Effect-directed analysis via hyphenated high-performance thin-layer chromatography for bioanalytical profiling of sunflower leaves

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Ágnes M. Móricz, Péter G. Ott, Imanuel Yüce, András Darcsi, Szabolcs Béni, Gertrud E. Morlock
High-performance thin-layer chromatography (HPTLC) coupled with effect-directed analysis was used for non-targeted screening of sunflower leaf extract for components exhibiting antioxidant, antibacterial and/or cholinesterase enzyme inhibitory effects. The active compounds were characterized by HPTLC-electrospray ionization-high resolution mass spectrometry (ESI-HRMS) and HPTLC-Direct Analysis in Real Time (DART)-MS/MS. The latter ambient ionization technique (less soft than ESI) resulted in oxidation and fragmentation products and characteristic fragment ions. NMR spectroscopy after targeted isolation via preparative normal phase flash chromatography and semi-preparative reversed phase high-performance liquid chromatography supported the identification of two diterpenes to be (-)-kaur-16-en-19-oic acid and 15-α-angeloyloxy-ent-kaur-16-en-19-oic acid. Both compounds found to be multi-potent as they inhibited acetylcholinesterase and butyrylcholinesterase and showed antibacterial effects against Gram-positive Bacillus subtilis and Gram-negative Aliivibrio fischeri bacteria. Kaurenoic acid was also active against the Gram-negative pepper pathogenic Xanthomonas euvesicatoria bacteria.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Ágnes M. Móricz, Péter G. Ott, Imanuel Yüce, András Darcsi, Szabolcs Béni, Gertrud E. Morlock

High-performance thin-layer chromatography (HPTLC) coupled with effect-directed analysis was used for non-targeted screening of sunflower leaf extract for components exhibiting antioxidant, antibacterial and/or cholinesterase enzyme inhibitory effects. The active compounds were characterized by HPTLC-electrospray ionization-high resolution mass spectrometry (ESI-HRMS) and HPTLC-Direct Analysis in Real Time (DART)-MS/MS. The latter ambient ionization technique (less soft than ESI) resulted in oxidation and fragmentation products and characteristic fragment ions. NMR spectroscopy after targeted isolation via preparative normal phase flash chromatography and semi-preparative reversed phase high-performance liquid chromatography supported the identification of two diterpenes to be (-)-kaur-16-en-19-oic acid and 15-α-angeloyloxy-ent-kaur-16-en-19-oic acid. Both compounds found to be multi-potent as they inhibited acetylcholinesterase and butyrylcholinesterase and showed antibacterial effects against Gram-positive Bacillus subtilis and Gram-negative Aliivibrio fischeri bacteria. Kaurenoic acid was also active against the Gram-negative pepper pathogenic Xanthomonas euvesicatoria bacteria.





Determination of caffeine, theobromine and theophylline in Mate beer and Mate soft drinks by high-performance thin-layer chromatography

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Claudia Oellig, Jacob Schunck, Wolfgang Schwack
Mate beer and Mate soft drinks are beverages produced from the dried leaves of Ilex paraguariensis (Yerba Mate). In Yerba Mate, the xanthine derivatives caffeine, theobromine and theophylline, also known as methylxanthines, are important active components. The presented method for the determination of caffeine, theobromine and theophylline in Mate beer and Mate soft drinks by high-performance thin-layer chromatography with ultraviolet detection (HPTLC–UV) offers a fully automated and sensitive determination of the three methylxanthines. Filtration of the samples was followed by degassing, dilution with acetonitrile in the case of Mate beers for protein precipitation, and centrifugation before the extracts were analyzed by HPTLC–UV on LiChrospher silica gel plates with fluorescence indicator and acetone/toluene/chloroform (4:3:3, v/v/v) as the mobile phase. For quantitation, the absorbance was scanned at 274nm. Limits of detection and quantitation were 1 and 4ng/zone, respectively, for caffeine, theobromine and theophylline. With recoveries close to 100% and low standard deviations reliable results were guaranteed. Experimental Mate beers as well as Mate beers and Mate soft drinks from the market were analyzed for their concentrations of methylxanthines.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Claudia Oellig, Jacob Schunck, Wolfgang Schwack

Mate beer and Mate soft drinks are beverages produced from the dried leaves of Ilex paraguariensis (Yerba Mate). In Yerba Mate, the xanthine derivatives caffeine, theobromine and theophylline, also known as methylxanthines, are important active components. The presented method for the determination of caffeine, theobromine and theophylline in Mate beer and Mate soft drinks by high-performance thin-layer chromatography with ultraviolet detection (HPTLC–UV) offers a fully automated and sensitive determination of the three methylxanthines. Filtration of the samples was followed by degassing, dilution with acetonitrile in the case of Mate beers for protein precipitation, and centrifugation before the extracts were analyzed by HPTLC–UV on LiChrospher silica gel plates with fluorescence indicator and acetone/toluene/chloroform (4:3:3, v/v/v) as the mobile phase. For quantitation, the absorbance was scanned at 274nm. Limits of detection and quantitation were 1 and 4ng/zone, respectively, for caffeine, theobromine and theophylline. With recoveries close to 100% and low standard deviations reliable results were guaranteed. Experimental Mate beers as well as Mate beers and Mate soft drinks from the market were analyzed for their concentrations of methylxanthines.





Thin-layer chromatography combined with diode laser thermal vaporization inductively coupled plasma mass spectrometry for the determination of selenomethionine and selenocysteine in algae and yeast

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Antonín Bednařík, Jan Kuta, Dai Long Vu, Karolína Ranglová, Pavel Hrouzek, Viktor Kanický, Jan Preisler
In this work we present a simple and cost-effective approach for the determination of selenium species in algae and yeast biomass, based on a combination of thin-layer chromatography (TLC) with diode laser thermal vaporization inductively coupled plasma mass spectrometry (DLTV ICP MS). Extraction of freeze-dried biomass was performed in 4M methanesulphonic acid and the selenium species were vaporized from cellulose TLC plates employing a continuous-wave infrared diode laser with power up to 4 W using a simple laboratory-built apparatus. Selenomethionine and selenocysteine were quantified with limits of detection 3 μg L−1 in a Se-enriched microalgae Chlorella vulgaris and yeast certified reference material SELM-1. Results delivered by TLC-DLTV ICP MS were consistent with those obtained by a routine coupling of high-performance liquid chromatography (HPLC) to ICP MS. In addition, the TLC approach is capable of analyzing extract containing even undiluted crude hydrolysates that could damage HPLC columns.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Antonín Bednařík, Jan Kuta, Dai Long Vu, Karolína Ranglová, Pavel Hrouzek, Viktor Kanický, Jan Preisler

In this work we present a simple and cost-effective approach for the determination of selenium species in algae and yeast biomass, based on a combination of thin-layer chromatography (TLC) with diode laser thermal vaporization inductively coupled plasma mass spectrometry (DLTV ICP MS). Extraction of freeze-dried biomass was performed in 4M methanesulphonic acid and the selenium species were vaporized from cellulose TLC plates employing a continuous-wave infrared diode laser with power up to 4 W using a simple laboratory-built apparatus. Selenomethionine and selenocysteine were quantified with limits of detection 3 μg L−1 in a Se-enriched microalgae Chlorella vulgaris and yeast certified reference material SELM-1. Results delivered by TLC-DLTV ICP MS were consistent with those obtained by a routine coupling of high-performance liquid chromatography (HPLC) to ICP MS. In addition, the TLC approach is capable of analyzing extract containing even undiluted crude hydrolysates that could damage HPLC columns.





À côté calibration – Making optimal use of time and space in quantitative high performance thin layer chromatography

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Josua Timotheus Oberlerchner, Christina Fuchs, Heinrich Grausgruber, Antje Potthast, Stefan Böhmdorfer
Quantitative High Performance Thin Layer Chromatography (HPTLC) requires the application of several standards to each plate, reducing the number of actual samples that can be analyzed in a single run. Using pure standard compounds and a selective detection method, the standards for quantitation can be applied besides – à côté – the chromatography area. This frees the sample application space to accommodate the maximal number of sample on each plate. Also, analysis time is spent exclusively on samples, drastically shortening the effective analysis time per sample and increasing sample throughput.Using this new calibration approach, the sample capacity of regular HPTLC methods can be increased or their scope be extended by an additional quantitative analysis. As a limitation, changes to the distribution of samples and standards within the plate as well as interferences from matrix compounds must be observed.We demonstrate the feasibility of this method by complementing an HPTLC method with a quantitative analysis of total anthocyanin content in colored wheat varieties. The quantitation was validated and compared to the conventional photometric analysis. As outcome, the additional photometric analysis could be replaced and rendered unnecessary, saving time, effort and equipment. This approach could also be employed to quantify highly retained substances, which are usually inaccessible for quantitative analysis.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Josua Timotheus Oberlerchner, Christina Fuchs, Heinrich Grausgruber, Antje Potthast, Stefan Böhmdorfer

Quantitative High Performance Thin Layer Chromatography (HPTLC) requires the application of several standards to each plate, reducing the number of actual samples that can be analyzed in a single run. Using pure standard compounds and a selective detection method, the standards for quantitation can be applied besides – à côté – the chromatography area. This frees the sample application space to accommodate the maximal number of sample on each plate. Also, analysis time is spent exclusively on samples, drastically shortening the effective analysis time per sample and increasing sample throughput. Using this new calibration approach, the sample capacity of regular HPTLC methods can be increased or their scope be extended by an additional quantitative analysis. As a limitation, changes to the distribution of samples and standards within the plate as well as interferences from matrix compounds must be observed. We demonstrate the feasibility of this method by complementing an HPTLC method with a quantitative analysis of total anthocyanin content in colored wheat varieties. The quantitation was validated and compared to the conventional photometric analysis. As outcome, the additional photometric analysis could be replaced and rendered unnecessary, saving time, effort and equipment. This approach could also be employed to quantify highly retained substances, which are usually inaccessible for quantitative analysis.





Bioprofiling of Salvia miltiorrhiza via planar chromatography linked to (bio)assays, high resolution mass spectrometry and nuclear magnetic resonance spectroscopy

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Ebrahim Azadniya, Gertrud E. Morlock
An affordable bioanalytical workflow supports the collection of data on active ingredients, required for the understanding of health-related food, superfood and traditional medicines. Targeted effect-directed responses of single compounds in a complex sample highlight this powerful bioanalytical hyphenation of planar chromatography with (bio)assays. Among many reports about biological properties of Salvia miltiorrhiza Bunge root (Danshen) and their analytical methods, the highly efficient direct bioautography (DB) workflow has not been considered so far. There was just one TLC-acetylcholinesterase (AChE) method with a poor zone resolution apart from our two HPTLC-DB studies, however, all methods were focused on the nonpolar extracts of Danshen (tanshinones) only. The current study on HPTLC-UV/Vis/FLD-(bio)assay-HRMS, followed by streamlined scale-up to preparative layer chromatography (PLC)-1H-NMR, aimed at an even more streamlined, yet comprehensive bioanalytical workflow. It comprised effect-directed screening of both, its polar (containing phenolics) and nonpolar extracts (containing tanshinones) on the same HPTLC plate, the biochemical and biological profiling with four different (bio)assays and elucidation of structures of known and unidentified active compounds. The five AChE inhibitors, salvianolic acid B (SAB), lithiospermic acid (LSA) and rosmarinic acid (RA) as well as cryptotanshinone (CT) and 15,16-dihydrotanshinone I (DHTI) were confirmed, but also unidentified inhibitors were observed. In the polar extracts, SAB, LSA and RA exhibited free radical scavenging properties in the 2,2-diphenyl-1-picrylhydrazyl assay. CT, DHTI and some unidentified nonpolar compounds were found active against Gram-positive Bacillus subtilis and Gram-negative Aliivibrio fischeri (LOD 12 ng/band for CT, and 5 ng/band for DHTI). For the first time, the most multipotent unidentified active compound zone in the B. subtilis, A. fischeri and AChE fingerprints of the nonpolar Danshen extract was identified as co-eluted band of 1,2-dihydrotanshinone and methylenetanshinquinone in the ratio of 2:1.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Ebrahim Azadniya, Gertrud E. Morlock

An affordable bioanalytical workflow supports the collection of data on active ingredients, required for the understanding of health-related food, superfood and traditional medicines. Targeted effect-directed responses of single compounds in a complex sample highlight this powerful bioanalytical hyphenation of planar chromatography with (bio)assays. Among many reports about biological properties of Salvia miltiorrhiza Bunge root (Danshen) and their analytical methods, the highly efficient direct bioautography (DB) workflow has not been considered so far. There was just one TLC-acetylcholinesterase (AChE) method with a poor zone resolution apart from our two HPTLC-DB studies, however, all methods were focused on the nonpolar extracts of Danshen (tanshinones) only. The current study on HPTLC-UV/Vis/FLD-(bio)assay-HRMS, followed by streamlined scale-up to preparative layer chromatography (PLC)-1H-NMR, aimed at an even more streamlined, yet comprehensive bioanalytical workflow. It comprised effect-directed screening of both, its polar (containing phenolics) and nonpolar extracts (containing tanshinones) on the same HPTLC plate, the biochemical and biological profiling with four different (bio)assays and elucidation of structures of known and unidentified active compounds. The five AChE inhibitors, salvianolic acid B (SAB), lithiospermic acid (LSA) and rosmarinic acid (RA) as well as cryptotanshinone (CT) and 15,16-dihydrotanshinone I (DHTI) were confirmed, but also unidentified inhibitors were observed. In the polar extracts, SAB, LSA and RA exhibited free radical scavenging properties in the 2,2-diphenyl-1-picrylhydrazyl assay. CT, DHTI and some unidentified nonpolar compounds were found active against Gram-positive Bacillus subtilis and Gram-negative Aliivibrio fischeri (LOD 12 ng/band for CT, and 5 ng/band for DHTI). For the first time, the most multipotent unidentified active compound zone in the B. subtilis, A. fischeri and AChE fingerprints of the nonpolar Danshen extract was identified as co-eluted band of 1,2-dihydrotanshinone and methylenetanshinquinone in the ratio of 2:1.





Extrathermodynamic parameters of sorption of light hydrocarbons on stationary phases prepared from tricyclononene polymers

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): A.A. Korolev, V.E. Shiryaeva, T.P. Popova, M.V. Bermeshev, A.Yu. Kanateva, A.A. Kurganov
Enthalpy and entropy of adsorption of polar and non-polar solutes were measured by chromatographic technique for new stationary phases prepared from membrane polymers based on tricyclonones. Data obtained within temperature interval from 40 to 150 °C were used to create extrathermodynamic dependences (compensation plots, dependences of enthalpy and entropy changes on solute carbon number). Compensation plots were very similar for all the stationary phases indicating similar adsorption mechanisms. The difference between the stationary phases was elucidated using dependences of enthalpy and entropy changes on solute carbon number. Higher retentivity of the stationary phase based on polymer 1 was explained by higher both enthalpy and entropy of solute adsorption on the stationary phase.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): A.A. Korolev, V.E. Shiryaeva, T.P. Popova, M.V. Bermeshev, A.Yu. Kanateva, A.A. Kurganov

Enthalpy and entropy of adsorption of polar and non-polar solutes were measured by chromatographic technique for new stationary phases prepared from membrane polymers based on tricyclonones. Data obtained within temperature interval from 40 to 150 °C were used to create extrathermodynamic dependences (compensation plots, dependences of enthalpy and entropy changes on solute carbon number). Compensation plots were very similar for all the stationary phases indicating similar adsorption mechanisms. The difference between the stationary phases was elucidated using dependences of enthalpy and entropy changes on solute carbon number. Higher retentivity of the stationary phase based on polymer 1 was explained by higher both enthalpy and entropy of solute adsorption on the stationary phase.





Graphical statistical approach to soil organic matter resilience using analytical pyrolysis data

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Gonzalo Almendros, Zulimar Hernández, Jesús Sanz, Sonia Rodríguez-Sánchez, Marco A. Jiménez-González, José A. González-Pérez
Pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) of humic acids (HAs) from 30 agricultural soils from a volcanic island (Tenerife, Spain) was used to discern the molecular characteristics of soil organic matter (SOM) associated to resilience. For faster perceptual identification of the results, the yields of the pyrolysis products in the form of surface density plots were compared in an update of the Van Krevelen graphical statistical method. This approach, with respect to data reduction and visualization, was also used to collectively represent statistical indices that were obtained after simple and partial least squares (PLS) regression. The resulting plots illustrate different SOM structural domains (for example, carbohydrate- and lignin-derived and condensed lipid). The content of SOM and total mineralization coefficient (TMC) values can be well estimated from the relative abundance of 57 major pyrolysis compounds: SOM content and composition parallels the accumulation of lignin- and carbohydrate-derived structures (lignocellulosic material) and the depletion of condensed polyalkyl structures. In other words, in the volcanic ash soils that were studied, we found that the higher the amount of SOM, the lower its quality in terms of resilience. Although no cause-and-effect is inferred from this fact, it is evident that the resistance to biodegradation of the SOM is related to its molecular composition.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Gonzalo Almendros, Zulimar Hernández, Jesús Sanz, Sonia Rodríguez-Sánchez, Marco A. Jiménez-González, José A. González-Pérez

Pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) of humic acids (HAs) from 30 agricultural soils from a volcanic island (Tenerife, Spain) was used to discern the molecular characteristics of soil organic matter (SOM) associated to resilience. For faster perceptual identification of the results, the yields of the pyrolysis products in the form of surface density plots were compared in an update of the Van Krevelen graphical statistical method. This approach, with respect to data reduction and visualization, was also used to collectively represent statistical indices that were obtained after simple and partial least squares (PLS) regression. The resulting plots illustrate different SOM structural domains (for example, carbohydrate- and lignin-derived and condensed lipid). The content of SOM and total mineralization coefficient (TMC) values can be well estimated from the relative abundance of 57 major pyrolysis compounds: SOM content and composition parallels the accumulation of lignin- and carbohydrate-derived structures (lignocellulosic material) and the depletion of condensed polyalkyl structures. In other words, in the volcanic ash soils that were studied, we found that the higher the amount of SOM, the lower its quality in terms of resilience. Although no cause-and-effect is inferred from this fact, it is evident that the resistance to biodegradation of the SOM is related to its molecular composition.





An alternative method for calibration of flow field flow fractionation channels for hydrodynamic radius determination: The nanoemulsion method (featuring multi angle light scattering)

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Hans Bolinsson, Yi Lu, Stephen Hall, Lars Nilsson, Andreas Håkansson
This study suggests a novel method for determination of the channel height in asymmetrical flow field-flow fractionation (AF4), which can be used for calibration of the channel for hydrodynamic radius determinations. The novel method uses an oil-in-water nanoemulsion together with multi angle light scattering (MALS) and elution theory to determine channel height from an AF4 experiment. The method is validated using two orthogonal methods; first, by using standard particle elution experiments and, secondly, by imaging an assembled and carrier liquid filled channel by x-ray computed tomography (XCT). It is concluded that the channel height can be determined with approximately the same accuracy as with the traditional channel height determination technique. However, the nanoemulsion method can be used under more challenging conditions than standard particles, as the nanoemulsion remains stable in a wider pH range than the previously used standard particles. Moreover, the novel method is also more cost effective.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Hans Bolinsson, Yi Lu, Stephen Hall, Lars Nilsson, Andreas Håkansson

This study suggests a novel method for determination of the channel height in asymmetrical flow field-flow fractionation (AF4), which can be used for calibration of the channel for hydrodynamic radius determinations. The novel method uses an oil-in-water nanoemulsion together with multi angle light scattering (MALS) and elution theory to determine channel height from an AF4 experiment. The method is validated using two orthogonal methods; first, by using standard particle elution experiments and, secondly, by imaging an assembled and carrier liquid filled channel by x-ray computed tomography (XCT). It is concluded that the channel height can be determined with approximately the same accuracy as with the traditional channel height determination technique. However, the nanoemulsion method can be used under more challenging conditions than standard particles, as the nanoemulsion remains stable in a wider pH range than the previously used standard particles. Moreover, the novel method is also more cost effective.





Identification and quantification of linear and branched isomers of perfluorooctanoic and perfluorooctane sulfonic acids in contaminated groundwater in the veneto region

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Alessandro Pellizzaro, Alessandro Zaggia, Massimo Fant, Lino Conte, Luigi Falletti
Perfluoroalkylated acids (PFAAs) are ubiquitous xenobiotic substances characterized by high persistency, bioaccumulation potential and toxicity. They have generated global concern because of their widespread presence both in water and biota compartments. In the past four years, alarming levels of these pollutants have been found in both surface and groundwater collected in an area covering more than 150 square kilometers in the south-western part of the province of Vicenza (Veneto region, Italy). One of the sources of the contamination recognized by local authorities is a fluorochemicals production plant that produced PFAAs since late sixties by electrochemical fluorination involving the obtainment of a complex mixture of linear and branched isomers. Branched isomers account for a significant part of total long chain homologues (22%–35%). Because of the potential threat to public health and the absence of specific limits set for these pollutants by Directive 98/83/EC, local authorities have established the following performance limits for drinking water: 90 ng L−1 for PFOA + PFOS, (reduced to 40 ng L−1 in the most contaminated municipalities), 30 ng L−1 for PFOS and 300 ng L−1 for the sum of all other PFAAs. Given the non-negligible incidence of branched isomers, it appears very important to correctly identify and quantify their contribution to total PFAAs. A liquid chromatography-electrospray ionization tandem spectrometry LC–MS/MS method, coupled with solid phase extraction, was developed to identify and quantify 25 PFAAs including six branched isomers of PFOS and four branched isomers of PFOA. Expanded uncertainty, recovery and precision were determined and found to agree with the reference EPA method 537:2009. The quantification limit is comprised in the 1–5 ng L−1 range.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Alessandro Pellizzaro, Alessandro Zaggia, Massimo Fant, Lino Conte, Luigi Falletti

Perfluoroalkylated acids (PFAAs) are ubiquitous xenobiotic substances characterized by high persistency, bioaccumulation potential and toxicity. They have generated global concern because of their widespread presence both in water and biota compartments. In the past four years, alarming levels of these pollutants have been found in both surface and groundwater collected in an area covering more than 150 square kilometers in the south-western part of the province of Vicenza (Veneto region, Italy). One of the sources of the contamination recognized by local authorities is a fluorochemicals production plant that produced PFAAs since late sixties by electrochemical fluorination involving the obtainment of a complex mixture of linear and branched isomers. Branched isomers account for a significant part of total long chain homologues (22%–35%). Because of the potential threat to public health and the absence of specific limits set for these pollutants by Directive 98/83/EC, local authorities have established the following performance limits for drinking water: 90 ng L−1 for PFOA + PFOS, (reduced to 40 ng L−1 in the most contaminated municipalities), 30 ng L−1 for PFOS and 300 ng L−1 for the sum of all other PFAAs. Given the non-negligible incidence of branched isomers, it appears very important to correctly identify and quantify their contribution to total PFAAs. A liquid chromatography-electrospray ionization tandem spectrometry LC–MS/MS method, coupled with solid phase extraction, was developed to identify and quantify 25 PFAAs including six branched isomers of PFOS and four branched isomers of PFOA. Expanded uncertainty, recovery and precision were determined and found to agree with the reference EPA method 537:2009. The quantification limit is comprised in the 1–5 ng L−1 range.





Dual polyhedral oligomeric silsesquioxanes polymerization approach to mutually-mediated separation mechanisms of hybrid monolithic stationary and mobile phases towards small molecules

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Jiao Su, Limin Yang, Qiuquan Wang
Hybrid monolithic stationary phase based HPLC is a typical example of practices in separation science. In this study, we developed a dual polyhedral oligomeric silsesquioxanes (POSS) polymerization approach to the preparation of a hybrid monolithic stationary phase of tri-porous structure and various surface chemistry. N-phenylaminopropyl-POSS (PA-POSS) and glycidyl-POSS (EP-POSS) were exemplified to demonstrate effective mutually-mediated separation mechanisms of the hybrid monolithic stationary phase and mobile phase towards diverse small molecules. PA-POSS and EP-POSS can be the monomer and/or crosslinker each other. They were polymerized via the epoxy-ring opening reaction to form the poly[(PA-POSS)-(EP-POSS)] (polyPOSS) monolithic stationary phase of 110.6/164.6 Å3 micropore (as a cube/ball), 10 nm mesopore and 0.95 μm macropore with the native siloxane cage and remaining phenyl/epoxy as well as chemically generated positive-chargeable tertiary phenylamine and hydrophilic hydroxyl groups. Such pore-structure and surface chemistry allow us to perform the effective separation of targeted small molecules, such as alkylbenzenes and alkylbenzene ketones, nucleic acid bases and amino acids, as well as phenols and phenolic acids, under reversed-phase, HILIC and mixed mode (polarity, size-exclusion and hydrogen-bonding) by just changing the molar ratio of POSS-precursors, and the composition and pH of a mobile phase as well. We believe that the approach developed herein can be extended to fabricate other kinds of hybrid monolithic stationary phases that are suitable for the separation of biomacromolecules and chiral molecules when choosing the existed POSS and/or designing new POSS with the substituted pendant groups of different physicochemical properties.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Jiao Su, Limin Yang, Qiuquan Wang

Hybrid monolithic stationary phase based HPLC is a typical example of practices in separation science. In this study, we developed a dual polyhedral oligomeric silsesquioxanes (POSS) polymerization approach to the preparation of a hybrid monolithic stationary phase of tri-porous structure and various surface chemistry. N-phenylaminopropyl-POSS (PA-POSS) and glycidyl-POSS (EP-POSS) were exemplified to demonstrate effective mutually-mediated separation mechanisms of the hybrid monolithic stationary phase and mobile phase towards diverse small molecules. PA-POSS and EP-POSS can be the monomer and/or crosslinker each other. They were polymerized via the epoxy-ring opening reaction to form the poly[(PA-POSS)-(EP-POSS)] (polyPOSS) monolithic stationary phase of 110.6/164.6 Å3 micropore (as a cube/ball), 10 nm mesopore and 0.95 μm macropore with the native siloxane cage and remaining phenyl/epoxy as well as chemically generated positive-chargeable tertiary phenylamine and hydrophilic hydroxyl groups. Such pore-structure and surface chemistry allow us to perform the effective separation of targeted small molecules, such as alkylbenzenes and alkylbenzene ketones, nucleic acid bases and amino acids, as well as phenols and phenolic acids, under reversed-phase, HILIC and mixed mode (polarity, size-exclusion and hydrogen-bonding) by just changing the molar ratio of POSS-precursors, and the composition and pH of a mobile phase as well. We believe that the approach developed herein can be extended to fabricate other kinds of hybrid monolithic stationary phases that are suitable for the separation of biomacromolecules and chiral molecules when choosing the existed POSS and/or designing new POSS with the substituted pendant groups of different physicochemical properties.





Chromatographic performance of microfluidic liquid chromatography devices: Experimental evaluation of straight versus serpentine packed channels

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Martin Gilar, Thomas S. McDonald, Fabrice Gritti, Gregory T. Roman, Jay S. Johnson, Bernard Bunner, Joseph D. Michienzi, Robert A. Collamati, Jim P. Murphy, Devesh D. Satpute, Matthew P. Bannon, Dennis DellaRovere, Robert A. Jencks, Tad A. Dourdeville, Keith E. Fadgen, Geoff C. Gerhardt
We prepared a series of planar titanium microfluidic (μLC) columns, each 100 mm long, with 0.15, 0.3 and 0.5 mm i.d.’s. The microfluidic columns were packed with 1.8 μm C18 sorbent and tested under isocratic and gradient conditions. The efficiency and peak capacity of these devices were monitored using a micro LC instrument with minimal extra column dispersion. Columns with serpentine channels were shown to perform worse than those with straight channels. The loss of efficiency and peak capacity was more prominent for wider i.d. columns, presumably due to on-column band broadening imparted by the so-called “race-track” effect. The loss of chromatographic performance was partially mitigated by tapering the turns (reduction in i.d. through the curved region). While good performance was obtained for 0.15 mm i.d. devices even without turn tapering, the performance of 0.3 mm i.d. columns could be brought on par with capillary LC devices by tapering down to 2/3 of the nominal channel width in the turn regions. The loss of performance was not fully compensated for in 0.5 mm devices even when tapering was employed; 30% loss in efficiency and 10% loss in peak capacity was observed. The experimental data for various devices were compared using the expected theoretical relationship between peak capacity Pc and efficiency N; (Pc−1) = N0.5 × const. While straight μLC columns showed the expected behavior, the devices with serpentine channels did not adhere to the plot. The results suggest that the loss of efficiency due to the turns is more pronounced than the corresponding loss of peak capacity.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Martin Gilar, Thomas S. McDonald, Fabrice Gritti, Gregory T. Roman, Jay S. Johnson, Bernard Bunner, Joseph D. Michienzi, Robert A. Collamati, Jim P. Murphy, Devesh D. Satpute, Matthew P. Bannon, Dennis DellaRovere, Robert A. Jencks, Tad A. Dourdeville, Keith E. Fadgen, Geoff C. Gerhardt

We prepared a series of planar titanium microfluidic (μLC) columns, each 100 mm long, with 0.15, 0.3 and 0.5 mm i.d.’s. The microfluidic columns were packed with 1.8 μm C18 sorbent and tested under isocratic and gradient conditions. The efficiency and peak capacity of these devices were monitored using a micro LC instrument with minimal extra column dispersion. Columns with serpentine channels were shown to perform worse than those with straight channels. The loss of efficiency and peak capacity was more prominent for wider i.d. columns, presumably due to on-column band broadening imparted by the so-called “race-track” effect. The loss of chromatographic performance was partially mitigated by tapering the turns (reduction in i.d. through the curved region). While good performance was obtained for 0.15 mm i.d. devices even without turn tapering, the performance of 0.3 mm i.d. columns could be brought on par with capillary LC devices by tapering down to 2/3 of the nominal channel width in the turn regions. The loss of performance was not fully compensated for in 0.5 mm devices even when tapering was employed; 30% loss in efficiency and 10% loss in peak capacity was observed. The experimental data for various devices were compared using the expected theoretical relationship between peak capacity Pc and efficiency N; (Pc −1) = N 0.5 × const. While straight μLC columns showed the expected behavior, the devices with serpentine channels did not adhere to the plot. The results suggest that the loss of efficiency due to the turns is more pronounced than the corresponding loss of peak capacity.





On the relationship between radial structure heterogeneities and efficiency of chromatographic columns

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Fabrice Gritti
The general dispersion theory of Aris is applied to predict the virtual asymptotic dispersion behavior of packed columns. The derived model is also used to estimate the actual pre-asymptotic dispersion behavior of modern 2.1 mm × 50 mm columns packed with sub-2 μm fully porous particles (FPPs) during the transient dispersion regime. The model accounts for the actual radial distribution of the flow velocity across the column diameter. From the wall to the center of the column, focused-ion-beam scanning electron microscopy (FIB-SEM) experiments were recently performed to reveal the existence of a thin (0.15dp wide, dp is the average particle diameter) hydrodynamic boundary layer (THBL), a thin (3dp wide) and loose orderly packed layer (TLOPL), a 60dp wide and dense randomly packed layer (WDRPL), and a large (≃460dp) randomly packed bulk central region [1].The theoretical calculations of the actual pre-asymptotic reduced van Deemter curves (2.1 mm × 50 mm column, sub-2 μm BEH-C18 FPPs, n-hexanophenone analyte, acetonitrile/water eluent, 80/20, v/v, flow rate from 0.05 to 0.35 mL/min) confirm that the impact of the sole THBL on column dispersion can be neglected. In contrast, the contribution of the TLOPL to the reduced plate height (RPH) is about 0.2 h unit at optimum reduced velocity. Most remarkably, the negative impact of the TLOPL on column performance may be fully compensated by the presence of the adjacent WDRPL if the depth of the velocity well were to be 5% of the bulk velocity. In actual 2.1 mm × 50 mm columns packed with sub-2 μm FPPs, this velocity depth is as large as 25% of the bulk velocity causing a significant RPH deviation of 0.7 h unit from the RPH of the bulk packing free from wall effects. Maximum column performance is expected for a reduction of WDRPL density. This suggests optimizing the packing process by finding the proper balance between the stress gradient across the WDRPL (responsible for the deep velocity well) and the friction forces between the packed particles (responsible for the rearrangement of the particles during bed consolidation). Past and recently reported RPH data support the theoretical insights: the stress gradient/particle friction balance in the WDRPL is better realized when packing superficially porous particles (SPPs) rather than FPPs in 2.1–4.6 mm i.d. columns (the RPH deviation is reduced to 0.4 h unit) or sub-2 μm particles in 100 cm × 75 μm i.d. capillaries combining high slurry concentrations and sonication (the RPH deviation is reduced to only 0.15 h unit).

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Fabrice Gritti

The general dispersion theory of Aris is applied to predict the virtual asymptotic dispersion behavior of packed columns. The derived model is also used to estimate the actual pre-asymptotic dispersion behavior of modern 2.1 mm × 50 mm columns packed with sub-2 μm fully porous particles (FPPs) during the transient dispersion regime. The model accounts for the actual radial distribution of the flow velocity across the column diameter. From the wall to the center of the column, focused-ion-beam scanning electron microscopy (FIB-SEM) experiments were recently performed to reveal the existence of a thin (0.15d p wide, d p is the average particle diameter) hydrodynamic boundary layer (THBL), a thin (3d p wide) and loose orderly packed layer (TLOPL), a 60d p wide and dense randomly packed layer (WDRPL), and a large (≃460d p ) randomly packed bulk central region [1]. The theoretical calculations of the actual pre-asymptotic reduced van Deemter curves (2.1 mm × 50 mm column, sub-2 μm BEH-C18 FPPs, n-hexanophenone analyte, acetonitrile/water eluent, 80/20, v/v, flow rate from 0.05 to 0.35 mL/min) confirm that the impact of the sole THBL on column dispersion can be neglected. In contrast, the contribution of the TLOPL to the reduced plate height (RPH) is about 0.2 h unit at optimum reduced velocity. Most remarkably, the negative impact of the TLOPL on column performance may be fully compensated by the presence of the adjacent WDRPL if the depth of the velocity well were to be 5% of the bulk velocity. In actual 2.1 mm × 50 mm columns packed with sub-2 μm FPPs, this velocity depth is as large as 25% of the bulk velocity causing a significant RPH deviation of 0.7 h unit from the RPH of the bulk packing free from wall effects. Maximum column performance is expected for a reduction of WDRPL density. This suggests optimizing the packing process by finding the proper balance between the stress gradient across the WDRPL (responsible for the deep velocity well) and the friction forces between the packed particles (responsible for the rearrangement of the particles during bed consolidation). Past and recently reported RPH data support the theoretical insights: the stress gradient/particle friction balance in the WDRPL is better realized when packing superficially porous particles (SPPs) rather than FPPs in 2.1–4.6 mm i.d. columns (the RPH deviation is reduced to 0.4 h unit) or sub-2 μm particles in 100 cm × 75 μm i.d. capillaries combining high slurry concentrations and sonication (the RPH deviation is reduced to only 0.15 h unit).





A double sealing technique for increasing the precision of headspace-gas chromatographic analysis

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Wei-Qi Xie, Kong-Xian Yu, Yi-Xian Gong
This paper investigates a new double sealing technique for increasing the precision of the headspace gas chromatographic method. The air leakage problem caused by the high pressure in the headspace vial during the headspace sampling process has a great impact to the measurement precision in the conventional headspace analysis (i.e., single sealing technique). The results (using ethanol solution as the model sample) show that the present technique is effective to minimize such a problem. The double sealing technique has an excellent measurement precision (RSD < 0.15%) and accuracy (recovery = 99.1%–100.6%) for the ethanol quantification. The detection precision of the present method was 10–20 times higher than that in earlier HS-GC work that use conventional single sealing technique. The present double sealing technique may open up a new avenue, and also serve as a general strategy for improving the performance (i.e., accuracy and precision) of headspace analysis of various volatile compounds.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Wei-Qi Xie, Kong-Xian Yu, Yi-Xian Gong

This paper investigates a new double sealing technique for increasing the precision of the headspace gas chromatographic method. The air leakage problem caused by the high pressure in the headspace vial during the headspace sampling process has a great impact to the measurement precision in the conventional headspace analysis (i.e., single sealing technique). The results (using ethanol solution as the model sample) show that the present technique is effective to minimize such a problem. The double sealing technique has an excellent measurement precision (RSD < 0.15%) and accuracy (recovery = 99.1%–100.6%) for the ethanol quantification. The detection precision of the present method was 10–20 times higher than that in earlier HS-GC work that use conventional single sealing technique. The present double sealing technique may open up a new avenue, and also serve as a general strategy for improving the performance (i.e., accuracy and precision) of headspace analysis of various volatile compounds.





Simultaneous analysis of opioid analgesics and their metabolites in municipal wastewaters and river water by liquid chromatography–tandem mass spectrometry

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Ivona Krizman-Matasic, Petra Kostanjevecki, Marijan Ahel, Senka Terzic
Although published literature provides a clear demonstration of widespread occurrence of opioid analgesics (OAs) in the aquatic environment, analytical methods suitable for a systematic study of this pharmaceutical class, which would include a broad spectrum of opioid analgesics and their metabolites, are still missing. In this work, a comprehensive multiresidue method for quantitative analysis of 27 opioid analgesics and their metabolites, including 2 morphine glucuronide conjugates, was developed and validated for three matrices: raw wastewater (RW), secondary effluent (SE) and river water. The method comprised different classes of opioid analgesics, including natural opiates (morphine and codeine), their semi-synthetic derivatives (hydrocodone, hydromorphone, oxycodone, oxymorphone and buprenorphine) as well as fully synthetic opioids such as methadone, fentanyl, sufentanil, propoxyphene and tramadol. The optimized enrichment procedure involved mixed-mode, strong cation-exchange sorbent in combination with a sequential elution procedure. The extracts were analyzed by reversed-phase liquid chromatography using a Synergy Polar column coupled to electrospray ionization tandem mass spectrometry (LC–MS/MS). Accurate quantification of target OAs was achieved using 19 deuterated analogues as surrogate standards. Method accuracies for RW, SE and river water varied in the range from 91 to 126%, 74 to 120% and 75 to 116%, respectively. Careful optimization of the procedure allowed reliable determination of OAs with method quantification limits in the low ng/L range (RW: 0.3-3.5 ng/L; SE: 0.2-1.9 ng/L, river water: 0.1-0.8 ng/L. The developed method was applied for analysis of RW, SE and river water samples from Croatia. The concentrations of individual OAs in municipal wastewater varied in a wide range (from < QL to 859 ng/L) and the most prevalent representatives were tramadol, codeine, morphine and methadone and their derivatives. Elevated concentrations of morphine glucuronides (up to 370 ng/L) found in raw municipal wastewater indicated their importance in the overall morphine mass balance.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Ivona Krizman-Matasic, Petra Kostanjevecki, Marijan Ahel, Senka Terzic

Although published literature provides a clear demonstration of widespread occurrence of opioid analgesics (OAs) in the aquatic environment, analytical methods suitable for a systematic study of this pharmaceutical class, which would include a broad spectrum of opioid analgesics and their metabolites, are still missing. In this work, a comprehensive multiresidue method for quantitative analysis of 27 opioid analgesics and their metabolites, including 2 morphine glucuronide conjugates, was developed and validated for three matrices: raw wastewater (RW), secondary effluent (SE) and river water. The method comprised different classes of opioid analgesics, including natural opiates (morphine and codeine), their semi-synthetic derivatives (hydrocodone, hydromorphone, oxycodone, oxymorphone and buprenorphine) as well as fully synthetic opioids such as methadone, fentanyl, sufentanil, propoxyphene and tramadol. The optimized enrichment procedure involved mixed-mode, strong cation-exchange sorbent in combination with a sequential elution procedure. The extracts were analyzed by reversed-phase liquid chromatography using a Synergy Polar column coupled to electrospray ionization tandem mass spectrometry (LC–MS/MS). Accurate quantification of target OAs was achieved using 19 deuterated analogues as surrogate standards. Method accuracies for RW, SE and river water varied in the range from 91 to 126%, 74 to 120% and 75 to 116%, respectively. Careful optimization of the procedure allowed reliable determination of OAs with method quantification limits in the low ng/L range (RW: 0.3-3.5 ng/L; SE: 0.2-1.9 ng/L, river water: 0.1-0.8 ng/L. The developed method was applied for analysis of RW, SE and river water samples from Croatia. The concentrations of individual OAs in municipal wastewater varied in a wide range (from < QL to 859 ng/L) and the most prevalent representatives were tramadol, codeine, morphine and methadone and their derivatives. Elevated concentrations of morphine glucuronides (up to 370 ng/L) found in raw municipal wastewater indicated their importance in the overall morphine mass balance.





Evaluation of mutual interference between bovine α-lactalbumin peptide and its isotope-labeled peptide in whey protein analysis using liquid chromatography-tandem mass spectrometry

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Zhen Wang, Qi Chen, Qiong Wu, Qifeng Li, Da Chen, Xiaogang Chu
Internal standard (IS) method is commonly used to correct the matrix effect of samples in the liquid chromatography-tandem mass spectrometric (LC–MS/MS) analysis of whey proteins. However, the presence of mutual interference between some peptides and their isotope-labeled peptides distorts the MS signals, requiring a fundamental evaluation to understand the phenomenon of signal variations. In this study, a simple strategy is proposed to evaluate the effects of sample pretreatment, materials and dilution of solutions on the MS signals of α-lactalbumin (VGINYWLAHK) and β-lactoglobulin peptides using two typical LC–MS/MS systems, Q-Trap and Q-Orbitrap. The strategy adapts the experimental MS data to optimize methods, thus providing meaningful solutions to suppress the mutual interference presented in the analysis of peptides. As a result, the strategy through the combination of 100-fold dilution and plastic injection vial improves the quantitation results of α-lactalbumin peptide significantly. While the β-lactoglobulin peptide presents different phenomenon of signal variations when compared with that of α-lactalbumin peptide, revealing that each peptide needs to be optimized individually. The calibration effect of different IS was also studied in fifteen infant milk powders to confirm the mutual interference impact to quantification result. These results indicated that a simple strategy through the combination of sample dilution and plastic injection vial could be well extended to quantitative analysis of any other peptide in the complex systems.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Zhen Wang, Qi Chen, Qiong Wu, Qifeng Li, Da Chen, Xiaogang Chu

Internal standard (IS) method is commonly used to correct the matrix effect of samples in the liquid chromatography-tandem mass spectrometric (LC–MS/MS) analysis of whey proteins. However, the presence of mutual interference between some peptides and their isotope-labeled peptides distorts the MS signals, requiring a fundamental evaluation to understand the phenomenon of signal variations. In this study, a simple strategy is proposed to evaluate the effects of sample pretreatment, materials and dilution of solutions on the MS signals of α-lactalbumin (VGINYWLAHK) and β-lactoglobulin peptides using two typical LC–MS/MS systems, Q-Trap and Q-Orbitrap. The strategy adapts the experimental MS data to optimize methods, thus providing meaningful solutions to suppress the mutual interference presented in the analysis of peptides. As a result, the strategy through the combination of 100-fold dilution and plastic injection vial improves the quantitation results of α-lactalbumin peptide significantly. While the β-lactoglobulin peptide presents different phenomenon of signal variations when compared with that of α-lactalbumin peptide, revealing that each peptide needs to be optimized individually. The calibration effect of different IS was also studied in fifteen infant milk powders to confirm the mutual interference impact to quantification result. These results indicated that a simple strategy through the combination of sample dilution and plastic injection vial could be well extended to quantitative analysis of any other peptide in the complex systems.





Use of an online extraction liquid chromatography quadrupole time-of-flight tandem mass spectrometry method for the characterization of polyphenols in Citrus paradisi cv. Changshanhuyu peel

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Chaoying Tong, Mijun Peng, Runna Tong, Ruyi Ma, Keke Guo, Shuyun Shi
Chemical profiling of natural products by high performance liquid chromatography (HPLC) was critical for understanding of their clinical bioactivities, and sample pretreatment steps have been considered as a bottleneck for analysis. Currently, concerted efforts have been made to develop sample pretreatment methods with high efficiency, low solvent and time consumptions. Here, a simple and efficient online extraction (OLE) strategy coupled with HPLC–diode array detector–quadrupole time-of-flight tandem mass spectrometry (HPLC–DAD–QTOF-MS/MS) was developed for rapid chemical profiling. For OLE strategy, guard column inserted with ground sample (2 mg) instead of sample loop was connected with manual injection valve, in which components were directly extracted and transferred to HPLC–DAD–QTOF-MS/MS system only by mobile phase without any extra time, solvent, instrument and operation. By comparison with offline heat-reflux extraction of Citrus paradisi cv. Changshanhuyu (Changshanhuyu) peel, OLE strategy presented higher extraction efficiency perhaps because of the high pressure and gradient elution mode. A total of twenty-two secondary metabolites were detected according to their retention times, UV spectra, exact mass, and fragmentation ions in MS/MS spectra, and nine of them were discovered in Changshanhuyu peel for the first time to our knowledge. It is concluded that the developed OLE–HPLC–DAD–QTOF-MS/MS system offers new perspectives for rapid chemical profiling of natural products.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Chaoying Tong, Mijun Peng, Runna Tong, Ruyi Ma, Keke Guo, Shuyun Shi

Chemical profiling of natural products by high performance liquid chromatography (HPLC) was critical for understanding of their clinical bioactivities, and sample pretreatment steps have been considered as a bottleneck for analysis. Currently, concerted efforts have been made to develop sample pretreatment methods with high efficiency, low solvent and time consumptions. Here, a simple and efficient online extraction (OLE) strategy coupled with HPLC–diode array detector–quadrupole time-of-flight tandem mass spectrometry (HPLC–DAD–QTOF-MS/MS) was developed for rapid chemical profiling. For OLE strategy, guard column inserted with ground sample (2 mg) instead of sample loop was connected with manual injection valve, in which components were directly extracted and transferred to HPLC–DAD–QTOF-MS/MS system only by mobile phase without any extra time, solvent, instrument and operation. By comparison with offline heat-reflux extraction of Citrus paradisi cv. Changshanhuyu (Changshanhuyu) peel, OLE strategy presented higher extraction efficiency perhaps because of the high pressure and gradient elution mode. A total of twenty-two secondary metabolites were detected according to their retention times, UV spectra, exact mass, and fragmentation ions in MS/MS spectra, and nine of them were discovered in Changshanhuyu peel for the first time to our knowledge. It is concluded that the developed OLE–HPLC–DAD–QTOF-MS/MS system offers new perspectives for rapid chemical profiling of natural products.





Evaluation of adsorption selectivity of immunoglobulins M, A and G and purification of immunoglobulin M with mixed-mode resins

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Ying-Di Luo, Qi-Lei Zhang, Shan-Jing Yao, Dong-Qiang Lin
This study investigated adsorption selectivity of immunoglobulin M (IgM), immunoglobulin A (IgA) and immunoglobulin (IgG) on four mixed-mode resins with the functional ligands of 4-mercatoethyl-pyridine (MEP), 2-mercapto-1-methylimidazole (MMI), 5-aminobenzimidazole (ABI) and tryptophan-5-aminobenzimidazole (W-ABI), respectively. IgM purification processes with mixed-mode resins were also proposed. All resins showed typical pH-dependent adsorption, and high adsorption capacity was found at pH 5.0–8.0 with low adsorption capacity under acidic conditions. Meanwhile, high selectivity of IgM/IgA and IgM/IgG was obtained with ABI-4FF and MMI-4FF resins at pH 4.0–5.0, which was used to develop a method for IgM, IgA and IgG separation by controlling loading and elution pH. Capture of monoclonal IgM from cell culture supernatant with ABI-4FF resins was studied and high purity (∼99%) and good recovery (80.8%) were obtained. Moreover, IgM direct separation from human serum with combined two-step chromatography (ABI-4FF and MMI-4FF) was investigated, and IgM purity of 65.2% and a purification factor of 28.3 were obtained after optimization. The antibody activity of IgM was maintained after purification. The results demonstrated that mixed-mode chromatography with specially-designed ligands is a promising way to improve adsorption selectivity and process efficiency of IgM purification from complex feedstock.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Ying-Di Luo, Qi-Lei Zhang, Shan-Jing Yao, Dong-Qiang Lin

This study investigated adsorption selectivity of immunoglobulin M (IgM), immunoglobulin A (IgA) and immunoglobulin (IgG) on four mixed-mode resins with the functional ligands of 4-mercatoethyl-pyridine (MEP), 2-mercapto-1-methylimidazole (MMI), 5-aminobenzimidazole (ABI) and tryptophan-5-aminobenzimidazole (W-ABI), respectively. IgM purification processes with mixed-mode resins were also proposed. All resins showed typical pH-dependent adsorption, and high adsorption capacity was found at pH 5.0–8.0 with low adsorption capacity under acidic conditions. Meanwhile, high selectivity of IgM/IgA and IgM/IgG was obtained with ABI-4FF and MMI-4FF resins at pH 4.0–5.0, which was used to develop a method for IgM, IgA and IgG separation by controlling loading and elution pH. Capture of monoclonal IgM from cell culture supernatant with ABI-4FF resins was studied and high purity (∼99%) and good recovery (80.8%) were obtained. Moreover, IgM direct separation from human serum with combined two-step chromatography (ABI-4FF and MMI-4FF) was investigated, and IgM purity of 65.2% and a purification factor of 28.3 were obtained after optimization. The antibody activity of IgM was maintained after purification. The results demonstrated that mixed-mode chromatography with specially-designed ligands is a promising way to improve adsorption selectivity and process efficiency of IgM purification from complex feedstock.





An integrated precipitation and ion-exchange chromatography process for antibody manufacturing: Process development strategy and continuous chromatography exploration

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Steffen Großhans, Gang Wang, Christian Fischer, Jürgen Hubbuch
In the past decades, research was carried out to find cost-efficient alternatives to Protein A chromatography as a capture step in monoclonal antibody (mAb) purification processes. In this work, polyethylene glycol (PEG) precipitation has shown promising results in the case of mAb yield and purity. Especially with respect to continuous processing, PEG precipitation has many advantages, like low cost of goods, simple setup, easy scalability, and the option to handle perfusion reactors.Nevertheless, replacing Protein A has the disadvantage of renouncing a platform unit operation as well. Furthermore, PEG precipitation is not capable of reducing high molecular weight impurities (HMW) like aggregates or DNA. To overcome these challenges, an integrated process strategy combining PEG precipitation with cation-exchange chromatography (CEX) for purification of a mAb is presented.This work discusses the process strategy as well as the associated fast, easy, and material-saving process development platform. These were implemented through the combination of high-throughput methods with empirical and mechanistic modeling. The strategy allows the development of a common batch process. Additionally, it is feasible to develop a continuous process. In the presented case study, a mAb provided from cell culture fluid (HCCF) was purified. The precipitation and resolubilization conditions as well as the chromatography method were optimized, and the mutual influence of all steps was investigated. A mAb yield of over 95.0% and a host cell protein (HCP) reduction of over 99.0% could be shown. At the same time, the aggregate level was reduced from 3.12% to 1.20% and the DNA level was reduced by five orders of magnitude. Furthermore, the mAb was concentrated three times to a final concentration of 11.9mg/mL.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Steffen Großhans, Gang Wang, Christian Fischer, Jürgen Hubbuch

In the past decades, research was carried out to find cost-efficient alternatives to Protein A chromatography as a capture step in monoclonal antibody (mAb) purification processes. In this work, polyethylene glycol (PEG) precipitation has shown promising results in the case of mAb yield and purity. Especially with respect to continuous processing, PEG precipitation has many advantages, like low cost of goods, simple setup, easy scalability, and the option to handle perfusion reactors. Nevertheless, replacing Protein A has the disadvantage of renouncing a platform unit operation as well. Furthermore, PEG precipitation is not capable of reducing high molecular weight impurities (HMW) like aggregates or DNA. To overcome these challenges, an integrated process strategy combining PEG precipitation with cation-exchange chromatography (CEX) for purification of a mAb is presented. This work discusses the process strategy as well as the associated fast, easy, and material-saving process development platform. These were implemented through the combination of high-throughput methods with empirical and mechanistic modeling. The strategy allows the development of a common batch process. Additionally, it is feasible to develop a continuous process. In the presented case study, a mAb provided from cell culture fluid (HCCF) was purified. The precipitation and resolubilization conditions as well as the chromatography method were optimized, and the mutual influence of all steps was investigated. A mAb yield of over 95.0% and a host cell protein (HCP) reduction of over 99.0% could be shown. At the same time, the aggregate level was reduced from 3.12% to 1.20% and the DNA level was reduced by five orders of magnitude. Furthermore, the mAb was concentrated three times to a final concentration of 11.9mg/mL.





Simultaneous determination of 12 vitamin D compounds in human serum using online sample preparation and liquid chromatography-tandem mass spectrometry

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Nur Sofiah Abu Kassim, Paul Nicholas Shaw, Amitha K. Hewavitharana
The development and validation of a method to simultaneously quantify 12 vitamin D compounds in human serum by LC–MS/MS is described. The main challenge was that of extracting and chromatographing vitamin D compounds with a range of polarities, both lipophilic and hydrophilic, in a single analytical procedure. The extractions of all 12 vitamin D compounds were achieved by an optimised protein precipitation method using acetonitrile as the precipitant, and the separation was accomplished by using a pentafluorophenyl (PFP) column. The sensitivity was increased by minimising matrix effects in MS detector rather than using a lengthy derivatisation procedure; an online solid phase extraction (SPE) using a PFP guard column was used for cleanup. Detection limits for all compounds were in the picomole range when using a 500μL sample volume. Recovery percentages ranged from 92% to 99%. LC–MS/MS resolution of all 12 vitamin D compounds, including the chromatographic separation of 25(OH)D3 from the isomer 3-epi-25(OH)D3 was achieved. Stable isotope labelled vitamin D compounds were used as internal standards for the quantification of all 12 vitamin D compounds. This is a simple yet accurate, selective, and sensitive method for the quantification of 12 major vitamin D compounds, including the sulfated forms, in human serum. The method is sufficiently robust to offer potential for use in routine analysis in a pathology laboratory setting.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Nur Sofiah Abu Kassim, Paul Nicholas Shaw, Amitha K. Hewavitharana

The development and validation of a method to simultaneously quantify 12 vitamin D compounds in human serum by LC–MS/MS is described. The main challenge was that of extracting and chromatographing vitamin D compounds with a range of polarities, both lipophilic and hydrophilic, in a single analytical procedure. The extractions of all 12 vitamin D compounds were achieved by an optimised protein precipitation method using acetonitrile as the precipitant, and the separation was accomplished by using a pentafluorophenyl (PFP) column. The sensitivity was increased by minimising matrix effects in MS detector rather than using a lengthy derivatisation procedure; an online solid phase extraction (SPE) using a PFP guard column was used for cleanup. Detection limits for all compounds were in the picomole range when using a 500μL sample volume. Recovery percentages ranged from 92% to 99%. LC–MS/MS resolution of all 12 vitamin D compounds, including the chromatographic separation of 25(OH)D3 from the isomer 3-epi-25(OH)D3 was achieved. Stable isotope labelled vitamin D compounds were used as internal standards for the quantification of all 12 vitamin D compounds. This is a simple yet accurate, selective, and sensitive method for the quantification of 12 major vitamin D compounds, including the sulfated forms, in human serum. The method is sufficiently robust to offer potential for use in routine analysis in a pathology laboratory setting.





Determination of household and industrial chemicals, personal care products and hormones in leafy and root vegetables by liquid chromatography-tandem mass spectrometry

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Irene Aparicio, Julia Martín, Concepción Abril, Juan Luis Santos, Esteban Alonso
A multiresidue method has been developed for the determination of emerging pollutants in leafy and root vegetables. Selected compounds were 6 perfluoroalkyl compounds (5 perfluorocarboxylic acids and perfluorooctanesulfonic acid), 3 non-ionic surfactants (nonylphenol and nonylphenolethoxylates), 8 anionic surfactants (4 alkylsulfates and 4 linear alkylbenzene sulfonates), 4 preservatives (parabens), 2 biocides (triclosan and triclocarban), 2 plasticizers (bisphenol A and di-(2-ethylhexyl)phthalate), 6 UV-filters (benzophenones) and 4 hormones. The method is based on ultrasound-assisted extraction, clean-up by dispersive solid-phase extraction (d-SPE) and liquid chromatography-tandem mass spectrometry analysis. Due to the diversity of the physico-chemical properties of the target compounds, and to better evaluate the influence of sample treatment variables in extraction efficiencies, Box-Behnken design was applied to optimize extraction solvent volume, number of extraction cycles and d-SPE sorbent amount. Linearity (R2) higher than 0.992, accuracy (expressed as relative recoveries) in the range from 81 to 126%, precision (expressed as relative standard deviation) lower than 19% and limits of detection between 0.025 and 12.5ngg−1 dry weight were achieved. The method was applied to leafy vegetables (lettuce, spinach and chard) and root vegetables (carrot, turnip and potato) from a local market. The highest concentrations corresponded to the surfactants reaching levels up to 114ngg−1 (dry weight), in one of the lettuce samples analyzed.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Irene Aparicio, Julia Martín, Concepción Abril, Juan Luis Santos, Esteban Alonso

A multiresidue method has been developed for the determination of emerging pollutants in leafy and root vegetables. Selected compounds were 6 perfluoroalkyl compounds (5 perfluorocarboxylic acids and perfluorooctanesulfonic acid), 3 non-ionic surfactants (nonylphenol and nonylphenolethoxylates), 8 anionic surfactants (4 alkylsulfates and 4 linear alkylbenzene sulfonates), 4 preservatives (parabens), 2 biocides (triclosan and triclocarban), 2 plasticizers (bisphenol A and di-(2-ethylhexyl)phthalate), 6 UV-filters (benzophenones) and 4 hormones. The method is based on ultrasound-assisted extraction, clean-up by dispersive solid-phase extraction (d-SPE) and liquid chromatography-tandem mass spectrometry analysis. Due to the diversity of the physico-chemical properties of the target compounds, and to better evaluate the influence of sample treatment variables in extraction efficiencies, Box-Behnken design was applied to optimize extraction solvent volume, number of extraction cycles and d-SPE sorbent amount. Linearity (R2) higher than 0.992, accuracy (expressed as relative recoveries) in the range from 81 to 126%, precision (expressed as relative standard deviation) lower than 19% and limits of detection between 0.025 and 12.5ngg−1 dry weight were achieved. The method was applied to leafy vegetables (lettuce, spinach and chard) and root vegetables (carrot, turnip and potato) from a local market. The highest concentrations corresponded to the surfactants reaching levels up to 114ngg−1 (dry weight), in one of the lettuce samples analyzed.