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1.
J Agric Food Chem ; 68(10): 3171-3183, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32052628

RESUMO

Reversed-phase liquid chromatography with electrospray ionization-high-resolution/accuracy Fourier transform mass spectrometry (RPC-ESI-FTMS) and chemometrics were exploited to evaluate the influence of horizontal centrifugation by two- or three-phase decanters on the content of major phenolic secoiridoids in extravirgin olive oils (EVOOs). Despite the occurrence of other potential sources of variability typical of commercial olive oils, horizontal centrifugation was found to play a primary role, with a general increase of secoiridoid content occurring when two-phase decanters were used. As emphasized by principal component analysis (PCA), the increase involved preferentially oleacin and oleocanthal, when oxidative deterioration was purposely minimized during and/or after production, and oleuropein and ligstroside aglycones, when no vertical centrifugation was performed at the end of the productive cycle. The influence of the type of horizontal centrifugation was also emphasized by the elaboration of RPC-ESI-FTMS data based on hierarchical cluster analysis (HCA) and linear discriminant analysis (LDA).


Assuntos
Centrifugação/métodos , Manipulação de Alimentos/métodos , Iridoides/química , Azeite de Oliva/química , Fenóis/química , Cromatografia de Fase Reversa , Frutas/química , Olea/química , Oxirredução , Espectrometria de Massas por Ionização por Electrospray
2.
J Agric Food Chem ; 68(10): 3121-3131, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32053364

RESUMO

A new method to simultaneously analyze various glucosinolates (GSLs) and isothiocyanates (ITCs) by reversed-phase ultra-high-performance liquid chromatography-electron spray ionization-tandem mass spectrometry has been developed and validated for 14 GSLs and 15 ITCs. It involved derivatization of ITCs with N-acetyl-l-cysteine (NAC). The limits of detection were 0.4-1.6 µM for GSLs and 0.9-2.6 µM for NAC-ITCs. The analysis of Sinapis alba, Brassica napus, and Brassica juncea extracts spiked with 14 GSLs and 15 ITCs indicated that the method generally had good intraday (≤10% RSD) and interday precisions (≤16% RSD). Recovery of the method was unaffected by the extracts and within 71-110% for GSLs and 66-122% for NAC-ITCs. The method was able to monitor the enzymatic hydrolysis of standard GSLs to ITCs in mixtures. Furthermore, GSLs and ITCs were simultaneously determined in Brassicaceae plant extracts before and after myrosinase treatment. This method can be applied to further investigate the enzymatic conversion of GSLs to ITCs in complex mixtures.


Assuntos
Brassicaceae/química , Cromatografia Líquida de Alta Pressão/métodos , Glucosinolatos/química , Isotiocianatos/química , Extratos Vegetais/química , Sinapis/química , Espectrometria de Massas em Tandem/métodos , Cromatografia de Fase Reversa/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos
3.
Artigo em Inglês | MEDLINE | ID: mdl-31915109

RESUMO

An liquid chromatography-mass spectrometry (LC-MS/MS) assay was developed for the combined analysis of the five poly (ADP-ribose) polymerase (PARP) inhibitors niraparib, olaparib, rucaparib talazoparib and veliparib. A simple and fast sample pre-treatment method was used by protein precipitating of plasma samples with acetonitrile and dilution of the supernatant with formic acid (0.1% v/v in water). This was followed by chromatographic separation on a reversed-phase UPLC BEH C18 column and detection with a triple quadrupole mass spectrometer operating in the positive mode. A simplified validation procedure specifically designed for bioanalytical methods for clinical therapeutic drug monitoring (TDM) purposes, was applied. This included assessment of the calibration model, accuracy and precision, lower limit of quantification (LLOQ), specificity and selectivity, carry-over and stability. The validated range was 30-3000 ng/mL for niraparib, 100-10,000 ng/mL for olaparib, 50-5000 ng/mL for rucaparib, 0.5-50 ng/mL for talazoparib and 50-5000 for veliparib. All results were within the criteria of the US Food and Drug Administration (FDA) guidance and European Medicines Agency (EMA) guidelines on method validation. The assay has been successfully implemented in our laboratory.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Monitoramento de Medicamentos/métodos , Inibidores de Poli(ADP-Ribose) Polimerases/sangue , Espectrometria de Massas em Tandem/métodos , Benzimidazóis/sangue , Cromatografia de Fase Reversa/métodos , Compostos Heterocíclicos com 1 Anel/sangue , Humanos , Indóis/sangue , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes
4.
Biomed Chromatogr ; 34(1): e4719, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31634417

RESUMO

A quality by design (QbD) based high-resolution HPLC method is described for determination of impurities in apixaban (APX) in the tablet dosage form. Employing a simple and stability-indicating HPLC method, nine known impurities were quantified with good peak resolution. Mobile phase A (MP-A) was prepared with buffer and acetonitrile 90:10 v/v, while mobile phase B (MP-B) contained water and acetonitrile 10:90 v/v. The gradient program was 0 min, MP-A 75%, B 25%; 20 min, MP-A 65%, B 35%; 30 min, MP-A 40%, B 60%; 40min, MP-A 40%, B 60%; 42 min, MP-A 75%, B 25%; and 50 min, MP-A 75%, B 25%. The chromatographic separation was achieved using a Zorbax RX C18 250 × 4.6 mm column, 5 µm (1.0 ml min-1 , 280 nm, 50 µl) and a column temperature of 40°C. Several separation studies were carried out using design of experiments to optimize the method. Validation results confirm the applicability of the developed method for quality analysis and stability studies of the regular product on the manufacturing stream.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Contaminação de Medicamentos , Pirazóis/análise , Pirazóis/química , Piridonas/análise , Piridonas/química , Estabilidade de Medicamentos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Comprimidos
5.
Anal Bioanal Chem ; 412(2): 425-438, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31776639

RESUMO

Dynamic light scattering (DLS), transmission electron microscopy (TEM), and reversed phase-high performance liquid chromatography (RP-HPLC) are staples of nanoparticle characterization for size distribution, shape/morphology, and composition, respectively. These techniques are simple and provide important details on sample characteristics. However, DLS and TEM are routinely done in batch-mode, while RP-HPLC affords separation of components within the entire sample population, regardless of sample polydispersity. While batch-mode analysis is informative and should be a first-step analysis for any material, it may not be ideal for polydisperse formulations, such as many nanomedicines. Herein, we describe the utility of asymmetric flow field-flow fractionation (AF4) as a useful tool for a more thorough understanding of these inherently polydisperse materials. AF4 was coupled with in-line DLS for an enhanced separation and resolution of various size populations in a nanomaterial. Additionally, the various size populations were collected for offline analysis by TEM for an assessment of different shape populations, or RP-HPLC to provide a compositional analysis of each individual size population. This technique was also extended to assess nanoparticle stability, i.e., drug release, both in buffer and physiologically relevant matrix, as well as qualitatively evaluate the protein binding capacity of nanomedicines. Overall, AF4 is proven to be a very versatile technique and can provide a wealth of information on a material's polydispersity and stability. Moreover, the ability to conduct analysis in physiological matrices provides an advantage that many other routine analytical techniques do not. Graphical Abstract.


Assuntos
Fracionamento por Campo e Fluxo/métodos , Nanomedicina , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Difusão Dinâmica da Luz/métodos , Humanos , Microscopia Eletrônica de Transmissão/métodos , Nanopartículas/química
6.
J Chromatogr A ; 1609: 460508, 2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31530383

RESUMO

Quantitative structure-retention relationships (QSRR) predicting the values of solute "hydrophobicity" coefficient η' in the approximate hydrophobic subtraction model (HSM) can be used to predict retention times of compounds on numerous reversed-phase (RP) columns, provided that column parameters on the corresponding stationary phases are available. In the present study, we propose a new dual clustering-based localised QSRR approach, combining P-ratio clustering (where P is the octanol-water partition coefficient) with second dominant interaction (SDI)-based clustering, to produce predictive models with an acceptable level of prediction accuracy for in silico column scoping in RP method development. QSRR models for η' values were derived for 49 compounds out of 63 in a dataset extracted from the literature, where retention data were measured under one isocratic mobile phase condition (i.e., acetonitrile-water, 50:50 [v/v]). These models gave a predictive squared correlation coefficient Qext(F2)2 of 0.83 and a root mean square error of prediction (RMSEP) of 0.14. For the modelling, a genetic algorithm-partial least square regression (GA-PLS) approach was performed using the η' values and their relevant molecular descriptors. The corresponding retention times were predicted by applying the predicted η' values of the models and the stationary phase "hydrophobicity" parameter H values for the corresponding columns to the approximate HSM, resulting in excellent accuracy and predictability (Qext(F2)2 of 0.90 and RMSEP of 0.72 min). The established QSRR approach was experimentally verified for six Thermo Scientific columns (Acclaim™ 120 C18, Acclaim Polar Advantage, Acclaim Polar Advantage II, Accucore™ aQ, Accucore Phenyl-X, and Hypersil Gold C18 columns) using two types of datasets. The first dataset consisted of eight model compounds extracted from the original dataset and retention time predictions for those compounds were then evaluated on the above columns. The result showed good agreement between predicted and observed retention times with an acceptable error in retention time predictions (slope of 0.97, Qext(F2)2 of 0.95, a mean absolute error (MAE) of 0.43 min and RMSEP of 0.61 min). The second dataset included eight test compounds not included in the original dataset, which were all classified into the η' cluster by applying a Tanimoto similarity (TS) threshold of 0.7. Similarly, predicted retention times of the test compounds were compared with their corresponding observed retention times, resulting in acceptable retention time predictions with the slope of 0.99, Qext(F2)2 of 0.93 and RMSEP of 0.52 min. Comparisons of resolution values between columns were utilised to select the most suitable columns for separations of the compounds in the respective test sets. Actual chromatograms obtained on the chosen columns showed the feasibility for effective column scoping without experimentation on numerous RP stationary phases available in the USP website, based on the predicted resolution values.


Assuntos
Cromatografia de Fase Reversa/métodos , Modelos Químicos , Relação Quantitativa Estrutura-Atividade , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , Simulação por Computador , Bases de Dados como Assunto , Análise dos Mínimos Quadrados
7.
J Chromatogr Sci ; 57(10): 931-938, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31642896

RESUMO

Paclitaxel (PTX) and sulforaphane (SFN) are known anticancer molecules. Their activity was found to be potentiated when tested concurrently. Only recently, however, a novel SFN enabled PTX self-microemulsifying formulation (SMEDDS) was developed for their simultaneous delivery. This necessitated the development of an analytical method for the simultaneous detection and quantitation of PTX and SFN. In this study, a simple and sensitive isocratic high performance liquid chromatography-ultraviolet (HPLC-UV) analytical method was developed and validated per International Conference on Harmonization guidelines to satisfy this objective. Its application was demonstrated when quantifying the amount of PTX and SFN released from the SMEDDS in various dissolution media. The separation of the analytes was performed with the aid of a reversed phase C18 column at ambient temperature using a 60:40 mixture of acetonitrile and KH2PO4 buffer (pH 5.0) as the mobile phase. PTX and SFN peaks were detected at 202 nm with high resolution without interference from excipients. This method showed linearity within 2.5-100 µg/mL range with r2 > 0.999. The limit of detection and lower limit of quantitation were 0.1638 and 0.4964 µg/mL for PTX and 0.4419 and 1.3389 µg/mL for SFN, respectively. A total of 98-101% of the injected samples was recovered with RSD of 0.06-0.68% indicating the suitability of the method for the simultaneous detection and quantitation of the molecules in dissolution media.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Emulsões/química , Isotiocianatos/análise , Lipídeos/química , Paclitaxel/análise , Cromatografia de Fase Reversa , Composição de Medicamentos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes
8.
J Chromatogr Sci ; 57(10): 867-873, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31602483

RESUMO

Nadifloxacin, mometasone furoate and miconazole nitrate are formulated together as a topical antifungal dosage form. In this work, a reversed-phase ultra-performance liquid chromatographic method coupled with a diode array detector (RP-UPLC-DAD) was developed and validated to determine nadifloxacin, mometasone furoate and miconazole nitrate simultaneously in their bulk powder, in pharmaceutical preparation and in spiked human plasma samples. Separation was achieved on an ACQUITY UPLC C18 column of 2.2 µm particle size (2.1 × 100 mm) via isocratic elution using a mobile phase consisting of methanol, acetonitrile and water with ratio (50:20:30; v/v/v) and 0.1 g ammonium acetate, then pH was adjusted to (7.00) using acetic acid, flow rate 0.6 mL/min, temperature 30°C and UV detection at 220 nm. The method is linear in a range from 5 to 400 µg/mL for both nadifloxacin and miconazole nitrate and from 20 to 500 µg/mL for mometasone furoate. The method was validated according to the ICH guidelines then applied successfully to determine the mentioned drugs in their pharmaceutical preparation and spiked human plasma samples. For plasma samples, the results showed that the method can determine nadifloxacin, mometasone furoate and miconazole nitrate in human plasma samples with high accuracy and precision.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fluoroquinolonas/análise , Miconazol/análise , Furoato de Mometasona/análise , Quinolizinas/análise , Cromatografia de Fase Reversa , Fluoroquinolonas/sangue , Fluoroquinolonas/química , Humanos , Limite de Detecção , Modelos Lineares , Miconazol/sangue , Miconazol/química , Furoato de Mometasona/sangue , Furoato de Mometasona/química , Quinolizinas/sangue , Quinolizinas/química , Reprodutibilidade dos Testes
9.
Biomed Chromatogr ; 34(1): e4699, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31524294

RESUMO

In this study, we developed and validated two reliable high-performance liquid chromatography (HPLC) methods for the qualitative detection of six oral ß-lactams, which are commonly used in pediatric patients with acute respiratory infections (ARIs). Two distinct reverse-phase chromatographic separations of six ß-lactams were obtained. Four ß-lactams (cefadroxil, cephalexin, cefaclor and cefixime) in urine were separated using a gradient program with a mobile phase consisting of K2 HPO4 buffer (20 mm, pH 2.8) and acetonitrile on a LichroCART 250 × 4.6 mm, Purospher STAR C18 end-capped (5 µm) column. Two remained ß-lactams (amoxicillin and cefuroxime) were analyzed using a gradient elution with the mobile phase containing K2 HPO4 buffer (20 mm, pH 3.0) and acetonitrile on a LichroCart® Purospher Star C8 end-capped column (5 µm, 125 × 4.6 mm). Good linearity within the range of 0.3-30 µg/ml for cefadroxil, cephalexin, cefaclor and cefixime, and 0.2-20 µg/ml for amoxicillin and cefuroxime, was attained. The precisions were <14%. The accuracies ranged from 85.87 to 102.8%. The two validated methods were then applied to determine these six antibiotics in 553 urine samples of pediatric patients with ARIs. As a result, 32.2% were positive with one or more of six tested ß-lactams. Cefixime was the most commonly detected agent, accounting for 9.8% of enrolled patients.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Infecções Respiratórias/tratamento farmacológico , beta-Lactamas/urina , Doença Aguda , Adolescente , Criança , Pré-Escolar , Cromatografia de Fase Reversa/métodos , Humanos , Lactente , Recém-Nascido , Limite de Detecção , Modelos Lineares , Uso Excessivo de Medicamentos Prescritos , Reprodutibilidade dos Testes , beta-Lactamas/uso terapêutico
10.
J Chromatogr A ; 1609: 460512, 2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31542208

RESUMO

The simultaneous use of nitrite and sorbate as preservatives in meat products may produce mutagenic compounds such as the ethylnitrolic acid and 2-methyl-1,4-dinitro-pyrrole. We developed a sensitive analytical method with high metrological reliability. After assessing several extraction approaches and chromatographic separation modes, a modified Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) approach was chosen for sample preparation, which were analyzed by reversed-phase liquid chromatography (with C18 as stationary phase) coupled to tandem mass spectrometry. After validation, we confirmed that this method is fit-for-purpose, since it was applied to the analysis of several meat products. Limits of detection were set from 5 to 20 µg kg-1. Satisfactory results were obtained for both compounds, such as precision (CV > 20%) and recoveries (77-92%). This method determine these carcinogenic compounds in processed meats, contributing to the preservation of public health and the improvement of food regulation and control.


Assuntos
Métodos Analíticos de Preparação de Amostras , Hidroxilaminas/análise , Produtos da Carne/análise , Mutagênicos/análise , Nitrilos/análise , Pirróis/química , Espectrometria de Massas em Tandem/métodos , Calibragem , Cromatografia Líquida , Cromatografia de Fase Reversa , Reprodutibilidade dos Testes
11.
J Chromatogr A ; 1609: 460437, 2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31409488

RESUMO

Thermal papers (e.g. point of sale receipts, adhesive labels, tickets) significantly contribute to contamination of paper material cycles and the environment with substances of (eco-) toxicological concern. In particular, they contain color developers like endocrine disrupting bisphenols in typical concentrations of about 1-2 percent per weight (wt%). Bisphenol A (BPA) was used as the common color developer over the last decades, but it will be restricted for thermal paper application in the European Union to a limit of 0.02 wt% from 2020 onwards. Consequently, a variety of BPA substituents such as bisphenol S (BPS) and its derivatives gain importance in thermal paper application. In this study, a rapid, reliable and cost-effective method for identification and quantification of BPA, alternative color developers and related substances like sensitizers is presented based on HPLC separation coupled with diode array detection (DAD) and Corona charged aerosol detection (CAD). Quantification was performed with regard to the intended use of the substances in thermal papers. Besides traditional UV external calibration using reference standards, alternative quantification approaches, in particular UV chromophore concentration for BPS derivatives and CAD universal response technique for low-volatile color developers, were applied and compared in order to allow quantification without reference substances. A market analysis for intended used color developers and sensitizers was performed on thermal paper samples (n = 211) collected in Germany during 2018 and 2019. Pergafast 201 (in 41.7% of the samples) was the most common color developer with concentrations above 0.02 wt%, followed by BPA (36.0%), BPS (13.3%) and other BPS derivatives known as D8, D-90, BPS-MAE and TGSA, that are mainly present in adhesive labels. Sensitizers were determined in over 90% of the samples.


Assuntos
Aerossóis/análise , Compostos Benzidrílicos/análise , Marketing , Papel , Fenóis/análise , Temperatura Ambiente , Compostos Benzidrílicos/química , Calibragem , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Cor , Disruptores Endócrinos/análise , União Europeia , Alemanha , Limite de Detecção , Modelos Lineares , Fenóis/química , Padrões de Referência , Sulfonas
12.
J Chromatogr A ; 1609: 460449, 2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31443968

RESUMO

The application of supercritical fluid chromatography with mass spectrometric (MS) detection (SFC-MS) was compared towards generic reversed phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) for the analysis of urine with regards of ionization performance and analyte identification. The different chromatographic conditions were characterized with a selected set of 51 metabolites from different classes reported in the Human Metabolome DataBase (HMDB) and previously detected in human urine and/or plasma. SFC using a diol column with a gradient of carbon dioxide (CO2) and methanol with 10 mM ammonium hydroxide as modifier was able to retain and separate 20 polar analytes co-eluting in the RPLC eluent front. In the conditions investigated and compared to HILIC where many metabolites were also co-eluting, SFC showed a different ratio between elution domain and analysis time. Similar peak width and symmetry were observed, while retention time variability was slightly lower compared to that of HILIC (0.15% versus 0.24% and 1.26% for RPLC and HILIC, respectively). In SFC-MS, a significant signal enhancement (2-150 times, average of about 10 times) was measured after post-column make-up addition (MeOH/H2O, 95/5, v/v + 25 mM ammonium acetate) for 28 analytes. Nine analytes measured by LC-MS could not be detected in SFC-MS. Applicability of SFC-MS for metabolomics was investigated with the analysis of urine samples using data independent acquisition (DIA) and more specifically Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH/MS). Using a metabolomics library, 74 metabolites from human urine could be identified in positive mode in a single SFC-MS analysis of 15 min.


Assuntos
Cromatografia com Fluido Supercrítico/métodos , Espectrometria de Massas/métodos , Metaboloma , Urina/química , Cromatografia de Fase Reversa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Metabolômica
13.
J Chromatogr A ; 1609: 460427, 2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31439441

RESUMO

Multi-linear gradients are a convenient solution to get separation of complex samples by modulating carefully the gradient slope, in order to accomplish the local selectivity needs for each particular solute cluster. These gradients can be designed by trial-and-error according to the chromatographer experience, but this strategy becomes quickly inappropriate for complex separations. More evolved solutions imply the sequential construction of multi-segmented gradients. However, this strategy discards part of the search space in each step of the construction and, again, cannot deal properly with very complex samples. When the complexity is too large, the only valid alternative for finding the best gradient is the use of global search methods, such as genetic algorithms (GAs). Recently, a new global approach where the level of detail is increased along the search has been proposed, namely Multi-scale optimisation (MSO). In this strategy, cubic splines are applied to build intermediate curves to define any arbitrary solvent variation function. Subdivision schemes are used to generate the cubic splines and control their level of detail. The search was subjected to a number of restrictions, such as avoiding long elution and favouring a balanced peak distribution. The aim of this work is evaluating and comparing the results of GAs and MSO. Both approaches were tested with a set of 14 diuretics and probenecid, eluted with acetonitrile-water mixtures using a C18 column. Satisfactory baseline resolution was obtained with an analysis time of 15-16 min. We found that GAs optimisation offered results equivalent to those provided by MSO, when the penalisation parameters were included in the cost function.


Assuntos
Algoritmos , Cromatografia de Fase Reversa/métodos , Diuréticos/isolamento & purificação , Acetonitrilos/química , Solventes , Água/química
14.
J Chromatogr A ; 1609: 460439, 2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31405573

RESUMO

A novel approach based on the use of desirability functions is presented for the robustness assessment of liquid chromatographic separations as derived from computer-assisted methods development processes. The approach is based on generally accepted hypothesis that a robust separation procedure will be inert to small random variations of the operational variables, typically encountered in the day-to-day routine analytical practice. This means that peak positions along the chromatograms must keep standstill or move insignificantly when operational variables are not intentionally changed. Thus, the degree of peak positions variation as evaluated from mathematical retention models can be used to assess the robustness of the developed procedures before testing the actual performance experimentally. In the approach proposed, this assessment is obtained by fixing a bilateral partial desirability window around each peak in the simulated chromatogram. The whole chromatogram robustness is characterized by an overall desirability value calculated as the geometric mean of the partial desirability windows evaluation. An added advantage of this approach is that the robustness value calculated is normalized between zero and one and thus, easy to interpret. Thus, when chromatograms are simulated and small random variations are introduced into the operational factors of the model, values for the overall desirability close to one means that the procedure performs robustly. On the contrary, low values for the overall desirability clearly indicated a serious lack of robustness. When used in conjunction with the Pareto optimality approach, as shown here, this robustness assessment strategy allows testing several Pareto front solutions before the final experimental testing which is always needed. In this way, a dramatical reduction of the experimental effort is obtained. Although the approach is theoretically applicable to any chromatographic separation, examples of reversed phase liquid chromatographic procedures are used to show the performance of the proposed methodology.


Assuntos
Cromatografia Líquida/métodos , Computadores , Algoritmos , Cromatografia de Fase Reversa , Probabilidade , Reprodutibilidade dos Testes
15.
J Chromatogr A ; 1609: 460505, 2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31500881

RESUMO

Biochemical and thermochemical processes are two pathways to convert lignocellulosic biomass into fuels and chemicals. Both conversion types produce aqueous complex samples containing many oxygenated chemical functions over a wide range of masses. Nowadays, composition of these biomass products is still largely unknown, especially their nonvolatile part (300-1000 Da) mostly made of carbohydrates and their derivatives. In the present study, size exclusion chromatography (SEC) was investigated and applied on water soluble phase of a fast pyrolysis bio-oil (thermochemical conversion) and on aqueous phase of pretreated wheat straw (biochemical conversion). An optimization of mobile phase composition using model molecules was necessary to limit non-steric interactions and elute all chemical families. At the end, separation of carbohydrates, heterosides and aromatic species was performed. The chemical organization of SEC chromatograms was confirmed by coupling SEC with a Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS) using electrospray ionization (ESI) in the negative mode. On-line SEC-UV/FT-ICR MS hyphenation was a powerful tool to provide exact mass distribution of samples and get molecular formulae classed by chemical family. To go further, the complementarity of SEC with reversed-phase liquid chromatography (RPLC) was established with an off-line comprehensive 2D-LC analysis of the two samples. First, 140 fractions were collected physically from SEC separation for each sample, then each fraction was analyzed by RPLC hyphenated to an Ion Trap - Time of Flight mass spectrometer (SEC × RPLC-UV/IT-TOF MS) using ESI in both positive and negative modes. This comprehensive approach combining 2D-LC and high resolution mass spectrometry nearly doubled the number of peaks detected in comparison with 1D RPLC analysis and thus offered well resolved 2D contour plots, considered as relevant analytical fingerprints of the aqueous phase of biomass samples.


Assuntos
Biomassa , Cromatografia em Gel , Cromatografia de Fase Reversa/métodos , Lignina/análise , Espectrometria de Massas/métodos , Cromatografia Líquida , Sistemas On-Line
16.
Anal Bioanal Chem ; 412(2): 299-309, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31814048

RESUMO

Oligonucleotide drugs represent an emerging area in the pharmaceutical industry. Solid-phase synthesis generates many structurally closely related impurities, making efficient separation systems for purification and analysis a key challenge during pharmaceutical drug development. To increase the fundamental understanding of the important preparative separation step, mass-overloaded injections of a fully phosphorothioated 16mer, i.e., deoxythymidine oligonucleotide, were performed on a C18 and a phenyl column. The narrowest elution profiles were obtained using the phenyl column, and the 16mer could be collected with high purity and yield on both columns. The most likely contribution to the successful purification was the quantifiable displacement of the early-eluting shortmers on both columns. In addition, the phenyl column displayed better separation of later-eluting impurities, such as the 17mer impurity. The mass-overloaded injections resulted in classical Langmuirian elution profiles on all columns, provided the concentration of the ion-pairing reagent in the eluent was sufficiently high. Two additional column chemistries, C4 and C8, were also investigated in terms of their selectivity and elution profile characteristics for the separation of 5-20mers fully phosphorothioated deoxythymidine oligonucleotides. When using triethylamine as ion-pairing reagent to separate phosphorothioated oligonucleotides, we observed peak broadening caused by the partial separation of diastereomers, predominantly seen on the C4 and C18 columns. When using the ion-pair reagent tributylamine, to suppress diastereomer separation, the greatest selectivity was found using the phenyl column followed by C18. The present results will be useful when designing and optimizing efficient preparative separations of synthetic oligonucleotides.


Assuntos
Indicadores e Reagentes/química , Oligonucleotídeos Fosforotioatos/análise , Oligonucleotídeos Fosforotioatos/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos
17.
Chem Biodivers ; 16(12): e1900373, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31654549

RESUMO

Phytochemicals, which are commonly found at different levels in many medicinal plants, are natural strong antioxidants used in traditional medicine. In this research, determination of differences of phytochemical compositions and biological properties were aimed as periodically (pre-, full and post flowering) and daily (6 am, 1 pm and 8 pm) in Achillea gypsicola Hub.-Mor. The volatile oils belonging to A. gypsicola were obtained by hydrodistillation and analyzed by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC/MS). The antimicrobial activities of the volatile oils were determined with disc diffusion method. The microdilution method was used to determine minimum inhibitory concentration (MIC). Total phenolic and flavonoid contents were determined by spectrophotometric methods and antioxidant capacities were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical, reducing power (RP) and metal chelating activity (MCA) assay. In addition, the phenolic acid and flavonoid compositions were evaluated by reversed phase-high-performance liquid chromatography (RP-HPLC). This study presented a comprehensive report for the first time on evaluation of the phytochemical composition and the biological properties of A. gypsicola at different phenological stages. Thirty-two compounds, containing the major component as camphor, 1,8-cineole and borneol, were detected. Designated harvest time for the highest yield of volatile oils was found to be at full flowering stage-1 pm. It has been observed that the volatile oil composition changes periodically and even daily. Also, in this research, menthol and menthone were found as the composition of volatile oil in Achillea species for the first time. Full flowering stage was found as the richest period in terms of phenolic acid and flavonoid compositions of A. gypsicola for the first time. The species examined in this research showed a high antioxidant and antimicrobial activity in comparison to other studies with Achillea species. The volatile oils exhibited high performances with range of inhibition zones (8.3-42.3 mm) and minimum inhibitory concentration values (2.25-144 µg/ml). Besides, a high correlation between antioxidant activity and phenolic content of A. gypsicola was found. These results suggest that A. gypsicola can be used as a safe source in the cosmetic, food and pharmaceutical industries.


Assuntos
Achillea/química , Óleos Voláteis/química , Compostos Fitoquímicos/química , Achillea/metabolismo , Antioxidantes/química , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Flavonoides/análise , Flavonoides/química , Flavonoides/farmacologia , Flores/química , Flores/metabolismo , Fungos/efeitos dos fármacos , Cromatografia Gasosa-Espectrometria de Massas , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Óleos Voláteis/farmacologia , Fenóis/análise , Fenóis/química , Fenóis/farmacologia , Compostos Fitoquímicos/farmacologia , Análise de Componente Principal
18.
Se Pu ; 37(8): 878-886, 2019 Aug 08.
Artigo em Chinês | MEDLINE | ID: mdl-31642259

RESUMO

One of the major shortcomings in top-down proteomics is the lack of efficient separations for intact proteins that can be effectively coupled to mass spectrometry. Capillary zone electrophoresis (CZE) and nanoflow reversed-phase liquid chromatography (nanoRPLC) are two methods that can be coupled to mass spectrometry directly and have been recently advanced in terms of their ability to separate intact proteins in complex biological mixtures. In this work, for the first time, we compared the state-of-the-art nanoRPLC-MS/MS and CZE-MS/MS platforms for top-down characterization of a standard protein mixture and an Escherichia coli (E. coli) proteome sample. CZE-MS produced comparable signals of standard proteins to RPLC-MS with 10-times less sample consumption. Interestingly, the proteins in RPLC-MS tended to have higher charge states than in CZE-MS, most likely due to the high acetonitrile concentration in RPLC mobile phase, leading to the more extensive unfolding of proteins in RPLC compared to in CZE. CZE-MS/MS identified 159 proteins and 513 proteoforms using 1-µg E. coli proteins in a single run and outperformed RPLC-MS/MS using 1-µg E. coli proteins in terms of protein and proteoform identifications (159 vs. 105 proteins and 513 vs. 277 proteoforms). The RPLC-MS/MS using 8-µg E. coli proteins identified 245 proteins and 1004 proteoforms in a single run, and the data was much better than that from CZE-MS/MS (1-µg E. coli proteins) regarding the number of identifications because of the 8-times higher sample loading amount and significantly wider separation window of RPLC-MS/MS compared to CZE-MS/MS.


Assuntos
Cromatografia de Fase Reversa , Eletroforese Capilar , Proteômica/métodos , Espectrometria de Massas em Tandem , Escherichia coli , Proteínas de Escherichia coli/análise
19.
Anal Chim Acta ; 1088: 150-167, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31623711

RESUMO

In this study, we report a methodology based on reversed phase LC (RP-LC) and hydrophilic interaction chromatography (HILIC) separations coupled to ion mobility (IM) and high resolution mass spectrometry (HR-MS) for the detailed analysis of hydrolysable tannins. The application of this approach to the analysis of an industrial chestnut (Castanea sativa, wood chips) tannin extract is demonstrated. A total of 38 molecular species, including a large number or isomers, were identified in this sample based on HR-MS(E) and UV absorption spectral information as well as retention behaviour in both separation modes. In total, 128 and 90 isomeric species were resolved by RP- and HILIC-LC-IM-TOF-MS, respectively. The combination of low- and high collision energy mass spectral data with complementary chromatographic separations allowed tentative and putative identification of twenty molecular species, comprising 78 isomers, in chestnut for the first time. Ion mobility resolved six new dimeric and trimeric vescalagin conformers with unique arrival (drift) times, including new conformers of roburin A-D which were not separated using either RP-LC or HILIC. HILIC was found to be the preferred separation mode for the analysis of vescalagin derivatives, while RP-LC is preferred for the analysis of ellagitannins with a cyclic glucose core. For the complete separation of the galloyl glucose species, comprehensive HILIC × RP-LC separation would be required.


Assuntos
Cromatografia de Fase Reversa , Fagaceae/química , Análise de Alimentos/métodos , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas , Taninos/análise , Taninos/isolamento & purificação , Ácido Gálico/química , Glucose/química , Hidrólise , Taninos Hidrolisáveis/química , Limite de Detecção , Taninos/química
20.
Pak J Pharm Sci ; 32(4): 1607-1614, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31608880

RESUMO

The paper aims to establish a RP-HPLC method for the simultaneous determination of six related substances in ketoprofen injection. The separation was performed on a VP-ODS C18 column (4.6mm×250mm, 5µm) with the mobile phase of 6.8% phosphate buffer solution (adjusted to pH3.5 with 85% phosphoric acid)-acetonitrile-water (2:43:55,v/v/v) at a flow rate of 1.2mL•min-1. The detection wavelength and the injection volume were set at 233nm and 20µL, respectively. Impurity A and C were calculated by external standard method. Main component self-compare method with calibration factor was used to calculate impurity B, D, E, F and main component self-compare method without calibration factor was used to calculate unspecified impurity. Related substances and degraded substances were completely separated from ketoprofen. For impurity A and C, the linear range of determination were separately 0.06 µg•mL-1 ~ 3.6µg•mL-1 and 0.036µg•mL-1 ~ 2.4µg•mL-1 with the correlation coefficient of 0.9999. The average recoveries (n=9) were 98.13% (RSD=0.35%) and 96.32% (RSD=0.43%). The precision and repeatability for method were good. With reference to ketoprofen (retention time =10.06 min), the relative retention time of impurity B, D, E, F were 0.71, 1.46, 0.59, 2.13, respectively, and the relative correction factors were 0.962, 0.938, 0.957, 0.960, respectively. Finally, determined that the contents of impurity A could not be more than 0.3%, any of the contents of impurity B, C, D, E, F and unspecified impurities could not be more than 0.2%, sum of the contents of impurities other than A and C couldn't be more than 0.5%. The method was proved to be simple, rapid, accurate, sensitive and suitable for the simultaneous determination of six related substances in ketoprofen injection.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Contaminação de Medicamentos , Cetoprofeno/análise , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia de Fase Reversa , Injeções , Cetoprofeno/administração & dosagem , Cetoprofeno/normas , Limite de Detecção , Controle de Qualidade , Análise de Regressão , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo
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