Off-line Coupling Research Articles

Effective separation of maslinic acid and oleanolic acid from olive pomace using high-speed shear off-line coupled with high-speed countercurrent chromatography and their antibacterial activity test

In this work, a high-speed shear extraction off-line coupling high-speed countercurrent chromatography method was developed to separate maslinic acid and oleanolic acid from olive pomace. To improve extraction efficiency, the polar disparity between maslinic acid and oleanolic acid necessitated the concurrent utilization of both polar and non-polar solvents during high-speed shear extraction. Then, the high-speed shear extraction was directly feed to high-speed countercurrent chromatography for subsequently separation. A total of 250 min were needed to complete the extraction and separation process. This yielded two molecules from 3.3 g of defatted olive pomace: 7.2 mg of 93.8 % pure maslinic acid and 2.3 mg of 90.1 % pure oleanolic acid, both determined by HPLC at 210 nm. Furthermore, the compounds exhibited inhibitory activity against Escherichia coli and Staphylococcus aureus. At a concentration of 100 μg/mL, its efficacy in inhibiting hyaluronidase was comparable to that of the standard drug indomethacin. Compared with the conventional separation method, this coupled technique reduced the whole time due to the direct injection of sample extraction solution. This technique provides a useful approach for the separation of natural products with significant polarity differences.

Determination of bupropion by off-line coupling Fe3O4@CuO&GO nanocomposite and ion mobility spectrometry with application to biological samples.

In this study, an off-line coupling of dispersive solid-phase extraction (DSPE) and ion mobility spectrometry (IMS) was introduced to extract and determine bupropion (BUP). A magnetic nanocomposite adsorbent (Fe3O4@CuO&GO) was fabricated by combining graphene oxide (GO) sheets with Fe3O4 and CuO through coprecipitation method. The synthesized adsorbent was characterized and analyzed using the analytical techniques. The effect of extraction parameters including desorption solvent (type and volume), pH, adsorbent amount, contact time, temperature, and the volume of analyte solution on the extraction efficiency was investigated and optimized. The operational parameters of IMS method were also investigated. Under the optimum conditions (DSPE-IMS), the proposed method provided a linear range 4.0-24.0ng for BUP with a determination coefficient R2 ≥ 0.98. LOD and LOQ values were 0.7 and 2.2ng for BUP. The repeatability of proposed method was evaluated and reported as relative standard deviation (RSD% ≤ 5.5). The developed method was applied to determine BUP in different biological samples, in which satisfactory results were obtained (93.0-98.0%).

Development of Two-Dimensional Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for the Separation of a Five-Component Submicrometer Particle Mixture.

Submicrometer colloidal particles are widely applied in a variety of industrial products. While precise size and surface charge control is crucial to the stability and functionality of these materials, a tool to determine these properties with sufficient resolution, detection sensitivity, and robustness is still not available. The recently reported offline coupling of asymmetrical flow field-flow fractionation and capillary electrophoresis (AF4 × CE) shows success in improving the separation resolution for nanoparticles; however, challenges remain for sensitive multiple-component submicrometer particle analysis because of wide size and mobility distributions. We here report offline coupling of an AF4 method and a CE method, which utilized the online reversed electrode polarity stacking mode, to successfully characterize a five-component, submicrometer particle mixture. The mixture was successfully separated and detected with an improved inter- and intracomponent resolution. Therefore, our developed platform holds great potential for industrial applications involving multiple-component particle mixtures.

Two-dimensional liquid chromatography-mass spectrometry for lipidomics using off-line coupling of hydrophilic interaction liquid chromatography with 50 cm long reversed phase capillary columns

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.

Off-line vs. semi-implicit TH-TH coupling schemes: A BEPU comparison

Several TH-TH code coupling methods are used in the nuclear industry to model the core thermal–hydraulic conditions and the system behaviour. In some cases, the boundary conditions obtained by system codes are applied to sub-channel codes by table (off-line coupling). Even though this approach is in general considered valid, some boundary parameters will present inconsistencies. Alternatively, system and sub-channel codes are coupled using different coupling methods (semi-implicit coupling). Recent studies have shown a strong influence of the boundary conditions uncertainty on the sub-channel code results. The present study aims to evaluate the differences produced by the coupling methods by performing a best-estimate plus uncertainties (BEPU) comparison to the following cases: a complete loss of forced flow and a pressurizer relief valve opening. Results show that BEPU analysis presents good agreement with some discrepancies that can be explained and correlated to the boundary conditions deviations between codes.

Novel Gradient Elution Chromatography Atomic Force Microscopic Hyphenated Offline Coupling Technique for Copolymers Morphology Investigation

Novel Gradient Elution Chromatography Atomic Force Microscopic Hyphenated Offline Coupling Technique for Copolymers Morphology Investigation

Strategy for stereospecific characterization of natural triacylglycerols using multidimensional chromatography and mass spectrometry

Stereoisomeric determination of individual triacylglycerols (TAGs) in natural oils and fats is a challenge due to similar physicochemical properties of TAGs with different fatty acid combinations. In this study, we present a strategy to resolve the enantiomeric composition of nutritionally important TAGs in sea buckthorn (Hippophaë rhamnoides) as an example food matrix. The targeted strategy combines 1) fatty acid profiling with GC, 2) separation of TAGs with RP-HPLC, 3) stereospecific separation with chiral-phase HPLC and 4) structural characterization with MS. Three major asymmetric diacid- and triacid-TAG species were analyzed in sea buckthorn pulp oil. Off-line coupling of RP-HPLC and chiral-phase HPLC allowed separation of several TAG regioisomers and enantiomers, which could not be resolved using one-dimensional techniques. Enantiomeric ratios were determined and specific structural analysis of separated TAGs was performed using direct inlet ammonia negative ion chemical ionization method.Of the TAG 16:0/16:1/16:1 palmitic acid (C16:0) was located predominantly in a primary position and the enantiomeric ratio of TAG sn-16:1-16:1-16:0 to sn-16:0-16:1-16:1 was 70.5/29.5. Among the TAGs 16:0/16:0/18:2 and 16:0/16:0/16:1, only ca 5% had C16:0 in the sn-2 position, thus, ca 95% were symmetric sn-16:0-18:2-16:0 and sn-16:0-16:1-16:0. The enantiomeric ratio of triacid-TAGs containing C16:0 and two unsaturated fatty acids (palmitoleic C16:1, oleic C18:1 or linoleic acids C18:2) could not be resolved due to lack of commercial enantiopure reference compounds. However, it became clear that the targeted strategy presented offer unique and convenient method to study the enantiomeric structure of individual TAGs.

Harnessing the power of electrophoresis and chromatography: Offline coupling of reverse phase liquid chromatography-capillary zone electrophoresis-tandem mass spectrometry for analysis of host cell proteins in monoclonal antibody producing CHO cell line.

Host cell proteins (HCPs) are widely regarded as a critical quality attribute for a biotherapeutic product. Bottom up MS is the present gold standard for HCP analysis but suffers from incomplete protein identification due to complex nature of the HCP mixture and limited separation efficiency of the preceding LC-based systems. In this paper, we present for the first time an application involving use of LC-CE-MS/MS platform for analysis of HCPs. It has been demonstrated that the proposed platform has been able to successfully identify 397 HCPs from the supernatants of recombinant Chinese hamster ovary cells, twice and thrice the number of proteins identified by the state-of-the-art LC-MS/MS (189 HCPs) and CE-MS/MS (128 HCPs) analyses, respectively. Of these, 225 HCPs were unique to the LC-CE-MS/MS approach and were not identified by either LC-MS/MS or CE-MS/MS. It is observed that the LC-CE-MS/MS platform combines the benefits of LC-MS/MS and CE-MS/MS techniques and identifies peptides in a wider range of size, pI, and hydrophobicity. Additionally, LC-CE-MS/MS also identified more HCPs associated with cellular components, molecular functions, biological processes, peptidases, and secretory proteins. The proposed approach would thus be a useful addition in HCP analysis and secretome studies of mAb-producing Chinese hamster ovary cells.

Development of an Input File Preparation Tool for Offline Coupling of DNDC and DSSAT Models

Development of an Input File Preparation Tool for Offline Coupling of DNDC and DSSAT Models

Atmospheric Simulations of Total Column CO2 Mole Fractions from Global to Mesoscale within the Carbon Monitoring System Flux Inversion Framework

Quantifying the uncertainty of inversion-derived CO2 surface fluxes and attributing the uncertainty to errors in either flux or atmospheric transport simulations continue to be challenges in the characterization of surface sources and sinks of carbon dioxide (CO2). Despite recent studies inferring fluxes while using higher-resolution modeling systems, the utility of regional-scale models remains unclear when compared to existing coarse-resolution global systems. Here, we present an off-line coupling of the mesoscale Weather Research and Forecasting (WRF) model to optimized biogenic CO2 fluxes and mole fractions from the global Carbon Monitoring System inversion system (CMS-Flux). The coupling framework consists of methods to constrain the mass of CO2 introduced into WRF, effectively nesting our regional domain covering most of North America (except the northern half of Canada) within the CMS global model. We test the coupling by simulating Greenhouse gases Observing SATellite (GOSAT) column-averaged dry-air mole fractions (XCO2) over North America for 2010. We find mean model-model differences in summer of ∼0.12 ppm, significantly lower than the original coupling scheme (from 0.5 to 1.5 ppm, depending on the boundary). While 85% of the XCO2 values are due to long-range transport from outside our North American domain, most of the model-model differences appear to be due to transport differences in the fraction of the troposphere below 850 hPa. Satellite data from GOSAT and tower and aircraft data are used to show that vertical transport above the Planetary Boundary Layer is responsible for significant model-model differences in the horizontal distribution of column XCO2 across North America.

Mass Spectrometry-Based Analytical Strategy for Comprehensive Molecular Characterization of Biodegradable Poly(lactic-co-glycolic Acid) Copolymers.

An analytical methodology with mass spectrometry as the core technique was developed for precise characterization of end groups, size, and co-monomeric composition of poly(lactic-co-glycolic acid) (PLGA) copolymers, as a preliminary step to qualify their biodegradability. Four PLGA samples were studied, with GA molar content varying from 0 to 50% and Mw ranging from 18 to 75 kg mol-1 according to the supplier. Size exclusion chromatography (SEC) and liquid state nuclear magnetic resonance (NMR) were used as either complementary or validation techniques. As confirmed by tandem mass spectrometry (MS/MS) experiments, macrocycles were most prominent in the low mass range. Nevertheless, elemental compositions derived from high resolution (HR) mass measurements of linear species were consistent with chain terminations revealed by NMR. Off-line coupling of SEC with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) permitted calibration curves to be built based on absolute molecular weights and, although slightly overestimated, so-obtained Mn and Mw values compared well with SEC and NMR results. Homogeneity of the co-monomeric content of all chains within each PLGA sample was demonstrated using surface-assisted laser desorption/ionization in a reactive mode (reactive-SALDI), a newly developed technique that takes advantage of residual acid on desorption ionization using through-hole alumina membrane (DIUTHAME) chips to induce dissociation of high-molecular-weight polymers containing cleavable C-O bonds. All HRMS data were best handled with Kendrick analysis, which helped reveal minor species and allowed automated computation of congested mass spectra.

Preparative isolation and purification of 12 main antioxidants from the roots of Polygonum multiflorum Thunb. using high-speed countercurrent chromatography and preparative HPLC guided by 1,1'-diphenyl-2-picrylhydrazyl-HPLC.

A hyphenated strategy by off-line coupling of 1,1'-diphenyl-2-picrylhydrazyl-high-performance liquid chromatography, high-speed countercurrent chromatography, and preparative high-performance liquid chromatography was established to screen and separate antioxidants from ethyl acetate fraction of the roots of Polygonum multiflorum. Under the targeted guidance of 1,1'-diphenyl-2-picrylhydrazyl-high-performance liquid chromatography experiment, 12 compounds were identified as potential antioxidants and readily isolated by high-speed counter-current chromatography and preparative high-performance liquid chromatography. Ultraviolet spectroscopy, mass spectrometry, and 1 H NMR spectroscopy were employed to identify their structures, which were assigned as gallic acid (1, 6.2mg, 98.28%), catechin (2, 8.8mg, 90.69%), epicatechin (3, 4.1mg, 96.71%), polydatin (4, 5.3mg, 94.91%), 2,3,5,4'-tetrahydroxy stilbene-2-Ο-β-D-glucoside (5, 20.2mg, 95.23%), piceatannol (6, 5.3mg, 96.85%), rutin (7, 5.4mg, 97.92%), resveratrol (8, 5.2mg, 96.94%), isorhapontigenin (9, 11.4mg, 94.81%), hyperoside (10, 9.7mg, 98.52%), rhein (11, 4.9mg, 97.46%), and emodin (12, 8.2mg, 95.74%). Notably, compounds 6 and 9 were isolated from Polygonum multiflorum for the first time. In addition, antioxidant activity of compounds 1-12 were evaluated, and compounds 1-8 and 10 exhibited stronger antioxidant activity than ascorbic acid (positive control). These results indicated that the proposed method is a highly efficient strategy to screen and isolate antioxidants from complex natural products.

Homoisoflavonoids profiling of Ophiopogon japonicus by off-line coupling high-speed countercurrent chromatography with high-performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry.

Roots of Ophiopogon japonicus have been used as a functional food ingredient and traditional Chinese medicine for a long time in China. Homoisoflavonoids are one of the major kinds of bioactive compounds in O. japonicus; however, literature data about its homoisoflavonoids profile are scarce because of the complex ingredients with low abundance. Here, homoisoflavonoid fraction was prepared by petroleum ether extraction. Then, a high-speed countercurrent chromatography off-line coupling with high-performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry was developed for systematic identification of homoisoflavonoids. After that, 39 homoisoflavonoids, including 29 homoisoflavanone and 10 homoisoflavone, were unambiguously or tentatively identified, while 12 of them were reported in O. japonicus for the first time. Finally, eight available homoisoflavonoids were sensitively, precisely, and accurately determined by standard calibration curves, with limit of detection and limit of quantification in the range of 0.05-0.30 μg/mL and 0.12-0.66 μg/mL, relative standard deviation less than 7.3% for intra- and interday variations, and recovery at 94.5-105.2%. Collectively, our developed method is efficient, reliable, and valuable to profile chemical components of complex natural products.

Harnessing the power of electrophoresis and chromatography: Offline coupling of reverse phase liquid chromatography-capillary zone electrophoresis-tandem mass spectrometry for peptide mapping for monoclonal antibodies

RPLC-MS/MS is the present workhorse for bottom-up proteomics-based characterization. However it suffers from limited peak capacity and challenges with respect to detection of small and hydrophilic peptides. CZE-MS/MS offers an orthogonal alternative to RPLC-MS/MS but has limited sensitivity due to low sample loading. In the present work, for the first time, offline coupling of an RPLC–CZE–MS/MS has been demonstrated for peptide mapping of a monoclonal antibody-based therapeutic product. The performance of this platform has been compared to the state-of-the-art RPLC-MS/MS and CZE-MS/MS approaches. Fifteen fractions were isolated from a mAb tryptic digest using RPLC, and each fraction was analyzed by CZE-ESI-MS/MS. Results indicate that not only the RPLC–CZE–MS/MS platform identified a larger number of distinct peptides (372) than the RPLC-MS/MS (219) and CZE-MS/MS (177) platforms, it also offered significantly superior sequence coverage (99.55% vs. 89.76% and 94.21% for the heavy chain and 98.6% vs. 92.06% and 95.79% for the light chain). Also, the distribution of amino acid residues with post translational modifications was 413 for RPLC–CZE–MS/MS, 150 for RPLC-MS/MS and 94 for CZE-MS/MS. In fact, the RPLC–CZE–MS/MS system performed better than the combined datasets from RPLC-MS/MS or CZE-MS/MS. Our study shows that the traditionally used one-dimensional (1D) RPLC-MS/MS and CZE-MS/MS-based platforms may not be enough for characterization of complex molecules such as monoclonal antibodies. The proposed approach should be an essential addition to the analytical toolkit for in-depth primary structural characterization of mAbs.

Comprehensive profiling of α-glucosidase inhibitors from the leaves of Rubus suavissimus using an off-line hyphenation of HSCCC, ultrafiltration HPLC-UV-MS and prep-HPLC

The leaves of Rubus suavissimus (also called “Chinese sweet tea”) is used not only as a beverage tea and food additive but also as a folk herbal medicine for the treatment of diabetes mellitus. To systematically investigate its material foundation of efficacy for treating diabetes, herein, a hyphenated strategy by off-line coupling high-speed countercurrent chromatography, ultrafiltration HPLC-UV-MS and prep-HPLC was developed. And thus, α-glucosidase inhibitors from Rubus suavissimus leaves was comprehensively profiled, purified and characterized. As a result, twenty-six compounds were identified as potential α-glucosidase inhibitors and favorably isolated by prep-HPLC. Their structures were identified via UV, MS, and 1H-NMR. Notably, fourteen compounds, including protocatechuic acid (1), myketin (3), epicatechin (4), vanillic acid (6), apigenin (11), catechin (12), ferulic acid (15), luteolin (16), 3, 3′-di-O-methylellagic acid (17), chlorogenic acid (19), 3, 3′-di-O-methylellagic acid-4′-O-β-D-glucoside (20), cinnamic acid (21), syringate (24) and ethylbrevifolin-carboxylate (25), were reported in leaves of Rubus suavissimus for the first time. In addition, α-glucosidase inhibitory activities of compounds 1–26 were evaluated, and eighteen compounds exhibited stronger α-glucosidase inhibitory activities than acarbose (IC50 value at 214.24 ± 3.48 μg/mL, positive control). The results indicated that the proposed method is a highly efficient strategy for comprehensive profiling and purification of bioactive target compounds, including both major and minor components, from natural products.

Off-line TLC-IR (UATR) characterization of additives in EPDM rubber

Abstract Sulfur donor accelerators and vulcanization agents, such as peroxides, due to their chemical characteristics and good thermal stability properties in rubber formulations, are essential to provide solutions in different sectors, such as aerospace and automotive, since they reduce the polymer degradation process (reversal), which can influence the performance of the elastomeric material. In this way, the use of methodologies for the components knowledge of a formulation is imperative for the structure proper establishment/property relationship of the elastomeric artifacts. The development of methodology that can separate and characterize the rubber formulation components is a constant challenge, still allows gaps, and is usually done by conventional but not coupled instrumental techniques. In this scenario, this paper aimed to contribute to the use of the off-line coupling of thin-layer or thin-layer chromatography (TLC)/infrared spectroscopy (FT-IR), by universal attenuated total reflection (UATR) for N-cyclohexyl-2-benzothiazolesulfenamide (CBS), tetramethyl thiuram disulphide (TMTD) and dicumyl peroxide (DCP), in formulations of ethylene and propylene diene monomer (EPDM) rubber containing the plasticizer naphthenic oil. This plasticizer was characterized in formulations that presented higher content of this compound, but the eluting systems were not effective in separating DCP. However, better separation of CBS and TMTD was observed.

Separation of polystyrene nanoparticles with different coatings using two-dimensional off-line coupling of asymmetrical flow field flow fractionation and capillary electrophoresis

The successful off-line coupling of asymmetrical flow field flow fractionation (AF4) and capillary electrophoresis (CE) for separation of nanoparticles (NPs) with different surface coatings was shown. We could successfully demonstrate that, in a certain NP size range, hyphenation of both techniques significantly improved the separation of differently coated NPs. Three mixtures of polystyrene nanoparticles (PS-NPs) with comparable core sizes but different coatings (no coating/carboxyl-coated) were studied. Separation in either method resulted in non-baseline resolved or non-separated peaks. In contrast, two-dimensional off-line coupling of AF4 and CE resulted in clearly separated regions in their 2 D plots in case of 20 and 50 nm particle mixtures, whereas the 100 nm NP mixture could not be separated at all. Various factors affecting the separation like hydrodynamic diameter or SDS concentration were discussed.

A generic workflow for the characterization of therapeutic monoclonal antibodies-application to daratumumab.

In the present analytical workflow, chromatographic methods have been developed and hyphenated to mass spectrometry (MS) for the characterization of protein size, charge, hydrophobic, and hydrophilic variants of daratumumab. Multiple critical quality attributes (CQAs) were characterized in forced degraded daratumumab sample, using size exclusion, ion exchange (IEX), and hydrophobic interaction (HIC) chromatography coupled to fluorescence detection for relative quantification and fractionation. Mass assignment was performed by using a fast, non-denaturing and universal size exclusion chromatography (SEC) method prior to native MS analysis of the collected fractions (off-line approach). This allowed the identification of N-terminal lysine clipping, and the extent of glycation and oxidation at intact protein level. Finally, middle-up analysis of daratumumab was performed using reversed phase (RPLC) and hydrophilic interaction (HILIC) chromatography coupled to MS to obtain a comprehensive overview of all PTMs after the forced stressed conditions and a fine characterization of the glycosylation profile. Conveniently, the presented workflow maintains the established golden standard non-denaturing chromatography techniques and additionally introduces a straightforward and automated desalting procedure prior to MS analysis. Therefore, it is expected that the off-line coupling of SEC, IEX, and HIC to SEC-MS has great potential to be implemented in routine characterization of mAbs. Graphical abstract ᅟ.

Improving Fractionation of Human Milk Proteins through Calcium Phosphate Coprecipitation and Their Rapid Characterization by Capillary Electrophoresis.

This work describes a simple sample pretreatment method for the fractionation of human milk proteins (into their two main groups, whey and caseins) prior to their analysis. The protein-extraction protocol is based on the addition of calcium phosphate to nonadjusted pH human milk. The combination of calcium ions with phosphate results in an effective coprecipitation of caseins. To assess the suitability of this fractionation protocol, the protein extracts were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), LC-MS/MS, and capillary electrophoresis (CE) analysis. The results evidence a significant decrease in contamination of casein fraction with whey proteins and vice versa compared with the conventional isoelectric precipitation of caseins. In addition, CE fraction collection coupled to LC-MS/MS (off-line coupling) has been successfully applied to the identification of minor proteins in this complex matrix. The methodology presented here constitutes a promising tool to enlarge the knowledge of human milk proteome.

Characterization of additives in NR formulations by TLC-IR (UATR)

It is a well-established fact that rubber accelerator is essential to provide solution in different sectors. However, there is a reversal process which can reduce the material performance. Sulfur accelerators donors and organic peroxides have been presented as a solution to the problem. The methodology development that can separate or characterize those components is a challenge and still allows gaps, explained by the application of conventional technique to reach this goal. This study aimed at contributing to the use of off-line coupling of thin layer chromatography (TLC)/infrared spectroscopy (IR) by Universal Attenuated Total Reflection (UATR) for analysis of N-cyclohexyl-2-benzotiazolsulfenamide (CBS), tetraethylthiuram disulfide (TMTD) and dicumyl peroxide (DCP), in natural poly-cis-isoprene (NR) formulations, containing naphthenic oil. The best results were obtained for the plasticizer and DCP, in formulations that had a greater proportion of these compounds. The separation of CBS and TMTD was made with less effectiveness, due to bands overlapping.