissions from anthropogenic sources. Nitrogen concentrations, 13C, 13N and PAH levels were significantly higher in 19th century samples with respect to the present century samples. Moreover, PAH distribution varied over the centuries, following a tendency of light PAH enrichment. The carbon, nitrogen and PAH levels measured in the mosses tally with the historic evolution of anthropogenic emissions in the area, mainly influenced by changes of economic activities, domestic heating and road traffic density. Mosses provided by herbaria seem to offer the possibility to study long-term temporal evolution of atmospheric PAH deposition

Development and Validation of a Stability-Indicating HPLC Method for the Simultaneous Determination of Florfenicol and Flunixin Meglumine Combination in an Injectable Solution

Thecombination of the powerful antimicrobial agent florfenicol and the nonsteroidal anti-inflammatory flunixin meglumine is used for the treatment of bovine respiratory disease (BRD) and control of BRD-associated pyrexia, in beef and nonlactating dairy cattle. This study describes the development and validation of an HPLC-UVmethod for the simultaneous determination of florfenicol and flunixin, in an injectable preparation with a mixture of excipients.The proposed RP-HPLC method was developed by a reversed phase- (RP-) C18e (250mm× 4.6 mm, 5 m) column at roomtemperature, with an isocraticmobile phase of acetonitrile and water mixture, and pH was adjusted to 2.8 using diluted phosphoric acid, a flow rate of 1.0 mL/min, and ultraviolet detection at 268 nm. The stability-indicating method was developed by exposing the drugs to stress conditions of acid and base hydrolysis, oxidation, photodegradation, and thermal degradation; the obtained degraded products were successfully separated from the APIs. This method was validated in accordance with FDA and ICH guidelines and showed excellent linearity, accuracy, precision, specificity, robustness, LOD, LOQ, and system suitability results within the acceptance criteria.This research was supported by the Advanced Veterinary Manufacturing Company (Palestine)

Development of ultra-high performance liquid chromatographic and fluorescent method for the analysis of insect chitin

A precise quantification of insect chitin is needed in order to avoid overestimation of crude protein due to chitin-bound nitrogen. An UPLC/FLR method was optimized and validated for the determination of glucosamine (GlcN) hydrolyzed from chitin in insect materials. The method was applied for quantifying the chitin content in mealworms (Tenebrio molitor) and crickets (Acheta domesticus). A baseline separation was obtained using an Acquity HSS T3 C18 column, with an external calibration curve of excellent linearity, and a low limit of de-tection and quantification of GlcN. Even though the recovery of GlcN from spiked cricket material was slightly lower compared to that using spectrophotometric method, the UPLC/FLR method proved a sensitive and specific method of quantification of insect chitin. Chitin contents in T. molitor and A. domesticus were 4.6 +/- 0.1% and 4.5 +/- 0.0% on dry matter basis, respectively. Less than 0.01% of chitin was present in insect protein-enriched fractions extracted with 0.1 N NaCl at pH 10.Peer reviewe

Mycotoxins in Brewing Materials and Beer

Vypracovaná disertační práce se zabývá problematikou mykotoxinů v pivovarských surovinách a pivu. Pozornost byla věnována především vybraným fusariovým mykotoxinům (deoxynivalenol, zearalenol, T-2 toxin a HT-2 toxin), ochratoxinu A a aflatoxinům B1, B2, G1 a G2. Náplní disertační práce bylo optimalizovat a validovat analytické metody pro stanovení výše uvedených mykotoxinů v pivovarských surovinách a v pivu. Pro separaci analytů byla použita metoda vysokoúčinné kapalinové chromatografie s hmotnostně – spektrometrickou detekcí (HPLC-MS/MS) a metoda ultravysokoúčinné kapalinové chromatografie s fluorescenční detekcí (UPLC/FLR). Tyto analytické metody byly následně aplikovány při zmapování výskytu fusariových mykotoxinů ve sklizních sladovnického ječmene v České republice a v sladařském a pivovarském průmyslu při sledování úrovně kontaminace mykotoxiny. Dále byly realizovány experimenty týkající se přepěňování piva, neboť primární gushing – přepěňování piva – souvisí stejně jako mykotoxiny s přítomností mikroskopických vláknitých hub v surovinách pro výrobu piva. Práce, ve kterých jsou tyto metody podrobně uvedeny, jsou součástí disertace (Příloha I – V). Ze všech publikovaných výsledků je zřejmé, že výskyt mykotoxinů v obilovinách včetně ječmene je přirozený a nelze mu zcela zabránit ani při dodržení podmínek správné zemědělské praxe. Je známo, že určitý podíl mykotoxinů přítomných v kontaminovaném sladovnickém ječmeni může přejít díky svým chemickým a fyzikálním vlastnostem do finálního produktu – piva. Nalezené koncentrace mykotoxinů však nepředstavují významné zdravotní ohrožení spotřebitelů.The presented thesis deals with the issue of mycotoxins in brewing materials and beer. Attention was devoted mainly to the selected fusarium mycotoxins (deoxynivalenol, zearalenol, T-2 toxin, and HT-2 toxin) ochratoxin A and aflatoxins B1, B2, G1, and G2. The aim of the thesis was to optimize and validate analytical methods for the determination of the above mentioned mycotoxins in the brewing materials and beer. Analytes were separated using high-performance liquid chromatography with mass – spectrometric detection (HPLC-MS/MS) and ultra-performance liquid chromatography with fluorescence detection (UPLC/FLR). These analytical methods were then applied for mapping the occurrence of fusarium mycotoxins in malting barley crops in the Czech Republic and monitoring the level of contamination with mycotoxins in malting and brewing industries. In addition, experiments studying over-foaming of beer were conducted as primary gushing – over-foaming of beer – is connected, similarly as mycotoxins, with the presence of microscopic filamentous fungi in the raw materials for beer production. Studies describing in detail these methods are part of this thesis (Annex I – V). From all published results, it is evident that the occurrence of mycotoxins in cereals including barley is natural and cannot be completely prevented, not even if all conditions of correct agricultural practice are observed. It is known that some mycotoxins present in contaminated malting barley pass to the final product – beer due to their chemical and physical properties. However, the mycotoxin concentrations found do not mean any significant health risk for consumers.

Impact of diversity of Mycoplasma hyopneumoniae strains on lung lesions in slaughter pigs

The importance of diversity of Mycoplasma hyopneumoniae (M. hyopneumoniae) strains is not yet fully known. This study investigated the genetic diversity of M. hyopneumoniae strains in ten pig herds, and assessed associations between the presence of different strains of M. hyopneumoniae and lung lesions at slaughter. Within each herd, three batches of slaughter pigs were investigated. At slaughter, from each batch, 20 post mortem bronchoalveolar lavage fluid samples were collected for multiple locus variable-number tandem repeat analysis (MLVA), and lung lesions (Mycoplasma-like lesions, fissures) were examined. Multivariable analyses including potential risk factors for respiratory disease were performed to assess associations between the number of different strains per batch (three categories: one strain, two-six strains, >= seven strains), and the lung lesions as outcome variables. In total, 135 different M. hyopneumoniae strains were found. The mean (min.-max.) number of different strains per batch were 7 (1-13). Batches with two-six strains or more than six strains had more severe Mycoplasma-like lesions (P = 0.064 and P = 0.012, respectively), a higher prevalence of pneumonia [odds ratio (OR): 1.30, P = 0.33 and OR: 2.08, P = 0.012, respectively], and fissures (OR = 1.35, P = 0.094 and OR = 1.70, P = 0.007, respectively) compared to batches with only one strain. In conclusion, many different M. hyopneumoniae strains were found, and batches of slaughter pigs with different M. hyopneumoniae strains had a higher prevalence and severity of Mycoplasma-like lung lesions at slaughter, implying that reducing the number of different strains may lead to less lung lesions at slaughter and better respiratory health of the pigs

Spatial distribution of PAH concentrations and stable isotope signatures (δ13C, δ15N) in mosses from three European areas – Characterization by multivariate analysis

Polycyclic aromatic hydrocarbon (PAH) concentrations and N, C stable isotope signatures were determined in mosses Hypnum cupressiforme Hedw. from 61 sites of 3 European regions: Île-de-France (France); Navarra (Spain); the Swiss Plateau and Basel area (Switzerland). Total PAH concentrations of 100-700 ng g-1, as well as δ13C values of -32 to -29‰ and δ15N values of -11 to -3‰ were measured. Pearson correlation tests revealed opposite trends between high molecular weight PAH (4-6 aromatic rings) content and δ13C values. Partial Least Square regressions explained the very significant correlations (r > 0.91, p < 0.001) between high molecular weight PAH concentrations by local urban land use (<10 km) and environmental factors such as elevation and pluviometry. Finally, specific correlations between heavy metal and PAH concentrations were attributed to industrial emissions in Switzerland and road traffic emissions in Spain

Coiled-Coil-Templated Acyl Transfer Reactions on the Surface of Living Cells

Fluoreszenzmarkierungstechniken für lebende Zellen ermöglichen es Biologen, einen Blick in eine komplexe biologische Umgebung zu werfen und Informationen über ein bestimmtes Ziel in einer nahezu natürlichen Umgebung zu erhalten. Dank der konzertierten Bemühungen der wissenschaftlichen Gemeinschaft gibt es eine Fülle von kommerziell erhältlichen, genetisch kodierbaren Markern und Reportern für die Fluoreszenzmikroskopie. Allerdings gibt es nur wenige Lebendzellmethoden, die eine direkte Konjugation von Nukleinsäuren mit Proteinen erlauben, obwohl es robuste DNA-Technologien gibt, die mit Oligo-Antikörper-Konjugaten auf Zelloberflächen durchgeführt werden. Ein weiterer, oft einschränkender Aspekt der Markierung ist die Fähigkeit, Ziele selektiv zu multiplexen. In dieser Studie wurde eine Methode der Tag-Probe-Markierung entwickelt, die eine selektive, gleichzeitige Markierung von zwei verschiedenen Zielen mit zwei Peptid-Nukleinsäure-Strängen (PNA) ermöglicht. Diese Methode verwendet ein Paar von Coiled-Coil-Peptiden, um die Konjugation einer PNA-Gruppe an ein Zielprotein zu steuern, das ein Peptid-Tag exprimiert. Die Verwendung orthogonaler Coiled-Coils ermöglicht Multiplexing. Die Markierung von synthetischen Tag-Peptiden, die mittels Flüssigchromatographie analysiert wurden, hat gezeigt, dass der orthogonale duale Transfer von PNA selektiv, quantitativ und schnell ist. Die PNA-Konjugation von exemplarischen Membranrezeptoren, gefolgt von der Hybridisierung mit komplementären Fluorophor-DNAs, ermöglichte eine unkomplizierte Visualisierung von dualen Rezeptoren in lebenden Zellen. Durch den Einsatz einfacher molekularer Hilfsmittel, die die Grundlage der DNA-Nanotechnologie bilden, konnte durch die Rekrutierung mehrerer DNAs eine zunehmend hellere Markierung erreicht werden und die löschbare Oberflächenmarkierung ermöglichte eine quantitative Untersuchung der Rezeptorinternalisierung.Live-cell fluorescent labelling techniques allow biologists to glimpse into a complex biological environment and derive information about a specific target in a near-native environment. Thanks to a concerted effort from the scientific community, a plethora of commercially available, genetically encodable tags and reporters for fluorescence microscopy exist. However, few live-cell methods allow direct conjugation of nucleic acids with proteins despite the robust DNA technologies carried out on cell surfaces using oligo-antibody conjugates. Another aspect of labelling which is often limiting is the ability to selectively multiplex targets. In this study, a method of tag–probe labelling was developed that accomplishes selective, simultaneous labelling of two distinct targets with two peptide nucleic acid (PNA) strands. The technique uses a pair of coiled-coil peptides to guide conjugation of a PNA group to a target protein expressing a peptide tag and using orthogonal coiled-coil enables multiplexing. Initially, the labelling of synthetic tag-peptides analysed by liquid chromatography revealed the orthogonal dual transfer of PNA to be selective, quantitative, and rapid. PNA conjugation of exemplar membrane receptors followed by hybridization with complementary fluorophore-DNAs achieved straightforward live-cell dual receptor visualization. Finally, using simple molecular tools that form the basis of DNA nanotechnology, recruitment of multiple DNAs facilitated progressively brighter labelling, and erasable surface labelling allowed quantitative study of receptor internalisation

Application of Switchable Hydrophobicity Solvents for Extraction of Emerging Contaminants in Wastewater Samples

In the present work, the effectiveness of switchable hydrophobicity solvents (SHSs) as extraction solvent (N,N-Dimethylcyclohexylamine (DMCA), N,N-Diethylethanamine (TEA), and N,N-Benzyldimethylamine (DMBA)) for a variety of emerging pollutants was evaluated. Different pharmaceutical products (nonsteroidal anti-inflammatory drugs (NSAIDs), hormones, and triclosan) were selected as target analytes, covering a range of hydrophobicity (LogP) of 3.1 to 5.2. The optimized procedure was used for the determination of the target pharmaceutical analytes in wastewater samples as model analytical problem. Absolute extraction recoveries were in the range of 51% to 103%. The presented method permits the determination of the target analytes at the low ng mL−1 level, ranging from 0.8 to 5.9 (except for Triclosan, 106 ng mL−1) with good precision (relative standard deviation lower than 6%) using high-pressure liquid chromatography (HPLC) combined with ultraviolet (DAD) and fluorescence (FLR) detection. The microextraction alternative resulted in a fast, simple, and green method for a wide variety of analytes in environmental water sample. The results suggest that this type of solvent turns out to be a great alternative for the determination of different analytes in relatively complex water samples