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1.
J Vis Exp ; (162)2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32894271

RESUMO

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.


Assuntos
Proteoma/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Cromatografia de Fase Reversa , Biologia Computacional , Proteoma/química , Proteoma/metabolismo
2.
J Chromatogr A ; 1629: 461510, 2020 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-32889298

RESUMO

A two-dimensional system composed of supercritical fluid chromatography (SFC) and reverse phase liquid chromatography (RPLC) coupled a tandem mass spectrometry (MS) was developed for the quantitative analysis of vitamin D in daily oily supplements. Two six-port switching valves are configured, allowing four different valve positions. When the valve positions were fixed at Position A, this system worked at SFC-MS mode. When the valve positions switched between Position B and C, this system worked at a SFC-LC-MS switching mode. Vitamin D3 in two kinds of oily drops, Baby Ddrops and Vitamin AD drops, was determined at both SFC-MS and SFC-LC-MS switching modes by using the same system. The linearity, repeatability and recovery were investigated using the internal and external standard methods for the two modes. The results obtained from the internal standard method are better than those of the external standard method at either mode. The coefficient of determination (r2) for the internal standard method is more than 0.999, with a linear range of 20-1000 µg/L. Both Baby Ddrops and Vitamin AD drops were analyzed with good repeatability (<3.21%) and recovery (94.1%-116.1%) using internal standard method. When calculated by the external standard method, as compared to SFC-MS mode, SFC-LC-MS switching mode has better linearity (r2>0.999), repeatability (Baby Ddrops: 4.45%, Vitamin AD drops: 1.12%) and recovery (86.3%-107.1%). The results indicate that the two-dimensional SFC-MS/SFC-LC-MS system is useful for determination of vitamin D in oily drops. If it works at SFC-MS mode, the internal standard is required. When SFC-LC-MS switching mode is used, external standard method can also obtain the accurate results with good precision.


Assuntos
Colecalciferol/análise , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia com Fluido Supercrítico/métodos , Suplementos Nutricionais/análise , Colecalciferol/isolamento & purificação , Cromatografia de Fase Reversa , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos
3.
J Chromatogr A ; 1628: 461450, 2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32822989

RESUMO

For choosing an optimal column for a particular separation by reversed-phase liquid chromatography (RPLC), it is essential to quantitatively understand the effects of the chemical structure of hydrophobic bonded layer derived onto silica particles on the distribution equilibrium of a solute compound at the interface between the aqueous mobile phase and the packing material. However, there is still a lack of understanding of the solute distribution equilibrium in RPLC separation due to the complexities of the chemistry at the interface between the mobile phase and the bonded layer. We successfully determined the distribution coefficients of various organic compounds concerning to their accumulation onto the water/bonded layer interface and into the bonded layer from bulk water using surface-bubble-modulated liquid chromatography with octadecyl- and phenyl hexyl-bonded silica columns. The water/phenyl hexyl-bonded layer interface accumulates organic compounds much less than the water/octadecyl-bonded layer interface due to its lower interfacial tension, and this result suggests that phenyl hexyl group orient their benzene ring facing toward water. On the other hand, aromatic moiety of phenyl hexyl group enhances partitioning of the organic compounds into the bonded layer. Experimental findings in the present work demonstrated that the water/bonded layer interface and the bonded layer itself have independent contributions to the solute distribution and the water/phenyl hexyl-bonded layer interface shows quite different solute retention selectivity from the water/octadecyl-bonded layer interface.


Assuntos
Cromatografia de Fase Reversa/métodos , Dióxido de Silício/química , Água/química , Cromatografia Líquida , Interações Hidrofóbicas e Hidrofílicas , Termodinâmica
4.
Artigo em Inglês | MEDLINE | ID: mdl-32854235

RESUMO

Saliva is an interesting, non-conventional, valuable diagnostic fluid. It can be collected using standardized sampling device; thus, its sampling is easy and non-invasive, it contains a variety of organic metabolites that reflect blood composition. The aim of this study was to validate a user-friendly method for the simultaneous determination of low molecular weight metabolites in saliva. We have optimized and validated a high throughput, direct, low-cost reversed phase liquid chromatographic method with diode array detection method without any pre- or post-column derivatization. We indexed salivary biomolecules in 35 whole non-stimulated saliva samples collected in 8 individuals in different days, including organic acids and amino acids and other carbonyl compounds. Among these, 16 whole saliva samples were collected by a single individual over three weeks before, during and after treatment with antibiotic in order to investigate the dynamics of metabolites. The concentrations of the metabolites were compared with the literature data. The multianalyte method here proposed requires a minimal sample handling and it is cost-effectiveness as it makes possible to analyze a high number of samples with basic instrumentation. The identification and quantitation of salivary metabolites may allow the definition of potential biomarkers for non-invasive "personal monitoring" during drug treatments, work out, or life habits over time.


Assuntos
Cromatografia de Fase Reversa/métodos , Peso Molecular , Saliva/química , Fluorescência , Humanos , Estrutura Molecular , Reprodutibilidade dos Testes
5.
J Chromatogr A ; 1629: 461505, 2020 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-32861092

RESUMO

Immobilized metal affinity chromatography (IMAC) is a technique primarily used in research and development laboratories to purify proteins containing engineered histidine tags. Although this type of chromatography is commonly used, it can be problematic as differing combinations of resins and metal chelators can result in highly variable chromatographic performance and product quality results. To generate a robust IMAC purification process, the binding differences of resin and metal chelator combinations were studied by generating breakthrough curves with a poly-histidine tagged bispecific protein. The optimal binding combination was statistically analyzed to determine the impact of chromatographic parameters on the operation. Additionally, equilibrium uptake isotherms were created to further elucidate the impact of chromatographic parameters on the binding of protein. It was found that for protein expressed in CHO cells, Millipore Sigma's Fractogel EMD Chelate (M) charged with Zn2+ and GE's pre-charged Ni Sepharose Excel displayed the highest binding capacities. When the protein was expressed in HEK-293, GE's IMAC Sepharose 6 Fast Flow charged with either Co2+ or Zn2+ bound the greatest amount of protein. The study further identified the metal binding capacity of the resin lot, the protein capacity to which the resin is loaded, and the ratio of poly-histidine tag residues on the protein all impacted the chromatographic performance and product quality. These findings enabled the development of a robust and scalable process. The CHO expressed cell culture product was directly loaded at a high capacity onto variable metal binding affinity Fractogel EMD Chelate (M). A 250 mM imidazole elution condition ensured the product contained monomeric 4 and 6-histidine tagged bispecific proteins. The optimized IMAC process conditions determined in this study can be applied to a wide variety of poly-histidine tagged proteins in research and development laboratories as various poly-histidine tagged proteins of differing molecular weights and formats expressed in either HEK-293 or CHO cells were successfully purified.


Assuntos
Cromatografia de Afinidade/métodos , Histidina/metabolismo , Metais/química , Proteínas Recombinantes/isolamento & purificação , Animais , Células CHO , Quelantes/química , Cromatografia de Fase Reversa , Cobalto/química , Cricetinae , Cricetulus , Células HEK293 , Histidina/genética , Humanos , Proteínas Recombinantes/biossíntese , Zinco/química
6.
J Chromatogr A ; 1626: 461427, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32739066

RESUMO

The physicochemical properties of small molecules that can be determined by retention measurements in reversed-phase liquid chromatography include solvent-based properties inferred from equilibrium processes occurring predominantly in the mobile phase and sorption properties for materials which can be used as stationary phases inferred from solute-stationary phase interactions. In addition, physicochemical properties can be estimated from correlation models based on surrogate chromatographic systems with a similar capability for intermolecular interactions to the chemical or biological system. Examples of properties determined by direct methods include molecular descriptors (solvation parameter model), acid dissociation constants, formation constants, and surface properties of solids determined by inverse liquid chromatography. Examples of properties estimated by indirect methods include hydrophobicity, lipophilicity, n-octanol-water partition constant, soil-water sorption constant, non-specific toxicity to fish and microorganisms, and permeation coefficients for the blood-brain and skin-water barriers. Since all approaches depend on an accurate measurement of chromatographic retention parameters typical operational and mechanistic problems are discussed from the perspective of data quality. Fundamentally the accuracy of direct methods is limited by stationary phase heterogeneity and indirect methods by the limited number of suitable surrogate chromatographic models.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa , Interações Hidrofóbicas e Hidrofílicas , Membranas Artificiais , Octanóis/química , Solo/química , Solventes/química , Água/química
7.
J Chromatogr A ; 1626: 461388, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797859

RESUMO

A reversed-phase high performance liquid chromatographic method was developed and validated for the simultaneous determination of the related substances of S-dapoxetine, including R-dapoxetine, (3S)-3-(dimethylamino-3-phenyl-1-propanol), S-3-amino-3-phenyl-1-propanol, 1-naphtol, 4-phenyl-2H,3H,4H-naphtho[1,2-b]pyran and 1-(2E)-Cinnamyloxynaphthalene. During the screening experiments seven different polysaccharide-type chiral stationary phases (amylose-based Lux-Amylose-1, Lux-i-Amylose-1 and Lux-Amylose-2, as well as cellulose-based Lux-Cellulose-1, Lux-Cellulose-2, Lux-Cellulose-3 and Lux-Cellulose-4) were tested in polar organic mode using a mobile phase consisting of 0.1% diethylamine in methanol, ethanol, 2-propanol and acetonitrile with 0.5 mL min-1 flow rate at 20 °C. Best results were obtained on Lux Cellulose-3 column with the ethanol-based mobile phase. To increase the retention factor of two, early-eluting impurities, water was added to the mobile phase. In order to counterbalance the increased total analysis time, higher column temperature (40 °C) and gradient elution, combined with flow-programming` was applied. Using the optimized conditions baseline separations were achieved for all compounds within 30 min. The method was validated according to the International Council on Harmonization guideline Q2(R1) and applied to the analysis of an approved, tablet formulation and dapoxetine-containing products sold on the internet. As expected, in the case of the pharmacy-acquired product, all of the monitored impurities were below 0.1%. However, interesting results were obtained when internet-acquired samples were analyzed. These tablets contained racemic dapoxetine and/or high concentration of R-dapoxetine impurity. Based on this work polysaccharide-based chiral stationary phases can be successfully applied for the simultaneous determination of achiral and chiral impurities in reversed-phase mode applying gradient elution and flow-rate programs. The study further underlines the importance of not only achiral, but also enantiomeric quality control, whenever counterfeiting of a single enantiomeric agent is suspected.


Assuntos
Benzilaminas/análise , Cromatografia Líquida de Alta Pressão/métodos , Naftalenos/análise , Cromatografia de Fase Reversa , Limite de Detecção , Espectrometria de Massas , Preparações Farmacêuticas/química , Estereoisomerismo , Comprimidos/química , Temperatura
8.
J Chromatogr A ; 1627: 461404, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823109

RESUMO

Liamocin biosurfactants and structurally related exophilins secreted by the Aureobasidium pullulans (A. pullulans) strain NRRL62031 were firstly analyzed by hyphenation of high-performance liquid chromatography (HPLC) with high-resolution mass spectrometry (HRMS). Ten different analytes were detected and identified by their accurate masses and divided into subclasses according to their different head groups: three liamocins with arabitol as head group, three mannitol liamocins, and four exophilins. A baseline separation of congeners within the subclasses was achieved by reversed phase HPLC on a C18 stationary phase, whereas an overlap of subclasses occurred. The structures were simultaneously confirmed by online tandem mass spectrometry (MS/MS) experiments in positive and negative ionization mode. The assigned polyol head groups and thus the feasibility of this method were confirmed by gas chromatography (GC)-MS data obtained after hydrolysis and derivatization of the liamocins. Based on the varying structural characteristics of liamocins, e.g. the polyol head group (or even none for exophilins) and the degree of acetylation, different detector response in LC-MS was expected, impairing relative quantification of congeners. Therefore, a complementary quantification method was developed using HPLC coupled to charged-aerosol detection (CAD), which allows the determination of the amount of the individual liamocin species without authentic liamocin standards. Hence, the here presented hyphenated techniques facilitate comprehensive analysis of liamocin biosurfactants.


Assuntos
Aerossóis/análise , Cromatografia de Fase Reversa/métodos , Manitol/análise , Tensoativos/análise , Espectrometria de Massas em Tandem , Ascomicetos/química , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Manitol/química , Álcoois Açúcares/análise , Álcoois Açúcares/química
9.
J Chromatogr A ; 1627: 461405, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823110

RESUMO

This study focused on the measurements and validity of relative distribution constants of vaporized hydrocarbons between air and polydimethylsiloxane (PDMS) using commercially available capillary columns. Capillary column gas chromatography (CCGC) measurements, using two columns containing a PDMS stationary phase with different film thicknesses, were conducted to determine the relative distribution constants of n-heptane, toluene, n-octane, p-xylene, n-nonane, and 1,2,4-trimethylbenzene between air and PDMS at 90 and 120 °C. To validate the accuracy of the relative distribution constants via CCGC, the compositions of three headspace samples containing different amounts of hydrocarbons were calculated using the relative distribution constants via CCGC and extracted amounts via PDMS solid phase microextraction (SPME) at 90 and 120 °C. It was found that calculated hydrocarbon compositions of headspace samples were comparable to true headspace hydrocarbon compositions via direct vapor analysis, with an average absolute relative error of 3.2%. Our results indicate that CCGC is an alternative method that can provide a reliable and convenient method to determine the relative distribution constants of various hydrocarbons between air and PDMS for quantitative chemical analysis of headspace.


Assuntos
Ar , Dimetilpolisiloxanos/química , Hidrocarbonetos/análise , Cromatografia de Fase Reversa , Hidrocarbonetos/química , Microextração em Fase Sólida , Volatilização
10.
J Chromatogr A ; 1627: 461421, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823116

RESUMO

Herein we present an efficient, column-switching method that relies on a custom-made T-union passive diffusion micromixer to assist water dilution and promote trap solute focusing of a high sample volume dissolved in pure organic solvent using a 0.075 mm i.d. nano-LC column. This method allows injecting 20 µL (or higher) of sample volume, speeding up the analysis time, with a 400-fold increase of the limits of quantitation for selected compounds. Five pesticides in different media were used as model compounds, and the analyses were carried out with a triple quadrupole mass spectrometer equipped with a Liquid Electron Ionization (LEI) LC-MS interface working in multiple reaction monitoring (MRM) mode. The system microfluidics were investigated using COMSOL modeling software. Robustness of the entire system was evaluated using a post-extraction addition soil extracts with limits of detection values spanning from 0.10 to 0.45 µg/L. Reproducible results in terms of peak area, peak shape, and retention times were achieved in soil matrix. Repeatability test on peak area variations were lower than 10%.


Assuntos
Cromatografia de Fase Reversa/métodos , Elétrons , Microfluídica/métodos , Nanopartículas/química , Espectrometria de Massas em Tandem/métodos , Água/química , Acetonitrilos/química , Difusão , Limite de Detecção , Praguicidas/análise , Reprodutibilidade dos Testes , Solventes/química
11.
J Chromatogr A ; 1627: 461423, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823118

RESUMO

A novel stationary phase co-modified with N-isopropyl acrylamide (NIPAM) and 3-aminophenylboronic acid copolymer on the silica was synthesized through atom transfer radical polymerization (ATRP) reaction for performing mixed-mode and boronate affinity chromatography. The prepared functionalized silica was characterized using Fourier transform infrared spectrometry (FT-IR), elemental analysis (EA) and thermogravimetric analysis (TGA), scanning electron micrographs (SEM) and Brunauer-Emmett-Teller (BET) measurements. The prepared column named Sil-PBA-NIPAM showed great separation performance for hydrophobic, hydrophilic, positional isomer, acidic and alkaline compounds. Besides, the mixture of cis-diol and non-cis-diol compounds was used to prove that the developed column also has potential to capture and enrich cis-diol compounds. The prepared column possesses merits of time-saving, high selectivity to cis-diol compounds and molecular-planarity selectivity compared with two commercial single-mode columns. The theoretical plates of material can reach to 57472 and the column has good hydrolysis stability and batch-to-batch reproducibility. In summary, the prepared column possesses good hydrophilicity, hydrophobicity, molecular-planarity selectivity and boronate affinity abilities for the analysis of various compounds.


Assuntos
Acrilamidas/química , Ácidos Borônicos/química , Cromatografia/métodos , Polímeros/química , Ácido Benzoico/análise , Cromatografia de Fase Reversa , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Fenóis/análise , Polimerização , Reprodutibilidade dos Testes , Dióxido de Silício/química , Espectroscopia de Infravermelho com Transformada de Fourier , Termogravimetria
12.
J Chromatogr A ; 1627: 461430, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823119

RESUMO

There is a huge, still increasing market for synthetic and therapeutic peptides. Their quality control is commonly based on a generic reversed-phase liquid chromatography (RPLC) method with C18 stationary phase and acetonitrile gradient with 0.1% trifluoroacetic acid in the mobile phase. It performs exceptionally well for a wide variety of impurities, yet structurally closely related impurities with similar sequences, not resolved in preparative RPLC, may easily coelute in the corresponding QC run as well. To address this problem an advanced generic 2D-LC impurity profiling method was developed in this work. It employs a selective comprehensive (high resolution sampling) RP×RP 2D-LC separation using a 100×2.1 mm ID column with the common acidic generic gradient in the first dimension, while RPLC under basic pH on a short 30×3 mm ID column is used in the second dimension. Recording data with a UV detector at 215 nm after 1D separation provides the common generic 1D chromatogram. However, after the 2D separation a flow splitter enabled recording of the signals of complementary detectors comprising a diode array detector (DAD) in-line with a charged aerosol detector (CAD) and a quadrupole-time-of-flight (QTOF) mass spectrometer (MS) with an electrospray ionization (ESI) source. Generic conditions of this 2D-LC method have been established through optimization of 2D stationary and mobile phase considering different pH values and buffer concentrations. The orthogonal separation principle has been documented by a number of therapeutic peptides including Exenatide, Octreotide, Cyclosporine A and Oxytocin as well as some other proprietary synthetic peptides. The information density can be further enhanced by using the QTOF-MS detector by data independent acquisition with SWATH. Through this sequential window acquisition of all theoretical fragment ion mass spectra it became possible to collect MS/MS data comprehensively in the high-resolution sampling window, thus enabling the extraction of 2D-EICs from fragment ions and the generation of 2D-contour plots of all product ions. Using Oxytocin as an example for an important therapeutic peptide, the ability of this advanced generic sRP-UV×RP-DAD-CAD-ESI-QTOF-MS/MS method with SWATH for peptide quality control is discussed.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Peptídeos/análise , Acetonitrilos/química , Tampões (Química) , Concentração de Íons de Hidrogênio , Ocitocina/análise , Controle de Qualidade , Solventes/química , Espectrometria de Massas em Tandem
13.
J Chromatogr A ; 1626: 461351, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797831

RESUMO

Traditional polymer-separation methods, such as size-exclusion chromatography and (gradient) liquid adsorption chromatography, cannot provide separations exclusively based on the number of deprotonated carboxylic-acid groups along the backbone chain of polymers. A novel separation method, based on non-aqueous ion-exchange chromatography (NAIEX), was developed, which allows such a separation of acid-functional polymers that are soluble in organic solvents. The polar, aprotic N-methyl-2-pyrrolidone was found to be a suitable solvent. It features a high relative permittivity (favouring dissociation of ion pairs into free ions) and it is a good solvent for polymers and organic salts, such as triethyl-ammonium formate. A negative charge is established on these polymers by deprotonation of the carboxylic-acid groups in the presence of an organic superbase (tetramethyl guanidine). Traditional potent organic bases, such as triethylamine, do not possess the base strength to compensate for the increase in pKa of polymeric carboxylic acid groups in non-aqueous conditions. Triethyl-ammonium formate is proposed as an alternative to traditional salts used for elution in aqueous ion-exchange chromatography. Separation was performed on an industry-standard strong-anion-exchange column and (near-)universal detection of the polymers was performed by high-temperature evaporative-light-scattering detection. The NAIEX method yielded a separation based on the acid-functionality distribution of the polymer. NAIEX was compared with traditional normal- and reversed-phase liquid-chromatography approaches for the separation of acid-functional copolymers.


Assuntos
Cromatografia por Troca Iônica/métodos , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Polímeros/química , Espectroscopia de Prótons por Ressonância Magnética , Sais/química , Solventes/química
14.
J Chromatogr A ; 1626: 461353, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797833

RESUMO

This paper reports the development of an LC-ESI-MS2 method for the sensitive determination of hydroxylated polychlorinated biphenyls (OH-PCBs) in human serum samples. Congener-specific separation was achieved by using a polar-embedded stationary phase, previously optimized for the working group, which provided better separation of isobaric compounds than the common octadecylsilane phases. MS fragmentation patterns and energies showed differences among OH-PCB congeners, mainly depending on the position of OH-group and the number of chlorine atoms in the molecule, although the most intense transitions were always those corresponding to the neutral loss of an HCl group from the quasi-molecular ion cluster. The method allowed the determination of OH-PCBs with good linearity (dynamic linear range of four orders of magnitude with R2 higher than 0.995) and precision (relative standard deviations of absolute areas lower than 10%), and with better sensitivity than other similar methods previously described in the literature. Matrix effect has been evaluated and reduced to less than 10% by the addition of isotopically labeled standards and a 10-fold dilution of the final sample extract. The low iLODs provided by the developed method (from 1.2 to 5.4 fg µL-1 for all the OH-PCBs studied, except 4'-OHCB108, whose iLOD was 61 fg µL-1) allows dilution without losses of detected peaks. Finally, the applicability of the method has been demonstrated by analyzing human serum samples belonging to an interlaboratory exercise.


Assuntos
Cromatografia Líquida/métodos , Cromatografia de Fase Reversa/métodos , Bifenilos Policlorados/análise , Espectrometria de Massas em Tandem/métodos , Humanos , Hidroxilação , Bifenilos Policlorados/sangue , Bifenilos Policlorados/química , Espectrometria de Massas por Ionização por Electrospray
15.
J Chromatogr A ; 1626: 461308, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797813

RESUMO

The Wayne State University (WSU) experimental descriptor database is utilized to bench mark the current capability of the solvation parameter model for use as a quantitative structure-retention relationship tool for estimating retention in gas and reversed-phase liquid chromatography. The prediction error for the retention factors of varied compounds on six open-tubular columns for gas chromatography (Rtx-5 SIL MS, DB-35 ms, RtxCLPesticides, HP-88, HP-INNOWAX and SLB-IL76) and three packed columns for reversed-phase liquid chromatography (SunFire C18, XBridge Shield RP18, and XBridge Phenyl) is used to establish expectations related to current practices. Each column data set was divided into a training set for calibration and a test set for validation employing a roughly 1 to 2 split, such that each test set contained about 40 to 80 varied compounds. The average absolute error for the prediction of retention factors by gas chromatography varied from about 0.1 to 0.4 on the retention factor scale with the larger error typical of stationary phases ranked as the most polar (or cohesive). For reversed-phase liquid chromatography the average error for the prediction of retention factors was 0.3 to 0.5 and generally larger than for gas chromatography. Statistical filters where utilized to identify a group of polycyclic aromatic compounds without hydrogen-bonding functional groups with a larger prediction error on the SunFire C18 column than for other compounds of smaller size, flexible structure or containing hydrogen-bonding functional groups. The heterogeneity of the retention mechanism is speculated to be the main contribution to the prediction error for both gas and liquid chromatography using the solvation parameter model.


Assuntos
Cromatografia Gasosa/métodos , Cromatografia de Fase Reversa/métodos , Ligação de Hidrogênio , Cinética , Solventes/química
16.
J Chromatogr A ; 1626: 461320, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797816

RESUMO

BMS-986142 is a Bruton's tyrosine kinase inhibitor under development to treat several disease types. The compound contains three chiral elements: one chiral center and two chiral axes, resulting in three potential atropisomeric impurities in its drug substance and drug products. Separation of BMS-986142 atropisomers has been successfully achieved on an achiral polar-embedded C18 column in reversed-phase liquid chromatography (RPLC) and on polysaccharide-based chiral columns in RPLC and supercritical fluid chromatography (SFC). Compared to the RPLC chiral separation, the SFC atropisomeric separation on a sub-2 µm immobilized cellulose-based column is much more efficient and environmentally friendly. The analysis time in SFC was reduced by 8-fold compared to that in RPLC, and the method sensitivity in SFC on the sub-2 µm chiral column in 3.0 mm I.D. was 2 to 4-fold better than that on 3 µm chiral columns in 4.6 mm I.D.. Furthermore, our study suggests that the contribution to band broadening from the extra column volume (ECV) of modern commercial SFC instrument was not negligible for a 3.0 mm I.D. × 100 mm column packed with 1.6 µm particles. This result reaffirms that there is a great need for further improvement of SFC instrument design in order to realize the full theoretical efficiency of both sub-2 µm achiral and chiral columns.


Assuntos
Cromatografia com Fluido Supercrítico/métodos , Polissacarídeos/química , Inibidores de Proteínas Quinases/análise , Cromatografia de Fase Reversa , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/isolamento & purificação , Estereoisomerismo
17.
J Chromatogr A ; 1628: 461435, 2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32822975

RESUMO

We report on the performance of three classes of evolutionary algorithms (genetic algorithms (GA), evolution strategies (ES) and covariance matrix adaptation evolution strategy (CMA-ES)) as a means to enhance searches in the method development spaces of 1D- and 2D-chromatography. After optimisation of the design parameters of the different algorithms, they were benchmarked against the performance of a plain grid search. It was found that all three classes significantly outperform the plain grid search, especially in terms of the number of search runs needed to achieve a given separation quality. As soon as more than 100 search runs are needed, the ES algorithm clearly outperforms the GA and CMA-ES algorithms, with the latter performing very well for short searches (<50 search runs) but being susceptible to convergence to local optima for longer searches. It was also found that the performance of the ES and GA algorithms, as well as the grid search, follow a hyperbolic law in the large search run number limit, such that the convergence rate parameter of this hyperbolic function can be used to quantify the difference in required number of search runs for these algorithms. In agreement with one's physical expectations, it was also found that the general advantage of the GA and ES algorithms over the grid search, as well as their mutual performance differences, grow with increasing difficulty of the separation problem.


Assuntos
Algoritmos , Cromatografia/métodos , Cromatografia de Fase Reversa , Simulação por Computador
18.
J Chromatogr A ; 1625: 461301, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709344

RESUMO

In this work we utilized basic and acidic analytes to investigate the ionic interaction participation in retention behavior of selected reversed-phase and polar columns. The test analytes included nitrate, benzenesulfonate and trimethylphenylammonium ions. The fully aqueous mobile phase comprising 10 mM dichloroacetic acid buffered with ammonia solution to desirable pH was used for retention experiments. Developed method was utilized to study the ionic interactions of stationary phases in pH range between 2.5 and 9.0. We demonstrate that selected sorbents used for reversed-phase and hydrophilic interaction chromatography separations exhibit cation- or anion-exchange interactions. We compare the results to novel Atlantis PREMIER BEH C18 AX mixed-mode column that combines reversed-phase and anion-exchange interaction modes. We evaluated the relative retention strength of selected columns for anionic and cationic analytes.


Assuntos
Cromatografia Líquida/métodos , Adsorção , Ânions , Cromatografia de Fase Reversa , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Dióxido de Silício/química , Água/química
19.
J Chromatogr A ; 1625: 461332, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709358

RESUMO

Two structural isomeric pentacyclic triterpenes, oleanolic acid and ursolic acid, were considered as the models for the quality control of many traditional Chinese herbal medicines and they have been proved to own important pharmacological activities. In the present work, liquid chromatographic and liquid-liquid chromatographic separation with high peak resolution of structural isomeric oleanolic acid and ursolic acid using hydroxypropyl-ß-cyclodextrin as mobile phase additive was successfully achieved, respectively. A high peak resolution, RS=8.143, was achieved for the two structural isomeric compounds by conventional reverse phase high performance liquid chromatography, which was greatly improved compared with the published values. Meanwhile, a biphasic solvent system composed of n-hexane-ethyl acetate-0.1 mol/L hydroxypropyl-ß-cyclodextrin (9:1:10, v/v) was selected for liquid-liquid chromatography, which provided a high peak resolution, RS = 6.573, for analytical apparatus and Rs = 8.500 for semi-preparative apparatus after optimization by liquid-liquid extractions. Two elution modes including reverse phase mode and normal phase mode were investigated for preparative separation of two acids from crude exact of Eriobotrya japonica Thunb. Furthermore, the inclusion complex between each of the two structural isomers and hydroxypropyl-ß-cyclodextrin were also investigated for high performance liquid chromatography and liquid-liquid chromatography, respectively, in which formation constants were determined for oleanolic acid and ursolic acid.


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/química , Cromatografia Líquida/métodos , Ácido Oleanólico/química , Ácido Oleanólico/isolamento & purificação , Triterpenos/química , Triterpenos/isolamento & purificação , Cromatografia de Fase Reversa , Eriobotrya/química , Isomerismo , Solventes , Temperatura , Termodinâmica , beta-Ciclodextrinas/química
20.
J Chromatogr A ; 1625: 461334, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709359

RESUMO

A comprehensive study on the chiral separation of bupivacaine, mepivacaine, prilocaine and propanocaine with eight commercial polysaccharide-based chiral stationary phases (CSPs) in reversed phase conditions compatible with MS detection is performed. Methanol and acetonitrile are used as organic modifiers. Retention and resolution values obtained for each compound in the different CSPs and mobile phases are compared. The polysaccharide-based CSPs tested present different enantioselectivity towards the analytes. From the results, the experimental conditions for determining the enantiomers of bupivacaine, mepivacaine, prilocaine and propanocaine in saline aqueous samples using MS detection are used, for the first time, to perform an enantioselective biodegradability study.


Assuntos
Anestésicos Locais/isolamento & purificação , Cromatografia de Fase Reversa/métodos , Polissacarídeos/química , Acetonitrilos/química , Biodegradação Ambiental , Cromatografia Líquida de Alta Pressão , Metanol/química , Peso Molecular , Estereoisomerismo , Água/química
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