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1.
Methods Mol Biol ; 2477: 293-309, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524124

RESUMO

Especially in eukaryotes, the N-terminal acetylation status of a protein reveals translation initiation sites and substrate specificities and activities of N-terminal acetyltransferases (NATs). Here, we discuss a bottom-up proteomics protocol for the enrichment of N-terminal peptides via strong cation exchange chromatography. This protocol is based on depleting internal tryptic peptides from proteome digests through their retention on strong cation exchangers, leaving N-terminally acetylated/blocked peptides enriched among the nonretained peptides. As such, one can identify novel N-terminal proteoforms and quantify the degree of N-terminal protein acetylation.


Assuntos
Proteoma , Proteômica , Acetilação , Cromatografia , Peptídeos/química , Proteômica/métodos
2.
Anal Chim Acta ; 1208: 339826, 2022 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-35525597

RESUMO

We here propose an efficient solvent-switching preconcentration method for the ion-chromatographic (IC) determination of halide impurities contained ionic liquids (ILs). Because halide impurities strongly affect the physicochemical properties of ILs, their analysis is an important task for the successful utilization of ILs. Although IC is an efficient method for this purpose, its application still involves significant challenges. The major halide impurities, such as F- and Cl-, show much smaller retention in aqueous anion-exchange chromatography than IL component anions. Therefore, if an IL sample is directly analyzed by IC with aqueous mobile phases, the halide impurities are eluted earlier, whereas the IL component anion is hardly eluted and gives a large peak once eluted. Thus, the introduction of the IL component anions into the IC separation column should be avoided for efficient analyses and also for preventing the degradation of the column by the accumulation of the IL anions in it. This problem, which arises from the ion-exchange selectivity in aqueous media, is solved by a solvent switching preconcentration method. The anion-exchange selectivity in aqueous media is reversed by a use of an aprotic solvent, such as acetonitrile (MeCN). Hence, we have come up with the idea of preconcentrating anions in MeCN and stripping them with an aqueous mobile phase for IC analysis. The introduction of the IL component anions into the IC separation column is substantially reduced while maintaining high sensitivity for the halide impurities. Sub µM impurities are detectable in the mM level of ILs.


Assuntos
Líquidos Iônicos , Ânions/química , Cromatografia , Líquidos Iônicos/química , Solventes/química , Água
3.
Protein Expr Purif ; 195-196: 106092, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35430350

RESUMO

Mutations in PARK7, the gene encoding the DJ-1 protein, are associated with early onset of Parkinson's disease. The C106 residue of DJ-1 is highly susceptible to oxidation, and its oxidation status is essential for various in vivo neuroprotective roles. Since C106 is readily oxidized to sulfinic acid that is not reduced by dithiothreitol, no method to separate native DJ-1 protein from the oxidized one creates challenges in the in vitro study of the biological relevance of C106-oxidation state. Here, we report an efficient column chromatography method to purify native, C106-sulfinic, and mixed (combination of the priors) forms of DJ-1. This method will be useful for systematic in vitro studies of DJ-1 functions by providing specific native and C106-sulfinic DJ-1 proteins.


Assuntos
Proteínas Oncogênicas , Doença de Parkinson , Cromatografia , Humanos , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Oxirredução , Estresse Oxidativo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/metabolismo
4.
J Chromatogr A ; 1671: 462999, 2022 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35381559

RESUMO

Support vector regression models are created and used to predict the retention times of oligonucleotides separated using gradient ion-pair chromatography with high accuracy. The experimental dataset consisted of fully phosphorothioated oligonucleotides. Two models were trained and validated using two pseudo-orthogonal gradient modes and three gradient slopes. The results show that the spread in retention time differs between the two gradient modes, which indicated varying degree of sequence dependent separation. Peak widths from the experimental dataset were calculated and correlated with the guanine-cytosine content and retention time of the sequence for each gradient slope. This data was used to predict the resolution of the n - 1 impurity among 250 000 random 12- and 16-mer sequences; showing one of the investigated gradient modes has a much higher probability of exceeding a resolution of 1.5, particularly for the 16-mer sequences. Sequences having a high guanine-cytosine content and a terminal C are more likely to not reach critical resolution. The trained SVR models can both be used to identify characteristics of different separation methods and to assist in the choice of method conditions, i.e. to optimize resolution for arbitrary sequences. The methodology presented in this study can be expected to be applicable to predict retention times of other oligonucleotide synthesis and degradation impurities if provided enough training data.


Assuntos
Cromatografia , Oligonucleotídeos , Cromatografia/métodos , Cromatografia Líquida de Alta Pressão/métodos , Citosina , Guanina , Aprendizado de Máquina , Oligonucleotídeos/análise
5.
J Chromatogr A ; 1671: 462976, 2022 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35381560

RESUMO

The pH transition method, developed for the determination of the ion-exchange group density on chromatographic stationary phase, was used for the quantification of immobilized protein A. Monolithic epoxy polyHIPE and particulate CNBr-Sepharose supports were used for immobilization. A lactate buffer was selected, having a buffer capacity peak approximately 0.5 pH units below the maximum buffer capacity of protein A. The pH transition measurements were performed at pH 4.3, where protein A exhibits maximum buffer capacity, with a lactate buffer concentration of 1 mM for protein A immobilized on polyHIPE monoliths and of 5 mM for protein A immobilized on CNBr-Sepharose. The pH transition height and full width at half maximum for the particulate support and the height for the polyHIPE matrix, showed a linear correlation with the amount of immobilized protein A determined from the absorbance difference before and after immobilization for both supports. The developed method allows a simple, non-invasive on-line determination of immobilized protein A using biological buffers, even for chromatographic columns with an amount of immobilized protein A as low as 0.25 mg. In addition, its sensitivity and duration can be easily adjusted by varying the buffer concentration and pH.


Assuntos
Cromatografia , Proteína Estafilocócica A , Cromatografia/métodos , Enzimas Imobilizadas/química , Concentração de Íons de Hidrogênio , Lactatos , Sefarose/química
6.
Sci Rep ; 12(1): 6572, 2022 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-35449227

RESUMO

Large-scale target cell isolation from patient blood preparations is one of the critical operations during drug product manufacturing for personalized cell therapy in immuno-oncology. Use of high-affinity murine antibody coated magnetic nanoparticles that remain on isolated cells is the current standard applied for this purpose. Here, we present the transformation of previously described technology - non-magnetic immunoaffinity column chromatography-based cell selection with reversible reagents into a new clinical-grade cell isolation platform called Automated Traceless Cell affinity chromatography (ATC). ATC is a fully closed and GMP-compliant cell selection and manufacturing system. Reversibility of reagents enables (sequential) positive cell selection, optionally in combination with depletion columns, enabling capture of highly specific cell subsets. Moreover, synergy with other Streptamer-based technologies allows novel uses beyond cell isolation including integrated and automated on-column target cell activation. In conclusion, ATC technology is an innovative as well as versatile platform to select, stimulate and modify cells for clinical manufacturing and downstream therapies.


Assuntos
Cromatografia , Animais , Separação Celular/métodos , Humanos , Camundongos
7.
Bioresour Technol ; 352: 127041, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35318144

RESUMO

Generation of specific xylooligosaccharides (XOS) is attractive to the pharmaceutical and food industries due to the importance of their structure upon their application. This study used chemometrics to develop a comprehensive computational modelling set to predict the parameters maximising the generation of the desired XOS during enzymatic hydrolysis. The evaluated parameters included pH, temperature, substrate concentration, enzyme dosage and reaction time. A Box-Behnken design was combined with response surface methodology to develop the models. High-performance anion-exchange chromatography coupled with triple-quadrupole mass spectrometry (HPAEC-QqQ-MS) allowed the identification of 22 XOS within beechwood xylan hydrolysates. These data were used to validate the developed models and demonstrated their accuracy in predicting the parameters maximising the generation of the desired XOS. The maximum yields for X2-X6 were 314.2 ± 1.2, 76.6 ± 4.5, 38.4 ± 0.4, 17.8 ± 0.7, and 5.3 ± 0.2 mg/g xylan, respectively. These values map closely to the model predicted values 311.7, 92.6, 43.0, 16.3, and 4.9 mg/g xylan, respectively.


Assuntos
Xilanos , Cromatografia , Endo-1,4-beta-Xilanases/química , Glucuronatos/química , Hidrólise , Oligossacarídeos/química , Xilanos/química
8.
Anal Chem ; 94(10): 4343-4351, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35245040

RESUMO

O-Glycosylation is an omnipresent modification of the human proteome affecting many cellular functions, including protein cleavage, protein folding, and cellular signaling, interactions, and trafficking. The functions are governed by differentially regulated O-glycan types and terminal structures. It is therefore essential to develop analytical methods that facilitate the annotation of O-glycans in biological material. While various successful strategies for the in-depth profiling of released O-glycans have been reported, these methods are often limitedly accessible to the nonspecialist or challenged by the high abundance of O-glycan structural isomers. Here, we developed a high-throughput sample preparation approach for the nonreductive release and characterization of O-glycans from human cell material. Reducing-end labeling allowed efficient isomer separation and detection using C18 nanoliquid chromatography coupled to Orbitrap mass spectrometry. Using the method in combination with a library of genetically glycoengineered cells displaying defined O-glycan types and structures, we were able to annotate individual O-glycan structural isomers from a complex mixture. Applying the method in a model system of human keratinocytes, we found a wide variety of O-glycan structures, including O-fucose, O-glucose, O-GlcNAc, and O-GalNAc glycosylation, with the latter carrying both elongated core1 and core2 structures and varying numbers of fucoses and sialic acids. The method, including the now well-characterized standards, provides the opportunity to study glycomic changes in human tissue and disease models using rather mainstream analytical equipment.


Assuntos
Cromatografia , Polissacarídeos , Glicosilação , Humanos , Isomerismo , Espectrometria de Massas , Polissacarídeos/química
9.
Anal Chim Acta ; 1201: 339605, 2022 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-35300799

RESUMO

The objective of the present work was to make a quantitative and critical comparison of a number of drift and noise-removal algorithms, which were proven useful by other researchers, but which had never been compared on an equal basis. To make a rigorous and fair comparison, a data generation tool is developed in this work, which utilizes a library of experimental backgrounds, as well as peak shapes obtained from curve fitting on experimental data. Several different distribution functions are used, such as the log-normal, bi-Gaussian, exponentially convoluted Gaussian, exponentially modified Gaussian and modified Pearson VII distributions. The tool was used to create a set of hybrid (part experimental, part simulated) data, in which the background and all peak profiles and areas are known. This large data set (500 chromatograms) was analysed using seven different drift-correction and five different noise-removal algorithms (35 combinations). Root-mean square errors and absolute errors in peak area were determined and it was shown that in most cases the combination of sparsity-assisted signal smoothing and asymmetrically reweighted penalized least-squares resulted in the smallest errors for relatively low-noise signals. However, for noisier signals the combination of sparsity-assisted signal smoothing and a local minimum value approach to background correction resulted in lower absolute errors in peak area. The performance of correction algorithms was studied as a function of the density and coverage of peaks in the chromatogram, shape of the background signal, and noise levels. The developed data-generation tool is published along with this article, so as to allow similar studies with other simulated data sets and possibly other algorithms. The rigorous assessment of correction algorithms in this work may facilitate further automation of data-analysis workflows.


Assuntos
Algoritmos , Cromatografia , Análise dos Mínimos Quadrados
10.
J Chromatogr A ; 1668: 462924, 2022 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-35259648

RESUMO

This study outlines the development and subsequent validation of a method using chelation ion chromatography (CIC) pretreatment followed by traditional ion chromatography (IC) and post column UV/vis detection to measure transition metals in fossil fuel wastewaters, such as oil & gas (O&G) brines and coal mine drainage (CMD) waters. Measurement of transition metals is often an important characterization step in the research of environmental and energy systems. IC represents one way to measure these metals with the advantages of being versatile, simple and relatively low cost compared to other analytical methods. However, high concentrations of alkali and alkaline earth metals present in fossil fuel wastewaters will decrease IC detectability of transition metals in these waters. In this study, a CIC method was developed for the analysis of transition metal ions (Fe3+, Cu2+, Ni2+, Zn2+, Co2+, Mn2+, and Fe2+) in fossil fuel associated wastewaters such as Appalachian CMD and O&G wastewaters from the Permian and Bakken shale basins in the United States. CIC system incorporated an on-line chelator column (e.g., the MetPac CC-1) with high selectivity for transition metals over alkali and alkaline earth metals for salt matrix removal prior to transition metal separation and detection. Additional method developments also included acidifying all samples to 2% v/v HCl and using gradient elution rather than isocratic. The recoverability of transition metals in simple salt solutions commonly found in CMD and brine samples (e.g. NaCl, Na2SO4, CaCl2) using CIC was evaluated and compared to that using traditional IC. Our results found that the CIC system significantly improved transition metal recoveries for samples in 10,000 mg/L CaCl2 matrix, reaching 87%-108% recovery for all analytes, as opposed to 2-323% recovery in traditional IC. The limits of detection in this study achieved 10.09-161.2 µg/L, comparable to reported values in similar IC studies. The developed method was also verified with certified water samples, resulting in 89%-111% recoveries in samples with higher analyte concentrations (i.e. >4x the LoDs). The developed method achieved 87%-112% recoveries for most analytes in CMD samples and 72%-138% recoveries for Bakken shale samples, relative to ICP-MS values. Overall, the current IC method can be a very good screening tool for fast and cheap analysis for transition metals at mg/L level, to facilitate selection of samples for more detailed ICP-MS analysis.


Assuntos
Combustíveis Fósseis , Águas Residuárias , Quelantes/química , Cromatografia , Íons , Águas Residuárias/química
11.
J Chromatogr A ; 1668: 462918, 2022 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-35259649

RESUMO

The combination of mixed-mode chromatography (MMC) and molecular imprinting technology (MIT) has been proven to be successful for protein separation, but suffered from cumbersome material preparation and limited performance. In this work, a new modification method marrying atom transfer radical polymerization (ATRP) and multicomponent reaction was proposed to simplify the preparation process. Using regenerated cellulose (RC) membrane as the substrate, immunoglobulin G (IgG) as the template protein and tryptamine as the ligand, a dual-recognition membrane adsorbers (MIM) was prepared by mild Ugi four-component reaction (Ugi-4CR) and surface initiated ATRP. Control the ATRP time is the key for surface imprinting. The static IgG uptake and selectivity of UGI membrane were 45 mg/mL and 1.8, respectively, while those of MIM-0.5 were 42.5 mg/mL and 14, indicating that the introduction of molecular imprinting technology significantly improved the selectivity of the membrane to IgG. The MIM-0.5 membrane retains the pH-dependent and salt-tolerant of HCIC. The dynamic flow-through results showed that the MIM-0.5 membrane could effectively separate IgG from IgG/BSA mixed solution with the purity of 88% and retained its bioactivity. This work demonstrated the feasibility of bonding HCIC and MIT to the membrane surface by Ugi-4CR and ATRP.


Assuntos
Cromatografia , Imunoglobulina G , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina G/química , Ligantes , Polimerização
12.
Artigo em Inglês | MEDLINE | ID: mdl-35240429

RESUMO

A well-defined and controlled glycosylation pattern is important to maintain quality and safety of therapeutic proteins. Glycosylation is strongly dependent on the host cell line used for recombinant protein expression. Cetuximab, which is produced in mouse myeloma cells has been shown to harbour Fab glycans, which contain non-human like features and hence, can potentially cause an immunogenic response in patients. In light of the advent of biosimilar and biobetter development, we produced cetuximab variants in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. A combination of orthogonal chromatographic modes such as hydrophilic interaction, size exclusion and strong cation exchange chromatography with various detection strategies was employed to characterise the three different cetuximab variants and to compare the in-house produced HEK and CHO variants with the reference drug product. While Fc galactosylation and sialic acid content of the drug product and the HEK variant were highly similar, the CHO product showed lower galactosylation on Fc glycans and a comparatively low sialic acid content in the Fab region. The elevated high-mannose content of CHO cetuximab also suggests potential rapid clearence from circulation. The combination of multiple chromatographic separation modes has proven powerful for the characterisation of expression system dependent protein quality attributes such as N-glycosylation.


Assuntos
Cetuximab/genética , Cetuximab/metabolismo , Polissacarídeos/metabolismo , Animais , Células CHO , Linhagem Celular/microbiologia , Cetuximab/química , Cromatografia , Cricetinae , Cricetulus , Expressão Gênica , Glicosilação , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Camundongos , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/química , Processamento de Proteína Pós-Traducional
13.
J Chromatogr A ; 1670: 462955, 2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35320751

RESUMO

In this study, new chiral ionic liquids based on the non-protein amino acid L-carnitine as cationic chiral counterpart and several anions (bis(trifluoromethane)sulfonimide (NTf2-), L-lactate- or Cl-) as counterions were synthesized. Moreover, three different salts based on L-carnitine were also synthesized and the other three were commercially acquired and used for comparison. The synthesized ionic liquids and salts were characterized by nuclear magnetic resonance, fourier transform infrared spectroscopy, high-performance liquid chromatography-mass spectrometry, and elemental analysis. Subsequently, they were used as additives to establish a γ-CD-based dual system for the enantiomeric separation of cysteine and homocysteine (previously derivatized with fluorenylmethoxycarbonyl chloride) by capillary electrokinetic chromatography. The effect of the nature of the anionic counterions and the presence of different substituents on the L-carnitine molecule on the chiral separation of both amino acids was investigated. The enantioseparations obtained with each dual system studied were compared in terms of the enantiomer effective mobilities (µeff) and the effective electrophoretic selectivity (αeff). Practically, all the dual systems evaluated exhibited substantially improved enantioseparations for the two amino acids compared with the single γ-CD system.


Assuntos
Ciclodextrinas , Líquidos Iônicos , Aminoácidos/química , Carnitina , Cromatografia , Ciclodextrinas/química , Eletroforese Capilar/métodos , Líquidos Iônicos/química , Sais , Estereoisomerismo , Compostos de Sulfidrila
14.
J Chromatogr A ; 1670: 462970, 2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35339019

RESUMO

Acoustically induced secondary flows are applied to enhance lateral mass transfer beyond the relatively slow diffusion. This has the goal to reduce convective axial dispersion and the resulting band broadening which, in turn, limits the performance of column chromatography. Traditional approaches based on Taylor-Aris model are limited to one-dimensional rectilinear (unidirectional) tube- or channel-flows. We therefore apply the generalized dispersion theory (GDT) allowing for prediction of the dependence of potentially improved performance on the characteristics of the induced secondary flow, channel geometry and solute properties as well as providing qualitative physical insight into the role of lateral flows. Results corroborate agreement with our experimental observations (residual standard deviation, Sres = 3.88) and demonstrate the advantage of applying GDT relative to 3D time-dependent simulations.


Assuntos
Cromatografia , Cromatografia/métodos , Difusão , Soluções
15.
J Inorg Biochem ; 230: 111747, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35151102

RESUMO

The reduction of vanadate (+V) in the presence of 3-mercaptopropionic acid was studied using a chromatographic method for the determination of vanadate (+V) versus vanadyl (+IV) species. Ion chromatography was combined with spectrophotometric investigation of the absorption properties of the solution. The chromatographic method for the separation of vanadate (+V) and vanadyl (+IV) was carried out with an anion exchange column. In the initial reaction mixture containing vanadate (+V) and 3-mercaptopropionic acid, ethylenediaminetetraacetic acid - EDTA was added in an excessive amount relative to the concentration of reactants in the solution. After the ligand exchange reaction, the added EDTA terminates the reduction, allowing redox speciation in the solutions. A strong pH dependence of the reduction rates in the investigated solution was observed. The vanadate reduction seems to proceed in 2 steps: 1) formation of the intermediate vanadate (+V)-thioester; 2) reduction reaction and formation of the vanadyl (+IV)-thiol complex. The obtained results strongly suggest that the reaction of vanadate (+V)-thioester formation is proton catalyzed. It was observed that the overall reduction rates are pH dependent due to the complex vanadate (+V) solution speciation and changes in the ionic form of 3-mercaptopropionic acid.


Assuntos
Ácido 3-Mercaptopropiônico , Vanadatos , Cromatografia , Ácido Edético , Oxirredução , Vanadatos/química , Vanádio
16.
J Chromatogr A ; 1667: 462881, 2022 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-35149414

RESUMO

We describe and discuss a simple dry-compression technique for preparing a flat cuboid chromatography device containing a shallow packed-bed of crystalline hydroxyapatite nanoparticles. We then discuss the use of this device for fast protein separation in the bind-and-elute mode. Such separation could be carried out at quite low pressures, making it possible to use inexpensive low pressure chromatography systems. In the flow rate range examined in this study, the pressure-drop across the device increased linearly with flow rate, indicating negligible media compaction during use. Using this device, binary protein mixtures could be separated in about a minute. Contrary to that observed in most packed-bed chromatographic separations, the width of the flow through and eluted peaks decreased with increase in flow rate. Therefore, both productivity and purity could be simultaneously increased by increasing flow rate. The suitability of this device for preparative protein separations was demonstrated by carrying out purification of a monoclonal antibody (Trastuzumab) from mammalian cell culture supernatant. This study opens up the possibility of developing dry-compression based flat cuboid packed-bed chromatography devices for fast preparative protein separation.


Assuntos
Durapatita , Nanopartículas , Animais , Anticorpos Monoclonais , Cromatografia/métodos , Pressão
17.
Food Chem ; 382: 132280, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35131694

RESUMO

A simple ionic chromatography method for nitrite analysis in processed food products was developed and validated. Nitrite in the sample was extracted using 80 °C distilled deionized water and centrifuged. Purification of nitrite from sample solution was performed using OnGuard II Ag, OnGuard II RP and OnGuard II Na cartridge connected in order. Determination of nitrite was carried out using IonPac AG9-HC (4 × 50 mm) and IonPac AS9-HC (4 × 250 mm) columns and a 9 mM sodium carbonate mobile phase. The validated results showed good linearity (r2 > 0.999), recoveries (83.7-107.6%) and precision (1.3-5.1%). The levels of nitrite in processed food products were between n.d. to 33.5 mg/kg, and nitrite was detected in ham, sausage and bacon products. The mean nitrite intake was 2.7% of the Acceptable Daily Intake (ADI, 0.07 mg/kg bw/day) for the Korean population. The method was suitable for the analysis of nitrite in processed foods.


Assuntos
Produtos da Carne , Nitritos , Cromatografia , Fast Foods/análise , Produtos da Carne/análise , Nitritos/análise , República da Coreia
18.
Anal Bioanal Chem ; 414(7): 2419-2430, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35175389

RESUMO

There is an increasing concern about the use of synthetic acaricides to fight the ectoparasitic mite Varroa destructor. Natural products such as formic acid (FA) and oxalic acid (OA) have emerged as a possible alternative control strategy. However, given the difficulty of analysing these highly polar compounds and the lack of robust and reliable methods, there are very few studies of the concentration and distribution of these natural acaricides in the beehive compartments. We present a reliable and simple analytical methodology, based on sample extraction with modified quick polar pesticide (QuPPe) methods followed by ion chromatography coupled to a quadrupole Orbitrap mass analyser for the analysis of FA and OA in honeybees, honey, beeswax, and beebread. The developed methods have been used in a field study for the evaluation of the presence and distribution of FA and OA in the beehive products, as well as in adult bees and bee brood samples, before, during, and up to 3 months after the application of the treatments by the beekeeper. Beebread and honey samples presented the highest concentration levels of OA and FA, respectively, mainly due to their natural presence. As expected, the organic acids showed low persistence in wax after the treatments. The natural acaricides were found in adult and developing bees at concentration levels below the reported LD50 in all the cases; however, residue levels of OA in larvae during the treatment application were very close to the reported LD50.


Assuntos
Mel , Praguicidas , Varroidae , Animais , Abelhas , Cromatografia , Mel/análise , Ácido Oxálico , Praguicidas/análise
19.
J Chromatogr A ; 1666: 462836, 2022 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-35108629

RESUMO

High-performance anion-exchange chromatography (HPAEC) coupled with triple quadrupole mass spectrometry (HPAEC-QqQ-MS) was applied to the determination of xylooligosaccharides (XOS) derived from enzymatically hydrolysed commercial xylan from beechwood and the analytical performance and advantages of the method explored. Separation, eluent suppression, electrospray ionisation, and detection options to enhance XOS sensitivity and selectivity were evaluated, delivering a new simple, fast, selective, and sensitive solution for the characterisation of these complex compounds. The method was fully validated in terms of its analytical performance for those XOS for which standards were available, i.e., degree of polymerisation from 1 to 6. The new method was applied to the analysis of xylan hydrolysates obtained by different enzymatic hydrolysis treatments using endo-xylanase from Thermomyces lanuginosus, characterising 25 different XOS and demonstrating the method's utility for future tailoring of enzymatic hydrolysis conditions to obtain desired XOS profiles in such hydrolysates. Linear XOS and 4-O-methyl glucuronic acid (MeGluA) branched XOS were detected by direct injection of the xylan hydrolysates after a simple 10-fold sample dilution and filtration. Identification of XOS detected by HPAEC-QqQ-MS was additionally confirmed using high-resolution orbitrap mass spectrometry (HR-orbitrap-MS). Further, an ultra-sensitive and -selective method was developed by using selected reaction monitoring acquisition mode (SRM), increasing signal-to noise ratio and decreasing the limits of detection, opening future applications to low concentrated sample analysis.


Assuntos
Espectrometria de Massas em Tandem , Xilanos , Ânions , Cromatografia , Glucuronatos/química , Hidrólise , Oligossacarídeos/química , Xilanos/química
20.
Sci Rep ; 12(1): 2653, 2022 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-35173260

RESUMO

Therapeutic drug monitoring (TDM) is an effective pharmacological approach for controlling drug concentration in a patient's serum. Herein, a new two-dimensional chromatography system was developed using two poly(N-isopropylacrylamide) (PNIPAAm)-modified bead-packed columns for effective and safe drug monitoring. PNIPAAm-modified silica beads were prepared as packing materials using atom transfer radical polymerization of NIPAAm. The increase in the retention times of the drugs requiring TDM with increasing temperature, was attributed to enhanced hydrophobic interactions at elevated temperatures. The drugs and serum proteins were separated on the prepared column at 40 °C using an all-aqueous mobile phase. Differences in the hydrophobic interactions accounted for the elution of the serum proteins and drugs at short and long retention times, respectively, and a primary column was employed to separate the serum proteins and drugs. After eluting the serum proteins from the column, the drug was introduced into the secondary column, leading to a peak of its purified form and enabling determination of the drug concentration. Two-dimensional temperature-responsive chromatography can benefit TDM by allowing the drug concentration in the serum to be measured in all-aqueous mobile phases without sample preparation.


Assuntos
Resinas Acrílicas , Cromatografia/métodos , Monitoramento de Medicamentos/métodos , Preparações Farmacêuticas/sangue , Temperatura , Proteínas Sanguíneas , Interações Hidrofóbicas e Hidrofílicas , Polimerização
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