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The development and validation of a turbulent flow chromatography–tandem mass spectrometry method for the endogenous steroid profiling of equine serum
07 September 2012,
08:54:55
Publication year:
2012
Source:Journal of Chromatography B, Volume 905
Benjamin C. Moeller, Scott D. Stanley
A method for the detection and quantitation of 35 endogenous steroids in equine serum was developed and validated. Androgens, estrogens, progestins and their metabolites potentially present in serum were simultaneously monitored in one method using on-line sample extraction by turbulent flow chromatography (TFC) on a 2-dimensional liquid chromatography system and detected on a triple-stage quadrupole mass spectrometer by electrospray ionization. Analytes were detected and quantitated by single-reaction monitoring or selected-ion monitoring. Limits of detection (range 0.025–10ngmL−1) and quantitation (range 0.125–25ngmL−1) along with recovery and matrix effects were determined for each analyte. Inter- and intra-day accuracy and precision was assessed for with the majority of analytes having %CV less than 20% and accuracy within 20% of the expected concentrations. Eight of the 35 analytes were unable to meet these guidelines across all of the quality control concentrations monitored for each analyte. This method was used to determine the endogenous steroid profiles of Thoroughbred horses and has been modified for use in non-human primates and cell culture.
Source:Journal of Chromatography B, Volume 905
Benjamin C. Moeller, Scott D. Stanley
A method for the detection and quantitation of 35 endogenous steroids in equine serum was developed and validated. Androgens, estrogens, progestins and their metabolites potentially present in serum were simultaneously monitored in one method using on-line sample extraction by turbulent flow chromatography (TFC) on a 2-dimensional liquid chromatography system and detected on a triple-stage quadrupole mass spectrometer by electrospray ionization. Analytes were detected and quantitated by single-reaction monitoring or selected-ion monitoring. Limits of detection (range 0.025–10ngmL−1) and quantitation (range 0.125–25ngmL−1) along with recovery and matrix effects were determined for each analyte. Inter- and intra-day accuracy and precision was assessed for with the majority of analytes having %CV less than 20% and accuracy within 20% of the expected concentrations. Eight of the 35 analytes were unable to meet these guidelines across all of the quality control concentrations monitored for each analyte. This method was used to determine the endogenous steroid profiles of Thoroughbred horses and has been modified for use in non-human primates and cell culture.
Highlights
► Validated method for analysis of 35 endogenous steroids in equine serum. ► Androgens, estrogens, and progestins are monitored in one method. ► Turbulent flow chromatography allows for minimal sample processing. ► Allows for analysis of multiple steroids in one run from 300μL serum.Liquid chromatographic mass spectrometric (LC/MS/MS) determination of plasma hydroxocobalamin and cyanocobalamin concentrations after hydroxocobalamin antidote treatment for cyanide poisoning
07 September 2012,
08:54:55
Publication year:
2012
Source:Journal of Chromatography B, Volume 905
Harvey A. Schwertner, Sandra Valtier, Vikhyat S. Bebarta
Cyanide poisoning occurs in individuals after fire smoke inhalation and after oral ingestion of cyanide. Hydroxocobalamin (HOCbl), a hydroxylated form of vitamin B12, is often used as an antidote to treat cyanide toxicity. It has a high affinity for cyanide and rapidly removes cyanide from tissue by forming cyanocobalamin (CNCbl). Little information is available on the pharmacokinetics of HOCbl and CNCbl largely because of the lack of analytical methods for analyzing HOCbl and CNCbl. In this study, we developed a new liquid chromatographic mass spectrometric (LC/MS/MS) method for the quantitative analysis of plasma HOCbl and CNCbl in the porcine (Sus scrofa) model. The method uses on-column extraction, reversed phase gradient chromatography, and multiple reaction monitoring (MRM) for quantitation. MRM transitions monitored were 664.7→147.3 and 664.7→359.2 for HOCbl and 678.8→147.3, 678.8→359.1 678.8→457.1 for CNCbl. The limit of detection (LOD) and the lower limit of quantitation (LLOQ) were 1.0 and 1.0μmole/L, respectively, for plasma HOCbl and 0.1 and 0.5μmole/L for plasma CNCbl. The within-day and between-day CVs were 4.3 and 6.4% for plasma HOCbl at 500.0μmole/L and 5.5 and 5.7% for CNCbl at 100.0μmole/L (n =6). The plasma HOCbl and CNCbl calibrations curves were linear from 100.0 to 2000.0 and 50.0 to 500.0μmole/L, respectively. Based on 6 separate calibration curves the average linear regression coefficient (R 2) for both HOCbl and CNCbl was 0.992. The LC/M/MS method was found to be accurate and precise and has been validated by determining the plasma HOCbl and CNCbl concentrations in 11 pigs that were treated with HOCbl for cyanide poisoning.
Source:Journal of Chromatography B, Volume 905
Harvey A. Schwertner, Sandra Valtier, Vikhyat S. Bebarta
Cyanide poisoning occurs in individuals after fire smoke inhalation and after oral ingestion of cyanide. Hydroxocobalamin (HOCbl), a hydroxylated form of vitamin B12, is often used as an antidote to treat cyanide toxicity. It has a high affinity for cyanide and rapidly removes cyanide from tissue by forming cyanocobalamin (CNCbl). Little information is available on the pharmacokinetics of HOCbl and CNCbl largely because of the lack of analytical methods for analyzing HOCbl and CNCbl. In this study, we developed a new liquid chromatographic mass spectrometric (LC/MS/MS) method for the quantitative analysis of plasma HOCbl and CNCbl in the porcine (Sus scrofa) model. The method uses on-column extraction, reversed phase gradient chromatography, and multiple reaction monitoring (MRM) for quantitation. MRM transitions monitored were 664.7→147.3 and 664.7→359.2 for HOCbl and 678.8→147.3, 678.8→359.1 678.8→457.1 for CNCbl. The limit of detection (LOD) and the lower limit of quantitation (LLOQ) were 1.0 and 1.0μmole/L, respectively, for plasma HOCbl and 0.1 and 0.5μmole/L for plasma CNCbl. The within-day and between-day CVs were 4.3 and 6.4% for plasma HOCbl at 500.0μmole/L and 5.5 and 5.7% for CNCbl at 100.0μmole/L (n =6). The plasma HOCbl and CNCbl calibrations curves were linear from 100.0 to 2000.0 and 50.0 to 500.0μmole/L, respectively. Based on 6 separate calibration curves the average linear regression coefficient (R 2) for both HOCbl and CNCbl was 0.992. The LC/M/MS method was found to be accurate and precise and has been validated by determining the plasma HOCbl and CNCbl concentrations in 11 pigs that were treated with HOCbl for cyanide poisoning.
Highlights
► LC/MS/MS method for analyzing plasma hydroxocobalamin and cyanocobalamin. ► Use of hydroxocobalamin in porcine animal models with induced cyanide poisoning. ► Plasma hydroxocobalamin and cyanocobalamin levels after hydroxocobalamin treatment.Breath biomarkers of liver cirrhosis
07 September 2012,
08:54:55
Publication year:
2012
Source:Journal of Chromatography B, Volume 905
Jesica Dadamio, Sandra Van den Velde, Wim Laleman, Paul Van Hee, Wim Coucke, Frederik Nevens, Marc Quirynen
The diagnosis of asymptomatic cirrhosis in patients with liver disease is of importance to start screening for complications in due time. Liver biopsy is neither sensitive nor practical enough to be used as a frequent follow-up test in patients with chronic liver disease. The volatile organic compounds present in exhaled breath offer the possibility of exploring internal physiologic and pathologic process in a non invasive way. This study examined whether a specific pattern of biomarkers can be found in breath samples of patients with cirrhosis. To this aim samples of alveolar breath from patients with cirrhosis and healthy volunteers were analyzed using gas chromatography–mass spectrometry. When linear discriminant analysis was used to search for a model(s)/pattern of compounds characteristic for liver cirrhosis, 24 models of 8 independent compounds could distinguish between the groups. The sensitivity and specificity (between 82% and 88%, and 96% and 100%, respectively) of the models suggest that a specific pattern of breath biomarkers can be found in patients with cirrhosis, which may allow detecting this complication of chronic liver disease in an early stage.
Source:Journal of Chromatography B, Volume 905
Jesica Dadamio, Sandra Van den Velde, Wim Laleman, Paul Van Hee, Wim Coucke, Frederik Nevens, Marc Quirynen
The diagnosis of asymptomatic cirrhosis in patients with liver disease is of importance to start screening for complications in due time. Liver biopsy is neither sensitive nor practical enough to be used as a frequent follow-up test in patients with chronic liver disease. The volatile organic compounds present in exhaled breath offer the possibility of exploring internal physiologic and pathologic process in a non invasive way. This study examined whether a specific pattern of biomarkers can be found in breath samples of patients with cirrhosis. To this aim samples of alveolar breath from patients with cirrhosis and healthy volunteers were analyzed using gas chromatography–mass spectrometry. When linear discriminant analysis was used to search for a model(s)/pattern of compounds characteristic for liver cirrhosis, 24 models of 8 independent compounds could distinguish between the groups. The sensitivity and specificity (between 82% and 88%, and 96% and 100%, respectively) of the models suggest that a specific pattern of breath biomarkers can be found in patients with cirrhosis, which may allow detecting this complication of chronic liver disease in an early stage.
Highlights
► We used TD/GC/MS to analyze the breath composition of patients with cirrhosis. ► We compared their breath profiles with healthy volunteers. ► Linear discriminant analysis identified 24 models. ► Sensitivity and specificity varied between 82% and 88%, and 96% and 100%, respectively. ► Patients with cirrhosis have a specific pattern of breath biomarkers.Concentration and selective fractionation of an antihypertensive peptide from an alfalfa white proteins hydrolysate by mixed ion-exchange centrifugal partition chromatography
07 September 2012,
08:54:55
Publication year:
2012
Source:Journal of Chromatography B, Volume 905
Leslie Boudesocque, Romain Kapel, Cedric Paris, Pascal Dhulster, Ivan Marc, Jean-Hugues Renault
This article reports a promising use of the mixed ion-exchange centrifugal partition chromatography (MIXCPC) technique in the field of downstream processes. A complex alfalfa white protein concentrate hydrolysate (AWPC hydrolysate) showing anti-hypertensive properties was successfully fractionated by MIXCPC to yield a l-valyl-l-tryptophan (VW) enriched fraction in one run. This dipeptide shows an interesting anti-angiotensin converting enzyme (anti-ACE) activity. An analytical method based on RP-LC/MS-MS was developed to quantify the target VW peptide in both the starting material and the enriched fractions. The best results for the MIXCPC fractionation were obtained by the combined use of the quaternary biphasic solvent system, methyl-tert-butylether/acetonitrile/n-butanol/water (2:1:2:5, v/v) in the descending mode, of the lipophilic di(2-ethylhexyl)phosphoric acid (DEHPA) cation-exchanger with an exchanger (DEHPA)/peptides ratio of 15, and of two displacers: calcium chloride and hydrochloric acid. The complexity of the starting material involved the selectivity optimization by splitting the stationary phase into two sections that differed by their triethylamine concentration. From 1g of AWPC hydrolysate containing 0.26% of VW, 30.7mg of a VW enriched fraction were recovered with a purity of 10.9%, corresponding to a purification factor of 41 and a recovery of 97%.
Source:Journal of Chromatography B, Volume 905
Leslie Boudesocque, Romain Kapel, Cedric Paris, Pascal Dhulster, Ivan Marc, Jean-Hugues Renault
This article reports a promising use of the mixed ion-exchange centrifugal partition chromatography (MIXCPC) technique in the field of downstream processes. A complex alfalfa white protein concentrate hydrolysate (AWPC hydrolysate) showing anti-hypertensive properties was successfully fractionated by MIXCPC to yield a l-valyl-l-tryptophan (VW) enriched fraction in one run. This dipeptide shows an interesting anti-angiotensin converting enzyme (anti-ACE) activity. An analytical method based on RP-LC/MS-MS was developed to quantify the target VW peptide in both the starting material and the enriched fractions. The best results for the MIXCPC fractionation were obtained by the combined use of the quaternary biphasic solvent system, methyl-tert-butylether/acetonitrile/n-butanol/water (2:1:2:5, v/v) in the descending mode, of the lipophilic di(2-ethylhexyl)phosphoric acid (DEHPA) cation-exchanger with an exchanger (DEHPA)/peptides ratio of 15, and of two displacers: calcium chloride and hydrochloric acid. The complexity of the starting material involved the selectivity optimization by splitting the stationary phase into two sections that differed by their triethylamine concentration. From 1g of AWPC hydrolysate containing 0.26% of VW, 30.7mg of a VW enriched fraction were recovered with a purity of 10.9%, corresponding to a purification factor of 41 and a recovery of 97%.
Highlights
► Fractionation of bioactive peptides by centrifugal partition chromatography (CPC). ► Efficiency of the ion-exchange CPC mode in the field of downstream processes. ► Optimization of the exchanger/analyte ratio. ► Quantification of the target dipeptide by RP-HPLC–MS/MS.High sensitivity measurement of amino acid isotope enrichment using liquid chromatography–mass spectrometry
07 September 2012,
08:54:55
Publication year:
2012
Source:Journal of Chromatography B, Volume 905
Hans M.H. van Eijk, Karolina A.P. Wijnands, Babs A.F.M. Bessems, Steven W. Olde Damink, Cornelis H.C. Dejong, Martijn Poeze
Measurement of the incorporation or conversion of infused stable isotope enriched metabolites in vivo such as amino acids plays a key role in metabolic research. Specific routes are frequently probed in knockout mouse models limiting the available amount of sample. Although less precise as compared to combustion-isotope ratio mass spectrometry (C-IRMS), gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS) techniques are therefore often the method of choice to measure isotopic enrichment of target metabolites. However, under conditions of metabolic depletion, the precision of these systems becomes limiting. In this paper, studies were performed to enhance the sensitivity and precision of isotope enrichment measurements using LC–MS. Ion-statistics and resolution were identified as critical factors for this application when using a linear trap mass spectrometer. The combination with an automated pre-column derivatization and a carefully selected solvent mix allowed us to measure isotopic enrichments down to 0.005% at plasma concentrations as low as 5μmol/l, an improvement by a factor of 100 compared to alternative methods. The resulting method now allowed measurement of the in vivo conversion of the amino acid arginine into citrulline as a marker for the production of nitric oxide in an in vivo murine endotoxemia model with depleted plasma levels of arginine and citrulline.
Source:Journal of Chromatography B, Volume 905
Hans M.H. van Eijk, Karolina A.P. Wijnands, Babs A.F.M. Bessems, Steven W. Olde Damink, Cornelis H.C. Dejong, Martijn Poeze
Measurement of the incorporation or conversion of infused stable isotope enriched metabolites in vivo such as amino acids plays a key role in metabolic research. Specific routes are frequently probed in knockout mouse models limiting the available amount of sample. Although less precise as compared to combustion-isotope ratio mass spectrometry (C-IRMS), gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS) techniques are therefore often the method of choice to measure isotopic enrichment of target metabolites. However, under conditions of metabolic depletion, the precision of these systems becomes limiting. In this paper, studies were performed to enhance the sensitivity and precision of isotope enrichment measurements using LC–MS. Ion-statistics and resolution were identified as critical factors for this application when using a linear trap mass spectrometer. The combination with an automated pre-column derivatization and a carefully selected solvent mix allowed us to measure isotopic enrichments down to 0.005% at plasma concentrations as low as 5μmol/l, an improvement by a factor of 100 compared to alternative methods. The resulting method now allowed measurement of the in vivo conversion of the amino acid arginine into citrulline as a marker for the production of nitric oxide in an in vivo murine endotoxemia model with depleted plasma levels of arginine and citrulline.
Highlights
► Amino acid (AA) isotope enrichment down to 0.005% at 5μM concentrations. ► Special focus on low arg and cit due to LPS treatment. ► Use OPA derivatization of AA combined with RP-HPLC and MS analysis.A HILIC–MS/MS method for the simultaneous determination of seven organic acids in rat urine as biomarkers of exposure to realgar
07 September 2012,
08:54:55
Publication year:
2012
Source:Journal of Chromatography B, Volume 905
Yin Huang, Yuan Tian, Zunjian Zhang, Can Peng
Realgar (As4S4) is a traditional medicine used in China and Europe for thousands of years. As an arsenical, the toxicity from realgar has raised public concern. Several organic acids in urine are found to be potential biomarkers of realgar exposure, including taurine, citric, glutamic, lactic, pyruvic, succinic and uric acid. In this study, using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS), a rapid and sensitive method was developed to separate and quantify these compounds in urine. A ZIC®-HILIC column was used for the separation at an isocratic condition of acetonitrile and 10mM ammonium acetate in water. Analytes were detected in multiple-reaction monitoring with negative ionization mode, using ibuprofen as internal standard. Good line arities (R 2 >0.996) were obtained for all analytes with the limits of detection from 0.2 to 0.7μg/mL. The intra-day and inter-day accuracy ranged from 89.1 to 104.4% and the relative standard deviation (RSD) did not exceed 15.0%. The recovery was more than 80%with RSD less than 14.0%. The validated method was applied to analyze the urine samples of control and reaglar treated rats, and significant changes of these organic acids were observed.
Source:Journal of Chromatography B, Volume 905
Yin Huang, Yuan Tian, Zunjian Zhang, Can Peng
Realgar (As4S4) is a traditional medicine used in China and Europe for thousands of years. As an arsenical, the toxicity from realgar has raised public concern. Several organic acids in urine are found to be potential biomarkers of realgar exposure, including taurine, citric, glutamic, lactic, pyruvic, succinic and uric acid. In this study, using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS), a rapid and sensitive method was developed to separate and quantify these compounds in urine. A ZIC®-HILIC column was used for the separation at an isocratic condition of acetonitrile and 10mM ammonium acetate in water. Analytes were detected in multiple-reaction monitoring with negative ionization mode, using ibuprofen as internal standard. Good line arities (R 2 >0.996) were obtained for all analytes with the limits of detection from 0.2 to 0.7μg/mL. The intra-day and inter-day accuracy ranged from 89.1 to 104.4% and the relative standard deviation (RSD) did not exceed 15.0%. The recovery was more than 80%with RSD less than 14.0%. The validated method was applied to analyze the urine samples of control and reaglar treated rats, and significant changes of these organic acids were observed.
Post acquisition data processing techniques for lipid analysis by quadrupole time-of-flight mass spectrometry
07 September 2012,
08:54:55
Publication year:
2012
Source:Journal of Chromatography B, Volume 905
Tong Xie, Yan Liang, Jiye A, Haiping Hao, Linsheng Liu, Xiao Zheng, Chen Dai, Yuanyuan Zhou, Tianye Guan, Yanna Liu, Lin Xie, Guangji Wang
This study describes the effectiveness of post-acquisition data processing techniques in detecting the lipid species rapidly from the massive data generated by high resolution mass spectrometry. The filtering approaches by product ions or neutral losses enabled glycerophospholipids and sterol conjugates to be identified based on the investigation of their fragmentation patterns, and the filtration by mass defect facilitated the detection of fatty acyl residues and bile acids by limiting the range of mass defect values. After application of these filtering techniques to mass spectra, the background noise was significantly filtered out and characteristic peaks of lipid species were efficiently sorted out. Totally 145 individual lipids were identified and structurally elucidated. Validation results of the LCMS-Q-TOF-based quantitative performance for all the peaks showed that the accuracy, expressed as relative errors (RE%), was lower than ±15%, and values (RSD%) of the inter-batch and intra-batch precision were lower than 15% in the assay. The developed method was integrated to the evaluation of plasma lipid profile from high fat diet versus energy restricted diet fed rats. A unique discrimination of the groups was successfully achieved through a principal component analysis (PCA).
Source:Journal of Chromatography B, Volume 905
Tong Xie, Yan Liang, Jiye A, Haiping Hao, Linsheng Liu, Xiao Zheng, Chen Dai, Yuanyuan Zhou, Tianye Guan, Yanna Liu, Lin Xie, Guangji Wang
This study describes the effectiveness of post-acquisition data processing techniques in detecting the lipid species rapidly from the massive data generated by high resolution mass spectrometry. The filtering approaches by product ions or neutral losses enabled glycerophospholipids and sterol conjugates to be identified based on the investigation of their fragmentation patterns, and the filtration by mass defect facilitated the detection of fatty acyl residues and bile acids by limiting the range of mass defect values. After application of these filtering techniques to mass spectra, the background noise was significantly filtered out and characteristic peaks of lipid species were efficiently sorted out. Totally 145 individual lipids were identified and structurally elucidated. Validation results of the LCMS-Q-TOF-based quantitative performance for all the peaks showed that the accuracy, expressed as relative errors (RE%), was lower than ±15%, and values (RSD%) of the inter-batch and intra-batch precision were lower than 15% in the assay. The developed method was integrated to the evaluation of plasma lipid profile from high fat diet versus energy restricted diet fed rats. A unique discrimination of the groups was successfully achieved through a principal component analysis (PCA).
Highlights
► Lipidomic analysis of rats’ plasma was studied. ► 145 lipids were detected by data processing techniques. ► Thirteen differential features could be highlighted upon using the described methodology.Simultaneous quantification of Polyphyllin D and Paris H, two potential antitumor active components in Paris polyphylla by liquid chromatography–tandem mass spectrometry and the application to pharmacokinetics in rats
07 September 2012,
08:54:55
Publication year:
2012
Source:Journal of Chromatography B, Volume 905
Shanshan Wu, Wenyuan Gao, Feng Qiu, Shuli Man, Shujun Fu, Changxiao Liu
Polyphyllin D and Paris H are two potential antitumor active components in Paris polyphylla, one of the Traditional Chinese Medicines (TCMs). The present study details the development and validation of a rapid, sensitive and accurate LC–ESI-MS/MS method for the separation and simultaneous determination of Polyphyllin D and Paris H in rat plasma using Ginsenoside Rh2 as the internal standard (IS). A simple protein precipitation method was used for the preparation of plasma sample. Chromatographic separation was successfully achieved on an Agilent Zorbax C18 column using a step gradient program with the mobile phase of 10mmol/L aqueous ammonium acetate and acetonitrile. Both analytes were detected by negative mode electrospray ionization mass spectrometry. Selected reaction monitoring (SRM) was applied for all monitored analytes. This method demonstrated good linearity and did not show any endogenous interference. The lower limits of quantification (LLOQs) of Polyphyllin D and Paris H were both 1.0ng/mL using 100μL rat plasma. The average recoveries of Polyphyllin D and Paris H from rat plasma were both above 80%. The inter-day precisions (%RSD) of both analytes determined in five days were all below 15%. The developed and validated method has been successfully applied in the simultaneous quantification and pharmacokinetic studies of Polyphyllin D and Paris H in rats.
Source:Journal of Chromatography B, Volume 905
Shanshan Wu, Wenyuan Gao, Feng Qiu, Shuli Man, Shujun Fu, Changxiao Liu
Polyphyllin D and Paris H are two potential antitumor active components in Paris polyphylla, one of the Traditional Chinese Medicines (TCMs). The present study details the development and validation of a rapid, sensitive and accurate LC–ESI-MS/MS method for the separation and simultaneous determination of Polyphyllin D and Paris H in rat plasma using Ginsenoside Rh2 as the internal standard (IS). A simple protein precipitation method was used for the preparation of plasma sample. Chromatographic separation was successfully achieved on an Agilent Zorbax C18 column using a step gradient program with the mobile phase of 10mmol/L aqueous ammonium acetate and acetonitrile. Both analytes were detected by negative mode electrospray ionization mass spectrometry. Selected reaction monitoring (SRM) was applied for all monitored analytes. This method demonstrated good linearity and did not show any endogenous interference. The lower limits of quantification (LLOQs) of Polyphyllin D and Paris H were both 1.0ng/mL using 100μL rat plasma. The average recoveries of Polyphyllin D and Paris H from rat plasma were both above 80%. The inter-day precisions (%RSD) of both analytes determined in five days were all below 15%. The developed and validated method has been successfully applied in the simultaneous quantification and pharmacokinetic studies of Polyphyllin D and Paris H in rats.
Highlights
► Polyphyllin D and Paris H are two new potential antitumor active components in Paris polyphylla, one of the Traditional Chinese Medicines (TCMs). ► A rapid, sensitive and accurate LC–ESI-MS/MS method for simultaneous determination of Polyphyllin D and Paris H in rat plasma was firstly established and fully validated. ► The lower limits of quantification (LLOQs) of Polyphyllin D and Paris H were both 1.0ng/mL using 100μL rat plasma. ► The developed and validated method has been successfully applied in the simultaneous quantification and pharmacokinetic studies of Polyphyllin D and Paris H in rats.Determination of glycine in biofluid by hydrophilic interaction chromatography coupled with tandem mass spectrometry and its application to the quantification of glycine released by embryonal carcinoma stem cells
07 September 2012,
08:54:55
Publication year:
2012
Source:Journal of Chromatography B, Volume 905
Ya-Bin Tang, Lin Teng, Fan Sun, Xiao-Lin Wang, Liang Peng, Yong-Yao Cui, Jin-Jia Hu, Xin Luan, Liang Zhu, Hong-Zhuan Chen
Because glycine plays a prominent role in living creatures, an accurate and precise quantitative analysis method for the compound is needed. Herein, a new approach to analyze glycine by hydrophilic interaction chromatography (HILIC) coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) was developed. This method avoids the use of derivatization and/or ion-pairing reagents. N-methyl-d-aspartate (NMDA) is used as the internal standard (IS). The mobile phase for the isocratic elution consisted of 10mM ammonium formate in acetonitrile–water (70:30, v/v, adjusted to pH 2.8 with formic acid), and a flow rate of 250μL/min was used. Two microliters of sample was injected for analysis. The signal was monitored in the positive multiple reaction monitoring (MRM) mode. The total run time was 5min. The dynamic range was 40–2000ng/mL for glycine in the biological matrix. The LLOQ (lower limit of quantification) of this method was 40ng/mL (80pg on column). The validated method was applied to determine the dynamic release of glycine from P19 embryonal carcinoma stem cells (ECSCs). Glycine spontaneously released from the ECSCs into the intercellular space gradually increased from 331.02±60.36ng/mL at 2min in the beginning to 963.52±283.80ng/mL at 60min and 948.27±235.09ng/mL at 120min, finally reaching a plateau, indicating that ECSCs consecutively release glycine until achieving equilibration between the release and the reuptake of the compound; on the contrary, the negative control NIH/3T3 embryonic fibroblast cells did not release glycine. This finding will help to improve our understanding of the novel effects of neurotransmitters, including glycine, on non-neural systems.
Source:Journal of Chromatography B, Volume 905
Ya-Bin Tang, Lin Teng, Fan Sun, Xiao-Lin Wang, Liang Peng, Yong-Yao Cui, Jin-Jia Hu, Xin Luan, Liang Zhu, Hong-Zhuan Chen
Because glycine plays a prominent role in living creatures, an accurate and precise quantitative analysis method for the compound is needed. Herein, a new approach to analyze glycine by hydrophilic interaction chromatography (HILIC) coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) was developed. This method avoids the use of derivatization and/or ion-pairing reagents. N-methyl-d-aspartate (NMDA) is used as the internal standard (IS). The mobile phase for the isocratic elution consisted of 10mM ammonium formate in acetonitrile–water (70:30, v/v, adjusted to pH 2.8 with formic acid), and a flow rate of 250μL/min was used. Two microliters of sample was injected for analysis. The signal was monitored in the positive multiple reaction monitoring (MRM) mode. The total run time was 5min. The dynamic range was 40–2000ng/mL for glycine in the biological matrix. The LLOQ (lower limit of quantification) of this method was 40ng/mL (80pg on column). The validated method was applied to determine the dynamic release of glycine from P19 embryonal carcinoma stem cells (ECSCs). Glycine spontaneously released from the ECSCs into the intercellular space gradually increased from 331.02±60.36ng/mL at 2min in the beginning to 963.52±283.80ng/mL at 60min and 948.27±235.09ng/mL at 120min, finally reaching a plateau, indicating that ECSCs consecutively release glycine until achieving equilibration between the release and the reuptake of the compound; on the contrary, the negative control NIH/3T3 embryonic fibroblast cells did not release glycine. This finding will help to improve our understanding of the novel effects of neurotransmitters, including glycine, on non-neural systems.
Highlights
► The analysis of glycine in biological matrix by HILIC–MS/MS is firstly reported. ► The method avoids the usage of derivatization or/and ion-pairing reagents. ► The method features the direct injection of samples, with no sample pretreatment. ► ECSCs are firstly discovered to release glycine by the validated method. ► The discovery helps to improve our understanding of the novel effects of glycine.Highly sensitive and quantitative profiling of acidic phytohormones using derivatization approach coupled with nano-LC–ESI-Q-TOF-MS analysis
07 September 2012,
08:54:55
Publication year:
2012
Source:Journal of Chromatography B, Volume 905
Ming-Luan Chen, Xiao-Meng Fu, Jia-Qi Liu, Tian-Tian Ye, Sheng-Yu Hou, Yun-Qing Huang, Bi-Feng Yuan, Yan Wu, Yu-Qi Feng
In current study, we developed a highly sensitive method for the quantitative profiling of acidic phytohormones. Tandem solid-phase extraction (SPE) and liquid–liquid extraction (LLE) was employed to efficiently purify acidic phytohormones, which were further derived by 3-bromoactonyltrimethylammonium bromide (BTA) to increase the ionization efficiency in electrospray ionization-mass spectrometry detection. Additionally, fifteen BTA-derived acidic phytohormones, including ten gibberellins (GAs), were well separated with a salt gradient on poly(methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-co-EDMA) monolithic column. By employing online trapping system, the signal intensities of the analytes were significantly improved. The limits of detection (LODs, Signal/Noise=3) of targeted phytohormones ranged from 1.05 to 122.4pg/mL, which allowed the highly sensitive determination of low abundant acidic phytohormones with tiny amount plant sample. Good reproducibility was obtained by evaluating the intra- and inter-day precisions with relative standard deviations (RSDs) less than 10.9 and 11.9%, respectively. Recoveries of the target analytes from spiked rice leave samples ranged from 88.3 to 104.3%. By employing the method developed here, we were able to simultaneously determine 11 endogenous acidic phytohormones from only 5mg of rice leave sample, which dramatically decreased the required sample amount (three orders of magnitude lower) for the profiling of low abundant acidic phytohormones compared to previous reports. Taken together, the method provided a good solution for the highly sensitive and quantitative profiling of endogenous acidic phytohormones.
Source:Journal of Chromatography B, Volume 905
Ming-Luan Chen, Xiao-Meng Fu, Jia-Qi Liu, Tian-Tian Ye, Sheng-Yu Hou, Yun-Qing Huang, Bi-Feng Yuan, Yan Wu, Yu-Qi Feng
In current study, we developed a highly sensitive method for the quantitative profiling of acidic phytohormones. Tandem solid-phase extraction (SPE) and liquid–liquid extraction (LLE) was employed to efficiently purify acidic phytohormones, which were further derived by 3-bromoactonyltrimethylammonium bromide (BTA) to increase the ionization efficiency in electrospray ionization-mass spectrometry detection. Additionally, fifteen BTA-derived acidic phytohormones, including ten gibberellins (GAs), were well separated with a salt gradient on poly(methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-co-EDMA) monolithic column. By employing online trapping system, the signal intensities of the analytes were significantly improved. The limits of detection (LODs, Signal/Noise=3) of targeted phytohormones ranged from 1.05 to 122.4pg/mL, which allowed the highly sensitive determination of low abundant acidic phytohormones with tiny amount plant sample. Good reproducibility was obtained by evaluating the intra- and inter-day precisions with relative standard deviations (RSDs) less than 10.9 and 11.9%, respectively. Recoveries of the target analytes from spiked rice leave samples ranged from 88.3 to 104.3%. By employing the method developed here, we were able to simultaneously determine 11 endogenous acidic phytohormones from only 5mg of rice leave sample, which dramatically decreased the required sample amount (three orders of magnitude lower) for the profiling of low abundant acidic phytohormones compared to previous reports. Taken together, the method provided a good solution for the highly sensitive and quantitative profiling of endogenous acidic phytohormones.
Highlights
► Using polymer monolithic column, 15 targeted hormones are well separated. ► High sensitivity of pg/mL is obtained, combined with online trapping process. ► Tandem SPE followed by LLE is developed for purification of hormones from matrix. ► Developed method is applied in the analysis of acidic phytohormones in 5mg rice.Validation of determination of plasma metabolites derived from thyme bioactive compounds by improved liquid chromatography coupled to tandem mass spectrometry
07 September 2012,
08:54:55
Publication year:
2012
Source:Journal of Chromatography B, Volume 905
Laura Rubió, Aida Serra, Alba Macià, Xenia Borràs, Maria-Paz Romero, Maria-José Motilva
In the present study, a selective and sensitive method, based on microelution solid-phase extraction (μSPE) plate and ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS) was validated and applied to determine the plasma metabolites of the bioactive compounds of thyme. For validation process, standards of the more representative components of the phenolic and monoterpene fractions of thyme were spiked in plasma samples and then the quality parameters of the method were studied. Extraction recoveries (%R) of the studied compounds were higher than 75%, and the matrix effect (%ME) was lower than 18%. The LODs ranged from 1 to 65μg/L, except for the thymol sulfate metabolite, which was 240μg/L. This method was then applied for the analysis of rat plasma obtained at different times, from 0 to 6h, after an acute intake of thyme extract (5g/kg body weight). Different thyme metabolites were identified and were mainly derived from rosmarinic acid (coumaric acid sulfate, caffeic acid sulfate, ferulic acid sulfate, hydroxyphenylpropionic acid sulfate, dihydroxyphenylpropionic acid sulfate and hydroxybenzoic acid) and thymol (thymol sulfate and thymol glucuronide). The most abundant thyme metabolites generated were hydroxyphenylpropionic acid sulfate and thymol sulfate, their respective concentrations in plasma being 446 and 8464μM 1h after the intake of the thyme extract.
Source:Journal of Chromatography B, Volume 905
Laura Rubió, Aida Serra, Alba Macià, Xenia Borràs, Maria-Paz Romero, Maria-José Motilva
In the present study, a selective and sensitive method, based on microelution solid-phase extraction (μSPE) plate and ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS) was validated and applied to determine the plasma metabolites of the bioactive compounds of thyme. For validation process, standards of the more representative components of the phenolic and monoterpene fractions of thyme were spiked in plasma samples and then the quality parameters of the method were studied. Extraction recoveries (%R) of the studied compounds were higher than 75%, and the matrix effect (%ME) was lower than 18%. The LODs ranged from 1 to 65μg/L, except for the thymol sulfate metabolite, which was 240μg/L. This method was then applied for the analysis of rat plasma obtained at different times, from 0 to 6h, after an acute intake of thyme extract (5g/kg body weight). Different thyme metabolites were identified and were mainly derived from rosmarinic acid (coumaric acid sulfate, caffeic acid sulfate, ferulic acid sulfate, hydroxyphenylpropionic acid sulfate, dihydroxyphenylpropionic acid sulfate and hydroxybenzoic acid) and thymol (thymol sulfate and thymol glucuronide). The most abundant thyme metabolites generated were hydroxyphenylpropionic acid sulfate and thymol sulfate, their respective concentrations in plasma being 446 and 8464μM 1h after the intake of the thyme extract.
Highlights
► A rapid and sensitive method for analysis of plasma thyme metabolites is proposed. ► Method combined micro elution solid-phase extraction and UPLC–MS/MS. ► LODs ranged from 1 to 65μg/L plasma, except thymol sulfate metabolite (240μg/L). ► Main thyme metabolites were hydroxyphenylpropionic acid sulfate and thymol sulfate.Fast liquid chromatography/tandem mass spectrometry determination of cannabinoids in micro volume blood samples after dabsyl derivatization
07 September 2012,
08:54:55
Publication year:
2012
Source:Journal of Chromatography B, Volume 905
C. Lacroix, E. Saussereau
Due to the non-polar nature and the absence of an ionizable group on the cannabinoids, the ionization efficiency in electrospray is low and leads to poor limits of detection (LOD). The reaction of chloride dabsyl with the phenolic OH group of cannabinoids results in a product containing a tertiary amine, which is easily protonated in positive electrospray mode and can significantly improve the cannabinoids LOD. A rapid, selective and sensitive LC/MS-MS method was developed for quantitative determination of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH–THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC–COOH), cannabinol (CBN) and cannabidiol (CBD), in micro volume blood samples following dabsyl derivatization to enhance signal intensity. The method comprised protein precipitation followed by derivatization with dabsyl chloride and subsequent analysis using liquid chromatography–tandem mass spectrometry (LC/MS-MS). Chromatographic separation was achieved using a 150mm×2.1mm C18 analytical column maintained at 65°C and eluted with a gradient of water and acetonitrile, both containing 0.2% formic acid. The run time was 8min. The assay was successfully validated using the approach based on the accuracy profile. Lower limits of quantification (LOQ) were 0.25ng/mL for THC and THC–COOH, 0.30ng/mL for 11-OH–THC, 0.40ng/mL for CBN and 0.80ng/mL for CBD. A comparative study of cannabinoids in blood and plasma, as determined by the developed LC/MS-MS method or the in-house GC/MS-MS technique, demonstrated an excellent correlation between the two methods. Dabsylation was also tested on-line with a spiral of peek tubing placed in the LC/MS-MS column heater at 65°C before the analytical column. The results obtained with on-line dabsyl derivatization were similar to those observed off-line.
Source:Journal of Chromatography B, Volume 905
C. Lacroix, E. Saussereau
Due to the non-polar nature and the absence of an ionizable group on the cannabinoids, the ionization efficiency in electrospray is low and leads to poor limits of detection (LOD). The reaction of chloride dabsyl with the phenolic OH group of cannabinoids results in a product containing a tertiary amine, which is easily protonated in positive electrospray mode and can significantly improve the cannabinoids LOD. A rapid, selective and sensitive LC/MS-MS method was developed for quantitative determination of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH–THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC–COOH), cannabinol (CBN) and cannabidiol (CBD), in micro volume blood samples following dabsyl derivatization to enhance signal intensity. The method comprised protein precipitation followed by derivatization with dabsyl chloride and subsequent analysis using liquid chromatography–tandem mass spectrometry (LC/MS-MS). Chromatographic separation was achieved using a 150mm×2.1mm C18 analytical column maintained at 65°C and eluted with a gradient of water and acetonitrile, both containing 0.2% formic acid. The run time was 8min. The assay was successfully validated using the approach based on the accuracy profile. Lower limits of quantification (LOQ) were 0.25ng/mL for THC and THC–COOH, 0.30ng/mL for 11-OH–THC, 0.40ng/mL for CBN and 0.80ng/mL for CBD. A comparative study of cannabinoids in blood and plasma, as determined by the developed LC/MS-MS method or the in-house GC/MS-MS technique, demonstrated an excellent correlation between the two methods. Dabsylation was also tested on-line with a spiral of peek tubing placed in the LC/MS-MS column heater at 65°C before the analytical column. The results obtained with on-line dabsyl derivatization were similar to those observed off-line.
Highlights
► Cannabinoids dabsyl derivatization allows LC/MS-MS determination in microvolume sample. ► Cannabinoids dabsylation requires only a protein precipitation step as pre-treatment. ► Sensitivity is similar as in GC–MS, but using only 50μL without extraction step. ► The method lies in rapid, specific, sensitive cannabinoids measurement in DRUID cases.Protein pre-fractionation with a mixed-bed ion exchange column in 3D LC–MS/MS proteome analysis
07 September 2012,
08:54:55
Publication year:
2012
Source:Journal of Chromatography B, Volume 905
Luofu Zhang, Ling Yao, Yan Zhang, Ting Xue, Guangchen Dai, Keying Chen, Xiaofang Hu, Lisa X. Xu
The fractionation of complex samples at the protein level prior to shotgun proteomics analysis is an efficient means to more comprehensive analysis of samples. A mixed-bed ion-exchange (IEX) column, packed with both weak anion exchange (WAX) and weak cation exchange (WCX) materials, was used for the first dimensional separation of complex samples at the protein level using volatile solvents. The peptides from digestion of each fraction were then identified by 2D SCX-RP-LC–MS/MS. We applied this 3D strategy to mouse mammary tumor 4T1 cell lysate and identified a total of 3084 proteins in a typical experiment. The moderate separation performance of the mixed-bed IEX column facilitated the in-depth identification of the proteins in the complex sample. There were some acceptable inter-fraction overlaps. Nearly half (45.8%) of the proteins were only identified in single fractions, while 82.3% were identified in no more than 3 fractions. The identified proteins covered a broad range of pI, size and grand average hydrophobicity (GRAVY) values. Detailed analysis of proteins identified in each fraction elucidated the separation characteristics of mixed-bed IEX. Retention on mixed-bed IEX was associated, but not restricted to the extreme pI values (pI <5, pI >10) and to the percentage of charged residues of both signs. In conclusion, we have exploited the mixed-bed IEX column to establish an efficient and comprehensive identification method for complex samples.
Source:Journal of Chromatography B, Volume 905
Luofu Zhang, Ling Yao, Yan Zhang, Ting Xue, Guangchen Dai, Keying Chen, Xiaofang Hu, Lisa X. Xu
The fractionation of complex samples at the protein level prior to shotgun proteomics analysis is an efficient means to more comprehensive analysis of samples. A mixed-bed ion-exchange (IEX) column, packed with both weak anion exchange (WAX) and weak cation exchange (WCX) materials, was used for the first dimensional separation of complex samples at the protein level using volatile solvents. The peptides from digestion of each fraction were then identified by 2D SCX-RP-LC–MS/MS. We applied this 3D strategy to mouse mammary tumor 4T1 cell lysate and identified a total of 3084 proteins in a typical experiment. The moderate separation performance of the mixed-bed IEX column facilitated the in-depth identification of the proteins in the complex sample. There were some acceptable inter-fraction overlaps. Nearly half (45.8%) of the proteins were only identified in single fractions, while 82.3% were identified in no more than 3 fractions. The identified proteins covered a broad range of pI, size and grand average hydrophobicity (GRAVY) values. Detailed analysis of proteins identified in each fraction elucidated the separation characteristics of mixed-bed IEX. Retention on mixed-bed IEX was associated, but not restricted to the extreme pI values (pI <5, pI >10) and to the percentage of charged residues of both signs. In conclusion, we have exploited the mixed-bed IEX column to establish an efficient and comprehensive identification method for complex samples.
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