RESUMO
NeuroEPO plus is a recently developed recombinant human erythropoietin (rhEPO) without erythropoietic activity and shorter plasma half-life due to its low sialic acid content. This novel rhEPO product is under investigation as therapeutic protein in the treatment of neurodegenerative diseases owing to its neuroprotective and neurodegenerative properties. In this study, an in-depth characterization of NeuroEPO plus N-glycans was performed by a glycan isotope [12C6]/[13C6] coded aniline labeling strategy followed by capillary zwitterionic hydrophilic interaction liquid chromatography-mass spectrometry (CapZIC-HILIC-MS). A superior amount of low sialylated glycans and less branched structures were detected in NeuroEPO plus compare to other commercial rhEPOs. At the intact glycoprotein level, NeuroEPO plus glycoforms were separated by capillary zone electrophoresis with ultraviolet detection (CE-UV), optimizing the composition and pH of the separation electrolyte. Moreover, an isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) method was also optimized for the simultaneous analysis of this basic rhEPO and conventional acidic rhEPO products. The proposed glycomic and intact glycoprotein methods provide a robust and reliable analytical platform for NeuroEPO plus characterization and for its future implementation as biopharmaceutical in neurodegenerative diseases.
Assuntos
Eritropoetina , Eritropoetina/química , Glicoproteínas/química , Humanos , Espectrometria de Massas/métodos , Polissacarídeos/análise , Proteínas Recombinantes/químicaRESUMO
Snakebites were classified by WHO as neglected diseases, and the study of venom content from snakes that cause accidents is very important and has a great impact on public health. Venomics comprises a set of analytical techniques that allows the study of the toxins present in the venom, understanding their structures and their biological functions and evolutionary relationships. However, when it comes to low mass molecules little is known about this fraction due to the lack of effective approaches for their study. Thus, the present study aimed to apply different methods of preparation and separation for the study of low mass molecules (<10kDa), to suggest alternatives for a better characterization of this little explored fraction. Viperidae of different species/genus and biomes were processed by ultrafiltration and subjected to hydrophilic interaction liquid chromatography (HILIC), mass spectrometry and antimicrobial activity screening. This is the first work to use the HILIC method for low molecular mass characterization of snake venoms, which showed better separation and molecular diversity than the classical RP-HPLC method. The fractions showed similar chromatographic profiles for snakes of the same genus, but genus-specific features were also detected. Mass spectrometry analyses revealed the presence of canonical BPPs, new peptides, and protein fragments (possible cryptides), after HILIC fractionation. the low molecular fractions from Agkistrodon bilineatus, Bitis gabonica, Bitis arietans and Bitis nasicornis showed antimicrobial activities against bacteria and fungi. In summary, the present study shows that HILIC is an efficient approach for the biochemical characterization of low mass molecules showing relevant biological activities from snake venoms, that could also be a new source of candidate-molecules with pharmacological potential.
Os acidentes ofídicos foram classificados pela OMS (Organização Mundial da Saúde) como doenças negligenciadas, e o estudo do conteúdo das peçonhas de serpentes que causam acidentes são muito importantes e tem grande impacto na saúde pública. A venômica compreende um conjunto de técnicas analíticas que permitem o estudo das toxinas presentes na peçonha, compreendendo suas estruturas, funções biológicas e relações evolutivas. No entanto, quando se trata de moléculas de baixa massa pouco se sabe sobre esta fração devido à falta de abordagens eficientes para o seu estudo. Assim, o presente estudo teve como objetivo aplicar diferentes métodos de preparação e separação para o estudo de moléculas de baixa massa (<10kDa), para sugerir alternativas para uma melhor caracterização desta fração pouco explorada. Viperídeos de diferentes espécies/gêneros e biomas (N=10) foram processados por ultrafiltração e submetidos a cromatografia líquida de interação hidrofílica (HILIC), espectrometria de massas e triagem de atividade antimicrobiana. Este é o primeiro trabalho a utilizar o método HILIC para caracterização de baixa massa molecular de peçonhas de serpentes, que apresentou melhor separação e diversidade molecular do que o método clássico de RP-HPLC. As frações apresentaram perfis cromatográficos semelhantes para serpentes do mesmo gênero, mas características específicas de gênero também foram detectadas. Análises de espectrometria de massa revelaram a presença de BPPs canônicos, novos peptídeos e fragmentos de proteínas (possíveis criptídeos), após fracionamento HILIC. as frações de baixo peso molecular de Agkistrodon bilineatus, Bitis gabonica, Bitis arietans e Bitis nasicornis apresentaram atividade antimicrobiana contra bactérias e fungos. Em resumo, o presente estudo mostra que HILIC é uma abordagem eficiente para a caracterização bioquímica de moléculas de baixa massa e que essa fração pode ser uma nova fonte de moléculas candidatas com potencial farmacológico.
RESUMO
Abstract The objective of the present study is to develop and validate a simple, selective and accurate hydrophilic interaction liquid chromatography - a high performance liquid chromatography incorporating an evaporative light scattering detector (HILIC-HPLC-ELSD) method for simultaneously determining glucosamine hydrochloride and chondroitin sulfate in dietary supplements. The chromatographic separation was carried out on a ZIC-HILIC column (150 mm x 4.6 mm x 5µm) in isocratic system mode with a mobile phase of acetonitrile, 30 mM ammonium formate and water (77:20:3, v/v/v) at pH 4.5, a column temperature of 35°C, a flow rate of 1 mL.min-1, and an injection volume of 5 µL. An evaporative light scattering (ELS) detector was used. Effective separation was achieved by means of analyte resolution of more than 1.5 with an analysis run time of approximately 20 minutes. The linearity of glucosamine hydrochloride and chondroitin sulfate ranged from 0.4 to 2.5 mg.mL-1. The limits of the detection and quantification of glucosamine hydrochloride were 20 and 80 mg.mL-1 respectively, while for chondroitin sulfate they were 80 and 400 mg.mL-1. All validation parameters satisfied the acceptance criteria in accordance with International Conference on Harmonisation (ICH) guidelines. The method was successfully applied to the assay of commercial dietary supplement samples
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Suplementos Nutricionais/análise , Estudo de Validação , Glucosamina/agonistasRESUMO
Metabolomics studies rely on the availability of suitable analytical platforms to determine a vast collection of chemically diverse metabolites in complex biospecimens. Liquid chromatography-mass spectrometry operated under reversed-phase conditions is the most commonly used platform in metabolomics, which offers extensive coverage for nonpolar and moderately polar compounds. However, complementary techniques are required to obtain adequate separation of polar and ionic metabolites, which are involved in several fundamental metabolic pathways. This chapter focuses on the main mass-spectrometry-based analytical platforms used to determine polar and/or ionizable compounds in metabolomics (GC-MS, HILIC-MS, CE-MS, IPC-MS, and IC-MS). Rather than comprehensively describing recent applications related to GC-MS, HILIC-MS, and CE-MS, which have been covered in a regular basis in the literature, a brief discussion focused on basic principles, main strengths, limitations, as well as future trends is presented in this chapter, and only key applications with the purpose of illustrating important analytical aspects of each platform are highlighted. On the other hand, due to the relative novelty of IPC-MS and IC-MS in the metabolomics field, a thorough compilation of applications for these two techniques is presented here.
Assuntos
Redes e Vias Metabólicas , Metabolômica , Cromatografia Líquida , Cromatografia Gasosa-Espectrometria de Massas , Espectrometria de MassasRESUMO
Posttranslational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation are an essential regulatory mechanism of protein function and interaction, and they are associated with a wide range of biological processes. Since most PTMs alter the molecular mass of a protein, mass spectrometry (MS) is the ideal analytical tool for studying various PTMs. However, PTMs are often present in substoichiometric levels, and therefore their unmodified counterpart often suppresses their signal in MS. Consequently, PTM analysis by MS is a challenging task, requiring highly specialized and sensitive PTM-specific enrichment methods. Currently, several methods have been implemented for PTM enrichment, and each of them has its drawbacks and advantages as they differ in selectivity and specificity toward specific protein modifications. Unfortunately, for the vast majority of more than 400 known modifications, we have no or poor tools for selective enrichment.Here, we describe a comprehensive workflow to simultaneously study phosphorylation, acetylation, and N-linked sialylated glycosylation from the same biological sample. The protocol involves an initial titanium dioxide (TiO2) step to enrich for phosphopeptides and sialylated N-linked glycopeptides followed by glycan release and post-fractionation using sequential elution from immobilized metal affinity chromatography (SIMAC) to separate mono-phosphorylated and deglycosylated peptides from multi-phosphorylated ones. The IMAC flow-through and acidic elution are subsequently subjected to a next round of TiO2 enrichment for further separation of mono-phosphopeptides from deglycosylated peptides. Furthermore, the lysine-acetylated peptides present in the first TiO2 flow-through fraction are enriched by immunoprecipitation (IP) after peptide cleanup. Finally, the samples are fractionated by high pH reversed phase chromatography (HpH) or hydrophilic interaction liquid chromatography (HILIC ) to reduce sample complexity and increase the coverage in the subsequent LC-MS /MS analysis. This allows the analysis of multiple types of modifications from the same highly complex biological sample without decreasing the quality of each individual PTM study.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas/análise , Proteômica , Acetilação , Cromatografia de Afinidade , Cromatografia de Fase Reversa , Glicosilação , Imunoprecipitação , Fosforilação , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Titânio/química , Fluxo de TrabalhoRESUMO
Amongst functional foods, garlic and its by-products stand out given their rich phytochemical profile. A comprehensive analytical approach becomes necessary to fully address garlic preparations health-promoting activities, considering the coexistence of several active ingredients from different chemical families. For this, we developed a multi-phytochemical protocol combining Ultrasound and Dispersive Liquid-Liquid Microextraction, coupled to Liquid Chromatography, for the determination of flavonols, organosulfur compounds, and inulin. Hydrophilic interaction chromatography showed an adequate resolution of flavonols and sugars in a shorter time. The protocol showed a suitable performance and acceptable quantitative yields for garlic powder, cooked garlic, black garlic, and liquid garlic flavouring samples. Additionally, the proposed methodology represented a useful tool to assess how the different garlic products related to functional properties, taking into account the various phytochemical families present in each sample. This is the first time a comprehensive and multi-phytochemical validated analysis of garlic preparations is proposed.
Assuntos
Cromatografia Líquida/métodos , Suplementos Nutricionais/análise , Alho/química , Microextração em Fase Líquida/métodos , Compostos Fitoquímicos/análise , Flavonóis/análise , Análise de Alimentos/métodos , Alimento Funcional/análise , Interações Hidrofóbicas e Hidrofílicas , Inulina/análise , Compostos de Enxofre/análise , UltrassomRESUMO
An accurate, sensitive and selective analytical method is proposed for sulfonamide residues analysis in infant formulas based on hydrophilic interaction liquid chromatography (HILIC) and quadrupole time-of-flight mass spectrometry in full scan mode. The sample preparation approach involves low-temperature lipid precipitation followed by dispersive solid-phase extraction with PSA and C18 sorbents, which was successfully optimized using Plackett-Burman design. In order to achieve high analytical sensitivity, the influence of HILIC conditions on sulfonamide ionization was investigated, such as the mobile phase composition, buffer concentration, and sample diluent for injection. The method performance characteristics, including linearity (range 5-120 µg kg-1), reliable limits of quantification (between 5 and 20 µg kg-1), recovery (72.9-109.2%) and precision (coefficient of variation values ≤ 19.8%) under repeatability and within-laboratory reproducibility conditions, were in accordance with the Codex Alimentarius Commission CAC/GL 71-2009 for quantitative analytical methods for veterinary drug residues in foods. Moreover, adequate identification of the compounds was provided with accurate mass measurement of both precursor and fragment ions in one single run. Finally, the developed method was applied to thirty-five powdered milk-based infant formula samples available in the Brazilian market.
Assuntos
Cromatografia Líquida , Resíduos de Drogas/análise , Análise de Alimentos/métodos , Fórmulas Infantis/química , Espectrometria de Massas , Sulfonamidas/análise , Brasil , Contaminação de Alimentos/análise , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lactente , Reprodutibilidade dos Testes , Extração em Fase SólidaRESUMO
In recent years there has been increasing interest in the use of HILIC separations in two-dimensional liquid chromatography (2D-LC), mainly because the selectivity of HILIC separations complement that of reversed-phase separations for a variety of molecules. Historically, the re-equilibration of HILIC phases following gradient elution has been perceived as too slow to be useful in the second dimension of 2D-LC separations in particular. Recent studies of re-equilibration of HILIC phases by McCalley and coworkers using a limited set of conditions showed that highly repeatable gradient separations could be obtained with re-equilibration times as short as 4.3 min [1,2]. In this study we aimed to study re-equilibration of HILIC phases under a broader set of conditions, and at much shorter re-equilibration times, in the interest of determining whether or not HILIC separations can be generally considered as a viable option for use in the second dimension of 2D-LC separations. To this end we studied the effects of mobile phase pH, buffer concentration, and preparation method, flow rate, analyte and stationary phase chemistry, column length, and re-equilibration time on retention of a variety of small molecule probe solutes following gradient elution. In general, we have found that excellent separation repeatability can be obtained with quite short (âª10â¯min) re-equilibration times, even when progress toward full equilibration of the column is quite slow (â«10â¯min). In other words, even if the stationary phase is not fully equilibrated, as long as it is partially equilibrated in a highly precise manner, highly repeatable retention times can be obtained. Higher flow rate has a positive effect on both the rate of progress toward full equilibration and the repeatability of separation. No significant, consistent effects of eluent pH or buffer concentration on repeatability of gradient separation were observed for the stationary phases studied. Excellent gradient separation repeatability was obtained with shorter columns (30â¯mm length) with re-equilibration times as short as 3â¯s. A proof-of-concept 2D-LC separation of several small molecule probes using HILIC separations in both dimensions was performed to illustrate that re-equilibration of these columns can be fast enough for HILIC columns to be considered as a viable option for the second dimension of comprehensive 2D-LC separations.
Assuntos
Cromatografia Líquida/métodos , Interações Hidrofóbicas e Hidrofílicas , Concentração de Íons de Hidrogênio , Reprodutibilidade dos Testes , Reologia , Solventes , Fatores de TempoRESUMO
In this study, a modified Quick Polar Pesticides (QuPPe) method, optimized by a central composite design, was developed to determine quaternary ammonium pesticides (QUATs) residues in barley and wheat by ultra-high-performance liquid chromatographic tandem mass spectrometry (UHPLC-MS/MS) using a hydrophilic interaction chromatography (HILIC) column. Considering the high polarity of these compounds, special conditions of sample preparation and analysis are required. Different mobile phases, extraction procedure and clean-up were evaluated. An isocratic elution with aqueous solution of ammonium formate 60 mmol L-1 (pH 3.7) and acetonitrile, 40:60 (v/v), was selected. Water and acidified methanol as extraction solvent, without heating, and a clean-up with dichloromethane, chitosan and acetonitrile presented good results. The validated method presented satisfactory selectivity, linearity, matrix effect, trueness and precision, providing recoveries from 93 to 110% with RSD < 13% for barley, and 70 to 115% with RSD < 18% for wheat. The complexity of these matrices requires the calibration in matrix and the diluted standard addition calibration (DSAC) procedure has been shown to be an excellent option to compensate for the matrix effect and the losses of the analytes in the extraction. Real samples of barley and wheat were analyzed and 60% presented concentrations of paraquat above the maximum limits allowed by the European Union. The modified QuPPe method combined with DSAC and HILIC-UHPLC-MS/MS demonstrated to be an effective approach to determine QUATs in barley and wheat, and is a good alternative for routine analysis. The use of the biosorbent chitosan is effective, low cost and more ecological when compared to others conventional sorbents.
Assuntos
Cromatografia Líquida , Análise de Alimentos/instrumentação , Análise de Alimentos/métodos , Hordeum/química , Praguicidas/análise , Espectrometria de Massas em Tandem , Triticum/química , Calibragem , Clormequat/análise , Diquat/análise , Paraquat/análise , Piperidinas/análise , Compostos de Amônio Quaternário/análiseRESUMO
A fast and facile hydrophilic interaction liquid chromatography (HILIC) method was developed and applied to quantify physiologically important ppGpp and its analogues in a tough sample, the astaxanthin-accumulating alga Hameatococcus pluvialis. The method is able to analyze simultaneously seven nucleotides, including ppGpp at the order of pmolâ¯g-1 cells within 12â¯min. Mechanism on the elution order was investigated. It was found that 1) phosphate salt competed for the amide groups on the HILIC column with the phosphate groups of the nucleotides; 2) intramolecular hydrogen bonds might contribute to the elution order by offsetting and reducing the number of free hydrogen acceptor/donor of the nucleotide molecules interacting with the amide groups. This is the first HILIC method for ppGpp, which is feasible and applicable to a wide range of samples, especially tough samples, e.g., algae and plants.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Guanosina Tetrafosfato/análise , Volvocida/química , Acetonitrilas , Guanosina Tetrafosfato/química , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Reprodutibilidade dos TestesRESUMO
We identified a plasma signature of 11 C14 to C26 ceramides and 1 C16 dihydroceramide predictive of major adverse cardiovascular events in patients with acute myocardial infarction (AMI). Among patients undergoing coronary artery bypass surgery, those with recent AMI, compared with those without recent AMI, showed a significant increase in 5 of the signature's 12 ceramides in plasma but not simultaneously-biopsied aortic tissue. In contrast, a rat AMI model, compared with sham control, showed a significant increase in myocardial concentrations of all 12 ceramides and up-regulation of 3 ceramide-producing enzymes, suggesting ischemic myocardium as a possible source of this ceramide signature.
RESUMO
A method based on matrix-solid phase dispersion (MSPD), focused on the principles of green analytical chemistry, aimed at the use of alternative solid supports and less toxic solvents, was developed for the simultaneous determination of 19 pharmaceuticals, 4 personal care products (PPCPs) and 4 degradation products in sewage sludge samples. Higher recoveries were achieved when 2â¯g sample was macerated for 5â¯min in a glass mortar, transferred to a centrifuge tube, and 1â¯min vortex agitation with 5â¯mL methanol. The performance of the method was evaluated through linearity, recovery, precision (intra-day), method detection and quantification limits (MDL and MQL) and matrix effect. The calibration curves prepared in methanol and in the matrix extract showed a correlation coefficient ranging from 0.98 to 0.99. MQL values ranged from 1.25 to 1250â¯ngâ¯g-1. Recoveries between 50 and 120% were reached with RSDs lower than 20% for most compounds. The method presented low and medium matrix effects for most analytes. This method was successfully applied to real samples and of the 27 compounds determined, amitriptyline, carbamazepine, diclofenac, haloperidol, ketoconazole, miconazole, albendazole, mebendazole, thiabendazole, triclosan and triclocarban were detected in concentrations between 2.5 and 5400â¯ngâ¯g-1.
Assuntos
Esgotos/química , Extração em Fase Sólida/métodos , Poluentes Químicos da Água/química , Poluentes Químicos da Água/análiseRESUMO
A method for the simultaneous analysis of veterinary drug residues (spectinomycin, halquinol, and zilpaterol) and contaminants (melamine) in feedingstuffs by liquid chromatography-tandem mass spectrometry was developed. Method performance for all analytes was evaluated by reversed-phase liquid chromatography, reversed-phase with altered chemical equilibrium, and hydrophilic interaction (HILIC) as chromatographic modes. Validation was in accordance to Commission Decision 657/2002/CE, by considering the best chromatographic approach. Ion-pair liquid chromatography with C18 as stationary phase led to the lowest random uncertainties, effective analyte separation and shorter time of analysis. Low precision deviations and good recovery rates were obtained and thus method reliability and sensitivity could be consolidated. Method applicability was evaluated by the analysis of samples of feedingstuffs, such as cattle, pig, and poultry feeds, feed ingredients of both animal and vegetable origins, and mineral feeds. Some samples showed quantifiable concentrations of halquinol and zilpaterol, reinforcing the importance of this new analytical control method.
Assuntos
Ração Animal/análise , Cloroquinolinóis/análise , Cromatografia Líquida/métodos , Espectinomicina/análise , Espectrometria de Massas em Tandem/métodos , Triazinas/análise , Compostos de Trimetilsilil/análise , Animais , Cloroquinolinóis/química , Cromatografia de Fase Reversa , Resíduos de Drogas/análise , Interações Hidrofóbicas e Hidrofílicas , Íons , Reprodutibilidade dos Testes , Espectinomicina/química , Compostos de Trimetilsilil/química , IncertezaRESUMO
Lippia origanoides (Verbenaceae) is an important Brazilian medicinal plant, also used for culinary purposes. Most chemical studies with this plant have been focused on its volatile composition. In this work, we combined High-Speed Counter-current Chromatography (HSCCC) and High Performance Liquid Chromatography coupled to Ultra Violet detection and High Resolution Mass Spectrometry (HPLC-UV-HRMSn) methodologies to access the non-volatile chemical composition of L. origanoides. The crude ethanol extract of L. origanoides (LOEF) was first analyzed by HPLC-UV-HRMSn and allowed the identification of 7 major compounds. Among them, eriodictyol, naringenin and pinocembrin, were determined and are phytochemical markers of this plant. However, owing to the complexity of this plant matrix, LOEF was fractionated by HSCCC (hexane-ethanol-water, 4:3:1) as a tool for preparative pre-purification, affording a flavonoid-rich fraction. A column screening with the chromatographic stationary phases ZIC-HILIC, monolithic and particulate RP18 was performed. The best column separation was achieved with a Purospher STAR RP18e, which was used for HPLC-DAD-HRMSn studies. By this approach 12 compounds were further identified in addition to the major ones identified in the raw extract. Two of them, 6,8-di-C-hexosyl-luteolin and 6,8-di-C-glucosyl-apigenin, are being reported for the first time in the family Verbenaceae. This work shows the integration of HSCCC as a preparative tool for the fractionation and purification of natural products from a complex plant extract with other analytical techniques, with the purpose of showing each technique's potential.
Assuntos
Técnicas de Química Analítica/métodos , Cromatografia Líquida de Alta Pressão , Distribuição Contracorrente , Lippia/química , Espectrometria de Massas , Fenóis/análise , Brasil , Fracionamento Químico , Extratos Vegetais/química , Plantas Medicinais/químicaRESUMO
O-GlcNAcylation is a kind of dynamic O-linked glycosylation of nucleocytoplasmic and mitochondrial proteins. It serves as a major nutrient sensor to regulate numerous biological processes including transcriptional regulation, cell metabolism, cellular signaling, and protein degradation. Dysregulation of cellular O-GlcNAcylated levels contributes to the etiologies of many diseases such as diabetes, neurodegenerative disease and cancer. However, deeper insight into the biological mechanism of O-GlcNAcylation is hampered by its extremely low stoichiometry and the lack of efficient enrichment approaches for large-scale identification by mass spectrometry. Herein, we developed a novel strategy for the global identification of O-GlcNAc proteins and peptides using selective enzymatic deglycosylation, HILIC enrichment and mass spectrometry analysis. Standard O-GlcNAc peptides can be efficiently enriched even in the presence of 500-fold more abundant non-O-GlcNAc peptides and identified by mass spectrometry with a low nanogram detection sensitivity. This strategy successfully achieved the first large-scale enrichment and characterization of O-GlcNAc proteins and peptides in human urine. A total of 474 O-GlcNAc peptides corresponding to 457 O-GlcNAc proteins were identified by mass spectrometry analysis, which is at least three times more than that obtained by commonly used enrichment methods. A large number of unreported O-GlcNAc proteins related to cell cycle, biological regulation, metabolic and developmental process were found in our data. The above results demonstrated that this novel strategy is highly efficient in the global enrichment and identification of O-GlcNAc peptides. These data provide new insights into the biological function of O-GlcNAcylation in human urine, which is correlated with the physiological states and pathological changes of human body and therefore indicate the potential of this strategy for biomarker discovery from human urine.
Assuntos
Acetilglucosamina/química , Cromatografia Líquida de Alta Pressão/métodos , Fragmentos de Peptídeos/urina , Processamento de Proteína Pós-Traducional , Proteínas/análise , Proteoma/análise , Espectrometria de Massas em Tandem/métodos , Adulto , Biomarcadores/urina , Feminino , Glicosilação , Humanos , Masculino , Fragmentos de Peptídeos/química , Proteínas/química , ProteômicaRESUMO
This work reports a simple isocratic hydrophilic interaction liquid chromatographic (HILIC) method for the simultaneous quantification of iodixanol and of its related impurities C, D and E in drug substance. The chromatographic separation was carried out with a Kinetex™ HILIC column, using acetonitrile and formic acid aqueous solution (1.0mmol/L, pH 3.2) (92:08, v/v) as eluent at a flow rate of 0.8mL/min. The autosampler and column temperature were maintained at 20°C and UV detection was set at 243nm. The method was validated in accordance to the ICH guideline and employed for the analysis of two different lots of iodixanol drug substance. The developed method is presented as a valuable alternative to the current methods described in the USP monograph.
Assuntos
Ácidos Tri-Iodobenzoicos/análise , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Interações Hidrofóbicas e Hidrofílicas , Indicadores e Reagentes , Reprodutibilidade dos TestesRESUMO
This is the first report on the determination of nicotine in third-hand smoke (THS) in outdoor communal areas. The term THS can be defined as the contamination of surfaces by second-hand smoke. This can remain for extended periods of time and undergo further chemical reactions to produce further pollutants which can be re-suspended in dust or re-emitted into the gas phase. As THS is a rather complex mixture, studies have focused on using nicotine as a marker of THS, as it is the most abundant organic compound emitted during smoking. In this present study, the extraction of dust-wipe samples and the subsequent chromatographic conditions required for the separation of nicotine by hydrophilic interaction liquid chromatography were optimized. The optimum chromatographic conditions were identified as a 150 mm x 4.6 mm, 5 µm Zorbax Carbohydrate Analysis column with a mobile phase consisting of 90 % acetonitrile, 10 % water at a flow rate of 1.0 mL/min with UV detection at 259 nm. Further investigations were made on samples collected from surfaces of public entrance ways. Under these conditions, a linear range for nicotine of 0.05 to 24 µg/mL (1.0-480 ng on column) was obtained, with a detection limit of 1.0 ng on column based on a signal-to-noise ratio of three. Acetone, naphthalene, phenol, musk ketone, and palmitic acid were found not to interfere. Communal entrance ways were found to be contaminated with THS nicotine levels of between 5.09 µg/m(2) and 309 µg/m(2) comparable to that found in other previous studies of indoor environments. Copyright © 2015 John Wiley & Sons, Ltd.
Assuntos
Poluentes Atmosféricos/análise , Cromatografia Líquida de Alta Pressão/métodos , Nicotina/análise , Poluição por Fumaça de Tabaco/análise , Poluentes Atmosféricos/isolamento & purificação , Fracionamento Químico/métodos , Poeira/análise , Limite de Detecção , Nicotina/isolamento & purificação , Razão Sinal-RuídoRESUMO
A new HILIC stationary phase comprised of native cyclofructan-6 (CF6) bonded to superficially porous silica particles (2.7µm) was developed. Its performance was evaluated and compared to fully porous silica particles with 5µm (commercially available as FRULIC-N) and 3µm diameters. Faster and more efficient chromatography was achieved with the superficially porous particles (SPPs). The columns were also evaluated in the normal phase mode. The peak efficiency, analysis time, resolution, and overall separation capabilities in both HILIC and normal phase modes were compared. The analysis times using the superficially porous based column in HILIC mode were shorter and the theoretical plates/min were higher over the entire range of flow rates studied. The column containing the superficially porous particles demonstrated higher optimum flow rates than the fully porous particle packed columns. At higher flow rates, the advantages of the superficially porous particles was more pronounced in normal phase separations than in HILIC, clearly demonstrating the influence that the mode of chromatography has on band broadening. However, the minimum reduced plate heights (hmin) were typically lower in HILIC than in the normal phase mode. Overall, the superficially porous particle based CF6 column showed clear advantages over the fully porous particle columns, in terms of high throughput and efficient separations of polar compounds in the HILIC mode.
Assuntos
Cromatografia Líquida/instrumentação , Frutanos/química , Dióxido de Silício/química , Interações Hidrofóbicas e Hidrofílicas , PorosidadeRESUMO
Monitoring of the plasmatic levels of levodopa (LEV) and carbidopa (CAR) is necessary to adjust the dose of these drugs according to the individual needs of Parkinson's disease patients. To support drug therapeutic monitoring, a method using HILIC mode and LC-MS/MS was developed for the simultaneous determination of carbidopa, levodopa, and its metabolites (3-o-methyldopa (3-OMD) and dopamine (DOPA)) in human plasma. A triple quadrupole mass spectrometry was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. After straightforward sample preparation via protein precipitation, an Atlantis HILIC (150 × 2.1 mm, 3 µm, Waters, USA) column were used for separation under the isocratic condition of acetonitrile/water (79:21, v/v) containing 0.05% formic acid and 3 mmol/L ammonium formate and the total run time was 7 min. Deuterated LEV was used as internal standard for quantification. The developed method was validated in human plasma with a lower limit of quantitation of 75 ng/mL for LEV, 65 ng/mL for CAR and 3-OMD, and 20 ng/mL for DOPA. The calibration curve was linear within the concentration range of 75-800 ng/mL for LEV, 65-800 ng/mL for CAR and 3-OMD, and 20-400 ng/mL for DOPA (r>0.99). The assay was accurate and precise, with inter-assay and intra-assay accuracies within ±13.44% of nominal and inter-assay and intra-assay precision≤13.99%. All results were within the acceptance criteria of the US FDA and ANVISA guidelines for method validation. LEV, CAR, 3-OMD and DOPA were stable in the battery of stability studies, long-term, bench-top, autosampler, and freeze/thaw cycles. Samples from patients undergoing treatment were analyzed, and the results indicated that this new method is suitable for therapeutic drug monitoring in Parkinson's disease patients.