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Highly sensitive trivalent copper chelate-luminol chemiluminescence system for capillary electrophoresis detection of epinephrine in the urine of smoker
30 November 2012,
03:09:23
Publication year:
2012
Source:Journal of Chromatography B, Volume 911
Tao Li, Zuorong Wang, Haoyue Xie, Zhifeng Fu
Epinephrine (EP) is one of the most important neurotransmitters and hormones. Some previous literatures show that there is a close relation between its release and smoking. To compare the levels of EP in urines of smokers and nonsmokers, a sensitive chemiluminescence (CL) system, luminol-diperiodatocuprate (III) (K5[Cu(HIO6)2], DPC), has been developed and validated for the determination of EP after CE separation. The DPC-luminol-EP CL reaction showed very intensive emission and fast kinetic characteristics, thus led to a high sensitivity in the flow-through detection mode for capillary electrophoresis. With the peak height as a quantitative parameter, the relative CL intensity was linear with the EP concentration in the range of 2.0–400ng/mL, with a limit of detection of 0.82ng/mL (S/N=3). The reproducibility was assessed by intra- and inter-day relative standard deviations (RSDs) for 11 replicate determinations of EP standard samples at low, medium and high concentrations. The intra- and inter-day RSDs for CL signals were 5.5%–6.6% and 6.1%–7.5%, respectively, and those for migration times were 3.4%–5.8% and 4.3%–6.3%, respectively. The presented method was successfully applied to the determination of EP in EP injection and urine samples of smokers and nonsmokers. The recovery test results for urine samples ranged from 86.5 to 112.0%, which demonstrated the reliability of this method. The results for urine sample detection indicate that the average level of EP in the urines of the smoker group is obviously higher than that in the urines of the nonsmoker group, which may demonstrate that smoking can stimulate the release of EP in human body.
Source:Journal of Chromatography B, Volume 911
Tao Li, Zuorong Wang, Haoyue Xie, Zhifeng Fu
Epinephrine (EP) is one of the most important neurotransmitters and hormones. Some previous literatures show that there is a close relation between its release and smoking. To compare the levels of EP in urines of smokers and nonsmokers, a sensitive chemiluminescence (CL) system, luminol-diperiodatocuprate (III) (K5[Cu(HIO6)2], DPC), has been developed and validated for the determination of EP after CE separation. The DPC-luminol-EP CL reaction showed very intensive emission and fast kinetic characteristics, thus led to a high sensitivity in the flow-through detection mode for capillary electrophoresis. With the peak height as a quantitative parameter, the relative CL intensity was linear with the EP concentration in the range of 2.0–400ng/mL, with a limit of detection of 0.82ng/mL (S/N=3). The reproducibility was assessed by intra- and inter-day relative standard deviations (RSDs) for 11 replicate determinations of EP standard samples at low, medium and high concentrations. The intra- and inter-day RSDs for CL signals were 5.5%–6.6% and 6.1%–7.5%, respectively, and those for migration times were 3.4%–5.8% and 4.3%–6.3%, respectively. The presented method was successfully applied to the determination of EP in EP injection and urine samples of smokers and nonsmokers. The recovery test results for urine samples ranged from 86.5 to 112.0%, which demonstrated the reliability of this method. The results for urine sample detection indicate that the average level of EP in the urines of the smoker group is obviously higher than that in the urines of the nonsmoker group, which may demonstrate that smoking can stimulate the release of EP in human body.
Highlights
► DPC was synthesized and used to develop a highly sensitive CL detection system. ► A CE-CL method was established for highly sensitive assay of EP in biological sample. ► Using this CE-CL method, smoking was found to stimulate the release of EP.Shotgun analysis of membrane proteomes by an improved SDS-assisted sample preparation method coupled with liquid chromatography–tandem mass spectrometry
30 November 2012,
03:09:23
Publication year:
2012
Source:Journal of Chromatography B, Volume 911
Yong Lin, Huajun Jiang, Yujun Yan, Bin Peng, Jinhua Chen, Haiyan Lin, Zhonghua Liu
Analysis of the membrane proteins, particularly the integral membrane proteins, is limited by the inherent membrane hydrophobicity. Sodium dodecyl sulfate (SDS) is one of the most efficient reagents used for the extraction of membrane proteins, but its presence in samples interferes with LC–MS-based proteomic analyses because it affects RP-LC separations and electrospray ionization. In this paper, we present an improved sample preparation strategy based on SDS-assisted digestion and peptide-level SDS-removal using an optimized potassium dodecyl sulfate (KDS) precipitation method (SSDP method) for shotgun analysis of the membrane proteome. This method utilizes a high concentration of SDS (1.0%) to lyse the membranes and to solubilize the hydrophobic membrane proteins, resulting in a more complete protein digestion in the diluted SDS buffer (0.1% SDS), and a high efficiency of SDS removal and peptide recovery by the optimized KDS precipitation for protein identification. The SSDP method provides evidence that proteins can be efficiently digested, and the SDS can be decreased to <0.01% allowing >95% peptide recovery. Compared to other sample preparation methods commonly used in shotgun membrane proteomics, the newly developed method not only increased the identified number of the total proteins, membrane proteins and integral membrane proteins by an average of 33.1%, 37.2% and 40.5%, respectively, but also leading to the identification of highest number of matching peptides. All the results showed that the method yielded better recovery and reliability in the identification of the proteins especially the highly hydrophobic integral membrane proteins, and thus providing a promising tool for the shotgun analysis of membrane proteome.
Source:Journal of Chromatography B, Volume 911
Yong Lin, Huajun Jiang, Yujun Yan, Bin Peng, Jinhua Chen, Haiyan Lin, Zhonghua Liu
Analysis of the membrane proteins, particularly the integral membrane proteins, is limited by the inherent membrane hydrophobicity. Sodium dodecyl sulfate (SDS) is one of the most efficient reagents used for the extraction of membrane proteins, but its presence in samples interferes with LC–MS-based proteomic analyses because it affects RP-LC separations and electrospray ionization. In this paper, we present an improved sample preparation strategy based on SDS-assisted digestion and peptide-level SDS-removal using an optimized potassium dodecyl sulfate (KDS) precipitation method (SSDP method) for shotgun analysis of the membrane proteome. This method utilizes a high concentration of SDS (1.0%) to lyse the membranes and to solubilize the hydrophobic membrane proteins, resulting in a more complete protein digestion in the diluted SDS buffer (0.1% SDS), and a high efficiency of SDS removal and peptide recovery by the optimized KDS precipitation for protein identification. The SSDP method provides evidence that proteins can be efficiently digested, and the SDS can be decreased to <0.01% allowing >95% peptide recovery. Compared to other sample preparation methods commonly used in shotgun membrane proteomics, the newly developed method not only increased the identified number of the total proteins, membrane proteins and integral membrane proteins by an average of 33.1%, 37.2% and 40.5%, respectively, but also leading to the identification of highest number of matching peptides. All the results showed that the method yielded better recovery and reliability in the identification of the proteins especially the highly hydrophobic integral membrane proteins, and thus providing a promising tool for the shotgun analysis of membrane proteome.
Highlights
► Developed a simple SDS-assisted sample preparation method for shotgun analysis of membrane proteomes. ► Utilized the strong solubilization ability of SDS for the extraction and solubilization of membrane proteins. ► Optimized KDS precipitation for high efficiency of SDS-removal and peptide recovery. ► The developed method obviously improves the identification of membrane proteins particularly IMPs. ► The developed method is easy to operate at low cost.Methodology for a rapid and simultaneous determination of total cysteine, homocysteine, cysteinylglycine and glutathione in plasma by isocratic RP-HPLC
30 November 2012,
03:09:23
Publication year:
2012
Source:Journal of Chromatography B, Volume 911
Rita Ferin, Maria Leonor Pavão, José Baptista
Alterations in the plasma aminothiols levels can be considered as important biomarkers for the diagnosis and screening of several human disorders, namely cardiovascular diseases. We aimed to optimize a rapid, sensitive and accurate RP-HPLC methodology with fluorescence detection, for the simultaneous determination of the total concentrations of cysteine, homocysteine, cysteinylglycine and glutathione in blood plasma, as well as its application to the evaluation of those thiols levels in plasma of a group of Azorean subjects. Aminothiols were reduced with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic reagent ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate. The thiols adducts were separated by an isocratic elution on a Platinum EPS C18 analytical column (53mm×7mm I.D., 3μm) using a phosphate buffer containing 4% of acetonitrile as a mobile phase. Results indicated an excellent linearity for all the analytes over their respective concentration ranges with correlation coefficients (r 2) ≥0.99. The LOD for the four plasma thiols was lower than 0.10μmol/L, while LOQ varied from 0.5 to 15μmol/L. For both intra- and inter-day precision, the RSD (%) values were lower than 1.9%, and the CV (%) values were found under 0.5%. The recovery ranged from 92% to 100% indicating a high degree of the method's accuracy. This method allows a simultaneous, complete analysis of the four plasma aminothiols and the internal standard in 6min. By reducing the total run time, a larger number of analysis can be performed daily.
Source:Journal of Chromatography B, Volume 911
Rita Ferin, Maria Leonor Pavão, José Baptista
Alterations in the plasma aminothiols levels can be considered as important biomarkers for the diagnosis and screening of several human disorders, namely cardiovascular diseases. We aimed to optimize a rapid, sensitive and accurate RP-HPLC methodology with fluorescence detection, for the simultaneous determination of the total concentrations of cysteine, homocysteine, cysteinylglycine and glutathione in blood plasma, as well as its application to the evaluation of those thiols levels in plasma of a group of Azorean subjects. Aminothiols were reduced with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic reagent ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate. The thiols adducts were separated by an isocratic elution on a Platinum EPS C18 analytical column (53mm×7mm I.D., 3μm) using a phosphate buffer containing 4% of acetonitrile as a mobile phase. Results indicated an excellent linearity for all the analytes over their respective concentration ranges with correlation coefficients (r 2) ≥0.99. The LOD for the four plasma thiols was lower than 0.10μmol/L, while LOQ varied from 0.5 to 15μmol/L. For both intra- and inter-day precision, the RSD (%) values were lower than 1.9%, and the CV (%) values were found under 0.5%. The recovery ranged from 92% to 100% indicating a high degree of the method's accuracy. This method allows a simultaneous, complete analysis of the four plasma aminothiols and the internal standard in 6min. By reducing the total run time, a larger number of analysis can be performed daily.
Highlights
► Rapid and simultaneous determination of four total aminothiols levels in plasma. ► RP-HPLC isocratic elution within 6min of all analytes, including the IS. ► Validated method exhibits an excellent precision and recovery for all aminothiols. ► Method appropriated for high-throughput routine clinical analysis.Development and validation of a LC–MS/MS method for the quantification of the regioisomers of dihydroxybutylether in human plasma
30 November 2012,
03:09:23
Publication year:
2012
Source:Journal of Chromatography B, Volume 911
Bo Yuan, Li Li, Yao Fu, Yi Jin, Lixin Guo, Haiyan Xu
Dihydroxybutylether (DHBE), a strong choleretic drug, is a mixture of three regioisomers: 4-(3-hydroxybutoxy)-2-butanol (I), 3-(4-hydroxy-2-butoxy)-1-butanol (II) and 3-(3-hydroxylbutoxy)-1-butanol (III). A liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of dihydroxybutylether (DHBE) regioisomers in human plasma. After plasma samples were deproteinized with 10% perchloric acid, the post-treatment samples were analyzed on a Capcell Pak C18 MGII column interfaced with a triple quadrupole tandem mass spectrometer in positive electrospray ionization mode. Methanol and water was used as the mobile phase with a gradient elution at a flow rate of 1mL/min. Acetaminophen was used as an internal standard (IS). Multiple selected reaction monitoring was performed using the transitions m/z 163→55 and m/z 152→110 to quantify DHBE regioisomers and IS, respectively. Five DHBE isomers (a, b, c, d and e) were separated under the present chromatographic condition. The assay was linear over the concentration range of 5.0–200ng/mL for DHBE isomers a, b and c, and 10.0–400ng/mL for DHBE isomers d and e. The intra- and inter-day precision was within 13.6% in terms of relative standard deviation (RSD%) and the accuracy within 7.3% in terms of relative error. This simple and sensitive and easily reproducible LC–MS/MS method was successfully applied to the pharmacokinetic study of DHBE regioisomers in healthy male Chinese volunteers after an oral dose of 1.0g DHBE.
Source:Journal of Chromatography B, Volume 911
Bo Yuan, Li Li, Yao Fu, Yi Jin, Lixin Guo, Haiyan Xu
Dihydroxybutylether (DHBE), a strong choleretic drug, is a mixture of three regioisomers: 4-(3-hydroxybutoxy)-2-butanol (I), 3-(4-hydroxy-2-butoxy)-1-butanol (II) and 3-(3-hydroxylbutoxy)-1-butanol (III). A liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of dihydroxybutylether (DHBE) regioisomers in human plasma. After plasma samples were deproteinized with 10% perchloric acid, the post-treatment samples were analyzed on a Capcell Pak C18 MGII column interfaced with a triple quadrupole tandem mass spectrometer in positive electrospray ionization mode. Methanol and water was used as the mobile phase with a gradient elution at a flow rate of 1mL/min. Acetaminophen was used as an internal standard (IS). Multiple selected reaction monitoring was performed using the transitions m/z 163→55 and m/z 152→110 to quantify DHBE regioisomers and IS, respectively. Five DHBE isomers (a, b, c, d and e) were separated under the present chromatographic condition. The assay was linear over the concentration range of 5.0–200ng/mL for DHBE isomers a, b and c, and 10.0–400ng/mL for DHBE isomers d and e. The intra- and inter-day precision was within 13.6% in terms of relative standard deviation (RSD%) and the accuracy within 7.3% in terms of relative error. This simple and sensitive and easily reproducible LC–MS/MS method was successfully applied to the pharmacokinetic study of DHBE regioisomers in healthy male Chinese volunteers after an oral dose of 1.0g DHBE.
Highlights
► This is the first LC–MS/MS method to determine DHBE regioisomers in human plasma. ► Five DHBE isomers were separated and determined by this method within 16.5min. ► This method is simple and sensitive and easily reproducible. ► It was applied to a pharmacokinetic study of DHBE regioisomers in humans.Identification and assay of 3′-O-methyltaxifolin by UPLC–MS in rat plasma
30 November 2012,
03:09:23
Publication year:
2012
Source:Journal of Chromatography B, Volume 911
Xiaodan Wang, Hui Zhou, Su Zeng
A new metabolite of taxifolin: 3′-O-methyltaxifolin (3′-O-MTAX) in Caco-2 cells and in rat plasma was identified. The chemical structure of 3′-O-MTAX was determined by MS and 1H NMR. A rapid, sensitive and specific UPLC–MS method to determine 3′-O-MTAX in rat plasma was also developed. Following ethyl acetate extraction, 3′-O-MTAX in plasma was separated on a Sunfire™ (2.1mm×50mm, 3.5μm) column and analyzed in the selected ion recording with a negative electrospray ionization mode using puerarin as the internal standard. The lower limit of quantification (LLOQ) was 2.75ng/mL. Intra- and inter-day precisions (% RSD) were all within 7.2% and accuracy (% deviation) ranged from −5.0 to 4.7%. The overall recoveries at four concentrations were all >72.0%. This validated method was successfully applied to measure 3′-O-MTAX in rat plasma after oral administration of taxifolin.
Source:Journal of Chromatography B, Volume 911
Xiaodan Wang, Hui Zhou, Su Zeng
A new metabolite of taxifolin: 3′-O-methyltaxifolin (3′-O-MTAX) in Caco-2 cells and in rat plasma was identified. The chemical structure of 3′-O-MTAX was determined by MS and 1H NMR. A rapid, sensitive and specific UPLC–MS method to determine 3′-O-MTAX in rat plasma was also developed. Following ethyl acetate extraction, 3′-O-MTAX in plasma was separated on a Sunfire™ (2.1mm×50mm, 3.5μm) column and analyzed in the selected ion recording with a negative electrospray ionization mode using puerarin as the internal standard. The lower limit of quantification (LLOQ) was 2.75ng/mL. Intra- and inter-day precisions (% RSD) were all within 7.2% and accuracy (% deviation) ranged from −5.0 to 4.7%. The overall recoveries at four concentrations were all >72.0%. This validated method was successfully applied to measure 3′-O-MTAX in rat plasma after oral administration of taxifolin.
Highlights
► Identified a new metabolite of taxifolin in Caco-2 cells and in rat plasma. ► Prepared the metabolite using Caco-2 cells. ► Evaluated the pharmacokinetic studies of 3′-O-methyltaxifolin in rats.Expression and purification of a chimeric protein consisting of the ectodomains of M and GP5 proteins of porcine reproductive and respiratory syndrome virus (PRRSV)
30 November 2012,
03:09:23
Publication year:
2012
Source:Journal of Chromatography B, Volume 911
Jianzhong Hu, Yanyan Ni, X.J. Meng, Chenming Zhang
Porcine reproductive and respiratory syndrome (PRRS) is the most economically important infectious disease currently affecting the swine industry worldwide. In the US alone, it causes economic losses of more than 560 million dollars every year. Although killed-virus and modified-live PRRS vaccines are commercially available, the unsatisfactory efficacy and safety of current vaccines drives the impetus of developing novel PRRSV vaccines. To fulfill this purpose, we designed a chimeric protein consisting of the ectodomains of viral GP5 and M protein, the two most widely studied subunit vaccine targets, and expressed it in E. coli. An optimized purification/refolding process composed of immobilized metal ion affinity chromatography, dialysis refolding and anion exchange chromatography was developed to purify the chimeric protein from the inclusion bodies. This process could recover approximately 12mgprotein/l E. coli broth with near 100% purity and very low endotoxin level. In addition, the purified protein is antigenic, can bind to a cellular receptor for the virus (heparan sulfate), and can block virus infection of susceptible cells. Therefore, the chimeric protein is a promising subunit vaccine candidate against PRRSV.
Source:Journal of Chromatography B, Volume 911
Jianzhong Hu, Yanyan Ni, X.J. Meng, Chenming Zhang
Porcine reproductive and respiratory syndrome (PRRS) is the most economically important infectious disease currently affecting the swine industry worldwide. In the US alone, it causes economic losses of more than 560 million dollars every year. Although killed-virus and modified-live PRRS vaccines are commercially available, the unsatisfactory efficacy and safety of current vaccines drives the impetus of developing novel PRRSV vaccines. To fulfill this purpose, we designed a chimeric protein consisting of the ectodomains of viral GP5 and M protein, the two most widely studied subunit vaccine targets, and expressed it in E. coli. An optimized purification/refolding process composed of immobilized metal ion affinity chromatography, dialysis refolding and anion exchange chromatography was developed to purify the chimeric protein from the inclusion bodies. This process could recover approximately 12mgprotein/l E. coli broth with near 100% purity and very low endotoxin level. In addition, the purified protein is antigenic, can bind to a cellular receptor for the virus (heparan sulfate), and can block virus infection of susceptible cells. Therefore, the chimeric protein is a promising subunit vaccine candidate against PRRSV.
Highlights
► A novel chimeric protein was expressed in E. coli inclusion bodies. ► A process to purify and refold the protein was developed. ► The protein is effective in blocking the infcetion of PRRSV. ► The protein can be a promising vaccine candidate against PRRSV.On-line clean-up and determination of tramadol in human plasma and urine samples using molecularly imprinted monolithic column coupling with HPLC
30 November 2012,
03:09:23
Publication year:
2012
Source:Journal of Chromatography B, Volume 911
Mehran Javanbakht, Mohammad Mahdi Moein, Behrouz Akbari-adergani
The applicability of an on-line solid phase extraction method using molecularly imprinted monolithic column was developed for the assay of tramadol (TRD) in urine and plasma samples. The monolithic column was prepared by using TRD as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker and chloroform as the porogen with in situ molecular imprinting polymerization technique. Various parameters affecting the extraction efficiency of the monolithic column were evaluated. Chromatographic analysis of TRD after on-line clean-up of samples was performed by reversed-phase HPLC on an ACE column with ultraviolet detection at 218nm. The present work was successfully applied for automated simple analysis of TRD in urine and plasma samples with high recoveries between 90.5–93.1% and 93.3–96.0%, respectively. The results revealed that in concentration up to 500ng/mL of dextromethorphan (DEX), timolol (TMO) and O-desmethyltramadol (M1), the recoveries were not reduced more than 4.3% and 4.0% for plasma and urine samples, respectively. The limit of detection (S/N=3) and limit of quantification (S/N=10) for TRD in urine samples were 0.03ng/mL and 0.10ng/mL, and in plasma samples were 0.3 and 1.0ng/mL, respectively. Inter-column precision of the assays (n =3) for urine and plasma samples at the 100ng/mL TRD level were 4.0% and 4.2%, respectively.
Source:Journal of Chromatography B, Volume 911
Mehran Javanbakht, Mohammad Mahdi Moein, Behrouz Akbari-adergani
The applicability of an on-line solid phase extraction method using molecularly imprinted monolithic column was developed for the assay of tramadol (TRD) in urine and plasma samples. The monolithic column was prepared by using TRD as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker and chloroform as the porogen with in situ molecular imprinting polymerization technique. Various parameters affecting the extraction efficiency of the monolithic column were evaluated. Chromatographic analysis of TRD after on-line clean-up of samples was performed by reversed-phase HPLC on an ACE column with ultraviolet detection at 218nm. The present work was successfully applied for automated simple analysis of TRD in urine and plasma samples with high recoveries between 90.5–93.1% and 93.3–96.0%, respectively. The results revealed that in concentration up to 500ng/mL of dextromethorphan (DEX), timolol (TMO) and O-desmethyltramadol (M1), the recoveries were not reduced more than 4.3% and 4.0% for plasma and urine samples, respectively. The limit of detection (S/N=3) and limit of quantification (S/N=10) for TRD in urine samples were 0.03ng/mL and 0.10ng/mL, and in plasma samples were 0.3 and 1.0ng/mL, respectively. Inter-column precision of the assays (n =3) for urine and plasma samples at the 100ng/mL TRD level were 4.0% and 4.2%, respectively.
Highlights
► We prepared molecularly imprinted monolithic column for the assay of tramadol. ► The column showed highly specific recognition for the template. ► The work was applied for automated analysis of tramadol in urine and plasma.Automated multi-step purification protocol for Angiotensin-I-Converting-Enzyme (ACE)
30 November 2012,
03:09:23
Publication year:
2012
Source:Journal of Chromatography B, Volume 911
Thomas Eisele, Timo Stressler, Bertolt Kranz, Lutz Fischer
Highly purified proteins are essential for the investigation of the functional and biochemical properties of proteins. The purification of a protein requires several steps, which are often time-consuming. In our study, the Angiotensin-I-Converting-Enzyme (ACE; EC 3.4.15.1) was solubilised from pig lung without additional detergents, which are commonly used, under mild alkaline conditions in a Tris–HCl buffer (50mM, pH 9.0) for 48h. An automation of the ACE purification was performed using a multi-step protocol in less than 8h, resulting in a purified protein with a specific activity of 37Umg−1 (purification factor 308) and a yield of 23.6%. The automated ACE purification used an ordinary fast-protein-liquid-chromatography (FPLC) system equipped with two additional switching valves. These switching valves were needed for the buffer stream inversion and for the connection of the Superloop™ used for the protein parking. Automated ACE purification was performed using four combined chromatography steps, including two desalting procedures. The purification methods contained two hydrophobic interaction chromatography steps, a Cibacron 3FG-A chromatography step and a strong anion exchange chromatography step. The purified ACE was characterised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE. The estimated monomer size of the purified glycosylated ACE was determined to be ∼175kDa by SDS-PAGE, with the dimeric form at ∼330kDa as characterised by a native PAGE using a novel activity staining protocol. For the activity staining, the tripeptide l-Phe-Gly-Gly was used as the substrate. The ACE cleaved the dipeptide Gly-Gly, releasing the l-Phe to be oxidised with l-amino acid oxidase. Combined with peroxidase and o-dianisidine, the generated H2O2 stained a brown coloured band. This automated purification protocol can be easily adapted to be used with other protein purification tasks.
Source:Journal of Chromatography B, Volume 911
Thomas Eisele, Timo Stressler, Bertolt Kranz, Lutz Fischer
Highly purified proteins are essential for the investigation of the functional and biochemical properties of proteins. The purification of a protein requires several steps, which are often time-consuming. In our study, the Angiotensin-I-Converting-Enzyme (ACE; EC 3.4.15.1) was solubilised from pig lung without additional detergents, which are commonly used, under mild alkaline conditions in a Tris–HCl buffer (50mM, pH 9.0) for 48h. An automation of the ACE purification was performed using a multi-step protocol in less than 8h, resulting in a purified protein with a specific activity of 37Umg−1 (purification factor 308) and a yield of 23.6%. The automated ACE purification used an ordinary fast-protein-liquid-chromatography (FPLC) system equipped with two additional switching valves. These switching valves were needed for the buffer stream inversion and for the connection of the Superloop™ used for the protein parking. Automated ACE purification was performed using four combined chromatography steps, including two desalting procedures. The purification methods contained two hydrophobic interaction chromatography steps, a Cibacron 3FG-A chromatography step and a strong anion exchange chromatography step. The purified ACE was characterised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE. The estimated monomer size of the purified glycosylated ACE was determined to be ∼175kDa by SDS-PAGE, with the dimeric form at ∼330kDa as characterised by a native PAGE using a novel activity staining protocol. For the activity staining, the tripeptide l-Phe-Gly-Gly was used as the substrate. The ACE cleaved the dipeptide Gly-Gly, releasing the l-Phe to be oxidised with l-amino acid oxidase. Combined with peroxidase and o-dianisidine, the generated H2O2 stained a brown coloured band. This automated purification protocol can be easily adapted to be used with other protein purification tasks.
Highlights
► Detergent free solubilisation of Angiotensin-I-Converting-Enzyme (ACE). ► Development of an automated multi-step purification protocol for ACE. ► ACE purification about 300-fold to a specific activity of 37Umg−1 in less than 8h. ► Development of a novel activity staining protocol for ACE. ► The automated protein purification protocol can be easily applied to other proteins.Development and validation of a HPLC method for the assay of dapivirine in cell-based and tissue permeability experiments
30 November 2012,
03:09:23
Publication year:
2012
Source:Journal of Chromatography B, Volume 911
José das Neves, Bruno Sarmento, Mansoor Amiji, Maria Fernanda Bahia
Dapivirine, a non-nucleoside reverse transcriptase inhibitor, is being currently used for the development of potential anti-HIV microbicide formulations and delivery systems. A new high-performance liquid chromatography (HPLC) method with UV detection was developed for the assay of this drug in different biological matrices, namely cell lysates, receptor media from permeability experiments and homogenates of mucosal tissues. The method used a reversed-phase C18 column with a mobile phase composed of trifluoroacetic acid solution (0.1%, v/v) and acetonitrile in a gradient mode. Injection volume was 50μL and the flow rate 1mL/min. The total run time was 12min and UV detection was performed at 290nm for dapivirine and the internal standard (IS) diphenylamine. A Box-Behnken experimental design was used to study different experimental variables of the method, namely the ratio of the mobile phase components and the gradient time, and their influence in responses such as the retention factor, tailing factor, and theoretical plates for dapivirine and the IS, as well as the peak resolution between both compounds. The optimized method was further validated and its usefulness assessed for in vitro and ex vivo experiments using dapivirine or dapivirine-loaded nanoparticles. The method showed to be selective, linear, accurate and precise in the range of 0.02–1.5μg/mL. Other chromatographic parameters, namely carry-over, lower limit of quantification (0.02μg/mL), limit of detection (0.006μg/mL), recovery (equal or higher than 90.7%), and sample stability at different storage conditions, were also determined and found adequate for the intended purposes. The method was successfully used for cell uptake assays and permeability studies across cell monolayers and pig genital mucosal tissues. Overall, the proposed method provides a simple, versatile and reliable way for studying the behavior of dapivirine in different biological matrices and assessing its potential as an anti-HIV microbicide drug.
Source:Journal of Chromatography B, Volume 911
José das Neves, Bruno Sarmento, Mansoor Amiji, Maria Fernanda Bahia
Dapivirine, a non-nucleoside reverse transcriptase inhibitor, is being currently used for the development of potential anti-HIV microbicide formulations and delivery systems. A new high-performance liquid chromatography (HPLC) method with UV detection was developed for the assay of this drug in different biological matrices, namely cell lysates, receptor media from permeability experiments and homogenates of mucosal tissues. The method used a reversed-phase C18 column with a mobile phase composed of trifluoroacetic acid solution (0.1%, v/v) and acetonitrile in a gradient mode. Injection volume was 50μL and the flow rate 1mL/min. The total run time was 12min and UV detection was performed at 290nm for dapivirine and the internal standard (IS) diphenylamine. A Box-Behnken experimental design was used to study different experimental variables of the method, namely the ratio of the mobile phase components and the gradient time, and their influence in responses such as the retention factor, tailing factor, and theoretical plates for dapivirine and the IS, as well as the peak resolution between both compounds. The optimized method was further validated and its usefulness assessed for in vitro and ex vivo experiments using dapivirine or dapivirine-loaded nanoparticles. The method showed to be selective, linear, accurate and precise in the range of 0.02–1.5μg/mL. Other chromatographic parameters, namely carry-over, lower limit of quantification (0.02μg/mL), limit of detection (0.006μg/mL), recovery (equal or higher than 90.7%), and sample stability at different storage conditions, were also determined and found adequate for the intended purposes. The method was successfully used for cell uptake assays and permeability studies across cell monolayers and pig genital mucosal tissues. Overall, the proposed method provides a simple, versatile and reliable way for studying the behavior of dapivirine in different biological matrices and assessing its potential as an anti-HIV microbicide drug.
Highlights
► A HPLC-UV method was developed for assaying dapivirine in biological matrices. ► Box-Behnken experimental design was successfully used to optimize the method. ► Validation according to FDA and EMA guidelines on bioanalytical assays was performed. ► Method was applied to cell-based/mucosal tissues uptake and permeability assays.Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography–tandem mass spectrometry
30 November 2012,
03:09:23
Publication year:
2012
Source:Journal of Chromatography B, Volume 911
Ulla-Maja Bailey, Chamindie Punyadeera, Justin J. Cooper-White, Benjamin L. Schulz
Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high diversity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid sample preparation and non-targeted LC–ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme diversity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva.
Source:Journal of Chromatography B, Volume 911
Ulla-Maja Bailey, Chamindie Punyadeera, Justin J. Cooper-White, Benjamin L. Schulz
Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high diversity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid sample preparation and non-targeted LC–ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme diversity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva.
Highlights
► Rapid and straightforward saliva sample preparation and LC–ESI-MS/MS detection. ► Untargeted analysis detected physiological proteolysis events. ► Extreme proteolytic diversity of salivary alpha-amylase isoforms detected. ► Implications for biomarker discovery, validation and clinical application.Separation of intermediates of iron-catalyzed dopamine oxidation reactions using reversed-phase ion-pairing chromatography coupled in tandem with UV–visible and ESI-MS detections
30 November 2012,
03:09:23
Publication year:
2012
Source:Journal of Chromatography B, Volume 911
Lin Zhang, Gargey Yagnik, Dianlu Jiang, Shuyun Shi, Peter Chang, Feimeng Zhou
Reversed-phase ion-pairing chromatography (RP-IPC) is coupled on-line with electrospray ionization-mass spectrometry (ESI-MS) through an interface comprising a four-way switch valve and an anion exchange column. Regeneration of the anion exchange column can be accomplished on-line by switching the four-way switch valve to interconnect the column to a regeneration solution. Positioning the anion exchange column between the RP-IPC and ESI-MS instruments allows the ion-pairing reagent (IPR) sodium octane sulfonate to be removed. The IPC–ESI-MS method enabled us to separate and detect four intermediates of the Fe(III)-catalyzed dopamine oxidation. In particular, 6-hydroxydopamine, which is short-lived and highly neurotoxic, was detected and quantified. Together with the separation of other intermediates, gaining insight into the mechanism and kinetics of the Fe(III)-catalyzed dopamine oxidation becomes possible.
Source:Journal of Chromatography B, Volume 911
Lin Zhang, Gargey Yagnik, Dianlu Jiang, Shuyun Shi, Peter Chang, Feimeng Zhou
Reversed-phase ion-pairing chromatography (RP-IPC) is coupled on-line with electrospray ionization-mass spectrometry (ESI-MS) through an interface comprising a four-way switch valve and an anion exchange column. Regeneration of the anion exchange column can be accomplished on-line by switching the four-way switch valve to interconnect the column to a regeneration solution. Positioning the anion exchange column between the RP-IPC and ESI-MS instruments allows the ion-pairing reagent (IPR) sodium octane sulfonate to be removed. The IPC–ESI-MS method enabled us to separate and detect four intermediates of the Fe(III)-catalyzed dopamine oxidation. In particular, 6-hydroxydopamine, which is short-lived and highly neurotoxic, was detected and quantified. Together with the separation of other intermediates, gaining insight into the mechanism and kinetics of the Fe(III)-catalyzed dopamine oxidation becomes possible.
Highlights
► Intermediates of iron-catalyzed dopamine oxidation were identified by IPC–ESI-MS. ► The short-lived intermediate, 6-hydroxydopamine, was detected and quantified. ► Regenerable anion exchange column is used for on-line ion-pairing reagent removal. ► Mechanism and kinetics of complex reactions can be studied with IPC–ESI-MS.Determination of tolperisone in human plasma by liquid chromatography/tandem mass spectrometry for clinical application
30 November 2012,
03:09:23
Publication year:
2012
Source:Journal of Chromatography B, Volume 911
Chang-Ik Choi, Jung-In Park, Hye-In Lee, Yun-Jeong Lee, Choon-Gon Jang, Jung-Woo Bae, Seok-Yong Lee
We have developed and validated a simple, rapid, and sensitive liquid chromatography analytical method employing tandem mass spectrometry (LC–MS/MS) for the determination of tolperisone, a centrally acting muscle relaxant, in human plasma. After liquid–liquid extraction with methyl t-butyl ether, chromatographic separation of tolperisone was performed using a reversed-phase Luna C18 column (2.0mm×50mm, 5μm particles) with a mobile phase of 10mM ammonium formate buffer (pH 3.5) – methanol (12:88, v/v) and quantified by tandem mass detection in ESI positive ion mode. The flow rate of the mobile phase was 250μL/min and the retention times of tolperisone and the internal standard (IS, dibucaine) were both 0.6min. The calibration curves were linear over a range of 0.5–300ng/mL (r >0.999). The lower limit of quantification, using 200μL human plasma, was 0.5ng/mL. The mean accuracy and precision for intra- and inter-day validation of tolperisone were within acceptable limits. The LC–MS/MS method reported here showed improved sensitivity for quantification of tolperisone in human plasma compared with previously described analytical methods. Lastly, the validated method was successfully applied to a pharmacokinetic study in humans.
Source:Journal of Chromatography B, Volume 911
Chang-Ik Choi, Jung-In Park, Hye-In Lee, Yun-Jeong Lee, Choon-Gon Jang, Jung-Woo Bae, Seok-Yong Lee
We have developed and validated a simple, rapid, and sensitive liquid chromatography analytical method employing tandem mass spectrometry (LC–MS/MS) for the determination of tolperisone, a centrally acting muscle relaxant, in human plasma. After liquid–liquid extraction with methyl t-butyl ether, chromatographic separation of tolperisone was performed using a reversed-phase Luna C18 column (2.0mm×50mm, 5μm particles) with a mobile phase of 10mM ammonium formate buffer (pH 3.5) – methanol (12:88, v/v) and quantified by tandem mass detection in ESI positive ion mode. The flow rate of the mobile phase was 250μL/min and the retention times of tolperisone and the internal standard (IS, dibucaine) were both 0.6min. The calibration curves were linear over a range of 0.5–300ng/mL (r >0.999). The lower limit of quantification, using 200μL human plasma, was 0.5ng/mL. The mean accuracy and precision for intra- and inter-day validation of tolperisone were within acceptable limits. The LC–MS/MS method reported here showed improved sensitivity for quantification of tolperisone in human plasma compared with previously described analytical methods. Lastly, the validated method was successfully applied to a pharmacokinetic study in humans.
