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Comparison of GC/MS and LC/MS methods for the analysis of propofol and its metabolites in urine
27 June 2012,
08:45:31
Publication year:
2012
Source:Journal of Chromatography B, Volume 900
Sun Young Lee, Na-Hyun Park, Eun-Kyung Jeong, Jae-Woo Wi, Chang-Ju Kim, Jin Young Kim, Moon Kyo In, Jongki Hong
Gas chromatography–mass spectrometry (GC/MS) and liquid chromatography–mass spectrometry (LC/MS) were compared for their capacity to metabolite identification, sensitivity, and speed of analysis for propofol and its metabolites in urine samples. Acidic hydrolysis, liquid–liquid extraction (LLE), and trimethylsilyl (TMS) derivatization procedures were applied for GC/MS analysis. The LC/MS analysis used a simple sample pretreatment based on centrifugation and dilution. Propofol and four metabolites were successfully analyzed by GC/MS following TMS derivatization. One compound, di-isopropanolphenol was tentatively characterized as a new metabolite observed for the first time in human urine. The TMS derivatization greatly improved the chromatographic properties and detection sensitivity, especially for hydroxylated metabolites. The lower limits of quantitation (LLOQ) of propofol were about 325 and 0.51ng/mL for the GC/MS scan mode and selected ion monitoring (SIM) mode, respectively. In addition, five conjugated propofol metabolites were successfully analyzed by LC–MS/MS in negative ion mode. The detection sensitivity for these conjugated metabolites could be greatly enhanced by the addition of triethylamine to the mobile phase without any loss of LC resolution capacity. The LLOQs of propofol-glucuronide (PG) were about 1.17 and 2.01ng/mL for the LC–MS-selected ion monitoring (SIM) and multiple reaction monitoring (MRM) mode, respectively. Both GC/MS and LC/MS methods sensitively detected nine metabolites of propofol and could be used to provide complementary data for the reasonable propofol metabolism study. Urinary excretion profiles for propofol and its metabolites following administration to human were suggested based on the total ion chromatograms obtained by GC/MS and LC/MS methods, respectively.
Source:Journal of Chromatography B, Volume 900
Sun Young Lee, Na-Hyun Park, Eun-Kyung Jeong, Jae-Woo Wi, Chang-Ju Kim, Jin Young Kim, Moon Kyo In, Jongki Hong
Gas chromatography–mass spectrometry (GC/MS) and liquid chromatography–mass spectrometry (LC/MS) were compared for their capacity to metabolite identification, sensitivity, and speed of analysis for propofol and its metabolites in urine samples. Acidic hydrolysis, liquid–liquid extraction (LLE), and trimethylsilyl (TMS) derivatization procedures were applied for GC/MS analysis. The LC/MS analysis used a simple sample pretreatment based on centrifugation and dilution. Propofol and four metabolites were successfully analyzed by GC/MS following TMS derivatization. One compound, di-isopropanolphenol was tentatively characterized as a new metabolite observed for the first time in human urine. The TMS derivatization greatly improved the chromatographic properties and detection sensitivity, especially for hydroxylated metabolites. The lower limits of quantitation (LLOQ) of propofol were about 325 and 0.51ng/mL for the GC/MS scan mode and selected ion monitoring (SIM) mode, respectively. In addition, five conjugated propofol metabolites were successfully analyzed by LC–MS/MS in negative ion mode. The detection sensitivity for these conjugated metabolites could be greatly enhanced by the addition of triethylamine to the mobile phase without any loss of LC resolution capacity. The LLOQs of propofol-glucuronide (PG) were about 1.17 and 2.01ng/mL for the LC–MS-selected ion monitoring (SIM) and multiple reaction monitoring (MRM) mode, respectively. Both GC/MS and LC/MS methods sensitively detected nine metabolites of propofol and could be used to provide complementary data for the reasonable propofol metabolism study. Urinary excretion profiles for propofol and its metabolites following administration to human were suggested based on the total ion chromatograms obtained by GC/MS and LC/MS methods, respectively.
Highlights
► Comprehensive GC/MS and LC/MS methods were proposed for analysis of propofol and metabolites. ► Both methods provided excellent sensitivity and selectivity in urine samples. ► 2,6-ω-Di-isopropanol phenol as new metabolite was first observed in human. ► LLOQ was 0.51ng/mL for propofol and 2.01ng/mL for propofol-glucuronide.Quantitative analysis of the opioid peptide DAMGO in rat plasma and microdialysis samples using liquid chromatography–tandem mass spectrometry
27 June 2012,
08:45:31
Publication year:
2012
Source:Journal of Chromatography B, Volume 900
Annika Lindqvist, Britt Jansson, Margareta Hammarlund-Udenaes
A liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) method for the quantification of the opioid peptide DAMGO in rat plasma, as well as DAMGO and the microdialysis recovery calibrator [13C2,15N]-DAMGO in microdialysis samples, is described. The microdialysis samples consisted of 15μL Ringer solution containing 0.5% bovine serum albumin. Pretreatment of the samples involved protein precipitation with acetonitrile followed by dilution with 0.01% formic acid. The lower limits of quantification were 0.52ng/mL and 0.24ng/mL for DAMGO and [13C2,15N]-DAMGO respectively and the response was linear up to 5000 fold higher concentrations. The plasma samples (50μL) were precipitated with acetonitrile containing the isotope labeled analog [13C2,15N]-DAMGO as internal standard. The method was linear in the range of 11–110,000ng/mL. The separations were conducted on a HyPurity C18 column, 50×4.6mm, 3μm particle size, with a mobile phase consisting of acetonitrile, water and formic acid to the proportions of 17.5:82.5:0.01. Low energy collision dissociation tandem mass spectrometric (CID-MS/MS) analysis was carried out in the positive ion mode using multiple reaction monitoring (MRM) of the following mass transitions: m/z 514.2→453.2 for DAMGO and m/z 517.2→456.2 for [13C2,15N]-DAMGO. The intra-day precision and accuracy did not exceed 5.2% and 93–104% for both compounds and sample types described. The inter-day precision an accuracy were <6.8% and 95–105% respectively. The method described is simple, reproducible and suitable for the analysis of small sample volumes at low concentrations.
Source:Journal of Chromatography B, Volume 900
Annika Lindqvist, Britt Jansson, Margareta Hammarlund-Udenaes
A liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) method for the quantification of the opioid peptide DAMGO in rat plasma, as well as DAMGO and the microdialysis recovery calibrator [13C2,15N]-DAMGO in microdialysis samples, is described. The microdialysis samples consisted of 15μL Ringer solution containing 0.5% bovine serum albumin. Pretreatment of the samples involved protein precipitation with acetonitrile followed by dilution with 0.01% formic acid. The lower limits of quantification were 0.52ng/mL and 0.24ng/mL for DAMGO and [13C2,15N]-DAMGO respectively and the response was linear up to 5000 fold higher concentrations. The plasma samples (50μL) were precipitated with acetonitrile containing the isotope labeled analog [13C2,15N]-DAMGO as internal standard. The method was linear in the range of 11–110,000ng/mL. The separations were conducted on a HyPurity C18 column, 50×4.6mm, 3μm particle size, with a mobile phase consisting of acetonitrile, water and formic acid to the proportions of 17.5:82.5:0.01. Low energy collision dissociation tandem mass spectrometric (CID-MS/MS) analysis was carried out in the positive ion mode using multiple reaction monitoring (MRM) of the following mass transitions: m/z 514.2→453.2 for DAMGO and m/z 517.2→456.2 for [13C2,15N]-DAMGO. The intra-day precision and accuracy did not exceed 5.2% and 93–104% for both compounds and sample types described. The inter-day precision an accuracy were <6.8% and 95–105% respectively. The method described is simple, reproducible and suitable for the analysis of small sample volumes at low concentrations.
Highlights
► Analysis of DAMGO in microdialysis samples and plasma using LC–MS/MS is described. ► Plasma method was linear between 11 and 110,000ng/mL. Sample volume was 50μL. ► Microdialysis method was linear between 0.52 and 2600ng/mL. Sample volume was 15μL. ► The intra-day and inter-day precision and accuracy did not exceed 6.8% and 93–105%. ► The method was applied to determine the brain distribution of DAMGO in rat.Screening, recognition and quantitation of salbutamol residues in ham sausages by molecularly imprinted solid phase extraction coupled with high-performance liquid chromatography–ultraviolet detection
27 June 2012,
08:45:31
Publication year:
2012
Source:Journal of Chromatography B, Volume 900
Hongyuan Yan, Ruiling Wang, Yehong Han, Suting Liu
A highly selective molecularly imprinted solid phase extraction (MISPE) coupled with liquid chromatography–ultraviolet detection was developed for the determination of salbutamol (SAL) in ham sausages. New molecularly imprinted polymers (MIPs) were synthesized with phenylephrine as dummy template and it revealed good affinity to SAL in methanol–acetonitrile system. Adsorption capacity of the MIPs was evaluated by dynamic adsorption experiments. The MIPs were used as SPE sorbent for the selective clean-up and pre-concentration of SAL in ham sausage samples. The results showed that the matrix compounds presented in ham sausage samples could be removed effectively and the recoveries of SAL at three spiked levels were ranged from 82.6 to 100.5% with the relative standard deviation (RSD) of less than 3.6%. This method is simple and sensitive, and is therefore an alternative tool to the existing methods for analyzing residual SAL in biological samples.
Source:Journal of Chromatography B, Volume 900
Hongyuan Yan, Ruiling Wang, Yehong Han, Suting Liu
A highly selective molecularly imprinted solid phase extraction (MISPE) coupled with liquid chromatography–ultraviolet detection was developed for the determination of salbutamol (SAL) in ham sausages. New molecularly imprinted polymers (MIPs) were synthesized with phenylephrine as dummy template and it revealed good affinity to SAL in methanol–acetonitrile system. Adsorption capacity of the MIPs was evaluated by dynamic adsorption experiments. The MIPs were used as SPE sorbent for the selective clean-up and pre-concentration of SAL in ham sausage samples. The results showed that the matrix compounds presented in ham sausage samples could be removed effectively and the recoveries of SAL at three spiked levels were ranged from 82.6 to 100.5% with the relative standard deviation (RSD) of less than 3.6%. This method is simple and sensitive, and is therefore an alternative tool to the existing methods for analyzing residual SAL in biological samples.
Highlights
► A simple and rapid MISPE–HPLC method for determination of salbutamol in ham sausages. ► The new MIPs were obtained by phenylephrine as dummy template in methanol–acetonitrile solution. ► Rapid screening of salbutamol in ham sausages, while interferences from milk matrix were eliminated. ► Extraction efficiency was markedly increased with reduced equilibrium time. ► This method improved the selectivity and eliminated the effect of template leakage on quantitative analysis.Detection of Δ4-3-oxo-steroid 5β-reductase deficiency by LC–ESI-MS/MS measurement of urinary bile acids
27 June 2012,
08:45:31
Publication year:
2012
Source:Journal of Chromatography B, Volume 900
Akina Muto, Hajime Takei, Atsushi Unno, Tsuyoshi Murai, Takao Kurosawa, Shoujiro Ogawa, Takashi Iida, Shigeo Ikegawa, Jun Mori, Akira Ohtake, Takayuki Hoshina, Tatsuki Mizuochi, Akihiko Kimura, Alan F. Hofmann, Lee R. Hagey, Hiroshi Nittono
The synthesis of bile salts from cholesterol is a complex biochemical pathway involving at least 16 enzymes. Most inborn errors of bile acid biosynthesis result in excessive formation of intermediates and/or their metabolites that accumulate in blood and are excreted in part in urine. Early detection is important as oral therapy with bile acids results in improvement. In the past, these intermediates in bile acid biosynthesis have been detected in neonatal blood or urine by screening with FAB-MS followed by detailed characterization using GC–MS. Both methods have proved difficult to automate, and currently most laboratories screen candidate samples using LC–MS/MS. Here, we describe a new, simple and sensitive analytical method for the identification and characterization of 39 conjugated and unconjugated bile acids, including Δ4-3-oxo- and Δ4,6-3-oxo-bile acids (markers for Δ4-3-oxo-steroid 5β-reductase deficiency), using liquid chromatography-electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). In this procedure a concentrated, desalted urinary sample (diluted with ethanol) is injected directly into the LC–ESI-MS/MS, operated with ESI and in the negative ion mode; quantification is obtained by selected reaction monitoring (SRM). To evaluate the performance of our new method, we compared it to a validated method using GC–MS, in the analysis of urine from two patients with genetically confirmed Δ4-3-oxo-steroid 5β-reductase deficiency as well as a third patient with an elevated concentration of abnormal conjugated and unconjugated Δ4-3-oxo-bile acids. The Δ4-3-oxo-bile acids concentration recovered in three patients with 5β-reductase deficiency were 48.8, 58.9, and 49.4μmol/mmol creatinine, respectively by LC–ESI-MS/MS.
Source:Journal of Chromatography B, Volume 900
Akina Muto, Hajime Takei, Atsushi Unno, Tsuyoshi Murai, Takao Kurosawa, Shoujiro Ogawa, Takashi Iida, Shigeo Ikegawa, Jun Mori, Akira Ohtake, Takayuki Hoshina, Tatsuki Mizuochi, Akihiko Kimura, Alan F. Hofmann, Lee R. Hagey, Hiroshi Nittono
The synthesis of bile salts from cholesterol is a complex biochemical pathway involving at least 16 enzymes. Most inborn errors of bile acid biosynthesis result in excessive formation of intermediates and/or their metabolites that accumulate in blood and are excreted in part in urine. Early detection is important as oral therapy with bile acids results in improvement. In the past, these intermediates in bile acid biosynthesis have been detected in neonatal blood or urine by screening with FAB-MS followed by detailed characterization using GC–MS. Both methods have proved difficult to automate, and currently most laboratories screen candidate samples using LC–MS/MS. Here, we describe a new, simple and sensitive analytical method for the identification and characterization of 39 conjugated and unconjugated bile acids, including Δ4-3-oxo- and Δ4,6-3-oxo-bile acids (markers for Δ4-3-oxo-steroid 5β-reductase deficiency), using liquid chromatography-electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). In this procedure a concentrated, desalted urinary sample (diluted with ethanol) is injected directly into the LC–ESI-MS/MS, operated with ESI and in the negative ion mode; quantification is obtained by selected reaction monitoring (SRM). To evaluate the performance of our new method, we compared it to a validated method using GC–MS, in the analysis of urine from two patients with genetically confirmed Δ4-3-oxo-steroid 5β-reductase deficiency as well as a third patient with an elevated concentration of abnormal conjugated and unconjugated Δ4-3-oxo-bile acids. The Δ4-3-oxo-bile acids concentration recovered in three patients with 5β-reductase deficiency were 48.8, 58.9, and 49.4μmol/mmol creatinine, respectively by LC–ESI-MS/MS.
Highlights
► We analyze 39 bile acids including abnormal bile acids by LC–ESI-MS/MS. ► We analyze urine from patients with 5β-reductase deficiency by LC–ESI-MS/MS. ► The urine from those patients elevated concentration of Δ4-3-oxo-bile acids. ► The results obtained with LC–ESI-MS/MS agree quite well with those obtained by GC–MS.Production, recovery and purification of a recombinant β-galactosidase by expanded bed anion exchange adsorption
27 June 2012,
08:45:31
Publication year:
2012
Source:Journal of Chromatography B, Volume 900
Valeria Boeris, Izabella Balce, Rami Reddy Vennapusa, Miguel Arévalo Rodríguez, Guillermo Picó, Marcelo Fernández Lahore
β-Galactosidase is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides; its major application in the food industry is to reduce the content of lactose in lactic products. The aim of this work is to recover this enzyme from a cell lysate by adsorption onto Streamline-DEAE in an expanded bed, avoiding, as much as possible, biomass deposition onto the adsorbent matrix. So as to achieve less cell debris–matrix interaction, the adsorbent surface was covered with polyvinyl pyrrolidone. The enzyme showed to bind in the same extent to naked and covered Streamline-DEAE (65mg β-gal/g matrix) in batch mode in the absence of any biomass. The kinetics of the adsorption process was studied and no effect of the polyvinyl pyrrolidone covering was found. The optimal conditions for the recovery were achieved by using a lysate made of 40% wet weight of cells, a polyvinyl pyrrolidone-covered matrix/lysate ratio of 10% and carrying out the adsorption process in expanded bed with recirculation over 2h in 20mM phosphate buffer pH 7.4. The fraction recovered after the elution contained 65% of the initial amount of enzyme with a 12.6-fold increased specific activity with respect to the lysate. The polyvinyl pyrrolidone content in the eluate was determined and found negligible. The remarkable point of this work is that it was possible to partially purify the enzyme using a feedstock containing an unusually high biomass concentration in the presence of polyvinyl pyrrolidone onto weak anion exchangers.
Source:Journal of Chromatography B, Volume 900
Valeria Boeris, Izabella Balce, Rami Reddy Vennapusa, Miguel Arévalo Rodríguez, Guillermo Picó, Marcelo Fernández Lahore
β-Galactosidase is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides; its major application in the food industry is to reduce the content of lactose in lactic products. The aim of this work is to recover this enzyme from a cell lysate by adsorption onto Streamline-DEAE in an expanded bed, avoiding, as much as possible, biomass deposition onto the adsorbent matrix. So as to achieve less cell debris–matrix interaction, the adsorbent surface was covered with polyvinyl pyrrolidone. The enzyme showed to bind in the same extent to naked and covered Streamline-DEAE (65mg β-gal/g matrix) in batch mode in the absence of any biomass. The kinetics of the adsorption process was studied and no effect of the polyvinyl pyrrolidone covering was found. The optimal conditions for the recovery were achieved by using a lysate made of 40% wet weight of cells, a polyvinyl pyrrolidone-covered matrix/lysate ratio of 10% and carrying out the adsorption process in expanded bed with recirculation over 2h in 20mM phosphate buffer pH 7.4. The fraction recovered after the elution contained 65% of the initial amount of enzyme with a 12.6-fold increased specific activity with respect to the lysate. The polyvinyl pyrrolidone content in the eluate was determined and found negligible. The remarkable point of this work is that it was possible to partially purify the enzyme using a feedstock containing an unusually high biomass concentration in the presence of polyvinyl pyrrolidone onto weak anion exchangers.
Highlights
► β-Galactosidase production from recombinant Saccharomyces cerevisiae. ► Cell lysate and adsorption onto Streamline-DEAE in an expanded bed. ► Adsorption of target proteins from unclarified feedstock. ► Cell debris–matrix interaction, and the effect of polyvinyl pyrrolidone on the enzyme adsorption.Simultaneous determination of asymmetric and symmetric dimethylarginine, l-monomethylarginine, l-arginine, and l-homoarginine in biological samples using stable isotope dilution liquid chromatography tandem mass spectrometry
27 June 2012,
08:45:31
Publication year:
2012
Source:Journal of Chromatography B, Volume 900
Mariska Davids, Eliane Swieringa, Fredrik Palm, Desirée E.C. Smith, Yvo M. Smulders, Peter G. Scheffer, Henk J. Blom, Tom Teerlink
Production of the endogenous vasodilator nitric oxide (NO) from l-arginine by NO synthase is modulated by l-homoarginine, l-monomethylargine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Here we report on a stable isotope dilution liquid chromatography tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of these metabolites in plasma, cells and tissues. After addition of the internal standards (D7-ADMA, D4-l-homoarginine and 13C6-l-arginine), analytes were extracted from the samples using Waters Oasis MCX solid phase extraction cartridges. Butylated analytes were separated isocratically on a Waters XTerra MS C18 column (3.5μm, 3.9mm×100mm) using 600mg/L ammonium formate in water - acetonitrile (95.5:4.5, v/v) containing 0.1vol% formic acid, and subsequently measured on an AB Sciex API 3000 triple quadrupole mass spectrometer. Multiple reaction monitoring in positive mode was used for analyte quantification. Validation was performed in plasma. Calibration lines were linear (r 2 ≥0.9979) and lower limits of quantification in plasma were 0.4nM for ADMA and SDMA and 0.8nM for the other analytes. Accuracy (% bias) was <3% except for MMA (<7%), intra-assay precision (expressed as CV) was <3.5%, inter-assay precision <9.6%, and recovery 92.9–103.2% for all analytes. The method showed good correlation (r 2 ≥0.9125) with our previously validated HPLC-fluorescence method for measurement in plasma, and was implemented with good performance for measurement of tissue samples. Application of the method revealed the remarkably fast (i.e. within 60min) appearance in plasma of stable isotope-labeled ADMA, SDMA, and MMA during infusion of D3-methyl-1-13C-methionine in healthy volunteers.
Source:Journal of Chromatography B, Volume 900
Mariska Davids, Eliane Swieringa, Fredrik Palm, Desirée E.C. Smith, Yvo M. Smulders, Peter G. Scheffer, Henk J. Blom, Tom Teerlink
Production of the endogenous vasodilator nitric oxide (NO) from l-arginine by NO synthase is modulated by l-homoarginine, l-monomethylargine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Here we report on a stable isotope dilution liquid chromatography tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of these metabolites in plasma, cells and tissues. After addition of the internal standards (D7-ADMA, D4-l-homoarginine and 13C6-l-arginine), analytes were extracted from the samples using Waters Oasis MCX solid phase extraction cartridges. Butylated analytes were separated isocratically on a Waters XTerra MS C18 column (3.5μm, 3.9mm×100mm) using 600mg/L ammonium formate in water - acetonitrile (95.5:4.5, v/v) containing 0.1vol% formic acid, and subsequently measured on an AB Sciex API 3000 triple quadrupole mass spectrometer. Multiple reaction monitoring in positive mode was used for analyte quantification. Validation was performed in plasma. Calibration lines were linear (r 2 ≥0.9979) and lower limits of quantification in plasma were 0.4nM for ADMA and SDMA and 0.8nM for the other analytes. Accuracy (% bias) was <3% except for MMA (<7%), intra-assay precision (expressed as CV) was <3.5%, inter-assay precision <9.6%, and recovery 92.9–103.2% for all analytes. The method showed good correlation (r 2 ≥0.9125) with our previously validated HPLC-fluorescence method for measurement in plasma, and was implemented with good performance for measurement of tissue samples. Application of the method revealed the remarkably fast (i.e. within 60min) appearance in plasma of stable isotope-labeled ADMA, SDMA, and MMA during infusion of D3-methyl-1-13C-methionine in healthy volunteers.
Highlights
► The endogenous vasodilator nitric oxide (NO) is produced from arginine by NO synthase. ► Homoarginine and the methylated arginines MMA, ADMA, and SDMA modulate NO production. ► An LC–MS/MS method for the simultaneous measurement of these compounds was developed. ► Stable isotope labeled arginine, homoarginine and ADMA served as internal standards. ► The method was validated for plasma and is also suitable for analysis of tissue samples.Three-phase hollow fiber liquid-phase microextraction of organophosphorous nerve agent degradation products from complex samples
27 June 2012,
08:45:31
Publication year:
2012
Source:Journal of Chromatography B, Volume 900
Charlotte Desoubries, Florence Chapuis-Hugon, Anne Bossée, Valérie Pichon
Degradation products of chemical warfare agents are considered as important environmental and biological markers of chemical attacks. Alkyl methylphosphonic acids (AMPAs), resulting from the fast hydrolysis of nerve agents, such as sarin and soman, and the methylphosphonic acid (MPA), final degradation product of AMPAs, were determined from complex matrices by using an emergent and miniaturized extraction technique, the hollow fiber liquid-phase microextraction (HF-LPME), before their analysis by liquid chromatography coupled to mass spectrometry (LC–MS). After studying different conditions of separation in the reversed phase LC–MS analysis, the sample treatment method was set up. The three-phase HF-LPME was carried out by using a porous polypropylene (PP) hollow fiber impregnated with 1-octanol that separates the donor and acceptor aqueous media. Various extraction parameters were evaluated such as the volume of the sample, the effect of the pH and the salt addition to the sample, the pH of the acceptor phase, the extraction temperature, the stirring speed of the sample, the immersion time in the organic solvent and the time of extraction. The optimum conditions were applied to the determination of MPA and five AMPAs in real samples, such as surface waters and urine. Compounds were extracted from a 3mL acidified sample into only 6μL of alkaline water without any other pretreatment of the complex matrices. Enrichment factors (EFs) higher than 170 were obtained for three less polar AMPAs. Limits of quantification (LOQs) in the 0.013–5.3ngmL−1 range were obtained after microextraction of AMPAs from river water and in the range of 0.056–4.8ngmL−1 from urine samples with RSD values between 1 and 9%.
Source:Journal of Chromatography B, Volume 900
Charlotte Desoubries, Florence Chapuis-Hugon, Anne Bossée, Valérie Pichon
Degradation products of chemical warfare agents are considered as important environmental and biological markers of chemical attacks. Alkyl methylphosphonic acids (AMPAs), resulting from the fast hydrolysis of nerve agents, such as sarin and soman, and the methylphosphonic acid (MPA), final degradation product of AMPAs, were determined from complex matrices by using an emergent and miniaturized extraction technique, the hollow fiber liquid-phase microextraction (HF-LPME), before their analysis by liquid chromatography coupled to mass spectrometry (LC–MS). After studying different conditions of separation in the reversed phase LC–MS analysis, the sample treatment method was set up. The three-phase HF-LPME was carried out by using a porous polypropylene (PP) hollow fiber impregnated with 1-octanol that separates the donor and acceptor aqueous media. Various extraction parameters were evaluated such as the volume of the sample, the effect of the pH and the salt addition to the sample, the pH of the acceptor phase, the extraction temperature, the stirring speed of the sample, the immersion time in the organic solvent and the time of extraction. The optimum conditions were applied to the determination of MPA and five AMPAs in real samples, such as surface waters and urine. Compounds were extracted from a 3mL acidified sample into only 6μL of alkaline water without any other pretreatment of the complex matrices. Enrichment factors (EFs) higher than 170 were obtained for three less polar AMPAs. Limits of quantification (LOQs) in the 0.013–5.3ngmL−1 range were obtained after microextraction of AMPAs from river water and in the range of 0.056–4.8ngmL−1 from urine samples with RSD values between 1 and 9%.
Highlights
► High potential of HF-LPME applied to reduced size samples. ► High enrichment factor for the extraction of polar compounds from water and urine. ► Quantitative extraction of AMPAs from surface waters and urine samples. ► High sensitivity for the LC/MS analysis of AMPAs without any derivatization step.Simultaneous determination of amoxicillin and prednisolone in bovine milk using ultra-high performance liquid chromatography tandem mass spectrometry
27 June 2012,
08:45:31
Publication year:
2012
Source:Journal of Chromatography B, Volume 900
Hui Li, Xi Xia, Yanan Xue, Shusheng Tang, Xilong Xiao, Jiancheng Li, Jianzhong Shen
A rapid and sensitive ultra-high performance liquid chromatography tandem mass spectrometric method was developed for simultaneous quantification of amoxicillin and prednisolone in bovine milk. In this method, amoxicillin, prednisolone and the internal standards penicillin G-d7 (for amoxicillin) and prednisolone-d6 were extracted from bovine milk using acetonitrile. The C18 solid phase extraction cartridges were selected for cleaning-up the extracts. The analytes were determined using a triple quadrupole mass spectrometry in positive electrospray ionization and multiple reaction monitoring mode. Calibration curves were linear over a concentration range of 2–1000μg/kg for the analytes. The mean recoveries were 89.2–92.3% for amoxicillin and 98.7–102.3% for prednisolone. Limits of detection were 0.5μg/kg for the analytes, and the limits of quantitation were 2μg/kg. Decision limit (CCα) and detection capability (CCβ) have also been estimated for each analyte. The method was validated according to the Commission Decision 2002/657/EC and successfully applied to the analysis of amoxicillin and prednisolone in real samples.
Source:Journal of Chromatography B, Volume 900
Hui Li, Xi Xia, Yanan Xue, Shusheng Tang, Xilong Xiao, Jiancheng Li, Jianzhong Shen
A rapid and sensitive ultra-high performance liquid chromatography tandem mass spectrometric method was developed for simultaneous quantification of amoxicillin and prednisolone in bovine milk. In this method, amoxicillin, prednisolone and the internal standards penicillin G-d7 (for amoxicillin) and prednisolone-d6 were extracted from bovine milk using acetonitrile. The C18 solid phase extraction cartridges were selected for cleaning-up the extracts. The analytes were determined using a triple quadrupole mass spectrometry in positive electrospray ionization and multiple reaction monitoring mode. Calibration curves were linear over a concentration range of 2–1000μg/kg for the analytes. The mean recoveries were 89.2–92.3% for amoxicillin and 98.7–102.3% for prednisolone. Limits of detection were 0.5μg/kg for the analytes, and the limits of quantitation were 2μg/kg. Decision limit (CCα) and detection capability (CCβ) have also been estimated for each analyte. The method was validated according to the Commission Decision 2002/657/EC and successfully applied to the analysis of amoxicillin and prednisolone in real samples.
Highlights
► Amoxicillin and prednisolone were determined simultaneously using UPLC–MS/MS. ► Deuterated internal standards were applied to accurate quantification. ► LODs for two analytes were far below MRLs set by EU in developed method. ► Recoveries in raw milk were good with a simple sample preparation.Development of a three-steps derivatization assay for the localization of double bond in monounsaturated monomers of poly-beta-hydroxyalkanoates by GC–MS
27 June 2012,
08:45:31
Publication year:
2012
Source:Journal of Chromatography B, Volume 900
Christelle Simon-Colin, Christelle Gouin, Pierre Lemechko, Nelly Kervarec, Jean Guezennec
A new gas chromatography–mass spectrometry (GC–MS) method for the localization of double bond in monounsaturated 3-hydroxyalkenoic acids monomers has been developed. A three steps derivation assay was used including a methanolysis, then acetylation and dimethyldisulfide (DMDS) addition to alkene groups. Electron impact GC–MS analysis of such derivatives offers characteristic fragments allowing the unambiguous determination of double bond position in side chain. This novel method is well-suited for the routine analysis of poly-beta-hydroxyalkanoates (PHAs), and was used to characterize monounsaturated monomers in both 3-hydroxyalkenoic acids standards as well as in mcl-PHAs and poly(3-hydroxyoctanoate-co-3-hydroxyundecenoate) (PHOU) produced by bacterial strain Pseudomonas guezennei from glucose or a mixture of sodium octanoate plus 10-undecenoic acid, respectively.
Source:Journal of Chromatography B, Volume 900
Christelle Simon-Colin, Christelle Gouin, Pierre Lemechko, Nelly Kervarec, Jean Guezennec
A new gas chromatography–mass spectrometry (GC–MS) method for the localization of double bond in monounsaturated 3-hydroxyalkenoic acids monomers has been developed. A three steps derivation assay was used including a methanolysis, then acetylation and dimethyldisulfide (DMDS) addition to alkene groups. Electron impact GC–MS analysis of such derivatives offers characteristic fragments allowing the unambiguous determination of double bond position in side chain. This novel method is well-suited for the routine analysis of poly-beta-hydroxyalkanoates (PHAs), and was used to characterize monounsaturated monomers in both 3-hydroxyalkenoic acids standards as well as in mcl-PHAs and poly(3-hydroxyoctanoate-co-3-hydroxyundecenoate) (PHOU) produced by bacterial strain Pseudomonas guezennei from glucose or a mixture of sodium octanoate plus 10-undecenoic acid, respectively.
Highlights
► Localization of double bond in monounsaturated 3-hydroxyalkenoic acids by GC–MS. ► The new method implies methanolysis, then acetylation and thiomethylation. ► The method was successfully applied to bacterial poly-beta-hydroxyalcanoates.Simultaneous clarification of Escherichia coli culture and purification of extracellularly produced penicillin G acylase using tangential flow filtration and anion-exchange membrane chromatography (TFF-AEMC)
27 June 2012,
08:45:31
Publication year:
2012
Source:Journal of Chromatography B, Volume 900
Valerie Orr, Jeno Scharer, Murray Moo-Young, C. Howie Honeyman, Drew Fenner, Lisa Crossley, Shing-Yi Suen, C. Perry Chou
Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins since conventional purification processes are lengthy, technically complicated, and time-consuming. To address this issue, herein we demonstrated the simultaneous clarification and purification of the extracellularly produced recombinant protein by Escherichia coli using an integrated system of tangential flow filtration and anion exchange membrane chromatography (TFF-AEMC). After cultivation in a bench-top bioreactor with 1L working volume using the developed host/vector system for high-level expression and effective secretion of recombinant penicillin G acylase (PAC), the whole culture broth was applied directly to the established system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry) and cross-flow operations. Most contaminant proteins in the extracellular medium were captured by the anion-exchange membrane and cells remained in the retentate, whereas extracellular PAC was purified and collected in the filtrate. The batch time for both cultivation and purification was less than 24h and recombinant PAC with high purity (19U/mg), yield (72% recovery), and productivity (41mg of purified PAC per liter of culture) was obtained. Due to the nature of the non-selective protein secretion system and the versatility of ion-exchange membrane chromatography, the developed system can be widely applied for effective production and purification of recombinant proteins.
Source:Journal of Chromatography B, Volume 900
Valerie Orr, Jeno Scharer, Murray Moo-Young, C. Howie Honeyman, Drew Fenner, Lisa Crossley, Shing-Yi Suen, C. Perry Chou
Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins since conventional purification processes are lengthy, technically complicated, and time-consuming. To address this issue, herein we demonstrated the simultaneous clarification and purification of the extracellularly produced recombinant protein by Escherichia coli using an integrated system of tangential flow filtration and anion exchange membrane chromatography (TFF-AEMC). After cultivation in a bench-top bioreactor with 1L working volume using the developed host/vector system for high-level expression and effective secretion of recombinant penicillin G acylase (PAC), the whole culture broth was applied directly to the established system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry) and cross-flow operations. Most contaminant proteins in the extracellular medium were captured by the anion-exchange membrane and cells remained in the retentate, whereas extracellular PAC was purified and collected in the filtrate. The batch time for both cultivation and purification was less than 24h and recombinant PAC with high purity (19U/mg), yield (72% recovery), and productivity (41mg of purified PAC per liter of culture) was obtained. Due to the nature of the non-selective protein secretion system and the versatility of ion-exchange membrane chromatography, the developed system can be widely applied for effective production and purification of recombinant proteins.
Highlights
► A novel protein separation system of TFF-AEMC was developed. ► The system was applied for effective culture clarification and protein purification. ► The batch time for combined cultivation and purification was less than 24h. ► Recombinant protein with a high purity and yield was obtained.Determination of arachidonic acid by on-line solid-phase extraction HPLC with UV detection for screening of cytosolic phospholipase A2α inhibitors
27 June 2012,
08:45:31
Publication year:
2012
Source:Journal of Chromatography B, Volume 900
Walburga Hanekamp, Matthias Lehr
An on-line solid-phase extraction (SPE)–liquid chromatographic method with ultraviolet detection at 200nm for screening of inhibitors of cytosolic phospholipase A2α (cPLA2α) was developed and validated. cPLA2α was isolated from porcine platelets. Enzyme activity was determined by measuring the release of arachidonic acid from a phospholipid substrate using automated on-line sample clean up on a trap column followed by isocratic back-flush elution on a RP18 analytical column. While the use of a conventional RP18 column for trapping the analyte led to peak broadening only after a few runs due to pollution of the column by binding of components present in the enzyme preparation, the application of a turbulent flow column (TurboFlow Cyclone™) resulted in sharp peaks even after a plurality of injections. Interestingly, for sample introduction a turbulent flow of the mobile phase produced by high flow rates was not necessary to maintain good peak shapes. The same result could also be achieved applying low flow rates (0.5mL/min). Several known cPLA2α inhibitors were used to validate the test system.
Source:Journal of Chromatography B, Volume 900
Walburga Hanekamp, Matthias Lehr
An on-line solid-phase extraction (SPE)–liquid chromatographic method with ultraviolet detection at 200nm for screening of inhibitors of cytosolic phospholipase A2α (cPLA2α) was developed and validated. cPLA2α was isolated from porcine platelets. Enzyme activity was determined by measuring the release of arachidonic acid from a phospholipid substrate using automated on-line sample clean up on a trap column followed by isocratic back-flush elution on a RP18 analytical column. While the use of a conventional RP18 column for trapping the analyte led to peak broadening only after a few runs due to pollution of the column by binding of components present in the enzyme preparation, the application of a turbulent flow column (TurboFlow Cyclone™) resulted in sharp peaks even after a plurality of injections. Interestingly, for sample introduction a turbulent flow of the mobile phase produced by high flow rates was not necessary to maintain good peak shapes. The same result could also be achieved applying low flow rates (0.5mL/min). Several known cPLA2α inhibitors were used to validate the test system.
Highlights
► Automated on-line solid-phase extraction–HPLC/UV method for cPLA2α inhibitor screening. ► Application of a turbulent flow column for trapping of the enzyme product arachidonic acid. ► A turbulent flow of the mobile phase is not necessary for effective separation of proteins.Use of hollow fiber liquid phase microextraction and HPLC for extraction and determination of apigenin in human urine after consumption of Satureja sahendica Bornm.
27 June 2012,
08:45:31
Publication year:
2012
Source:Journal of Chromatography B, Volume 900
Mohammad Reza Hadjmohammadi, Mona Soltani, Vahid sharifi
The applicability of hollow fiber liquid phase microextraction (HF-LPME) was evaluated for extraction and preconcentration of apigenin prior to its determination by HPLC. Different parameters affecting the HF-LPME recovery such as nature of organic solvent, pH of donor and acceptor phases, extraction time, stirring speed, salt addition were optimized. Under optimum conditions (1-octanol as organic solvent, pH of the donor phase=3 and pH of acceptor phase=11.5, extraction time of 75min, stirring speed of 1000rpm) limit of detection (LOD) of 0.1ng/mL, linear range of 0.5–300ng/mL and correlation of determination (R 2) of 0.9956 were obtained. The relative intra and inter-day standard deviations (RSD%) based on five replicate measurement were 3.5% and 10.7% respectively. Enrichment factor of 315 and recovery 85% were achieved. Finally, the applicability of the proposed method was evaluated by extraction and determination of apigenin in urine sample after consumption of Satureja sahendica Bornm. which is a native medicinal plant from Iran. Concentration of apigenin in urine sample was found to be 6.20ng/mL.
Source:Journal of Chromatography B, Volume 900
Mohammad Reza Hadjmohammadi, Mona Soltani, Vahid sharifi
The applicability of hollow fiber liquid phase microextraction (HF-LPME) was evaluated for extraction and preconcentration of apigenin prior to its determination by HPLC. Different parameters affecting the HF-LPME recovery such as nature of organic solvent, pH of donor and acceptor phases, extraction time, stirring speed, salt addition were optimized. Under optimum conditions (1-octanol as organic solvent, pH of the donor phase=3 and pH of acceptor phase=11.5, extraction time of 75min, stirring speed of 1000rpm) limit of detection (LOD) of 0.1ng/mL, linear range of 0.5–300ng/mL and correlation of determination (R 2) of 0.9956 were obtained. The relative intra and inter-day standard deviations (RSD%) based on five replicate measurement were 3.5% and 10.7% respectively. Enrichment factor of 315 and recovery 85% were achieved. Finally, the applicability of the proposed method was evaluated by extraction and determination of apigenin in urine sample after consumption of Satureja sahendica Bornm. which is a native medicinal plant from Iran. Concentration of apigenin in urine sample was found to be 6.20ng/mL.
Highlights
► A simple and sensitive method for determination of apigenin has been introduced. ► Hollow fiber liquid phase microextraction was used for extraction of apigenin. ► Trace amount of apigenin in biological sample was preconcentrated using this method. ► The HF-LPME method has excellent clean-up and high-preconcentration factor.A liquid chromatography–tandem mass spectrometric method for quantification of curcumin-O-glucuronide and curcumin in human plasma
27 June 2012,
08:45:31
Publication year:
2012
Source:Journal of Chromatography B, Volume 900
Wei Chen, Patty Fan-Havard, Lisa D. Yee, Yu Cao, Gary D. Stoner, Kenneth K. Chan, Zhongfa Liu
Curcumin is a widely used herbal medicine for various human diseases including inflammation and cancer. The demonstration and optimization of curcumin's activities in the clinical setting, however, have been compromised by its poor bioavailability and the lack of analytic methods to monitor its absorption. In this paper, we report the first validated liquid chromatography–tandem mass spectrometric method for simultaneous quantification of curcumin and its major metabolite: curcumin-O-glucuronide (COG), in the linear range of 2.0–2000ng/mL in human plasma. The intra-day and inter-day accuracies of curcumin and COG in human plasma were in the range of 91.3–111.5% and 82.7–109.2% and their co-efficiency of variations were in the range of 3.5–12.7% and 3.1–11.3%, respectively. This method was capable of detecting only COG in human plasma samples from two healthy volunteers after an oral ingestion of curcumin.
Source:Journal of Chromatography B, Volume 900
Wei Chen, Patty Fan-Havard, Lisa D. Yee, Yu Cao, Gary D. Stoner, Kenneth K. Chan, Zhongfa Liu
Curcumin is a widely used herbal medicine for various human diseases including inflammation and cancer. The demonstration and optimization of curcumin's activities in the clinical setting, however, have been compromised by its poor bioavailability and the lack of analytic methods to monitor its absorption. In this paper, we report the first validated liquid chromatography–tandem mass spectrometric method for simultaneous quantification of curcumin and its major metabolite: curcumin-O-glucuronide (COG), in the linear range of 2.0–2000ng/mL in human plasma. The intra-day and inter-day accuracies of curcumin and COG in human plasma were in the range of 91.3–111.5% and 82.7–109.2% and their co-efficiency of variations were in the range of 3.5–12.7% and 3.1–11.3%, respectively. This method was capable of detecting only COG in human plasma samples from two healthy volunteers after an oral ingestion of curcumin.
Highlights
► First direct method to measure curcumin-O-glucuronide (COG) in human plasma. ► The LLOQ is 2ng/mL with CVs (<15%) and accuracy (85–115%). ► Capable of characterizing the pharmacokinetics of COG up to 24h in human.Determination of phenylethanolamine A in animal hair, tissues and feeds by reversed phase liquid chromatography tandem mass spectrometry with QuEChERS
27 June 2012,
08:45:31
Publication year:
2012
Source:Journal of Chromatography B, Volume 900
Ming-Xia Zhang, Cun Li, Yin-Liang Wu
A simple, sensitive and reliable analytical method was developed for the determination of a new beta-agonist phenylethanolamine A in animal hair, tissues and animal feeds by ultra high performance liquid chromatography–positive electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS) with QuEChERS. Samples were extracted with acetonitrile/water (80:20, v/v). The extract was purified through QuEChERS method, then was dried with nitrogen and residues were redissolved in mobile phase for hair sample or directly diluted with 0.1% formic acid in water for other samples, and analyzed by LC–MS/MS on a Waters Acquity BEH C18 column with 0.1% formic acid in water/methanol as mobile phase with gradient elution. The samples were quantified using phenylethanolamine A-D3 as internal standards. The proposed method was validated according to the European Commission Decision 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), recovery, precision, linearity, robustness and stability. The CCα values ranged from 0.10 to 0.26μg/kg. The CCβ values ranged from 0.20 to 0.37μg/kg. The mean recoveries of 95.4–108.9% with intra-day CVs of 2.2–5.6% and inter-day CVs of 3.1–6.2% were obtained. The method is demonstrated to be suitable for the determination of phenylethanolamine A in animal hair, tissues and animal feeds. The total time required for the analysis of one sample except animal hair sample, including sample preparation, was about 25min.
Source:Journal of Chromatography B, Volume 900
Ming-Xia Zhang, Cun Li, Yin-Liang Wu
A simple, sensitive and reliable analytical method was developed for the determination of a new beta-agonist phenylethanolamine A in animal hair, tissues and animal feeds by ultra high performance liquid chromatography–positive electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS) with QuEChERS. Samples were extracted with acetonitrile/water (80:20, v/v). The extract was purified through QuEChERS method, then was dried with nitrogen and residues were redissolved in mobile phase for hair sample or directly diluted with 0.1% formic acid in water for other samples, and analyzed by LC–MS/MS on a Waters Acquity BEH C18 column with 0.1% formic acid in water/methanol as mobile phase with gradient elution. The samples were quantified using phenylethanolamine A-D3 as internal standards. The proposed method was validated according to the European Commission Decision 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), recovery, precision, linearity, robustness and stability. The CCα values ranged from 0.10 to 0.26μg/kg. The CCβ values ranged from 0.20 to 0.37μg/kg. The mean recoveries of 95.4–108.9% with intra-day CVs of 2.2–5.6% and inter-day CVs of 3.1–6.2% were obtained. The method is demonstrated to be suitable for the determination of phenylethanolamine A in animal hair, tissues and animal feeds. The total time required for the analysis of one sample except animal hair sample, including sample preparation, was about 25min.
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