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Internal standards in the quantitative determination of protein biopharmaceuticals using liquid chromatography coupled to mass spectrometry
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Kees J. Bronsema, Rainer Bischoff, Nico C. van de Merbel
Following the increase in development of protein biopharmaceuticals, there is a growing demand for the sensitive and reliable quantification of these proteins in complex biological matrices such as plasma and serum to support (pre)-clinical research. In this field, ligand binding assays (LBAs) are currently the standard analytical technique, but in recent years, there is a trend towards the use of liquid chromatography hyphenated with (tandem) mass spectrometry (LC–MS/MS). One of the reasons for this trend is the possibility to use internal standards to correct for analytical variability and thus improve the precision and accuracy of the results. In the LC–MS/MS bioanalysis of small molecules, internal standardization is quite straightforward: either a stable-isotope labeled (SIL) form of the analyte or a structural analogue is used. For the quantification of biopharmaceutical proteins, the situation is more complex. Since the protein of interest is digested to a mixture of peptides, one of which is subsequently used for quantification, there are more options for internal standardization. A SIL form or a structural analogue of either the intact protein or the signature peptide can be used. In addition, a modified form of the SIL-peptide internal standard, containing one or more cleavable groups is a possibility, and an internal standard can be generated during the analysis by using differential derivatization techniques. In this paper we provide an overview of the different options for internal standardization in the field of absolute targeted quantification of protein biopharmaceuticals using LC–MS/MS, based on literature from 2003 to 2011. The advantages and disadvantages of the different approaches are evaluated both with regard to the correction they provide for the variability of the different steps of the analysis and with regard to their generic availability. As most of the approaches used lead to acceptable results in terms of accuracy and precision, we conclude that there currently is no clear preferable method for internal standardization in the field of protein quantification by LC–MS/MS. It is essential, however, that any step in the analysis that is not covered by the internal standard chosen, should be carefully optimized and controlled.
Source:Journal of Chromatography B, Volumes 893–894
Kees J. Bronsema, Rainer Bischoff, Nico C. van de Merbel
Following the increase in development of protein biopharmaceuticals, there is a growing demand for the sensitive and reliable quantification of these proteins in complex biological matrices such as plasma and serum to support (pre)-clinical research. In this field, ligand binding assays (LBAs) are currently the standard analytical technique, but in recent years, there is a trend towards the use of liquid chromatography hyphenated with (tandem) mass spectrometry (LC–MS/MS). One of the reasons for this trend is the possibility to use internal standards to correct for analytical variability and thus improve the precision and accuracy of the results. In the LC–MS/MS bioanalysis of small molecules, internal standardization is quite straightforward: either a stable-isotope labeled (SIL) form of the analyte or a structural analogue is used. For the quantification of biopharmaceutical proteins, the situation is more complex. Since the protein of interest is digested to a mixture of peptides, one of which is subsequently used for quantification, there are more options for internal standardization. A SIL form or a structural analogue of either the intact protein or the signature peptide can be used. In addition, a modified form of the SIL-peptide internal standard, containing one or more cleavable groups is a possibility, and an internal standard can be generated during the analysis by using differential derivatization techniques. In this paper we provide an overview of the different options for internal standardization in the field of absolute targeted quantification of protein biopharmaceuticals using LC–MS/MS, based on literature from 2003 to 2011. The advantages and disadvantages of the different approaches are evaluated both with regard to the correction they provide for the variability of the different steps of the analysis and with regard to their generic availability. As most of the approaches used lead to acceptable results in terms of accuracy and precision, we conclude that there currently is no clear preferable method for internal standardization in the field of protein quantification by LC–MS/MS. It is essential, however, that any step in the analysis that is not covered by the internal standard chosen, should be carefully optimized and controlled.
Rapid and reliable quantitation of amino acids and myo-inositol in mouse brain by high performance liquid chromatography and tandem mass spectrometry
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Sai P. Bathena, Jiangeng Huang, Adrian A. Epstein, Howard E. Gendelman, Michael D. Boska, Yazen Alnouti
Amino acids and myo-inositol have long been proposed as putative biomarkers for neurodegenerative diseases. Accurate measures and stability have precluded their selective use. To this end, a sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method based on multiple reaction monitoring was developed to simultaneously quantify glutamine, glutamate, γ-aminobutyric acid (GABA), aspartic acid, N-acetyl aspartic acid, taurine, choline, creatine, phosphocholine and myo-inositol in mouse brain by methanol extractions. Chromatography was performed using a hydrophilic interaction chromatography silica column within in a total run time of 15min. The validated method is selective, sensitive, accurate, and precise. The method has a limit of quantification ranging from 2.5 to 20ng/ml for a range of analytes and a dynamic range from 2.5–20 to 500–4000ng/ml. This LC–MS/MS method was validated for biomarker discovery in models of human neurological disorders.
Source:Journal of Chromatography B, Volumes 893–894
Sai P. Bathena, Jiangeng Huang, Adrian A. Epstein, Howard E. Gendelman, Michael D. Boska, Yazen Alnouti
Amino acids and myo-inositol have long been proposed as putative biomarkers for neurodegenerative diseases. Accurate measures and stability have precluded their selective use. To this end, a sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method based on multiple reaction monitoring was developed to simultaneously quantify glutamine, glutamate, γ-aminobutyric acid (GABA), aspartic acid, N-acetyl aspartic acid, taurine, choline, creatine, phosphocholine and myo-inositol in mouse brain by methanol extractions. Chromatography was performed using a hydrophilic interaction chromatography silica column within in a total run time of 15min. The validated method is selective, sensitive, accurate, and precise. The method has a limit of quantification ranging from 2.5 to 20ng/ml for a range of analytes and a dynamic range from 2.5–20 to 500–4000ng/ml. This LC–MS/MS method was validated for biomarker discovery in models of human neurological disorders.
Development of a fully automated on-line solid phase extraction and high-performance liquid chromatography with diode array detection method for the pharmacokinetic evaluation of bavachinin: A study on absolute bioavailability and dose proportionality
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Lei Liu, Kang-Ning Liu, Ya-Bin Wen, Han-Wen Zhang, Ya-Xin Lu, Zheng Yin
A fully automated on-line solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with diode array detection (DAD) method was developed for determination of bavachinin in mouse plasma. Analytical process was performed on two reversed-phase columns (SPE cartridge and analytical column) connected via a Valco 6-port switching valve. Plasma samples (10μL) were injected directly onto a C18 SPE cartridge (MF Ph-1 C18, 10mm×4mm, 5μm) and the biological matrix was washed out for 2min with the loading solvent (5mM NaH2PO4 buffer, pH 3.5) at a flow rate of 1mL/min. By rotation of the switching valve, bavachinin was eluted from the SPE cartridge in the back-flush mode and transferred to the analytical column (Venusil MP C18, 4.6mm×150mm, 5μm) by the chromatographic mobile phase consisted of acetonitrile-5mM NaH2PO4 buffer 65/35 (v/v, pH 3.5) at a flow rate of 1mL/min. The complete cycle of the on-line SPE purification and chromatographic separation of the analyte was 13min with UV detection performed at 236nm. Calibration curve with good linearity (r =0.9997) was obtained in the range of 20–4000ng/mL in mouse plasma. The intra-day and inter-day precisions (RSD) of bavachinin were in the range of 0.20–2.32% and the accuracies were between 98.47% and 102.95%. The lower limit of quantification (LLOQ) of the assay was 20ng/mL. In conclusion, the established automated on-line SPE-HPLC-DAD method demonstrated good performance in terms of linearity, specificity, detection and quantification limits, precision and accuracy, and was successfully utilized to quantify bavachinin in mouse plasma to support the pharmacokinetic (PK) studies. The PK properties of bavachinin were characterized as rapid oral absorption, high clearance, and poor absolute bioavailability.
Source:Journal of Chromatography B, Volumes 893–894
Lei Liu, Kang-Ning Liu, Ya-Bin Wen, Han-Wen Zhang, Ya-Xin Lu, Zheng Yin
A fully automated on-line solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with diode array detection (DAD) method was developed for determination of bavachinin in mouse plasma. Analytical process was performed on two reversed-phase columns (SPE cartridge and analytical column) connected via a Valco 6-port switching valve. Plasma samples (10μL) were injected directly onto a C18 SPE cartridge (MF Ph-1 C18, 10mm×4mm, 5μm) and the biological matrix was washed out for 2min with the loading solvent (5mM NaH2PO4 buffer, pH 3.5) at a flow rate of 1mL/min. By rotation of the switching valve, bavachinin was eluted from the SPE cartridge in the back-flush mode and transferred to the analytical column (Venusil MP C18, 4.6mm×150mm, 5μm) by the chromatographic mobile phase consisted of acetonitrile-5mM NaH2PO4 buffer 65/35 (v/v, pH 3.5) at a flow rate of 1mL/min. The complete cycle of the on-line SPE purification and chromatographic separation of the analyte was 13min with UV detection performed at 236nm. Calibration curve with good linearity (r =0.9997) was obtained in the range of 20–4000ng/mL in mouse plasma. The intra-day and inter-day precisions (RSD) of bavachinin were in the range of 0.20–2.32% and the accuracies were between 98.47% and 102.95%. The lower limit of quantification (LLOQ) of the assay was 20ng/mL. In conclusion, the established automated on-line SPE-HPLC-DAD method demonstrated good performance in terms of linearity, specificity, detection and quantification limits, precision and accuracy, and was successfully utilized to quantify bavachinin in mouse plasma to support the pharmacokinetic (PK) studies. The PK properties of bavachinin were characterized as rapid oral absorption, high clearance, and poor absolute bioavailability.
Development and validation of a rapid high-performance liquid chromatography-tandem mass spectrometry method for the determination of WJ-38, a novel aldose reductase inhibitor, in rat plasma and its application to a pharmacokinetic study
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Jing Lu, Youping Liu, Xin Wang, Shaojie Wang, Xin Di
WJ-38 is an aldose reductase inhibitor that is being developed for the treatment of diabetic complications. The present paper describes a sensitive and specific liquid chromatography-tandem mass spectrometry method for the determination of WJ-38 in rat plasma. Partial denaturation of plasma proteins with methanol followed by liquid–liquid extraction using ethyl acetate was used to extract strongly protein-bound WJ-38 from rat plasma. Chromatographic separation was performed on an Inertsil ODS-3 column with an isocratic mobile phase consisting of acetonitrile, water and formic acid (75:25:0.125, v/v/v). Mass spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an ESI interface operating in positive ionization mode. Quantitation was performed using selected reaction monitoring of precursor-product ion transitions at m/z 392→246 for WJ-38 and m/z 446→321 for glipizide (internal standard). A linear calibration curve was obtained over the concentration range of 10.0–10,000ng/mL for WJ-38 in rat plasma. The intra- and inter-day precisions were less than 13.6% and the accuracy was within ±5.3%. The extraction recovery of WJ-38 from rat plasma was over 66.0%. The validated method has been successfully applied to a pharmacokinetic study in rats after intragastrical administration of WJ-38.
Source:Journal of Chromatography B, Volumes 893–894
Jing Lu, Youping Liu, Xin Wang, Shaojie Wang, Xin Di
WJ-38 is an aldose reductase inhibitor that is being developed for the treatment of diabetic complications. The present paper describes a sensitive and specific liquid chromatography-tandem mass spectrometry method for the determination of WJ-38 in rat plasma. Partial denaturation of plasma proteins with methanol followed by liquid–liquid extraction using ethyl acetate was used to extract strongly protein-bound WJ-38 from rat plasma. Chromatographic separation was performed on an Inertsil ODS-3 column with an isocratic mobile phase consisting of acetonitrile, water and formic acid (75:25:0.125, v/v/v). Mass spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an ESI interface operating in positive ionization mode. Quantitation was performed using selected reaction monitoring of precursor-product ion transitions at m/z 392→246 for WJ-38 and m/z 446→321 for glipizide (internal standard). A linear calibration curve was obtained over the concentration range of 10.0–10,000ng/mL for WJ-38 in rat plasma. The intra- and inter-day precisions were less than 13.6% and the accuracy was within ±5.3%. The extraction recovery of WJ-38 from rat plasma was over 66.0%. The validated method has been successfully applied to a pharmacokinetic study in rats after intragastrical administration of WJ-38.
LC/LC–MS/MS of an innovative prostate human epithelial cancer (PHEC) in vitro model system
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
John D. Lapek, James L. McGrath, William A. Ricke, Alan E. Friedman
This work describes the proteomic characterization of a novel in vitro prostate cancer model system, the clonal prostatic human epithelial cancer (PHEC) cell lines. The model is composed of three cell lines representing the three progressive cancer states found in vivo: non-tumorigenic, tumorigenic, and metastatic. The cell lines were evaluated for differential protein expression between states using two dimensional liquid:liquid chromatographic separation followed by mass spectral identification. The proteins from cellular extracts were first separated using liquid:liquid primary separation based on their isoelectric points and hydrophobicity. The resulting peptide fractions were applied to liquid chromatography–mass spectrometry (LC–MS) separation for mass determination and protein identification based on Mascot database inquiry. Over 200 proteins that change expression over the course of progression of this in vitro prostate cancer model were discovered during the comparative analysis of the three cell lines. The importance of these proteins on prostate cancer progression remains to be elucidated with further characterizations. The combination of the two dimensional liquid:liquid separation and mass spectral identifications was used to successfully analyze differential protein expression between multiple cell lines.
Source:Journal of Chromatography B, Volumes 893–894
John D. Lapek, James L. McGrath, William A. Ricke, Alan E. Friedman
This work describes the proteomic characterization of a novel in vitro prostate cancer model system, the clonal prostatic human epithelial cancer (PHEC) cell lines. The model is composed of three cell lines representing the three progressive cancer states found in vivo: non-tumorigenic, tumorigenic, and metastatic. The cell lines were evaluated for differential protein expression between states using two dimensional liquid:liquid chromatographic separation followed by mass spectral identification. The proteins from cellular extracts were first separated using liquid:liquid primary separation based on their isoelectric points and hydrophobicity. The resulting peptide fractions were applied to liquid chromatography–mass spectrometry (LC–MS) separation for mass determination and protein identification based on Mascot database inquiry. Over 200 proteins that change expression over the course of progression of this in vitro prostate cancer model were discovered during the comparative analysis of the three cell lines. The importance of these proteins on prostate cancer progression remains to be elucidated with further characterizations. The combination of the two dimensional liquid:liquid separation and mass spectral identifications was used to successfully analyze differential protein expression between multiple cell lines.
Enrichment and purification of gardenia yellow from Gardenia jasminoides var. radicans Makino by column chromatography technique
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Jian-Fang Chen, Gui-Ming Fu, Yin Wan, Cheng-Mei Liu, Jian-Xin Chai, Hong-Ge Li, Jian-Tao Wang, Ming-Hu, Lun-Ning Zhang
In present study, the performance and separation characteristics of nine macroporous resins for the enrichment and purification of gardenia yellow from Gardenia jasminoides var. radicans Makino have been evaluated. The adsorption and desorption properties of crude gardenia yellow solution on macroporous resins including HPD722, HPD100, HPD100A, HPD400, HPD400A, D101, AB-8, XAD-16, and NKA-9 have been compared. Then, HPD722 was chosen to purify gardenia yellow because of its strong adsorption and desorption abilities as well as high selectivity. Column packed with HPD722 resin was used to perform dynamic adsorption and desorption tests to optimize the separation process of gardenia yellow. The optimal conditions were as follows: The crude gardenia yellow solution with concentration of 15mg/mL was loaded in column packed with HPD722 resin at the flow rate of 1.0mL/min, and the adsorbate-laden column was washed with 800mL water, 600mL 15% ethanol water solution respectively at the speed of 2.5mL/min, then desorbed with 200mL 80% ethanol water solution at the speed of 3.5mL/min. The colority of the product obtained were up to 300. The method developed in this study provides a new approach for scale-up separation and purification of gardenia yellow from G. jasminoides var. radicans Makino.
Source:Journal of Chromatography B, Volumes 893–894
Jian-Fang Chen, Gui-Ming Fu, Yin Wan, Cheng-Mei Liu, Jian-Xin Chai, Hong-Ge Li, Jian-Tao Wang, Ming-Hu, Lun-Ning Zhang
In present study, the performance and separation characteristics of nine macroporous resins for the enrichment and purification of gardenia yellow from Gardenia jasminoides var. radicans Makino have been evaluated. The adsorption and desorption properties of crude gardenia yellow solution on macroporous resins including HPD722, HPD100, HPD100A, HPD400, HPD400A, D101, AB-8, XAD-16, and NKA-9 have been compared. Then, HPD722 was chosen to purify gardenia yellow because of its strong adsorption and desorption abilities as well as high selectivity. Column packed with HPD722 resin was used to perform dynamic adsorption and desorption tests to optimize the separation process of gardenia yellow. The optimal conditions were as follows: The crude gardenia yellow solution with concentration of 15mg/mL was loaded in column packed with HPD722 resin at the flow rate of 1.0mL/min, and the adsorbate-laden column was washed with 800mL water, 600mL 15% ethanol water solution respectively at the speed of 2.5mL/min, then desorbed with 200mL 80% ethanol water solution at the speed of 3.5mL/min. The colority of the product obtained were up to 300. The method developed in this study provides a new approach for scale-up separation and purification of gardenia yellow from G. jasminoides var. radicans Makino.
Comparison of three derivatization reagents for the simultaneous determination of highly hydrophilic pyrimidine antitumor agents in human plasma by LC–MS/MS
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
He-ying Liu, Li Ding, Yong Yu, Yan Chu, He Zhu
A comparison of three derivatization reagents (dansyl chloride, diazomethane and p-bromophenacyl bromide) for the simultaneous quantitation of three anticancer chemicals (tegafur, 5-fluorouracil and gimeracil) and endogenous uracil in plasma using high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed and evaluated. Through a comprehensive consideration, p-bromophenacyl bromide (p-BPB) was finally selected as the derivatization reagent. Because it essentially changed the chromatographic behavior of the aforementioned highly hydrophilic compounds and significantly enhanced their sensitivities. The method was validated over the concentration ranges of 5–5000ng/ml for tegafur, 0.6–700ng/ml for 5-fluorouracil, 3–700ng/ml for gimeracil and 6–2000ng/ml for uracil. The method was successfully applied to the pharmacokinetics study of tegafur, 5-fluorouracil, gimeracil and uracil in cancer patients.
Source:Journal of Chromatography B, Volumes 893–894
He-ying Liu, Li Ding, Yong Yu, Yan Chu, He Zhu
A comparison of three derivatization reagents (dansyl chloride, diazomethane and p-bromophenacyl bromide) for the simultaneous quantitation of three anticancer chemicals (tegafur, 5-fluorouracil and gimeracil) and endogenous uracil in plasma using high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed and evaluated. Through a comprehensive consideration, p-bromophenacyl bromide (p-BPB) was finally selected as the derivatization reagent. Because it essentially changed the chromatographic behavior of the aforementioned highly hydrophilic compounds and significantly enhanced their sensitivities. The method was validated over the concentration ranges of 5–5000ng/ml for tegafur, 0.6–700ng/ml for 5-fluorouracil, 3–700ng/ml for gimeracil and 6–2000ng/ml for uracil. The method was successfully applied to the pharmacokinetics study of tegafur, 5-fluorouracil, gimeracil and uracil in cancer patients.
Development of a highly sensitive method for the quantification of estrone and estradiol in serum by liquid chromatography tandem mass spectrometry without derivatization
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Tom Fiers, Bruno Casetta, Brigitte Bernaert, Eric Vandersypt, Martine Debock, Jean-Marc Kaufman
Measurement of estrone (E1) and estradiol (E2) values <1pg/mL (3.7pmol/L) is necessary for postmenopausal, pediatric and male serum samples. Until now this was rarely reached and only through derivatization which can present problems for estradiol. A very sensitive LC–MS/MS method was developed avoiding derivatization, convenient for large-scale studies. The desired sensitivity and specificity were achieved using ESI negative mode, LLE and a 2D chromatography consisting of a trapping column and a second dimension reverse-phase C8 analytical column. A mixture of an aqueous solution of ammonium fluoride at 0.2mM and methanol was used on the analytical column to further increase the sensitivity. Serum LOQ was <0.5pg/mL (1.9pmol/L) for E2 and E1 and recoveries ranged from 95 to 105%. No carry-over was detectable. Inter assay CV's were 4.0% at 21pg/mL (77pmol/L) for E2, 7.6% at 25pg/mL (93pmol/L) for E1. Comparison with commercial direct estrogen assays (Roche Diagnostics E170 for E2, Bioline RIA for E1) exposed analytical unsuitability (due to a combined lack of sensitivity and specificity) for the assay of male, postmenopausal or pediatric samples.
Source:Journal of Chromatography B, Volumes 893–894
Tom Fiers, Bruno Casetta, Brigitte Bernaert, Eric Vandersypt, Martine Debock, Jean-Marc Kaufman
Measurement of estrone (E1) and estradiol (E2) values <1pg/mL (3.7pmol/L) is necessary for postmenopausal, pediatric and male serum samples. Until now this was rarely reached and only through derivatization which can present problems for estradiol. A very sensitive LC–MS/MS method was developed avoiding derivatization, convenient for large-scale studies. The desired sensitivity and specificity were achieved using ESI negative mode, LLE and a 2D chromatography consisting of a trapping column and a second dimension reverse-phase C8 analytical column. A mixture of an aqueous solution of ammonium fluoride at 0.2mM and methanol was used on the analytical column to further increase the sensitivity. Serum LOQ was <0.5pg/mL (1.9pmol/L) for E2 and E1 and recoveries ranged from 95 to 105%. No carry-over was detectable. Inter assay CV's were 4.0% at 21pg/mL (77pmol/L) for E2, 7.6% at 25pg/mL (93pmol/L) for E1. Comparison with commercial direct estrogen assays (Roche Diagnostics E170 for E2, Bioline RIA for E1) exposed analytical unsuitability (due to a combined lack of sensitivity and specificity) for the assay of male, postmenopausal or pediatric samples.
Semi-automated solid-phase extraction method for studying the biodegradation of ochratoxin A by human intestinal microbiota
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Valérie Camel, Minale Ouethrani, Cindy Coudray, Catherine Philippe, Sylvie Rabot
A simple and rapid semi-automated solid-phase (SPE) extraction method has been developed for the analysis of ochratoxin A in aqueous matrices related to biodegradation experiments (namely digestive contents and faecal excreta), with a view of using this method to follow OTA biodegradation by human intestinal microbiota. Influence of extraction parameters that could affect semi-automated SPE efficiency was studied, using C18-silica as the sorbent and water as the simplest matrix, being further applied to the matrices of interest. Conditions finally retained were as follows: 5-mL aqueous samples (pH 3) containing an organic modifier (20% ACN) were applied on 100-mg cartridges. After drying (9mL of air), the cartridge was rinsed with 5-mL H2O/ACN (80:20, v/v), before eluting the compounds with 3× 1mL of MeOH/THF (10:90, v/v). Acceptable recoveries and limits of quantification could be obtained considering the complexity of the investigated matrices and the low volumes sampled; this method was also suitable for the analysis of ochratoxin B in faecal extracts. Applicability of the method is illustrated by preliminary results of ochratoxin A biodegradation studies by human intestinal microbiota under simple in vitro conditions. Interestingly, partial degradation of ochratoxin A was observed, with efficiencies ranging from 14% to 47% after 72h incubation. In addition, three phase I metabolites could be identified using high resolution mass spectrometry, namely ochratoxin α, open ochratoxin A and ochratoxin B.
Source:Journal of Chromatography B, Volumes 893–894
Valérie Camel, Minale Ouethrani, Cindy Coudray, Catherine Philippe, Sylvie Rabot
A simple and rapid semi-automated solid-phase (SPE) extraction method has been developed for the analysis of ochratoxin A in aqueous matrices related to biodegradation experiments (namely digestive contents and faecal excreta), with a view of using this method to follow OTA biodegradation by human intestinal microbiota. Influence of extraction parameters that could affect semi-automated SPE efficiency was studied, using C18-silica as the sorbent and water as the simplest matrix, being further applied to the matrices of interest. Conditions finally retained were as follows: 5-mL aqueous samples (pH 3) containing an organic modifier (20% ACN) were applied on 100-mg cartridges. After drying (9mL of air), the cartridge was rinsed with 5-mL H2O/ACN (80:20, v/v), before eluting the compounds with 3× 1mL of MeOH/THF (10:90, v/v). Acceptable recoveries and limits of quantification could be obtained considering the complexity of the investigated matrices and the low volumes sampled; this method was also suitable for the analysis of ochratoxin B in faecal extracts. Applicability of the method is illustrated by preliminary results of ochratoxin A biodegradation studies by human intestinal microbiota under simple in vitro conditions. Interestingly, partial degradation of ochratoxin A was observed, with efficiencies ranging from 14% to 47% after 72h incubation. In addition, three phase I metabolites could be identified using high resolution mass spectrometry, namely ochratoxin α, open ochratoxin A and ochratoxin B.
Evaluation of human interferon adsorption performance of Cibacron Blue F3GA attached cryogels and interferon purification by using FPLC system
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Ali Doğan, Serpil Özkara, Müfrettin Murat Sarı, Lokman Uzun, Adil Denizli
In this study, we have focused our attention on preparing supermacroporous cryogels as a potential dye-affinity adsorbent for interferon purification. For this purpose, 2-hydroxyethyl methacrylate (HEMA) and Cibacron Blue F3GA (CB) were selected as main monomer and dye–ligand. Cibacron Blue F3GA attached supermacroporous poly(2-hydroxyethyl methacrylate) [poly(HEMA)/CB] cryogels were prepared and characterized by swelling test, scanning electron microscopy, elemental analysis, and FTIR. After that, the effecting factors such as pH, concentration, interaction time, and ionic strength on the interferon separation were evaluated. The maximum adsorption capacity of poly(HEMA)/CB cryogels was obtained as 38.2mg/g at pH 6.0. Fast protein liquid chromatography (FPLC) system was used for interferon purification from human gingival fibroblast extract. The chromatography parameters, capacity and selectivity factors, resolution and theoretical plate number were found as 7.79, 9.62, 4.23 and 554, respectively. Although some decreases in total protein content, from 320μg to 18μg, and interferon activity, from 2.6×103 IU to 2.2×103 IU, were determined, specific antiviral activity increased from 7.19IU/μg to 122.2IU/μg. The purified interferon samples have 97.6% purity determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After repeated ten adsorption–desorption cycles, no significant decrease was determined in adsorption capacity of cryogel. In result, poly(HEMA)/CB cryogels have an application potential for rapid, cheap and specific purification of interferon.
Source:Journal of Chromatography B, Volumes 893–894
Ali Doğan, Serpil Özkara, Müfrettin Murat Sarı, Lokman Uzun, Adil Denizli
In this study, we have focused our attention on preparing supermacroporous cryogels as a potential dye-affinity adsorbent for interferon purification. For this purpose, 2-hydroxyethyl methacrylate (HEMA) and Cibacron Blue F3GA (CB) were selected as main monomer and dye–ligand. Cibacron Blue F3GA attached supermacroporous poly(2-hydroxyethyl methacrylate) [poly(HEMA)/CB] cryogels were prepared and characterized by swelling test, scanning electron microscopy, elemental analysis, and FTIR. After that, the effecting factors such as pH, concentration, interaction time, and ionic strength on the interferon separation were evaluated. The maximum adsorption capacity of poly(HEMA)/CB cryogels was obtained as 38.2mg/g at pH 6.0. Fast protein liquid chromatography (FPLC) system was used for interferon purification from human gingival fibroblast extract. The chromatography parameters, capacity and selectivity factors, resolution and theoretical plate number were found as 7.79, 9.62, 4.23 and 554, respectively. Although some decreases in total protein content, from 320μg to 18μg, and interferon activity, from 2.6×103 IU to 2.2×103 IU, were determined, specific antiviral activity increased from 7.19IU/μg to 122.2IU/μg. The purified interferon samples have 97.6% purity determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After repeated ten adsorption–desorption cycles, no significant decrease was determined in adsorption capacity of cryogel. In result, poly(HEMA)/CB cryogels have an application potential for rapid, cheap and specific purification of interferon.
HPLC–DAD protein kinase inhibitor analysis in human serum
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Marek Dziadosz, Rüdiger Lessig, Heidemarie Bartels
We here describe an HPLC–DAD method to analyse different protein kinase inhibitors. Potential applications of this method are pharmacokinetic studies and therapeutic drug monitoring. Optimised chromatography conditions resulted in a very good separation of seven inhibitors (vatalanib, bosutinib, canertinib, tandutinib, pazopanib, dasatinib – internal standard and erlotinib). The good sensitivity makes this method competitive with LC/MS/MS. The separation was performed with a Lichrospher 100-5 RP8, 250mm×4mm column maintained at 30±1°C, and with a mobile phase of 0.05M H3PO4/KH2PO4 (pH=2.3)–acetonitrile (7:3, v/v) at a flow rate of 0.7mL/min. A simple and fast sample preparation sequence with liquid–liquid extraction led to good recoveries (73–90%) of all analytes. The recovery hardly reached 50% only for pazopanib. This method can also be used for targeted protein kinase inhibitor quantification. A perfect linearity in the validated range (20–10,000ng/mL) and an LOQ of 20ng/mL were achieved. The relative standard deviations and accuracies of all examined drug concentrations gave values much lower than 15% both for between- and within-batch calculations. All analysed PKIs were stable for 6 months in a 1mg/mL dimethyl sulfoxide stock solution. Vatalanib, bosutinib and erlotinib were also stable in human serum in the whole examined concentration range.
Source:Journal of Chromatography B, Volumes 893–894
Marek Dziadosz, Rüdiger Lessig, Heidemarie Bartels
We here describe an HPLC–DAD method to analyse different protein kinase inhibitors. Potential applications of this method are pharmacokinetic studies and therapeutic drug monitoring. Optimised chromatography conditions resulted in a very good separation of seven inhibitors (vatalanib, bosutinib, canertinib, tandutinib, pazopanib, dasatinib – internal standard and erlotinib). The good sensitivity makes this method competitive with LC/MS/MS. The separation was performed with a Lichrospher 100-5 RP8, 250mm×4mm column maintained at 30±1°C, and with a mobile phase of 0.05M H3PO4/KH2PO4 (pH=2.3)–acetonitrile (7:3, v/v) at a flow rate of 0.7mL/min. A simple and fast sample preparation sequence with liquid–liquid extraction led to good recoveries (73–90%) of all analytes. The recovery hardly reached 50% only for pazopanib. This method can also be used for targeted protein kinase inhibitor quantification. A perfect linearity in the validated range (20–10,000ng/mL) and an LOQ of 20ng/mL were achieved. The relative standard deviations and accuracies of all examined drug concentrations gave values much lower than 15% both for between- and within-batch calculations. All analysed PKIs were stable for 6 months in a 1mg/mL dimethyl sulfoxide stock solution. Vatalanib, bosutinib and erlotinib were also stable in human serum in the whole examined concentration range.
Sensitive quantification of roflumilast and roflumilast N-oxide in human plasma by LC–MS/MS employing parallel chromatography and electrospray ionisation
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Norbert G. Knebel, Rolf Herzog, Felix Reutter, Karl Zech
A high throughput bioanalytical method based on semi-automated liquid extraction and liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for the sensitive quantification of roflumilast and its metabolite roflumilast N-oxide, a phosphodiesterase (PDE) inhibitor in human plasma and serum. The sample work-up procedure comprised liquid extraction using penta-deuterated analogues of both analytes as internal standards. Chromatography was performed on C18 revered phase analytical columns at a flow rate of 0.5mL/min in the dual column mode employing a column switching technique and a linear gradient from 18% to 54% acetonitrile in 0.005M aqueous ammonium acetate containing 0.006% formic acid. Mass spectrometry was performed on an API 4000 instrument in the positive ion SRM-mode (selected reaction monitoring) with the Turbo-V® ionspray interface. The method showed linear detector responses over the entire calibration range between 0.1ng/mL (lower limit of quantification (LLOQ)) and 50ng/mL (upper limit of quantification (ULOQ)) for both analytes. Linear regression analysis with concentration-squared weighting (1/x 2 for roflumilast and 1/x for roflumilast N-oxide) yielded inaccuracy and precision values <15% and coefficients of correlation (r) for the calibration curves >0.99 for both analytes.
Source:Journal of Chromatography B, Volumes 893–894
Norbert G. Knebel, Rolf Herzog, Felix Reutter, Karl Zech
A high throughput bioanalytical method based on semi-automated liquid extraction and liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for the sensitive quantification of roflumilast and its metabolite roflumilast N-oxide, a phosphodiesterase (PDE) inhibitor in human plasma and serum. The sample work-up procedure comprised liquid extraction using penta-deuterated analogues of both analytes as internal standards. Chromatography was performed on C18 revered phase analytical columns at a flow rate of 0.5mL/min in the dual column mode employing a column switching technique and a linear gradient from 18% to 54% acetonitrile in 0.005M aqueous ammonium acetate containing 0.006% formic acid. Mass spectrometry was performed on an API 4000 instrument in the positive ion SRM-mode (selected reaction monitoring) with the Turbo-V® ionspray interface. The method showed linear detector responses over the entire calibration range between 0.1ng/mL (lower limit of quantification (LLOQ)) and 50ng/mL (upper limit of quantification (ULOQ)) for both analytes. Linear regression analysis with concentration-squared weighting (1/x 2 for roflumilast and 1/x for roflumilast N-oxide) yielded inaccuracy and precision values <15% and coefficients of correlation (r) for the calibration curves >0.99 for both analytes.
Development and validation of LC–MS/MS assays for the quantification of bendamustine and its metabolites in human plasma and urine
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
A.C. Dubbelman, M. Tibben, H. Rosing, A. Gebretensae, L. Nan, S.H. Gorman, P. Robertson, J.H.M. Schellens, J.H. Beijnen
A sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) assay is described for the quantification of the anti-cancer agent bendamustine and its phase I metabolites γ-hydroxy-bendamustine (M3) and N-des-methylbendamustine (M4) and for its product of two-fold hydrolysis, dihydroxy-bendamustine (HP2), in human plasma and urine. Like most alkylating nitrogen mustards, bendamustine is prone to chemical hydrolysis in aqueous solution. To minimize degradation of bendamustine, urine samples were stabilized by a 100-fold dilution with human plasma and then processed identically to plasma samples. Sample aliquots of 200μL were mixed with an internal standard solution and acidified before separation of the analytes from the biomatrix with solid phase extraction. Dried and reconstituted extracts were injected on a Synergi Hydro RP column for the analysis of bendamustine, M3 and M4 or a Synergi Polar RP column for the analysis of HP2. Gradient elution was applied using 5mM ammonium formate with 0.1% formic acid in water and methanol as mobile phases. Analytes were ionized using an electrospray ionisation source in positive mode and detected with a triple quadrupole mass spectrometer. The quantifiable range for bendamustine, M3 and M4 was 0.5–500ng/mL in plasma and 0.5–50μg/mL in urine, and that for HP2 was 1–500ng/mL in plasma and 0.1–50μg/mL in urine. The assays were accurate and precise, with inter-assay and intra-assay accuracies within ±20% of nominal and CV values below 20% at the lower limit of quantification and within ±15% of nominal and below 15% at the other concentration levels tested. These methods were successfully applied to evaluate the pharmacokinetic profile of bendamustine and its metabolites in cancer patients treated with bendamustine.
Source:Journal of Chromatography B, Volumes 893–894
A.C. Dubbelman, M. Tibben, H. Rosing, A. Gebretensae, L. Nan, S.H. Gorman, P. Robertson, J.H.M. Schellens, J.H. Beijnen
A sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) assay is described for the quantification of the anti-cancer agent bendamustine and its phase I metabolites γ-hydroxy-bendamustine (M3) and N-des-methylbendamustine (M4) and for its product of two-fold hydrolysis, dihydroxy-bendamustine (HP2), in human plasma and urine. Like most alkylating nitrogen mustards, bendamustine is prone to chemical hydrolysis in aqueous solution. To minimize degradation of bendamustine, urine samples were stabilized by a 100-fold dilution with human plasma and then processed identically to plasma samples. Sample aliquots of 200μL were mixed with an internal standard solution and acidified before separation of the analytes from the biomatrix with solid phase extraction. Dried and reconstituted extracts were injected on a Synergi Hydro RP column for the analysis of bendamustine, M3 and M4 or a Synergi Polar RP column for the analysis of HP2. Gradient elution was applied using 5mM ammonium formate with 0.1% formic acid in water and methanol as mobile phases. Analytes were ionized using an electrospray ionisation source in positive mode and detected with a triple quadrupole mass spectrometer. The quantifiable range for bendamustine, M3 and M4 was 0.5–500ng/mL in plasma and 0.5–50μg/mL in urine, and that for HP2 was 1–500ng/mL in plasma and 0.1–50μg/mL in urine. The assays were accurate and precise, with inter-assay and intra-assay accuracies within ±20% of nominal and CV values below 20% at the lower limit of quantification and within ±15% of nominal and below 15% at the other concentration levels tested. These methods were successfully applied to evaluate the pharmacokinetic profile of bendamustine and its metabolites in cancer patients treated with bendamustine.
Rapid and efficient purification of chrysophanol in Rheum Palmatum LINN by supercritical fluid extraction coupled with preparative liquid chromatography in tandem
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Tiffany Chien-Ting Lo, Hung-Chi Nian, Kong-Hwa Chiu, Ai-Yih Wang, Ben-Zen Wu
Chrysophanol has high pharmaceutical values. However, it was difficult to use the traditional extraction method to extract high-concentration chrysophanol. Therefore, the purpose of this study is to purify and separate chrysophanol in traditional herb, Rheum Palmatum LINN, by using supercritical fluid extraction (SFE) and preparative high-performance liquid chromatography (P-HPLC) for rapid and large-scale isolation. The method is efficient for selective extraction of chrysophanol from the herbs, which have complex compositions. The extraction efficiency of chrysophanol with SFE is 25× higher than that of boiled water extraction under the same extraction time. The optimal conditions for SFE were 210atm and 85°C for 30min; for P-HPLC, a C18 column was used with a gradient elution of methanol and 1% acetic acid at a flow rate of 10mL/min. According to 1H NMR and LC–MS analyses, the purity of the isolated chrysophanol was as high as 99%. The recovery for chrysophanol in Rheum after SPE/PHPLC processing was in the range of 88–91.5%. Compared with other extraction and purification methods, the sequential system (SFE/P-HPLC) achieved the highest amount of extracted chrysophanol from Rheum Palmatum LINN (0.38mg/g) and the shortest run time (3h). Hence, this rapid and environmentally friendly method can separate compounds based on polarity with high efficiencies and, coupled with P-HPLC, it may be applicable in the large-scale production of foods and medicines in the future.
Source:Journal of Chromatography B, Volumes 893–894
Tiffany Chien-Ting Lo, Hung-Chi Nian, Kong-Hwa Chiu, Ai-Yih Wang, Ben-Zen Wu
Chrysophanol has high pharmaceutical values. However, it was difficult to use the traditional extraction method to extract high-concentration chrysophanol. Therefore, the purpose of this study is to purify and separate chrysophanol in traditional herb, Rheum Palmatum LINN, by using supercritical fluid extraction (SFE) and preparative high-performance liquid chromatography (P-HPLC) for rapid and large-scale isolation. The method is efficient for selective extraction of chrysophanol from the herbs, which have complex compositions. The extraction efficiency of chrysophanol with SFE is 25× higher than that of boiled water extraction under the same extraction time. The optimal conditions for SFE were 210atm and 85°C for 30min; for P-HPLC, a C18 column was used with a gradient elution of methanol and 1% acetic acid at a flow rate of 10mL/min. According to 1H NMR and LC–MS analyses, the purity of the isolated chrysophanol was as high as 99%. The recovery for chrysophanol in Rheum after SPE/PHPLC processing was in the range of 88–91.5%. Compared with other extraction and purification methods, the sequential system (SFE/P-HPLC) achieved the highest amount of extracted chrysophanol from Rheum Palmatum LINN (0.38mg/g) and the shortest run time (3h). Hence, this rapid and environmentally friendly method can separate compounds based on polarity with high efficiencies and, coupled with P-HPLC, it may be applicable in the large-scale production of foods and medicines in the future.
Serum metabolomic profiles from patients with acute kidney injury: A pilot study
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Jinchun Sun, Melissa Shannon, Yosuke Ando, Laura K. Schnackenberg, Nasim A. Khan, Didier Portilla, Richard D. Beger
Low sensitivity of current clinical markers (serum creatinine and blood urea nitrogen (BUN)) in early stages of the development of acute kidney injury (AKI) limits their utility. Rapid LC/MS-based metabolic profiling of serum demonstrated in a pilot study that metabolomics could provide novel indicators of AKI. Metabolic profiles of serum samples from seventeen hospitalized patients with newly diagnosed AKI were compared with the profiles of serum from age-matched subjects with normal kidney function. Increases in acylcarnitines and amino acids (methionine, homocysteine, pyroglutamate, asymmetric dimethylarginine (ADMA), and phenylalanine) and a reduction in serum levels of arginine and several lysophosphatidyl cholines were observed in patients with AKI compared to healthy subjects. Increases in homocysteine, ADMA and pyroglutamate have been recognized as biomarkers of cardiovascular and renal disease, and acylcarnitines represent biomarkers of defective fatty acid oxidation. The results of this pilot study demonstrate the utility of metabolomics in the discovery of novel serum biomarkers that can facilitate the diagnosis and determine prognosis of AKI in hospitalized patients.
Source:Journal of Chromatography B, Volumes 893–894
Jinchun Sun, Melissa Shannon, Yosuke Ando, Laura K. Schnackenberg, Nasim A. Khan, Didier Portilla, Richard D. Beger
Low sensitivity of current clinical markers (serum creatinine and blood urea nitrogen (BUN)) in early stages of the development of acute kidney injury (AKI) limits their utility. Rapid LC/MS-based metabolic profiling of serum demonstrated in a pilot study that metabolomics could provide novel indicators of AKI. Metabolic profiles of serum samples from seventeen hospitalized patients with newly diagnosed AKI were compared with the profiles of serum from age-matched subjects with normal kidney function. Increases in acylcarnitines and amino acids (methionine, homocysteine, pyroglutamate, asymmetric dimethylarginine (ADMA), and phenylalanine) and a reduction in serum levels of arginine and several lysophosphatidyl cholines were observed in patients with AKI compared to healthy subjects. Increases in homocysteine, ADMA and pyroglutamate have been recognized as biomarkers of cardiovascular and renal disease, and acylcarnitines represent biomarkers of defective fatty acid oxidation. The results of this pilot study demonstrate the utility of metabolomics in the discovery of novel serum biomarkers that can facilitate the diagnosis and determine prognosis of AKI in hospitalized patients.
Hydrophilic interaction liquid chromatography/positive ion electrospray mass spectrometry for the quantification of deferasirox, an oral iron chelator, in human plasma
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Helen Pligoropoulou, Ariadni Vonaparti, Irene Panderi
A rapid hydrophilic interaction liquid chromatography/positive ion electrospray mass spectrometric assay (HILIC/ESI-MS) was developed, validated and applied to the determination of deferasirox, in human plasma. The sample preparation process involved liquid–liquid extraction of 50μL plasma sample using ethyl acetate as an extraction solvent. Chromatographic separation was performed on an XBridge®-HILIC analytical column (150.0mm×2.1mm i.d., particle size 3.5μm, 135Å) under isocratic elution. The mobile phase was composed of a 10% 8.0mM ammonium acetate water solution pH=5.0, adjusted with formic acid, in a binary mixture of acetonitrile/methanol (50:50, v/v) and pumped at a flow rate of 0.20mL/min. Quantitation of deferasirox was performed with selected ion monitoring (SIM) in positive ionization mode using electrospray ionization interface. The assay was found to be linear in the concentration range of 0.20–120.0μg/mL for deferasirox. Intermediate precision was found less than 3.9% over the tested concentration ranges. A run time of less than 6.0min for each sample made it possible to analyze a large number of human plasma samples per day. The method can be used to support a wide range of clinical studies concerning deferasirox monitoring and it was applied to the analysis of human plasma samples obtained from patients with β-thalassemia major.
Source:Journal of Chromatography B, Volumes 893–894
Helen Pligoropoulou, Ariadni Vonaparti, Irene Panderi
A rapid hydrophilic interaction liquid chromatography/positive ion electrospray mass spectrometric assay (HILIC/ESI-MS) was developed, validated and applied to the determination of deferasirox, in human plasma. The sample preparation process involved liquid–liquid extraction of 50μL plasma sample using ethyl acetate as an extraction solvent. Chromatographic separation was performed on an XBridge®-HILIC analytical column (150.0mm×2.1mm i.d., particle size 3.5μm, 135Å) under isocratic elution. The mobile phase was composed of a 10% 8.0mM ammonium acetate water solution pH=5.0, adjusted with formic acid, in a binary mixture of acetonitrile/methanol (50:50, v/v) and pumped at a flow rate of 0.20mL/min. Quantitation of deferasirox was performed with selected ion monitoring (SIM) in positive ionization mode using electrospray ionization interface. The assay was found to be linear in the concentration range of 0.20–120.0μg/mL for deferasirox. Intermediate precision was found less than 3.9% over the tested concentration ranges. A run time of less than 6.0min for each sample made it possible to analyze a large number of human plasma samples per day. The method can be used to support a wide range of clinical studies concerning deferasirox monitoring and it was applied to the analysis of human plasma samples obtained from patients with β-thalassemia major.
Separation and purification of phosvitin phosphopeptides using immobilized metal affinity nanoparticles
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Jing Zhang, Jun Sun, Yuntao Liu, Junhua Li, Yujie Su, Wenshui Xia, Yanjun Yang
Monodispersed and functional immobilized metal affinity magnetic chondroitin sodium sulfate nanoparticles (short as IMAN @ Fe (III)) were prepared and employed in extracting of Phosvitin Phosphopeptides (short as PPPs) from egg yolk. It was found that the diameter of the magnetic CS nanoparticles was about 20nm, and they could easily be aggregated by a magnet when suspending in the aqueous solution. The adsorption equilibrium of PPPs onto the obtained nanocarriers fitted well with the Langmuir model. The adsorption capacity of PPPs onto the superparamagnetic nanoparticles was influenced by pH and the initial concentration of the peptides solution. The final nitrogen/phosphorus molar ratios (short as N/P) of PPPs from crude egg yolk peptides and phosvitin peptides were low to 5.78 and 5.23, respectively. Compared with traditional methods, the need for preparation of phosvitin before purification is obviated and the higher purity of PPPs were obtained. In conclusion, this type of IMAN @ Fe (III) would bring advantages to the conventional separation techniques of PPPs from chicken egg yolk.
Source:Journal of Chromatography B, Volumes 893–894
Jing Zhang, Jun Sun, Yuntao Liu, Junhua Li, Yujie Su, Wenshui Xia, Yanjun Yang
Monodispersed and functional immobilized metal affinity magnetic chondroitin sodium sulfate nanoparticles (short as IMAN @ Fe (III)) were prepared and employed in extracting of Phosvitin Phosphopeptides (short as PPPs) from egg yolk. It was found that the diameter of the magnetic CS nanoparticles was about 20nm, and they could easily be aggregated by a magnet when suspending in the aqueous solution. The adsorption equilibrium of PPPs onto the obtained nanocarriers fitted well with the Langmuir model. The adsorption capacity of PPPs onto the superparamagnetic nanoparticles was influenced by pH and the initial concentration of the peptides solution. The final nitrogen/phosphorus molar ratios (short as N/P) of PPPs from crude egg yolk peptides and phosvitin peptides were low to 5.78 and 5.23, respectively. Compared with traditional methods, the need for preparation of phosvitin before purification is obviated and the higher purity of PPPs were obtained. In conclusion, this type of IMAN @ Fe (III) would bring advantages to the conventional separation techniques of PPPs from chicken egg yolk.
A new HPLC UV validated method for therapeutic monitoring of deferasirox in thalassaemic patients
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Silvia De Francia, Davide Massano, Francesca Maria Piccione, Elisa Pirro, Silvia Racca, Francesco Di Carlo, Antonio Piga
We describe a new high performance liquid chromatography coupled with ultraviolet detection method for the quantification of plasma concentration of oral iron chelating agent deferasirox. A simple protein precipitation extraction procedure was applied on 500μl of plasma aliquots. Chromatographic separation was achieved on a C18 reverse phase column and eluate was monitored at 295nm, with 8min of analytical run. This method has been validated following Food and Drug Administration procedures: mean intra and inter day variability was 4.64 and 10.55%; mean accuracy was 6.27%; mean extraction recovery 91.66%. Calibration curves ranged from 0.078125 to 40μg/ml. Limit of quantification was set at 0.15625 while limit of detection at 0.078125μg/ml. We applied methodology developed on plasma samples of thalassaemic patients treated with deferasirox, finding correlation between deferasirox plasma concentrations and serum ferritin levels. This methodology allowed a specific, sensitive and reliable determination of deferasirox, that could be useful to perform its therapeutic monitoring and pharmacokinetic studies in patients plasma.
Source:Journal of Chromatography B, Volumes 893–894
Silvia De Francia, Davide Massano, Francesca Maria Piccione, Elisa Pirro, Silvia Racca, Francesco Di Carlo, Antonio Piga
We describe a new high performance liquid chromatography coupled with ultraviolet detection method for the quantification of plasma concentration of oral iron chelating agent deferasirox. A simple protein precipitation extraction procedure was applied on 500μl of plasma aliquots. Chromatographic separation was achieved on a C18 reverse phase column and eluate was monitored at 295nm, with 8min of analytical run. This method has been validated following Food and Drug Administration procedures: mean intra and inter day variability was 4.64 and 10.55%; mean accuracy was 6.27%; mean extraction recovery 91.66%. Calibration curves ranged from 0.078125 to 40μg/ml. Limit of quantification was set at 0.15625 while limit of detection at 0.078125μg/ml. We applied methodology developed on plasma samples of thalassaemic patients treated with deferasirox, finding correlation between deferasirox plasma concentrations and serum ferritin levels. This methodology allowed a specific, sensitive and reliable determination of deferasirox, that could be useful to perform its therapeutic monitoring and pharmacokinetic studies in patients plasma.
Overcoming non-specific adsorption issues for AZD9164 in human urine samples: Consideration of bioanalytical and metabolite identification procedures
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Steve Silvester, Frank Zang
A key challenge in the development of robust bioanalytical methods, for the determination of drug analyte in human urine samples, is the elimination of potential analyte losses as a result of non-specific adsorption to container surfaces in which the samples are collected, stored or processed. A common approach to address adsorption issues is to treat the urine samples with additives that serve to increase analyte solubility and/or minimise interaction with the container surfaces. A series of adsorption experiments were performed on human urine samples containing an adsorption-prone in-house development compound (AZD9164). A roller-mixing methodology was employed to maximise sample interaction with container surfaces and quantification of analyte was performed by LC–MS/MS following minimal sample preparation. In the absence of any urine additive, adsorptive losses averaged 35% but were highly variable between different lots of urine. In the presence of a range of additives, including the surfactants Tween 80, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS) and sodium dodecylbenzenesulphonate (SDBS), analyte adsorption was shown to be eliminated. Of particular academic interest was the finding that adsorptive losses could also be reduced upon the addition of phospholipid. The presence of additive generally had no marked impact on the analyte MS response but the use of an isotopically labelled internal standard satisfactorily compensated for instances in which ion suppression was observed, e.g. in the presence of Tween 80. Since metabolite profiling/identification investigations are often performed on urine samples originating from early clinical pharmacology studies, the elution of selected additives was also monitored by MS. CHAPS, dimethylacetamide (DMA) and HP-β-cyclodextrin eluted as single chromatographic peaks in, or just after, the column void volume whilst polymeric Tween 80, and to a lesser extent SDBS, eluted over a wide retention time window. The potential of the latter surfactants to obscure the detection of unknown metabolites is significant and therefore their use in urine samples, upon which metabolite investigations are to be performed, is not recommended. Upon consideration of other factors such as additive cost and toxicity, CHAPS was selected for use in development of the validated assay.
Source:Journal of Chromatography B, Volumes 893–894
Steve Silvester, Frank Zang
A key challenge in the development of robust bioanalytical methods, for the determination of drug analyte in human urine samples, is the elimination of potential analyte losses as a result of non-specific adsorption to container surfaces in which the samples are collected, stored or processed. A common approach to address adsorption issues is to treat the urine samples with additives that serve to increase analyte solubility and/or minimise interaction with the container surfaces. A series of adsorption experiments were performed on human urine samples containing an adsorption-prone in-house development compound (AZD9164). A roller-mixing methodology was employed to maximise sample interaction with container surfaces and quantification of analyte was performed by LC–MS/MS following minimal sample preparation. In the absence of any urine additive, adsorptive losses averaged 35% but were highly variable between different lots of urine. In the presence of a range of additives, including the surfactants Tween 80, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS) and sodium dodecylbenzenesulphonate (SDBS), analyte adsorption was shown to be eliminated. Of particular academic interest was the finding that adsorptive losses could also be reduced upon the addition of phospholipid. The presence of additive generally had no marked impact on the analyte MS response but the use of an isotopically labelled internal standard satisfactorily compensated for instances in which ion suppression was observed, e.g. in the presence of Tween 80. Since metabolite profiling/identification investigations are often performed on urine samples originating from early clinical pharmacology studies, the elution of selected additives was also monitored by MS. CHAPS, dimethylacetamide (DMA) and HP-β-cyclodextrin eluted as single chromatographic peaks in, or just after, the column void volume whilst polymeric Tween 80, and to a lesser extent SDBS, eluted over a wide retention time window. The potential of the latter surfactants to obscure the detection of unknown metabolites is significant and therefore their use in urine samples, upon which metabolite investigations are to be performed, is not recommended. Upon consideration of other factors such as additive cost and toxicity, CHAPS was selected for use in development of the validated assay.
Sensitive determination of isoprostanes in exhaled breath condensate samples with use of liquid chromatography–tandem mass spectrometry
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Monika Janicka, Paweł Kubica, Agata Kot-Wasik, Jacek Kot, Jacek Namieśnik
Oxidative stress is the hallmark of various inflammatory lung diseases. Increased concentrations of reactive oxygen species in the lungs are reflected by elevated concentrations of oxidative stress markers in the breath, airways, lung tissue and blood. The aim of this work was to develop a method for the fast measurement of F2-isoprostanes in exhaled breath condensate (EBC) samples using equipment which is nowadays available and routinely exploited in analytical laboratories, liquid chromatography coupled with tandem mass spectrometry. Because of the limited volume of an EBC sample and the very low concentrations of biomarkers, we chose lyophilization as the preconcentration technique. The diastereoisomers determined show similar fragmentation patterns, which is why complete chromatographic separation with excellent peak shapes was essential for accurate quantitation. Isoprostanes were separated using a narrow-bore Agilent Extend C-18 column in isocratic elution mode using acetonitrile/methanol and water with the addition of 0.01%(v/v) formic acid. The limits of determination and quantitation for the determination of four isoprostanes in samples of EBC ranged from 1 to 3pg/ml. The recoveries of all isoprostanes ranged from 96.7 to 101.7, with a relative standard deviation of <7%. The stability of the isoprostanes at different temperatures was measured as well.
Source:Journal of Chromatography B, Volumes 893–894
Monika Janicka, Paweł Kubica, Agata Kot-Wasik, Jacek Kot, Jacek Namieśnik
Oxidative stress is the hallmark of various inflammatory lung diseases. Increased concentrations of reactive oxygen species in the lungs are reflected by elevated concentrations of oxidative stress markers in the breath, airways, lung tissue and blood. The aim of this work was to develop a method for the fast measurement of F2-isoprostanes in exhaled breath condensate (EBC) samples using equipment which is nowadays available and routinely exploited in analytical laboratories, liquid chromatography coupled with tandem mass spectrometry. Because of the limited volume of an EBC sample and the very low concentrations of biomarkers, we chose lyophilization as the preconcentration technique. The diastereoisomers determined show similar fragmentation patterns, which is why complete chromatographic separation with excellent peak shapes was essential for accurate quantitation. Isoprostanes were separated using a narrow-bore Agilent Extend C-18 column in isocratic elution mode using acetonitrile/methanol and water with the addition of 0.01%(v/v) formic acid. The limits of determination and quantitation for the determination of four isoprostanes in samples of EBC ranged from 1 to 3pg/ml. The recoveries of all isoprostanes ranged from 96.7 to 101.7, with a relative standard deviation of <7%. The stability of the isoprostanes at different temperatures was measured as well.
Validation of the PCR–dHPLC method for rapid identification of Candida glabrata phylogenetically related species in different biological matrices
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
O. Telleria, G. Ezpeleta, O. Herrero, I. Miranda-Zapico, G. Quindós, R. Cisterna
Since two new species phylogenetically related to Candida glabrata with slightly different phenotypes and antifungal susceptibility profiles have been described, it seems to be necessary from clinical point of view, to develop a rapid and accurate identification system in order to distinguish between these three fungal species. We studied the performance of denaturing high performance liquid chromatography (dHPLC) as a faster (less than 7min) and alternative novel technique for simultaneous analysis of Candida species in different biological matrices. The analyses show the good low limit of detection (LLOD) in all biological matrices studied (5.16–9.56ngμL−1, 4.14–4.70ngμL−1 and 3.99–4.66ngμL−1 for Candida bracarensis, Candida nivariensis and C. glabrata, respectively). 180 Candida isolates were analyzed in order to demonstrate the method suitability for screening analysis to identify C. glabrata and its cryptic species (C. bracarensis and C. nivariensis) in clinical routine.
Source:Journal of Chromatography B, Volumes 893–894
O. Telleria, G. Ezpeleta, O. Herrero, I. Miranda-Zapico, G. Quindós, R. Cisterna
Since two new species phylogenetically related to Candida glabrata with slightly different phenotypes and antifungal susceptibility profiles have been described, it seems to be necessary from clinical point of view, to develop a rapid and accurate identification system in order to distinguish between these three fungal species. We studied the performance of denaturing high performance liquid chromatography (dHPLC) as a faster (less than 7min) and alternative novel technique for simultaneous analysis of Candida species in different biological matrices. The analyses show the good low limit of detection (LLOD) in all biological matrices studied (5.16–9.56ngμL−1, 4.14–4.70ngμL−1 and 3.99–4.66ngμL−1 for Candida bracarensis, Candida nivariensis and C. glabrata, respectively). 180 Candida isolates were analyzed in order to demonstrate the method suitability for screening analysis to identify C. glabrata and its cryptic species (C. bracarensis and C. nivariensis) in clinical routine.
A rapid and sensitive LC/ESI–MS/MS method for quantitative analysis of docetaxel in human plasma and its application to a pharmacokinetic study
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Hiroaki Yamaguchi, Asuka Fujikawa, Hajime Ito, Nobuaki Tanaka, Ayako Furugen, Kazuaki Miyamori, Natsuko Takahashi, Jiro Ogura, Masaki Kobayashi, Takehiro Yamada, Nariyasu Mano, Ken Iseki
Docetaxel is a taxane family antineoplastic agent widely employed in cancer chemotherapy. We developed a liquid chromatography/tandem mass spectrometry method for the determination of docetaxel in human plasma. Plasma samples were deproteinized by acetonitrile containing internal standard paclitaxel. Chromatographic separation was performed on a TSKgel ODS-100V 3μm (50mm×2.0mm i.d.) column using a mobile phase composed of acetonitrile–methanol–water–formic acid (50:5:45:0.1, v/v/v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. This method covered a linearity range of 5–5000ng/mL with the lower limit of quantification of 5ng/mL. The intra-day precision and inter-day precision (R.S.D.) of analysis were less than 6.7%, and the accuracy (R.E.) was within ±9.0% at the concentrations of 5, 20, 200, and 2000ng/mL. The total run time was 5.0min. This method was successfully applied for clinical pharmacokinetic investigation.
Source:Journal of Chromatography B, Volumes 893–894
Hiroaki Yamaguchi, Asuka Fujikawa, Hajime Ito, Nobuaki Tanaka, Ayako Furugen, Kazuaki Miyamori, Natsuko Takahashi, Jiro Ogura, Masaki Kobayashi, Takehiro Yamada, Nariyasu Mano, Ken Iseki
Docetaxel is a taxane family antineoplastic agent widely employed in cancer chemotherapy. We developed a liquid chromatography/tandem mass spectrometry method for the determination of docetaxel in human plasma. Plasma samples were deproteinized by acetonitrile containing internal standard paclitaxel. Chromatographic separation was performed on a TSKgel ODS-100V 3μm (50mm×2.0mm i.d.) column using a mobile phase composed of acetonitrile–methanol–water–formic acid (50:5:45:0.1, v/v/v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. This method covered a linearity range of 5–5000ng/mL with the lower limit of quantification of 5ng/mL. The intra-day precision and inter-day precision (R.S.D.) of analysis were less than 6.7%, and the accuracy (R.E.) was within ±9.0% at the concentrations of 5, 20, 200, and 2000ng/mL. The total run time was 5.0min. This method was successfully applied for clinical pharmacokinetic investigation.
Simultaneous quantitative determination of paracetamol and its glucuronide conjugate in human plasma and urine by liquid chromatography coupled to electrospray tandem mass spectrometry: Application to a clinical pharmacokinetic study
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Qin-you Tan, Rong-hua Zhu, Huan-de Li, Feng Wang, Miao Yan, Li-bo Dai
A specific, sensitive and rapid method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC–MS/MS) was developed for the simultaneous determination of paracetamol (APAP) and its glucuronide conjugate (PG) in human plasma and urine. Plasma samples were precipitated with the mixture of acetonitrile and propylene glycol (90:10, v/v) solution and urine samples were diluted with the mobile phase, which were used to isolate the analytes from biological matrices followed by injection of the extracts onto a C18 column with isocratic elution. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI+). The method was validated over the concentration range of 10–30,000ng/mL and 100–6000ng/mL for APAP in human plasma and urine as well as 10–15,000ng/mL and 200–60,000ng/mL for PG in human plasma and urine, respectively. Inter- and intra-run precisions of APAP and PG were less than 15% and the accuracy was within 85–115% for both plasma and urine. The average extraction recoveries were 93.1% and 89.1% for APAP, and 93.7% and 92.3% for PG in human plasma and urine, respectively. The linearity, recovery and stability were validated for APAP and PG in human plasma and urine. The method proved to be simple, robust and time efficient.
Source:Journal of Chromatography B, Volumes 893–894
Qin-you Tan, Rong-hua Zhu, Huan-de Li, Feng Wang, Miao Yan, Li-bo Dai
A specific, sensitive and rapid method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC–MS/MS) was developed for the simultaneous determination of paracetamol (APAP) and its glucuronide conjugate (PG) in human plasma and urine. Plasma samples were precipitated with the mixture of acetonitrile and propylene glycol (90:10, v/v) solution and urine samples were diluted with the mobile phase, which were used to isolate the analytes from biological matrices followed by injection of the extracts onto a C18 column with isocratic elution. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI+). The method was validated over the concentration range of 10–30,000ng/mL and 100–6000ng/mL for APAP in human plasma and urine as well as 10–15,000ng/mL and 200–60,000ng/mL for PG in human plasma and urine, respectively. Inter- and intra-run precisions of APAP and PG were less than 15% and the accuracy was within 85–115% for both plasma and urine. The average extraction recoveries were 93.1% and 89.1% for APAP, and 93.7% and 92.3% for PG in human plasma and urine, respectively. The linearity, recovery and stability were validated for APAP and PG in human plasma and urine. The method proved to be simple, robust and time efficient.
Development of a fast and simple liquid chromatography–tandem mass spectrometry method for the quantitation of argatroban in patient plasma samples
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Jeanne M. Rhea, Marion L. Snyder, Anne M. Winkler, Charbel Abou-Diwan, Corinne R. Fantz, James C. Ritchie, Fania Szlam, Kenichi A. Tanaka, Ross J. Molinaro
An ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the direct measurement of argatroban in human plasma was developed and compared with the activity-based Hemoclot Thrombin Inhibitors assay. UPLC–MS/MS was performed using diclofenac as an internal standard. In summary, argatroban and diclofenac were extracted from 100μL of plasma using a methanol precipitation protocol, and chromatographic separation was performed on an ACQUITY™ TQD mass spectrometer using a UPLC C18 BEH 1.7μm column with a water and methanol gradient containing 0.1% formic acid. The detection and quantitation were performed using positive ion electrospray ionization and multiple reaction monitoring (MRM) mode. The UPLC–MS/MS method was linear over the concentration range of 0.003–3.0μg/mL, with a lower limit of quantitation for argatroban of 0.003μg/mL. The intra- and inter-assay imprecision was less than 12% at the plasma argatroban concentrations tested. Good correlation was demonstrated between the UPLC–MS/MS method and the indirect activity-based assay for determination of argatroban. However, increased plasma fibrinogen levels caused underestimation of argatroban levels using the indirect activity-based assay, whereas the UPLC–MS/MS method was unaffected. UPLC–MS/MS provides a relatively simple, sensitive, and rapid means of argatroban monitoring. It has successfully been applied to assess plasma argatroban concentrations in hospitalized patients and may provide a more accurate determination of argatroban concentrations than an activity-based assay in certain clinical conditions.
Source:Journal of Chromatography B, Volumes 893–894
Jeanne M. Rhea, Marion L. Snyder, Anne M. Winkler, Charbel Abou-Diwan, Corinne R. Fantz, James C. Ritchie, Fania Szlam, Kenichi A. Tanaka, Ross J. Molinaro
An ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the direct measurement of argatroban in human plasma was developed and compared with the activity-based Hemoclot Thrombin Inhibitors assay. UPLC–MS/MS was performed using diclofenac as an internal standard. In summary, argatroban and diclofenac were extracted from 100μL of plasma using a methanol precipitation protocol, and chromatographic separation was performed on an ACQUITY™ TQD mass spectrometer using a UPLC C18 BEH 1.7μm column with a water and methanol gradient containing 0.1% formic acid. The detection and quantitation were performed using positive ion electrospray ionization and multiple reaction monitoring (MRM) mode. The UPLC–MS/MS method was linear over the concentration range of 0.003–3.0μg/mL, with a lower limit of quantitation for argatroban of 0.003μg/mL. The intra- and inter-assay imprecision was less than 12% at the plasma argatroban concentrations tested. Good correlation was demonstrated between the UPLC–MS/MS method and the indirect activity-based assay for determination of argatroban. However, increased plasma fibrinogen levels caused underestimation of argatroban levels using the indirect activity-based assay, whereas the UPLC–MS/MS method was unaffected. UPLC–MS/MS provides a relatively simple, sensitive, and rapid means of argatroban monitoring. It has successfully been applied to assess plasma argatroban concentrations in hospitalized patients and may provide a more accurate determination of argatroban concentrations than an activity-based assay in certain clinical conditions.
Analysis of 8-hydroxy-2′-deoxyguanosine in human urine using hydrophilic interaction chromatography with tandem mass spectrometry
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Chiemi Hosozumi, Akira Toriba, Thanyarat Chuesaard, Takayuki Kameda, Ning Tang, Kazuichi Hayakawa
Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) is a widely used noninvasive biomarker of oxidative stress. A selective, sensitive and rapid method for determining 8-OHdG in human urine was developed using hydrophilic interaction chromatography–tandem mass spectrometry (HILIC–MS/MS) with electrospray ionization. 8-OHdG and isotopically labeled 8-OHdG (internal standard) were separated on a HILIC column with a mobile phase of 10mM ammonium acetate: acetonitrile (1:9, v/v) within 10min and detected by using a positive electrospray ionization interface under the selected reaction monitoring mode. The detection limits of 8-OHdG (corresponding to a signal-to-noise ratio of 3) for the HILIC-MS/MS system and the conventional method using a reversed-phase column with MS/MS were 1.0 and 26.0fmol/injection, respectively. The proposed method makes it possible to monitor the basal level of urinary 8-OHdG from non-exposed healthy subjects and can be used for large-scale human studies.
Source:Journal of Chromatography B, Volumes 893–894
Chiemi Hosozumi, Akira Toriba, Thanyarat Chuesaard, Takayuki Kameda, Ning Tang, Kazuichi Hayakawa
Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) is a widely used noninvasive biomarker of oxidative stress. A selective, sensitive and rapid method for determining 8-OHdG in human urine was developed using hydrophilic interaction chromatography–tandem mass spectrometry (HILIC–MS/MS) with electrospray ionization. 8-OHdG and isotopically labeled 8-OHdG (internal standard) were separated on a HILIC column with a mobile phase of 10mM ammonium acetate: acetonitrile (1:9, v/v) within 10min and detected by using a positive electrospray ionization interface under the selected reaction monitoring mode. The detection limits of 8-OHdG (corresponding to a signal-to-noise ratio of 3) for the HILIC-MS/MS system and the conventional method using a reversed-phase column with MS/MS were 1.0 and 26.0fmol/injection, respectively. The proposed method makes it possible to monitor the basal level of urinary 8-OHdG from non-exposed healthy subjects and can be used for large-scale human studies.
Selective derivatization of nucleotide diphosphate (NDP)-4-keto sugars for electrospray ionization-mass spectrometry (ESI-MS)
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Yun-Gon Kim, Hyung-Yeon Park, Dongwon Yoo, Changmin Sung, Eunjung Song, Jae-Hun Lee, Yun-Hui Choi, Yong-Hyun Kim, Chang-Soo Lee, Kyungmoon Park, Byung-Gee Kim, Yung-Hun Yang
Nucleotide diphosphate (NDP) sugars are widely present in antibiotics and glycoconjugates, such as protein- and lipid-linked oligosaccharides, where they act as substrates for glycosyltransferase in eukaryotes and prokaryotes. Among NDP sugars, NDP-4-keto sugars are key intermediates in the synthesis of structurally diverse NDP sugars with different functional groups. However, the structural identification of the NDP-4-keto sugars via mass spectrometry (electrospray ionization-mass spectrometry (ESI-MS)) continues to be a challenge because of the carbonyl group in these sugars interferes with ionization process. In this study, we evaluated various hydroxylamine compounds for the derivatization of NDP-4-keto sugars, so that the detection of the sugars by ESI-MS is more efficient. As a result, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine was found to be the most effective tagging molecule for the detection of NDP-4-keto sugars without being interfered by original MS. This method can be used for identifying NDP-4-keto sugars such as thymidine diphosphate (TDP)-, adenosine diphosphate (ADP)-, uridine diphosphate (UDP)-, and cytosine diphosphate (CDP)-4-keto sugars as well as new NDP-4-keto-dehydratases.
Source:Journal of Chromatography B, Volumes 893–894
Yun-Gon Kim, Hyung-Yeon Park, Dongwon Yoo, Changmin Sung, Eunjung Song, Jae-Hun Lee, Yun-Hui Choi, Yong-Hyun Kim, Chang-Soo Lee, Kyungmoon Park, Byung-Gee Kim, Yung-Hun Yang
Nucleotide diphosphate (NDP) sugars are widely present in antibiotics and glycoconjugates, such as protein- and lipid-linked oligosaccharides, where they act as substrates for glycosyltransferase in eukaryotes and prokaryotes. Among NDP sugars, NDP-4-keto sugars are key intermediates in the synthesis of structurally diverse NDP sugars with different functional groups. However, the structural identification of the NDP-4-keto sugars via mass spectrometry (electrospray ionization-mass spectrometry (ESI-MS)) continues to be a challenge because of the carbonyl group in these sugars interferes with ionization process. In this study, we evaluated various hydroxylamine compounds for the derivatization of NDP-4-keto sugars, so that the detection of the sugars by ESI-MS is more efficient. As a result, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine was found to be the most effective tagging molecule for the detection of NDP-4-keto sugars without being interfered by original MS. This method can be used for identifying NDP-4-keto sugars such as thymidine diphosphate (TDP)-, adenosine diphosphate (ADP)-, uridine diphosphate (UDP)-, and cytosine diphosphate (CDP)-4-keto sugars as well as new NDP-4-keto-dehydratases.
PEGylation, detection and chromatographic purification of site-specific PEGylated CD133-Biotin antibody in route to stem cell separation
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Mirna González-González, Karla Mayolo-Deloisa, Marco Rito-Palomares
Recovery and purification of stem cells are determining steps in order to obtain the purity and viability required for transplantation. In this context, immunochemical techniques have been widely preferred due to their high selectivity. CD133, a glycoprotein expressed by stem cells, is a well-used marker for isolation of neural stem cells. Transplantation of neural stem cells into patients can promote neural growth and improve neuronal functions. In this study, a new method for site-specific PEGylation of CD133-Biotin antibody is performed through streptavidin–biotin conjugation. Purification was carried out by ion-exchange chromatography. The characterization of the single PEGylated CD133-Biotin antibody was confirmed using electrophoresis with silver staining and I2–BaCl2 for PEG detection. Moreover, online PEG quantification directly after the chromatographic step was conducted (in each fraction) to detect exact elution times of PEG. In conclusion, the novel CD133-Biotin antibody PEGylation strategy conducted in this study could be used as a process step in route to neural stem cell recovery and purification via the modification of existing techniques such as aqueous two phase systems, PEGylated affinity columns or fluidized chromatography.
Source:Journal of Chromatography B, Volumes 893–894
Mirna González-González, Karla Mayolo-Deloisa, Marco Rito-Palomares
Recovery and purification of stem cells are determining steps in order to obtain the purity and viability required for transplantation. In this context, immunochemical techniques have been widely preferred due to their high selectivity. CD133, a glycoprotein expressed by stem cells, is a well-used marker for isolation of neural stem cells. Transplantation of neural stem cells into patients can promote neural growth and improve neuronal functions. In this study, a new method for site-specific PEGylation of CD133-Biotin antibody is performed through streptavidin–biotin conjugation. Purification was carried out by ion-exchange chromatography. The characterization of the single PEGylated CD133-Biotin antibody was confirmed using electrophoresis with silver staining and I2–BaCl2 for PEG detection. Moreover, online PEG quantification directly after the chromatographic step was conducted (in each fraction) to detect exact elution times of PEG. In conclusion, the novel CD133-Biotin antibody PEGylation strategy conducted in this study could be used as a process step in route to neural stem cell recovery and purification via the modification of existing techniques such as aqueous two phase systems, PEGylated affinity columns or fluidized chromatography.
Analysis of fatty acid composition in insulin secreting cells by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Amy L. Payeur, Matthew A. Lorenz, Robert T. Kennedy
A comprehensive two-dimensional gas chromatography (GC×GC) time-of-flight mass spectrometry method was developed for determination of fatty acids (irrespective of origin, i.e., both free fatty acids and fatty acids bound in sources such as triglycerides) in cultured mammalian cells. The method was applied to INS-1 cells, an insulin-secreting cell line commonly used as a model in diabetes studies. In the method, lipids were extracted and transformed to fatty acid methyl esters for analysis. GC×GC analysis revealed the presence of 30 identifiable fatty acids in the extract. This result doubles the number of fatty acids previously identified in these cells. The method yielded linear calibrations and an average relative standard deviation of 8.4% for replicate injections of samples and 12.4% for replicate analysis of different samples. The method was used to demonstrate changes in fatty acid content as a function of glucose concentration on the cells. These results demonstrate the utility of this method for analysis of fatty acids in mammalian cell cultures.
Source:Journal of Chromatography B, Volumes 893–894
Amy L. Payeur, Matthew A. Lorenz, Robert T. Kennedy
A comprehensive two-dimensional gas chromatography (GC×GC) time-of-flight mass spectrometry method was developed for determination of fatty acids (irrespective of origin, i.e., both free fatty acids and fatty acids bound in sources such as triglycerides) in cultured mammalian cells. The method was applied to INS-1 cells, an insulin-secreting cell line commonly used as a model in diabetes studies. In the method, lipids were extracted and transformed to fatty acid methyl esters for analysis. GC×GC analysis revealed the presence of 30 identifiable fatty acids in the extract. This result doubles the number of fatty acids previously identified in these cells. The method yielded linear calibrations and an average relative standard deviation of 8.4% for replicate injections of samples and 12.4% for replicate analysis of different samples. The method was used to demonstrate changes in fatty acid content as a function of glucose concentration on the cells. These results demonstrate the utility of this method for analysis of fatty acids in mammalian cell cultures.
Influence of ionization source design on matrix effects during LC–ESI-MS/MS analysis
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B, Volumes 893–894
Chinmoy Ghosh, Chandrakant P. Shinde, Bhaswat S. Chakraborty
Glycerophosphocholines (GPChos) are known to cause matrix ionization effects during the analysis of biological samples (i.e. plasma, urine, etc.) in LC–MS/MS. In general, such matrix effect is directly related to an insufficient sample clean-up of the biofluids. In addition to GPCho; design of ionization source and/or LC also plays a very important role in matrix effects. In this research paper, different types of matrix effects, i.e. ion suppression or enhancement were observed in differently designed ion sources coupled with different LCs, from the same molecule, acamprosate (ACM), under the same chromatographic conditions. ACM was analyzed in a negative polarity in electrospray ionization interface using Z-spray and orthogonal spray ion source design. The analyte showed almost complete ion suppression in the Z-spray ionization source coupled with UPLC/HPLC, whereas there was very little ion enhancement in the orthogonal spray ionization source coupled with HPLC. In both the cases different GPChos were responsible, as evident from the presence of m/z 815.4 in Z-spray ion source and m/z 759.0 in orthogonal spray ion source. Hence, this approach can be used to evaluate the matrix effects in plasma samples during development and validation of LC–MS/MS method of drugs and their metabolites in different biological matrixes.
Source:Journal of Chromatography B, Volumes 893–894
Chinmoy Ghosh, Chandrakant P. Shinde, Bhaswat S. Chakraborty
Glycerophosphocholines (GPChos) are known to cause matrix ionization effects during the analysis of biological samples (i.e. plasma, urine, etc.) in LC–MS/MS. In general, such matrix effect is directly related to an insufficient sample clean-up of the biofluids. In addition to GPCho; design of ionization source and/or LC also plays a very important role in matrix effects. In this research paper, different types of matrix effects, i.e. ion suppression or enhancement were observed in differently designed ion sources coupled with different LCs, from the same molecule, acamprosate (ACM), under the same chromatographic conditions. ACM was analyzed in a negative polarity in electrospray ionization interface using Z-spray and orthogonal spray ion source design. The analyte showed almost complete ion suppression in the Z-spray ionization source coupled with UPLC/HPLC, whereas there was very little ion enhancement in the orthogonal spray ionization source coupled with HPLC. In both the cases different GPChos were responsible, as evident from the presence of m/z 815.4 in Z-spray ion source and m/z 759.0 in orthogonal spray ion source. Hence, this approach can be used to evaluate the matrix effects in plasma samples during development and validation of LC–MS/MS method of drugs and their metabolites in different biological matrixes.
Computational design and synthesis of Molecular Imprinted Polymers for selective extraction of Allopurinol from human plasma
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B
Mehrdad Tabandeh, Soheila Ghassamipour, Heydar Aqbaba, Meisam Tabatabaei, eisam Hasheminejad
The present study was focused on the rational development of polymers for selective extraction of allopurinol (ALP) from human plasma. Therefore, a computational modeling approach was combined with the molecular imprinting technology to obtain the polymers. The computational approach was used in order to screen the functional monomers as well as the polymerization solvents for rational design of molecular imprinted polymers (MIPs). It was based on the comparison of the binding energy (ΔE) of the formed complexes between the template molecule and different functional monomers. In the design, the effect of the polymerization solvent was also included using the polarizable continuum model. The theoretical calculation results showed that among virtual solvents tested, acrylamide (AAM) gave the largest ΔE while acrylonitrile (ACN) gave the smallest ΔE in acetone. Therefore, the MIP prepared using AAM as functional monomer in acetone was desired. To examine the validity of this approach, three MIPs were synthesized with different functional monomers i.e. AAM, acrylic acid (AA), and ACN, and then evaluated using Langmuir–Freundlich (LF) isotherm. The results obtained from this experiment confirmed the computational results that the MIP prepared by AAM was the most appropriate adsorbent. Subsequently, the MIP was used to develop a molecular imprinted solid-phase extraction (MISPE) procedure. Finally, the MISPE procedure followed by HPLC was developed for selective extraction and determination of allopurinol in human plasma. For the proposed MISPE method, the linearity between peak area and concentration was found in the range of 0.100 to 25.000μM with a linear regression coefficient (R2) of 0.995. The limit of detection (LOD) and quantification (LOQ) in plasma were 0.028 and 0.093μM, respectively. The results of this study indicated the possibility of using computer aided design for rational selection of functional monomers and solvents for preparation of the MIPs capable of extracting allopurinol from human plasma.
Source:Journal of Chromatography B
Mehrdad Tabandeh, Soheila Ghassamipour, Heydar Aqbaba, Meisam Tabatabaei, eisam Hasheminejad
The present study was focused on the rational development of polymers for selective extraction of allopurinol (ALP) from human plasma. Therefore, a computational modeling approach was combined with the molecular imprinting technology to obtain the polymers. The computational approach was used in order to screen the functional monomers as well as the polymerization solvents for rational design of molecular imprinted polymers (MIPs). It was based on the comparison of the binding energy (ΔE) of the formed complexes between the template molecule and different functional monomers. In the design, the effect of the polymerization solvent was also included using the polarizable continuum model. The theoretical calculation results showed that among virtual solvents tested, acrylamide (AAM) gave the largest ΔE while acrylonitrile (ACN) gave the smallest ΔE in acetone. Therefore, the MIP prepared using AAM as functional monomer in acetone was desired. To examine the validity of this approach, three MIPs were synthesized with different functional monomers i.e. AAM, acrylic acid (AA), and ACN, and then evaluated using Langmuir–Freundlich (LF) isotherm. The results obtained from this experiment confirmed the computational results that the MIP prepared by AAM was the most appropriate adsorbent. Subsequently, the MIP was used to develop a molecular imprinted solid-phase extraction (MISPE) procedure. Finally, the MISPE procedure followed by HPLC was developed for selective extraction and determination of allopurinol in human plasma. For the proposed MISPE method, the linearity between peak area and concentration was found in the range of 0.100 to 25.000μM with a linear regression coefficient (R2) of 0.995. The limit of detection (LOD) and quantification (LOQ) in plasma were 0.028 and 0.093μM, respectively. The results of this study indicated the possibility of using computer aided design for rational selection of functional monomers and solvents for preparation of the MIPs capable of extracting allopurinol from human plasma.
Determination of four immunosuppressive drugs in whole blood using MEPS and LC-MS/MS allowing automated sample work-up and analysis
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B
Rana Said, Anton Pohanka, Mohamed Abdel-Rehim, Olof Beck
In treatment with immunosuppressive drugs, monitoring of blood drug concentration is needed. The aim of this work was to explore micro extraction by packed sorbent (MEPS) as a possible on-line sample preparation method in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantification of cyclosporine, everolimus, sirolimus and tacrolimus in whole blood. An automated on-line MEPS system connected with a LC-MS/MS instrument was set up. A C8 sorbent was used for the MEPS extraction. Subsequent analysis was performed with a gradient LC system. The adduct ions [M+NH4]+ of the analytes were monitored in SRM mode for quantification. Ascomycin and cyclosporine D were used as internal standards. The chromatographic run time 2.5min and the quantification ranges were 3-1500ng/mL (r2≥0.999, n=6) for cyclosporine and 0.5–50ng/mL for everolimus, sirolimus and tacrolimus (r2≥0.998, 0.994 and 0.993 respectively, n=6). Precision and accuracy were documented at three levels. Accuracy results were between 102-109% with precision between 2-13% and carry over <0.02%Matrix effects were characterized and found to be below 20%. The quantifications obtained were in agreement with a reference LC-MS/MS method based on protein precipitation, and results obtained from external proficiency test samples compared with the mean of all other LC- mass spectrometry methods showed good agreement. This method provides an accurate, precise and automated procedure that can be applied for therapeutic drug monitoring of immunosuppressive drugs in clinical laboratories equipped with LC-MS/MS.
Source:Journal of Chromatography B
Rana Said, Anton Pohanka, Mohamed Abdel-Rehim, Olof Beck
In treatment with immunosuppressive drugs, monitoring of blood drug concentration is needed. The aim of this work was to explore micro extraction by packed sorbent (MEPS) as a possible on-line sample preparation method in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantification of cyclosporine, everolimus, sirolimus and tacrolimus in whole blood. An automated on-line MEPS system connected with a LC-MS/MS instrument was set up. A C8 sorbent was used for the MEPS extraction. Subsequent analysis was performed with a gradient LC system. The adduct ions [M+NH4]+ of the analytes were monitored in SRM mode for quantification. Ascomycin and cyclosporine D were used as internal standards. The chromatographic run time 2.5min and the quantification ranges were 3-1500ng/mL (r2≥0.999, n=6) for cyclosporine and 0.5–50ng/mL for everolimus, sirolimus and tacrolimus (r2≥0.998, 0.994 and 0.993 respectively, n=6). Precision and accuracy were documented at three levels. Accuracy results were between 102-109% with precision between 2-13% and carry over <0.02%Matrix effects were characterized and found to be below 20%. The quantifications obtained were in agreement with a reference LC-MS/MS method based on protein precipitation, and results obtained from external proficiency test samples compared with the mean of all other LC- mass spectrometry methods showed good agreement. This method provides an accurate, precise and automated procedure that can be applied for therapeutic drug monitoring of immunosuppressive drugs in clinical laboratories equipped with LC-MS/MS.
On-fiber furan formation from volatile precursors: a critical example of artefact formation during Solid-Phase Microextraction
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B
An Adams, Fien Van Lancker, Bruno De Meulenaer, Agnieszka Owczarek-Fendor, Norbert De Kimpe
For the analysis of furan, a possible carcinogen formed during thermal treatment of food, Solid-Phase Microextraction (SPME) is a preferred and validated sampling method. However, when volatile furan precursors are adsorbed on the Carboxen/PDMS fiber, additional amounts of furan can be formed on the fiber during thermal desorption, as shown here for 2-butenal and furfural. No significant increase in furan amounts was found upon heating the furan precursor 2-butenal, indicating that the furan amounts formed during precursor heating experiments are negligible as compared to the additional amounts of furan formed during fiber desorption. This artefactual furan formation increased with increasing desorption time, but especially with increasing desorption temperature. Although this effect was most pronounced on the Car/PDMS SPME-fiber, it was also noted on two other SPME-fibers tested (PDMS and DVB/Car/PDMS). The general impact on furan data from food and model systems in literature will depend on the amounts of volatile precursors present, but will probably remain limited. However, considering the importance of this worldwide food contaminant, special care has to be taken during SPME-analysis of furan. Especially when performing precursor studies, static headspace sampling should preferably be applied for furan analysis.
Source:Journal of Chromatography B
An Adams, Fien Van Lancker, Bruno De Meulenaer, Agnieszka Owczarek-Fendor, Norbert De Kimpe
For the analysis of furan, a possible carcinogen formed during thermal treatment of food, Solid-Phase Microextraction (SPME) is a preferred and validated sampling method. However, when volatile furan precursors are adsorbed on the Carboxen/PDMS fiber, additional amounts of furan can be formed on the fiber during thermal desorption, as shown here for 2-butenal and furfural. No significant increase in furan amounts was found upon heating the furan precursor 2-butenal, indicating that the furan amounts formed during precursor heating experiments are negligible as compared to the additional amounts of furan formed during fiber desorption. This artefactual furan formation increased with increasing desorption time, but especially with increasing desorption temperature. Although this effect was most pronounced on the Car/PDMS SPME-fiber, it was also noted on two other SPME-fibers tested (PDMS and DVB/Car/PDMS). The general impact on furan data from food and model systems in literature will depend on the amounts of volatile precursors present, but will probably remain limited. However, considering the importance of this worldwide food contaminant, special care has to be taken during SPME-analysis of furan. Especially when performing precursor studies, static headspace sampling should preferably be applied for furan analysis.
Determination of imidacloprid in rice by molecularly imprinted-matrix solid-phase dispersion with liquid chromatography tandem mass spectrometry
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B
Ligang Chen, Bin Li
A new method based on matrix solid-phase dispersion (MSPD) coupled with liquid chromatography tandem mass spectrometry has been developed for the determination of imidacloprid in rice. The molecularly imprinted polymers were synthesized and applied as the dispersant of MSPD for selective extraction of imidacloprid from rice, while interferences originated from sample matrices were eliminated simultaneously. The satisfactory recovery of imidacloprid was obtained by the optimized extraction conditions: 1:2 as the ratio of sample to MIPs; 8min as the dispersion time; 20% aqueous methanol as washing solvent and methanol as elution solvent. Under the optimal conditions, the linearity of imidacloprid in rice sample was achieved in the range of 10–1000ng/g, and limit of detection was 2.4ng/g. The relative standard deviations of intra- and inter-day tests ranging from 4.5% to 5.9% and from 4.8% to 7.1% are obtained, respectively. The proposed method was applied to the determination of imidacloprid in eight rice samples with recoveries in the range of 83.8% - 92.5%.
Source:Journal of Chromatography B
Ligang Chen, Bin Li
A new method based on matrix solid-phase dispersion (MSPD) coupled with liquid chromatography tandem mass spectrometry has been developed for the determination of imidacloprid in rice. The molecularly imprinted polymers were synthesized and applied as the dispersant of MSPD for selective extraction of imidacloprid from rice, while interferences originated from sample matrices were eliminated simultaneously. The satisfactory recovery of imidacloprid was obtained by the optimized extraction conditions: 1:2 as the ratio of sample to MIPs; 8min as the dispersion time; 20% aqueous methanol as washing solvent and methanol as elution solvent. Under the optimal conditions, the linearity of imidacloprid in rice sample was achieved in the range of 10–1000ng/g, and limit of detection was 2.4ng/g. The relative standard deviations of intra- and inter-day tests ranging from 4.5% to 5.9% and from 4.8% to 7.1% are obtained, respectively. The proposed method was applied to the determination of imidacloprid in eight rice samples with recoveries in the range of 83.8% - 92.5%.
Determination of aniracetam's main metabolite, N-anisoyl-GABA, in human plasma by LC-MS/MS and its application to a pharmacokinetic study
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B
Shuang Cai, Lei Wang
A simple and rapid high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method has been developed and validated for the determination of 4-p-anisamidobutyric acid (ABA; or N-anysoyl-γ-aminobutiryc acid, N-anisoyl-GABA), a major active metabolite of aniracetam, in human plasma. After protein precipitation of plasma sample with methanol, ABA and the internal standard lisinopril were separated on a Venusil ASB C18 column at 25°C. The mobile phase consisted of methanol-ammonium acetate (10mmol/L) (30:70, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer with an ESI source in negative ion mode. Multiple reaction monitoring (MRM) using the precursor → product ion combinations of m/z 235.8→m/z 106.6, and m/z 403.8→m/z 113.6 was used to quantify ABA and lisinopril, respectively. This is the first LC-MS/MS method for ABA with advantages of short analysis time (4.5min per sample run) and high selectivity attributable to the MRM detection and optimized HPLC conditions. The response was linear in a concentration range of 0.0485 ∼ 19.4μg/ml in plasma. The extraction recovery of ABA was between 89.1% and 100.7%. The precision (R.S.D) and accuracy (R.E.) of the method were evaluated to be within 7.3% and from 2.5% to 6.9%. The validated method has been applied to the pharmacokinetic study after a single oral administration of aniracetam dispersible tablets to human beings.
Source:Journal of Chromatography B
Shuang Cai, Lei Wang
A simple and rapid high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method has been developed and validated for the determination of 4-p-anisamidobutyric acid (ABA; or N-anysoyl-γ-aminobutiryc acid, N-anisoyl-GABA), a major active metabolite of aniracetam, in human plasma. After protein precipitation of plasma sample with methanol, ABA and the internal standard lisinopril were separated on a Venusil ASB C18 column at 25°C. The mobile phase consisted of methanol-ammonium acetate (10mmol/L) (30:70, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer with an ESI source in negative ion mode. Multiple reaction monitoring (MRM) using the precursor → product ion combinations of m/z 235.8→m/z 106.6, and m/z 403.8→m/z 113.6 was used to quantify ABA and lisinopril, respectively. This is the first LC-MS/MS method for ABA with advantages of short analysis time (4.5min per sample run) and high selectivity attributable to the MRM detection and optimized HPLC conditions. The response was linear in a concentration range of 0.0485 ∼ 19.4μg/ml in plasma. The extraction recovery of ABA was between 89.1% and 100.7%. The precision (R.S.D) and accuracy (R.E.) of the method were evaluated to be within 7.3% and from 2.5% to 6.9%. The validated method has been applied to the pharmacokinetic study after a single oral administration of aniracetam dispersible tablets to human beings.
Rapid, simultaneous and nanomolar determination of pyroglutamic acid and cis-/trans-urocanic acid in human stratum corneum by hydrophilic interaction liquid chromatography (HILIC)–electrospray ionization tandem mass spectrometry
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B
Kyung-Mi Joo, Ji Yeon Han, Eui Dong Son, Gae-Won Nam, Han Young Chung, Hye-Jin Jeong, Jun-Cheol Cho, Kyung-Min Lim
A rapid, sensitive and specific hydrophilic interaction liquid chromatography coupled to tandem mass spectrometric (HILIC-MS/MS) method for the simultaneous determination of pyroglutamic acid, cis- and trans-urocanic acid in human skin stratum corneum (SC) were developed and validated. This method was carried out without derivatization or addition of ion-pair additives in mobile phase. The analytes were extracted by PBS buffer solution and analyzed using an electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode. Chromatographic separation was performed on an AQUITY UPLC amide column using gradient elution with the mobile phase of water and acetonitrile. The standard curves were linear over the concentration range of 1.0–250ng/mL with a correlation coefficient higher than 0.999 with an LLOQ of 0.5ng/mL. The lower limits of detection (LLOD) of these analytes were lower than 0.2ng/mL. The intra- and inter-day precisions were measured to be below 7.7% and accuracies were within the range of 94.3–102.6%. The validated method was successfully applied to determine the level of pyroglutamic acid and cis-/trans- urocanic acid in the SC samples from forearm and forehead region of 19 human volunteers.
Source:Journal of Chromatography B
Kyung-Mi Joo, Ji Yeon Han, Eui Dong Son, Gae-Won Nam, Han Young Chung, Hye-Jin Jeong, Jun-Cheol Cho, Kyung-Min Lim
A rapid, sensitive and specific hydrophilic interaction liquid chromatography coupled to tandem mass spectrometric (HILIC-MS/MS) method for the simultaneous determination of pyroglutamic acid, cis- and trans-urocanic acid in human skin stratum corneum (SC) were developed and validated. This method was carried out without derivatization or addition of ion-pair additives in mobile phase. The analytes were extracted by PBS buffer solution and analyzed using an electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode. Chromatographic separation was performed on an AQUITY UPLC amide column using gradient elution with the mobile phase of water and acetonitrile. The standard curves were linear over the concentration range of 1.0–250ng/mL with a correlation coefficient higher than 0.999 with an LLOQ of 0.5ng/mL. The lower limits of detection (LLOD) of these analytes were lower than 0.2ng/mL. The intra- and inter-day precisions were measured to be below 7.7% and accuracies were within the range of 94.3–102.6%. The validated method was successfully applied to determine the level of pyroglutamic acid and cis-/trans- urocanic acid in the SC samples from forearm and forehead region of 19 human volunteers.
Determination of bullatacin in rat plasma by liquid chromatography-mass spectrometry
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B
Yong Chen, Jian-wei Chen, Shi-jia Liu, Chun-lei Xu, Hui-qing Xu, Bao-chang Cai, Xiang Li, Wen-zheng Ju
A liquid chromatography-mass spectrometry method has been developed and validated for the quantification of bullatacin, a bistetrahydrofuran annonaceous acetogenin, in rat plasma. Squamostatin-A was selected as the internal standard. Analytes were extracted from rat plasma by liquid/liquid extraction using ethyl acetate with high efficiency. The chromatographical separation was performed on an Agilent Zorbax SB-C18 column (150mm×2.1mm, 5μm). The mobile phase consisted of methanol and deionized water (95:5, v/v) containing 0.01% (v/v) formic acid. The chromatographic run time was 7min per injection and flow rate was 0.2mL/min. The retention time was 3.22 and 5.23min for internal standard and bullatacin, respectively. The elutes were detected under positive electrospray ionization and the target analytes quantified by selected ion monitoring mode (645.9 m/z for bullatacin and 661.9 m/z for squamostatin-A). The method was sensitive with the limit of quantitation at 0.5ng/mL in 100μL of rat plasma. Good linearity (r 2 =0.9998) was obtained covering the concentration of 0.5–2000ng/mL. The intra- and inter-day assay precision ranged from 3.2 to 8.7% and 2.7 to 9.2%, respectively. In addition, the stability, extraction recovery and matrix effect involved in the method were also validated. This method was applied to measure the plasma bullatacin concentrations after a single tail vein intravenous administration of bullatacin in rats.
Source:Journal of Chromatography B
Yong Chen, Jian-wei Chen, Shi-jia Liu, Chun-lei Xu, Hui-qing Xu, Bao-chang Cai, Xiang Li, Wen-zheng Ju
A liquid chromatography-mass spectrometry method has been developed and validated for the quantification of bullatacin, a bistetrahydrofuran annonaceous acetogenin, in rat plasma. Squamostatin-A was selected as the internal standard. Analytes were extracted from rat plasma by liquid/liquid extraction using ethyl acetate with high efficiency. The chromatographical separation was performed on an Agilent Zorbax SB-C18 column (150mm×2.1mm, 5μm). The mobile phase consisted of methanol and deionized water (95:5, v/v) containing 0.01% (v/v) formic acid. The chromatographic run time was 7min per injection and flow rate was 0.2mL/min. The retention time was 3.22 and 5.23min for internal standard and bullatacin, respectively. The elutes were detected under positive electrospray ionization and the target analytes quantified by selected ion monitoring mode (645.9 m/z for bullatacin and 661.9 m/z for squamostatin-A). The method was sensitive with the limit of quantitation at 0.5ng/mL in 100μL of rat plasma. Good linearity (r 2 =0.9998) was obtained covering the concentration of 0.5–2000ng/mL. The intra- and inter-day assay precision ranged from 3.2 to 8.7% and 2.7 to 9.2%, respectively. In addition, the stability, extraction recovery and matrix effect involved in the method were also validated. This method was applied to measure the plasma bullatacin concentrations after a single tail vein intravenous administration of bullatacin in rats.
Graphical Abstract
Graphical abstract Highlights
► We developed a LC-MS method for the quantification of bullatacin in rat plasma. ► The method was fully validated. ► This method was successfully applied to pharmacokinetic studies in rats.Development and validation of a subcritical fluid extraction and high performance liquid chromatography assay for medroxyprogesterone in aquatic products
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B
Yuqian Han, Qinchuan Ma, Jie Lu, Yong Xue, Jie Xu, Changhu Xue
A simple, rapid and sensitive method was developed for the determination of medroxyprogesterone in aquatic products by extraction with subcritical 1,1,1,2-tetrafluoroethane (R134a) and high performance liquid chromatography (HPLC). A response surface methodology (RSM) was adopted to optimise extraction pressure, temperature and co-solvent volume. The optimum extraction conditions predicted within the experimental ranges were as follows: pressure, 3MPa; temperature, 25°C; and co-solvent volume, 6ml. The analysis was carried out on Zorbax SB-C18 column (4.6 mm×150mm, 5μm) with the mobile phase acetonitrile-water (55:45, v/v), flow rate 1.0ml/min, temperature 30°C and wavelength 240nm. Good linearity of detection was obtained for testosterone propionate between concentrations of 50-250ng/ml, r2 =0.999. The method was validated using samples fortified with medroxyprogesterone at levels of 10, 30 and 50ng/g, the mean recovery exceeds 90%, and the RSD values were less than 10%
Source:Journal of Chromatography B
Yuqian Han, Qinchuan Ma, Jie Lu, Yong Xue, Jie Xu, Changhu Xue
A simple, rapid and sensitive method was developed for the determination of medroxyprogesterone in aquatic products by extraction with subcritical 1,1,1,2-tetrafluoroethane (R134a) and high performance liquid chromatography (HPLC). A response surface methodology (RSM) was adopted to optimise extraction pressure, temperature and co-solvent volume. The optimum extraction conditions predicted within the experimental ranges were as follows: pressure, 3MPa; temperature, 25°C; and co-solvent volume, 6ml. The analysis was carried out on Zorbax SB-C18 column (4.6 mm×150mm, 5μm) with the mobile phase acetonitrile-water (55:45, v/v), flow rate 1.0ml/min, temperature 30°C and wavelength 240nm. Good linearity of detection was obtained for testosterone propionate between concentrations of 50-250ng/ml, r2 =0.999. The method was validated using samples fortified with medroxyprogesterone at levels of 10, 30 and 50ng/g, the mean recovery exceeds 90%, and the RSD values were less than 10%
Graphical Abstract
Graphical abstract Highlights
► A method for determination of medroxyprogesterone in aquatic products was developed. ► 1, 1, 1, 2-tetrafluoroethane (R134a) is used as subcritical fluid. ► Response surface methodology (RSM) was employed to optimize the conditions of the extraction. ► High extraction efficiency could be obtained at low pressure and temperature. ► The proposed method was successfully applied to the real samplesLC-MS/MS method for the quantitation of metabolites of eight commonly- used synthetic cannabinoids in human urine – an Australian perspective
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B
Andrew D. de Jager, Janet V. Warner, Michael Henman, Wendy Ferguson, Ashley Hall
AnLC-MS/MS method for the quantitation of urinary metabolites of eightJWH-type synthetic cannabinoids (SCs) has been developed and validated. Urine samples are subjected to deconjugation using β-glucuronidase, followed by a solvent extraction procedure.Compounds are separated on a reverse-phase HPLC columnwithin a 14minute cycle.Low assay limitsare required in order to demonstrate prior exposure to SCs. Matrix effects were studied and proved to be significant for selected analytes, and were challenging to circumvent as isotope-labelled internal standards are not available. An elimination profile from a naïve user following a single smoke of “Kronic” was constructed, showing urinary excretion over 2 – 3 days with peak concentrations of different metabolites 3 – 16.5hours after smoking. This method has been developed to process several hundred samples within a high-throughput drugs of abuse laboratory, with growing evidence that the use of synthetic cannabinoid blends is common within the Australian workforce.
Source:Journal of Chromatography B
Andrew D. de Jager, Janet V. Warner, Michael Henman, Wendy Ferguson, Ashley Hall
AnLC-MS/MS method for the quantitation of urinary metabolites of eightJWH-type synthetic cannabinoids (SCs) has been developed and validated. Urine samples are subjected to deconjugation using β-glucuronidase, followed by a solvent extraction procedure.Compounds are separated on a reverse-phase HPLC columnwithin a 14minute cycle.Low assay limitsare required in order to demonstrate prior exposure to SCs. Matrix effects were studied and proved to be significant for selected analytes, and were challenging to circumvent as isotope-labelled internal standards are not available. An elimination profile from a naïve user following a single smoke of “Kronic” was constructed, showing urinary excretion over 2 – 3 days with peak concentrations of different metabolites 3 – 16.5hours after smoking. This method has been developed to process several hundred samples within a high-throughput drugs of abuse laboratory, with growing evidence that the use of synthetic cannabinoid blends is common within the Australian workforce.
LC-ESI-MS Method for the Monitoring of Abl 1 Tyrosine Kinase
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B
Hui Chen, Erwin Adams, Ann Van Schepdael
A liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) method was developed and validated to study Abl 1 tyrosine kinase. An online desalting system was adopted, and a transformation of the ratio of product to substrate instead of a deuterated internal standard was introduced to calculate the concentration of product. In this study, the substrate used was Abltide (KKGEAIYAAPFA-NH2). The detection was performed by selected ion monitoring (SIM) mode via positive ESI interface. Chromatographic separation was achieved on a C18 column using an isocratic mobile phase system. The limit of quantification (LOQ) was 10nM for the product and 25nM for the substrate. The simple ratios of product to substrate maintained a linear relationship (R2= 0.9997) over the ratio of 0-50% product. Intra- and inter-day precision was less than 10% and accuracy was from -1.6 to +5.3%. The validated method was applied to the Abl 1 kinase kinetic study and the Km and Vmax constants obtained for Abltide were 34.78μM and 5.563μmol/mg/min and for adenosine triphosphate (ATP) were 43.61μM and 5.906μmol/mg/min. The enzymatic reaction of Abl 1 tyrosine kinase belongs to ternary-complex mechanism.
Source:Journal of Chromatography B
Hui Chen, Erwin Adams, Ann Van Schepdael
A liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) method was developed and validated to study Abl 1 tyrosine kinase. An online desalting system was adopted, and a transformation of the ratio of product to substrate instead of a deuterated internal standard was introduced to calculate the concentration of product. In this study, the substrate used was Abltide (KKGEAIYAAPFA-NH2). The detection was performed by selected ion monitoring (SIM) mode via positive ESI interface. Chromatographic separation was achieved on a C18 column using an isocratic mobile phase system. The limit of quantification (LOQ) was 10nM for the product and 25nM for the substrate. The simple ratios of product to substrate maintained a linear relationship (R2= 0.9997) over the ratio of 0-50% product. Intra- and inter-day precision was less than 10% and accuracy was from -1.6 to +5.3%. The validated method was applied to the Abl 1 kinase kinetic study and the Km and Vmax constants obtained for Abltide were 34.78μM and 5.563μmol/mg/min and for adenosine triphosphate (ATP) were 43.61μM and 5.906μmol/mg/min. The enzymatic reaction of Abl 1 tyrosine kinase belongs to ternary-complex mechanism.
Validation of a liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B
Annamaria Jakab, Serge Winter, Bertrand Guy, Marc Raccuglia, Frank Picard, John M Kovarik, Jayraj Chudasama, Swati Guttikar, Puran Singhal, Olivier Kretz
A liquid chromatography/tandem mass spectrometry (HPLC–MS/MS) method was validated for the quantification of Sotrastaurin (AEB071) and N-desmethyl-sotrastaurin in human blood. The validation of the analytical procedure was performed according to the latest Food and Drug Administration (FDA) “Guidance for Industry, Bioanalytical Method Validation”. Chromatographic separation was performed using an RP C18 (50×4.6mm, 5μm) column at 40±3.0 oC with a mobile phase consisted of 2mM ammonium acetate in water (pH 4.5): methanol: acetonitrile (25:15:60 v/v) of a flow rate of 1 mL/min followed by quantification with tandem mass spectrometer, operated in electrospray ionization (ESI) positive ion mode and applying multiple reaction monitoring (MRM). The validated method described in this paper presents high absolute recovery, with a sensitivity of 3.00ng/mL as lower limit of quantitation using a sample volume of 300μL, low inter-run bias and variability (for Sotrastaurin, 0.4 to −4.4% and 1.8 to 5.2% and for N-desmethyl-sotrastaurin, ranged from 2.3 to −1.6% and 3.9 to 2.7%, respectively) with a short runtime of 3.5min. The method was validated using K3EDTA as specific anticoagulant and cross-validated using Li-Heparin and Na-Heparin. The method was specific for Sotrastaurin and N-desmethyl-sotrastaurin within the given criteria of acceptance (apparent peak area for Sotrastaurin and N-desmethyl-sotrastaurin in zero samples ≤ 20% of mean peak area at LLOQ) in human blood. The method was fully validated for the quantitative determination of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood between the range of 3.00ng/mL and 1200ng/mL.
Source:Journal of Chromatography B
Annamaria Jakab, Serge Winter, Bertrand Guy, Marc Raccuglia, Frank Picard, John M Kovarik, Jayraj Chudasama, Swati Guttikar, Puran Singhal, Olivier Kretz
A liquid chromatography/tandem mass spectrometry (HPLC–MS/MS) method was validated for the quantification of Sotrastaurin (AEB071) and N-desmethyl-sotrastaurin in human blood. The validation of the analytical procedure was performed according to the latest Food and Drug Administration (FDA) “Guidance for Industry, Bioanalytical Method Validation”. Chromatographic separation was performed using an RP C18 (50×4.6mm, 5μm) column at 40±3.0 oC with a mobile phase consisted of 2mM ammonium acetate in water (pH 4.5): methanol: acetonitrile (25:15:60 v/v) of a flow rate of 1 mL/min followed by quantification with tandem mass spectrometer, operated in electrospray ionization (ESI) positive ion mode and applying multiple reaction monitoring (MRM). The validated method described in this paper presents high absolute recovery, with a sensitivity of 3.00ng/mL as lower limit of quantitation using a sample volume of 300μL, low inter-run bias and variability (for Sotrastaurin, 0.4 to −4.4% and 1.8 to 5.2% and for N-desmethyl-sotrastaurin, ranged from 2.3 to −1.6% and 3.9 to 2.7%, respectively) with a short runtime of 3.5min. The method was validated using K3EDTA as specific anticoagulant and cross-validated using Li-Heparin and Na-Heparin. The method was specific for Sotrastaurin and N-desmethyl-sotrastaurin within the given criteria of acceptance (apparent peak area for Sotrastaurin and N-desmethyl-sotrastaurin in zero samples ≤ 20% of mean peak area at LLOQ) in human blood. The method was fully validated for the quantitative determination of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood between the range of 3.00ng/mL and 1200ng/mL.
Study of the Photodegradation of 2-Bromophenol Under Uv and Sunlight by Spectroscopic, Chromatographic and Chemometric Techniques
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B
Anusha Jayaraman, S.ílvia Mas, Romà Tauler, Anna de Juan
This work is focused on the study of the photodegradation of 2-bromophenol under the action of UV light and sunlight. The photodegradation process has been monitored using UV-Vis spectroscopy and High Performance Liquid Chromatography coupled to diode array and mass spectrometry detectors in tandem (HPLC-DAD-MS). Multivariate resolution methods, such as Multivariate Curve Resolution–Alternating Least Squares (MCR-ALS) and Hybrid Soft- and Hard-Modeling–Multivariate Curve Resolution (HS-MCR), have been applied to the experimental data to obtain the information about the kinetic evolution and identification of the compounds involved in the photodegradation process. From the analysis of HPLC-DAD results, the complexity of the photodegradation process has been confirmed. Ten components were found to be involved in parallel, second- or higher-order reactions, which could not be ascertained from the spectroscopic results. The HPLC-MS results allowed postulating the identity of some of the compounds (such as hydroxyderivatives and bromophenol homologs) which resulted from the reactions of photohydrolysis, debromination and bromine transfer to different position of the phenol ring. The effect of the UV light and sunlight on the photodegradation process was found to affect mainly the rate of the reaction, but not the identity of the photoproducts formed. The advantages and limitations of the spectroscopic and chromatographic analysis were also discussed. The potential of combining spectroscopic and chromatographic data in a single multiset structure was also shown. This strategy, uses the advantage of the good definition of the process time axis from the spectroscopic experiment and the capability to distinguish among compounds, linked to the use of chromatographic information.
Source:Journal of Chromatography B
Anusha Jayaraman, S.ílvia Mas, Romà Tauler, Anna de Juan
This work is focused on the study of the photodegradation of 2-bromophenol under the action of UV light and sunlight. The photodegradation process has been monitored using UV-Vis spectroscopy and High Performance Liquid Chromatography coupled to diode array and mass spectrometry detectors in tandem (HPLC-DAD-MS). Multivariate resolution methods, such as Multivariate Curve Resolution–Alternating Least Squares (MCR-ALS) and Hybrid Soft- and Hard-Modeling–Multivariate Curve Resolution (HS-MCR), have been applied to the experimental data to obtain the information about the kinetic evolution and identification of the compounds involved in the photodegradation process. From the analysis of HPLC-DAD results, the complexity of the photodegradation process has been confirmed. Ten components were found to be involved in parallel, second- or higher-order reactions, which could not be ascertained from the spectroscopic results. The HPLC-MS results allowed postulating the identity of some of the compounds (such as hydroxyderivatives and bromophenol homologs) which resulted from the reactions of photohydrolysis, debromination and bromine transfer to different position of the phenol ring. The effect of the UV light and sunlight on the photodegradation process was found to affect mainly the rate of the reaction, but not the identity of the photoproducts formed. The advantages and limitations of the spectroscopic and chromatographic analysis were also discussed. The potential of combining spectroscopic and chromatographic data in a single multiset structure was also shown. This strategy, uses the advantage of the good definition of the process time axis from the spectroscopic experiment and the capability to distinguish among compounds, linked to the use of chromatographic information.
Sensitive and robust method for anabolic agents in human urine by gas chromatography–triple quadrupole mass spectrometry
13 April 2012,
11:55:02
Publication year:
2012
Source:Journal of Chromatography B
Miguel A. Delgadillo, Lorena Garrostas, Óscar J. Pozo, Rosa Ventura, Benjamín Velasco, Jordi Segura, Josep Marcos
A rapid, sensitive and robust gas chromatography- triple quadrupole mass spectrometry method was developed for the determination of seven anabolic agents in human urine. The selection of analytes includes the main metabolites of all anabolics with higher sensitivity requirements. After optimizing the fragmentation conditions for each compound, a validation procedure for qualitative analysis was performed. The selectivity of the method showed that no interfering peaks were observed at the retention time of the compound. Adequate intermediate precision, below 14%, was observed for all of the compounds at the lower concentration tested. The concentrations assayed were in accordance with the performance limits required by the World Anti-Doping Agency (WADA). Unlike a previously published GC/QqQ method, detection of 17α-methyl-5β-androstane-3α,17β-diol (the main metabolites of methyltestosterone) at 2ng/mL was accomplished under routine conditions. The qualitative method was applied to the analysis of 1367 samples in the span of two weeks, as part of the doping control of the XVI Pan American Games which took place in Mexico (14th -30th October, 2011). The high sensitivity was maintained during the analysis of all analytical batches, proving for the first time the excellent ruggedness of GC/QqQ methods.
Source:Journal of Chromatography B
Miguel A. Delgadillo, Lorena Garrostas, Óscar J. Pozo, Rosa Ventura, Benjamín Velasco, Jordi Segura, Josep Marcos
A rapid, sensitive and robust gas chromatography- triple quadrupole mass spectrometry method was developed for the determination of seven anabolic agents in human urine. The selection of analytes includes the main metabolites of all anabolics with higher sensitivity requirements. After optimizing the fragmentation conditions for each compound, a validation procedure for qualitative analysis was performed. The selectivity of the method showed that no interfering peaks were observed at the retention time of the compound. Adequate intermediate precision, below 14%, was observed for all of the compounds at the lower concentration tested. The concentrations assayed were in accordance with the performance limits required by the World Anti-Doping Agency (WADA). Unlike a previously published GC/QqQ method, detection of 17α-methyl-5β-androstane-3α,17β-diol (the main metabolites of methyltestosterone) at 2ng/mL was accomplished under routine conditions. The qualitative method was applied to the analysis of 1367 samples in the span of two weeks, as part of the doping control of the XVI Pan American Games which took place in Mexico (14th -30th October, 2011). The high sensitivity was maintained during the analysis of all analytical batches, proving for the first time the excellent ruggedness of GC/QqQ methods.
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