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Determination of deltonin in rat plasma by using HPLC–MS/MS and the application of this method in pharmacokinetic studies
05 June 2013,
09:36:17
Publication date: 15 July
2013
Source:Journal of Chromatography B, Volume 931
Author(s): Dan Du , Bo Gao , Guang Xin , Aimin Sun , Baozhan Huang , Rui Zhang , Zhihua Xing , Qianming Chen , Yang He , Wen Huang
Deltonin is a naturally occurring spirostanol glycoside from Dioscorea zingiberensis C.H. Wright, which is used in traditional Chinese medicine. It exerts strong cytotoxic effect on C26 cells, inhibits C26 derived-tumor growth, and prolongs the survival of tumor-bearing mice after its oral administration, indicating its potential for use as an anti-tumor drug. To investigate the pharmacokinetic profiles of deltonin, a rapid, sensitive, and simplified high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) assay was developed and validated for the determination of deltonin in rat plasma. After acetonitrile-mediated plasma protein precipitation, chromatographic separation of deltonin was achieved using a reversed phase Hypersil Gold column (150mm×2.1mm, 5μm), with gradient elution using 0.1% formic acid and acetonitrile. Thereafter, deltonin was quantified using MS/MS with electrospray ionization (ESI) in positive multiple reaction monitoring (MRM) mode. The flow rate of the mobile phase was 200μL/min, and the retention time was 9.03min for deltonin and 6.31min for the internal standard (IS: 20(S)-ginsenoside Rb1). The linear range of the calibration curve was 2–5000ng/mL (r 2 >0.99), and the limit of detection (LOD) was 0.46ng/mL. The intra- and inter-day accuracies ranged from −2.8% to 11.1% and precisions (RSD) were within 13.1%. Deltonin was found to be stable under short-term temperature conditions, post-preparative temperature conditions, and after 3 freeze–thaw cycles conditions. The validated method was successfully applied to a pharmacokinetic study in rats after oral administration of deltonin (50 and 100mg/kg). The pharmacokinetics is characterized by high apparent clearance (CL/F) and apparent volume of distribution (Vd/F).
Source:Journal of Chromatography B, Volume 931
Author(s): Dan Du , Bo Gao , Guang Xin , Aimin Sun , Baozhan Huang , Rui Zhang , Zhihua Xing , Qianming Chen , Yang He , Wen Huang
Deltonin is a naturally occurring spirostanol glycoside from Dioscorea zingiberensis C.H. Wright, which is used in traditional Chinese medicine. It exerts strong cytotoxic effect on C26 cells, inhibits C26 derived-tumor growth, and prolongs the survival of tumor-bearing mice after its oral administration, indicating its potential for use as an anti-tumor drug. To investigate the pharmacokinetic profiles of deltonin, a rapid, sensitive, and simplified high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) assay was developed and validated for the determination of deltonin in rat plasma. After acetonitrile-mediated plasma protein precipitation, chromatographic separation of deltonin was achieved using a reversed phase Hypersil Gold column (150mm×2.1mm, 5μm), with gradient elution using 0.1% formic acid and acetonitrile. Thereafter, deltonin was quantified using MS/MS with electrospray ionization (ESI) in positive multiple reaction monitoring (MRM) mode. The flow rate of the mobile phase was 200μL/min, and the retention time was 9.03min for deltonin and 6.31min for the internal standard (IS: 20(S)-ginsenoside Rb1). The linear range of the calibration curve was 2–5000ng/mL (r 2 >0.99), and the limit of detection (LOD) was 0.46ng/mL. The intra- and inter-day accuracies ranged from −2.8% to 11.1% and precisions (RSD) were within 13.1%. Deltonin was found to be stable under short-term temperature conditions, post-preparative temperature conditions, and after 3 freeze–thaw cycles conditions. The validated method was successfully applied to a pharmacokinetic study in rats after oral administration of deltonin (50 and 100mg/kg). The pharmacokinetics is characterized by high apparent clearance (CL/F) and apparent volume of distribution (Vd/F).
Pharmacokinetics of ganoderic acid D and its main metabolite by liquid chromatography–tandem mass spectrometry
05 June 2013,
09:36:17
Publication date: 1 July
2013
Source:Journal of Chromatography B, Volume 930
Author(s): Chun-Ru Cheng , Min Yang , Shu-Hong Guan , Xiao-Hui Wu , Xiao-Yan Pang , Yang Wang , Yi Yang , Jie Ding , De-An Guo
The present study aims to investigate the pharmacokinetics of ganoderic acid D (GD), a representative active triterpenoid from Ganoderma lucidum. A sensitive and selective liquid chromatography–tandem mass spectrometry method was developed for the simultaneous determination of the concentrations of GD and its main metabolite (ganoderic acid B) in rat plasma. Following protein precipitation, the analytes were separated on a reversed-phase C18 column. Acetonitrile–water–acetic acid (40:60:0.01) was used at a flow-rate of 0.2ml/min. A triple quadrupole mass spectrometer equipped with an electrospray ionization source was used as the detector and was operated in the negative ion mode. Multiple reaction monitoring using the characteristic transitions was performed to quantify the analytes. The method had a lower limit of quantification of 8.19ng/ml for GD, and 8.59ng/ml for ganoderic acid B (GB). The calibration curves were demonstrated to be linear over the concentration range of 8.19–4096ng/ml and 8.59–2149ng/ml, respectively. Variations within- and between-batch were less than 6.4% and 4.6%, respectively. The extraction recovery rates ranged from 98.8 to 105.2% and 100.7 to 113.6%, respectively. The validated method was successfully applied to the quantification of GD and GB concentrations in rat plasma after oral administration (or intravenous administration) of GD preparations at a dose of 15mg/kg. The data showed that the absolute bioavailability increased from 22% to 70% after the GD suspension was changed to GD loaded solid lipid nanoparticles. In the meantime, the C max increased from 107.2 to 1555.6ng/ml; the t max changed from 2.0h to 0.3h. These results are very helpful in the further studies.
Source:Journal of Chromatography B, Volume 930
Author(s): Chun-Ru Cheng , Min Yang , Shu-Hong Guan , Xiao-Hui Wu , Xiao-Yan Pang , Yang Wang , Yi Yang , Jie Ding , De-An Guo
The present study aims to investigate the pharmacokinetics of ganoderic acid D (GD), a representative active triterpenoid from Ganoderma lucidum. A sensitive and selective liquid chromatography–tandem mass spectrometry method was developed for the simultaneous determination of the concentrations of GD and its main metabolite (ganoderic acid B) in rat plasma. Following protein precipitation, the analytes were separated on a reversed-phase C18 column. Acetonitrile–water–acetic acid (40:60:0.01) was used at a flow-rate of 0.2ml/min. A triple quadrupole mass spectrometer equipped with an electrospray ionization source was used as the detector and was operated in the negative ion mode. Multiple reaction monitoring using the characteristic transitions was performed to quantify the analytes. The method had a lower limit of quantification of 8.19ng/ml for GD, and 8.59ng/ml for ganoderic acid B (GB). The calibration curves were demonstrated to be linear over the concentration range of 8.19–4096ng/ml and 8.59–2149ng/ml, respectively. Variations within- and between-batch were less than 6.4% and 4.6%, respectively. The extraction recovery rates ranged from 98.8 to 105.2% and 100.7 to 113.6%, respectively. The validated method was successfully applied to the quantification of GD and GB concentrations in rat plasma after oral administration (or intravenous administration) of GD preparations at a dose of 15mg/kg. The data showed that the absolute bioavailability increased from 22% to 70% after the GD suspension was changed to GD loaded solid lipid nanoparticles. In the meantime, the C max increased from 107.2 to 1555.6ng/ml; the t max changed from 2.0h to 0.3h. These results are very helpful in the further studies.
Quantitative analysis of autoinducing peptide I (AIP-I) from Staphylococcus aureus cultures using ultrahigh performance liquid chromatography–high resolving power mass spectrometry
05 June 2013,
09:36:17
Publication date: 1 July
2013
Source:Journal of Chromatography B, Volume 930
Author(s): Hiyas A. Junio , Daniel A. Todd , Keivan A. Ettefagh , Brandie M. Ehrmann , Jeffrey S. Kavanaugh , Alexander R. Horswill , Nadja B. Cech
Staphylococcus aureus infections acquired in hospitals now cause more deaths per annum in the US than does HIV/AIDS. Perhaps even more alarming is the rise in community associated methicillin-resistant S. aureus (CA-MRSA) infections, which have spread out of hospital settings and are infecting otherwise healthy individuals. The mechanism of enhanced pathogenesis in CA-MRSA remains unclear, but it has been postulated that high activity in the agr quorum-sensing system could be a contributing factor. The purpose of this study was to develop a quantitative method for analysis of autoinducing peptide I (AIP-I), the activating signal for the agr system in S. aureus. An effective method was developed using ultrahigh performance liquid chromatography (UHPLC) coupled to electrospray ionization mass spectrometry with an LTQ Orbitrap mass spectrometer. Relying on the exceptional resolving power and mass accuracy of this instrument configuration, it was possible to quantify AIP-I directly from the complex growth media of S. aureus cultures with a limit of detection (LOD) of 0.25μM and a linear dynamic range of 2.6 to 63μM. The method was then employed to monitor time-dependent production of AIP-I by S. aureus cultures, and it was observed that AIP-I production reached a maximum and leveled off after approximately 16h. Finally, it was determined that virulence of S. aureus was correlated with AIP-I production in some (but not all) strains analyzed.
Source:Journal of Chromatography B, Volume 930
Author(s): Hiyas A. Junio , Daniel A. Todd , Keivan A. Ettefagh , Brandie M. Ehrmann , Jeffrey S. Kavanaugh , Alexander R. Horswill , Nadja B. Cech
Staphylococcus aureus infections acquired in hospitals now cause more deaths per annum in the US than does HIV/AIDS. Perhaps even more alarming is the rise in community associated methicillin-resistant S. aureus (CA-MRSA) infections, which have spread out of hospital settings and are infecting otherwise healthy individuals. The mechanism of enhanced pathogenesis in CA-MRSA remains unclear, but it has been postulated that high activity in the agr quorum-sensing system could be a contributing factor. The purpose of this study was to develop a quantitative method for analysis of autoinducing peptide I (AIP-I), the activating signal for the agr system in S. aureus. An effective method was developed using ultrahigh performance liquid chromatography (UHPLC) coupled to electrospray ionization mass spectrometry with an LTQ Orbitrap mass spectrometer. Relying on the exceptional resolving power and mass accuracy of this instrument configuration, it was possible to quantify AIP-I directly from the complex growth media of S. aureus cultures with a limit of detection (LOD) of 0.25μM and a linear dynamic range of 2.6 to 63μM. The method was then employed to monitor time-dependent production of AIP-I by S. aureus cultures, and it was observed that AIP-I production reached a maximum and leveled off after approximately 16h. Finally, it was determined that virulence of S. aureus was correlated with AIP-I production in some (but not all) strains analyzed.
Para-aminobenzamidine linked regenerated cellulose membranes for plasminogen activator purification: Effect of spacer arm length and ligand density
05 June 2013,
09:36:17
Publication date: 1 July
2013
Source:Journal of Chromatography B, Volume 930
Author(s): Ezio Fasoli , Yiaslin Ruiz Reyes , Osiris Martinez Guzman , Alexandra Rosado , Vivian Rodriguez Cruz , Amaris Borges , Edmarie Martinez , Vibha Bansal
Despite membrane-based separations offering superior alternative to packed bed chromatographic processes, there has been a substantial lacuna in their actual application to separation processes. One of the major reasons behind this is the lack of availability of appropriately modified or end-group modifiable membranes. In this paper, an affinity membrane was developed using a commercially available serine protease inhibitor, para-aminobenzamidine (pABA). The membrane modification was optimized for protein binding capacity by varying: (i) the length of the spacer arm (SA; 5-atoms, 7-atoms, and 14-atoms) linking the ligand to membrane surface; (ii) the affinity ligand (pABA) density on membrane surface (5–25nmol/cm2). Resulting membranes were tested for their ability to bind plasminogen activators (PAs) from mono- and multi-component systems in batch mode. The membrane containing pABA linked through 7-atoms SA but similar ligand density as in the case of 5- or 14-atoms long SA was found to bind up to 1.6-times higher amounts of PA per nmoles of immobilized ligand from conditioned HeLa cell culture media. However, membranes with similar ligand densities but different lengths of SA, showed comparable binding capacities in mono-component system. In addition, the length of SA did not affect the selectivity of the ligand for PA. A clear inverse linear correlation was observed between ligand density and binding capacity until the point of PA binding optima was reached (11±1.0nmol/cm2) in mono- and multi-component systems for 7- as well as 14-atoms SA. Up to 200-fold purification was achieved in a single step separation of PA from HeLa conditioned media using these affinity membranes. The issues of ligand leaching and reuse of the membranes were also investigated. An extensive regeneration procedure allowed the preservation of approximately 95% of the PA binding capacity of the membranes even after five cycles of use.
Source:Journal of Chromatography B, Volume 930
Author(s): Ezio Fasoli , Yiaslin Ruiz Reyes , Osiris Martinez Guzman , Alexandra Rosado , Vivian Rodriguez Cruz , Amaris Borges , Edmarie Martinez , Vibha Bansal
Despite membrane-based separations offering superior alternative to packed bed chromatographic processes, there has been a substantial lacuna in their actual application to separation processes. One of the major reasons behind this is the lack of availability of appropriately modified or end-group modifiable membranes. In this paper, an affinity membrane was developed using a commercially available serine protease inhibitor, para-aminobenzamidine (pABA). The membrane modification was optimized for protein binding capacity by varying: (i) the length of the spacer arm (SA; 5-atoms, 7-atoms, and 14-atoms) linking the ligand to membrane surface; (ii) the affinity ligand (pABA) density on membrane surface (5–25nmol/cm2). Resulting membranes were tested for their ability to bind plasminogen activators (PAs) from mono- and multi-component systems in batch mode. The membrane containing pABA linked through 7-atoms SA but similar ligand density as in the case of 5- or 14-atoms long SA was found to bind up to 1.6-times higher amounts of PA per nmoles of immobilized ligand from conditioned HeLa cell culture media. However, membranes with similar ligand densities but different lengths of SA, showed comparable binding capacities in mono-component system. In addition, the length of SA did not affect the selectivity of the ligand for PA. A clear inverse linear correlation was observed between ligand density and binding capacity until the point of PA binding optima was reached (11±1.0nmol/cm2) in mono- and multi-component systems for 7- as well as 14-atoms SA. Up to 200-fold purification was achieved in a single step separation of PA from HeLa conditioned media using these affinity membranes. The issues of ligand leaching and reuse of the membranes were also investigated. An extensive regeneration procedure allowed the preservation of approximately 95% of the PA binding capacity of the membranes even after five cycles of use.
Rapid multi-method for the determination of growth promoters in bovine milk by liquid chromatography–tandem mass spectrometry
05 June 2013,
09:36:17
Publication date: 1 July
2013
Source:Journal of Chromatography B, Volume 930
Author(s): George Kaklamanos , Georgios Theodoridis
A rapid multi-method was developed for the determination of 21 growth promoters from different classes, including gestagens, corticosteroids, RALs, stilbenes, steroids in bovine milk by liquid chromatography–tandem mass spectrometry (LC–MS/MS). All compounds were eluted from the analytical column in less than 8.5min and were subsequently analyzed with atmospheric pressure chemical ionization (APCI) using both positive and negative mode. Sample preparation included extraction of the compounds with acetonitrile and purification with solid-phase extraction (SPE). The method was validated according to Commission Decision 2002/657/EC, at a validation level of 1ng/ml. The specificity, accuracy, precision, decision limit (CCα) and the detection capability (CCβ) were satisfactory evaluated. The recoveries ranged from 80.7% to 118.8% and reproducibility represented as coefficient of variance (CV) was from 1.8% to 13.0%. The CCα and CCβ values were in the ranges 0.06–0.10ng/ml and 0.11–0.17ng/ml, respectively. The developed method was applied in real samples proving its rapidness and sensitivity for the determination of the 21 growth promoters.
Source:Journal of Chromatography B, Volume 930
Author(s): George Kaklamanos , Georgios Theodoridis
A rapid multi-method was developed for the determination of 21 growth promoters from different classes, including gestagens, corticosteroids, RALs, stilbenes, steroids in bovine milk by liquid chromatography–tandem mass spectrometry (LC–MS/MS). All compounds were eluted from the analytical column in less than 8.5min and were subsequently analyzed with atmospheric pressure chemical ionization (APCI) using both positive and negative mode. Sample preparation included extraction of the compounds with acetonitrile and purification with solid-phase extraction (SPE). The method was validated according to Commission Decision 2002/657/EC, at a validation level of 1ng/ml. The specificity, accuracy, precision, decision limit (CCα) and the detection capability (CCβ) were satisfactory evaluated. The recoveries ranged from 80.7% to 118.8% and reproducibility represented as coefficient of variance (CV) was from 1.8% to 13.0%. The CCα and CCβ values were in the ranges 0.06–0.10ng/ml and 0.11–0.17ng/ml, respectively. The developed method was applied in real samples proving its rapidness and sensitivity for the determination of the 21 growth promoters.
Detection and quantification of quinolone signalling molecule: A third quorum sensing molecule of Pseudomonas aeruginosa by high performance-thin layer chromatography
05 June 2013,
09:36:17
Publication date: 1 July
2013
Source:Journal of Chromatography B, Volume 930
Author(s): Anju Bala , Ravi Kumar Gupta , Sanjay Chhibber , Kusum Harjai
Sophisticated network of quorum sensing involves the production of chemical signals which regulate the combined expression of virulence genes and biofilm formation in Pseudomonas aeruginosa. Two well-characterized acyl homoserine lactone based las and rhl systems together with alkyl quinolone based Pseudomonas quinolone signalling (PQS) are fundamental components of this network. Third signalling molecule, 2-heptyl-3-hydroxy-4-quinolone (PQS) is of paramount importance because of its interconnecting role in quorum sensing hierarchy in P. aeruginosa. Accurate detection of PQS molecule is very important to understand the involvement of this system in infection process of P. aeruginosa. In this study, high performance-thin layer chromatography (HP-TLC) method was developed for detection as well as quantification of PQS signal molecules in P. aeruginosa, which combines conventional method like TLC with sophisticated instrumentation. This method was validated using parameters like linearity, accuracy, precision, reproducibility and sensitivity. Intra- and inter-day accuracy and precision values were determined which were found to be within acceptable level and hence showed reproducibility. Measurement of PQS in the range of 0.01nmol indicated excellent sensitivity of this approach for quantifying PQS molecule. Automated sampling, rapid and simultaneous analysis of large number of samples and minimal errors make this method more suitable for analysis of PQS signalling molecules. Production of PQS was found to be strain dependent since variation in amount of PQS was observed among different P. aeruginosa isolates. Further, PQS production was also dependent on growth phase of P. aeruginosa with maximum production in late stationary phase.
Source:Journal of Chromatography B, Volume 930
Author(s): Anju Bala , Ravi Kumar Gupta , Sanjay Chhibber , Kusum Harjai
Sophisticated network of quorum sensing involves the production of chemical signals which regulate the combined expression of virulence genes and biofilm formation in Pseudomonas aeruginosa. Two well-characterized acyl homoserine lactone based las and rhl systems together with alkyl quinolone based Pseudomonas quinolone signalling (PQS) are fundamental components of this network. Third signalling molecule, 2-heptyl-3-hydroxy-4-quinolone (PQS) is of paramount importance because of its interconnecting role in quorum sensing hierarchy in P. aeruginosa. Accurate detection of PQS molecule is very important to understand the involvement of this system in infection process of P. aeruginosa. In this study, high performance-thin layer chromatography (HP-TLC) method was developed for detection as well as quantification of PQS signal molecules in P. aeruginosa, which combines conventional method like TLC with sophisticated instrumentation. This method was validated using parameters like linearity, accuracy, precision, reproducibility and sensitivity. Intra- and inter-day accuracy and precision values were determined which were found to be within acceptable level and hence showed reproducibility. Measurement of PQS in the range of 0.01nmol indicated excellent sensitivity of this approach for quantifying PQS molecule. Automated sampling, rapid and simultaneous analysis of large number of samples and minimal errors make this method more suitable for analysis of PQS signalling molecules. Production of PQS was found to be strain dependent since variation in amount of PQS was observed among different P. aeruginosa isolates. Further, PQS production was also dependent on growth phase of P. aeruginosa with maximum production in late stationary phase.
An improved GC–MS method in determining glycerol in different types of biological samples
05 June 2013,
09:36:17
Publication date: 1 July
2013
Source:Journal of Chromatography B, Volume 930
Author(s): Yixiao Shen , Zhimin Xu
Glycerol is an important compound participating in the lipid metabolism and energy conversion of body. Its level, especially in the blood circulation, has been considered as an index in assessing the triglycerides level, fat mobilization and potential risk of hyperlipidemia. In this gas chromatography mass spectrometry (GC–MS) method, 1,2,3-butanetriol was selected as an internal standard instead of isotope labeled glycerol. The internal standard and sample were derivatized by trimethylsilyl imidazole. The glycerol and internal standard derivatives in the sample were measured by a selected ion monitor mode of GC–MS. The sensitivity and repeatability of this method were examined by analyzing glycerol in eleven different types of biological tissue and fluid samples. The glycerol level in the measured mouse plasma sample was at 11.71±0.48μg/mL, while it was in a range of 0.15±0.01 (brain) to 0.39±0.02μg/mg (liver) in the tissue samples. It was 27.06±0.12 and 1.60±0.04μg/mL in the tested human blood and urine samples, respectively. Also, the glycerol recoveries of all samples were higher than 80% and over 90% for the fluid samples, especially. With the satisfactory repeatability and recovery and non-isotope internal standard, the GC–MS method could be a reliable technique in monitoring the glycerol status of biological samples regardless of whether they are from a study in which isotope labeled glycerol or other stable isotope materials were involved.
Source:Journal of Chromatography B, Volume 930
Author(s): Yixiao Shen , Zhimin Xu
Glycerol is an important compound participating in the lipid metabolism and energy conversion of body. Its level, especially in the blood circulation, has been considered as an index in assessing the triglycerides level, fat mobilization and potential risk of hyperlipidemia. In this gas chromatography mass spectrometry (GC–MS) method, 1,2,3-butanetriol was selected as an internal standard instead of isotope labeled glycerol. The internal standard and sample were derivatized by trimethylsilyl imidazole. The glycerol and internal standard derivatives in the sample were measured by a selected ion monitor mode of GC–MS. The sensitivity and repeatability of this method were examined by analyzing glycerol in eleven different types of biological tissue and fluid samples. The glycerol level in the measured mouse plasma sample was at 11.71±0.48μg/mL, while it was in a range of 0.15±0.01 (brain) to 0.39±0.02μg/mg (liver) in the tissue samples. It was 27.06±0.12 and 1.60±0.04μg/mL in the tested human blood and urine samples, respectively. Also, the glycerol recoveries of all samples were higher than 80% and over 90% for the fluid samples, especially. With the satisfactory repeatability and recovery and non-isotope internal standard, the GC–MS method could be a reliable technique in monitoring the glycerol status of biological samples regardless of whether they are from a study in which isotope labeled glycerol or other stable isotope materials were involved.
Determination of nitrite and nitrate in cerebrospinal fluid by microchip electrophoresis with microsolid phase extraction pre-treatment
05 June 2013,
09:36:17
Publication date: 1 July
2013
Source:Journal of Chromatography B, Volume 930
Author(s): Peter Troška , Richard Chudoba , Ladislav Danč , Róbert Bodor , Michal Horčičiak , Eva Tesařová , Marián Masár
A new method for the determination of nitrite and nitrate, indicators of various neurological diseases (meningitis, multiple sclerosis, Parkinson's disease) in cerebrospinal fluid (CSF) on an electrophoresis chip was developed. An on-line combination of isotachophoresis (ITP) with capillary electrophoresis (CE) on a poly(methylmethacrylate) chip assembled with coupled separation channels (CC) and contact conductivity detectors was employed. ITP separations performed at low pH (3.6) in the first separation channel enabled a highly selective transfer of the analytes to the second CE stage working under micellar conditions implemented by zwitterionic surfactant, 3-(N,N-dimethyldodecylammonio)-propanesulfonate. The proposed method achieved low limits of detection varied from 0.2 to 0.4μgL−1 when the sample volume injected onto the chip (9.9μl) was almost the same as the volume of both separation channels. Preferable working conditions on the CC chip (suppressed hydrodynamic and electroosmotic flow) contributed for reproducible migration velocities (intra-day reproducibility up to 2.1% RSD) and determinations of trace concentrations of nitrite and nitrate (intra-day precision up to 3.0% RSD). Huge amount of chloride present in CSF (approx. 4.5gL−1) was removed from analyzed CSF samples by microsolid phase extraction performed on silver-form resin prior to the ITP-CE analysis. Developed method provided fast (approx. 20min total analysis time) and reliable determinations of trace nitrite and nitrate and could be fully integrated into the analysis of CSF samples.
Source:Journal of Chromatography B, Volume 930
Author(s): Peter Troška , Richard Chudoba , Ladislav Danč , Róbert Bodor , Michal Horčičiak , Eva Tesařová , Marián Masár
A new method for the determination of nitrite and nitrate, indicators of various neurological diseases (meningitis, multiple sclerosis, Parkinson's disease) in cerebrospinal fluid (CSF) on an electrophoresis chip was developed. An on-line combination of isotachophoresis (ITP) with capillary electrophoresis (CE) on a poly(methylmethacrylate) chip assembled with coupled separation channels (CC) and contact conductivity detectors was employed. ITP separations performed at low pH (3.6) in the first separation channel enabled a highly selective transfer of the analytes to the second CE stage working under micellar conditions implemented by zwitterionic surfactant, 3-(N,N-dimethyldodecylammonio)-propanesulfonate. The proposed method achieved low limits of detection varied from 0.2 to 0.4μgL−1 when the sample volume injected onto the chip (9.9μl) was almost the same as the volume of both separation channels. Preferable working conditions on the CC chip (suppressed hydrodynamic and electroosmotic flow) contributed for reproducible migration velocities (intra-day reproducibility up to 2.1% RSD) and determinations of trace concentrations of nitrite and nitrate (intra-day precision up to 3.0% RSD). Huge amount of chloride present in CSF (approx. 4.5gL−1) was removed from analyzed CSF samples by microsolid phase extraction performed on silver-form resin prior to the ITP-CE analysis. Developed method provided fast (approx. 20min total analysis time) and reliable determinations of trace nitrite and nitrate and could be fully integrated into the analysis of CSF samples.
On-line screening of matrix metalloproteinase inhibitors by capillary electrophoresis coupled to ESI mass spectrometry
05 June 2013,
09:36:17
Publication date: 1 July
2013
Source:Journal of Chromatography B, Volume 930
Author(s): Xu Wang , Zhiying Dou , Yaozuo Yuan , Shuli Man , Kris Wolfs , Erwin Adams , Ann Van Schepdael
Capillary electrophoresis (CE) with the use of mass spectrometry (MS) has been considered as a unique tool for microscale enzyme assay and inhibitor screening. In this study, matrix metalloproteinase-9 (MMP-9) was selected as target enzyme due to its important role in tumor invasion and metastasis. In order to define the optimal MS parameters, a two level half fraction factorial experimental design was performed. A background electrolyte consisting of 20mM ammonium acetate (pH 6.8) and a sheath liquid of water–methanol (50:50, v/v) containing 0.05% formic acid at a flow rate of 4μl/min were selected. This system was operated in the positive ion mode with a detection-limit of 10nM for the MMP reaction product and provided 60 folds enhancement of sensitivity by using selected reaction monitoring detection compared with MS full scan mode, which significantly increased the detectability of the system and therefore reduced the enzyme reaction time in both off-line and in-line mode. Both electrophoretically mediated microanalysis and pressure mediated microanalysis combined with MS detection were investigated for MMP inhibitor screening. Good repeatability (RSD of peak area and migration time were lower than 5.0%) and linearity (R 2 >0.996) were obtained for both in-capillary approaches. Several tetracycline antibiotics and natural products were selected to test the system. The results indicated an agreement on the ranking of inhibitory potency for both in-capillary approaches.
Source:Journal of Chromatography B, Volume 930
Author(s): Xu Wang , Zhiying Dou , Yaozuo Yuan , Shuli Man , Kris Wolfs , Erwin Adams , Ann Van Schepdael
Capillary electrophoresis (CE) with the use of mass spectrometry (MS) has been considered as a unique tool for microscale enzyme assay and inhibitor screening. In this study, matrix metalloproteinase-9 (MMP-9) was selected as target enzyme due to its important role in tumor invasion and metastasis. In order to define the optimal MS parameters, a two level half fraction factorial experimental design was performed. A background electrolyte consisting of 20mM ammonium acetate (pH 6.8) and a sheath liquid of water–methanol (50:50, v/v) containing 0.05% formic acid at a flow rate of 4μl/min were selected. This system was operated in the positive ion mode with a detection-limit of 10nM for the MMP reaction product and provided 60 folds enhancement of sensitivity by using selected reaction monitoring detection compared with MS full scan mode, which significantly increased the detectability of the system and therefore reduced the enzyme reaction time in both off-line and in-line mode. Both electrophoretically mediated microanalysis and pressure mediated microanalysis combined with MS detection were investigated for MMP inhibitor screening. Good repeatability (RSD of peak area and migration time were lower than 5.0%) and linearity (R 2 >0.996) were obtained for both in-capillary approaches. Several tetracycline antibiotics and natural products were selected to test the system. The results indicated an agreement on the ranking of inhibitory potency for both in-capillary approaches.
Semi-preparative isolation of dihydroresveratrol-3-O-β-d-glucuronide and four resveratrol conjugates from human urine after oral intake of a resveratrol-containing dietary supplement
05 June 2013,
09:36:17
Publication date: 1 July
2013
Source:Journal of Chromatography B, Volume 930
Author(s): Yulia Radko , Kathrine B. Christensen , Lars P. Christensen
A method for semi-preparative isolation of major resveratrol metabolites from human urine after oral intake of a trans-resveratrol-containing dietary supplement was developed. Pretreatment of the urine (6L) by using solid-phase extraction gave a brown oily residue (9.3g), which was separated using a combination of normal phase column chromatography and reversed-phase flash column chromatography resulting in fractions containing 1.1g crude trans-resveratrol-3-O-sulfate (M1), 86mg of a crude mixture of trans-resveratrol-3,5-O-disulfate (M2) and trans-resveratrol-3,4′-O-disulfate (M3), and 568mg of a crude mixture of trans-resveratrol-3-O-β-d-glucuronide (M4) and dihydroresveratrol-3-O-β-d-glucuronide (M5). Purification of the crude metabolites was performed by semi-preparative reversed-phase HPLC using a gradient of aqueous ammonium acetate (2.5mmol/L, pH 6.7)/acetonitrile for purification of M1, M2 and M3 or trifluoroacetic acid in water (pH 2.5)/acetonitrile for purification of M4 and M5. From a part of the crude metabolites (50–75mg), 47mg M1 (purity 98.7%), 14mg M2 (purity 96.1%), 10mg M3 (purity 96.3%), 38mg M4 (purity 98.2%) and 18mg M5 (purity 97.8%) were obtained. The structures of all isolated resveratrol metabolites were elucidated by spectroscopic and spectrometric methods such as 1D and 2D NMR, UV, and LC–MS. This method represents a novel approach to obtain resveratrol metabolites being the first method describing the direct isolation of pure resveratrol metabolites from urine samples in quantities sufficient for full chemical characterization and testing in vitro and in preclinical trials.
Source:Journal of Chromatography B, Volume 930
Author(s): Yulia Radko , Kathrine B. Christensen , Lars P. Christensen
A method for semi-preparative isolation of major resveratrol metabolites from human urine after oral intake of a trans-resveratrol-containing dietary supplement was developed. Pretreatment of the urine (6L) by using solid-phase extraction gave a brown oily residue (9.3g), which was separated using a combination of normal phase column chromatography and reversed-phase flash column chromatography resulting in fractions containing 1.1g crude trans-resveratrol-3-O-sulfate (M1), 86mg of a crude mixture of trans-resveratrol-3,5-O-disulfate (M2) and trans-resveratrol-3,4′-O-disulfate (M3), and 568mg of a crude mixture of trans-resveratrol-3-O-β-d-glucuronide (M4) and dihydroresveratrol-3-O-β-d-glucuronide (M5). Purification of the crude metabolites was performed by semi-preparative reversed-phase HPLC using a gradient of aqueous ammonium acetate (2.5mmol/L, pH 6.7)/acetonitrile for purification of M1, M2 and M3 or trifluoroacetic acid in water (pH 2.5)/acetonitrile for purification of M4 and M5. From a part of the crude metabolites (50–75mg), 47mg M1 (purity 98.7%), 14mg M2 (purity 96.1%), 10mg M3 (purity 96.3%), 38mg M4 (purity 98.2%) and 18mg M5 (purity 97.8%) were obtained. The structures of all isolated resveratrol metabolites were elucidated by spectroscopic and spectrometric methods such as 1D and 2D NMR, UV, and LC–MS. This method represents a novel approach to obtain resveratrol metabolites being the first method describing the direct isolation of pure resveratrol metabolites from urine samples in quantities sufficient for full chemical characterization and testing in vitro and in preclinical trials.
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