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papers from the latest issue:
A validated LC–MS/MS method for rapid determination of methotrexate in human saliva and its application to an excretion evaluation study
05 September 2013,
09:18:04
Publication date: 15 October
2013
Source:Journal of Chromatography B, Volume 937
Author(s): Igor Rodin , Arkady Braun , Andrey Stavrianidi , Oleg Shpigun
A sensitive and simple method for the methotrexate quantification was developed using aminopterin as internal standard. Methotrexate is an anticancer agent that is widely used in a variety of human cancers including primary central nervous system lymphoma. The compound was quantified by liquid-chromatography coupled to electrospray ionization (positive ion-mode) low-energy collision dissociation-tandem mass spectrometry. Quantitative detection was by multiple reaction monitoring of the transitions of the [M+H]+ ion of MTX to its common product ion at m/z 308.4 and of aminopterin at m/z 441.2→ m/z 294.0. The method demonstrated linearity over at least three orders of magnitude and had a detection limit of 1ng/ml for methotrexate. A run time of less than 8.0min for each sample made it possible to analyze a large number of human saliva samples per day. Application of this procedure was demonstrated to a saliva excretion study of methotrexate on the samples obtained after an intravenously administration of 1mg/kg/dose of methotrexate to six patients with acute lymphoblastic leukemia
Source:Journal of Chromatography B, Volume 937
Author(s): Igor Rodin , Arkady Braun , Andrey Stavrianidi , Oleg Shpigun
A sensitive and simple method for the methotrexate quantification was developed using aminopterin as internal standard. Methotrexate is an anticancer agent that is widely used in a variety of human cancers including primary central nervous system lymphoma. The compound was quantified by liquid-chromatography coupled to electrospray ionization (positive ion-mode) low-energy collision dissociation-tandem mass spectrometry. Quantitative detection was by multiple reaction monitoring of the transitions of the [M+H]+ ion of MTX to its common product ion at m/z 308.4 and of aminopterin at m/z 441.2→ m/z 294.0. The method demonstrated linearity over at least three orders of magnitude and had a detection limit of 1ng/ml for methotrexate. A run time of less than 8.0min for each sample made it possible to analyze a large number of human saliva samples per day. Application of this procedure was demonstrated to a saliva excretion study of methotrexate on the samples obtained after an intravenously administration of 1mg/kg/dose of methotrexate to six patients with acute lymphoblastic leukemia
Centrifugal partition extraction in the pH-zone-refining displacement mode: An efficient strategy for the screening and isolation of biologically active phenolic compounds
05 September 2013,
09:18:04
Publication date: 15 October
2013
Source:Journal of Chromatography B, Volume 937
Author(s): Mahmoud Hamzaoui , Jean-Hugues Renault , Romain Reynaud , Jane Hubert
Centrifugal partition extraction (CPE) was developed for the first time in the pH-zone-refining mode to fractionate a crude bark extract of the African tree Anogeissus leiocarpus Guill. & Perr. (Combretaceae). The fractionation process was performed at a flow rate of 20mL/min using a biphasic solvent system composed of methyl tert-butyl ether/acetonitrile/water (4:1:5, v/v/v) in the ascending mode. Sodium hydroxide (40mM) and trifluoroacetic acid (30mM) were used as retainer and displacer agents, respectively. In a single run of 67min, 3g of the initial crude extract were successfully separated into fractions selectively enriched in ionizable triterpenes, ellagic acid derivatives and flavonoids. The antioxidant potential of the initial crude extract, isolated compounds and fraction pools was also evaluated by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) stable free radical scavenging assay, providing an interesting view about the effect of the degree of substitution of ellagic acid derivatives on their radical scavenging activity. This study will demonstrate that centrifugal partition extraction used in the pH-zone-refining mode can be proposed as an efficient strategy for the rapid screening of natural phenolic compounds.
Source:Journal of Chromatography B, Volume 937
Author(s): Mahmoud Hamzaoui , Jean-Hugues Renault , Romain Reynaud , Jane Hubert
Centrifugal partition extraction (CPE) was developed for the first time in the pH-zone-refining mode to fractionate a crude bark extract of the African tree Anogeissus leiocarpus Guill. & Perr. (Combretaceae). The fractionation process was performed at a flow rate of 20mL/min using a biphasic solvent system composed of methyl tert-butyl ether/acetonitrile/water (4:1:5, v/v/v) in the ascending mode. Sodium hydroxide (40mM) and trifluoroacetic acid (30mM) were used as retainer and displacer agents, respectively. In a single run of 67min, 3g of the initial crude extract were successfully separated into fractions selectively enriched in ionizable triterpenes, ellagic acid derivatives and flavonoids. The antioxidant potential of the initial crude extract, isolated compounds and fraction pools was also evaluated by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) stable free radical scavenging assay, providing an interesting view about the effect of the degree of substitution of ellagic acid derivatives on their radical scavenging activity. This study will demonstrate that centrifugal partition extraction used in the pH-zone-refining mode can be proposed as an efficient strategy for the rapid screening of natural phenolic compounds.
Simultaneous quantification of Kirenol and ent-16β,17-dihydroxy-kauran-19-oic acid from Herba Siegesbeckiae in rat plasma by liquid chromatography–tandem mass spectrometry and its application to pharmacokinetic studies
05 September 2013,
09:18:04
Publication date: 15 October
2013
Source:Journal of Chromatography B, Volume 937
Author(s): Linan Huo , Zhen Jiang , Ming Lei , Xintao Wang , Xingjie Guo
A rapid and specific liquid chromatography-electrospray ionization–tandem mass spectrometry (LC-ESI–MS/MS) method was developed for the simultaneous determination of two active diterpenoids: Kirenol and ent-16β,17-dihydroxy-kauran-19-oic acid (DHKA) from Herba Siegesbeckiae in rat plasma using osthole as an internal standard (IS). Plasma sample pretreatment involved a one-step liquid–liquid extraction with ethyl acetate. Chromatographic separation was performed on a Waters Symmetry C18 column (2.1mm×100mm, 3.5μm) with isocratic elution using methanol–5mmol/L aqueous ammonium acetate (80:20, v/v) as the mobile phase at a flow rate of 0.2mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode under positive and negative electrospray ionization. The calibration curves were linear over the range of 50.0–25,000ng/mL for Kirenol, and 25.0–12,500ng/mL for DHKA. The extraction recoveries of the two analytes and the IS were all over 85%. The intra- and inter-day precision (relative standard deviation) values were less than 16.8% and the accuracy (relative error) ranged from −10.7 to 10.6% at four quality control levels. The validated method was successfully applied to a comparative pharmacokinetic study of the two diterpenoids in rat plasma after intragastric administration of Kirenol, DHKA and Herba Siegesbeckiae extract. The results showed that there were obvious differences between the pharmacokinetic behaviors after oral administration of Herba Siegesbeckiae extract compared with each of the substances alone.
Source:Journal of Chromatography B, Volume 937
Author(s): Linan Huo , Zhen Jiang , Ming Lei , Xintao Wang , Xingjie Guo
A rapid and specific liquid chromatography-electrospray ionization–tandem mass spectrometry (LC-ESI–MS/MS) method was developed for the simultaneous determination of two active diterpenoids: Kirenol and ent-16β,17-dihydroxy-kauran-19-oic acid (DHKA) from Herba Siegesbeckiae in rat plasma using osthole as an internal standard (IS). Plasma sample pretreatment involved a one-step liquid–liquid extraction with ethyl acetate. Chromatographic separation was performed on a Waters Symmetry C18 column (2.1mm×100mm, 3.5μm) with isocratic elution using methanol–5mmol/L aqueous ammonium acetate (80:20, v/v) as the mobile phase at a flow rate of 0.2mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode under positive and negative electrospray ionization. The calibration curves were linear over the range of 50.0–25,000ng/mL for Kirenol, and 25.0–12,500ng/mL for DHKA. The extraction recoveries of the two analytes and the IS were all over 85%. The intra- and inter-day precision (relative standard deviation) values were less than 16.8% and the accuracy (relative error) ranged from −10.7 to 10.6% at four quality control levels. The validated method was successfully applied to a comparative pharmacokinetic study of the two diterpenoids in rat plasma after intragastric administration of Kirenol, DHKA and Herba Siegesbeckiae extract. The results showed that there were obvious differences between the pharmacokinetic behaviors after oral administration of Herba Siegesbeckiae extract compared with each of the substances alone.
Rapid separation and quantitation of curcuminoids combining pseudo two-dimensional liquid flash chromatography and NMR spectroscopy
05 September 2013,
09:18:04
Publication date: 15 October
2013
Source:Journal of Chromatography B, Volume 937
Author(s): G.K. Jayaprakasha , G.A. Nagana Gowda , Sixto Marquez , Bhimanagouda S. Patil
Rapid separation, characterization and quantitation of curcuminoids are important owing to their numerous pharmacological properties including antimicrobial, antiviral, antifungal, anticancer, and anti-inflammatory activities. In the present study, pseudo two-dimensional liquid flash chromatography was used for the separation of four curcuminoids (curcumin, demethoxy curcumin, bisdemethoxy curcumin and dihydro bisdemethoxy curcumin) for the first time. Silica and diol columns were used for separation of curcuminoids using gradient mobile phase. The separated peaks were monitored at 244, 360nm to obtain four compounds. The purity of compounds were determined by rapid quantitative 1H NMR (qNMR) using 3-(trimethylsilyl) propionic-(2,2,3,3-d4) acid sodium salt (TSP-d4) (0.012%) in D2O. These results were compared with those obtained by HPLC method. The purity of isolated curcuminoids using pseudo 2D chromatography was found to be in the range of 92.4–95.45%. The structures of these compounds were characterized unambiguously using 13C (APT) NMR spectra. The developed pseudo 2D separation technique has the advantage of simplified automation with shorter run time compared to conventional separation techniques. The method that combines rapid pseudo 2D separation and simple quantitation using qNMR reported herein can be of wide utility for routine analysis of curcuminoids in complex mixtures.
Source:Journal of Chromatography B, Volume 937
Author(s): G.K. Jayaprakasha , G.A. Nagana Gowda , Sixto Marquez , Bhimanagouda S. Patil
Rapid separation, characterization and quantitation of curcuminoids are important owing to their numerous pharmacological properties including antimicrobial, antiviral, antifungal, anticancer, and anti-inflammatory activities. In the present study, pseudo two-dimensional liquid flash chromatography was used for the separation of four curcuminoids (curcumin, demethoxy curcumin, bisdemethoxy curcumin and dihydro bisdemethoxy curcumin) for the first time. Silica and diol columns were used for separation of curcuminoids using gradient mobile phase. The separated peaks were monitored at 244, 360nm to obtain four compounds. The purity of compounds were determined by rapid quantitative 1H NMR (qNMR) using 3-(trimethylsilyl) propionic-(2,2,3,3-d4) acid sodium salt (TSP-d4) (0.012%) in D2O. These results were compared with those obtained by HPLC method. The purity of isolated curcuminoids using pseudo 2D chromatography was found to be in the range of 92.4–95.45%. The structures of these compounds were characterized unambiguously using 13C (APT) NMR spectra. The developed pseudo 2D separation technique has the advantage of simplified automation with shorter run time compared to conventional separation techniques. The method that combines rapid pseudo 2D separation and simple quantitation using qNMR reported herein can be of wide utility for routine analysis of curcuminoids in complex mixtures.
A validated assay for the simultaneous quantification of six tyrosine kinase inhibitors and two active metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry
05 September 2013,
09:18:04
Publication date: 15 October
2013
Source:Journal of Chromatography B, Volume 937
Author(s): Nielka P. van Erp , Djoeke de Wit , Henk-Jan Guchelaar , Hans Gelderblom , Trees J. Hessing , Jan den Hartigh
A sensitive, sophisticated and practical bioanalytical assay for the simultaneous determination of six tyrosine kinase inhibitors (imatinib, sunitinib, nilotinib, dasatinib, pazopanib, regorafenib) and two active metabolites (N-desmethyl imatinib and N-desethyl sunitinib) was developed and validated. For the quantitative assay, a mixture of three stable isotopes as internal standards was added to human serum, standards and controls. Thereafter, samples were pre-treated using protein precipitation with methanol. The supernatant was diluted with water and injected into an ultra pressure liquid chromatographic system with an Acquity TQ tandem mass spectrometry detector. The compounds were separated on an Acquity BEH C18 analytical column (100mm×2.1mm ID, 1.7μm particle size) and eluted with a linear gradient system. The ions were detected in the multiple reaction monitoring mode. The lower limit of quantification and the linearity of all compounds generously met with the concentrations that are to be expected in clinical practice. The developed bioanalytical assay can be used for guiding TKI therapy in daily clinical practice as well as for investigator-initiated research.
Source:Journal of Chromatography B, Volume 937
Author(s): Nielka P. van Erp , Djoeke de Wit , Henk-Jan Guchelaar , Hans Gelderblom , Trees J. Hessing , Jan den Hartigh
A sensitive, sophisticated and practical bioanalytical assay for the simultaneous determination of six tyrosine kinase inhibitors (imatinib, sunitinib, nilotinib, dasatinib, pazopanib, regorafenib) and two active metabolites (N-desmethyl imatinib and N-desethyl sunitinib) was developed and validated. For the quantitative assay, a mixture of three stable isotopes as internal standards was added to human serum, standards and controls. Thereafter, samples were pre-treated using protein precipitation with methanol. The supernatant was diluted with water and injected into an ultra pressure liquid chromatographic system with an Acquity TQ tandem mass spectrometry detector. The compounds were separated on an Acquity BEH C18 analytical column (100mm×2.1mm ID, 1.7μm particle size) and eluted with a linear gradient system. The ions were detected in the multiple reaction monitoring mode. The lower limit of quantification and the linearity of all compounds generously met with the concentrations that are to be expected in clinical practice. The developed bioanalytical assay can be used for guiding TKI therapy in daily clinical practice as well as for investigator-initiated research.
2-Methyltetrahydrofuran and cyclopentylmethylether: Two green solvents for efficient purification of membrane proteins like FhuA
05 September 2013,
09:18:04
Publication date: 15 October
2013
Source:Journal of Chromatography B, Volume 937
Author(s): Stefanie-Joana Tenne , Julia Kinzel , Marcus Arlt , Fabrizio Sibilla , Marco Bocola , Ulrich Schwaneberg
β-Barrel shaped membrane proteins are attractive hosts for hybrid catalysts in which reactions are controlled through space. Production and extraction of β-barrel shaped membrane proteins in gram scale is challenging due to their hydrophobicity. Solvent mixtures such as chloroform/methanol (CM) are widely used for membrane protein extraction but toxicity and mutagenicity were reported in several cases. 2-Methyltetrahydrofuran (2-MeTHF) and cyclopentylmethylether (CPME) are two green (reduction of solvent-related environmental damage in chemical production) and potentially efficient solvents for membrane protein purification. On the example of the ferric hydroxamate uptake protein component A (FhuA) a 4-Step method was developed to provide gram amounts of highly purified FhuA: cell disruption (Step 1), removal of membrane protein impurities with n-octyl-poly-oxyethylene (oPOE) (Step 2), dissolution of membranes and FhuA precipitation (Step 3), and refolding using urea and dialysis with polyethylene-polyethyleneglycol (PE-PEG; Step 4) resulted in high FhuA purity (95% 2-MeTHF, 80% CPME; 70mg FhuA per liter fermenter broth). Structural integrity of FhuA protein was confirmed by circular dichroism (CD) and a translocation functionality assay.
Source:Journal of Chromatography B, Volume 937
Author(s): Stefanie-Joana Tenne , Julia Kinzel , Marcus Arlt , Fabrizio Sibilla , Marco Bocola , Ulrich Schwaneberg
β-Barrel shaped membrane proteins are attractive hosts for hybrid catalysts in which reactions are controlled through space. Production and extraction of β-barrel shaped membrane proteins in gram scale is challenging due to their hydrophobicity. Solvent mixtures such as chloroform/methanol (CM) are widely used for membrane protein extraction but toxicity and mutagenicity were reported in several cases. 2-Methyltetrahydrofuran (2-MeTHF) and cyclopentylmethylether (CPME) are two green (reduction of solvent-related environmental damage in chemical production) and potentially efficient solvents for membrane protein purification. On the example of the ferric hydroxamate uptake protein component A (FhuA) a 4-Step method was developed to provide gram amounts of highly purified FhuA: cell disruption (Step 1), removal of membrane protein impurities with n-octyl-poly-oxyethylene (oPOE) (Step 2), dissolution of membranes and FhuA precipitation (Step 3), and refolding using urea and dialysis with polyethylene-polyethyleneglycol (PE-PEG; Step 4) resulted in high FhuA purity (95% 2-MeTHF, 80% CPME; 70mg FhuA per liter fermenter broth). Structural integrity of FhuA protein was confirmed by circular dichroism (CD) and a translocation functionality assay.
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