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Selected papers from the latest issue:
Karel Macek (1928–2011)
14 March 2012, 05:07:24
Publication year: 2012
Source:Journal of Chromatography B, Volumes 889–890
Jaroslav Janák
Source:Journal of Chromatography B, Volumes 889–890
Jaroslav Janák
Purification, identification and profiling of serum amyloid A proteins from sera of advanced-stage cancer patients
14 March 2012, 05:07:24
Publication year: 2012
Source:Journal of Chromatography B, Volumes 889–890
Jing Li, Zhensheng Xie, Linan Shi, Zhiqiang Zhao, Junjie Hou, Xiulan Chen, Ziyou Cui, Peng Xue, Tanxi Cai, Peng Wu, Sutang Guo, Fuquan Yang
Surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) is a powerful tool for screening potential biomarkers of various pathological conditions. However, low resolution and mass accuracy of SELDI-TOF-MS remain a major obstacle for determination of biological identities of potential protein biomarkers. We report here a refined workflow that combines ZipTip desalting, acetonitrile precipitation, high-performance liquid chromatography (HPLC) separation and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis for the profiling, purification and identification of the targeted serum proteins found by SELDI-TOF-MS. By using this workflow, we purified ten targeted proteins from the sera of patients with various types of advanced stage (stage III–IV) cancers. These proteins were identified as isoforms of the human serum amyloid protein A (SAA) family with or without truncations at their N-terminals. This was confirmed by Western blot analysis. Different SAA expression patterns were observed by MALDI-TOF-MS profiling. SAA has long been reported as a biomarker for various cancer types such as lung cancer, ovarian cancer, and breast cancer. However, in this study we found increased SAA expression in the sera of advanced-stage cancer patients with different cancer types. Our results suggest that maybe SAA should not be used alone as a biomarker for any specific cancer type.
Source:Journal of Chromatography B, Volumes 889–890
Jing Li, Zhensheng Xie, Linan Shi, Zhiqiang Zhao, Junjie Hou, Xiulan Chen, Ziyou Cui, Peng Xue, Tanxi Cai, Peng Wu, Sutang Guo, Fuquan Yang
Surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) is a powerful tool for screening potential biomarkers of various pathological conditions. However, low resolution and mass accuracy of SELDI-TOF-MS remain a major obstacle for determination of biological identities of potential protein biomarkers. We report here a refined workflow that combines ZipTip desalting, acetonitrile precipitation, high-performance liquid chromatography (HPLC) separation and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis for the profiling, purification and identification of the targeted serum proteins found by SELDI-TOF-MS. By using this workflow, we purified ten targeted proteins from the sera of patients with various types of advanced stage (stage III–IV) cancers. These proteins were identified as isoforms of the human serum amyloid protein A (SAA) family with or without truncations at their N-terminals. This was confirmed by Western blot analysis. Different SAA expression patterns were observed by MALDI-TOF-MS profiling. SAA has long been reported as a biomarker for various cancer types such as lung cancer, ovarian cancer, and breast cancer. However, in this study we found increased SAA expression in the sera of advanced-stage cancer patients with different cancer types. Our results suggest that maybe SAA should not be used alone as a biomarker for any specific cancer type.
Development and validation of a sensitive LC–MS/MS assay for simultaneous quantitation of ranolazine and its three metabolites in human plasma
14 March 2012, 05:07:24
Publication year: 2012
Source:Journal of Chromatography B, Volumes 889–890
Yuan Wang, Xiaoyan Chen, Zuoming Sun, Yong Yang, Ying Zhang, Wanhui Liu, Dafang Zhong
A rapid, sensitive and reliable LC–MS/MS method was developed and validated for the simultaneous determination of ranolazine and its three metabolites, CVT-2514, CVT-2738, and CVT-4786, in human plasma. The plasma samples were prepared by protein precipitation. Chromatographic separation was achieved on a Gemini C18 column (50mm×2.0mm, 5μm) using methanol: 5mM ammonium acetate as the mobile phase with gradient elution. Mass detection was carried out by electrospray ionization in both positive and negative ion multiple reaction monitoring (MRM) modes. The calibration curves were linear over a concentration range of 4–2000ng/mL for ranolazine, 4–1000ng/mL for CVT-2514 and CVT-2738 and 8–1000ng/mL for CVT-4786. The intra-day and inter-day accuracy and precision were within the acceptable limits of ±15% at all concentrations. The method was successfully applied for the simultaneous estimation of ranolazine and its three metabolites in human plasma from a clinical pharmacokinetics study.
Source:Journal of Chromatography B, Volumes 889–890
Yuan Wang, Xiaoyan Chen, Zuoming Sun, Yong Yang, Ying Zhang, Wanhui Liu, Dafang Zhong
A rapid, sensitive and reliable LC–MS/MS method was developed and validated for the simultaneous determination of ranolazine and its three metabolites, CVT-2514, CVT-2738, and CVT-4786, in human plasma. The plasma samples were prepared by protein precipitation. Chromatographic separation was achieved on a Gemini C18 column (50mm×2.0mm, 5μm) using methanol: 5mM ammonium acetate as the mobile phase with gradient elution. Mass detection was carried out by electrospray ionization in both positive and negative ion multiple reaction monitoring (MRM) modes. The calibration curves were linear over a concentration range of 4–2000ng/mL for ranolazine, 4–1000ng/mL for CVT-2514 and CVT-2738 and 8–1000ng/mL for CVT-4786. The intra-day and inter-day accuracy and precision were within the acceptable limits of ±15% at all concentrations. The method was successfully applied for the simultaneous estimation of ranolazine and its three metabolites in human plasma from a clinical pharmacokinetics study.
Quantification of carbamazepine and its active metabolite by direct injection of human milk serum using liquid chromatography tandem ion trap mass spectrometry
14 March 2012, 05:07:24
Publication year: 2012
Source:Journal of Chromatography B, Volumes 889–890
B.R. Lopes, J.C. Barreiro, P.T. Baraldi, Q.B. Cass
This work reports the use of a liquid chromatography ion trap tandem mass spectrometry (LC–IT-MS/MS) system for quantification in human milk samples of both carbamazepine (CBZ) and its active metabolite, carbamazepine 10,11-epoxide (CBZE). An octadecyl restricted-access media bovine serum albumin column (RAM-BSA C18) was used in single-column mode. Selectivity, extraction efficiency, accuracy and precision were achieved employing 100μL of the sample, without preparation, with detection limits of 20.0ng/mL for CBZ and 40.0ng/mL for CBZE. The matrix effect was investigated for the compounds by post-column infusion (qualitative) and by on-line extraction (quantitative). It was observed suppression effect for CBZ and CBZE by post-column infusion, ion suppression of 0.80 for CBZ, and enhancement of 1.28 for CBZE by on-line extraction. The developed method was validated and applied to analyze breast milk samples from one nursing mother. CBZ and CBZE were quantified in the concentrations of 2.26μg/mL and 1.54μg/mL, respectively. To our knowledge, this is the first report on the simultaneous determination of CBZ and its active metabolite by direct injection of human milk serum.
Source:Journal of Chromatography B, Volumes 889–890
B.R. Lopes, J.C. Barreiro, P.T. Baraldi, Q.B. Cass
This work reports the use of a liquid chromatography ion trap tandem mass spectrometry (LC–IT-MS/MS) system for quantification in human milk samples of both carbamazepine (CBZ) and its active metabolite, carbamazepine 10,11-epoxide (CBZE). An octadecyl restricted-access media bovine serum albumin column (RAM-BSA C18) was used in single-column mode. Selectivity, extraction efficiency, accuracy and precision were achieved employing 100μL of the sample, without preparation, with detection limits of 20.0ng/mL for CBZ and 40.0ng/mL for CBZE. The matrix effect was investigated for the compounds by post-column infusion (qualitative) and by on-line extraction (quantitative). It was observed suppression effect for CBZ and CBZE by post-column infusion, ion suppression of 0.80 for CBZ, and enhancement of 1.28 for CBZE by on-line extraction. The developed method was validated and applied to analyze breast milk samples from one nursing mother. CBZ and CBZE were quantified in the concentrations of 2.26μg/mL and 1.54μg/mL, respectively. To our knowledge, this is the first report on the simultaneous determination of CBZ and its active metabolite by direct injection of human milk serum.
Degradation of the tricyclic antipsychotic drug chlorpromazine under environmental conditions, identification of its main aquatic biotic and abiotic transformation products by LC–MSn and their effects on environmental bacteria
14 March 2012, 05:07:24
Publication year: 2012
Source:Journal of Chromatography B, Volumes 889–890
Christoph Trautwein, Klaus Kümmerer
The search for environmental transformation products of organic pollutants (like drugs) is a difficult task and usually only few compounds are detected. This might be due to effective degradation but could also be a result of analytical deficits dealing with complex matrices. Especially transformation products of very low concentrations in sludge were difficult to identify so far. Additionally, the use of standard separation techniques might lead to the loss of isomeric compounds, which possess identical spectroscopic and spectrometric properties. To date no complete study investigating the environmental fate of any tricyclic antipsychotic drug has been reported. Therefore, this study investigated the popular neuroleptic drug chlorpromazine and its potential transformation by all main environmental pathways: aerobic and anaerobic biodegradation as well as abiotic photolytic degradation by sunlight. Analysis of test samples by high performance liquid chromatography coupled to multiple stage mass-spectrometry (HPLC–MS n ) allowed the detection of numerous compounds. Further, the use of a special software allowed distinguishing between transformation products of small intensities and background “noise” caused by sludge or matrix. Three aerobic tests of different bacterial density (the Closed Bottle test, OECD 301D; the Manometric Respiratory test, OECD 301F; the modified Zahn–Wellens test, 302B; one anaerobic test (a modified anaerobic degradation test according to ISO 11734) as well as a photodegradation test were performed in the present study. According to the individual test guidelines, chlorpromazine had to be classified as not biodegradable in all of the biodegradation tests. However, a special chromatographic column and gradient along with mass spectrometric fragmentation experiments of higher order uncovered the presence of a total of 61 abiotic and biotic transformation products which where formed during the course of the tests. The structures of three aerobic and one anaerobic biotransformation products were elucidated by HPLC-UV-Flourescence–MS n . Photodegradation showed almost complete elimination of chlorpromazine after 4h of irradiation with a xenon arc lamp. 57 photoproducts were found and for 28 of them LC–MS n fragmentation experiments (n =4) were performed. The molecular structures of the three main photolysis products were elucidated. The identified transformation products are expected to be found in the aquatic environment, yet nothing is known about their ecotoxicological properties. As some of the performed tests showed toxic effects of chlorpromazine or its transformation products on bacteria, further risk assessment upon this drug and its fate is strongly recommended.
Source:Journal of Chromatography B, Volumes 889–890
Christoph Trautwein, Klaus Kümmerer
The search for environmental transformation products of organic pollutants (like drugs) is a difficult task and usually only few compounds are detected. This might be due to effective degradation but could also be a result of analytical deficits dealing with complex matrices. Especially transformation products of very low concentrations in sludge were difficult to identify so far. Additionally, the use of standard separation techniques might lead to the loss of isomeric compounds, which possess identical spectroscopic and spectrometric properties. To date no complete study investigating the environmental fate of any tricyclic antipsychotic drug has been reported. Therefore, this study investigated the popular neuroleptic drug chlorpromazine and its potential transformation by all main environmental pathways: aerobic and anaerobic biodegradation as well as abiotic photolytic degradation by sunlight. Analysis of test samples by high performance liquid chromatography coupled to multiple stage mass-spectrometry (HPLC–MS n ) allowed the detection of numerous compounds. Further, the use of a special software allowed distinguishing between transformation products of small intensities and background “noise” caused by sludge or matrix. Three aerobic tests of different bacterial density (the Closed Bottle test, OECD 301D; the Manometric Respiratory test, OECD 301F; the modified Zahn–Wellens test, 302B; one anaerobic test (a modified anaerobic degradation test according to ISO 11734) as well as a photodegradation test were performed in the present study. According to the individual test guidelines, chlorpromazine had to be classified as not biodegradable in all of the biodegradation tests. However, a special chromatographic column and gradient along with mass spectrometric fragmentation experiments of higher order uncovered the presence of a total of 61 abiotic and biotic transformation products which where formed during the course of the tests. The structures of three aerobic and one anaerobic biotransformation products were elucidated by HPLC-UV-Flourescence–MS n . Photodegradation showed almost complete elimination of chlorpromazine after 4h of irradiation with a xenon arc lamp. 57 photoproducts were found and for 28 of them LC–MS n fragmentation experiments (n =4) were performed. The molecular structures of the three main photolysis products were elucidated. The identified transformation products are expected to be found in the aquatic environment, yet nothing is known about their ecotoxicological properties. As some of the performed tests showed toxic effects of chlorpromazine or its transformation products on bacteria, further risk assessment upon this drug and its fate is strongly recommended.
Determination of sinomenine sustained-release capsules in healthy Chinese volunteers by liquid chromatography–tandem mass spectrometry
14 March 2012, 05:07:24
Publication year: 2012
Source:Journal of Chromatography B, Volumes 889–890
Meng-Xiang Su, Min Song, De-Zhu Sun, Hua Zhao, Xiao Gu, Ling Zhu, Xiao-Le Zhan, Zhong-Nan Xu, Ai-Dong Wen, Tai-Jun Hang
A sensitive and selective liquid chromatographic tandem mass spectrometric method was developed and validated for the determination of sinomenine in human plasma. Plasma samples were precipitated using methanol with metronidazole as internal standard. Separation was carried out on an Inertsil ODS-3 column using a mixture of 0.2% ammonium acetate solution (A) and methanol (B) as the mobile phase with linear gradient elution as follows: 0min (50%B)→1.5min (80%B)→4.5min (80%B)→4.6min (50%B)→6.0min (50%B). All mass data were obtained in the positive ion mode, and the fragmentation transitions for the selective multiple reaction monitoring were m/z 330→181 and 172→128 for sinomenine and metronidazole, respectively. The method was fully validated to be accurate and precise with a linear range of 0.5–500ng/mL and applied to a single- and multiple-dose pharmacokinetics study of sustained-release capsules of sinomenine hydrochloride in 20 healthy Chinese volunteers. After oral administration of a single 60-mg dose, the T max, C max, AUC0–96 and t 1/2 were 7.9±2.0h, 123±22ng/mL, 3032±682ngh/mL and 13.4±1.6h, respectively. After oral administration of the 60mg capsules twice-daily for 7 consecutive days, these parameters were 4.4±3.6h, 279±69ng/mL, 7333±2096ngh/mL and 15.1±1.3h, respectively. The AUC and C max values after multiple-dose treatment were significantly higher than those after a single-dose treatment (P <0.01), with an accumulation factor of 2.49±0.77.
Source:Journal of Chromatography B, Volumes 889–890
Meng-Xiang Su, Min Song, De-Zhu Sun, Hua Zhao, Xiao Gu, Ling Zhu, Xiao-Le Zhan, Zhong-Nan Xu, Ai-Dong Wen, Tai-Jun Hang
A sensitive and selective liquid chromatographic tandem mass spectrometric method was developed and validated for the determination of sinomenine in human plasma. Plasma samples were precipitated using methanol with metronidazole as internal standard. Separation was carried out on an Inertsil ODS-3 column using a mixture of 0.2% ammonium acetate solution (A) and methanol (B) as the mobile phase with linear gradient elution as follows: 0min (50%B)→1.5min (80%B)→4.5min (80%B)→4.6min (50%B)→6.0min (50%B). All mass data were obtained in the positive ion mode, and the fragmentation transitions for the selective multiple reaction monitoring were m/z 330→181 and 172→128 for sinomenine and metronidazole, respectively. The method was fully validated to be accurate and precise with a linear range of 0.5–500ng/mL and applied to a single- and multiple-dose pharmacokinetics study of sustained-release capsules of sinomenine hydrochloride in 20 healthy Chinese volunteers. After oral administration of a single 60-mg dose, the T max, C max, AUC0–96 and t 1/2 were 7.9±2.0h, 123±22ng/mL, 3032±682ngh/mL and 13.4±1.6h, respectively. After oral administration of the 60mg capsules twice-daily for 7 consecutive days, these parameters were 4.4±3.6h, 279±69ng/mL, 7333±2096ngh/mL and 15.1±1.3h, respectively. The AUC and C max values after multiple-dose treatment were significantly higher than those after a single-dose treatment (P <0.01), with an accumulation factor of 2.49±0.77.
Development and validation of a sensitive LC–MS/MS method for the simultaneous quantitation of theophylline and its metabolites in rat plasma
14 March 2012, 05:07:24
Publication year: 2012
Source:Journal of Chromatography B, Volumes 889–890
Jung-woo Chae, Dong-hyun Kim, Byung-yo Lee, Eun jung Kim, Kwang-il Kwon
A rapid, specific, and reliable LC–MS/MS-based bioanalytical method was developed and validated in rat plasma for the simultaneous quantitation of theophylline and its four metabolites: 1,3-dimethyluric acid (1,3-DMU), 3-methylxanthine (3-MX), 1-methylxanthine (1-MX), and 1-methyluric acid (1-MU). Chromatographic separation of these analytes was achieved on a Gemini C18 column (50mm×4.60mm, 5μm) using reversed phase chromatography. The analytes were monitored by electrospray ionization in negative ion multiple reaction monitoring mode. Modification of collision energies was performed in parallel with chromatographic separation to further eliminate interference peaks. The method was validated from 0.05 to 30μg/mL for 1-MX, 1,3-DMU, 1-MU, and theophylline and from 0.1 to 30μg/mL for 3-MX using 0.2mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations (RSD) of less than 13% and with relative error (RE) values of −8.8% to 9.7%. The method was successfully applied for the quantitation of theophylline and its metabolite in rat plasma samples.
Source:Journal of Chromatography B, Volumes 889–890
Jung-woo Chae, Dong-hyun Kim, Byung-yo Lee, Eun jung Kim, Kwang-il Kwon
A rapid, specific, and reliable LC–MS/MS-based bioanalytical method was developed and validated in rat plasma for the simultaneous quantitation of theophylline and its four metabolites: 1,3-dimethyluric acid (1,3-DMU), 3-methylxanthine (3-MX), 1-methylxanthine (1-MX), and 1-methyluric acid (1-MU). Chromatographic separation of these analytes was achieved on a Gemini C18 column (50mm×4.60mm, 5μm) using reversed phase chromatography. The analytes were monitored by electrospray ionization in negative ion multiple reaction monitoring mode. Modification of collision energies was performed in parallel with chromatographic separation to further eliminate interference peaks. The method was validated from 0.05 to 30μg/mL for 1-MX, 1,3-DMU, 1-MU, and theophylline and from 0.1 to 30μg/mL for 3-MX using 0.2mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations (RSD) of less than 13% and with relative error (RE) values of −8.8% to 9.7%. The method was successfully applied for the quantitation of theophylline and its metabolite in rat plasma samples.
Purification and characterization of catalase from sprouted black gram (Vigna mungo) seeds
14 March 2012, 05:07:24
Publication year: 2012
Source:Journal of Chromatography B, Volumes 889–890
Sai Srikar Kandukuri, Ayesha Noor, S. Shiva Ranjini, M.A. Vijayalakshmi
Black gram (Vigna mungo) is a legume which belongs to Fabaceae family. It is a rich source of protein. It has been known to have interesting small molecule antioxidant activity. However, its enzymatic antioxidant properties have not been explored much. In the present work we studied catalase, a principal antioxidant enzyme from black gram seeds. Day four sprouted black gram seeds were found to have a significant catalase content approximately of 15,240U/g seeds. IMAC (Seph 4B-IDA-Zn(II)) was used for purifying this catalase, a purification fold of 106 and a high specific activity of 25,704U/mg was obtained. The K m and V max of the purified catalase were found to be 16.2mM and 2.5μmol/min. The effect of inhibitors like Sodium azide (NaN3) and EDTA and different metal ions on catalase activity were studied. NaN3, Fe3+and Cu2+ were found to have profound inhibitory effects on the enzyme activity. Other metal ions like Ni2+, Ca2+, Mg2+ and Mn2+ had both enhancing and inhibitory effects. The enzyme showed optimal activity at a temperature of 40°C and pH 7.0. It was stable over a broad range of pH 6.0–10.0 and had a half life of 7h 30min at 50°C.
Source:Journal of Chromatography B, Volumes 889–890
Sai Srikar Kandukuri, Ayesha Noor, S. Shiva Ranjini, M.A. Vijayalakshmi
Black gram (Vigna mungo) is a legume which belongs to Fabaceae family. It is a rich source of protein. It has been known to have interesting small molecule antioxidant activity. However, its enzymatic antioxidant properties have not been explored much. In the present work we studied catalase, a principal antioxidant enzyme from black gram seeds. Day four sprouted black gram seeds were found to have a significant catalase content approximately of 15,240U/g seeds. IMAC (Seph 4B-IDA-Zn(II)) was used for purifying this catalase, a purification fold of 106 and a high specific activity of 25,704U/mg was obtained. The K m and V max of the purified catalase were found to be 16.2mM and 2.5μmol/min. The effect of inhibitors like Sodium azide (NaN3) and EDTA and different metal ions on catalase activity were studied. NaN3, Fe3+and Cu2+ were found to have profound inhibitory effects on the enzyme activity. Other metal ions like Ni2+, Ca2+, Mg2+ and Mn2+ had both enhancing and inhibitory effects. The enzyme showed optimal activity at a temperature of 40°C and pH 7.0. It was stable over a broad range of pH 6.0–10.0 and had a half life of 7h 30min at 50°C.
On-line simultaneous deproteinization of biological samples and trace enrichment of three dipine series using a poly(N-isopropylacrylamide-co-ethyleneglycol dimethacrylate) monolith
14 March 2012, 05:07:24
Publication year: 2012
Source:Journal of Chromatography B, Volumes 889–890
Haiyan Liu, Yanhui Duan, Yanhua Jia, Yanzhao Gu, Jia Li, Cuihong Yan, Gengliang Yang
A porous poly(N-isopropylacrylamide-co-ethyleneglycol dimethacrylate) [poly(NIPAAm-co-EDMA)] monolithic column was prepared by in situ free-radical polymerization. The morphology of monolithic column and pressure drop across the columns were characterized. The results showed excellent permeability and high selectivity. Nifedipine, nitrendipine and nisoldipine were simultaneously selected to validate the extraction efficiency of the prepared monolith both in plasma and urine. The extracted nifedipine, nitrendipine and nisoldipine from plasma and urine samples have been on-line tested quantitatively by using the prepared monolith connected with RP-C18 column. The total analytical run time was 38min. For all analytes, linear calibration curves were obtained over a range of 2–500ng/mL with coefficient of correlation>0.997. Precision for inter- and intra-day assay showed acceptable results for quantitative assay with relative standard deviation (RSD) less than 12%. The accuracy and recovery was found to be in the range of 89–109% and 88–106%. The results indicated that the prepared monolith was feasible to be used as an on-line SPE sorbent material and the method was especially appropriate for multi-analytes monitoring in plasma and urine samples. Finally, the proposed method was successfully applied to simultaneously screen nifedipine, nitrendipine and nisoldipine in plasma.
Source:Journal of Chromatography B, Volumes 889–890
Haiyan Liu, Yanhui Duan, Yanhua Jia, Yanzhao Gu, Jia Li, Cuihong Yan, Gengliang Yang
A porous poly(N-isopropylacrylamide-co-ethyleneglycol dimethacrylate) [poly(NIPAAm-co-EDMA)] monolithic column was prepared by in situ free-radical polymerization. The morphology of monolithic column and pressure drop across the columns were characterized. The results showed excellent permeability and high selectivity. Nifedipine, nitrendipine and nisoldipine were simultaneously selected to validate the extraction efficiency of the prepared monolith both in plasma and urine. The extracted nifedipine, nitrendipine and nisoldipine from plasma and urine samples have been on-line tested quantitatively by using the prepared monolith connected with RP-C18 column. The total analytical run time was 38min. For all analytes, linear calibration curves were obtained over a range of 2–500ng/mL with coefficient of correlation>0.997. Precision for inter- and intra-day assay showed acceptable results for quantitative assay with relative standard deviation (RSD) less than 12%. The accuracy and recovery was found to be in the range of 89–109% and 88–106%. The results indicated that the prepared monolith was feasible to be used as an on-line SPE sorbent material and the method was especially appropriate for multi-analytes monitoring in plasma and urine samples. Finally, the proposed method was successfully applied to simultaneously screen nifedipine, nitrendipine and nisoldipine in plasma.
Purification strategies, characteristics and thermodynamic analysis of a highly thermostable alkaline protease from a salt-tolerant alkaliphilic actinomycete, Nocardiopsis alba OK-5
14 March 2012, 05:07:24
Publication year: 2012
Source:Journal of Chromatography B, Volumes 889–890
Sangeeta D. Gohel, Satya P. Singh
An alkaline protease from salt tolerant alkaliphilic actinomycetes, Nocardiopsis alba strain OK-5 was purified to homogeneity by 27 and 13 fold with a yield of 35 and 13% using two-steps and one-step method, respectively. The purification methods involved hydrophobic interaction on phenyl sapharose matrix. The apparent molecular mass was 20kDa. The temperature optimum shifted from 70 to 80°C in 4M NaCl and 30% Na-glutamate, with significant stability at 60–80°C in Na-glutamate. Deactivation rate constant (K d) increased and half life (t 1/2) decreased with the increasing temperatures from 37 to 80°C. The order of stability was: 30% Na-glutamate>4M NaCl>2M NaCl>0M NaCl. The enzyme was stable even at 80°C in 30% Na-glutamate with K d 4.11 and t 1/2 168.64min. The activation energies (E), enthalpy (ΔH*) and entropy (ΔS*) for protease deactivation in with Na-glutamate were 31.97kJ/mole, 29.23kJ/mole and −211.83J/mole, respectively. The change in free energy (ΔG*) for protease deactivation at 60°C in 30% Na-glutamate was 101.70kJ/mole. Protease had the highest activity and stability at pH 10–11. While the enzyme was highly resistant against chemical denaturation, it had varied responses to metal ions. Complete inhibition by PMSF confirmed serine nature of the protease. Na-glutamate, H2O2, β-mercaptoethanol and different surfactants enhanced the activity.
Source:Journal of Chromatography B, Volumes 889–890
Sangeeta D. Gohel, Satya P. Singh
An alkaline protease from salt tolerant alkaliphilic actinomycetes, Nocardiopsis alba strain OK-5 was purified to homogeneity by 27 and 13 fold with a yield of 35 and 13% using two-steps and one-step method, respectively. The purification methods involved hydrophobic interaction on phenyl sapharose matrix. The apparent molecular mass was 20kDa. The temperature optimum shifted from 70 to 80°C in 4M NaCl and 30% Na-glutamate, with significant stability at 60–80°C in Na-glutamate. Deactivation rate constant (K d) increased and half life (t 1/2) decreased with the increasing temperatures from 37 to 80°C. The order of stability was: 30% Na-glutamate>4M NaCl>2M NaCl>0M NaCl. The enzyme was stable even at 80°C in 30% Na-glutamate with K d 4.11 and t 1/2 168.64min. The activation energies (E), enthalpy (ΔH*) and entropy (ΔS*) for protease deactivation in with Na-glutamate were 31.97kJ/mole, 29.23kJ/mole and −211.83J/mole, respectively. The change in free energy (ΔG*) for protease deactivation at 60°C in 30% Na-glutamate was 101.70kJ/mole. Protease had the highest activity and stability at pH 10–11. While the enzyme was highly resistant against chemical denaturation, it had varied responses to metal ions. Complete inhibition by PMSF confirmed serine nature of the protease. Na-glutamate, H2O2, β-mercaptoethanol and different surfactants enhanced the activity.
A method for the simultaneous determination of mercapturic acids as biomarkers of exposure to 2-chloroprene and epichlorohydrin in human urine
14 March 2012, 05:07:24
Publication year: 2012
Source:Journal of Chromatography B, Volumes 889–890
Elisabeth Eckert, Gabriele Leng, Wolfgang Gries, Thomas Göen
We developed and validated an analytical method for the simultaneous determination of several chlorine and non-chlorine containing mercapturic acids in urine as specific metabolites of the hazardous chemicals 2-chloroprene and epichlorohydrin. The method involves an online column switching arrangement for online solid phase extraction of the analytes with subsequent analytical separation and detection using LC–MS/MS. The developed method enables for the first time the determination of Cl-MA-I (4-chloro-3-oxobutyl mercapturic acid), Cl-MA-II (4-chloro-3-hydroxybutyl mercapturic acid), Cl-MA-III (3-chloro-2-hydroxy-3-butenyl mercapturic acid) and HOBMA (4-hydroxy-3-oxobutyl mercapturic acid) as potential biomarkers of 2-chloroprene in urine. Additionally, CHPMA (3-chloro-2-hydroxypropyl mercapturic acid) as a specific metabolite of epichlorohydrin in urine and DHBMA (3,4-dihydroxybutyl mercapturic acid) can be determined. The analytical method proved to be both sensitive and reliable with detection limits ranging from 1.4μg/L (for Cl-MA-III) to 4.2μg/L (for HOBMA). Intra- and interday imprecision was determined to range from 4.7 to 11.8%. Due to the good accuracy and precision and the low limits of detection the developed method is well suited for application in biomonitoring studies in order to determine occupational exposure to 2-chloroprene and epichlorohydrin.
Source:Journal of Chromatography B, Volumes 889–890
Elisabeth Eckert, Gabriele Leng, Wolfgang Gries, Thomas Göen
We developed and validated an analytical method for the simultaneous determination of several chlorine and non-chlorine containing mercapturic acids in urine as specific metabolites of the hazardous chemicals 2-chloroprene and epichlorohydrin. The method involves an online column switching arrangement for online solid phase extraction of the analytes with subsequent analytical separation and detection using LC–MS/MS. The developed method enables for the first time the determination of Cl-MA-I (4-chloro-3-oxobutyl mercapturic acid), Cl-MA-II (4-chloro-3-hydroxybutyl mercapturic acid), Cl-MA-III (3-chloro-2-hydroxy-3-butenyl mercapturic acid) and HOBMA (4-hydroxy-3-oxobutyl mercapturic acid) as potential biomarkers of 2-chloroprene in urine. Additionally, CHPMA (3-chloro-2-hydroxypropyl mercapturic acid) as a specific metabolite of epichlorohydrin in urine and DHBMA (3,4-dihydroxybutyl mercapturic acid) can be determined. The analytical method proved to be both sensitive and reliable with detection limits ranging from 1.4μg/L (for Cl-MA-III) to 4.2μg/L (for HOBMA). Intra- and interday imprecision was determined to range from 4.7 to 11.8%. Due to the good accuracy and precision and the low limits of detection the developed method is well suited for application in biomonitoring studies in order to determine occupational exposure to 2-chloroprene and epichlorohydrin.
Liquid chromatography and tandem mass spectrometry method for the quantitative determination of saxagliptin and its major pharmacologically active 5-monohydroxy metabolite in human plasma: Method validation and overcoming specific and non-specific binding at low concentrations
14 March 2012, 05:07:24
Publication year: 2012
Source:Journal of Chromatography B, Volumes 889–890
Xiaohui (Sophia) Xu, Roger Demers, Huidong Gu, Lisa J. Christopher, Hong Su, Laura Cojocaru, David W. Boulton, Mark Kirby, Bruce Stouffer, William G. Humphreys, Mark E. Arnold
A liquid chromatography and tandem mass spectrometry (LC–MS/MS) method was developed and validated to simultaneously determine the concentrations of saxagliptin (Onglyza™, BMS-477118) and its major active metabolite, 5-hydroxy saxagliptin to support pharmacokinetic analyses in clinical studies. The dynamic range of the assay was 0.1–50ng/mL for saxagliptin and 0.2–100ng/mL for 5-hydroxy saxagliptin. Protein precipitation (PPT) with acetonitrile was used to extract the analytes from plasma matrix before injecting on an Atlantis® dC18 column (50mm×2.1mm, 5μm) for LC–MS/MS analysis. The sample pre-treatment process was carefully controlled to disrupt DPP4-specific binding and non-specific binding observed at lower concentrations. The recoveries for both analytes were >90%. The assay was selective, rugged and reproducible; storage stability of at least 401 days at −20°C was demonstrated. Under these chromatographic conditions, the isomers of saxagliptin and 5-hydroxy saxagliptin were chromatographically separated from saxagliptin and 5-hydroxy saxagliptin. The assay has been used to support multiple clinical studies and regulatory approvals.
Source:Journal of Chromatography B, Volumes 889–890
Xiaohui (Sophia) Xu, Roger Demers, Huidong Gu, Lisa J. Christopher, Hong Su, Laura Cojocaru, David W. Boulton, Mark Kirby, Bruce Stouffer, William G. Humphreys, Mark E. Arnold
A liquid chromatography and tandem mass spectrometry (LC–MS/MS) method was developed and validated to simultaneously determine the concentrations of saxagliptin (Onglyza™, BMS-477118) and its major active metabolite, 5-hydroxy saxagliptin to support pharmacokinetic analyses in clinical studies. The dynamic range of the assay was 0.1–50ng/mL for saxagliptin and 0.2–100ng/mL for 5-hydroxy saxagliptin. Protein precipitation (PPT) with acetonitrile was used to extract the analytes from plasma matrix before injecting on an Atlantis® dC18 column (50mm×2.1mm, 5μm) for LC–MS/MS analysis. The sample pre-treatment process was carefully controlled to disrupt DPP4-specific binding and non-specific binding observed at lower concentrations. The recoveries for both analytes were >90%. The assay was selective, rugged and reproducible; storage stability of at least 401 days at −20°C was demonstrated. Under these chromatographic conditions, the isomers of saxagliptin and 5-hydroxy saxagliptin were chromatographically separated from saxagliptin and 5-hydroxy saxagliptin. The assay has been used to support multiple clinical studies and regulatory approvals.
Evaluation of enantioselective binding of propanocaine to human serum albumin by ultrafiltration and electrokinetic chromatography under intermediate precision conditions
14 March 2012, 05:07:24
Publication year: 2012
Source:Journal of Chromatography B, Volumes 889–890
María Amparo Martínez-Gómez, Laura Escuder-Gilabert, Rosa María Villanueva-Camañas, Salvador Sagrado, María José Medina-Hernández
Stereoselectivity in protein binding can have a significant effect on the pharmacokinetic and pharmacodynamic properties of chiral drugs. In this paper, the enantioselective binding of propanocaine (PRO) enantiomers to human serum albumin (HSA), the most relevant plasmatic protein in view of stereoselectivity, has been evaluated by incubation and ultrafiltration of racemic PRO–HSA mixtures and chiral analysis of the bound and unbound fractions by electrokinetic chromatography using HSA as chiral selector. Experimental conditions for the separation of PRO enantiomers using HSA as chiral selector and electrokinetic chromatography have been optimised. Affinity constants and protein binding in percentage (PB) were obtained for both enantiomers of PRO, as well as the enantioselectivity (ES) to HSA. Data were obtained in two independent working sessions (days). The influence of the session and fraction processed factors were examined. A univariate direct-estimation approach was used facilitating outliers’ identification and statistical comparison. Non-linear fitting of data was used to verify the stoichiometry and affinity estimations obtained by the direct approach. Robust statistics were applied to obtain reliable estimations of uncertainty, accounting for the factors (day and processed fraction), thus representing intermediate precision conditions. Mimicking in vivo experimental conditions, information unapproachable by in vivo experiments was obtained for PRO enantiomers interacting with HSA. For the first (E1) and the second (E2) eluted PRO enantiomers the results were: 1:1 stoichiometry, medium affinity constants, log K E1 =3.20±0.16 and log K E2 =3.40±0.14, medium protein binding percentage, PB=48.7 and 60.1% for E1 and E2, respectively, and moderate but significant enantioselectivity, ES= K E2/K E1 =1.5±0.3.
Source:Journal of Chromatography B, Volumes 889–890
María Amparo Martínez-Gómez, Laura Escuder-Gilabert, Rosa María Villanueva-Camañas, Salvador Sagrado, María José Medina-Hernández
Stereoselectivity in protein binding can have a significant effect on the pharmacokinetic and pharmacodynamic properties of chiral drugs. In this paper, the enantioselective binding of propanocaine (PRO) enantiomers to human serum albumin (HSA), the most relevant plasmatic protein in view of stereoselectivity, has been evaluated by incubation and ultrafiltration of racemic PRO–HSA mixtures and chiral analysis of the bound and unbound fractions by electrokinetic chromatography using HSA as chiral selector. Experimental conditions for the separation of PRO enantiomers using HSA as chiral selector and electrokinetic chromatography have been optimised. Affinity constants and protein binding in percentage (PB) were obtained for both enantiomers of PRO, as well as the enantioselectivity (ES) to HSA. Data were obtained in two independent working sessions (days). The influence of the session and fraction processed factors were examined. A univariate direct-estimation approach was used facilitating outliers’ identification and statistical comparison. Non-linear fitting of data was used to verify the stoichiometry and affinity estimations obtained by the direct approach. Robust statistics were applied to obtain reliable estimations of uncertainty, accounting for the factors (day and processed fraction), thus representing intermediate precision conditions. Mimicking in vivo experimental conditions, information unapproachable by in vivo experiments was obtained for PRO enantiomers interacting with HSA. For the first (E1) and the second (E2) eluted PRO enantiomers the results were: 1:1 stoichiometry, medium affinity constants, log K E1 =3.20±0.16 and log K E2 =3.40±0.14, medium protein binding percentage, PB=48.7 and 60.1% for E1 and E2, respectively, and moderate but significant enantioselectivity, ES= K E2/K E1 =1.5±0.3.
Molecular imprinting based composite cryogel membranes for purification of anti-hepatitis B surface antibody by fast protein liquid chromatography
14 March 2012, 05:07:24
Publication year: 2012
Source:Journal of Chromatography B, Volumes 889–890
Sevgi Asliyuce, Lokman Uzun, Abbas Yousefi Rad, Serhat Unal, Ridvan Say, Adil Denizli
In the present study, we have focused our attention to prepare molecular imprinted composite cryogel membranes for purification of hepatitis B surface antibody (anti-HBs) by fast protein liquid chromatography. Before the preparation of the molecular imprinted composite cryogel membranes (MI-CMs) by free radical polymerization at sub-zero temperature, we have synthesized and characterized the anti-HBs imprinted particles. Then, the cryogel membranes (CMs) were characterized by swelling test, scanning electron microscopy and Fourier transform infrared spectroscopy. Prior to chromatographic purification studies, the effective parameters on the anti-HBs adsorption process were evaluated by investigating the dependency of the adsorption capacity on flow-rate, anti-HBs concentration, contact time and ionic strength. The maximum anti-HBs adsorption capacity was calculated as 701.4mIU/g CM. The selectivity of the MI-CMs was shown by competitive adsorption of anti-HBs, total anti-hepatitis A antibody (anti-HAV) and total immunoglobulin E (IgE) adsorption studies. The MI-CMs have relative selectivity coefficients as 5.45 for anti-HBs/total anti-HAV and 9.05 for anti-HBs/total IgE, respectively. The phosphate buffer solution (pH 7.4) containing 1.0M NaCl was used for elution, almost completely, of adsorbed anti-HBs molecules. The MI-CMs could be used many times without any significant decrease in the adsorption capacity. The chromatographic purification performances of the MI-CMs were also investigated. The chromatographic parameters such as capacity and separation factors, the theoretical plate number and resolution of the MI-CMs were calculated as 5.48, 6.02, 1153.9, and 1.72 for anti-HBs molecules, respectively. As a conclusion, we can say that the MI-CMs could be used for specific purification of anti-HBs from anti-HBs positive human plasma.
Source:Journal of Chromatography B, Volumes 889–890
Sevgi Asliyuce, Lokman Uzun, Abbas Yousefi Rad, Serhat Unal, Ridvan Say, Adil Denizli
In the present study, we have focused our attention to prepare molecular imprinted composite cryogel membranes for purification of hepatitis B surface antibody (anti-HBs) by fast protein liquid chromatography. Before the preparation of the molecular imprinted composite cryogel membranes (MI-CMs) by free radical polymerization at sub-zero temperature, we have synthesized and characterized the anti-HBs imprinted particles. Then, the cryogel membranes (CMs) were characterized by swelling test, scanning electron microscopy and Fourier transform infrared spectroscopy. Prior to chromatographic purification studies, the effective parameters on the anti-HBs adsorption process were evaluated by investigating the dependency of the adsorption capacity on flow-rate, anti-HBs concentration, contact time and ionic strength. The maximum anti-HBs adsorption capacity was calculated as 701.4mIU/g CM. The selectivity of the MI-CMs was shown by competitive adsorption of anti-HBs, total anti-hepatitis A antibody (anti-HAV) and total immunoglobulin E (IgE) adsorption studies. The MI-CMs have relative selectivity coefficients as 5.45 for anti-HBs/total anti-HAV and 9.05 for anti-HBs/total IgE, respectively. The phosphate buffer solution (pH 7.4) containing 1.0M NaCl was used for elution, almost completely, of adsorbed anti-HBs molecules. The MI-CMs could be used many times without any significant decrease in the adsorption capacity. The chromatographic purification performances of the MI-CMs were also investigated. The chromatographic parameters such as capacity and separation factors, the theoretical plate number and resolution of the MI-CMs were calculated as 5.48, 6.02, 1153.9, and 1.72 for anti-HBs molecules, respectively. As a conclusion, we can say that the MI-CMs could be used for specific purification of anti-HBs from anti-HBs positive human plasma.
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