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1.
Anal Chim Acta ; 1324: 343086, 2024 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-39218572

RESUMEN

BACKGROUND: Polyesters are applied in high-end products in many industrial applications, including resins and powder-coating applications. The characterization of the chemical heterogeneities within a polyester is of utmost interest to develop new products or improve existing applications. Unfortunately, characterization is a difficult task, as polyesters may feature distributions in end-group functionality, molecular weight, chemical composition, and degree of branching. Currently, no analytical method can characterize all these interdependent distributions in a single analysis. RESULTS: We report the use of comprehensive normal-phase liquid chromatography × size-exclusion chromatography hyphenated with ultraviolet-light spectroscopy and high-resolution mass spectrometry in parallel (NPLC × SEC-UV/HRMS) to characterize polyesters according to their end-group-functionality and molecular-weight distributions. The chemical composition can be measured with HRMS, while relative quantitation can be performed with UV detection. A supercharging agent was used during ionization allowing to extend the molecular-weight range of the detected chemical species. SIGNIFICANCE: The presented platform allows characterization of polyesters with varying fractions of carboxyl or hydroxyl end-group functionalities and varying distributions of molecular weight, degree of branching, and chemical compositions. The number-average and weight-average molar masses are obtained in the same analysis. This information cannot be obtained by any one-dimensional technique. The developed NPLC × SEC-UV/HRMS platform is a valuable tool for characterizing polyesters in an industrial setting.

2.
Sci Total Environ ; 952: 175908, 2024 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-39218084

RESUMEN

To date, poly- and perfluoroalkyl substances (PFAS) represent a real threat for their environmental persistence, wide physicochemical variability, and their potential toxicity. Thus far a large portion of these chemicals remain structurally unknown. These chemicals, therefore, require the implementation of complex non-targeted analysis workflows using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) for their comprehensive detection and monitoring. This approach, even though comprehensive, does not always provide the much-needed analytical resolution for the analysis of complex PFAS mixtures such as fire-fighting aqueous film-forming foams (AFFFs). This study consolidates the advantages of the LC×LC technique hyphenated with high-resolution tandem mass spectrometry (HRMS/MS) for the identification of PFAS in AFFF mixtures. A total of 57 PFAS homolog series (HS) were identified in 3M and Orchidee AFFF mixtures thanks to the (i) high chromatographic peak capacity (n'2D,c ~ 300) and the (i) increased mass domain resolution provided by the "remainder of Kendrick Mass" (RKM) analysis on the HRMS data. Then, we attempted to annotate the PFAS of each HS by exploiting the available reference standards and the FluoroMatch workflow in combination with the RKM defect by different fluorine repeating units, such as CF2, CF2O, and C2F4O. This approach resulted in 12 identified PFAS HS, including compounds belonging to the HS of perfluoroalkyl carboxylic acids (PFACAs), perfluoroalkyl sulfonic acids (PFASAs), (N-pentafluoro(5)sulfide)-perfluoroalkane sulfonates (SF5-PFASAs), N-sulfopropyldimethylammoniopropyl perfluoroalkane sulfonamides (N-SPAmP-FASA), and N-carboxymethyldimethylammoniopropyl perfluoroalkane sulfonamide (N-CMAmP-FASA). The annotated categories of perfluoroalkyl aldehydes and chlorinated PFASAs represent the first record of PFAS HS in the investigated AFFF samples.

3.
Foods ; 13(15)2024 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-39123568

RESUMEN

In the melamine scandals of the early 2000s, different companies of the dairy industry cheated their products by applying chemical substances to feign a higher content of nitrogen. However, this had a severe toxic impact on the kidney health of consumers. As a result, tremendous effort was put into the prevention of further harm to the public. In the present study, a fast-screening method for the determination of melamine and cyanuric acid in infant formula was developed. While a 1D-LC approach is faster and easier to set up, a 2D-LC approach allows for a more accurate result with better selectivity and sensitivity. For both instrumental approaches, the signal ratio of the isotopologues was crucial and had a dominant effect on the results and the measurement uncertainty. For this reason, the different contributions to the measurement uncertainty were determined experimentally using Matched Standard Addition-IDMS and compared to the Exact Matching Double IDMS.

4.
Molecules ; 29(13)2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38999085

RESUMEN

Vitex negundo has strong antioxidant activity, but its primary antioxidant components are not clear. In this study, the antioxidant components were screened by offline two-dimensional liquid chromatography coupled with electrochemical detection (2D-LC-ECD) and subsequently assessed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification, radical scavenging capacity, and molecular docking. Various fractions were isolated from Vitex negundo leaves, and 39 antioxidant components were screened and identified. All of the fractions containing the antioxidant components exhibited certain antioxidant activity. Correlation analysis revealed a strong correlation between the response of LC-ECD and the in vitro antioxidant activity of the fractions. Molecular docking demonstrated that components with high response to LC-ECD exhibited robust interaction with antioxidant-related target proteins. The main antioxidant components of Vitex negundo leaves were isoorientin, chlorogenic acid, agnuside, cynaroside, and scutellarin. The 2D-LC-ECD combined with LC-MS/MS was rapid and effective in screening the antioxidant components in Vitex negundo leaves and could also provide technical support for the discovery of antioxidant components with different polarities and contents in other medicinal and edible plants.


Asunto(s)
Antioxidantes , Simulación del Acoplamiento Molecular , Hojas de la Planta , Espectrometría de Masas en Tándem , Vitex , Vitex/química , Hojas de la Planta/química , Espectrometría de Masas en Tándem/métodos , Antioxidantes/química , Antioxidantes/análisis , Cromatografía Liquida/métodos , Extractos Vegetales/química , Cromatografía Líquida con Espectrometría de Masas
5.
J Chromatogr A ; 1730: 465065, 2024 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-38879974

RESUMEN

In previous publications we have described the pISep dual simultaneous, independent gradients (DSIGs) liquid chromatography (LC) for uncoupling gradients of non-buffering solute (NaCl, urea or acetonitrile) from externally generated pH gradients. In DSIGs the shape and slope of the [salute] gradient does not depend on the shape and slope of the pH gradient. The technique allows in a single run true simultaneous two dimensional LC separation of complex protein mixtures on various stationary phases including anion, cation exchangers (AEX, CEX), reversed phase (RP), mixed mode and mixed bed. Using a humanized IgG1 (HIgG1) monoclonal antibody (MAb) and a variety of pH & [NaCl] DSIGs, we show that most of MAb isoforms can be successfully separated from each other. These experimental observations are supported by an initial theoretical argument presented here predicting an overall improvement of all MAb isoforms separation by DSIGs of pH & [NaCl]. Theoretical calculations predict that, in general, there exists an optimal non-zero isocratic salt concentration in a pH gradient separation that will resolve isoforms close in binding energy, but a wide range of salt concentrations will be required for acceptable resolution of all isoforms. Theory also predicts better separation of weaker rather than stronger binding isoforms. Experimentally, we have found that no one set of DSIGs LC conditions could optimally baseline resolve all identifiable MAb isoforms in a single run of reasonable duration. The versatility and simplicity of the pH & [NaCl] pISep DSIGs LC allows fast, automated scouting of protein separations over any range of pH from 2.4 to 10.8 and [NaCl] from 0 to 1 M without changing the chemistry of the buffering system. Due to the universal applicability of the pISep buffering system in IEX LC, the researcher is given a powerful tool to easily develop pH & [NaCl] DSIGs protocols that vary mobile phase compositions to achieve high resolution separations of targeted proteins.


Asunto(s)
Anticuerpos Monoclonales , Cloruro de Sodio , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Concentración de Iones de Hidrógeno , Cromatografía por Intercambio Iónico/métodos , Cloruro de Sodio/química , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina G/química , Humanos , Cromatografía Liquida/métodos , Fuerza Protón-Motriz , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/química
6.
J Chromatogr A ; 1730: 465076, 2024 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-38879975

RESUMEN

In recent years, many nucleic acid-based pharmaceuticals have been approved and entered the market, and even a larger number are in late stage clinical trials. Conventional oligonucleotides are facing issues in vivo like fast renal clearance and nuclease degradation. Therefore, to increase their stability, phosphorothioation is a frequent modification of therapeutic oligonucleotides (ONs) which also leads to improved binding affinity facilitating cell internalization and intracellular distribution. At the same time, by replacing a phosphodiester linkage with a phosphorothioate group, a phosphorous stereogenic center is generated which causes the formation of Rp- and Sp-diastereomers. It increases the structural diversity. For example, with 15 of those phosphorothioate (PS) linkages, 32,768 different diastereomers are expected. Since the phosphorothioate is introduced non-stereoselectively, the molecular complexity of the resultant phosphorothioate ON products is tremendously increased impeding the chromatographic separation in the course of quality control. Since distinct phosphorothioate diastereomers have different bioactivities and pharmacological properties, there is increasing interest in implications of stereoisomerism of phosphorothiate oligonucleotides. From a quality and regulatory viewpoint, batch-to-batch reproducibility of the diastereomer profile may be of significant concern. In order to address this issue, this study investigates the stereoselectivity of LC methods for two phosphorothioate oligonucleotide (PSO) compounds differing in their molecular size and numbers of PS linkages. Diastereoselectivity of ion-pairing reversed-phase liquid chromatography (IP-RPLC), RPLC without ion-pairing agents and LC with chiral polysaccharide-based column were evaluated for model PSOs and an active pharmaceutical ingredient (API) of PSO with trivalent N-acetylgalactosamine (GalNAc) conjugate. Due to the structural complexity of PSOs, the separation power for the diastereomer mixture was increased by using sequential selective comprehensive two-dimensional chromatography with an amylose tris(α-methylbenzylcarbamate)-immobilized chiral stationary phase (CSP) in the first dimension and ion-pair RPLC with ethylammonium acetate in the second dimension. Improved diastereomer selectivity was obtained and a larger number of peaks could be separated.


Asunto(s)
Cromatografía de Fase Inversa , Oligonucleótidos Fosforotioatos , Oligonucleótidos Fosforotioatos/química , Estereoisomerismo , Cromatografía de Fase Inversa/métodos , Reproducibilidad de los Resultados
7.
Talanta ; 276: 126232, 2024 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-38749159

RESUMEN

Robust monitoring of heterogeneity in biopharmaceutical development is crucial for producing safe and efficacious biotherapeutic products. Multiattribute monitoring (MAM) has emerged as an efficient tool for monitoring of mAb heterogeneities like deamidation, sialylation, glycosylation, and oxidation. Conventional biopharma analysis during mAb development relies on use of one-dimensional methods for monitoring titer and charge-based heterogeneity using non-volatile solvents without direct coupling with mass spectrometry (MS). This approach requires analysis of mAb harvest by ProA for titer estimation followed by separate cation exchange chromatography (CEX) analysis of the purified sample for estimating charge-based heterogeneity. This can take up to 60-90 min due to the required fraction collection and buffer exchange steps. In this work, a native two-dimensional liquid chromatography (2DLC) mass spectrometry method has been developed with Protein A chromatography in the first dimension for titer estimation and cation exchange chromatography (CEX) in the second dimension for charge variant analysis. The method uses volatile salts for both dimensions and enables easy coupling to MS. The proposed 2DLC method exhibits a charge variant profile that is similar to that observed via the traditional methods and takes only 15 min for mass identification of each variant. A total of six charge variants were separated by the CEX analysis after titer estimation, including linearity assessment from 5 µg to 160 µg of injected mAb sample. The proposed method successfully estimated charge variants for the mAb innovator and 4 of its biosimilars, showcasing its applicability for biosimilarity exercises. Hence, the 2D ProA CEX MS method allows direct titer and charge variant estimation of mAbs in a single workflow.


Asunto(s)
Anticuerpos Monoclonales , Cricetulus , Espectrometría de Masas , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/análisis , Espectrometría de Masas/métodos , Animales , Cromatografía por Intercambio Iónico/métodos , Células CHO , Técnicas de Cultivo de Célula
8.
J Sep Sci ; 47(9-10): e2300935, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38801757

RESUMEN

A common separation approach for polar compounds involves coupling reversed-phase liquid chromatography (RPLC) with hydrophilic interaction chromatography (HILIC) in two-dimensional chromatography. The higher proportion of acetonitrile used in the HILIC mobile phase, which enhances mass spectrometry detection, encourages its use in the second dimension. Previous studies demonstrated that the HILIC column can be partially equilibrated within very short timeframes without compromising retention time stability, rendering it suitable in online comprehensive two-dimensional liquid chromatography (LC×LC) setups. In addition, a specific number of conditioning cycles seems necessary to establish stable retention times. Here, the repeatability of HILIC when employed as second dimension in LC×LC was investigated, with a focus on determining the required number of conditioning cycles to achieve repeatable retention times. Various parameters influenced by the LC×LC online modulation system were studied, such as steep gradient slopes up to 8%, and very short equilibration times, less than or equal to dead time, as well as injection volume and solvent, which depend on the first dimension. Finally, the use of HILIC as a second dimension with tailored conditioning runs was applied to the analysis of hyaluronic acid hydrogel digests. The application of an RPLC×HILIC method using five conditioning runs yielded exceptional stability in second-dimension retention times.

9.
Sci Total Environ ; 927: 172199, 2024 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-38580108

RESUMEN

Effect-directed analysis (EDA) is a crucial tool in environmental toxicology, effectively integrating toxicity testing with chemical analysis. The conventional EDA approach, however, presents challenges such as significant solvent consumption, extended analysis time, labor intensity, and potential contamination risks. In response, we introduce an innovative alternative to the conventional EDA. This method utilizes the MTT bioassay and online two-dimensional liquid chromatography (2D LC) coupled with high-resolution mass spectrometry (HR-MS), significantly reducing the fractionation steps and leveraging the enhanced sensitivity of the bioassay and automated chemical analysis. In the chemical analysis phase, a switching valve interface is employed for comprehensive analysis. We tested the performance of both the conventional and our online 2D LC-based methods using a household product. Both methods identified the same number of toxicants in the sample. Our alternative EDA is 22.5 times faster than the conventional method, fully automated, and substantially reduces solvent consumption. This novel approach offers ease, cost-effectiveness, and represents a paradigm shift in EDA methodologies. By integrating a sensitive bioassay with online 2D LC, it not only enhances efficiency but also addresses the challenges associated with traditional methods, marking a significant advancement in environmental toxicology research.


Asunto(s)
Contaminantes Ambientales , Cromatografía Liquida/métodos , Contaminantes Ambientales/toxicidad , Contaminantes Ambientales/análisis , Pruebas de Toxicidad/métodos , Monitoreo del Ambiente/métodos , Espectrometría de Masas/métodos , Bioensayo/métodos , Ecotoxicología/métodos
10.
J Chromatogr A ; 1721: 464818, 2024 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-38564929

RESUMEN

Comprehensive two-dimensional liquid chromatography (LCxLC) represents a valuable alternative to conventional single column, or one-dimensional, liquid chromatography (1D-LC) for resolving multiple components in a complex mixture in a short time. However, developing LCxLC methods with trial-and-error experiments is challenging and time-consuming, which is why the technique is not dominant despite its significant potential. This work presents a novel shortcut model to in-silico predicting retention time and peak width within an RPLCxRPLC separation system (i.e., LCxLC systems that use reversed-phase columns (RPLC) in both separation dimensions). Our computationally effective model uses the hydrophobic-subtraction model (HSM) to predict retention and considers limitations due to the sample volume, undersampling and the maximum pressure drop. The shortcut model is used in a two-step strategy for sample-dependent optimization of RPLCxRPLC separation systems. In the first step, the Kendall's correlation coefficient of all possible combinations of available columns is evaluated, and the best column pair is selected accordingly. In the second step, the optimal values of design variables, flow rate, pH and sample loop volume, are obtained via multi-objective stochastic optimization. The strategy is applied to method development for the separation of 8, 12 and 16 component mixtures. It is shown that the proposed strategy provides an easy way to accelerate method development for full-comprehensive 2D-LC systems as it does not require any experimental campaign and an entire optimization run can take less than two minutes.


Asunto(s)
Cromatografía de Fase Inversa , Cromatografía Liquida/métodos , Cromatografía de Fase Inversa/métodos
11.
J Chromatogr A ; 1722: 464874, 2024 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-38598893

RESUMEN

Hydroxypropyl methyl cellulose (HPMC) is a type of cellulose derivative with properties that render it useful in e.g. food, cosmetics, and pharmaceutical industry. The substitution degree and composition of the ß-glucose subunits of HPMC affect its physical and functional properties, but HPMC characterization is challenging due to its high structural heterogeneity, including many isomers. In this study, comprehensive two-dimensional liquid chromatography-mass spectrometry was used to examine substituted glucose monomers originating from complete acid hydrolysis of HPMC. Resolution between the different monomers was achieved using a C18 and cyano column in the first and second LC dimension, respectively. The data analysis process was structured to obtain fingerprints of the monomers of interest. The results revealed that isomers of the respective monomers could be selectively separated based on the position of substituents. The examination of two industrial HPMC products revealed differences in overall monomer composition. While both products contained monomers with a similar degree of substitution, they exhibited distinct regioselectivity.


Asunto(s)
Derivados de la Hipromelosa , Glucosa/química , Glucosa/análisis , Hidrólisis , Derivados de la Hipromelosa/química , Isomerismo , Cromatografía Líquida con Espectrometría de Masas
12.
J Chromatogr A ; 1721: 464824, 2024 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-38522405

RESUMEN

Two-dimensional liquid chromatography (2D-LC), and in particular comprehensive two-dimensional liquid chromatography (LC×LC), offers increased peak capacity, resolution and selectivity compared to one-dimensional liquid chromatography. It is commonly accepted that the technique produces the best results when the separation mechanisms in the two dimensions are completely orthogonal; however, the use of similar separation mechanisms in both dimensions has been gaining popularity as it helps avoid difficulties related to mobile phase incompatibility and poor column efficiency. The remarkable advantages of using reversed phase in both dimensions (RPLC×RPLC) over other separation mechanisms made it a promising technique in the separation of complex samples. This review discusses some physical and practical considerations in method development for 2D-LC involving the use of RP in both dimensions. In addition, an extensive overview is presented of different applications that relied on RPLC×RPLC and 2D-LC with reversed phase column combinations to separate components of complex samples in different fields including food analysis, natural product analysis, environmental analysis, proteomics, lipidomics and metabolomics.


Asunto(s)
Cromatografía de Fase Inversa , Proteómica , Cromatografía Liquida/métodos , Cromatografía de Fase Inversa/métodos
13.
Foods ; 13(6)2024 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-38540862

RESUMEN

Sheep's milk is a significant source of nucleotide monophosphates (NMPs) but can also contain undesirable residues from veterinary drugs, posing a potential human health risk. This study introduces a novel application of two-dimensional liquid chromatography (2D-LC), in heart-cutting mode, for the simultaneous determination of nucleotides and veterinary drug residues in sheep's milk. 2D-LC allows for the separation of these compounds in a single chromatographic run despite their differing physicochemical properties. The proposed method separates six veterinary drug residues and five NMPs in a single injection. The compounds were separated using a C18 reversed-phase column in the first dimension and a Primesep SB analytical column in the second dimension. The method performance was evaluated in terms of linearity range, detection and quantification limits, matrix effects, precision, and accuracy. The results demonstrated good linearity and sensitivity, with quantification limits allowing for the quantification of veterinary drugs at the maximum residue level and nucleotides at typical levels found in milk samples. The method has been successfully applied to the analysis of sheep's milk samples acquired from local supermarkets, with recoveries within a range of 70-119% and 82-117% for veterinary residues and NMPs, respectively.

14.
Molecules ; 29(6)2024 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-38542930

RESUMEN

Rhizoma Panacis Japonici (RPJ) is an ancient herbal medicine from China that has long been employed for its medicinal benefits in relieving arthritis physical debility and diverse afflictions. The primary bioactive constituents found in RPJ are triterpene saponins, which exhibit numerous pharmacological actions, including anti-inflammatory, antioxidant, and immunomodulating effects. The present study established a straightforward and effective approach for characterizing triterpene saponins in RPJ. An offline HILIC × RP LC/QTOF-MS method was developed, along with a self-constructed in-house database containing 612 saponins reported in the Panax genus and 228 predicted metabolites. The approach achieved good chromatographic performance in isolating triterpene saponins of RPJ, with the HILIC column as the first dimension (1D) and the BEH C18 column as the second dimension (2D). The developed two-dimensional liquid chromatography system exhibited an orthogonality of 0.61 and a peak capacity of 1249. Detection was performed using a QTOF mass spectrometer in a data-independent manner (MSE) in a negative ion mode. Using the in-house database, the collected MS data were processed by an automatic workflow on UNIFI 1.8.2 software, which included data correction, matching of precursor and product ions, and peak annotation. In this study, 307 saponins were characterized from RPJ and 76 saponins were identified for the first time in Panax japonicus. This research not only enhances our understanding of the chemical characteristics of RPJ but also offers a simple and efficient method for analyzing the complex composition of herbal medicine.


Asunto(s)
Medicamentos Herbarios Chinos , Panax , Plantas Medicinales , Saponinas , Triterpenos , Saponinas/química , Triterpenos/química , Cromatografía Líquida de Alta Presión/métodos , Extractos Vegetales/química , Espectrometría de Masas , Plantas Medicinales/química
15.
Chin Herb Med ; 16(1): 27-41, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38375051

RESUMEN

Traditional Chinese medicines (TCMs) possess a rich historical background, unique theoretical framework, remarkable therapeutic efficacy, and abundant resources. However, the modernization and internationalization of TCMs have faced significant obstacles due to their diverse ingredients and unknown mechanisms. To gain deeper insights into the phytochemicals and ensure the quality control of TCMs, there is an urgent need to enhance analytical techniques. Currently, two-dimensional (2D) chromatography, which incorporates two independent separation mechanisms, demonstrates superior separation capabilities compared to the traditional one-dimensional (1D) separation system when analyzing TCMs samples. Over the past decade, new techniques have been continuously developed to gain actionable insights from complex samples. This review presents the recent advancements in the application of multidimensional chromatography for the quality evaluation of TCMs, encompassing 2D-gas chromatography (GC), 2D-liquid chromatography (LC), as well as emerging three-dimensional (3D)-GC, 3D-LC, and their associated data-processing approaches. These studies highlight the promising potential of multidimensional chromatographic separation for future phytochemical analysis. Nevertheless, the increased separation capability has resulted in higher-order data sets and greater demands for data-processing tools. Considering that multidimensional chromatography is still a relatively nascent research field, further hardware enhancements and the implementation of chemometric methods are necessary to foster its robust development.

16.
J Chromatogr A ; 1718: 464722, 2024 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-38359690

RESUMEN

Immunoglobulin G (IgG) is the most common monoclonal antibody (mAb) grown for therapeutic applications. While IgG is often selectively isolated from cell lines using protein A (ProA) chromatography, this is only a stepping stone for complete characterization. Further classification can be obtained from weak cation exchange chromatography (WCX) to determine IgG charge variant distributions. The charge variants of monoclonal antibodies can influence the stability and efficacy in vivo, and deviations in charge heterogeneity are often cell-specific and sensitive to upstream process variability. Current methods to characterize IgG charge variants are often performed off-line, meaning that the IgG eluate from the ProA separation is collected, diluted to adjust the pH, and then transferred to the WCX separation, adding time, complexity, and potential contamination to the sample analysis process. More recently, reports have appeared to streamline this separation using in-line two-dimensional liquid chromatography (2D-LC). Presented here is a novel, 2D-LC coupling of ProA in the first dimension (1D) and WCX in the second dimension (2D) chromatography. As anticipated, the initial direct column coupling proved to be challenging due to the pH incompatibility between the mobile phases for the two stages. To solve the solvent compatibility issue, a size exclusion column was placed in the switching valve loop of the 2D-LC instrument to act as a means for the on-line solvent exchange. The efficacy of the methodology presented was confirmed through a charge variant determination using the NIST monoclonal antibody standard (NIST mAb), yielding correct acidic, main, and basic variant compositions. The methodology was employed to determine the charge variant profile of IgG from an in-house cultured Chinese hamster ovary (CHO) cell supernatant. It is believed that this methodology can be easily implemented to provide higher-throughput assessment of IgG charge variants for process monitoring and cell line development.


Asunto(s)
Inmunoglobulina G , Proteína Estafilocócica A , Cricetinae , Animales , Cricetulus , Inmunoglobulina G/química , Cromatografía por Intercambio Iónico/métodos , Células CHO , Anticuerpos Monoclonales , Cationes , Técnicas de Cultivo de Célula , Solventes
17.
J Sep Sci ; 47(2): e2300624, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38286726

RESUMEN

The comprehensive and efficient characterization of components in traditional Chinese medicine is crucial for elucidating its active constituents and uncovering its mechanism. Identifying the compounds of the Bushen Huoxue Prescription (BHP) is difficult because of its complex composition and the large difference in concentration among its compounds. In this study, a hydrophilic interaction liquid chromatography coupled with reversed-phase LC (HILIC × RPLC) offline 2D-LC tandem high-resolution mass spectrometry method was established to analyze the total compounds of the BHP. Database screening and molecular networking were performed to identify the compounds. In contrast to conventional 1D chromatography, 2D chromatography increased peak capacity, enriched trace ingredients, and prevented the masking of high-abundance compounds. A total of 165 compounds were identified, and 14 potential compounds needed to be further identified. This study provided an effective method for comprehensively analyzing the complex system of traditional Chinese medicine compounds.


Asunto(s)
Medicamentos Herbarios Chinos , Cromatografía Líquida de Alta Presión/métodos , Medicamentos Herbarios Chinos/análisis , Espectrometría de Masas , Cromatografía Liquida , Tecnología , Cromatografía de Fase Inversa
18.
Molecules ; 29(2)2024 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-38257252

RESUMEN

Dianthus superbus L. has been extensively studied for its potential medicinal properties in traditional Chinese medicine and is often consumed as a tea by traditional folk. It has the potential to be exploited in the treatment of inflammation, immunological disorders, and diabetic nephropathy. Based on previous studies, this study continued the separation of another subfraction of Dianthus superbus and established reversed-phase/reversed-phase and reversed-phase/hydrophilic (RPLC) two-dimensional (2D) high-performance liquid chromatography (HPLC) modes, quickly separating two C-glycosylflavones, among which 2″-O-rhamnosyllutonarin was a new compound and isomer with 6‴-O-rhamnosyllutonarin. This is the first study to investigate the effects of 2″-O-rhamnosyllutonarin and 6‴-O-rhamnosyllutonarin on cellular glucose metabolism in vitro. First, molecular docking was used to examine the effects of 2″-O-rhamnosyllutonarin and 6″-O-rhamnosyllutonarin on AKT and AMPK; these two compounds exhibited relatively high activity. Following this, based on the HepG2 cell model of insulin resistance, it was proved that both of the 2″-O-rhamnosyllutonarin and 6‴-O-rhamnosyllutonarin demonstrated substantial efficacy in ameliorating insulin resistance and were found to be non-toxic. Simultaneously, it is expected that the methods developed in this study will provide a basis for future studies concerning the separation and pharmacological effects of C-glycosyl flavonoids.


Asunto(s)
Dianthus , Resistencia a la Insulina , Simulación del Acoplamiento Molecular , Metabolismo de los Hidratos de Carbono , Glucosa
19.
J Agric Food Chem ; 72(2): 1339-1353, 2024 Jan 17.
Artículo en Inglés | MEDLINE | ID: mdl-38183657

RESUMEN

Two offline multidimensional chromatography/high-resolution mass spectrometry systems (method 1: fractionation and online two-dimensional liquid chromatography, 2D-LC; method 2: fractionation and offline 2D-LC) were established to characterize the metabolites simultaneously from three Glycyrrhiza species. Ion exchange chromatography in the first-dimensional (1D) separation was well fractionated between the acidic (mainly triterpenoids) and weakly acidic components (flavonoids). These obtained subsamples got sophisticated separation by the second (2D) and third dimension (3D) of chromatography either by online reversed-phase chromatography × reversed-phase chromatography (RPC × RPC) or offline hydrophilic interaction chromatography × RPC (HILIC × RPC). Orthogonality for the 2D/3D separations reached 0.73 for method 1 and 0.81 for method 2, respectively. We could characterize 1097 compounds from three Glycyrrhiza species based on an in-house library and 33 reference standards, involving 618 by method 1 and 668 by method 2, respectively. They exhibited a differentiated performance and complementarity in identifying the multiple subclasses of Glycyrrhiza components.


Asunto(s)
Cromatografía de Fase Inversa , Glycyrrhiza , Espectrometría de Masas , Cromatografía de Fase Inversa/métodos , Cromatografía Líquida de Alta Presión/métodos , Flavonoides/análisis , Interacciones Hidrofóbicas e Hidrofílicas
20.
Biomed Chromatogr ; 38(2): e5778, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38073142

RESUMEN

Periprosthetic joint infection (PJI) is a catastrophic complication following joint replacement surgery. One potential treatment approach for PJI could be the combination of one-stage revision and intra-articular infusion of antibiotics. Meropenem is one of the commonly used intra-articular antibiotics in our institution. Determining the concentration of meropenem in the joint cavity could be crucial for optimizing its local application, effectively eradicating biofilm infection, and improving PJI treatment outcomes. In this study, we developed a simple, precise, and accurate method of two-dimensional liquid chromatography (2D-LC) for determining the concentration of meropenem in human synovial fluid. The method was then validated based on the guidelines of the Food and Drug Administration and the Chinese Pharmacopoeia. Meropenem showed good linearity in the range of 0.31-25.01 µg/mL (r ≥ .999). Selectivity, intra-day and inter-day precision and accuracy, extraction recovery, and stability validation results were all within the acceptance range. This method has been successfully applied to the determination of synovial fluid samples from PJI patients, providing a useful detection method for meropenem therapeutic drug monitoring (TDM) in PJI patients.


Asunto(s)
Artroplastia de Reemplazo de Cadera , Artroplastia de Reemplazo de Rodilla , Infecciones Relacionadas con Prótesis , Humanos , Meropenem , Artroplastia de Reemplazo de Cadera/efectos adversos , Artroplastia de Reemplazo de Rodilla/efectos adversos , Líquido Sinovial/química , Infecciones Relacionadas con Prótesis/tratamiento farmacológico , Infecciones Relacionadas con Prótesis/diagnóstico , Infecciones Relacionadas con Prótesis/etiología , Biomarcadores/análisis , Antibacterianos/análisis , Cromatografía Liquida
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