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Validation of an on-line solid-phase extraction method coupled to liquid chromatography–tandem mass spectrometry detection for the determination of Indacaterol in human serum
26 April 2012,
11:00:04
Publication year:
2012
Source:Journal of Chromatography B, Volumes 895–896
Corinne Emotte, Olivier Heudi, Fanny Deglave, Adrien Bonvie, Laurence Masson, Franck Picard, Animesh Chaturvedi, Tapan Majumdar, Ashish Agarwal, Ralph Woessner, Olivier Kretz
Indacaterol has been recently approved in Europe for the treatment of chronic obstructive pulmonary disease (COPD). In the present study, we have developed and validated a rapid and sensitive on-line solid phase extraction (SPE) method coupled to liquid chromatography–tandem mass spectrometry (LC–MS/MS) detection for the determination of Indacaterol in human serum. The sample preparation involves the serum dilution with a 0.2% acetic acid solution prior to the on-line SPE on a mixed-mode cationic (MCX) polymer based sorbent. The samples were then eluted on a reversed phase column with a mobile phase made of acidified water and methanol and detection was performed by MS using electrospay ionization in positive mode. The analysis time between 2 samples was 7.0min. Standard curves were linear over the range of 10.0pg/mL (LLOQ) to 1000pg/mL with correlation coefficient (r 2) greater than 0.990. The method specificity was demonstrated in six different batches of human serum. Intra-run and inter-run precision and accuracy within ±20% (at the LLOQ) and ±15% (other levels) were achieved during a 3-run validation for quality control samples (QCs). The stability at room temperature (38h) was determined and reported. In addition, the comparison between an off-line SPE procedure and our method gave equivalent results. The results of the present work demonstrated that our on-line SPE–LC–MS/MS method is rapid, sensitive, specific and could be applied to the quantitative analysis of Indacaterol in human serum samples. Our method effectively eliminated the tedious conditioning and rinsing steps associated with conventional off-line SPE and reduced the analysis time. The on-line SPE approach appears attractive for supporting the analysis of several hundreds of clinical samples.
Source:Journal of Chromatography B, Volumes 895–896
Corinne Emotte, Olivier Heudi, Fanny Deglave, Adrien Bonvie, Laurence Masson, Franck Picard, Animesh Chaturvedi, Tapan Majumdar, Ashish Agarwal, Ralph Woessner, Olivier Kretz
Indacaterol has been recently approved in Europe for the treatment of chronic obstructive pulmonary disease (COPD). In the present study, we have developed and validated a rapid and sensitive on-line solid phase extraction (SPE) method coupled to liquid chromatography–tandem mass spectrometry (LC–MS/MS) detection for the determination of Indacaterol in human serum. The sample preparation involves the serum dilution with a 0.2% acetic acid solution prior to the on-line SPE on a mixed-mode cationic (MCX) polymer based sorbent. The samples were then eluted on a reversed phase column with a mobile phase made of acidified water and methanol and detection was performed by MS using electrospay ionization in positive mode. The analysis time between 2 samples was 7.0min. Standard curves were linear over the range of 10.0pg/mL (LLOQ) to 1000pg/mL with correlation coefficient (r 2) greater than 0.990. The method specificity was demonstrated in six different batches of human serum. Intra-run and inter-run precision and accuracy within ±20% (at the LLOQ) and ±15% (other levels) were achieved during a 3-run validation for quality control samples (QCs). The stability at room temperature (38h) was determined and reported. In addition, the comparison between an off-line SPE procedure and our method gave equivalent results. The results of the present work demonstrated that our on-line SPE–LC–MS/MS method is rapid, sensitive, specific and could be applied to the quantitative analysis of Indacaterol in human serum samples. Our method effectively eliminated the tedious conditioning and rinsing steps associated with conventional off-line SPE and reduced the analysis time. The on-line SPE approach appears attractive for supporting the analysis of several hundreds of clinical samples.
Liquid chromatography–electrospray quadrupole linear ion trap mass spectrometry method for the quantitation of palonosetron in human plasma and urine: Application to a pharmacokinetic study
26 April 2012,
11:00:04
Publication year:
2012
Source:Journal of Chromatography B, Volumes 895–896
Pengfei Li, Ping Ma, Yan Wang, Weihang Tong, Jing Wang, Cheng Wu, Lihong Liu
The new analytical method for the determination of palonosetron in human plasma and urine has been developed based on liquid chromatography–mass spectrometry. The method utilized tramadol as the internal standard (IS). Separation was carried out on a Zorbax Eclipse TC-C18 column using methanol–1mM ammonium formate in water (containing 0.1% formic acid, v/v, pH=2.8) as mobile phase for gradient elution. Detection is carried out by multiple reaction monitoring (MRM) on 3200Qtrap™ mass spectrometry. The method has a chromatographic run time of 5.5min and is linear within the concentration range 0.01–5.00ng/mL for plasma and 0.10–30.00ng/mL for urine both with a LOD of 0.003ng/mL. Intra- and inter-day RSD of the concentration was 3.66–6.60%, 1.29–7.71% for plasma and 2.39–5.76%, 2.06–7.13% for urine. The relative error (RE) was −4.58% to 3.26% for plasma and −1.47% to 2.53% for urine. The recovery rates of palonosetron and IS both for plasma and urine were more than 90%. Palonosetron was stable under all the conditions tested. The method was successfully used to analyze palonosetron in human plasma and urine over a period of 168h after intravenously pumping a single dose of 0.25mg to volunteers. No significant differences were found between the pharmacokinetic parameters and urine accumulated excretory rate for male and female volunteers (P >0.05). A two-compartment model was obtained after administrations. Palonosetron was eliminated at a slow rate in volunteers. The mean urine accumulated excretory rate was 25.97±12.87%. Inter-individual differences could not be neglected due to the high coefficient of variety in several pharmacokinetic parameters and the urine accumulated excretion.
Source:Journal of Chromatography B, Volumes 895–896
Pengfei Li, Ping Ma, Yan Wang, Weihang Tong, Jing Wang, Cheng Wu, Lihong Liu
The new analytical method for the determination of palonosetron in human plasma and urine has been developed based on liquid chromatography–mass spectrometry. The method utilized tramadol as the internal standard (IS). Separation was carried out on a Zorbax Eclipse TC-C18 column using methanol–1mM ammonium formate in water (containing 0.1% formic acid, v/v, pH=2.8) as mobile phase for gradient elution. Detection is carried out by multiple reaction monitoring (MRM) on 3200Qtrap™ mass spectrometry. The method has a chromatographic run time of 5.5min and is linear within the concentration range 0.01–5.00ng/mL for plasma and 0.10–30.00ng/mL for urine both with a LOD of 0.003ng/mL. Intra- and inter-day RSD of the concentration was 3.66–6.60%, 1.29–7.71% for plasma and 2.39–5.76%, 2.06–7.13% for urine. The relative error (RE) was −4.58% to 3.26% for plasma and −1.47% to 2.53% for urine. The recovery rates of palonosetron and IS both for plasma and urine were more than 90%. Palonosetron was stable under all the conditions tested. The method was successfully used to analyze palonosetron in human plasma and urine over a period of 168h after intravenously pumping a single dose of 0.25mg to volunteers. No significant differences were found between the pharmacokinetic parameters and urine accumulated excretory rate for male and female volunteers (P >0.05). A two-compartment model was obtained after administrations. Palonosetron was eliminated at a slow rate in volunteers. The mean urine accumulated excretory rate was 25.97±12.87%. Inter-individual differences could not be neglected due to the high coefficient of variety in several pharmacokinetic parameters and the urine accumulated excretion.
Simultaneous determination of 45 pesticides in fruit and vegetable using an improved QuEChERS method and on-line gel permeation chromatography–gas chromatography/mass spectrometer
26 April 2012,
11:00:04
Publication year:
2012
Source:Journal of Chromatography B, Volumes 895–896
Dasheng Lu, Xinlei Qiu, Chao Feng, Yu’e Jin, Yuanjie Lin, Libei Xiong, Yimin Wen, Dongli Wang, Guoquan Wang
In this study, a method was developed to determine 45 selected pesticides (of different chemical families) in fruit and vegetable (including apple, spinach and cucumber). Samples were extracted using an improved QuEChERS method with salting out and phase separation in two steps. The target pesticides in concentrated extracts were analyzed by an on-line gel permeation chromatography–gas chromatography/mass spectrometer (online-GPC–GC/MS). Online GPC effectively removed matrix interferences and greatly improved the method sensitivity, recoveries and automation. Method limits of quantification were 10ng/g for uniconazole and metalaxyl, and 5ng/g for other 43 target analytes. In three fruit and vegetable matrices each spiked with 45 pesticides (0.01μg/g), mean recoveries ranged from 80 to 118% for most of the tested pesticides except for profenofos (77% in apple) and chlorpyrifos (68% in apple and 75% in cucumber), with relative standard deviations (RSDs) of less than 14%. The results of the proficiency testing showed that the method is very successful in measuring the certified pesticides with less than 1.3 of the absolute value of Z-score. This method has been applied for routinely monitoring pesticides in fresh fruit and vegetable.
Source:Journal of Chromatography B, Volumes 895–896
Dasheng Lu, Xinlei Qiu, Chao Feng, Yu’e Jin, Yuanjie Lin, Libei Xiong, Yimin Wen, Dongli Wang, Guoquan Wang
In this study, a method was developed to determine 45 selected pesticides (of different chemical families) in fruit and vegetable (including apple, spinach and cucumber). Samples were extracted using an improved QuEChERS method with salting out and phase separation in two steps. The target pesticides in concentrated extracts were analyzed by an on-line gel permeation chromatography–gas chromatography/mass spectrometer (online-GPC–GC/MS). Online GPC effectively removed matrix interferences and greatly improved the method sensitivity, recoveries and automation. Method limits of quantification were 10ng/g for uniconazole and metalaxyl, and 5ng/g for other 43 target analytes. In three fruit and vegetable matrices each spiked with 45 pesticides (0.01μg/g), mean recoveries ranged from 80 to 118% for most of the tested pesticides except for profenofos (77% in apple) and chlorpyrifos (68% in apple and 75% in cucumber), with relative standard deviations (RSDs) of less than 14%. The results of the proficiency testing showed that the method is very successful in measuring the certified pesticides with less than 1.3 of the absolute value of Z-score. This method has been applied for routinely monitoring pesticides in fresh fruit and vegetable.
Simultaneous determination of flumatinib and its two major metabolites in plasma of chronic myelogenous leukemia patients by liquid chromatography–tandem mass spectrometry
26 April 2012,
11:00:04
Publication year:
2012
Source:Journal of Chromatography B, Volumes 895–896
Yong Yang, Ke Liu, Dafang Zhong, Xiaoyan Chen
Flumatinib is an antineoplastic tyrosine kinase inhibitor used for the treatment of chronic myelogenous leukemia (CML). Its major metabolites in the circulation are N-desmethyl flumatinib (M1) and amide hydrolysis product (M3). To investigate the pharmacokinetics of flumatinib in CML patients, a simple, specific and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of flumatinib and its two major metabolites in patient plasma. After a simple, one-step protein precipitation with methanol, flumatinib, its two metabolites, and internal standard (HHGV-E) were separated on a C18 column using an isocratic mobile phase of methanol:5mM ammonium acetate:formic acid (60:40:0.4, v/v/v). A total chromatographic run time of 4.2min was achieved. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 563→ m/z 463 for flumatinib, m/z 549→ m/z 463 for M1, m/z 303→ m/z 175 for M3, and m/z 529→ m/z 429 for HHGV-E. The method was linear over the concentration ranges of 0.400–400ng/mL for flumatinib, 0.100–100ng/mL for M1, and 0.200–200ng/mL for M3, using only 50μL of plasma. The intra- and inter-day precisions were less than 8.5% for flumatinib, 9.8% for M1, and 10.6% for M3 in terms of the relative standard deviation. The accuracy was within ±2.2% for flumatinib, ±6.0% for M1, and ±9.9% for M3 in terms of relative error. The validated method was successfully applied to clinical pharmacokinetic studies of flumatinib mesylate in CML patients following oral administration at all dosage regimens.
Source:Journal of Chromatography B, Volumes 895–896
Yong Yang, Ke Liu, Dafang Zhong, Xiaoyan Chen
Flumatinib is an antineoplastic tyrosine kinase inhibitor used for the treatment of chronic myelogenous leukemia (CML). Its major metabolites in the circulation are N-desmethyl flumatinib (M1) and amide hydrolysis product (M3). To investigate the pharmacokinetics of flumatinib in CML patients, a simple, specific and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of flumatinib and its two major metabolites in patient plasma. After a simple, one-step protein precipitation with methanol, flumatinib, its two metabolites, and internal standard (HHGV-E) were separated on a C18 column using an isocratic mobile phase of methanol:5mM ammonium acetate:formic acid (60:40:0.4, v/v/v). A total chromatographic run time of 4.2min was achieved. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 563→ m/z 463 for flumatinib, m/z 549→ m/z 463 for M1, m/z 303→ m/z 175 for M3, and m/z 529→ m/z 429 for HHGV-E. The method was linear over the concentration ranges of 0.400–400ng/mL for flumatinib, 0.100–100ng/mL for M1, and 0.200–200ng/mL for M3, using only 50μL of plasma. The intra- and inter-day precisions were less than 8.5% for flumatinib, 9.8% for M1, and 10.6% for M3 in terms of the relative standard deviation. The accuracy was within ±2.2% for flumatinib, ±6.0% for M1, and ±9.9% for M3 in terms of relative error. The validated method was successfully applied to clinical pharmacokinetic studies of flumatinib mesylate in CML patients following oral administration at all dosage regimens.
Simultaneous determination and pharmacokinetics of protein unbound aspirin and salicylic acid in rat blood and brain by microdialysis: An application to herbal–drug interaction
26 April 2012,
11:00:04
Publication year:
2012
Source:Journal of Chromatography B, Volumes 895–896
Lee-Hsin Shaw, Tung-Hu Tsai
Aspirin is commonly used for the prevention of myocardial infarction and ischemic stroke; whereas the Chinese people employ the bu-yang-huan-wu-tang (BYHWT) as a routine herbal formulation for the treatment and prevention of transient ischemic stroke. The current study develops a microdialysis technique coupled to a validated liquid chromatography system to measure free-form aspirin and salicylic acid for herbal–drug interaction in rat blood and brain. The intra- and inter-day precisions in biological dialysates were within 0.1–9.4% in the concentration ranges of 0.1–50μg/mL and the accuracies ranged from −4.7 to 6.1%. The pharmacokinetic data demonstrate that the area under the concentration time curve (AUC) of the aspirin was 2031±266minμg/mL after aspirin administration (100mg/kg, i.v.). The AUC of salicylic acid was 12660±1799minμg/mL, which suggests that aspirin is quickly hydrolyzed to salicylic acid in blood and the metabolite can also be detected within 15min in brain dialysate. The herbal–drug pharmacokinetic interaction showed no significant effect in blood and brain. The results of pharmacodynamics for the bleeding time suggested that there were no significant differences between the aspirin alone group and the BYHWT pretreated group. However, the bleeding time has been prolonged when compared aspirin alone or the group pretreated with BYHWT to the blank control. The conclusion provides practical information for clinical practice for the herbal formulation BYHWT and aspirin used concurrently.
Source:Journal of Chromatography B, Volumes 895–896
Lee-Hsin Shaw, Tung-Hu Tsai
Aspirin is commonly used for the prevention of myocardial infarction and ischemic stroke; whereas the Chinese people employ the bu-yang-huan-wu-tang (BYHWT) as a routine herbal formulation for the treatment and prevention of transient ischemic stroke. The current study develops a microdialysis technique coupled to a validated liquid chromatography system to measure free-form aspirin and salicylic acid for herbal–drug interaction in rat blood and brain. The intra- and inter-day precisions in biological dialysates were within 0.1–9.4% in the concentration ranges of 0.1–50μg/mL and the accuracies ranged from −4.7 to 6.1%. The pharmacokinetic data demonstrate that the area under the concentration time curve (AUC) of the aspirin was 2031±266minμg/mL after aspirin administration (100mg/kg, i.v.). The AUC of salicylic acid was 12660±1799minμg/mL, which suggests that aspirin is quickly hydrolyzed to salicylic acid in blood and the metabolite can also be detected within 15min in brain dialysate. The herbal–drug pharmacokinetic interaction showed no significant effect in blood and brain. The results of pharmacodynamics for the bleeding time suggested that there were no significant differences between the aspirin alone group and the BYHWT pretreated group. However, the bleeding time has been prolonged when compared aspirin alone or the group pretreated with BYHWT to the blank control. The conclusion provides practical information for clinical practice for the herbal formulation BYHWT and aspirin used concurrently.
Multiresidue determination of veterinary drugs in aquaculture fish samples by ultra high performance liquid chromatography coupled to tandem mass spectrometry
26 April 2012,
11:00:04
Publication year:
2012
Source:Journal of Chromatography B, Volumes 895–896
Renata Pereira Lopes, Rocío Cazorla Reyes, Roberto Romero-González, José Luis Martínez Vidal, Antonia Garrido Frenich
A simple, selective and fast multiresidue method was developed for the determination of 32 veterinary drug residues belonging to several families, in gilthead sea bream (Sparus aurata) by ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS). The extraction was based on modified QuEChERS (quick, easy, cheap, effective, rugged and safe) procedure, using as extraction solution a mixture of acetonitrile and methanol (75:25, v/v), and it reduces sample handling, increasing sample throughput in relation to current methodologies. The developed method was validated and mean recovery ranged from 69% to 125% (at 10, 25, 50 and 100μg/kg). Intra and interday precision, estimated as the same levels and expressed as relative standard deviation, RSD, were lower than 20% and 30%, respectively. Limits of detection (LODs) and quantification (LOQs) were lower than 7.5 and 25μg/kg, respectively, except for danofloxacin, oxytetracycline and tetracycline (LOD and LOQ of 15.0 and 50μg/kg, respectively). Decision limit (CCα) and detection capability (CCβ) were also calculated and ranged from 16.7μg/kg (levamisole) to 605.0 (flumequine) μg/kg and from 23.5μg/kg (levamisole) to 611.5μg/kg (flumequine), respectively. The expanded uncertainty, U, was also evaluated ant it was below 25% at 100μg/kg level, except for tetracycline (28%). Finally, the method was applied to ten samples obtained from local supermarkets in Almería (Spain) and traces of some compounds were detected.
Source:Journal of Chromatography B, Volumes 895–896
Renata Pereira Lopes, Rocío Cazorla Reyes, Roberto Romero-González, José Luis Martínez Vidal, Antonia Garrido Frenich
A simple, selective and fast multiresidue method was developed for the determination of 32 veterinary drug residues belonging to several families, in gilthead sea bream (Sparus aurata) by ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS). The extraction was based on modified QuEChERS (quick, easy, cheap, effective, rugged and safe) procedure, using as extraction solution a mixture of acetonitrile and methanol (75:25, v/v), and it reduces sample handling, increasing sample throughput in relation to current methodologies. The developed method was validated and mean recovery ranged from 69% to 125% (at 10, 25, 50 and 100μg/kg). Intra and interday precision, estimated as the same levels and expressed as relative standard deviation, RSD, were lower than 20% and 30%, respectively. Limits of detection (LODs) and quantification (LOQs) were lower than 7.5 and 25μg/kg, respectively, except for danofloxacin, oxytetracycline and tetracycline (LOD and LOQ of 15.0 and 50μg/kg, respectively). Decision limit (CCα) and detection capability (CCβ) were also calculated and ranged from 16.7μg/kg (levamisole) to 605.0 (flumequine) μg/kg and from 23.5μg/kg (levamisole) to 611.5μg/kg (flumequine), respectively. The expanded uncertainty, U, was also evaluated ant it was below 25% at 100μg/kg level, except for tetracycline (28%). Finally, the method was applied to ten samples obtained from local supermarkets in Almería (Spain) and traces of some compounds were detected.
On-line two dimensional liquid chromatography/mass spectrometry for the analysis of triacylglycerides in peanut oil and mouse tissue
26 April 2012,
11:00:04
Publication year:
2012
Source:Journal of Chromatography B, Volumes 895–896
Qin Yang, Xianzhe Shi, Qun Gu, Sumin Zhao, Yuanhong Shan, Guowang Xu
Triacylglycerides (TAGs) are a large class of complex neutral lipids that naturally occur in both plants and animals. In the present work, an on-line comprehensive silver-ion liquid chromatography (silver-ion LC)×reversed-phase liquid chromatography (RPLC) system was constructed to analyze these compounds. A micro bore silver-ion modified column was employed in the first dimension with the commonly used hexane-based mobile phase. After a series of C18 columns were assessed, a wide bore column packed with 1.5μm particles was selected as the second dimension column to reduce the negative effect caused by the large volume and strong solvent injection in the second dimension. The system coupled with mass spectrometry was applied to the analysis of an edible peanut oil and a mouse liver extract. Twenty-eight TAGs from the peanut oil and forty-four from the mouse liver were identified based on the TAGs’ retention behaviors on the comprehensive two-dimensional LC system and their APCI MS fragments.
Source:Journal of Chromatography B, Volumes 895–896
Qin Yang, Xianzhe Shi, Qun Gu, Sumin Zhao, Yuanhong Shan, Guowang Xu
Triacylglycerides (TAGs) are a large class of complex neutral lipids that naturally occur in both plants and animals. In the present work, an on-line comprehensive silver-ion liquid chromatography (silver-ion LC)×reversed-phase liquid chromatography (RPLC) system was constructed to analyze these compounds. A micro bore silver-ion modified column was employed in the first dimension with the commonly used hexane-based mobile phase. After a series of C18 columns were assessed, a wide bore column packed with 1.5μm particles was selected as the second dimension column to reduce the negative effect caused by the large volume and strong solvent injection in the second dimension. The system coupled with mass spectrometry was applied to the analysis of an edible peanut oil and a mouse liver extract. Twenty-eight TAGs from the peanut oil and forty-four from the mouse liver were identified based on the TAGs’ retention behaviors on the comprehensive two-dimensional LC system and their APCI MS fragments.
High-sensitivity liquid chromatography–tandem mass spectrometry for the simultaneous determination of five drugs and their cytochrome P450-specific probe metabolites in human plasma
26 April 2012,
11:00:04
Publication year:
2012
Source:Journal of Chromatography B, Volumes 895–896
Kyung-Suk Oh, Su-Jin Park, Dhananjay D. Shinde, Jae-Gook Shin, Dong-Hyun Kim
A sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) method with electrospray ionization was developed for the simultaneous quantitation of five probe drugs and their metabolites in human plasma for assessing the in vivo activities of cytochrome P450 (CYP). CYP isoform specific substrates and their metabolites of CYP1A2 (caffeine), CYP2C9 (losartan), CYP2C19 (omeprazole), CYP2D6 (dextromethorphan) and CYP3A (midazolam) were all simultaneously analyzed using LC–MS/MS after administration of a mixture of five drugs (i.e., a “cocktail approach”) to healthy volunteers. The assay uses propranolol as an internal standard; dual liquid extraction; a Xbridge MS C18 (100mm×2.1mm, 3.5μm) column; a gradient mobile phase of 0.1% formic acid/acetonitrile (7/3→3/7); mass spectrometric detection in positive ion mode. The method was validated from 5 to 500ng/mL for caffeine and paraxanthine, 0.1–40ng/mL for losartan and EXP3174, 0.05–20ng/mL for omeprazole and 5-hydroxyomeprazole, 0.008–0.8ng/mL for dextromethorphan and dextrorphan, 0.01–1.0ng/mL for midazolam, and 0.04–4ng/mL for 1′-hydroxymidazolam. The intra- and inter-day precision over the concentration ranges for all analytes were lower than 12.5% and 13.8% (relative standard deviation, %RSD), and accuracy was between 86.5% and 108.4% and between 87.0% and 107.0%, respectively. This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10–100 times lower than typical therapeutic doses.
Source:Journal of Chromatography B, Volumes 895–896
Kyung-Suk Oh, Su-Jin Park, Dhananjay D. Shinde, Jae-Gook Shin, Dong-Hyun Kim
A sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) method with electrospray ionization was developed for the simultaneous quantitation of five probe drugs and their metabolites in human plasma for assessing the in vivo activities of cytochrome P450 (CYP). CYP isoform specific substrates and their metabolites of CYP1A2 (caffeine), CYP2C9 (losartan), CYP2C19 (omeprazole), CYP2D6 (dextromethorphan) and CYP3A (midazolam) were all simultaneously analyzed using LC–MS/MS after administration of a mixture of five drugs (i.e., a “cocktail approach”) to healthy volunteers. The assay uses propranolol as an internal standard; dual liquid extraction; a Xbridge MS C18 (100mm×2.1mm, 3.5μm) column; a gradient mobile phase of 0.1% formic acid/acetonitrile (7/3→3/7); mass spectrometric detection in positive ion mode. The method was validated from 5 to 500ng/mL for caffeine and paraxanthine, 0.1–40ng/mL for losartan and EXP3174, 0.05–20ng/mL for omeprazole and 5-hydroxyomeprazole, 0.008–0.8ng/mL for dextromethorphan and dextrorphan, 0.01–1.0ng/mL for midazolam, and 0.04–4ng/mL for 1′-hydroxymidazolam. The intra- and inter-day precision over the concentration ranges for all analytes were lower than 12.5% and 13.8% (relative standard deviation, %RSD), and accuracy was between 86.5% and 108.4% and between 87.0% and 107.0%, respectively. This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10–100 times lower than typical therapeutic doses.
Low density solvent based dispersive liquid–liquid microextraction with gas chromatography–electron capture detection for the determination of cypermethrin in tissues and blood of cypermethrin treated rats
26 April 2012,
11:00:04
Publication year:
2012
Source:Journal of Chromatography B, Volumes 895–896
Mohana Krishna Reddy Mudiam, Rajeev Jain, Shailendra Kumar Maurya, Haider A. Khan, Sanghamitra Bandyopadhyay, R.C. Murthy
A simple and rapid method to determine the cypermethrin (CYP) insecticide in rat tissues (kidney, liver and brain) and blood has been developed for the first time using low density solvent-dispersive liquid–liquid microextraction (LDS-DLLME) followed by gas chromatography–electron capture detector (GC–ECD) analysis. Initially, tissue samples containing CYP were homoginized in acetone. Subsequently, homogenate was mixed with n-hexane (extraction solvent) and the mixture was rapidly injected into water. The upper n-hexane layer was collected in a separate microtube and injected into GC–ECD for analysis. Blood samples were diluted with ultrapure water and subjected to DLLME through similar procedure. Parameters such as type and volume of disperser and extraction solvent, salting out effect and extraction time, which can affect the extraction efficiency of DLLME, were optimized. Method was validated by investigating linearity, precision, recovery, limit of detection (LOD) and quantification (LOQ). LODs in tissue were in the range of 0.043–0.314ngmg−1 and for blood it was 8.6ngmL−1 with a signal to noise ratio of 3:1. LOQs in tissue were in the range of 0.143–1.03ngmg−1 and for blood it was 28.3ngmL−1 with a signal to noise ratio of 10:1. Mean recoveries of CYP at three different concentation levels in all the matrices were found to be in the range of 81.6–103.67%. The results show that, LDS-DLLME coupled with GC–ECD offers a simple, rapid and efficient technique for extraction and determination of CYP in rat tissues and blood samples, which in turn would be useful for toxicological studies of CYP.
Source:Journal of Chromatography B, Volumes 895–896
Mohana Krishna Reddy Mudiam, Rajeev Jain, Shailendra Kumar Maurya, Haider A. Khan, Sanghamitra Bandyopadhyay, R.C. Murthy
A simple and rapid method to determine the cypermethrin (CYP) insecticide in rat tissues (kidney, liver and brain) and blood has been developed for the first time using low density solvent-dispersive liquid–liquid microextraction (LDS-DLLME) followed by gas chromatography–electron capture detector (GC–ECD) analysis. Initially, tissue samples containing CYP were homoginized in acetone. Subsequently, homogenate was mixed with n-hexane (extraction solvent) and the mixture was rapidly injected into water. The upper n-hexane layer was collected in a separate microtube and injected into GC–ECD for analysis. Blood samples were diluted with ultrapure water and subjected to DLLME through similar procedure. Parameters such as type and volume of disperser and extraction solvent, salting out effect and extraction time, which can affect the extraction efficiency of DLLME, were optimized. Method was validated by investigating linearity, precision, recovery, limit of detection (LOD) and quantification (LOQ). LODs in tissue were in the range of 0.043–0.314ngmg−1 and for blood it was 8.6ngmL−1 with a signal to noise ratio of 3:1. LOQs in tissue were in the range of 0.143–1.03ngmg−1 and for blood it was 28.3ngmL−1 with a signal to noise ratio of 10:1. Mean recoveries of CYP at three different concentation levels in all the matrices were found to be in the range of 81.6–103.67%. The results show that, LDS-DLLME coupled with GC–ECD offers a simple, rapid and efficient technique for extraction and determination of CYP in rat tissues and blood samples, which in turn would be useful for toxicological studies of CYP.
Residual metals cause variability in methionine oxidation measurements in protein pharmaceuticals using LC-UV/MS peptide mapping
26 April 2012,
11:00:04
Publication year:
2012
Source:Journal of Chromatography B, Volumes 895–896
Li Zang, Tyler Carlage, David Murphy, Ruth Frenkel, Peter Bryngelson, Mark Madsen, Yelena Lyubarskaya
Methionine oxidation has been demonstrated to play an important role in protein stability in vitro and in vivo. It may also cause changes in biological activity and immunogenicity profile of therapeutic proteins. Therefore, it is critical to monitor methionine oxidation in biopharmaceuticals during process and formulation development, as well as long-term stability studies. A common analytical method for methionine oxidation determination is peptide mapping analysis of protein enzymatic digests using UV detection with or without mass spectrometric detection. The quantitation of oxidation is performed based on the UV or extracted ion chromatographic peak areas of the oxidized and non-oxidized peptides. This method was found to be susceptible to significant variability over long-term use. Major factors leading to this variability included presence of low levels of metal ions, especially iron, in the digestion buffer, chromatographic column, LC injector, and other sample contact surfaces. Careful control of metal ion levels generally leads to less variability and long-term consistency of peptide mapping methods for oxidation determination.
Source:Journal of Chromatography B, Volumes 895–896
Li Zang, Tyler Carlage, David Murphy, Ruth Frenkel, Peter Bryngelson, Mark Madsen, Yelena Lyubarskaya
Methionine oxidation has been demonstrated to play an important role in protein stability in vitro and in vivo. It may also cause changes in biological activity and immunogenicity profile of therapeutic proteins. Therefore, it is critical to monitor methionine oxidation in biopharmaceuticals during process and formulation development, as well as long-term stability studies. A common analytical method for methionine oxidation determination is peptide mapping analysis of protein enzymatic digests using UV detection with or without mass spectrometric detection. The quantitation of oxidation is performed based on the UV or extracted ion chromatographic peak areas of the oxidized and non-oxidized peptides. This method was found to be susceptible to significant variability over long-term use. Major factors leading to this variability included presence of low levels of metal ions, especially iron, in the digestion buffer, chromatographic column, LC injector, and other sample contact surfaces. Careful control of metal ion levels generally leads to less variability and long-term consistency of peptide mapping methods for oxidation determination.
Application of high-speed counter-current chromatography coupled with a reverse micelle solvent system to separate three proteins from Momordica charantia
26 April 2012,
11:00:04
Publication year:
2012
Source:Journal of Chromatography B, Volumes 895–896
Yingnan Li, Lianhong Yin, Lingli Zheng, Lina Xu, Youwei Xu, Yanyan Zhao, Yan Qi, Jihong Yao, Xu Han, Kexin Liu, Jinyong Peng
High-speed counter-current chromatography (HSCCC) coupled with a reverse micelle solvent system was successfully developed to separate three proteins from Momordica charantia. Suitable HSCCC conditions were carefully optimized as follows: the stationary phase was a reverse micellar phase composed of isooctane and 50mM bis-(2-ethylhexyl)-1-sulfosuccinate sodium (AOT). The mobile phase contained mobile phase A (50mM Tris–HCl buffer containing 50mM KCl at pH 7.0) for forward-extraction and mobile phase B (50mM Tris–HCl buffer containing 0.5M KCl at pH 10.0) for back-extraction. The flow rate, detection wavelength and column temperature were set at 1.5ml/min, 280nm and 4°C, respectively. Under these conditions, three fractions (I, II and III) were separated, which showed high purity when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The structures of these proteins were then identified by MALDI-TOF/TOF-MS/MS and compared with the NCBInr database. Fractions I and III were identified as resistance-like protein P-B and pentatricopeptide repeat-containing protein, respectively, which were found in M. charantia for the first time. However, fraction II, which is thought to be a new protein, was not identified, and further investigations on this fraction are required. The anticancer activities of these three proteins on the human gastric cancer cell line SGC-7901 were evaluated in vitro. The results indicated that fraction II has excellent anticancer activity (IC50 =0.116mg/ml for 48h treatment). This is the first report on the use of HSCCC to isolate proteins from M. charantia.
Source:Journal of Chromatography B, Volumes 895–896
Yingnan Li, Lianhong Yin, Lingli Zheng, Lina Xu, Youwei Xu, Yanyan Zhao, Yan Qi, Jihong Yao, Xu Han, Kexin Liu, Jinyong Peng
High-speed counter-current chromatography (HSCCC) coupled with a reverse micelle solvent system was successfully developed to separate three proteins from Momordica charantia. Suitable HSCCC conditions were carefully optimized as follows: the stationary phase was a reverse micellar phase composed of isooctane and 50mM bis-(2-ethylhexyl)-1-sulfosuccinate sodium (AOT). The mobile phase contained mobile phase A (50mM Tris–HCl buffer containing 50mM KCl at pH 7.0) for forward-extraction and mobile phase B (50mM Tris–HCl buffer containing 0.5M KCl at pH 10.0) for back-extraction. The flow rate, detection wavelength and column temperature were set at 1.5ml/min, 280nm and 4°C, respectively. Under these conditions, three fractions (I, II and III) were separated, which showed high purity when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The structures of these proteins were then identified by MALDI-TOF/TOF-MS/MS and compared with the NCBInr database. Fractions I and III were identified as resistance-like protein P-B and pentatricopeptide repeat-containing protein, respectively, which were found in M. charantia for the first time. However, fraction II, which is thought to be a new protein, was not identified, and further investigations on this fraction are required. The anticancer activities of these three proteins on the human gastric cancer cell line SGC-7901 were evaluated in vitro. The results indicated that fraction II has excellent anticancer activity (IC50 =0.116mg/ml for 48h treatment). This is the first report on the use of HSCCC to isolate proteins from M. charantia.
Development and validation of a rapid HPLC method for the determination of cefdinir in beagle dog plasma integrated with an automatic on-line solid-phase extraction following protein precipitation in the 96-well plate format
26 April 2012,
11:00:04
Publication year:
2012
Source:Journal of Chromatography B, Volumes 895–896
Ji Li, Li Wang, Zhao Chen, Rui Xie, You Li, Taijun Hang, Guorong Fan
The high-performance liquid chromatography (HPLC) coupled with on-line solid phase extraction (SPE) and ultraviolet (UV) detection was developed for determining cefdinir in beagle dog plasma. After simple pretreatment for plasma with 6% perchloric acid, a volume of 100μL upper layer of the plasma sample was injected into the self-made on-line SPE column. The analytes were retained on the trap column (Lichrospher C18, 4.6mm×37mm, 25μm), and the biological matrix was washed out with the solvent (20mM KH2PO4 adjusted pH 3.0) at flow rate of 2mL/min. By rotation of the switching valve, the target analytes could be eluted from trap column to analytical column in the back-flush mode by the mobile phase (methanol–acetonitrile–20mM KH2PO4 adjusted pH 3.0, 11.25:6.75:82, v/v/v) at flow rate of 1.5mL/min, and then separated on the analytical column (Ultimate™ XB-C18, 4.6mm×50mm, 5μm). The complete cycle of the on-line SPE preconcentration, purification and HPLC separation of the analytes was 4min. The UV detection was performed at 286nm. The calibration curves showed excellent linear relationship (R 2 =0.9995) over the concentration range of 0.05–50μg/mL. The optimized method showed good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This method was successfully applied to quantify cefdinir in beagle dog plasma to support the pre-clinical pharmacokinetic trial.
Source:Journal of Chromatography B, Volumes 895–896
Ji Li, Li Wang, Zhao Chen, Rui Xie, You Li, Taijun Hang, Guorong Fan
The high-performance liquid chromatography (HPLC) coupled with on-line solid phase extraction (SPE) and ultraviolet (UV) detection was developed for determining cefdinir in beagle dog plasma. After simple pretreatment for plasma with 6% perchloric acid, a volume of 100μL upper layer of the plasma sample was injected into the self-made on-line SPE column. The analytes were retained on the trap column (Lichrospher C18, 4.6mm×37mm, 25μm), and the biological matrix was washed out with the solvent (20mM KH2PO4 adjusted pH 3.0) at flow rate of 2mL/min. By rotation of the switching valve, the target analytes could be eluted from trap column to analytical column in the back-flush mode by the mobile phase (methanol–acetonitrile–20mM KH2PO4 adjusted pH 3.0, 11.25:6.75:82, v/v/v) at flow rate of 1.5mL/min, and then separated on the analytical column (Ultimate™ XB-C18, 4.6mm×50mm, 5μm). The complete cycle of the on-line SPE preconcentration, purification and HPLC separation of the analytes was 4min. The UV detection was performed at 286nm. The calibration curves showed excellent linear relationship (R 2 =0.9995) over the concentration range of 0.05–50μg/mL. The optimized method showed good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This method was successfully applied to quantify cefdinir in beagle dog plasma to support the pre-clinical pharmacokinetic trial.
Antibody affinity purification using metallic nickel particles
26 April 2012,
11:00:04
Publication year:
2012
Source:Journal of Chromatography B, Volumes 895–896
Jun Gao, Zhijun Li, Thomas Russell, Zhiyu Li
Functionalized magnetic particles are emerging as a reliable and convenient technique in the purification of biomacromolecules (proteins and nucleic acids) and cell separation. In this study, we used novel solid nickel ferromagnetic particles coated with Protein A for the affinity purification of antibody. The study demonstrated that IgG can be purified from undiluted mouse serum in as few as 5min using Protein A-coated nickel particles. Further, protein crosslinking was shown to stabilize the Protein A on the nickel particle surfaces to minimize Protein A leaching during the affinity purification and elution of IgG. The separation procedure is gentle, scalable, automatable, efficient and economical. By modifying the functional groups of amino acids in the protein coating, crosslinked nickel particles can be used not only for protein affinity purification but for other biological sample preparation and chromatographic applications as well. Methods proposed and tested in this study can be easily modified for small and medium scale antibody purification in lab and pre-clinical research.
Source:Journal of Chromatography B, Volumes 895–896
Jun Gao, Zhijun Li, Thomas Russell, Zhiyu Li
Functionalized magnetic particles are emerging as a reliable and convenient technique in the purification of biomacromolecules (proteins and nucleic acids) and cell separation. In this study, we used novel solid nickel ferromagnetic particles coated with Protein A for the affinity purification of antibody. The study demonstrated that IgG can be purified from undiluted mouse serum in as few as 5min using Protein A-coated nickel particles. Further, protein crosslinking was shown to stabilize the Protein A on the nickel particle surfaces to minimize Protein A leaching during the affinity purification and elution of IgG. The separation procedure is gentle, scalable, automatable, efficient and economical. By modifying the functional groups of amino acids in the protein coating, crosslinked nickel particles can be used not only for protein affinity purification but for other biological sample preparation and chromatographic applications as well. Methods proposed and tested in this study can be easily modified for small and medium scale antibody purification in lab and pre-clinical research.
High-performance liquid chromatography quadrupole time-of-flight mass spectrometry method for the analysis of antidiabetic drugs in aqueous environmental samples
26 April 2012,
11:00:04
Publication year:
2012
Source:Journal of Chromatography B, Volumes 895–896
Julia Martín, Wolfgang Buchberger, Juan Luis Santos, Esteban Alonso, Irene Aparicio
Antidiabetic compounds are among the most prescribed pharmaceuticals. Nevertheless, their presence in the environment has been scarcely evaluated as there is no method for their determination in environmental samples. This paper reports the development of an analytical method for the determination of traditionally used antidiabetics (metformin and glibenclamide) and novel antidiabetics (vildagliptin, sitagliptin and pioglitazone). The method is based on solid-phase extraction and determination by high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. The method was applied to effluent wastewater, river water and tap water. Mean recoveries of glibenclamide, vildagliptin, sitagliptin and pioglitazone in the matrices evaluated were in the range 78–83%; limits of quantification were in the range 0.4–4.3ngL−1; and precision values were in the range 2.2–13%. The high hydrophilicity and polarity of metformin complicated its simultaneous extraction. Chromabond Tetracycline cartridges and sample pH 8.5 were applied to the extraction of glibenclamide, vildagliptin, sitagliptin and pioglitazone. Oasis HLB cartridges, neutral sample pH and SDS as ion-pair reagent were used for the extraction of metformin. Validation results of metformin were not as favorable as those of the other antidiabetic drugs but were comparable with others previously reported. The developed method was applied to the first-time determination of the concentrations of the five antidiabetic drugs in wastewater, river water and tap water. Metformin was the antidiabetic drug at the highest concentration in wastewater and surface water (up to 253ngL−1 and 104ngL−1, respectively). Two of the antidiabetic drugs of recent prescription, sitagliptin and vildagliptin, were found in effluent wastewater at concentrations of 117ngL−1 and 12ngL−1, respectively, and in river water at concentrations of 35ngL−1 and 6ngL−1, respectively, whereas the classic antidiabetic drug glibenclamide and the novel drug pioglitazone were not detected.
Source:Journal of Chromatography B, Volumes 895–896
Julia Martín, Wolfgang Buchberger, Juan Luis Santos, Esteban Alonso, Irene Aparicio
Antidiabetic compounds are among the most prescribed pharmaceuticals. Nevertheless, their presence in the environment has been scarcely evaluated as there is no method for their determination in environmental samples. This paper reports the development of an analytical method for the determination of traditionally used antidiabetics (metformin and glibenclamide) and novel antidiabetics (vildagliptin, sitagliptin and pioglitazone). The method is based on solid-phase extraction and determination by high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. The method was applied to effluent wastewater, river water and tap water. Mean recoveries of glibenclamide, vildagliptin, sitagliptin and pioglitazone in the matrices evaluated were in the range 78–83%; limits of quantification were in the range 0.4–4.3ngL−1; and precision values were in the range 2.2–13%. The high hydrophilicity and polarity of metformin complicated its simultaneous extraction. Chromabond Tetracycline cartridges and sample pH 8.5 were applied to the extraction of glibenclamide, vildagliptin, sitagliptin and pioglitazone. Oasis HLB cartridges, neutral sample pH and SDS as ion-pair reagent were used for the extraction of metformin. Validation results of metformin were not as favorable as those of the other antidiabetic drugs but were comparable with others previously reported. The developed method was applied to the first-time determination of the concentrations of the five antidiabetic drugs in wastewater, river water and tap water. Metformin was the antidiabetic drug at the highest concentration in wastewater and surface water (up to 253ngL−1 and 104ngL−1, respectively). Two of the antidiabetic drugs of recent prescription, sitagliptin and vildagliptin, were found in effluent wastewater at concentrations of 117ngL−1 and 12ngL−1, respectively, and in river water at concentrations of 35ngL−1 and 6ngL−1, respectively, whereas the classic antidiabetic drug glibenclamide and the novel drug pioglitazone were not detected.
UPLC–MS/MS determination of ractopamine residues in retinal tissue of treated food-producing pigs
26 April 2012,
11:00:04
Publication year:
2012
Source:Journal of Chromatography B, Volumes 895–896
Ana Vulić, Jelka Pleadin, Nina Perši, Dinka Milić, Wolfgang Radeck
Ractopamine is a β2-adrenergic agonist, which reduces fat deposition and promotes muscle growth in animals for meat production. In the European Union countries, systematic monitoring and control of this contaminant residue is regularly performed by use of validated analytical methods of detection in different biological materials. The aim of the present study was to assess persistence of ractopamine in retina as a pigmented tissue by determination of its residues using UPLC–MS/MS as a quantitative confirmatory method after pig exposure to a ractopamine dose of 0.51mg/kg b.w. Experimental group (n =9) of pigs were orally administered ractopamine for 28 days and then randomly sacrificed (n =3) on days 1, 3 and 8 of treatment discontinuation, whereas control animals (n =3) were left untreated. Study results showed mean ractopamine residue concentrations of 110.36μg/kg, 67.11μg/kg and 89.93μg/kg on days 1, 3 and 8 after withdrawal, respectively, indicating high accumulation of ractopamine in retina despite a low dose applied. These data pointed to high affinity of ractopamine for binding to the pigmented segment of the eye, thus supporting the use of pigmented tissues as matrices in the regulatory monitoring of this β2-adrenergic agonist.
Source:Journal of Chromatography B, Volumes 895–896
Ana Vulić, Jelka Pleadin, Nina Perši, Dinka Milić, Wolfgang Radeck
Ractopamine is a β2-adrenergic agonist, which reduces fat deposition and promotes muscle growth in animals for meat production. In the European Union countries, systematic monitoring and control of this contaminant residue is regularly performed by use of validated analytical methods of detection in different biological materials. The aim of the present study was to assess persistence of ractopamine in retina as a pigmented tissue by determination of its residues using UPLC–MS/MS as a quantitative confirmatory method after pig exposure to a ractopamine dose of 0.51mg/kg b.w. Experimental group (n =9) of pigs were orally administered ractopamine for 28 days and then randomly sacrificed (n =3) on days 1, 3 and 8 of treatment discontinuation, whereas control animals (n =3) were left untreated. Study results showed mean ractopamine residue concentrations of 110.36μg/kg, 67.11μg/kg and 89.93μg/kg on days 1, 3 and 8 after withdrawal, respectively, indicating high accumulation of ractopamine in retina despite a low dose applied. These data pointed to high affinity of ractopamine for binding to the pigmented segment of the eye, thus supporting the use of pigmented tissues as matrices in the regulatory monitoring of this β2-adrenergic agonist.
nice and worthy..
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