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Insulin related compounds and identification
17 September 2012,
09:17:14
Publication year:
2012
Source:Journal of Chromatography B
Cheol-Ki Min, Jin-Won Lee, Chang-Kyu Kim, Seong Hwan Kim, Sang Yeol Lee, Sang-Myung Lee, Young-Jin Son
Insulin-related compounds(IRCs), which originate during the expression and purification of human insulin using recombinant E. coli, were purified and identified. We investigated the identity of IRCs and their origin. We also presented methods for inhibiting IRC formation. The strains used in this report were E. coli B5K and E. coli H27R. E. coli B5K had a 6-amino acid-fused peptide at the N-terminus of proinsulin, and E. coli H27R had a 28-amino acid-fused peptide at the N-terminus of proinsulin. We investigated the identity of IRCs and their origin by mainly using High Performance Liquid Chromatography (HPLC). The well-known IRCs, desamido human insulin and desthreonine human insulin, formed in both strains. In addition to these two IRCs, the B5K strain produced three different IRCs, ArgA(0)-insulin (IRC 1), prepeptide-insulin (IRC 2), and GluA(22)-insulin (IRC 3). The amounts of IRC 1, IRC 2, IRC 3 were approximately 0.1 - 0.3% after final purification step. Among these IRCs, ArgA(0)-insulin, prepeptide-insulin, and desthreonine insulin originated from incomplete enzyme reaction. GluA(22)-insulin was formed when we used a double stop codon during the expression of preproinsulin; that is, it was formed by the misreading of the first stop codon through the amber mutation. The major IRCs of the H27R strain were human insulin fragment (B1-B21) (IRC 4), and A9(Ser→Asn) amino acid single mutation human insulin (IRC 5), ArgB(31)-insulin (IRC 6). Human insulin fragment (B1-B21) was formed by ß-mercaptoethanol, which was added during refolding. It formed when the disulfide bonds between A-chain and B-chain of human insulin were cut by ß-mercaptoethanol, followed by cleavage of the B-chain by trypsin and carboxypeptidase B. A9(Ser→Asn) amino acid single mutation human insulin originated from the mistranslation of A9 serine, such that asparagine was translated instead of serine. ArgB(31)-insulin originated from incomplete enzyme reaction. The amount of IRC 4 was 10–15% after enzyme reaction. The amounts of IRC 5, IRC 6 were around 0.2% after final purification step. We present methods for inhibiting the formation of IRCs by controlling the amount of enzyme, controlling the rate of enzyme reaction, using a single stop codon, using hydrogen peroxide(H2O2) to inhibit ß-mercaptoethanol, and modifying the A9 codon.
Source:Journal of Chromatography B
Cheol-Ki Min, Jin-Won Lee, Chang-Kyu Kim, Seong Hwan Kim, Sang Yeol Lee, Sang-Myung Lee, Young-Jin Son
Insulin-related compounds(IRCs), which originate during the expression and purification of human insulin using recombinant E. coli, were purified and identified. We investigated the identity of IRCs and their origin. We also presented methods for inhibiting IRC formation. The strains used in this report were E. coli B5K and E. coli H27R. E. coli B5K had a 6-amino acid-fused peptide at the N-terminus of proinsulin, and E. coli H27R had a 28-amino acid-fused peptide at the N-terminus of proinsulin. We investigated the identity of IRCs and their origin by mainly using High Performance Liquid Chromatography (HPLC). The well-known IRCs, desamido human insulin and desthreonine human insulin, formed in both strains. In addition to these two IRCs, the B5K strain produced three different IRCs, ArgA(0)-insulin (IRC 1), prepeptide-insulin (IRC 2), and GluA(22)-insulin (IRC 3). The amounts of IRC 1, IRC 2, IRC 3 were approximately 0.1 - 0.3% after final purification step. Among these IRCs, ArgA(0)-insulin, prepeptide-insulin, and desthreonine insulin originated from incomplete enzyme reaction. GluA(22)-insulin was formed when we used a double stop codon during the expression of preproinsulin; that is, it was formed by the misreading of the first stop codon through the amber mutation. The major IRCs of the H27R strain were human insulin fragment (B1-B21) (IRC 4), and A9(Ser→Asn) amino acid single mutation human insulin (IRC 5), ArgB(31)-insulin (IRC 6). Human insulin fragment (B1-B21) was formed by ß-mercaptoethanol, which was added during refolding. It formed when the disulfide bonds between A-chain and B-chain of human insulin were cut by ß-mercaptoethanol, followed by cleavage of the B-chain by trypsin and carboxypeptidase B. A9(Ser→Asn) amino acid single mutation human insulin originated from the mistranslation of A9 serine, such that asparagine was translated instead of serine. ArgB(31)-insulin originated from incomplete enzyme reaction. The amount of IRC 4 was 10–15% after enzyme reaction. The amounts of IRC 5, IRC 6 were around 0.2% after final purification step. We present methods for inhibiting the formation of IRCs by controlling the amount of enzyme, controlling the rate of enzyme reaction, using a single stop codon, using hydrogen peroxide(H2O2) to inhibit ß-mercaptoethanol, and modifying the A9 codon.
Highlights
► We report six human insulin-related compounds from two different expression strains. ► Insulin-related compounds were purified and identified using analytical methods. ► An analytical HPLC method was introduced to detect desthreonine human insulin, which is a major impurity during human insulin production. ► The formation mechanisms of insulin-related compounds were studied based on the identities of the related compounds. ► Taken together, our results present several insulin-related compounds and effective analytical methods for detecting them.A rapid validated UHPLC-PDA method for anthocyanins quantification from Euterpe oleracea fruits
17 September 2012,
09:17:14
Publication year:
2012
Source:Journal of Chromatography B
A.L.S. Dias, E. Rozet, G. Chataigné, A.C. Oliveira, C.A.S. Rabelo, Ph. Hubert, H. Rogez, J. Quetin-Leclercq
The aim of this work is to develop the first validated UHPLC-PDA method for major anthocyanins quantification in Euterpe oleracea fruits after fast extraction procedures and samples preparation. The separation was performed on HSS C18 column (1.8 μm) using a gradient elution with acetonitrile and 5% formic acid in a total run time of only 17min. Total error and accuracy profiles were used as criteria for the validation process. Calibration in the matrix was found to be more accurate than calibration without matrix. Trueness (<6.76% relative bias), repeatability (<4.6% RSD), intermediate precision (<5.3% RSD), selectivity, response function and linearity for major anthocyanins, cyanidin-3-glucoside and cyanidin-3-rutinoside, were evaluated. The concentration range validated was 1 to 48 μg/mL for both compounds. In addition two cyanidin-di-O-glycosides were detected for the fist time in this fruit. We also showed that a first extraction of the fruits with ethyl acetate removes the lipophilic compounds and allows an easier extraction by methanol and quantification of anthocyanins in this extract.
Source:Journal of Chromatography B
A.L.S. Dias, E. Rozet, G. Chataigné, A.C. Oliveira, C.A.S. Rabelo, Ph. Hubert, H. Rogez, J. Quetin-Leclercq
The aim of this work is to develop the first validated UHPLC-PDA method for major anthocyanins quantification in Euterpe oleracea fruits after fast extraction procedures and samples preparation. The separation was performed on HSS C18 column (1.8 μm) using a gradient elution with acetonitrile and 5% formic acid in a total run time of only 17min. Total error and accuracy profiles were used as criteria for the validation process. Calibration in the matrix was found to be more accurate than calibration without matrix. Trueness (<6.76% relative bias), repeatability (<4.6% RSD), intermediate precision (<5.3% RSD), selectivity, response function and linearity for major anthocyanins, cyanidin-3-glucoside and cyanidin-3-rutinoside, were evaluated. The concentration range validated was 1 to 48 μg/mL for both compounds. In addition two cyanidin-di-O-glycosides were detected for the fist time in this fruit. We also showed that a first extraction of the fruits with ethyl acetate removes the lipophilic compounds and allows an easier extraction by methanol and quantification of anthocyanins in this extract.
Highlights
► A first EtOAc extraction of EOF allows a better anthocyanins extraction by MeOH/HCl. ► 7 anthocyanins were separated in only 10min using UHPLC-PDA. ► Cy3glu and cy3rut quantification method was validated: dosing range 1 to 48μg/mL. ► Calibrations in the matrix were used for the quantifications. ► Accuracy profiles and total error were used as validation criteria.A method for determining the free (unbound) concentration of ten beta-lactam antibiotics in human plasma using high performance liquid chromatography with ultraviolet detection
17 September 2012,
09:17:14
Publication year:
2012
Source:Journal of Chromatography B
Scott E. Briscoe, Brett C. McWhinney, Jeffrey Lipman, Jason A. Roberts, Jacobus P.J. Ungerer
With the clinical imperative to further research in the area of optimising antibiotic dosing in the intensive care setting, a simple High Performance Liquid Chromatography method was developed and validated for routinely determining the free (unbound) concentration of ten beta-lactam antibiotics in 200μL of human plasma. Antibiotics determined include three cephalosporins (ceftriaxone, cephazolin and cephalotin); two carbapenems (meropenem and ertapenem); and five penicillins (ampicillin, piperacillin, benzylpenicillin, flucloxacillin and dicloxacillin). There was a single common sample preparation method involving ultracentrifugation and stabilisation. Chromatography was performed on a Waters XBridge C18 column with, depending on analytes, one of four acetonitrile-phosphate buffered mobile phases. Peaks of interest were detected via ultraviolet absorbance at 210, 260 and 304nm. The method has been validated and used in a pathology laboratory for therapeutic drug monitoring in critically ill patients. The significant variability in the level of protein binding that is common with antibiotics traditionally considered to have high protein binding (e.g. ceftriaxone, cephazolin, ertapenem, flucloxacillin and dicloxacillin) suggests that this assay should be preferred for measuring the pharmacologically active concentration of beta-lactam antibiotics in a therapeutic drug monitoring program.
Source:Journal of Chromatography B
Scott E. Briscoe, Brett C. McWhinney, Jeffrey Lipman, Jason A. Roberts, Jacobus P.J. Ungerer
With the clinical imperative to further research in the area of optimising antibiotic dosing in the intensive care setting, a simple High Performance Liquid Chromatography method was developed and validated for routinely determining the free (unbound) concentration of ten beta-lactam antibiotics in 200μL of human plasma. Antibiotics determined include three cephalosporins (ceftriaxone, cephazolin and cephalotin); two carbapenems (meropenem and ertapenem); and five penicillins (ampicillin, piperacillin, benzylpenicillin, flucloxacillin and dicloxacillin). There was a single common sample preparation method involving ultracentrifugation and stabilisation. Chromatography was performed on a Waters XBridge C18 column with, depending on analytes, one of four acetonitrile-phosphate buffered mobile phases. Peaks of interest were detected via ultraviolet absorbance at 210, 260 and 304nm. The method has been validated and used in a pathology laboratory for therapeutic drug monitoring in critically ill patients. The significant variability in the level of protein binding that is common with antibiotics traditionally considered to have high protein binding (e.g. ceftriaxone, cephazolin, ertapenem, flucloxacillin and dicloxacillin) suggests that this assay should be preferred for measuring the pharmacologically active concentration of beta-lactam antibiotics in a therapeutic drug monitoring program.
Highlights
► Clinical imperative to foster research in the area of optimising antibiotic dosing. ► Determined the free (unbound) concentration of ten beta-lactam antibiotics. ► Validated method used in pathology laboratory for TDM in critically ill patients. ► Suggest as preferred assay due to antibiotic protein binding variability. ► Method is highly advantageous from a laboratory and clinical perspective.
17 September 2012,
09:17:14
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
17 September 2012,
09:17:14
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
17 September 2012,
09:17:14
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
17 September 2012,
09:17:14
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
17 September 2012,
09:17:14
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
17 September 2012,
09:17:14
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
17 September 2012,
09:17:14
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
17 September 2012,
09:17:14
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.
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