A reliable and stable method for determination of brigatinib in rat plasma by UPLC–MS/MS: Application to a pharmacokinetic study

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069
Author(s): Jian Wen, Susu Bao, Yaoyao Cai, Bowen Zhang, Rongrong Wang, Chenchen Wang, Guoxin Hu
Brigatinib is the second-generation anaplastic lymphoma kinase – inhibitor in non-small cell lung cancer and it can overcome the crizotinib-resistance. Chromatographic separation was carried out on an Acquity Ultra Performance Liquid Chromatography (UPLC) unit with a BEH C18 column (2.1mm×50mm, 1.7μm). The mobile phase was composed of acetonitrile and 0.1% formic acid in water. No endogenous interfering compounds was discovered at retention time of brigatinib (0.56min) and imatinib (IS, 1.41min). MS/MS detection was performed in positive mode. And the MRM transitions were m/z 584.09→484.08 and m/z 494.3→394.2 for brigatinib and IS, respectively. This method was assessed to be stable, specificity, and no matrix effect in three concentrations (0.004, 0.4, 4μg/mL). The intra-day and inter-day precisions were less than 11.09% and 6.43%. And intra-day and inter-day accuracies were ranged from −3.88% to 5.44%. The recovery of brigatinib was from 85.26% to 96.05%. Additionally, the method had a good linearity in the range of 0.002–5μg/mL. The presented method was effectively implemented to determine the concentration of brigatinib in rat plasma.

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069

Author(s): Jian Wen, Susu Bao, Yaoyao Cai, Bowen Zhang, Rongrong Wang, Chenchen Wang, Guoxin Hu

Brigatinib is the second-generation anaplastic lymphoma kinase – inhibitor in non-small cell lung cancer and it can overcome the crizotinib-resistance. Chromatographic separation was carried out on an Acquity Ultra Performance Liquid Chromatography (UPLC) unit with a BEH C18 column (2.1mm×50mm, 1.7μm). The mobile phase was composed of acetonitrile and 0.1% formic acid in water. No endogenous interfering compounds was discovered at retention time of brigatinib (0.56min) and imatinib (IS, 1.41min). MS/MS detection was performed in positive mode. And the MRM transitions were m/z 584.09484.08 and m/z 494.3394.2 for brigatinib and IS, respectively. This method was assessed to be stable, specificity, and no matrix effect in three concentrations (0.004, 0.4, 4μg/mL). The intra-day and inter-day precisions were less than 11.09% and 6.43%. And intra-day and inter-day accuracies were ranged from −3.88% to 5.44%. The recovery of brigatinib was from 85.26% to 96.05%. Additionally, the method had a good linearity in the range of 0.002–5μg/mL. The presented method was effectively implemented to determine the concentration of brigatinib in rat plasma.





Fast and sensitive HPLC–MS/MS method for direct quantification of intracellular deoxyribonucleoside triphosphates from tissue and cells

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069
Author(s): Sigurast Olafsson, Dale Whittington, Jason Murray, Michael Regnier, Farid Moussavi-Harami
Deoxyribonucleoside triphosphates (dNTPs) are used in DNA synthesis and repair. Even slight imbalances can have adverse biological effects. This study validates a fast and sensitive HPLC–MS/MS method for direct quantification of intracellular dNTPs from tissue. Equal volumes of methanol and water were used for nucleotide extraction from mouse heart and gastrocnemius muscle and isolated cardiomyocytes followed by centrifugation to remove particulates. The resulting supernatant was analyzed on a porous graphitic carbon chromatography column using an elution gradient of ammonium acetate in water and ammonium hydroxide in acetonitrile with a run time of just 10min. Calibration curves of all dNTPs ranged from 62.5 to 2500fmol injections and demonstrated excellent linearity (r2>0.99). The within day and between day precision, as measured by the coefficient of variation (CV (%)), was <25% for all points, including the lower limit of quantification (LLOQ). The inter-day accuracy was within 12% of expected concentration for the LLOQ and within 7% for all other points on the calibration curve. The intra-day accuracy was within 22% for the LLOQ and within 11% for all points on the curve. Compared to existing methods, this study presents a faster and more sensitive method for dNTP quantification.

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069

Author(s): Sigurast Olafsson, Dale Whittington, Jason Murray, Michael Regnier, Farid Moussavi-Harami

Deoxyribonucleoside triphosphates (dNTPs) are used in DNA synthesis and repair. Even slight imbalances can have adverse biological effects. This study validates a fast and sensitive HPLC–MS/MS method for direct quantification of intracellular dNTPs from tissue. Equal volumes of methanol and water were used for nucleotide extraction from mouse heart and gastrocnemius muscle and isolated cardiomyocytes followed by centrifugation to remove particulates. The resulting supernatant was analyzed on a porous graphitic carbon chromatography column using an elution gradient of ammonium acetate in water and ammonium hydroxide in acetonitrile with a run time of just 10min. Calibration curves of all dNTPs ranged from 62.5 to 2500fmol injections and demonstrated excellent linearity (r2 >0.99). The within day and between day precision, as measured by the coefficient of variation (CV (%)), was <25% for all points, including the lower limit of quantification (LLOQ). The inter-day accuracy was within 12% of expected concentration for the LLOQ and within 7% for all other points on the calibration curve. The intra-day accuracy was within 22% for the LLOQ and within 11% for all points on the curve. Compared to existing methods, this study presents a faster and more sensitive method for dNTP quantification.





Simultaneous quantitative analysis of uremic toxins by LC–MS/MS with a reversed-phase/cation-exchange/anion-exchange tri-modal mixed-mode column

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069
Author(s): Yoshitomi Kanemitsu, Kei Asaji, Yotaro Matsumoto, Hiroki Tsukamoto, Daisuke Saigusa, Chikahisa Mukawa, Tatsuki Tachikawa, Takaaki Abe, Yoshihisa Tomioka
Column choice is crucial to the development of liquid chromatography/tandem mass spectrometry (LC–MS/MS) methods because analyte selectivity is dependent on the nature of the stationary phase. Recently, mixed-mode chromatography, which employs a combination of two or more stationary phases and solvent systems, has emerged as an alternative to multiple, complementary, single-column systems. This report describes the development and validation of a novel analytical method based on LC–MS/MS employing a reversed-phase/cation-exchange/anion-exchange tri-modal column (Scherzo SS-C18; Imtakt) for the simultaneous quantification of various uremic toxins (UTx), including creatinine, 1-methyladenosine, trimethylamine-N-oxide, indoxyl sulfate, p-cresyl sulfate, phenyl sulfate and 4-ethylphenyl sulfate. Stable isotope-labeled compounds were prepared as internal standards (ISs) for each analyte. Mobile phase optimization and appropriate gradient conditions resulted in satisfactory retention and peak resolution that could not have been attained with a single stationary phase LC system. The essential validation parameters, including intra- and inter-assay precision and accuracy, were adequate. The validated method was applied to measure serum levels of the aforementioned compounds in 19 patients with chronic kidney disease. This is the first report detailing the simultaneous quantification of these analytes using stable isotopes as ISs. Our results suggest that Scherzo SS-C18 columns will be considered breakthrough tools in the development of analytical methods for compounds that are difficult to quantify simultaneously in traditional LC systems.

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069

Author(s): Yoshitomi Kanemitsu, Kei Asaji, Yotaro Matsumoto, Hiroki Tsukamoto, Daisuke Saigusa, Chikahisa Mukawa, Tatsuki Tachikawa, Takaaki Abe, Yoshihisa Tomioka

Column choice is crucial to the development of liquid chromatography/tandem mass spectrometry (LC–MS/MS) methods because analyte selectivity is dependent on the nature of the stationary phase. Recently, mixed-mode chromatography, which employs a combination of two or more stationary phases and solvent systems, has emerged as an alternative to multiple, complementary, single-column systems. This report describes the development and validation of a novel analytical method based on LC–MS/MS employing a reversed-phase/cation-exchange/anion-exchange tri-modal column (Scherzo SS-C18; Imtakt) for the simultaneous quantification of various uremic toxins (UTx), including creatinine, 1-methyladenosine, trimethylamine-N-oxide, indoxyl sulfate, p-cresyl sulfate, phenyl sulfate and 4-ethylphenyl sulfate. Stable isotope-labeled compounds were prepared as internal standards (ISs) for each analyte. Mobile phase optimization and appropriate gradient conditions resulted in satisfactory retention and peak resolution that could not have been attained with a single stationary phase LC system. The essential validation parameters, including intra- and inter-assay precision and accuracy, were adequate. The validated method was applied to measure serum levels of the aforementioned compounds in 19 patients with chronic kidney disease. This is the first report detailing the simultaneous quantification of these analytes using stable isotopes as ISs. Our results suggest that Scherzo SS-C18 columns will be considered breakthrough tools in the development of analytical methods for compounds that are difficult to quantify simultaneously in traditional LC systems.





Simultaneous determination of five Alternaria toxins in cereals using QuEChERS-based methodology

Publication date: Available online 4 October 2017
Source:Journal of Chromatography B
Author(s): Jian Zhou, Jiao-Jiao Xu, Zeng-Xuan Cai, Bai-Fen Huang, Mi-Cong Jin, Yi-Ping Ren
Two analytical approaches, including ultra-high performance liquid chromatograph linked with photo-diode array/fluorescence detector, and ultra-high performance liquid chromatography-tandem mass spectrometry, have been proposed for simultaneous determination of five Alternaria toxins in cereals, which both based on QuEChERS strategy. After QuEChERS extraction and salting out, the collected supernatant was subjected to an extra dispersive liquid-liquid microextraction step prior to quantitative analysis. Response surface methodology based on central composite design was employed to optimize the micro-extraction conditions. During photo-diode array/fluorescence detector method validation, satisfactory analytical characteristics, in terms of selectivity, recovery (72.7%-109.1%), precision (inter-day RSDs <9.6%), sensitivity (limits of quantification ranged from 2.1μgkg−1 to 120.0μgkg−1) and linearity (R2 all higher than 0.9984), were achieved. Mass spectrometry method was employed as a certified method for accuracy. The two methodologies were successfully applied to 71 samples including three different matrices and the quantitative results were compared. As the result of non-parametric test shown, the established two analytical approach exhibited comparable quantitative accuracy to each other.

Graphical abstract

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Publication date: Available online 4 October 2017
Source:Journal of Chromatography B

Author(s): Jian Zhou, Jiao-Jiao Xu, Zeng-Xuan Cai, Bai-Fen Huang, Mi-Cong Jin, Yi-Ping Ren

Two analytical approaches, including ultra-high performance liquid chromatograph linked with photo-diode array/fluorescence detector, and ultra-high performance liquid chromatography-tandem mass spectrometry, have been proposed for simultaneous determination of five Alternaria toxins in cereals, which both based on QuEChERS strategy. After QuEChERS extraction and salting out, the collected supernatant was subjected to an extra dispersive liquid-liquid microextraction step prior to quantitative analysis. Response surface methodology based on central composite design was employed to optimize the micro-extraction conditions. During photo-diode array/fluorescence detector method validation, satisfactory analytical characteristics, in terms of selectivity, recovery (72.7%-109.1%), precision (inter-day RSDs <9.6%), sensitivity (limits of quantification ranged from 2.1μgkg−1 to 120.0μgkg−1) and linearity (R2 all higher than 0.9984), were achieved. Mass spectrometry method was employed as a certified method for accuracy. The two methodologies were successfully applied to 71 samples including three different matrices and the quantitative results were compared. As the result of non-parametric test shown, the established two analytical approach exhibited comparable quantitative accuracy to each other.

Graphical abstract

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Effects of Pu-erh ripened tea on hyperuricemic mice studied by serum metabolomics

Publication date: Available online 4 October 2017
Source:Journal of Chromatography B
Author(s): Ran Zhao, Dong Chen, Hualing Wu
To evaluate effects of Pu-erh ripened tea in hyperuricemic mice, a mouse hyperuricemia model was developed by oral administration of potassium oxonate for 7 d. Serum metabolomics, based on gas chromatography–mass spectrometry, was used to generate metabolic profiles from normal control, hyperuricemic and allopurinol-treated hyperuricemic mice, as well as hyperuricemic mice given Pu-erh ripened tea at three doses. Pu-erh ripened tea significantly lowered serum uric acid levels. Twelve potential biomarkers associated with hyperuricemia were identified. Pu-erh ripened tea and allopurinol differed in their metabolic effects in the hyperuricemic mice. Levels of glutamic acid, indolelactate, L-allothreonine, nicotinoylglycine, isoleucine, L-cysteine and glycocyamine, all involved in amino acid metabolism, were significantly changed in hyperuricemic mice treated Pu-erh ripened tea. Thus, modulating amino acid metabolism might be the primary mechanism of anti-hyperuricemia by Pu-erh ripened tea.

Publication date: Available online 4 October 2017
Source:Journal of Chromatography B

Author(s): Ran Zhao, Dong Chen, Hualing Wu

To evaluate effects of Pu-erh ripened tea in hyperuricemic mice, a mouse hyperuricemia model was developed by oral administration of potassium oxonate for 7 d. Serum metabolomics, based on gas chromatography–mass spectrometry, was used to generate metabolic profiles from normal control, hyperuricemic and allopurinol-treated hyperuricemic mice, as well as hyperuricemic mice given Pu-erh ripened tea at three doses. Pu-erh ripened tea significantly lowered serum uric acid levels. Twelve potential biomarkers associated with hyperuricemia were identified. Pu-erh ripened tea and allopurinol differed in their metabolic effects in the hyperuricemic mice. Levels of glutamic acid, indolelactate, L-allothreonine, nicotinoylglycine, isoleucine, L-cysteine and glycocyamine, all involved in amino acid metabolism, were significantly changed in hyperuricemic mice treated Pu-erh ripened tea. Thus, modulating amino acid metabolism might be the primary mechanism of anti-hyperuricemia by Pu-erh ripened tea.





A plasma lipidomics strategy reveals perturbed lipid metabolic pathways and potential lipid biomarkers of human colorectal cancer

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069
Author(s): Sensen Shen, Li Yang, Linnan Li, Yu Bai, Chun Cai, Huwei Liu
To explore underlying molecular mechanisms and identify novel lipid biomarkers promising for colorectal cancer (CRC) diagnosis, a continuous-flow two dimensional liquid chromatography–quadrupole time-of-flight mass spectrometry (2D LC-QToF/MS) method was employed to comprehensively measure lipid species in human plasma of CRC patients and healthy controls. With a total of 427 annotated lipid species, we identified 64 lipid species with corrected p value less than 0.05 and fold change more than 1.5. These significantly altered lipid species were mainly involved in glycerolipids and glycerophospholipids metabolism and sphingolipids metabolism. After the diagnosis ability evaluation based on the receiver operating characteristic (ROC) curve, phosphatidylglycerol (34:0), sphingomyelin (42:2), ceramide (44:5), lysophosphatidylcholine (18:3), lysophosphatidylcholine (18:2), phosphatidylethanolamine (O-36:3), phosphatidylethanolamine (O-38:3) and sphingomyelin (38:8) were finally proposed as the potential biomarkers with the area under the curve (AUC) more than 0.900. These results suggest that this 2D LC-QToF/MS-based lipidomics profiling has great potential as a noninvasive diagnostic method in detecting CRC and hopefully provide new clues to understand its underlying mechanism.

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069

Author(s): Sensen Shen, Li Yang, Linnan Li, Yu Bai, Chun Cai, Huwei Liu

To explore underlying molecular mechanisms and identify novel lipid biomarkers promising for colorectal cancer (CRC) diagnosis, a continuous-flow two dimensional liquid chromatography–quadrupole time-of-flight mass spectrometry (2D LC-QToF/MS) method was employed to comprehensively measure lipid species in human plasma of CRC patients and healthy controls. With a total of 427 annotated lipid species, we identified 64 lipid species with corrected p value less than 0.05 and fold change more than 1.5. These significantly altered lipid species were mainly involved in glycerolipids and glycerophospholipids metabolism and sphingolipids metabolism. After the diagnosis ability evaluation based on the receiver operating characteristic (ROC) curve, phosphatidylglycerol (34:0), sphingomyelin (42:2), ceramide (44:5), lysophosphatidylcholine (18:3), lysophosphatidylcholine (18:2), phosphatidylethanolamine (O-36:3), phosphatidylethanolamine (O-38:3) and sphingomyelin (38:8) were finally proposed as the potential biomarkers with the area under the curve (AUC) more than 0.900. These results suggest that this 2D LC-QToF/MS-based lipidomics profiling has great potential as a noninvasive diagnostic method in detecting CRC and hopefully provide new clues to understand its underlying mechanism.





Practical Aspects of the Automated Preparation of Aqueous Two Phase Systems for the Analysis of Biological Macromolecules

Publication date: Available online 3 October 2017 Source:Journal of Chromatography B Author(s): Rana Hameed, Jonathan Huddleston, Svetlana Ignatova A robust strategy for the automated preparation of aqueous two-phase systems (ATP…

Publication date: Available online 3 October 2017
Source:Journal of Chromatography B

Author(s): Rana Hameed, Jonathan Huddleston, Svetlana Ignatova

A robust strategy for the automated preparation of aqueous two-phase systems (ATPS) using a liquid handling sample processor was developed using gravimetric methods: to determine the accuracy of preparation. The major robotic control parameters requiring adjustment were; speed of aspiration and dispense; delay times following aspiration and dispense alongside measures to control cross-contamination during phase sampling. In general mixture compositions of both polymer/polymer and polymer/salt mixtures could be prepared with a target bias accuracy of less than 5%. However, we found that the bias accuracy with which systems of defined TLL and MR could be constructed was highly dependent on the tie line length of the ATPS and the geometrical form of the ATPS co-existence curve. For systems with a very low degree of curvature (PEG/salt systems here) increases in bias (accuracy) are appreciable at relatively long tie line lengths. Where the degree of curvature is more pronounced (PEG/dextran systems) closer approach to the critical point was possible without major effect on bias/accuracy. Application of the strategy to the measurement of the partitioning of phosphorylated and dephosphorylated forms of the model protein ovalbumin are reported. Differences in partition of phosphorylated (native) forms and dephosphorylated forms could be demonstrated. In a PEG/salt system this was manifest as a substantial decrease in solubility based on overall protein recovery derived from accurate knowledge of the system mass ratio. In a PEG/dextran system differences in partition coefficient could be demonstrated between phosphorylated and dephosphorylated forms.





A simple dilute and shoot approach incorporated with pentafluorophenyl (PFP) column based LC-MS/MS assay for the simultaneous determination of trimethylamine N-oxide and trimethylamine in spot urine samples with high throughput

Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067
Author(s): Xiaoguang (Sunny) Li, Shu Li, Gottfried Kellermann
Determination of trimethylamine N-oxide (TMAO) and trimethylamine (TMA) in biological and environmental samples has drawn great attention recently due to their increasing association with human health and disease. It remains a challenge to simultaneously quantify TMAO and TMA in a simple, fast and cost-effective manner due to pre-analytical and analytical constraints. For the first time, we describe a dilute and shoot approach combined with LC-MS/MS detection for the simultaneous measurement of the analytes in spot urine samples with high throughput. Compared to the existing methods, the merits of the proposed assay include the use of a simple dilute and shoot approach (100-fold), small sample volume (10μL), short LC run on a PFP column (4.0min) and multi-analyte MS detection without sample cleanup, derivatization, evaporation and a HILIC column. Dilution, LC and MS parameters were optimized in detail. Method validation yielded a wide linearity for TMAO (1.0–400μg/mL) and TMA (0.025–10μg/mL) with a respective limit of quantitation of 1.0 and 0.025μg/mL. The quantitation was not affected by 41 major urinary components, structurally-related drugs and metabolites. The intra- and inter-day assay precisions were ≤3.6% and recoveries were 93.3%–103.3% for spiked quality control samples. The clinical utility of the alternative spot urine sampling approach compared to conventional 24h urine collection was supported by a significant correlation between the two sampling strategies (n=20, p<0.0001, r=0.757–0.862; slope=0.687–1.170) and no statistical difference in day-to-day biological variability (n=20). The applicability and reliability of the assay was verified by the assessment of reference intervals in a cohort of 118 healthy people. The proposed assay would be beneficial for the rapid and accurate determination of the increasingly important TMAO and TMA demanded in clinical, environmental, pharmaceutical and nutritional fields.

Graphical abstract

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Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067

Author(s): Xiaoguang (Sunny) Li, Shu Li, Gottfried Kellermann

Determination of trimethylamine N-oxide (TMAO) and trimethylamine (TMA) in biological and environmental samples has drawn great attention recently due to their increasing association with human health and disease. It remains a challenge to simultaneously quantify TMAO and TMA in a simple, fast and cost-effective manner due to pre-analytical and analytical constraints. For the first time, we describe a dilute and shoot approach combined with LC-MS/MS detection for the simultaneous measurement of the analytes in spot urine samples with high throughput. Compared to the existing methods, the merits of the proposed assay include the use of a simple dilute and shoot approach (100-fold), small sample volume (10μL), short LC run on a PFP column (4.0min) and multi-analyte MS detection without sample cleanup, derivatization, evaporation and a HILIC column. Dilution, LC and MS parameters were optimized in detail. Method validation yielded a wide linearity for TMAO (1.0–400μg/mL) and TMA (0.025–10μg/mL) with a respective limit of quantitation of 1.0 and 0.025μg/mL. The quantitation was not affected by 41 major urinary components, structurally-related drugs and metabolites. The intra- and inter-day assay precisions were ≤3.6% and recoveries were 93.3%–103.3% for spiked quality control samples. The clinical utility of the alternative spot urine sampling approach compared to conventional 24h urine collection was supported by a significant correlation between the two sampling strategies (n=20, p< 0.0001, r =0.757–0.862; slope=0.687–1.170) and no statistical difference in day-to-day biological variability (n=20). The applicability and reliability of the assay was verified by the assessment of reference intervals in a cohort of 118 healthy people. The proposed assay would be beneficial for the rapid and accurate determination of the increasingly important TMAO and TMA demanded in clinical, environmental, pharmaceutical and nutritional fields.

Graphical abstract

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Choices of capture chromatography technology in antibody manufacturing processes

Publication date: Available online 3 October 2017 Source:Journal of Chromatography B Author(s): Michael DiLeo, Arthur Ley, Andrew E. Nixon, Jie Chen The capture process employed in monoclonal antibody downstream purification is…

Publication date: Available online 3 October 2017
Source:Journal of Chromatography B

Author(s): Michael DiLeo, Arthur Ley, Andrew E. Nixon, Jie Chen

The capture process employed in monoclonal antibody downstream purification is not only the most critically impacted process by increased antibody titer resulting from optimized mammalian cell culture expression systems, but also the most important purification step in determining overall process throughput, product quality, and economics. Advances in separation technology for capturing antibodies from complex feedstocks have been one focus of downstream purification process innovation for past 10 years. In this study, we evaluated new generation chromatography resins used in the antibody capture process including Protein A, cation exchange, and mixed mode chromatography to address the benefits and unique challenges posed by each chromatography approach. Our results demonstrate the benefit of improved binding capacity of new generation Protein A resins, address the concern of high concentration surge caused aggregation when using new generation cation exchange resins with over 100mg/mL binding capacity, and highlight the potential of multimodal cation exchange resins for capture process design. The new landscape of capture chromatography technologies provides options to achieve overall downstream purification outcome with high product quality and process efficiency.





Determination of quantitative retention-activity relationships between pharmacokinetic parameters and biological effectiveness fingerprints of Salvia miltiorrhiza constituents using biopartitioning and microemulsion high-performance liquid chromatography

Publication date: 1 November 2017 Source:Journal of Chromatography B, Volume 1067 Author(s): Haoshi Gao, Hongzhang Huang, Aini Zheng, Nuojun Yu, Ning Li In this study, we analyzed danshen (Salvia miltiorrhiza) constituents us…

Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067

Author(s): Haoshi Gao, Hongzhang Huang, Aini Zheng, Nuojun Yu, Ning Li

In this study, we analyzed danshen (Salvia miltiorrhiza) constituents using biopartitioning and microemulsion high-performance liquid chromatography (MELC). The quantitative retention-activity relationships (QRARs) of the constituents were established to model their pharmacokinetic (PK) parameters and chromatographic retention data, and generate their biological effectiveness fingerprints. A high-performance liquid chromatography (HPLC) method was established to determine the abundance of the extracted danshen constituents, such as sodium danshensu, rosmarinic acid, salvianolic acid B, protocatechuic aldehyde, cryptotanshinone, and tanshinone IIA. And another HPLC protocol was established to determine the abundance of those constituents in rat plasma samples. An experimental model was built in Sprague Dawley (SD) rats, and calculated the corresponding PK parameterst with 3P97 software package. Thirty-five model drugs were selected to test the PK parameter prediction capacities of the various MELC systems and to optimize the chromatographic protocols. QRARs and generated PK fingerprints were established. The test included water/oil-soluble danshen constituents and the prediction capacity of the regression model was validated. The results showed that the model had good predictability.





Determination of quinolones in wastewater by porous β-cyclodextrin polymer based solid-phase extraction coupled with HPLC

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069
Author(s): Jingyi Zhang, Donghao Liu, Yi Shi, Chen Sun, Muchuan Niu, Ruya Wang, Fan Hu, Deli Xiao, Hua He
In this research, a novel insoluble sorbent based on cyclodextrin and rigid aromatic groups tetrafluoroacetonitrile was designed for dispersive insoluble solid-phase extraction (DSPE). Due to its high adsorption capacity, this obtained polymer was applied to separation and concentration of trace quinolones in wastewater before HPLC determination. Various parameters influencing the extraction performance were studied and optimized. A DSPE approach coupled with high performance liquid chromatography was developed for the determination of four quinolones in wastewater samples. The limit of quantitation of fleroxacin, ciprofloxacin, gatifloxacin, norfloxacin were 2.67, 3.17, 4.75, 5.50ngmL−1, respectively. The recoveries of four quinolones range from 96.43 to 103.3% with relative standard deviations less than 4.5%. These results demonstrated that the proposed approach based on CDP was efficient, low-cost for extraction of quinolones from wastewater.

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069

Author(s): Jingyi Zhang, Donghao Liu, Yi Shi, Chen Sun, Muchuan Niu, Ruya Wang, Fan Hu, Deli Xiao, Hua He

In this research, a novel insoluble sorbent based on cyclodextrin and rigid aromatic groups tetrafluoroacetonitrile was designed for dispersive insoluble solid-phase extraction (DSPE). Due to its high adsorption capacity, this obtained polymer was applied to separation and concentration of trace quinolones in wastewater before HPLC determination. Various parameters influencing the extraction performance were studied and optimized. A DSPE approach coupled with high performance liquid chromatography was developed for the determination of four quinolones in wastewater samples. The limit of quantitation of fleroxacin, ciprofloxacin, gatifloxacin, norfloxacin were 2.67, 3.17, 4.75, 5.50ngmL−1, respectively. The recoveries of four quinolones range from 96.43 to 103.3% with relative standard deviations less than 4.5%. These results demonstrated that the proposed approach based on CDP was efficient, low-cost for extraction of quinolones from wastewater.





Quantification of xanthine- and uric acid-related compounds in urine using a “dilute-and-shoot” technique coupling ultra-high-performance liquid chromatography and high-resolution Orbitrap mass spectrometry

Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067
Author(s): Adrian Rodriguez, Rosa Maria Gomila, Gabriel Martorell, Antonia Costa-Bauza, Felix Grases
Quantitative analysis of relevant metabolites in biofluids such as urine is often a tedious procedure, since it usually requires extraction, purification or preconcentration. For instance, in the analysis of methylxanthines in urine, a solid-phase extraction is often required. In the current work, a rapid and highly sensitive “dilute-and-shoot” method combining ultra-high-performance liquid chromatography and high-resolution mass spectrometry (UHPLC/HRMS) was validated for urinary determination of twelve analytes: uric acid, hypoxanthine, xanthine, 1-methyluric acid, 1,3-dimethyluric acid, 1-methylxanthine, 3-methylxanthine, 7-methylxanthine, theophylline, theobromine, paraxanthine and caffeine. These analytes are the major physiological metabolites of caffeine, theobromine or theophylline, or final products of purine catabolism. The separation was carried out on a core-shell Kinetek EVO C18 column coupled to a Q Exactive Orbitrap high-resolution mass spectrometer equipped with a heated electrospray ionization (HESI) probe, that operated both in positive and negative ionization modes. The twelve analytes eluted from between 1.5 and 10.5min. The lower limit of quantification (LLOQ) values ranged from 0.25 to 2.5ng/mL, and the calibration curves were linear from the LLOQ to 100ng/mL. The only pretreatment needed was to dilute each urine sample (typically to 1/500) with 0.1% formic acid solution, and then filter the diluted sample before injecting it into the UHPLC system. With this high dilution, there were no significant matrix effects, and the intra- and inter-day precision and accuracy values were acceptable (coefficients of variance and relative errors below 15%, except for the LLOQ, for which they were below 20%). Furthermore, the analysis of spiked urine samples with 25ng/mL of the target analytes showed excellent recoveries and precision levels for the twelve analytes. To our knowledge, there is no other published method that allows for the simultaneous determination of the concentrations of these twelve compounds, nor has a previously reported method been indicated to show such low LLOQ values as we have for the majority of the analytes. We expect our protocol to be useful for nutritional assessments, interventional studies, kidney stone research, and purine metabolism studies.

Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067

Author(s): Adrian Rodriguez, Rosa Maria Gomila, Gabriel Martorell, Antonia Costa-Bauza, Felix Grases

Quantitative analysis of relevant metabolites in biofluids such as urine is often a tedious procedure, since it usually requires extraction, purification or preconcentration. For instance, in the analysis of methylxanthines in urine, a solid-phase extraction is often required. In the current work, a rapid and highly sensitive “dilute-and-shoot” method combining ultra-high-performance liquid chromatography and high-resolution mass spectrometry (UHPLC/HRMS) was validated for urinary determination of twelve analytes: uric acid, hypoxanthine, xanthine, 1-methyluric acid, 1,3-dimethyluric acid, 1-methylxanthine, 3-methylxanthine, 7-methylxanthine, theophylline, theobromine, paraxanthine and caffeine. These analytes are the major physiological metabolites of caffeine, theobromine or theophylline, or final products of purine catabolism. The separation was carried out on a core-shell Kinetek EVO C18 column coupled to a Q Exactive Orbitrap high-resolution mass spectrometer equipped with a heated electrospray ionization (HESI) probe, that operated both in positive and negative ionization modes. The twelve analytes eluted from between 1.5 and 10.5min. The lower limit of quantification (LLOQ) values ranged from 0.25 to 2.5ng/mL, and the calibration curves were linear from the LLOQ to 100ng/mL. The only pretreatment needed was to dilute each urine sample (typically to 1/500) with 0.1% formic acid solution, and then filter the diluted sample before injecting it into the UHPLC system. With this high dilution, there were no significant matrix effects, and the intra- and inter-day precision and accuracy values were acceptable (coefficients of variance and relative errors below 15%, except for the LLOQ, for which they were below 20%). Furthermore, the analysis of spiked urine samples with 25ng/mL of the target analytes showed excellent recoveries and precision levels for the twelve analytes. To our knowledge, there is no other published method that allows for the simultaneous determination of the concentrations of these twelve compounds, nor has a previously reported method been indicated to show such low LLOQ values as we have for the majority of the analytes. We expect our protocol to be useful for nutritional assessments, interventional studies, kidney stone research, and purine metabolism studies.





MOF-5(Zn)-Fe2O4 nanocomposite based magnetic solid-phase microextraction followed by HPLC-UV for efficient enrichment of colchicine in root of colchicium extracts and plasma samples

Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067
Author(s): Sonia Bahrani, Mehrorang Ghaedi, Kheibar Dashtian, Abbas Ostovan, Mohammad Javad Khoshnood Mansoorkhani, Amin Salehi
In present work, facile method is developed for determination of colchicine in human plasma sample, autumn and spring root of colchicium extracts by ultrasound assisted dispersive magnetic solid phase microextraction followed by HPLC-UV method (UAD-MSPME-HPLC-UV). Magnetic (Fe2O4-nanoparticles) metal organic framework-5, (MOF-5(Zn)-Fe2O4NPs) was synthesized by dispersing MOF-5 and Fe(NO3)3.9H2O in ethylene glycol (as capping agent) and NaOH (pH adjustment agent) by hydrothermal method. The prepared sorbent was characterized via XRD and SEM analysis and applied as magnetic solid phase in UAD-MSPME-HPLC-UV method. In this method, colchicine molecules were sorbed on MOF-5(Zn)-Fe2O4NPs sorbent by various mechanisms like ion exchange, hydrogen bonding and electrostatic, ᴨ-ᴨ, hard–hard and dipole-ion interaction followed by exposing sonication waves as incremental mass transfer agent and then the sorbent was separated from the sample matrix by an external magnetic fields. Subsequently, accumulated colchicine were eluted by small volume of desorption organic solvent. Influence of operational variables such as MOF-5(Zn)-Fe2O4NPs mass, volume of extracting solvent and sonication time on response property (recovery) were studied and optimized by central composite design (CCD) combined with desirability function (DF) approach. Under optimum condition, the method has wide linear calibration rang (0.5–1700ngmL−1) with reasonable detection limit (0.13ngmL−1) and R2=0.9971. Finally, the UAD-MSPME-HPLC-UV method was successfully applied for determination of colchicine autumn and spring root of colchicium extracts and plasma samples.

Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067

Author(s): Sonia Bahrani, Mehrorang Ghaedi, Kheibar Dashtian, Abbas Ostovan, Mohammad Javad Khoshnood Mansoorkhani, Amin Salehi

In present work, facile method is developed for determination of colchicine in human plasma sample, autumn and spring root of colchicium extracts by ultrasound assisted dispersive magnetic solid phase microextraction followed by HPLC-UV method (UAD-MSPME-HPLC-UV). Magnetic (Fe2O4-nanoparticles) metal organic framework-5, (MOF-5(Zn)-Fe2O4 NPs) was synthesized by dispersing MOF-5 and Fe(NO3)3.9H2O in ethylene glycol (as capping agent) and NaOH (pH adjustment agent) by hydrothermal method. The prepared sorbent was characterized via XRD and SEM analysis and applied as magnetic solid phase in UAD-MSPME-HPLC-UV method. In this method, colchicine molecules were sorbed on MOF-5(Zn)-Fe2O4 NPs sorbent by various mechanisms like ion exchange, hydrogen bonding and electrostatic, ᴨ-ᴨ, hard–hard and dipole-ion interaction followed by exposing sonication waves as incremental mass transfer agent and then the sorbent was separated from the sample matrix by an external magnetic fields. Subsequently, accumulated colchicine were eluted by small volume of desorption organic solvent. Influence of operational variables such as MOF-5(Zn)-Fe2O4 NPs mass, volume of extracting solvent and sonication time on response property (recovery) were studied and optimized by central composite design (CCD) combined with desirability function (DF) approach. Under optimum condition, the method has wide linear calibration rang (0.5–1700ngmL−1) with reasonable detection limit (0.13ngmL−1) and R2 =0.9971. Finally, the UAD-MSPME-HPLC-UV method was successfully applied for determination of colchicine autumn and spring root of colchicium extracts and plasma samples.





Optimization of a new method for extraction of cyanidin chloride and pelargonidin chloride anthocyanins with magnetic solid phase extraction and determination in fruit samples by HPLC with central composite design

Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067
Author(s): Abdollah Yari, Saba Rashnoo
Here, we are reporting a sensitive, simple and rapid method for the analysis of cyanidin chloride and pelargonidin chloride anthocyanins in cherry, sour cherry, pomegranate and barberry produced in Iran. The analytes were extracted with acetonitrile-hydrochloric acid (1% v/v) mixture under optimized pretreatment conditions. Clean-up of the extract from fruits was conducted by magnetic solid phase extraction using salicylic acid functionalized silica-coated magnetite nanoparticles (SCMNPs) as the adsorbent. The optimized conditions searched with central composite design. Working under optimum conditions specified as: SCMNPs modified with salicylic acid, sorbent contact time and sample 10min, mechanical stirring time 57.3min.HPLC with UV-detection was used for determination of the analytes. The limit of detection, LOD, obtained for the two anthocyanins were 0.02 and 0.03μgg−1, respectively. The ranges of the spiked recoveries were 80.0–97.6 and 72.9–97.2%, with the relative standard deviations (RSD) of 2.1 and 2.5%, respectively.

Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067

Author(s): Abdollah Yari, Saba Rashnoo

Here, we are reporting a sensitive, simple and rapid method for the analysis of cyanidin chloride and pelargonidin chloride anthocyanins in cherry, sour cherry, pomegranate and barberry produced in Iran. The analytes were extracted with acetonitrile-hydrochloric acid (1% v/v) mixture under optimized pretreatment conditions. Clean-up of the extract from fruits was conducted by magnetic solid phase extraction using salicylic acid functionalized silica-coated magnetite nanoparticles (SCMNPs) as the adsorbent. The optimized conditions searched with central composite design. Working under optimum conditions specified as: SCMNPs modified with salicylic acid, sorbent contact time and sample 10min, mechanical stirring time 57.3min. HPLC with UV-detection was used for determination of the analytes. The limit of detection, LOD, obtained for the two anthocyanins were 0.02 and 0.03μgg−1, respectively. The ranges of the spiked recoveries were 80.0–97.6 and 72.9–97.2%, with the relative standard deviations (RSD) of 2.1 and 2.5%, respectively.





Development of a high-performance liquid chromatography method for the determination of florfenicol in animal feedstuffs

Publication date: Available online 29 September 2017
Source:Journal of Chromatography B
Author(s): JinJing Yang, GuiZhi Sun, MingRong Qian, LingLi Huang, XianBing Ke, Bo Yang
An effective thin layer chromatography (TLC) purification procedure coupled to high-performance liquid chromatography (HPLC) method was developed for the determination of florfenicol (FF) in pig, chicken and fish feedstuffs. The feedstuff samples were extracted with ethyl acetate, defatted with n-hexane saturated with acetonitrile, and further purified by TLC. The chromatographic separation was performed on a Waters Symmetry C18 column using an isocratic procedure with acetonitrile-water (35:65, v/v) at 0.6mL/min. The ultraviolet (UV) detector was set at a wavelength of 225nm. The FF concentrations in feedstuff samples were quantified using a standard curve. Good linear correlations (y=159075x−15054, r>0.9999) were achieved within the concentration range of 0.05–200μg/mL. The recoveries of FF spiked at levels of 1, 100 and 1000μg/g ranged from 80.6% to 105.3% with the intra-day and inter-day relative standard deviation (RSD) less than 9.3%. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.02 and 0.06mg/kg for pig feedstuffs, 0.02 and 0.07mg/kg for chicken feedstuffs, and 0.02 and 0.05mg/kg for fish feedstuffs, respectively. This reliable, simple and cost-effective method could be applied to the routine monitoring of FF in animal feedstuffs.

Publication date: Available online 29 September 2017
Source:Journal of Chromatography B

Author(s): JinJing Yang, GuiZhi Sun, MingRong Qian, LingLi Huang, XianBing Ke, Bo Yang

An effective thin layer chromatography (TLC) purification procedure coupled to high-performance liquid chromatography (HPLC) method was developed for the determination of florfenicol (FF) in pig, chicken and fish feedstuffs. The feedstuff samples were extracted with ethyl acetate, defatted with n-hexane saturated with acetonitrile, and further purified by TLC. The chromatographic separation was performed on a Waters Symmetry C18 column using an isocratic procedure with acetonitrile-water (35:65, v/v) at 0.6mL/min. The ultraviolet (UV) detector was set at a wavelength of 225nm. The FF concentrations in feedstuff samples were quantified using a standard curve. Good linear correlations (y =159075 x 15054, r> 0.9999) were achieved within the concentration range of 0.05–200μg/mL. The recoveries of FF spiked at levels of 1, 100 and 1000μg/g ranged from 80.6% to 105.3% with the intra-day and inter-day relative standard deviation (RSD) less than 9.3%. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.02 and 0.06mg/kg for pig feedstuffs, 0.02 and 0.07mg/kg for chicken feedstuffs, and 0.02 and 0.05mg/kg for fish feedstuffs, respectively. This reliable, simple and cost-effective method could be applied to the routine monitoring of FF in animal feedstuffs.





Magnetic graphene dispersive solid phase extraction-ultra performance liquid chromatography tandem mass spectrometry for determination of β-agonists in urine

Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067
Author(s): Geng Nan Wang, Ning Peng Wu, Xin He, Hui Cai Zhang, Jing Liu, Jian Ping Wang
In this study, a magnetic graphene-based dispersive solid phase extraction method was first developed for extraction of β-agonists in urine. During the experiments, the absorbent amount, sample pH, extraction time, elution solution and elution time were optimized respectively. The optimized extraction method was finished within 10min, and showed high enrichment factors for 9 β-agonists (20–26 folds). Furthermore, this absorbent could be reused for at least 60 times. Then this extraction method was combined with ultra performance liquid chromatography triple quadrupole tandem mass spectrometry to determine the 9 drugs in urine. The limits of detection for the 9 drugs were in a range of 0.015–0.023ngmL−1, and the recoveries from the standards fortified blank urine were in a range of 60.2%–109.4%. Therefore, this method could be used as a simple, rapid, sensitive and accurate tool to determine trace level of β-agonists in urine.

Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067

Author(s): Geng Nan Wang, Ning Peng Wu, Xin He, Hui Cai Zhang, Jing Liu, Jian Ping Wang

In this study, a magnetic graphene-based dispersive solid phase extraction method was first developed for extraction of β-agonists in urine. During the experiments, the absorbent amount, sample pH, extraction time, elution solution and elution time were optimized respectively. The optimized extraction method was finished within 10min, and showed high enrichment factors for 9 β-agonists (20–26 folds). Furthermore, this absorbent could be reused for at least 60 times. Then this extraction method was combined with ultra performance liquid chromatography triple quadrupole tandem mass spectrometry to determine the 9 drugs in urine. The limits of detection for the 9 drugs were in a range of 0.015–0.023ngmL−1, and the recoveries from the standards fortified blank urine were in a range of 60.2%–109.4%. Therefore, this method could be used as a simple, rapid, sensitive and accurate tool to determine trace level of β-agonists in urine.





An improved method for fast and selective separation of carotenoids by LC–MS

Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067
Author(s): Daniel Abate-Pella, Dana M. Freund, Janet P. Slovin, Adrian D. Hegeman, Jerry D. Cohen
Carotenoids are a large class of compounds that are biosynthesized by condensation of isoprene units in plants, fungi, bacteria, and some animals. They are characteristically highly conjugated through double bonds, which lead to many isomers as well susceptibility to oxidation and other chemical modifications. Carotenoids are important because of their potent antioxidant activity and are the pigments responsible for color in a wide variety of foods. Human consumption is correlated to many health benefits including prevention of cancer, cardiovascular disease, and age-related disease. Extreme hydrophobicity, poor stability, and low concentration in biological samples make these compounds difficult to analyze and difficult to develop analytical methods for aimed towards identification and quantification. Examples in the literature frequently report the use of exotic stationary phases, solvents, and additives, such as ethyl acetate, dichloromethane, and methyl tert-butyl ether that are incompatible with liquid chromatography mass spectrometry (LC–MS). In order to address these issues, we implemented the use of LC–MS friendly conditions using a low-hydrophobicity cyano-propyl column (Agilent Zorbax SB-CN). We successfully differentiated between isomeric carotenoids by optimizing two gradient methods and using a mixture of 11 standards and LC–MS in positive ionization mode. Three complex biological samples from strawberry leaf, chicken feed supplement, and the photosynthetic bacterium Chloroflexus aurantiacus were analyzed and several carotenoids were resolved in these diverse backgrounds. Our results show this methodology is a significant improvement over other alternatives for analyzing carotenoids because of its ease of use, rapid analysis time, high selectivity, and, most importantly, its compatibility with typical LC–MS conditions.

Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067

Author(s): Daniel Abate-Pella, Dana M. Freund, Janet P. Slovin, Adrian D. Hegeman, Jerry D. Cohen

Carotenoids are a large class of compounds that are biosynthesized by condensation of isoprene units in plants, fungi, bacteria, and some animals. They are characteristically highly conjugated through double bonds, which lead to many isomers as well susceptibility to oxidation and other chemical modifications. Carotenoids are important because of their potent antioxidant activity and are the pigments responsible for color in a wide variety of foods. Human consumption is correlated to many health benefits including prevention of cancer, cardiovascular disease, and age-related disease. Extreme hydrophobicity, poor stability, and low concentration in biological samples make these compounds difficult to analyze and difficult to develop analytical methods for aimed towards identification and quantification. Examples in the literature frequently report the use of exotic stationary phases, solvents, and additives, such as ethyl acetate, dichloromethane, and methyl tert-butyl ether that are incompatible with liquid chromatography mass spectrometry (LC–MS). In order to address these issues, we implemented the use of LC–MS friendly conditions using a low-hydrophobicity cyano-propyl column (Agilent Zorbax SB-CN). We successfully differentiated between isomeric carotenoids by optimizing two gradient methods and using a mixture of 11 standards and LC–MS in positive ionization mode. Three complex biological samples from strawberry leaf, chicken feed supplement, and the photosynthetic bacterium Chloroflexus aurantiacus were analyzed and several carotenoids were resolved in these diverse backgrounds. Our results show this methodology is a significant improvement over other alternatives for analyzing carotenoids because of its ease of use, rapid analysis time, high selectivity, and, most importantly, its compatibility with typical LC–MS conditions.





Development and validation of a sensitive assay for analysis of midazolam, free and conjugated 1-hydroxymidazolam and 4-hydroxymidazolam in pediatric plasma: Application to Pediatric Pharmacokinetic Study

Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067
Author(s): Ganesh S. Moorthy, Harini Jogiraju, Christina Vedar, Athena F. Zuppa
Pharmacokinetic, pharmacodynamic and pharmacogenomic studies of midazolam are currently being performed in critically ill children to find suitable dose regimens. Sensitive assays using small volumes of plasma are necessary to determine the concentrations of midazolam and its respective metabolites in pediatric studies. Midazolam is metabolized to hydroxylated midazolam isomers, which are present as free as well as the corresponding glucuronide conjugates. A high-performance liquid chromatographic method with tandem mass spectrometry has been developed and validated for the quantification of midazolam, and free and total 1-hydroxymidazolam and 4-hydroxymidazolam metabolites in small volumes of plasma. Cleanup consisted of 96-well μ-elution solid phase extraction (SPE). The analytes were separated by gradient elution using a C18 analytical column with a total run time of 5min. Multiple reaction monitoring was employed using precursor to product ion transitions of m/z 326.2→291.3 for midazolam, m/z 342.1→203.0 for 1-hydroxymidazolam, m/z 342.1→325.1 for 4-hydroxymidazolam and m/z 330.2→295.3 for 2H4-midazolam (internal standard). Since authentic hydroxymidazolamglucuronide standards are not available, samples were hydrolyzed with β-glucuronidase under optimized conditions. Assay conditions were modified and optimized to provide appropriate recovery and stability because 4-hydroxymidazolam was very acid sensitive. Standard curves were linear from 0.5 to 1000ng/mL for all three analytes. Intra- and inter day accuracy and precision for quality control samples (2, 20, 200 and 800ng/mL) were within 85–115% and 15% (coefficient of variation), respectively. Stability in plasma and extracts were sufficient under assay conditions. Plasma samples were processed and analyzed for midazolam, and free 1-hydroxymidazolam and 4-hydroxymidazolam metabolites. Plasma samples that were hydrolyzed with β-glucuronidase were processed and analyzed for midazolam, and total 1-hydroxymidazolam and 4-hydroxymidazolam metabolites under the same assay conditions. The difference in concentration between the total and free hydroxymidazolam metabolites provided an estimate of conjugated hydroxymidazolam metabolites. The combination of 96-well μ-elution SPE and LC–MS/MS allows reliable quantification of midazolam and its metabolites in small volumes of plasma for pediatric patients. This assay is currently being successfully utilized for analysis of samples from ongoing clinical trials.

Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067

Author(s): Ganesh S. Moorthy, Harini Jogiraju, Christina Vedar, Athena F. Zuppa

Pharmacokinetic, pharmacodynamic and pharmacogenomic studies of midazolam are currently being performed in critically ill children to find suitable dose regimens. Sensitive assays using small volumes of plasma are necessary to determine the concentrations of midazolam and its respective metabolites in pediatric studies. Midazolam is metabolized to hydroxylated midazolam isomers, which are present as free as well as the corresponding glucuronide conjugates. A high-performance liquid chromatographic method with tandem mass spectrometry has been developed and validated for the quantification of midazolam, and free and total 1-hydroxymidazolam and 4-hydroxymidazolam metabolites in small volumes of plasma. Cleanup consisted of 96-well μ-elution solid phase extraction (SPE). The analytes were separated by gradient elution using a C18 analytical column with a total run time of 5min. Multiple reaction monitoring was employed using precursor to product ion transitions of m/z 326.2291.3 for midazolam, m/z 342.1203.0 for 1-hydroxymidazolam, m/z 342.1325.1 for 4-hydroxymidazolam and m/z 330.2295.3 for 2H4-midazolam (internal standard). Since authentic hydroxymidazolamglucuronide standards are not available, samples were hydrolyzed with β-glucuronidase under optimized conditions. Assay conditions were modified and optimized to provide appropriate recovery and stability because 4-hydroxymidazolam was very acid sensitive. Standard curves were linear from 0.5 to 1000ng/mL for all three analytes. Intra- and inter day accuracy and precision for quality control samples (2, 20, 200 and 800ng/mL) were within 85–115% and 15% (coefficient of variation), respectively. Stability in plasma and extracts were sufficient under assay conditions. Plasma samples were processed and analyzed for midazolam, and free 1-hydroxymidazolam and 4-hydroxymidazolam metabolites. Plasma samples that were hydrolyzed with β-glucuronidase were processed and analyzed for midazolam, and total 1-hydroxymidazolam and 4-hydroxymidazolam metabolites under the same assay conditions. The difference in concentration between the total and free hydroxymidazolam metabolites provided an estimate of conjugated hydroxymidazolam metabolites. The combination of 96-well μ-elution SPE and LC–MS/MS allows reliable quantification of midazolam and its metabolites in small volumes of plasma for pediatric patients. This assay is currently being successfully utilized for analysis of samples from ongoing clinical trials.





Efficient and relatively safe emulsification microextraction using a deep eutectic solvent for influential enrichment of trace main anti-depressant drugs from complicated samples

Publication date: Available online 28 September 2017
Source:Journal of Chromatography B
Author(s): Ahmad Ghoochani Moghadam, Maryam Rajabi, Alireza Asghari
In this research work, an efficient, facile, prompt, and relatively safe enrichment procedure, named as air agitated-emulsification microextraction based on a low density-deep eutectic solvent (AA-EME-LD-DES), was applied for the first time to separate trace amounts of the drugs escitalopram, desipramine, and imipramine from complex sample solutions. This bio-degradable and cheap choline chloride-based extracting agent was readily prepared by the easy blending process at the ambient temperature, resulting in a eutectic liquid mixture with distinct features. Also the subsequent usage of an effective proceeding of the current microextraction procedure without a vital requirement for a further purification was adopted as another impressive benefit. Investigation of the main parameters influencing the multivariate technique based on the central composite design (CCD) combined with the desirability function (DF) revealed that pH12.0, 200μL of the extraction solvent, 430μL of the emulsifier solvent, and 14 air agitation cycles led to maximum extraction efficiencies with the DF value close to 0.97. Under the optimal experimental conditions, the wide linear dynamic ranges (LDRs) of 10.0–5000, 15.0–8000, and 15.0–6000ngmL−1 for escitalopram, desipramine, and imipramine were accurately obtainable, respectively, with the determination coefficients (R2s) higher than 0.98 and the low limits of detection (LODs) of 3.0–4.5ngmL−1. The percent extraction recoveries and enrichment factors were found to be adequately quantitative in the spans of 42–68% and 25–40, respectively, possessing good relative standard deviations (%RSDs, n=3) in the range of 3.6–5.7%. Finally, accurate analyses at therapeutically low ranges for the human plasma sample and trace levels for the pharmaceutical wastewater sample were successfully obtained, certifying the appropriate pre-concentration and enrichment capabilities of the proposed microextraction approach.

Graphical abstract

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Publication date: Available online 28 September 2017
Source:Journal of Chromatography B

Author(s): Ahmad Ghoochani Moghadam, Maryam Rajabi, Alireza Asghari

In this research work, an efficient, facile, prompt, and relatively safe enrichment procedure, named as air agitated-emulsification microextraction based on a low density-deep eutectic solvent (AA-EME-LD-DES), was applied for the first time to separate trace amounts of the drugs escitalopram, desipramine, and imipramine from complex sample solutions. This bio-degradable and cheap choline chloride-based extracting agent was readily prepared by the easy blending process at the ambient temperature, resulting in a eutectic liquid mixture with distinct features. Also the subsequent usage of an effective proceeding of the current microextraction procedure without a vital requirement for a further purification was adopted as another impressive benefit. Investigation of the main parameters influencing the multivariate technique based on the central composite design (CCD) combined with the desirability function (DF) revealed that pH12.0, 200μL of the extraction solvent, 430μL of the emulsifier solvent, and 14 air agitation cycles led to maximum extraction efficiencies with the DF value close to 0.97. Under the optimal experimental conditions, the wide linear dynamic ranges (LDRs) of 10.0–5000, 15.0–8000, and 15.0–6000ngmL−1 for escitalopram, desipramine, and imipramine were accurately obtainable, respectively, with the determination coefficients (R2s) higher than 0.98 and the low limits of detection (LODs) of 3.0–4.5ngmL−1. The percent extraction recoveries and enrichment factors were found to be adequately quantitative in the spans of 42–68% and 25–40, respectively, possessing good relative standard deviations (%RSDs, n=3) in the range of 3.6–5.7%. Finally, accurate analyses at therapeutically low ranges for the human plasma sample and trace levels for the pharmaceutical wastewater sample were successfully obtained, certifying the appropriate pre-concentration and enrichment capabilities of the proposed microextraction approach.

Graphical abstract

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Pipette tip dummy molecularly imprinted solid-phase extraction of Bisphenol A from urine samples and analysis by gas chromatography coupled to mass spectrometry

Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067
Author(s): Tamires Amabile Valim Brigante, Luis Felippe Cabral Miranda, Israel Donizeti de Souza, Vinicius Ricardo Acquaro Junior, Maria Eugênia Costa Queiroz
Molecularly imprinted polymers (MIPs) were synthesized and used as sorbent for Bisphenol A (BPA) pipette tip solid-phase microextraction from urine samples and BPA analysis by gas chromatography coupled to mass spectrometry (GC–MS). The MIPs were synthesized by the sol–gel methodology. Aminopropyltriethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) were used as functional monomer and cross-linking reagent, respectively. BPA and tetrabromobisphenol A (TBBPA) were evaluated as template during MIP synthesis. The BPA-based MIP displayed slightly higher extraction efficiency than the TBBPA-based dummy molecularly imprinted polymer (DMIP), but the TBBPA-based DMIP was selected as sorbent to minimize interference from leaked template. Comparison of the TBBPA-based DMIP, BPA-based MIP, and non-imprinted polymer (NIP) extraction efficiencies attested that the TBBPA-based DMIP was selective. The synthesized polymers were characterized by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared spectroscopy (FTIR). The TBBPA-based DMIP was reused for over 100 times, which confirmed its robustness. The developed method was linear from 50 to 500ngmL−1. Precision values had coefficient of variation (CV) ranging from 4 to 14%. The accuracy relative standard deviation values (RSD) varied from −13.6 to 12.3%.

Publication date: 1 November 2017
Source:Journal of Chromatography B, Volume 1067

Author(s): Tamires Amabile Valim Brigante, Luis Felippe Cabral Miranda, Israel Donizeti de Souza, Vinicius Ricardo Acquaro Junior, Maria Eugênia Costa Queiroz

Molecularly imprinted polymers (MIPs) were synthesized and used as sorbent for Bisphenol A (BPA) pipette tip solid-phase microextraction from urine samples and BPA analysis by gas chromatography coupled to mass spectrometry (GC–MS). The MIPs were synthesized by the sol–gel methodology. Aminopropyltriethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) were used as functional monomer and cross-linking reagent, respectively. BPA and tetrabromobisphenol A (TBBPA) were evaluated as template during MIP synthesis. The BPA-based MIP displayed slightly higher extraction efficiency than the TBBPA-based dummy molecularly imprinted polymer (DMIP), but the TBBPA-based DMIP was selected as sorbent to minimize interference from leaked template. Comparison of the TBBPA-based DMIP, BPA-based MIP, and non-imprinted polymer (NIP) extraction efficiencies attested that the TBBPA-based DMIP was selective. The synthesized polymers were characterized by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared spectroscopy (FTIR). The TBBPA-based DMIP was reused for over 100 times, which confirmed its robustness. The developed method was linear from 50 to 500ngmL−1. Precision values had coefficient of variation (CV) ranging from 4 to 14%. The accuracy relative standard deviation values (RSD) varied from −13.6 to 12.3%.