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1.
J Chromatogr A ; 1689: 463747, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36621106

RESUMO

As green, less toxic, and abundant ligands with rich functional groups, natural products are widely used in synthesis of chromatographic stationary phases. In this work, dodecyl imide maleopimaric acid glycidyl methacrylate ester (C12-MPAGN) was prepared from maleopimaric acid through the imidization and ring-opening based esterification reaction. By using "thiol-ene" click chemistry, it was chemically bonded to the silica and (3-mercaptopropyl) trimethoxysilane (γ-MPS) was used as the coupling agent to obtain dodecyl imide maleopimaric acid glycidyl methacrylate ester bonded silica stationary phase (Sil-C12-MPAGN). Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), scanning electron microscopies (SEM), and elemental analysis (EA) were utilized to verify that the Sil-C12-MPAGN stationary phase was successfully prepared with C12-MPAGN immobilized on the silica surface. In order to evaluate the chromatographic performance and retention mechanisms of the Sil-C12-MPAGN column and compared with C18 column, a variety of compounds were used, including stander mixture of Tanaka, alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs), phenols and flavonoids. Based on these multiple interactions, including hydrophobic, hydrogen-bonding, and π-π interactions, high selectivity and superior separation performance were demonstrated by the Sil-C12-MPAGN column for probe molecules what had previously been mentioned. In addition, the quantitative determination of paclitaxel content in Yew bark extract was conducted with this column, which was found that the concentration was 83.67 mg/L, respectively. In short, the present study proposes a new strategy for introducing rosin to liquid chromatography with high selectivity and separation performance.


Assuntos
Cromatografia de Fase Reversa , Ésteres , Cromatografia de Fase Reversa/métodos , Espectroscopia de Infravermelho com Transformada de Fourier , Imidas , Dióxido de Silício/química , Interações Hidrofóbicas e Hidrofílicas , Cromatografia Líquida de Alta Pressão/métodos
2.
Molecules ; 28(2)2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36677532

RESUMO

The analytical quality by design (AQbD) approach is utilized for developing and validating the simple, sensitive, cost-effective reverse-phase high performance liquid chromatographic method for the estimation of xanthohumol (XH) in bulk and nanoformulations. The Box-Behnken design (BBD) is applied for method optimization. The mobile phase ratio, pH and flow rate were selected as independent variables, whereas retention time, peak area, peak height, tailing factor, and theoretical plates were selected as dependent variables. The chromatogram of XH obtained under optimized conditions has given optimum conditions such as retention time (5.392 min), peak area (1,226,737 mAU), peak height (90,121 AU), tailing factor (0.991) and theoretical plates (4446.667), which are contoured in the predicted values shown by BBD. Validation of the method has been performed according to ICH Q2(R1) recommendations, using optimized conditions for linearity, limit of detection (LOD) and limit of quantification (LOQ), accuracy, precision, robustness and system suitability. All the values of validation parameters lie within the acceptable limits prescribed by ICH. Therefore, the developed and validated method of XH by the AQbD approach can be applied for the estimation of XH in bulk and various nanoformulations.


Assuntos
Cromatografia de Fase Reversa , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Limite de Detecção
3.
Methods Mol Biol ; 2625: 79-88, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36653634

RESUMO

Phospholipids are essential components of membrane lipid bilayers and serve as precursors of multiple signaling molecules, so alterations in their homeostasis are associated with the pathogenesis of numerous diseases. In this context, the application of mass spectrometry-based metabolomics has demonstrated great potential to comprehensively characterize the human phospholipidome. In this chapter, we describe an untargeted method for the determination of phospholipids and other related metabolites in a variety of biological matrices, including plasma/serum, erythrocytes, and tissues, based on the combination of high-throughput direct mass spectrometry fingerprinting and subsequent profiling by ultra-high-performance reversed-phase liquid chromatography coupled to mass spectrometry. Furthermore, we also review the characteristic fragmentation patterns of phospholipids with the aim of providing simple guidelines for their straightforward annotation.


Assuntos
Metabolômica , Fosfolipídeos , Humanos , Fosfolipídeos/química , Cromatografia Líquida de Alta Pressão/métodos , Metabolômica/métodos , Espectrometria de Massas , Cromatografia de Fase Reversa/métodos
4.
Methods Mol Biol ; 2625: 65-69, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36653632

RESUMO

This chapter focuses on identifying gangliosides in the optic nerve of the mouse using mass spectrometry techniques. The described protocol will also permit the characterization of the sample's lipidome. Two deuterium-labeled ganglioside standards and a general lipid class standard will be utilized for extraction efficiency and quantification. Using reversed-phase high-performance liquid chromatography (HPLC) coupled to a Q Exactive mass spectrometer, the samples will be analyzed. The method will consist of both an untargeted approach and a targeted approach with a ganglioside-specific inclusion list.


Assuntos
Cromatografia de Fase Reversa , Gangliosídeos , Camundongos , Animais , Gangliosídeos/química , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Cromatografia de Fase Reversa/métodos , Nervo Óptico/química
5.
Talanta ; 255: 124228, 2023 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-36587429

RESUMO

In this work, a retention behavior based on mixed-mode reversed-phase (RP)/hydrophilic interaction liquid chromatography (HILIC) was observed for benzalkonium chloride (BAK) using a core-shell column functionalized with biphenyl groups. Although in the literature, the U-shaped retention was reported for polar compounds in mixed functionalized phases, in the present work, the behavior was dependent upon the chemical structure of the analyte with mixed functionality (ammonium group, a benzyl group and an alkyl chain) and on the high selectivity of the chromatographic column. The bimodal retention was observed for the four BAK homologues using a content of acetonitrile from 65 to 95% in the mobile phase. The data were adjusted to polynomial equations which allow for modeling and predicting the U-shaped retention. The salt concentration (50 and 100 mM), anion (formate and acetate) and cation (ammonium and triethylammonium) of the salt, pH (4 and 5) in the mobile phase were studied in order to understand their influence on the two retention modes. Significant electrostatic interactions were involved in the two retention modes, especially with a content of acetonitrile higher that 90%. Linear relationships between the retention factors of the four homologues were found in a wide range of %acetonitrile when the salt and triethylamine concentration, pH and nature of salt were changed. The differences found on the retention of the homologues, when increasing the alkyl chain length, were more significant in the RP mode due to predominant hydrophobic interactions. A pH decrease and a salt concentration increase caused a retention decrease for both modes. A decrease on of the retention was observed when acetate anion was replaced by formate anion. The different order of the polynomial equations according to the used mobile phase confirmed its relevant role in the interactions with the analytes and stationary phase. A mobile phase was selected (85% acetonitrile, pH 4 and 100 mM ammonium formate) for the BAK determination in cutaneous, otic and ophthalmic formulations with different active pharmaceutical ingredients and excipients. Low sample volume (500 µL) and short analysis time (<5 min) were some of the advantages of the proposed method. In addition, good analytical performance (R2 > 0.999, % RSD <4.5% for intra-day precision and <5.8% for inter-day precision, and recoveries in the 92-105% range) was obtained.


Assuntos
Compostos de Benzalcônio , Cromatografia de Fase Reversa , Composição de Medicamentos , Cromatografia de Fase Reversa/métodos , Acetonitrilas/química , Interações Hidrofóbicas e Hidrofílicas , Ânions
6.
J Chromatogr A ; 1689: 463758, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36592481

RESUMO

Cellulose ethers (CEs) are semi-synthetic polymers produced by derivatization of natural cellulose, yielding highly substituted products such as ethyl hydroxyethyl cellulose (EHEC) or methyl ethyl hydroxyethyl cellulose (MEHEC). CEs are commonly applied as pharmaceutical excipients and thickening agents in paints and drymix mortars. CE properties, such as high viscosity in solution, solubility, and bio-stability are of high interest to achieve required product qualities, which may be strongly affected by the substitution pattern obtained after derivatization. The average and molar degree of substitution often cannot explain functional differences observed among CE batches, and more in-depth analysis is needed. In this work, a new method was developed for the comprehensive mapping of the substitution degree and composition of ß-glucose monomers of CE samples. To this end, CEs were acid-hydrolyzed and then analyzed by gradient reversed-phase liquid chromatography-mass spectrometry (LC-MS) using an acid-stable LC column and time-of-flight (TOF) mass spectrometer. LC-MS provided monomer resolution based on ethylene oxide, hydroxyl, and terminating methyl/ethyl content, allowing the assignment of detailed compositional distributions. An essential further distinction of constitutional isomer distributions was achieved using an in-house developed probability-based deconvolution algorithm. Aided by differential heat maps for visualization and straightforward interpretation of the measured LC-MS data, compositional variation between bio-stable and non-bio-stable CEs could be identified using this new approach. Moreover, it disclosed unexpected methylations in EHEC samples. Overall, the obtained molecular information on relevant CE samples demonstrated the method's potential for the study of CE structure-property relationships.


Assuntos
Celulose , Éter , Espectrometria de Massas , Cromatografia Líquida/métodos , Celulose/química , Cromatografia de Fase Reversa
7.
J Chromatogr A ; 1690: 463785, 2023 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-36641941

RESUMO

Hydrophilic interaction chromatography (HILIC) has been proposed as a valuable alternative to ion-pairing reversed-phase chromatography (IP-RPLC) for oligonucleotide (ON) analysis. In this context, the potential of seven zwitterionic HILIC columns has been evaluated against amide- and poly-hydroxy fructan-functionalized HILIC columns and a C18 column operated under IP-RPLC mode. Based on the retention characteristics of key small molecule pairs, each zwitterionic HILIC column showed a unique radar-shaped profile, suggesting different selectivities for distinct structural differences. Unmodified DNA and RNA samples were then evaluated, and the columns classified based on their retentivity. Two zwitterionic columns were particularly promising in terms of overall resolution, especially for the largest ONs (> 40-mer). Finally, separations between a chemically modified drug-like ON and its closely related impurities were performed. Although the ZIC-cHILIC column showed similar selectivity values as compared to the reference IP-RPLC technique, all columns demonstrated a general decrease in selectivity due to the minor structural differences present in the highly complex samples. This work highlights the utility of zwitterionic HILIC mode for ON analysis and it reveals the importance of understanding columns characteristics - in terms of retention and selectivity - when selecting a stationary phase for specific ON applications.


Assuntos
Cromatografia de Fase Reversa , Oligonucleotídeos , Cromatografia Líquida/métodos , Cromatografia de Fase Reversa/métodos , Interações Hidrofóbicas e Hidrofílicas , Íons/química
8.
J Chromatogr A ; 1690: 463786, 2023 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-36641939

RESUMO

In this work, a solute retention optimization method (SRO) was proposed to exploit the purification potential of two-dimensional liquid chromatography (2D-LC). According to our findings, the complementarity of 2D-LC correlates with some specific impurities. In the two methods used in 2D-LC, the retention order of these impurities and target compound is completely opposite. Taking full advantage of the complementarity is crucial to enhance the saturation capacity (wmax) of 2D-LC by SRO. For the purpose of validating the effectiveness of SRO, a reverse-phase liquid chromatography (RPLC) coupled with hydrophilic interaction chromatography (HILIC) was developed to purify p-chlorobenzoic acid from substituted benzenes. By using the overloading effects of analytes as indicators, the wmax of RPLC × HILIC was determined by the bisection method, and finally defined by the extremely high loading volume of 4.9 mL. A touch-peak separation of impurities and the target compound occurred precisely during the secondary separation. The effectiveness of SRO was also verified by the greater purification efficiency of RPLC × HILIC than that of HILIC × RPLC. Subsequently, a RPLC × RPLC method was developed by SRO to prepare the reference materials of caffeine from tea extracts. Only by an analytical C18 column, 15.6 mg of caffeine with the purity of 98.3% was obtained at once with the recovery up to 82.3%. However, without the aid of SRO, the purity rapidly decreased to 62.0%. Compared to other methods, SRO-based 2D-LC offers certain advantages in terms of purity, recovery, and the purification efficiency, suggesting that it is particularly effective in developing preparative 2D-LC facing complex matrices.


Assuntos
Cafeína , Cromatografia de Fase Reversa , Cromatografia de Fase Reversa/métodos , Interações Hidrofóbicas e Hidrofílicas
9.
J Chromatogr A ; 1690: 463779, 2023 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-36681007

RESUMO

Untargeted metabolomic studies require an extensive set of analyte (metabolic) information to be obtained from each analyzed sample. Thus, highly selective, and efficient analytical methodologies together with reversed-phase (RP) or hydrophilic interaction liquid chromatography (HILIC) are usually applied in these approaches. Here, we present a performance comparison of five different chromatographic columns (C18, C8, RP Amide, zicHILIC, OH5 HILIC phases) to evaluate their sufficiency of analysis for a large analyte library, consisting of 817 authentic standards. By taking into account experimental chromatographic parameters (i.e. retention time, peak tailing and asymmetry, FWHM, signal-to-noise ratio and peak area and intensity), the proposed column scoring approach provides a simple criterion that may assist analysis in the select of a stationary phase for those metabolites of interest. RPLC methods offered better results regarding metabolic library coverage, while the zicHILIC stationary phase delivered a bigger number of properly eluted compounds. This study demonstrates the importance of choosing the most suitable configuration for the analysis of different metabolic classes.


Assuntos
Metaboloma , Metabolômica , Cromatografia Líquida/métodos , Metabolômica/métodos , Espectrometria de Massas/métodos , Interações Hidrofóbicas e Hidrofílicas , Cromatografia de Fase Reversa/métodos
10.
J Chromatogr A ; 1690: 463801, 2023 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-36702058

RESUMO

Three mixed mobile phase organic modifiers, tetrahydrofuran: 2-propanol 1:1 (v/v), tetrahydrofuran: 2-propanol: 1:3 (v/v), and tetrahydrofuran: 2-propanol: 3:1 (v/v) were studied at 20-70% (v/v) total organic solvent compositions. The solvent strength parameters for the three mixed organic modifiers and system properties were compared to those of more established binary solvent systems, acetonitrile-water and methanol-water. To interpret intermolecular interactions responsible for retention for the three mixed mobile phase organic modifiers, system maps were constructed and compared with acetonitrile and methanol. Three mixed organic mobile phase modifiers on one stationary phase chemistry (Kinetex C18) provide different selectivity than the more established acetonitrile and methanol mobile phase modifiers on the same stationary phase (Kinetex C18) as well as different stationary phase chemistries (Kinetex Biphenyl, Kinetex Phenyl-Hexyl, Kinetex F5, Kinetex XB-C18, and Kinetex EVO C18). The solvation parameter models for all three mixed mobile phase systems the coefficient of determination ranged from 0.991 to 0.999, the Fisher statistic from 338 to 1850, and the standard error of the estimate ranged from 0.024 to 0.097.


Assuntos
Cromatografia de Fase Reversa , Água , Água/química , Cromatografia de Fase Reversa/métodos , Metanol/química , 2-Propanol , 1-Propanol , Porosidade , Solventes/química , Propanóis , Acetonitrilas/química , Indicadores e Reagentes
11.
J Chromatogr A ; 1688: 463718, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36565652

RESUMO

To address the chemical complexity is indispensable in a number of research fields. Herb metabolome is typically composed by more than one class of structure analogs produced via different biosynthetic pathways. Multidimensional chromatography (MDC), due to the greatly enhanced separation space, offers the potential solution to comprehensive characterization of herbal metabolites. Here, we presented a strategy, by integrating MDC and quadrupole time-of-flight mass spectrometry (QTOF-MS), to accomplish the in-depth herbal metabolites characterization. Using the metabolome of two Astragalus species (A. membranaceus var. mongholicus,AMM; A. membranaceus, AM) as the case, an off-line three-dimensional liquid chromatography (3D-LC) system was established: hydrophilic interaction chromatography using an XAmide column as the first dimension (1D) for fractionating the total extract, on-line reversed-phase × reversed-phase liquid chromatography separately configuring a CSH Fluoro-Phenyl column and a Cosmocore C18 column as the second dimension (2D) and the third dimension (3D) of chromatography to enable the explicit separation of three well fractionated samples. Moreover, the negative-mode collision-induced dissociation by QTOF-MS under the optimized condition could provide diversified fragments that were useful for the structural elucidation of AMM and AM. An in-house library (composed by 247 known compounds) and comparison with 43 reference standards were utilized to assist more reliable characterization. We could characterize 513 compounds from two Astragalus species (344 from AMM and 323 from AM), including 236 flavonoids, 150 triterpenoids, 18 organic acids, and 109 others. Conclusively, the established MDC approach gained excellent performance favoring the analogs-oriented in-depth characterization of herbal metabolites, but received uncompromising analytical efficiency.


Assuntos
Cromatografia de Fase Reversa , Flavonoides , Espectrometria de Massas/métodos , Análise Espectral , Flavonoides/análise , Metaboloma , Cromatografia Líquida de Alta Pressão/métodos
12.
J Chromatogr A ; 1689: 463711, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36586280

RESUMO

A polyethyleneimine (PEI)-functionalized polymer substrate polar stationary phase was prepared for hydrophilic interaction chromatography (HILIC) by grafting PEI onto poly(styrene-divinylbenzene) (PS-DVB) microspheres. The phase shows a U-shape retention profile and it exhibits typical hydrophilic characteristic when the organic solvent fraction in the mobile phase is > 60%. Hydrogen bonding, anion exchange, and hydrophobic interaction are involved in the retention mechanism. Good separation and unique selectivity for acidic, basic and neutral polar analytes were achieved. It showed extremely low column bleed (comparable to that of blank) under gradient elution mode (even to 50% fraction of water) and wide pH tollerance range (at least 1-13).


Assuntos
Polietilenoimina , Polímeros , Polietilenoimina/química , Cromatografia Líquida/métodos , Cromatografia de Fase Reversa/métodos , Solventes , Interações Hidrofóbicas e Hidrofílicas
13.
J Chromatogr A ; 1687: 463707, 2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36516490

RESUMO

Comprehensive characterization of the lipidome remains a challenge requiring development of new analytical approaches to expand lipid coverage in complex samples. In this work, offline two-dimensional liquid chromatography-mass spectrometry was investigated for lipidomics from human plasma. Hydrophilic interaction liquid chromatography was implemented in the first dimension to fractionate lipid classes. Nine fractions were collected and subjected to a second-dimension separation utilizing 50 cm capillary columns packed with 1.7 µm C18 particles operated on custom-built instrumentation at 35 kpsi. Online coupling with time-of-flight mass spectrometry allowed putative lipid identification from precursor-mass based library searching. The method had good orthogonality (fractional coverage of ∼40%), achieved a peak capacity of approximately 1900 in 600 min, and detected over 1000 lipids from a 5 µL injection of a human plasma extract while consuming less than 3 mL of solvent. The results demonstrate the expected gains in peak capacity when employing long columns and two-dimensional separations and illustrate practical approaches for improving lipidome coverage from complex biological samples.


Assuntos
Lipidômica , Lipídeos , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Interações Hidrofóbicas e Hidrofílicas , Lipídeos/química , Cromatografia de Fase Reversa/métodos , Cromatografia Líquida de Alta Pressão/métodos
14.
Int J Mol Sci ; 23(24)2022 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-36555119

RESUMO

Oligonucleotides (OGNs) are relatively new modalities that offer unique opportunities to expand the therapeutic targets. Reliable and high-throughput bioanalytical methods are pivotal for preclinical and clinical investigations of therapeutic OGNs. Liquid chromatography-mass spectrometry (LC-MS) is now evolving into being the method of choice for the bioanalysis of OGNs. Ion paring reversed-phase liquid chromatography (IP-RPLC) has been widely used in sample preparation and LC-MS analysis of OGNs; however, there are technical issues associated with these methods. IP-free methods, such as hydrophilic interaction liquid chromatography (HILIC) and anion-exchange techniques, have emerged as promising approaches for the bioanalysis of OGNs. In this review, the state-of-the-art IP-RPLC-MS bioanalytical methods of OGNs and their metabolites published in the past 10 years (2012-2022) are critically reviewed. Recent advances in IP-reagent-free LC-MS bioanalysis methods are discussed. Finally, we describe future opportunities for developing new methods that can be used for the comprehensive bioanalysis of OGNs.


Assuntos
Oligonucleotídeos , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Oligonucleotídeos/química , Cromatografia de Fase Reversa , Íons
15.
Int J Mol Sci ; 23(23)2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36499291

RESUMO

The aim of this research was to develop a simple and efficient ion-pair reagent-free chromatographic method for the separation and qualitative determination of oligonucleotide impurities, exemplified by synthesis of raw products of the two single strands of patisiran siRNA. The stationary phases with mixed hydrophobic/hydrophilic properties (cholesterol and alkylamide) were firstly used for this purpose with reversed-phased high-performance liquid chromatography. Several different chromatographic parameters were tested for their impact on impurities separation: type, concentration, pH of salt, as well as organic solvent type in the mobile phase. The pH was the most influential factor on the separation and signal intensities in mass spectrometry detection. Finally, the optimized method included the application of cholesterol stationary phase, with mobile phase containing 20 mM ammonium formate (pH 6.5) and methanol. It allowed good separation and the identification of most impurities within 25 min. Since not all closely related impurities could be fully resolved from the main peak in this oligonucleotide impurity profiling, two-dimensional liquid chromatography was used for peak purity determination of the target oligonucleotides. The Ethylene Bridged Hybrid (BEH) Amide column in hydrophilic interaction liquid chromatography was applied in the second dimension, allowing additional separation of three closely related impurities.


Assuntos
Cromatografia de Fase Reversa , Oligonucleotídeos , Cromatografia Líquida , Espectrometria de Massas , Cromatografia de Fase Reversa/métodos , Interações Hidrofóbicas e Hidrofílicas , Oligonucleotídeos/química , Indicadores e Reagentes , Colesterol , Cromatografia Líquida de Alta Pressão/métodos
16.
Molecules ; 27(23)2022 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-36500399

RESUMO

In the pharmaceutical field, and more precisely in quality control laboratories, robust liquid chromatographic methods are needed to separate and analyze mixtures of compounds. The development of such chromatographic methods for new mixtures can result in a long and tedious process even while using the design of experiments methodology. However, developments could be accelerated with the help of in silico screening. In this work, the usefulness of a strategy combining response surface methodology (RSM) followed by multicriteria decision analysis (MCDA) applied to predictions from a quantitative structure-retention relationship (QSRR) model is demonstrated. The developed strategy shows that selecting equations for the retention time prediction models based on the pKa of the compound allows flexibility in the models. The MCDA developed is shown to help to make decisions on different criteria while being robust to the user's decision on the weights for each criterion. This strategy is proposed for the screening phase of the method lifecycle. The strategy offers the possibility to the user to select chromatographic conditions based on multiple criteria without being too sensitive to the importance given to them. The conditions with the highest desirability are defined as the starting point for further optimization steps.


Assuntos
Cromatografia de Fase Reversa , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida , Preparações Farmacêuticas
17.
Anal Chem ; 94(46): 16142-16150, 2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36356979

RESUMO

Two-dimensional liquid chromatography (2D-LC) is a powerful technique used to characterize complex samples such as synthetic polymers, biomacromolecules, and hybrid modalities (conjugates, oligonucleotides, nanoparticles, etc., which fall between traditional small molecules and large molecules). Characterizing such molecules often requires a highly orthogonal 2D-LC workflow to resolve structurally similar impurities. However, it remains a challenge to achieve truly orthogonal 2D-LC coupling due to incompatibility of the chromatographic conditions used in each dimension. In this work, we present a facile strategy of connecting an in-line mixer, in-line mixing modulation (ILMM), to realize challenging 2D-LC workflows: (1) coupling gel permeation chromatography (GPC) with reversed-phase liquid chromatography (RPLC) for hydrophobic oligomer analysis and (2) coupling ion-pair reversed-phase (IPRP) with hydrophilic interaction liquid chromatography (HILIC) for polar antisense oligonucleotide (ASO) analysis. Compared with the state-of-the-art commercially available active solvent modulation (ASM), engaging the ILMM significantly reduces the peak distortion in GPC-RPLC, allowing an at least 67% higher transfer volume from the primary to secondary dimension, and resolves the ASO sample breakthrough in selective comprehensive IPRP×HILIC. Also remarkably, ILMM demonstrated superiority in comprehensive RPLC×RPLC analysis in comparison with ASM, suggesting its potential in broader 2D-LC applications. In addition to chromatography improvement, ILMM offers several advantages over benchmark modulation approaches in regard to alleviating the need of an additional dilution flow and a simple as well as flexible system configuration, opening many opportunities to establish innovative and versatile multidimensional workflows for characterizing compounds with increasing complexity.


Assuntos
Cromatografia de Fase Reversa , Oligonucleotídeos , Solventes , Cromatografia de Fase Reversa/métodos , Cromatografia Líquida/métodos , Interações Hidrofóbicas e Hidrofílicas
18.
J Chromatogr A ; 1685: 463627, 2022 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-36370628

RESUMO

An alternative method to the classical fit of semi-empirical, statistical, or artificial intelligence-based models to retention data is proposed to predict surface excess adsorption and retention factors in liquid chromatography. The approach is based on a fundamental, microscopic description of the liquid-to-solid adsorption of analytes taking place at the interface between a bulk liquid phase and a solid surface. Molecular dynamics (MD) simulations are performed at T=300 K in a 100 Å wide slit-pore model (ß-cristobalite-C18 surface in contact with an acetonitrile/water mobile phase) to quantify a priori the retention factors of small molecules expected in reversed phase liquid chromatography (RPLC). Uracil is chosen as the reference "non-retained" marker, whereas benzyl alcohol, acetophenone, benzene, and ethylbenzene are four selected retained, neutral compounds. The MD simulations allow to determine the pore-level density profiles of these five compounds, i.e., the variation of the analyte concentration as a function of distance from the silica surface. The retention factors of the retained analytes are expressed using their respective calculated surface excess adsorption relative to uracil. By definition, the retention factors are proportional to the surface excess adsorbed and the proportionality constant is directly scaled to the retention time of the "non-retained" marker. Experimentally, a 4.6 mm × 150 mm RPLC-C18 column packed with 5 µm 100 Å High Strength Silica (HSS)-C18 particles is used and the retention times of these five compounds are measured. The volume fraction of acetonitrile in water increases from 20 to 90% generating a wide range of retention factors from 0.15 to 183 at T=300 K. The results demonstrate very good agreement between the MD-predicted surface excess adsorption data and measured retention factors (R2> 0.985). A systematic error is observed as the proportionality constant is not exactly scaled to the retention time of uracil. This is most likely caused by the differences between the chemical and morphological features of the slit-pore model adopted in the MD simulations and those of the actual HSS-C18 particles: the average surface coverage with C18 chains, the geometry of the mesopores, and the pore size distribution. Specifically, the impact on RPLC retention of slight, local variations in surface chemistry (e.g., functional group density and uniformity) and how this aspect is affected by the pore space morphology (e.g., pore curvature and size) is worth investigating by future MD simulations.


Assuntos
Cromatografia de Fase Reversa , Simulação de Dinâmica Molecular , Cromatografia de Fase Reversa/métodos , Adsorção , Inteligência Artificial , Acetonitrilas/química , Água/química , Dióxido de Silício/química , Uracila
19.
J Chromatogr A ; 1685: 463646, 2022 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-36401912

RESUMO

The properties of ionic liquids (ILs) greatly influenced the retention of solutes on IL-functionalized stationary phases. In this work, three IL-functionalized stationary phases (Sil-C4Im, Sil-C7Im, and Sil-C10Im) were prepared by modifying silica gel with three ionic liquid silane reagents differing in spacer alkyl chain lengths, which refers to the alkyl chain between the imidazolium and the sulfur atom. The preparation was proved through a range of characterization techniques, including elemental analysis (EA), Fourier transform infrared spectroscopy (FT-IR), and thermogravimetric analysis (TGA). The effects of chromatographic conditions, such as acetonitrile content, salt concentration, pH, and column temperature, were studied to explore the retention mechanism of three stationary phases, which indicates that three IL-stationary phases provide interactions with solutes in reverse-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), and ion exchange chromatography (IEC). The column efficiency is 83,686 plates/m for aminobenzoic acid, and the column also has excellent repeatability of retention time with relative standard deviations (RSDs) between 0.24% and 0.34% (n = 12). A variety of analytes, including five nucleosides and nucleobases, six alkylbenzenes, two polycyclic aromatic hydrocarbons (PAHs), and four inorganic anions, were separated well on three IL-functionalized stationary phases. It was found that the spacer alkyl chain length influenced selectivity by comparing the retention of the three IL-functionalized stationary phases.


Assuntos
Líquidos Iônicos , Dióxido de Silício , Cromatografia Líquida de Alta Pressão/métodos , Espectroscopia de Infravermelho com Transformada de Fourier , Dióxido de Silício/química , Cromatografia de Fase Reversa , Líquidos Iônicos/química
20.
J Chromatogr A ; 1685: 463632, 2022 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-36347071

RESUMO

The separation and characterization of small polar impurities in polar drugs such as calcium gluconate products are always challenging, due to their poor retention on traditional reversed phase (RP) columns. Although ion-pair reversed-phase liquid chromatography (IPRP-LC) and hydrophilic interaction liquid chromatography (HILIC) are commonly used methods for polar compound analysis, both methods have some drawbacks. For example, IPRP-LC is incompatible with mass spectrometry (MS) due to the presence of non-volatile salts in its mobile phase and HILIC has limited sensitivity due to the poor solubility of polar drugs in the organic-rich sample diluents used in HILIC separations. In order to characterize the highly polar impurities in calcium gluconate injections, a heart-cutting two-dimensional liquid chromatography (2D-LC) method coupled with quadrupole time-of-flight mass spectrometry (Q-TOF/MS) was developed in this study. An IPRP-LC method in the first dimension (1D) provided the selectivity for the separation of polar analytes, using a 100% aqueous mobile phase containing phosphate buffer and ion-pair reagent. Heart cuts of target peaks were collected with sample loops and transferred to the second dimension (2D) HILIC column using an organic-rich mobile phase. In order to solve the mobile phase mismatch problem between the two dimensions, a make-up flow module was introduced in the 2D-LC system to dilute the 1D-water-rich fractions with acetonitrile before entering the sample loops. By optimizing the loop size and dilution factor, good retention and peak shape of the highly polar impurities were obtained on the 2D-HILIC column, and the ion suppression effect for MS detection from the ion-pair reagent and non-volatile salt in the 1D-effluent was minimized. A total of five impurities were identified through fragmentation studies by Q-TOF/MS analysis and their fragmentation pathways were proposed. Four of them were further confirmed by reference substances. This study not only provided useful information for quality control of calcium gluconate injections, but also provided an alternative method for polar impurity characterization in pharmaceuticals.


Assuntos
Gluconato de Cálcio , Cromatografia de Fase Reversa , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Cromatografia de Fase Reversa/métodos , Interações Hidrofóbicas e Hidrofílicas , Água/química
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