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Selected papers from the latest issue:
Combined quantification of faecal sterols, stanols, stanones and bile acids in soils and terrestrial sediments by gas chromatography–mass spectrometry
23 May 2012,
08:19:04
Publication year:
2012
Source:Journal of Chromatography A, Volume 1242
Jago Jonathan Birk, Michaela Dippold, Guido L.B. Wiesenberg, Bruno Glaser
Faeces incorporation can alter the concentration patterns of stanols, stanones, Δ5-sterols and bile acids in soils and terrestrial sediments. A joint quantification of these substances would give robust and specific information about the faecal input. Therefore, a method was developed for their purification and determination via gas chromatography–mass spectrometry (GC–MS) based on a total lipid extract (TLE) of soils and terrestrial sediments. Stanols, stanones, Δ5-steroles and bile acids were extracted by a single Soxhlet extraction yielding a TLE. The TLE was saponified with KOH in methanol. Sequential liquid–liquid extraction was applied to recover the biomarkers from the saponified extract and to separate the bile acids from the neutral stanoles, stanones and Δ5-steroles. The neutral fraction was directly purified using solid phase extraction (SPE) columns packed with 5% deactivated silica gel. The bile acids were methylated in dry HCl in methanol and purified on SPE columns packed with activated silica gel. A mixture of hexamethyldisilazane (HMDS), trimethylchlorosilane (TMCS) and pyridine was used to silylate the hydroxyl groups of the stanols and Δ5-sterols avoiding a silylation of the keto groups of the stanones in their enol-form. Silylation of the bile acids was carried out with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing N-trimethylsilylimidazole (TSIM). TLEs from a set of soils with different physico-chemical properties were used for method evaluation and for comparison of amounts of faecal biomarkers analysed with saponification and without saponification of the TLE. Therefore, a Regosol, a Podzol and a Ferralsol were sampled. To proof the applicability of the method for faecal biomarker analyses in archaeological soils and sediments, additional samples were taken from pre-Columbian Anthrosols in Amazonia and an Anthrosol from a site in central Europe settled since the Neolithic. The comparison of the amounts of steroids in combination with and without saponification of the TLE showed that high amounts of faecal biomarkers occur bound to other lipids and were liberated by saponification. The method was evaluated by standard addition. The standard contained 5β-stanols, 5β-stanones and their 5α-isomers together with Δ5-sterols and bile acids (19 substances). The standard addition revealed mean recoveries of individual substances ≥85%. The recoveries of biomarkers within each biomarker group did not differ significantly. Precisions were ≤0.22 (RSD) and quantification limits were between 1.3 and 10ngg−1 soil. These data showed that the method can be applied for quantification of trace amounts of faecal steroids and for the analyses of steroid patterns to detect enhanced faeces deposition in soils and sediments.
Source:Journal of Chromatography A, Volume 1242
Jago Jonathan Birk, Michaela Dippold, Guido L.B. Wiesenberg, Bruno Glaser
Faeces incorporation can alter the concentration patterns of stanols, stanones, Δ5-sterols and bile acids in soils and terrestrial sediments. A joint quantification of these substances would give robust and specific information about the faecal input. Therefore, a method was developed for their purification and determination via gas chromatography–mass spectrometry (GC–MS) based on a total lipid extract (TLE) of soils and terrestrial sediments. Stanols, stanones, Δ5-steroles and bile acids were extracted by a single Soxhlet extraction yielding a TLE. The TLE was saponified with KOH in methanol. Sequential liquid–liquid extraction was applied to recover the biomarkers from the saponified extract and to separate the bile acids from the neutral stanoles, stanones and Δ5-steroles. The neutral fraction was directly purified using solid phase extraction (SPE) columns packed with 5% deactivated silica gel. The bile acids were methylated in dry HCl in methanol and purified on SPE columns packed with activated silica gel. A mixture of hexamethyldisilazane (HMDS), trimethylchlorosilane (TMCS) and pyridine was used to silylate the hydroxyl groups of the stanols and Δ5-sterols avoiding a silylation of the keto groups of the stanones in their enol-form. Silylation of the bile acids was carried out with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing N-trimethylsilylimidazole (TSIM). TLEs from a set of soils with different physico-chemical properties were used for method evaluation and for comparison of amounts of faecal biomarkers analysed with saponification and without saponification of the TLE. Therefore, a Regosol, a Podzol and a Ferralsol were sampled. To proof the applicability of the method for faecal biomarker analyses in archaeological soils and sediments, additional samples were taken from pre-Columbian Anthrosols in Amazonia and an Anthrosol from a site in central Europe settled since the Neolithic. The comparison of the amounts of steroids in combination with and without saponification of the TLE showed that high amounts of faecal biomarkers occur bound to other lipids and were liberated by saponification. The method was evaluated by standard addition. The standard contained 5β-stanols, 5β-stanones and their 5α-isomers together with Δ5-sterols and bile acids (19 substances). The standard addition revealed mean recoveries of individual substances ≥85%. The recoveries of biomarkers within each biomarker group did not differ significantly. Precisions were ≤0.22 (RSD) and quantification limits were between 1.3 and 10ngg−1 soil. These data showed that the method can be applied for quantification of trace amounts of faecal steroids and for the analyses of steroid patterns to detect enhanced faeces deposition in soils and sediments.
Highlights
► Method allows quantification of steroids in order to detect faeces deposition. ► Saponification yields higher amounts of steroids than analyses without saponification. ► Standard addition to soils samples was used for method evaluation. ► Method was tested in soils with different physico-chemical properties. ► Mean recoveries of individual steroids were ≥85%.Hydrophobic interaction chromatography for purification of monoPEGylated RNase A
23 May 2012,
08:19:04
Publication year:
2012
Source:Journal of Chromatography A, Volume 1242
Karla Mayolo-Deloisa, Ma. Elena Lienqueo, Barbara Andrews, Marco Rito-Palomares, Juan A. Asenjo
The chromatographic methods used for the purification of PEGylated proteins are mainly Size Exclusion (SEC) and Ion Exchange Chromatography (IEX). Although the PEGylation affects the protein hydrophobicity, Hydrophobic Interaction Chromatography (HIC) has not been extensively applied for the separation of these proteins. Purification of monoPEGylated Ribonuclease A (RNase A) using HIC is studied in this work. The products of the PEGylation reaction of RNase A with 20kDa methoxy-poly(ethylene glycol) were separated using three resins with different degrees of hydrophobicity: Butyl, Octyl and Phenyl sepharose. The effects of resin type, concentration and salt type (ammonium sulphate or sodium chloride), and gradient length on the separation performance were evaluated. Yield and purity were calculated using the plate model. Under all conditions assayed the native protein was completely separated from PEGylated species. The best conditions for the purification of monoPEGylated RNase A were: Butyl sepharose, 1M ammonium sulphate and 35 column volumes (CVs); this resulted in a yield as high as 85% with a purity of 97%. The purity of monoPEGylated RNase A is comparable to that obtained when the separation is performed using SEC, but the yield increases from 65% with SEC to ∼85% with HIC. This process represents a viable alternative for the separation of PEGylated proteins.
Source:Journal of Chromatography A, Volume 1242
Karla Mayolo-Deloisa, Ma. Elena Lienqueo, Barbara Andrews, Marco Rito-Palomares, Juan A. Asenjo
The chromatographic methods used for the purification of PEGylated proteins are mainly Size Exclusion (SEC) and Ion Exchange Chromatography (IEX). Although the PEGylation affects the protein hydrophobicity, Hydrophobic Interaction Chromatography (HIC) has not been extensively applied for the separation of these proteins. Purification of monoPEGylated Ribonuclease A (RNase A) using HIC is studied in this work. The products of the PEGylation reaction of RNase A with 20kDa methoxy-poly(ethylene glycol) were separated using three resins with different degrees of hydrophobicity: Butyl, Octyl and Phenyl sepharose. The effects of resin type, concentration and salt type (ammonium sulphate or sodium chloride), and gradient length on the separation performance were evaluated. Yield and purity were calculated using the plate model. Under all conditions assayed the native protein was completely separated from PEGylated species. The best conditions for the purification of monoPEGylated RNase A were: Butyl sepharose, 1M ammonium sulphate and 35 column volumes (CVs); this resulted in a yield as high as 85% with a purity of 97%. The purity of monoPEGylated RNase A is comparable to that obtained when the separation is performed using SEC, but the yield increases from 65% with SEC to ∼85% with HIC. This process represents a viable alternative for the separation of PEGylated proteins.
Highlights
► Purification of monoPEGylated Ribonuclease A (RNase A) using HIC is reported. ► Products of PEGylation reaction of RNase A were processed using three resins with different degrees of hydrophobicity. ► This process represents an alternative to separate PEGylated proteins. ► monoPEGylated RNase A was obtained with a yield and purity of 85% and 97%, respectively.Comprehensive analysis of dipeptides in alcoholic beverages by tag-based separation and determination using liquid chromatography/electrospray ionization tandem mass spectrometry and quadrupole-time-of-flight mass spectrometry
23 May 2012,
08:19:04
Publication year:
2012
Source:Journal of Chromatography A, Volume 1242
Kei Takahashi, Masafumi Tokuoka, Hiromi Kohno, Nobuko Sawamura, Yuka Myoken, Akihiro Mizuno
Fermented foods and beverages contain several different types of dipeptides, which are believed to be important components for taste. To date, however, a method for the comprehensive analysis of dipeptides in these products has not yet been established. In this study, comprehensive analysis of dipeptides in alcoholic beverages was performed by a high-resolution separation method based on the structural characteristics of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-derivatized dipeptides as well as dipeptide quantification and structural estimation using ultra-high-pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) and UHPLC-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS), respectively. Dipeptide content was found to differ considerably among Japanese sake, beer, and wine; UHPLC–MS/MS analysis revealed that many types of dipeptides are present in sake. Dipeptide quantification analysis identified 32 types of dipeptides within the concentration range of 1.1–97.2μM in sake. The analysis was validated by dipeptide recovery of 64.0–107.2% (2.5μM of standard) with a relative standard deviation of ≤33.2% from an actual alcoholic sample. Furthermore, UHPLC-Q-TOFMS analysis suggested the existence of more than 35 types of dipeptides in sake. Thus, by the combined analysis methods, we discovered that more than 60 dipeptides are present in sake. This research is the first report of dipeptide profiling of fermented alcoholic beverages by comprehensive analysis.
Source:Journal of Chromatography A, Volume 1242
Kei Takahashi, Masafumi Tokuoka, Hiromi Kohno, Nobuko Sawamura, Yuka Myoken, Akihiro Mizuno
Fermented foods and beverages contain several different types of dipeptides, which are believed to be important components for taste. To date, however, a method for the comprehensive analysis of dipeptides in these products has not yet been established. In this study, comprehensive analysis of dipeptides in alcoholic beverages was performed by a high-resolution separation method based on the structural characteristics of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-derivatized dipeptides as well as dipeptide quantification and structural estimation using ultra-high-pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) and UHPLC-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS), respectively. Dipeptide content was found to differ considerably among Japanese sake, beer, and wine; UHPLC–MS/MS analysis revealed that many types of dipeptides are present in sake. Dipeptide quantification analysis identified 32 types of dipeptides within the concentration range of 1.1–97.2μM in sake. The analysis was validated by dipeptide recovery of 64.0–107.2% (2.5μM of standard) with a relative standard deviation of ≤33.2% from an actual alcoholic sample. Furthermore, UHPLC-Q-TOFMS analysis suggested the existence of more than 35 types of dipeptides in sake. Thus, by the combined analysis methods, we discovered that more than 60 dipeptides are present in sake. This research is the first report of dipeptide profiling of fermented alcoholic beverages by comprehensive analysis.
Highlights
► A separation method of AQC-derivatized dipeptide was developed by UHPLC using two sub-3μm core-shell type columns. ► Quantification and structural estimation of dipeptide using MS/MS and Q-TOFMS were examined. ► Dipeptide content in alcoholic beverages was quite differ among Japanese sake, beer, and wine. ► Sake contains many species and significant quantities of dipeptides. More than 60 species of dipeptides were found in sake.Design of countercurrent separation of Ginkgo biloba terpene lactones by nuclear magnetic resonance
23 May 2012,
08:19:04
Publication year:
2012
Source:Journal of Chromatography A, Volume 1242
Feng Qiu, J. Brent Friesen, James B. McAlpine, Guido F. Pauli
Terpene lactones such as bilobalide, ginkgolides A, B, C, and J are major bioactive compounds of Ginkgo biloba L. Purification of these compounds is tedious due to their similar chemical properties. For the purpose of developing an effective and efficient method for both analytical and preparative separation of terpene lactones in G. biloba, an innovative orthogonality-enhanced high-speed countercurrent chromatography (HSCCC) method was established. Taking advantage of quantitative 1H NMR (qHNMR) methodology, partition coefficients (K) of individual terpene lactones were calculated directly from crude G. biloba leaf extract, using their H-12 signals as distinguishing feature. The partitioning experiment assisted the design of a two dimensional (2D) HSCCC procedure using a pair of orthogonal HSCCC solvent systems (SSs), ChMWat +4 and HEMSoWat +3/0.05%. It was surprising that the resolution of ginkgolides A and B was improved by 25% in the HEMWat +3 SS modified with 0.5% DMSO. Consequently, all five terpene lactones could be well separated with qHNMR purity>95% from G. biloba leaf extract. The separation was further evaluated by offline qHNMR analysis of HSCCC fractions associated with Gaussian curve fitting. The results showed less than 2% error in HSCCC retention predicted from the partitioning experiment. This compelling consistency demonstrates that qHNMR-derived K determination (“K-by-NMR”) can be used to predict CCC fractionation and target purification of analytes from complex mixtures. Furthermore, Gaussian curve fitting enabled an accurate prediction of less than 2% impurity in the CCC fraction, which demonstrates its potential as a powerful tool to study the presence of minor constituents, especially when they are beyond the detection limit of conventional spectroscopic detectors.
Source:Journal of Chromatography A, Volume 1242
Feng Qiu, J. Brent Friesen, James B. McAlpine, Guido F. Pauli
Terpene lactones such as bilobalide, ginkgolides A, B, C, and J are major bioactive compounds of Ginkgo biloba L. Purification of these compounds is tedious due to their similar chemical properties. For the purpose of developing an effective and efficient method for both analytical and preparative separation of terpene lactones in G. biloba, an innovative orthogonality-enhanced high-speed countercurrent chromatography (HSCCC) method was established. Taking advantage of quantitative 1H NMR (qHNMR) methodology, partition coefficients (K) of individual terpene lactones were calculated directly from crude G. biloba leaf extract, using their H-12 signals as distinguishing feature. The partitioning experiment assisted the design of a two dimensional (2D) HSCCC procedure using a pair of orthogonal HSCCC solvent systems (SSs), ChMWat +4 and HEMSoWat +3/0.05%. It was surprising that the resolution of ginkgolides A and B was improved by 25% in the HEMWat +3 SS modified with 0.5% DMSO. Consequently, all five terpene lactones could be well separated with qHNMR purity>95% from G. biloba leaf extract. The separation was further evaluated by offline qHNMR analysis of HSCCC fractions associated with Gaussian curve fitting. The results showed less than 2% error in HSCCC retention predicted from the partitioning experiment. This compelling consistency demonstrates that qHNMR-derived K determination (“K-by-NMR”) can be used to predict CCC fractionation and target purification of analytes from complex mixtures. Furthermore, Gaussian curve fitting enabled an accurate prediction of less than 2% impurity in the CCC fraction, which demonstrates its potential as a powerful tool to study the presence of minor constituents, especially when they are beyond the detection limit of conventional spectroscopic detectors.
Highlights
► Novel qHNMR-based approach to countercurrent partition coefficients (K values). ► Enabled targeted countercurrent separation (CS) of Ginkgo biloba terpene lactones. ► Established orthogonal solvent systems (SSs) for separation of Ginkgo lactones. ► Developed DMSO-modified SSs as a new means of resolution enhancement in CS. ► Evaluated ID and residual complexity of eluents by qHNMR with Gaussian fitting.Preparation and characterization of agarose–nickel nanoporous composite particles customized for liquid expanded bed adsorption
23 May 2012,
08:19:04
Publication year:
2012
Source:Journal of Chromatography A, Volume 1242
F. Asghari, M. Jahanshahi, A.A. Ghoreyshi
Agarose–nickel nanoporous composite matrices with a series of densities, named Ag–Ni, were prepared herein for expanded bed adsorption of nanobioproduct/bioproduct by a water-in-oil emulsification method. The optical microscope (OM), scanning electronic microscope (SEM) and particle size analyzer (PSA) were utilized in order to characterize the structure and morphology of the agarose–nickel composite. The results indicated that the matrices prepared had a spherical appearance, appropriate wet density of 1.73–2.56g/ml, water content of 32.2–58.5% and porosity of 79.4–96.37% and pore size of about 100–150nm. All the Ag–Ni beads follow logarithmic normal size distribution with the range of 60–230μm and average diameter of 133.68–148.4μm. One of the useful properties of the Ag–Ni particles is the high wet density up to 2.56g/ml, which shows a potential for the operation in an expanded bed at high flow rate. The impact of nickel powder addition on the physical and hydrodynamic properties was also investigated. In addition, the fluidization behavior of the Ag–Ni particles under various conditions was characterized by the measurement of bed expansion and axial dispersion coefficients for the liquid phase when operated in a standard fluidized bed contactor. It was observed that the expansion factors were decreased with the increasing matrix density under the same velocity. The bed expansion and fluid velocity were correlated with Richardson–Zaki equation for all particles prepared and the correlation parameters (the terminal settling velocity U t and expansion index n) were investigated. Using measurements of residence time distributions, hydrodynamic properties in the expanded beds were investigated and were compared with reported matrices in other literatures. In addition, the impact of the flow velocity, bed expansion degree and density of adsorbent on hydrodynamic properties in the expanded beds were investigated. The results indicated that the expansion factor showed little effect on the hydrodynamic properties while the fluid velocity was the most essential factor on this regard. Furthermore, the results indicated that the heavy matrices of Ag–Ni-3, Ag–Ni-4 and Ag–Ni-5, were more suited for high operation fluid velocity. In addition, even the light matrices, i.e. Ag–Ni-1 and Ag–Ni-2, seem to be superior to other matrices in hydrodynamic properties, which made them promising adsorbents for further use in EBA processes.
Source:Journal of Chromatography A, Volume 1242
F. Asghari, M. Jahanshahi, A.A. Ghoreyshi
Agarose–nickel nanoporous composite matrices with a series of densities, named Ag–Ni, were prepared herein for expanded bed adsorption of nanobioproduct/bioproduct by a water-in-oil emulsification method. The optical microscope (OM), scanning electronic microscope (SEM) and particle size analyzer (PSA) were utilized in order to characterize the structure and morphology of the agarose–nickel composite. The results indicated that the matrices prepared had a spherical appearance, appropriate wet density of 1.73–2.56g/ml, water content of 32.2–58.5% and porosity of 79.4–96.37% and pore size of about 100–150nm. All the Ag–Ni beads follow logarithmic normal size distribution with the range of 60–230μm and average diameter of 133.68–148.4μm. One of the useful properties of the Ag–Ni particles is the high wet density up to 2.56g/ml, which shows a potential for the operation in an expanded bed at high flow rate. The impact of nickel powder addition on the physical and hydrodynamic properties was also investigated. In addition, the fluidization behavior of the Ag–Ni particles under various conditions was characterized by the measurement of bed expansion and axial dispersion coefficients for the liquid phase when operated in a standard fluidized bed contactor. It was observed that the expansion factors were decreased with the increasing matrix density under the same velocity. The bed expansion and fluid velocity were correlated with Richardson–Zaki equation for all particles prepared and the correlation parameters (the terminal settling velocity U t and expansion index n) were investigated. Using measurements of residence time distributions, hydrodynamic properties in the expanded beds were investigated and were compared with reported matrices in other literatures. In addition, the impact of the flow velocity, bed expansion degree and density of adsorbent on hydrodynamic properties in the expanded beds were investigated. The results indicated that the expansion factor showed little effect on the hydrodynamic properties while the fluid velocity was the most essential factor on this regard. Furthermore, the results indicated that the heavy matrices of Ag–Ni-3, Ag–Ni-4 and Ag–Ni-5, were more suited for high operation fluid velocity. In addition, even the light matrices, i.e. Ag–Ni-1 and Ag–Ni-2, seem to be superior to other matrices in hydrodynamic properties, which made them promising adsorbents for further use in EBA processes.
Highlights
► Agarose–nickel nanoporous particles have been fabricated with high wet density of 1.73–2.56g/ml and pore size of 100–150nm. ► The influence of matrix density on the performance of expanded bed adsorption has been studied. ► The results indicated that the matrix with small density can form a better stable expanded bed. ► As compared to HETP, B 0 and D ax analysis, it was observed that D ax is the best parameter for characterizing the bed stability.Simultaneous metabolite fingerprinting of hydrophilic and lipophilic compounds in Echinacea pallida by high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometry detection
23 May 2012,
08:19:04
Publication year:
2012
Source:Journal of Chromatography A, Volume 1242
Federica Pellati, Giulia Orlandini, Stefania Benvenuti
In this study, a detailed phytochemical characterization of Echinacea pallida (Nutt.) Nutt. root extracts and dietary supplements was carried out for the first time by developing advanced chromatographic techniques, based on HPLC with diode array (DAD) and electrospray ionization-mass spectrometry (ESI-MS) detection (with ion trap and triple quadrupole mass analyzers), for the simultaneous analysis of hydrophilic and lipophilic secondary metabolites. The HPLC analyses were carried out on an Ascentis C18 column (250mm×4.6mm I.D., 5μm), with a mobile phase composed by H2O and ACN both containing 0.1% formic acid, under gradient elution. The UV spectra, in combination with MS and MS/MS data, allowed the identification of fourteen compounds, including caffeic acid derivatives, polyacetylenes and polyenes, in the analyzed samples. MS and MS/MS data were discussed in detail and the typical fragmentation patterns of each class of secondary metabolites were identified. For the first time, a hydroperoxide intermediate was characterized as an oxidation product of one of E. pallida monocarbonylic acetylenes, providing a confirmation of the mechanism that leads to the generation of hydroxylated derivatives. The HPLC method was fully validated in agreement with ICH guidelines and then applied to real samples. The quantitative analysis indicated that there was a great variability in the amount of the active compounds in the dietary supplements containing E. pallida root extracts: the content of total caffeic acid derivatives ranged from 2.31 to 11.45mg/g and the amount of total polyacetylenes and polyenes from 6.38 to 30.54mg/g. In the analyzed samples, the most abundant caffeic acid derivative was found to be echinacoside. Regarding polyacetylenes and polyenes, the most representative compounds were found to be tetradec-(8Z)-ene-11,13-diyn-2-one, pentedeca-(8Z,11Z)-dien-2-one and pentadec-(8Z)-en-2-one. The developed method can be considered suitable for metabolite fingerprinting and quality control of E. pallida plant material and natural products.
Source:Journal of Chromatography A, Volume 1242
Federica Pellati, Giulia Orlandini, Stefania Benvenuti
In this study, a detailed phytochemical characterization of Echinacea pallida (Nutt.) Nutt. root extracts and dietary supplements was carried out for the first time by developing advanced chromatographic techniques, based on HPLC with diode array (DAD) and electrospray ionization-mass spectrometry (ESI-MS) detection (with ion trap and triple quadrupole mass analyzers), for the simultaneous analysis of hydrophilic and lipophilic secondary metabolites. The HPLC analyses were carried out on an Ascentis C18 column (250mm×4.6mm I.D., 5μm), with a mobile phase composed by H2O and ACN both containing 0.1% formic acid, under gradient elution. The UV spectra, in combination with MS and MS/MS data, allowed the identification of fourteen compounds, including caffeic acid derivatives, polyacetylenes and polyenes, in the analyzed samples. MS and MS/MS data were discussed in detail and the typical fragmentation patterns of each class of secondary metabolites were identified. For the first time, a hydroperoxide intermediate was characterized as an oxidation product of one of E. pallida monocarbonylic acetylenes, providing a confirmation of the mechanism that leads to the generation of hydroxylated derivatives. The HPLC method was fully validated in agreement with ICH guidelines and then applied to real samples. The quantitative analysis indicated that there was a great variability in the amount of the active compounds in the dietary supplements containing E. pallida root extracts: the content of total caffeic acid derivatives ranged from 2.31 to 11.45mg/g and the amount of total polyacetylenes and polyenes from 6.38 to 30.54mg/g. In the analyzed samples, the most abundant caffeic acid derivative was found to be echinacoside. Regarding polyacetylenes and polyenes, the most representative compounds were found to be tetradec-(8Z)-ene-11,13-diyn-2-one, pentedeca-(8Z,11Z)-dien-2-one and pentadec-(8Z)-en-2-one. The developed method can be considered suitable for metabolite fingerprinting and quality control of E. pallida plant material and natural products.
Highlights
► Echinacea pallida plant material and dietary supplements were analyzed. ► Analysis of both hydrophilic and lipophilic secondary metabolites was performed. ► HPLC-UV/DAD, ESI-MS and MS/MS allowed the identification of fourteen compounds. ► The typical fragmentation patterns of each class of constituents were identified. ► Great variability was observed in the content of E. pallida dietary supplements.Chemical anchoring of lauryl methacrylate-based reversed phase monolith to 1/16″ o.d. polyetheretherketone tubing
23 May 2012,
08:19:04
Publication year:
2012
Source:Journal of Chromatography A, Volume 1242
Shin Shu, Hiroharu Kobayashi, Masaki Okubo, Akhmad Sabarudin, Michio Butsugan, Tomonari Umemura
In this paper, we describe a method for the preparation of easy-to-use reversed-phase monolithic microbore columns. Polyetheretherketone (PEEK) tubing with an outer diameter of 1/16″ and an inner diameter of 1.0mm was used as a column housing (empty column), and in it lauryl methacrylate (LMA) was copolymerized with ethylene dimethacrylate (EDMA). In order to chemically anchor the polymer monolith to the tube wall, the inner wall surface was pretreated by the following two-step procedure. (1) 50% sulfuric acid was filled into the PEEK tubing and left to stand for 6h to generate sulfonate groups on the surface. (2) After washing with Milli-Q water, the sulfonated PEEK surface was brought into contact with 1M glycidyl methacrylate in dichloromethane (or acetone) at 40°C for 4h to introduce methacryloyl groups via the reaction of sulfonate groups and epoxy groups. Mechanical strength and column efficiency of the resulting monoliths were evaluated through the separation of a series of alkylbenzenes in acetonitrile–water (50:50, v/v) eluent over the flow rate range of 50–750μL/min (corresponding to 1.7–25.5mm/s). The poly(LMA-co-EDMA) monolith provided acceptable column efficiency of 2000 theoretical plates/10cm (HETP value of 50μm) for amylbenzene (separation factor k =40) and low flow resistance of 0.5MPa/10cm at a normal flow rate of 50μL/min. The methacryloylated PEEK tubing tightly held the monolith, and the monolithic column exhibited good pressure resistance up to 15MPa, allowing rapid separation at a 15–20 fold higher flow rate than normal.
Source:Journal of Chromatography A, Volume 1242
Shin Shu, Hiroharu Kobayashi, Masaki Okubo, Akhmad Sabarudin, Michio Butsugan, Tomonari Umemura
In this paper, we describe a method for the preparation of easy-to-use reversed-phase monolithic microbore columns. Polyetheretherketone (PEEK) tubing with an outer diameter of 1/16″ and an inner diameter of 1.0mm was used as a column housing (empty column), and in it lauryl methacrylate (LMA) was copolymerized with ethylene dimethacrylate (EDMA). In order to chemically anchor the polymer monolith to the tube wall, the inner wall surface was pretreated by the following two-step procedure. (1) 50% sulfuric acid was filled into the PEEK tubing and left to stand for 6h to generate sulfonate groups on the surface. (2) After washing with Milli-Q water, the sulfonated PEEK surface was brought into contact with 1M glycidyl methacrylate in dichloromethane (or acetone) at 40°C for 4h to introduce methacryloyl groups via the reaction of sulfonate groups and epoxy groups. Mechanical strength and column efficiency of the resulting monoliths were evaluated through the separation of a series of alkylbenzenes in acetonitrile–water (50:50, v/v) eluent over the flow rate range of 50–750μL/min (corresponding to 1.7–25.5mm/s). The poly(LMA-co-EDMA) monolith provided acceptable column efficiency of 2000 theoretical plates/10cm (HETP value of 50μm) for amylbenzene (separation factor k =40) and low flow resistance of 0.5MPa/10cm at a normal flow rate of 50μL/min. The methacryloylated PEEK tubing tightly held the monolith, and the monolithic column exhibited good pressure resistance up to 15MPa, allowing rapid separation at a 15–20 fold higher flow rate than normal.
Highlights
► The inner wall of a PEEK tubing was sulfonated by exposing to sulfuric acid. ► The sulfonated PEEK tube wall was subsequently methacryloylated by exposing to GMA. ► Polymer monolith was in situ synthesized in the methacryloylated PEEK tubing. ► The monolithic column exhibited good pressure resistance.Comprehensive two-dimensional HepG2/cell membrane chromatography/monolithic column/time-of-flight mass spectrometry system for screening anti-tumor components from herbal medicines
23 May 2012,
08:19:04
Publication year:
2012
Source:Journal of Chromatography A, Volume 1242
Xiaofei Chen, Yan Cao, Diya Lv, Zhenyu Zhu, Junping Zhang, Yifeng Chai
Cell membrane chromatography (CMC) is a biological affinity chromatographic method using specific cell membrane as stationary phase. It has been proved to be a practical tool for investigating binding interactions between drugs and membrane receptors. In this study, a novel comprehensive two-dimensional (2D) chromatography approach was established for screening anti-tumor components from herbal medicines (HMs). HepG2/CMC model was first developed and applied as the first dimensional column. Using an automatic ten-port switching valve equipped with two sample loops, the fractions of the first-dimension were introduced in the second-dimension consists of a monolithic column and a time-of-flight mass spectrometry (TOFMS) with high resolving ability. Based on the stability, selectivity and suitability assays of the HepG2/CMC/monolithic column/TOFMS system, berberine (BBR) and tetrahydropalmatine (THP) from Cortex phellodendri amurensis, oxymatrine and matrine from Radix sophorae flavescentis were screened and identified as potential active components. The competitive displacement assay suggested that the four components could act on epidermal growth factor receptor region on the HepG2 cell membrane in similar manner of gefitinib. Furthermore, their inhibiting effects on cell proliferation in vitro were also confirmed and, BBR and THP showed concentration dependently inhibitory ability on HepG2 cell proliferation (p <0.05). The result demonstrated that the proposed comprehensive 2D HepG2/CMC/monolithic column/TOFMS system has the advantages of strong recognition and rapid analysis abilities for the total screening procedure, which will be selectable and practical in drug discovery from complex HM samples and can also be applied to other biochromatography models.
Source:Journal of Chromatography A, Volume 1242
Xiaofei Chen, Yan Cao, Diya Lv, Zhenyu Zhu, Junping Zhang, Yifeng Chai
Cell membrane chromatography (CMC) is a biological affinity chromatographic method using specific cell membrane as stationary phase. It has been proved to be a practical tool for investigating binding interactions between drugs and membrane receptors. In this study, a novel comprehensive two-dimensional (2D) chromatography approach was established for screening anti-tumor components from herbal medicines (HMs). HepG2/CMC model was first developed and applied as the first dimensional column. Using an automatic ten-port switching valve equipped with two sample loops, the fractions of the first-dimension were introduced in the second-dimension consists of a monolithic column and a time-of-flight mass spectrometry (TOFMS) with high resolving ability. Based on the stability, selectivity and suitability assays of the HepG2/CMC/monolithic column/TOFMS system, berberine (BBR) and tetrahydropalmatine (THP) from Cortex phellodendri amurensis, oxymatrine and matrine from Radix sophorae flavescentis were screened and identified as potential active components. The competitive displacement assay suggested that the four components could act on epidermal growth factor receptor region on the HepG2 cell membrane in similar manner of gefitinib. Furthermore, their inhibiting effects on cell proliferation in vitro were also confirmed and, BBR and THP showed concentration dependently inhibitory ability on HepG2 cell proliferation (p <0.05). The result demonstrated that the proposed comprehensive 2D HepG2/CMC/monolithic column/TOFMS system has the advantages of strong recognition and rapid analysis abilities for the total screening procedure, which will be selectable and practical in drug discovery from complex HM samples and can also be applied to other biochromatography models.
Highlights
► A new HepG2 cell membrane model was developed. ► Novel comprehensive two-dimensional chromatography approach was established and optimized for fast screening procedure. ► Four anti-tumor active components from herbal medicines were screened and reliably identified by TOFMS. ► EGFR was preliminary selected as the target membrane receptor via competitive displacement assays. ► BBR and THP revealed inhibiting effects on cell proliferation in vitro.Comparison of electrospray ionization, atmospheric pressure chemical ionization and atmospheric pressure photoionization for a lipidomic analysis of Leishmania donovani
23 May 2012,
08:19:04
Publication year:
2012
Source:Journal of Chromatography A, Volume 1242
Laurent Imbert, Mathieu Gaudin, Danielle Libong, David Touboul, Sonia Abreu, Philippe M. Loiseau, Olivier Laprévote, Pierre Chaminade
A comparison of electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) for the analysis of a wide range of lipids has been performed on standard mixtures and extracts of Leishmania donovani promastigotes resistant to Amphotericin B (AmB). Calibration model, precision, limits of detection and quantification (LOD and LOQ) were assessed for each source. APPI provided the highest signal, signal-to-noise (S/N), and sensitivity for non-polar and low-polarity lipids, while ESI and APCI gave better results for the most polar ones. The linear model was valid for all lipids, except for one class with APPI, six classes with ESI, and eleven classes with APCI. LODs ranged from 0.2 to 20μgmL−1 for ESI, from 0.1 to 10μgmL−1 for APCI, and from 0.02 to 9.5μgmL−1 for APPI. LOQs ranged from 0.2 to 61μgmL−1 for ESI, from 0.4 to 31μgmL−1 for APCI, and from 0.1 to 29μgmL−1 for APPI. Each source provided similar lipid composition and variations in a comparison of three different L. donovani samples: miltefosine-treated, miltefosine-resistant and treated miltefosine-resistant parasites. A treated miltefosine-resistant sample was finally analyzed with each ion source in order to verify that the same lipid molecular species are detected.
Source:Journal of Chromatography A, Volume 1242
Laurent Imbert, Mathieu Gaudin, Danielle Libong, David Touboul, Sonia Abreu, Philippe M. Loiseau, Olivier Laprévote, Pierre Chaminade
A comparison of electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) for the analysis of a wide range of lipids has been performed on standard mixtures and extracts of Leishmania donovani promastigotes resistant to Amphotericin B (AmB). Calibration model, precision, limits of detection and quantification (LOD and LOQ) were assessed for each source. APPI provided the highest signal, signal-to-noise (S/N), and sensitivity for non-polar and low-polarity lipids, while ESI and APCI gave better results for the most polar ones. The linear model was valid for all lipids, except for one class with APPI, six classes with ESI, and eleven classes with APCI. LODs ranged from 0.2 to 20μgmL−1 for ESI, from 0.1 to 10μgmL−1 for APCI, and from 0.02 to 9.5μgmL−1 for APPI. LOQs ranged from 0.2 to 61μgmL−1 for ESI, from 0.4 to 31μgmL−1 for APCI, and from 0.1 to 29μgmL−1 for APPI. Each source provided similar lipid composition and variations in a comparison of three different L. donovani samples: miltefosine-treated, miltefosine-resistant and treated miltefosine-resistant parasites. A treated miltefosine-resistant sample was finally analyzed with each ion source in order to verify that the same lipid molecular species are detected.
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