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Selected papers from the latest issue:
Design of a microfluidic platform for monoclonal antibody extraction using an aqueous two-phase system
09 July 2012,
08:33:15
Publication year:
2012
Source:Journal of Chromatography A, Volume 1249
D.F.C. Silva, A.M. Azevedo, P. Fernandes, V. Chu, J.P. Conde, M.R. Aires-Barros
The use of monoclonal antibodies (mAbs) in medical treatments and in laboratory techniques has a very important impact in the battle against many diseases, namely in the treatment of cancer, autoimmune diseases and neural disorders. Thus these biopharmaceuticals have become increasingly important, reinforcing the demand for efficient, scalable and cost-effective techniques for providing pure antibodies. Aqueous two-phase systems (ATPS) have shown potential for downstream processing of mAbs. In this work, an ATPS in a microfluidic platform was designed and tested for mAbs extraction. The system demonstrated the potential to be an effective tool to accelerate bioprocess design and optimization. The partition of immunoglobulin G (IgG) tagged with fluorescein isothiocyanate (FITC) in an ATPS of polyethylene-glycol (PEG)/phosphate buffer with NaCl was investigated using a PDMS microfluidic device fabricated using soft lithography techniques. Different structures were tested with different values of microchannel length (3.14–16.8cm) and flow rates of the salt (1–2μL/min) and PEG-rich phases (0.2–0.5μL/min). A stable interphase between the phases was obtained and the phenomena of diffusion and of partition of the IgG from the salt-rich phase to the PEG-rich phase were measured by fluorescence microscopy. Process simulation allowed the modeling of the IgG diffusion and partitioning behavior observed in the microstructure. The reduction to the microscale does not greatly affect the antibody extraction yield when compared with macroscale results, but it does reduce the operation time, demonstrating the potentiality of this approach to process optimization.
Source:Journal of Chromatography A, Volume 1249
D.F.C. Silva, A.M. Azevedo, P. Fernandes, V. Chu, J.P. Conde, M.R. Aires-Barros
The use of monoclonal antibodies (mAbs) in medical treatments and in laboratory techniques has a very important impact in the battle against many diseases, namely in the treatment of cancer, autoimmune diseases and neural disorders. Thus these biopharmaceuticals have become increasingly important, reinforcing the demand for efficient, scalable and cost-effective techniques for providing pure antibodies. Aqueous two-phase systems (ATPS) have shown potential for downstream processing of mAbs. In this work, an ATPS in a microfluidic platform was designed and tested for mAbs extraction. The system demonstrated the potential to be an effective tool to accelerate bioprocess design and optimization. The partition of immunoglobulin G (IgG) tagged with fluorescein isothiocyanate (FITC) in an ATPS of polyethylene-glycol (PEG)/phosphate buffer with NaCl was investigated using a PDMS microfluidic device fabricated using soft lithography techniques. Different structures were tested with different values of microchannel length (3.14–16.8cm) and flow rates of the salt (1–2μL/min) and PEG-rich phases (0.2–0.5μL/min). A stable interphase between the phases was obtained and the phenomena of diffusion and of partition of the IgG from the salt-rich phase to the PEG-rich phase were measured by fluorescence microscopy. Process simulation allowed the modeling of the IgG diffusion and partitioning behavior observed in the microstructure. The reduction to the microscale does not greatly affect the antibody extraction yield when compared with macroscale results, but it does reduce the operation time, demonstrating the potentiality of this approach to process optimization.
Highlights
► We developed an ATPS microfluidic device for mAbs extraction. ► PDMS microfluidic device fabricated using soft lithography techniques with three inlets/three outlets design was used. ► A stable interphase between the phases was obtained. ► The diffusion of IgG into PEG-rich phase was complete for a length of 16.8cm. ► The partition behavior of IgG at the macroscale can be translated into the microscale.Experimental design applied to the optimization of microwave-assisted DNA hydrolysis
09 July 2012,
08:33:15
Publication year:
2012
Source:Journal of Chromatography A, Volume 1249
Giorgio Marrubini, Paolo Fattorini, Carlo Previderé, Silvia Goi, Solange Sorçaburu Cigliero, Pierangela Grignani, Massimo Serra, Raffaela Biesuz, Gabriella Massolini
The assessment of the integrity of the DNA primary structure and the identification of canonical and modified bases are useful tools in medical, pharmaceutical, and forensic applications. In this article we report on the first microwave-assisted hydrolyses of deoxyribonucleoside-triphosphates (dNTPs) and human DNA using “Design of Experiments” methodology. We use hydrophilic interaction chromatography (HILIC) and UV detection at 260nm for the determination of purinic and pyrimidinic bases at levels of 0.5μM. We use a ZIC-HILIC® 150mm×2.1mm i.d., 5μm particle size column and ammonium formate buffers in acetonitrile for gradient separation of the analytes. We then compare the final concentrations of Thymine, Adenine, Cytosine, and Guanine with the nominal amounts of such bases in the dNTPs and DNA submitted to hydrolysis. After optimization of the hydrolysis (11.5min, 0.15M aqueous HCl, 150°C), the method turns out to be suitable for the determination of products released from quantities of human DNA as low as 500ng with precision (RSD<10%) and accuracy (REC 97–104%). These results confirm that the kinetics of the release of the bases depends on their molecular structure and show that the concentration of the substrate plays a relevant role. We conclude with a discussion of the method and a comparison to the methods described in previous studies.
Source:Journal of Chromatography A, Volume 1249
Giorgio Marrubini, Paolo Fattorini, Carlo Previderé, Silvia Goi, Solange Sorçaburu Cigliero, Pierangela Grignani, Massimo Serra, Raffaela Biesuz, Gabriella Massolini
The assessment of the integrity of the DNA primary structure and the identification of canonical and modified bases are useful tools in medical, pharmaceutical, and forensic applications. In this article we report on the first microwave-assisted hydrolyses of deoxyribonucleoside-triphosphates (dNTPs) and human DNA using “Design of Experiments” methodology. We use hydrophilic interaction chromatography (HILIC) and UV detection at 260nm for the determination of purinic and pyrimidinic bases at levels of 0.5μM. We use a ZIC-HILIC® 150mm×2.1mm i.d., 5μm particle size column and ammonium formate buffers in acetonitrile for gradient separation of the analytes. We then compare the final concentrations of Thymine, Adenine, Cytosine, and Guanine with the nominal amounts of such bases in the dNTPs and DNA submitted to hydrolysis. After optimization of the hydrolysis (11.5min, 0.15M aqueous HCl, 150°C), the method turns out to be suitable for the determination of products released from quantities of human DNA as low as 500ng with precision (RSD<10%) and accuracy (REC 97–104%). These results confirm that the kinetics of the release of the bases depends on their molecular structure and show that the concentration of the substrate plays a relevant role. We conclude with a discussion of the method and a comparison to the methods described in previous studies.
Highlights
► New protocol for DNA microwave-assisted hydrolysis. ► Optimization of the hydrolysis studied using Design of Experiments. ► Hydrolysis products separated by HPLC in HILIC mode and UV detection. ► Method tested on human DNA. ► Sensitivity, precision, and accuracy of the hydrolysis of DNA documented for the first time.On-line large-volume electroextraction coupled to liquid chromatography–mass spectrometry to improve detection of peptides
09 July 2012,
08:33:15
Publication year:
2012
Source:Journal of Chromatography A, Volume 1249
Petrus W. Lindenburg, F.W. Alexander Tempels, Ubbo R. Tjaden, Jan van der Greef, Thomas Hankemeier
Electroextraction (EE) takes place in a two-phase liquid–liquid system, consisting of an aqueous and an organic phase, where an applied electric field causes ions to be extracted from one phase into the other, to be concentrated close after the liquid–liquid interface. The extraction takes place in a wide-bore capillary that is connected to a 2-way 10-port switching valve, which serves to couple capillary EE (cEE) with LC–MS. In this set-up, volumes as high as 100μL can be extracted, which is a ten times larger volume than has been reported, earlier. After a feasibility study using the cationic purple dye crystal violet, the method was coupled to LC–MS and large volume cEE of several model peptides was optimized. The cEE-LC–MS method had good repeatability, good linearity and LODs between 0.5 and 10nM. The whole procedure was automated and could be used routinely. Finally, the method was applied to plasma analysis and calibration curves of the relevant plasma peptides angiotensin 1 and 2 as well as the fragment angiotensin 2 (3–8) showed good linearity and repeatability; LOD values were 10–50nM. Analysis of unspiked plasma resulted in 60 putative endogenous peptides, underlining the great potential of EE as on-line sample concentrating technique. On-line large volume cEE-LC–MS allows for enrichment, separation and detection of plasma peptides from large sample volumes, minimizes sample handling and can be an important step in full automation of analytical procedures.
Source:Journal of Chromatography A, Volume 1249
Petrus W. Lindenburg, F.W. Alexander Tempels, Ubbo R. Tjaden, Jan van der Greef, Thomas Hankemeier
Electroextraction (EE) takes place in a two-phase liquid–liquid system, consisting of an aqueous and an organic phase, where an applied electric field causes ions to be extracted from one phase into the other, to be concentrated close after the liquid–liquid interface. The extraction takes place in a wide-bore capillary that is connected to a 2-way 10-port switching valve, which serves to couple capillary EE (cEE) with LC–MS. In this set-up, volumes as high as 100μL can be extracted, which is a ten times larger volume than has been reported, earlier. After a feasibility study using the cationic purple dye crystal violet, the method was coupled to LC–MS and large volume cEE of several model peptides was optimized. The cEE-LC–MS method had good repeatability, good linearity and LODs between 0.5 and 10nM. The whole procedure was automated and could be used routinely. Finally, the method was applied to plasma analysis and calibration curves of the relevant plasma peptides angiotensin 1 and 2 as well as the fragment angiotensin 2 (3–8) showed good linearity and repeatability; LOD values were 10–50nM. Analysis of unspiked plasma resulted in 60 putative endogenous peptides, underlining the great potential of EE as on-line sample concentrating technique. On-line large volume cEE-LC–MS allows for enrichment, separation and detection of plasma peptides from large sample volumes, minimizes sample handling and can be an important step in full automation of analytical procedures.
Highlights
► Large volume cEE is an electromigration-based online sample concentration technique. ► Large volume cEE takes place in a two-phase liquid–liquid system. ► Large volume cEE is capable of fast online peptide concentration (2–3 orders of magnitude). ► Large volume cEE can be coupled online to LC–MS. ► Large volume cEE is applicable to bioanalysis.Determination of ultraviolet filters in water samples by vortex-assisted dispersive liquid–liquid microextraction followed by gas chromatography–mass spectrometry
09 July 2012,
08:33:15
Publication year:
2012
Source:Journal of Chromatography A, Volume 1249
Yufeng Zhang, Hian Kee Lee
For the first time, a simple solvent microextraction method termed vortex-assisted liquid–liquid microextraction (VADLLME) coupled with gas chromatography–mass spectrometry (GC–MS) has been developed and used for the analysis of six benzophenone ultraviolet (UV) filters (i.e. benzhydrol, 2,4-dihydroxybenzophenone, benzophenone, 2-hydroxy-4-methoxybenzophenone, ethylhexyl salicylate and homosalate) in water samples. The most favorable extraction variables in the VADLLME process were determined. In the extraction procedure, 40μL of tetrachloroethene as extraction solvent were directly injected into a 15-mL centrifuge tube containing 10mL of aqueous sample, adjusted to pH 4 for VADLLME. After VADLLME, the extract was evaporated under a gentle nitrogen gas stream and then reconstituted with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA), thus allowing the target analytes to be converted into their trimethylsilyl derivatives to optimize the GC–MS analysis. No centrifugation and disperser solvent were required in this microextraction procedure. Significantly, short extraction time and high extraction efficiency were achieved. This method opens up a potentially new horizon for on-site dispersive liquid–liquid microextraction. Under the optimum conditions, the proposed method provided good enrichment factors up to 310, with relative standard deviations ranging from 6.1 to 12.9%. The limits of quantification were in the range of 20–100ng/L, depending on the analytes. The linearities were between 0.05 and 10μg/L and 0.1 and 10μg/L for different UV filters. Finally, the proposed method was successfully applied to the determination of UV filters from spiked genuine water samples and acceptable recoveries over the range of 71.0–120.0% were obtained.
Source:Journal of Chromatography A, Volume 1249
Yufeng Zhang, Hian Kee Lee
For the first time, a simple solvent microextraction method termed vortex-assisted liquid–liquid microextraction (VADLLME) coupled with gas chromatography–mass spectrometry (GC–MS) has been developed and used for the analysis of six benzophenone ultraviolet (UV) filters (i.e. benzhydrol, 2,4-dihydroxybenzophenone, benzophenone, 2-hydroxy-4-methoxybenzophenone, ethylhexyl salicylate and homosalate) in water samples. The most favorable extraction variables in the VADLLME process were determined. In the extraction procedure, 40μL of tetrachloroethene as extraction solvent were directly injected into a 15-mL centrifuge tube containing 10mL of aqueous sample, adjusted to pH 4 for VADLLME. After VADLLME, the extract was evaporated under a gentle nitrogen gas stream and then reconstituted with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA), thus allowing the target analytes to be converted into their trimethylsilyl derivatives to optimize the GC–MS analysis. No centrifugation and disperser solvent were required in this microextraction procedure. Significantly, short extraction time and high extraction efficiency were achieved. This method opens up a potentially new horizon for on-site dispersive liquid–liquid microextraction. Under the optimum conditions, the proposed method provided good enrichment factors up to 310, with relative standard deviations ranging from 6.1 to 12.9%. The limits of quantification were in the range of 20–100ng/L, depending on the analytes. The linearities were between 0.05 and 10μg/L and 0.1 and 10μg/L for different UV filters. Finally, the proposed method was successfully applied to the determination of UV filters from spiked genuine water samples and acceptable recoveries over the range of 71.0–120.0% were obtained.
Highlights
► VADLLME-GC–MS was for the first time applied to the determination of UV filters. ► High extraction efficiency with good enrichment factors up to 310 were achieved. ► The new method did not need centrifugation or disperser solvent. ► Fast extraction time relative to other methods was another advantage. ► Simple and efficient method with low LODs, good repeatability and linearity.Generic sample treatment method for simultaneous determination of multiclass pesticides and mycotoxins in wines by liquid chromatography–mass spectrometry
09 July 2012,
08:33:15
Publication year:
2012
Source:Journal of Chromatography A, Volume 1249
Patricia Pérez-Ortega, Bienvenida Gilbert-López, Juan F. García-Reyes, Natividad Ramos-Martos, Antonio Molina-Díaz
In this work, a generic sample treatment method for simultaneous determination of multiclass pesticides and mycotoxins in wines is presented. The proposed method is based on solid-phase extraction (SPE) using polymeric-type SPE cartridges. To evaluate the proposed sample treatment, a liquid chromatography electrospray time-of-flight mass spectrometry method was used for testing 60 selected representative multiclass pesticides and 9 mycotoxins. Two different polymeric sorbents were evaluated, with hydrophilic–lipophilic-balanced (HLB) polymer cartridges being selected (Oasis™ HLB) as the most suitable for the present study. The identification and confirmation of the compounds was based on retention time and accurate mass measurements of the protonated molecules ([M+H]+). Limits of detection were below 1μgL−1 for the 87% of the studied compounds. With the selected 4:1 preconcentration factor, 70% of the target compounds showed relatively low matrix effects, corresponding to signal suppressions lower than 30%. Recovery studies (n =10) were carried out at two concentration levels, 2.5μgL−1 and 25μgL−1, obtaining mean recovery rates between 70 and 120% for the 90% of studied analytes. The relative standard deviation (RSD%) values of the entire procedure were below 15% in most cases (97% of the studied analytes). The proposed method was successfully applied to 24 red wine samples produced in different regions of Spain. The concentration levels of the target compounds found in the studied samples were in compliance with the current regulations. Aflatoxin B2 and metalaxyl were the most detected compounds (75% and 50% of the studied samples, respectively).
Source:Journal of Chromatography A, Volume 1249
Patricia Pérez-Ortega, Bienvenida Gilbert-López, Juan F. García-Reyes, Natividad Ramos-Martos, Antonio Molina-Díaz
In this work, a generic sample treatment method for simultaneous determination of multiclass pesticides and mycotoxins in wines is presented. The proposed method is based on solid-phase extraction (SPE) using polymeric-type SPE cartridges. To evaluate the proposed sample treatment, a liquid chromatography electrospray time-of-flight mass spectrometry method was used for testing 60 selected representative multiclass pesticides and 9 mycotoxins. Two different polymeric sorbents were evaluated, with hydrophilic–lipophilic-balanced (HLB) polymer cartridges being selected (Oasis™ HLB) as the most suitable for the present study. The identification and confirmation of the compounds was based on retention time and accurate mass measurements of the protonated molecules ([M+H]+). Limits of detection were below 1μgL−1 for the 87% of the studied compounds. With the selected 4:1 preconcentration factor, 70% of the target compounds showed relatively low matrix effects, corresponding to signal suppressions lower than 30%. Recovery studies (n =10) were carried out at two concentration levels, 2.5μgL−1 and 25μgL−1, obtaining mean recovery rates between 70 and 120% for the 90% of studied analytes. The relative standard deviation (RSD%) values of the entire procedure were below 15% in most cases (97% of the studied analytes). The proposed method was successfully applied to 24 red wine samples produced in different regions of Spain. The concentration levels of the target compounds found in the studied samples were in compliance with the current regulations. Aflatoxin B2 and metalaxyl were the most detected compounds (75% and 50% of the studied samples, respectively).
Highlights
► Generic sample treatment for simultaneous determination of pesticides and mycotoxins in wines. ► An LC–TOFMS method was used for testing 69 selected representative multiclass contaminants. ► Two different polymeric sorbents were evaluated. ► The method was successfully applied to 24 Spanish red wine samples. ► Aflatoxin B2 and metalaxyl were the most often detected compounds.Negative electrospray ionization ion mobility spectrometry combined with microextraction in packed syringe for direct analysis of phenoxyacid herbicides in environmental waters
09 July 2012,
08:33:15
Publication year:
2012
Source:Journal of Chromatography A, Volume 1249
Mohammad T. Jafari, Mohammad Saraji, Shila Yousefi
A novel method based on microextraction in packed syringe (MEPS) coupled with negative electrospray ionization-ion mobility spectrometry (ESI-IMS) was developed for simultaneous and direct determination of phenoxyacid herbicides in water samples. The effects of various parameters such as pH of the sample solution, sample loading rate, the nature of elution solvent and its volume, and the number of extraction cycles were investigated. The method was exhaustively validated in terms of sensitivity, recovery, reproducibility, and enrichment factor. The detection limits of the method for 2,4-D, silvex, and haloxyfop were found to be 60, 70, and 90ngL−1, respectively. The recovery of the analyzed herbicides was in the range 73–102%, and the relative standard deviation (RSD) was lower than 10% for all the experiments. Various real samples were analyzed with the proposed method, and the obtained results revealed the efficient extraction of the analytes using MEPS extraction before the analysis by negative ESI-IMS.
Source:Journal of Chromatography A, Volume 1249
Mohammad T. Jafari, Mohammad Saraji, Shila Yousefi
A novel method based on microextraction in packed syringe (MEPS) coupled with negative electrospray ionization-ion mobility spectrometry (ESI-IMS) was developed for simultaneous and direct determination of phenoxyacid herbicides in water samples. The effects of various parameters such as pH of the sample solution, sample loading rate, the nature of elution solvent and its volume, and the number of extraction cycles were investigated. The method was exhaustively validated in terms of sensitivity, recovery, reproducibility, and enrichment factor. The detection limits of the method for 2,4-D, silvex, and haloxyfop were found to be 60, 70, and 90ngL−1, respectively. The recovery of the analyzed herbicides was in the range 73–102%, and the relative standard deviation (RSD) was lower than 10% for all the experiments. Various real samples were analyzed with the proposed method, and the obtained results revealed the efficient extraction of the analytes using MEPS extraction before the analysis by negative ESI-IMS.
Highlights
► Microextraction in packed syringe was coupled with negative ESI-IMS for the first time. ► Very fast screening of chemical compounds by the developed method is expected. ► Direct and simultaneous analysis of 2,4-D, silvex, and haloxyfop in water samples was accomplished.Hollow-fiber liquid phase microextraction for lignin pyrolysis acids in aerosol samples and gas chromatography–mass spectrometry analysis
09 July 2012,
08:33:15
Publication year:
2012
Source:Journal of Chromatography A, Volume 1249
Murtaza Hyder, Jan Åke Jönsson
A method based on three-phase hollow fiber liquid phase microextraction was developed and successfully applied to aerosols for the analysis of lignin pyrolysis acids such as syringic acid, vanillic acid and p-salicylic acid. Important parameters related to extraction process like organic solvent for membrane phase, tri-n-octylphosphine (TOPO) oxide contents in organic solvent, stirring speed, extraction time etc. were optimized. 6-Undecanone with 15% TOPO contents (w/v) was found a suitable solvent for organic liquid membrane, 900rpm was the optimum stirring speed and time of 4h was found optimum extraction time. Donor phase pH was 1.3 while acceptor phase pH was adjusted to 9.5. The optimized extraction method was used for the extraction of real aerosol samples. Analytes were derivatized using BSTFA containing 1% trimethylsilyl chloride and gas chromatography mass spectrometry was used for analysis. Very low limits of detection in the range 0.2–1.0ngL−1 were found, corresponding to 10–50pgm−3 of analytes in aerosols. Extraction efficiency obtained ranged 60.3–71.7% and enrichment factors ranged 3015–3585 times. The optimized method was successfully applied to aerosol samples and all of the selected analytes were detected in the analyzed samples.
Source:Journal of Chromatography A, Volume 1249
Murtaza Hyder, Jan Åke Jönsson
A method based on three-phase hollow fiber liquid phase microextraction was developed and successfully applied to aerosols for the analysis of lignin pyrolysis acids such as syringic acid, vanillic acid and p-salicylic acid. Important parameters related to extraction process like organic solvent for membrane phase, tri-n-octylphosphine (TOPO) oxide contents in organic solvent, stirring speed, extraction time etc. were optimized. 6-Undecanone with 15% TOPO contents (w/v) was found a suitable solvent for organic liquid membrane, 900rpm was the optimum stirring speed and time of 4h was found optimum extraction time. Donor phase pH was 1.3 while acceptor phase pH was adjusted to 9.5. The optimized extraction method was used for the extraction of real aerosol samples. Analytes were derivatized using BSTFA containing 1% trimethylsilyl chloride and gas chromatography mass spectrometry was used for analysis. Very low limits of detection in the range 0.2–1.0ngL−1 were found, corresponding to 10–50pgm−3 of analytes in aerosols. Extraction efficiency obtained ranged 60.3–71.7% and enrichment factors ranged 3015–3585 times. The optimized method was successfully applied to aerosol samples and all of the selected analytes were detected in the analyzed samples.
Highlights
► Lignin pyrolysis acids are contaminants that exist at trace levels in organic aerosol. ► Using hollow liquid phase microextraction (HF-LPME) enables very high enrichment of analytes and good clean up of sample. ► HF-LPME can also be applied to aerosol samples. ► LOD are down to pgm−3 and high enrichment factors obtained when HF-LPME is coupled with GC–MS analysis.Simultaneous determination of cork taint and Brett character responsible compounds in wine using ultrasound-assisted emulsification microextraction with solidification of floating organic drop
09 July 2012,
08:33:15
Publication year:
2012
Source:Journal of Chromatography A, Volume 1249
C. Pizarro, C. Sáenz-González, N. Pérez-del-Notario, J.M. González-Sáiz
In this study, ultrasound-assisted emulsification microextraction combined with solidification of floating organic drop method (USAEME–SFOD) has been proposed as a novel approach for the sensitive determination of haloanisoles and volatile phenols in wines. For this purpose, the influence of the different parameters affecting the procedure (type and volume of extraction solvent, temperature, time and ionic strength) was evaluated in order to optimise the efficiency of the process. Subsequently, the linearity, detection and quantification limits, precision, recoveries and applicability to real samples were studied, obtaining excellent method performance results. Moreover, USAEME–SFOD was compared with other liquid–liquid microextraction methods such as dispersive liquid–liquid microextraction (DLLME) and ultrasound-assisted emulsification microextraction (USAEME). This comparison study proved the suitability of USAEME–SFOD as an alternative to previously reported methods for the simultaneous determination of cork taint and Brett character responsible compounds in wines.
Source:Journal of Chromatography A, Volume 1249
C. Pizarro, C. Sáenz-González, N. Pérez-del-Notario, J.M. González-Sáiz
In this study, ultrasound-assisted emulsification microextraction combined with solidification of floating organic drop method (USAEME–SFOD) has been proposed as a novel approach for the sensitive determination of haloanisoles and volatile phenols in wines. For this purpose, the influence of the different parameters affecting the procedure (type and volume of extraction solvent, temperature, time and ionic strength) was evaluated in order to optimise the efficiency of the process. Subsequently, the linearity, detection and quantification limits, precision, recoveries and applicability to real samples were studied, obtaining excellent method performance results. Moreover, USAEME–SFOD was compared with other liquid–liquid microextraction methods such as dispersive liquid–liquid microextraction (DLLME) and ultrasound-assisted emulsification microextraction (USAEME). This comparison study proved the suitability of USAEME–SFOD as an alternative to previously reported methods for the simultaneous determination of cork taint and Brett character responsible compounds in wines.
Highlights
► Haloanisoles and volatile phenosl have been analysed in wine using USAEME–SFOD. ► Optimal conditions of the process were found using experimental design methodology. ► In line with green chemistry, little volume of a low toxic solvent has been used. ► The proposed method achieves suitable detection limits, recoveries and precision. ► USAEME–SFOD method achieve LODs lower than previously reported methods.Identifying orthogonal and similar reversed phase liquid chromatography stationary phases using the system selectivity cube and the hydrophobic subtraction model
09 July 2012,
08:33:15
Publication year:
2012
Source:Journal of Chromatography A, Volume 1249
Andrew R. Johnson, Carrie M. Johnson, Dwight R. Stoll, Mark F. Vitha
We have compared over 500 RPLC columns characterized by the hydrophobic subtraction model using the system selectivity cube (SSC). We have shown numerous differences in column selectivity even among columns in the same class (e.g., alkyl-silica, cyano, or embedded polar groups). We also illustrate the utility of our method for selecting alternative columns with different selectivities for problematic separations and for selecting orthogonal columns for use in two-dimensional separations. The system selectivity cube offers a visual way to easily compare many columns simultaneously and select those columns offering the desired selectivity.
Source:Journal of Chromatography A, Volume 1249
Andrew R. Johnson, Carrie M. Johnson, Dwight R. Stoll, Mark F. Vitha
We have compared over 500 RPLC columns characterized by the hydrophobic subtraction model using the system selectivity cube (SSC). We have shown numerous differences in column selectivity even among columns in the same class (e.g., alkyl-silica, cyano, or embedded polar groups). We also illustrate the utility of our method for selecting alternative columns with different selectivities for problematic separations and for selecting orthogonal columns for use in two-dimensional separations. The system selectivity cube offers a visual way to easily compare many columns simultaneously and select those columns offering the desired selectivity.
Highlights
► The system selectivity cube (SSC) guides RPLC column selection. ► The SSC can identify RPLC columns with similar and different selectivities. ► Column comparisons are shown within and between classes (e.g., C18, cyano, etc.). ► This application of the SSC is based on the Dolan–Snyder hydrophobic subtraction model (HSM). ► The HSM database includes over 500 commercial columns from a range of classes.Retrospective screening of relevant pesticide metabolites in food using liquid chromatography high resolution mass spectrometry and accurate-mass databases of parent molecules and diagnostic fragment ions
09 July 2012,
08:33:15
Publication year:
2012
Source:Journal of Chromatography A, Volume 1249
László Polgár, Juan F. García-Reyes, Péter Fodor, Attila Gyepes, Mihály Dernovics, László Abrankó, Bienvenida Gilbert-López, Antonio Molina-Díaz
In recent years, the detection and characterization of relevant pesticide metabolites in food is an important task in order to evaluate their formation, kinetics, stability, and toxicity. In this article, a methodology for the systematic screening of pesticides and their main metabolites in fruit and vegetable samples is described, using LC–HRMS and accurate-mass database search of parent compounds and their diagnostic fragment ions. The approach is based on (i) search for parent pesticide molecules; (ii) search for their metabolites in the positive samples, assuming common fragmentation pathways between the metabolites and parent pesticide molecules; and (iii) search for pesticide conjugates using the data from both parent species and diagnostic fragment ions. An accurate-mass database was constructed consisting of 1396 compounds (850 parent compounds, 447 fragment ions and 99 metabolites). The screening process was performed by the software in an automated fashion. The proposed methodology was evaluated with 29 incurred samples and the output obtained was compared to standard pesticide testing methods (targeted LC–MS/MS). Examples on the application of the proposed approach are shown, including the detection of several pesticide glycosides derivatives, which were found with significantly relevant intensities. Glucose-conjugated forms of parent compounds (e.g., fenhexamid-O-glucoside) and those of metabolites (e.g., despropyl-iprodione-N-glycoside) were detected. Facing the lack of standards for glycosylated pesticides, the study was completed with the synthesis of fenhexamid-O-glucoside for quantification purposes. In some cases the pesticide derivatives were found in a relatively high ratio, drawing the attention to these kinds of metabolites and showing that they should not be neglected in multi-residue methods. The global coverage obtained on the 29 analyzed samples showed the usefulness and benefits of the proposed approach and highlights the practical benefit obtained when the so-called screening methods are used as a complementary tool to standard targeted LC–MS/MS methods.
Source:Journal of Chromatography A, Volume 1249
László Polgár, Juan F. García-Reyes, Péter Fodor, Attila Gyepes, Mihály Dernovics, László Abrankó, Bienvenida Gilbert-López, Antonio Molina-Díaz
In recent years, the detection and characterization of relevant pesticide metabolites in food is an important task in order to evaluate their formation, kinetics, stability, and toxicity. In this article, a methodology for the systematic screening of pesticides and their main metabolites in fruit and vegetable samples is described, using LC–HRMS and accurate-mass database search of parent compounds and their diagnostic fragment ions. The approach is based on (i) search for parent pesticide molecules; (ii) search for their metabolites in the positive samples, assuming common fragmentation pathways between the metabolites and parent pesticide molecules; and (iii) search for pesticide conjugates using the data from both parent species and diagnostic fragment ions. An accurate-mass database was constructed consisting of 1396 compounds (850 parent compounds, 447 fragment ions and 99 metabolites). The screening process was performed by the software in an automated fashion. The proposed methodology was evaluated with 29 incurred samples and the output obtained was compared to standard pesticide testing methods (targeted LC–MS/MS). Examples on the application of the proposed approach are shown, including the detection of several pesticide glycosides derivatives, which were found with significantly relevant intensities. Glucose-conjugated forms of parent compounds (e.g., fenhexamid-O-glucoside) and those of metabolites (e.g., despropyl-iprodione-N-glycoside) were detected. Facing the lack of standards for glycosylated pesticides, the study was completed with the synthesis of fenhexamid-O-glucoside for quantification purposes. In some cases the pesticide derivatives were found in a relatively high ratio, drawing the attention to these kinds of metabolites and showing that they should not be neglected in multi-residue methods. The global coverage obtained on the 29 analyzed samples showed the usefulness and benefits of the proposed approach and highlights the practical benefit obtained when the so-called screening methods are used as a complementary tool to standard targeted LC–MS/MS methods.
Highlights
► Novel workflow for systematic screening of pesticides and their metabolites in food. ► An LC–HRMS accurate-mass database developed comprising 1396 species. ► The proposed methodology was successfully tested with 29 market-purchased samples. ► Several novel pesticide metabolites were identified for the first time. ► Analyte coverage obtained shows the benefits of the proposed approach.Modeling of ion-pairing effect in peptide reversed-phase chromatography
09 July 2012,
08:33:15
Publication year:
2012
Source:Journal of Chromatography A, Volume 1249
David Gétaz, Subrahmaniam B. Hariharan, Alessandro Butté, Massimo Morbidelli
The modeling of counterion and organic modifier concentration effects in peptide APIs reversed-phase preparative chromatography is discussed in this manuscript. A stoichiometric retention model based on the counterion binding to the charged functional groups of the peptide is proposed. The model parameters were evaluated using a rather large set of retention data measured in mobile phases with various counterions and acetonitrile concentrations. The model parameters were experimentally validated by a new counterion binding measurement technique. The n max model parameter value was found to be equal to the peptide net charge, whereas the K model parameter value was found to be specific to the counterion type (i.e. AcO− <H2PO4 − <TFA− <PFPA−). The impact of the mobile phase composition on the peptide saturation capacity was also investigated. It was shown that, at low acetonitrile concentration, the peptide saturation capacity was constant for all investigated counterion types and concentrations. On the other hand, at intermediate acetonitrile concentration, the peptide saturation capacity was significantly lower and with a tendency to increase with the counterion concentration. On the whole, the developed model provides a reliable a reliable tool for the design and development of peptide purification processes at the preparative and industrial scale.
Source:Journal of Chromatography A, Volume 1249
David Gétaz, Subrahmaniam B. Hariharan, Alessandro Butté, Massimo Morbidelli
The modeling of counterion and organic modifier concentration effects in peptide APIs reversed-phase preparative chromatography is discussed in this manuscript. A stoichiometric retention model based on the counterion binding to the charged functional groups of the peptide is proposed. The model parameters were evaluated using a rather large set of retention data measured in mobile phases with various counterions and acetonitrile concentrations. The model parameters were experimentally validated by a new counterion binding measurement technique. The n max model parameter value was found to be equal to the peptide net charge, whereas the K model parameter value was found to be specific to the counterion type (i.e. AcO− <H2PO4 − <TFA− <PFPA−). The impact of the mobile phase composition on the peptide saturation capacity was also investigated. It was shown that, at low acetonitrile concentration, the peptide saturation capacity was constant for all investigated counterion types and concentrations. On the other hand, at intermediate acetonitrile concentration, the peptide saturation capacity was significantly lower and with a tendency to increase with the counterion concentration. On the whole, the developed model provides a reliable a reliable tool for the design and development of peptide purification processes at the preparative and industrial scale.
Highlights
► The effect of the counterion on the elution of a polypeptide is investigated. ► A retention model based on the counterion binding to the peptide charges is proposed. ► The model is validated through two independent sets of experiments. ► The model developed can be used for preparative process optimization.Proteomics-based, multivariate random forest method for prediction of protein separation behavior during cation-exchange chromatography
09 July 2012,
08:33:15
Publication year:
2012
Source:Journal of Chromatography A, Volume 1249
Ryan K. Swanson, Ruo Xu, Dan Nettleton, Charles E. Glatz
The most significant cost of recombinant protein production lies in the optimization of the downstream purification methods, mainly due to a lack of knowledge of the separation behavior of the host cell proteins (HCP). To reduce the effort required for purification process development, this work was aimed at modeling the separation behavior of a complex mixture of proteins in cation-exchange chromatography (CEX). With the emergence of molecular pharming as a viable option for the production of recombinant pharmaceutical proteins, the HCP mixture chosen was an extract of corn germ. Aqueous two phase system (ATPS) partitioning followed by two-dimensional electrophoresis (2DE) provided data on isoelectric point, molecular weight and surface hydrophobicity of the extract and step-elution fractions. A multivariate random forest (MVRF) method was then developed using the three characterization variables to predict the elution pattern of individual corn HCP. The MVRF method achieved an average root mean squared error (RMSE) value of 0.0406 (fraction of protein eluted in each CEX elution step) for all the proteins that were characterized, providing evidence for the effectiveness of both the characterization method and the analysis approach for protein purification applications.
Source:Journal of Chromatography A, Volume 1249
Ryan K. Swanson, Ruo Xu, Dan Nettleton, Charles E. Glatz
The most significant cost of recombinant protein production lies in the optimization of the downstream purification methods, mainly due to a lack of knowledge of the separation behavior of the host cell proteins (HCP). To reduce the effort required for purification process development, this work was aimed at modeling the separation behavior of a complex mixture of proteins in cation-exchange chromatography (CEX). With the emergence of molecular pharming as a viable option for the production of recombinant pharmaceutical proteins, the HCP mixture chosen was an extract of corn germ. Aqueous two phase system (ATPS) partitioning followed by two-dimensional electrophoresis (2DE) provided data on isoelectric point, molecular weight and surface hydrophobicity of the extract and step-elution fractions. A multivariate random forest (MVRF) method was then developed using the three characterization variables to predict the elution pattern of individual corn HCP. The MVRF method achieved an average root mean squared error (RMSE) value of 0.0406 (fraction of protein eluted in each CEX elution step) for all the proteins that were characterized, providing evidence for the effectiveness of both the characterization method and the analysis approach for protein purification applications.
Highlights
► Complex mixture of proteins characterized by 3 properties: pI, MW, hydrophobicity. ► Characterized proteins’ separation behavior during cation-exchange chromatography. ► Multivariate statistical method used to predict protein's separation behavior. ► Could be applied to other separation methods aiding purification process selection.Using chiral liquid chromatography quadrupole time-of-flight mass spectrometry for the analysis of pharmaceuticals and illicit drugs in surface and wastewater at the enantiomeric level
09 July 2012,
08:33:15
Publication year:
2012
Source:Journal of Chromatography A, Volume 1249
J.P. Bagnall, S.E. Evans, M.T. Wort, A.T. Lubben, B. Kasprzyk-Hordern
This paper presents and compares for the first time two chiral LC–QTOF-MS methodologies (utilising CBH and Chirobiotic V columns with cellobiohydrolase and vancomycin as chiral selectors) for the quantification of amphetamine, methamphetamine, MDA (methylenedioxyamphetamine), MDMA (methylenedioxymethamphetamine), propranolol, atenolol, metoprolol, fluoxetine and venlafaxine in river water and sewage effluent. The lowest MDLs (0.3–5.0ngL−1 and 1.3–15.1ngL−1 for river water and sewage effluent respectively) were observed using the chiral column Chirobiotic V. This is with the exception of methamphetamine and MDMA which had lower MDLs using the CBH column. However, the CBH column resulted in better resolution of enantiomers (R s =2.5 for amphetamine compared with R s =1.2 with Chirobiotic V). Method recovery rates were typically >80% for both methodologies. Pharmaceuticals and illicit drugs detected and quantified in environmental samples were successfully identified using MS/MS confirmation. In sewage effluent, the total beta-blocker concentrations of propranolol, atenolol and metoprolol were on average 77.0, 1091.0 and 3.6ngL−1 thus having EFs (Enantiomeric Fractions) of 0.43, 0.55 and 0.54 respectively. In river water, total propranolol and atenolol was quantified on average at <10.0ngL−1. Differences in EF between sewage and river water matrices were evident: venlafaxine was observed with respective EF of 0.43±0.02 and 0.58±0.02.
Source:Journal of Chromatography A, Volume 1249
J.P. Bagnall, S.E. Evans, M.T. Wort, A.T. Lubben, B. Kasprzyk-Hordern
This paper presents and compares for the first time two chiral LC–QTOF-MS methodologies (utilising CBH and Chirobiotic V columns with cellobiohydrolase and vancomycin as chiral selectors) for the quantification of amphetamine, methamphetamine, MDA (methylenedioxyamphetamine), MDMA (methylenedioxymethamphetamine), propranolol, atenolol, metoprolol, fluoxetine and venlafaxine in river water and sewage effluent. The lowest MDLs (0.3–5.0ngL−1 and 1.3–15.1ngL−1 for river water and sewage effluent respectively) were observed using the chiral column Chirobiotic V. This is with the exception of methamphetamine and MDMA which had lower MDLs using the CBH column. However, the CBH column resulted in better resolution of enantiomers (R s =2.5 for amphetamine compared with R s =1.2 with Chirobiotic V). Method recovery rates were typically >80% for both methodologies. Pharmaceuticals and illicit drugs detected and quantified in environmental samples were successfully identified using MS/MS confirmation. In sewage effluent, the total beta-blocker concentrations of propranolol, atenolol and metoprolol were on average 77.0, 1091.0 and 3.6ngL−1 thus having EFs (Enantiomeric Fractions) of 0.43, 0.55 and 0.54 respectively. In river water, total propranolol and atenolol was quantified on average at <10.0ngL−1. Differences in EF between sewage and river water matrices were evident: venlafaxine was observed with respective EF of 0.43±0.02 and 0.58±0.02.
Highlights
► Here two chiral LC–QTOF-MS methods are detailed for the analysis of pharmaceuticals and illicit drugs. ► Methods are suitable for resolving and quantifying chiral compounds in river and wastewater. ► Chiral compounds quantified in environment samples were confirmed using MS/MS confirmation. ► Differences in EF between compounds in sewage and river water were evident.Identification of flavonoid glycosides in Rosa chinensis flowers by liquid chromatography–tandem mass spectrometry in combination with 13C nuclear magnetic resonance
09 July 2012,
08:33:15
Publication year:
2012
Source:Journal of Chromatography A, Volume 1249
Lin-Sen Qing, Ying Xue, Jian-Guang Zhang, Zhi-Feng Zhang, Jian Liang, Yan Jiang, Yi-Ming Liu, Xun Liao
Flowers of Rosa chinensis are widely used in traditional Chinese medicine as well as in food industry. Flavonoid glycosides are believed to be the major components in R. chinensis that are responsible for its antioxidant activities. In this work, a liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for analysis of flavonoid glycosides presented in ethyl acetate extract of dried R. chinensis flowers. Twelve flavonoid glycosides were separated and detected. By comparing the retention times, UV spectra, and tandem MS fragments with those of respective authentic compounds, eight flavonoid glycosides were unequivocally identified. Although the other four were also identified as flavonoid glycosides, the glycosylation positions could not be determined due to lack of authentic compounds. Fortunately, the glycosylation effects were clearly observed in the 13C NMR spectrum of the extract. The detailed structural information was, therefore, obtained to identify the four flavonoid glycosides as quercetin-3-O-d-glucoside, quercetin-3-O-d-xyloside, kaempferol-3-O-d-xyloside and quercetin-3-O-d-(6″-coumaroyl)-galactoside. These flavonoid glycosides were detected and identified for the first time in this botanic material. This work reports on the first use of 13C NMR of a mixture to enhance a rapid HPLC–MS/MS analysis. The proposed analytical protocol was validated with a mixture of authentic flavonoid glycosides.
Source:Journal of Chromatography A, Volume 1249
Lin-Sen Qing, Ying Xue, Jian-Guang Zhang, Zhi-Feng Zhang, Jian Liang, Yan Jiang, Yi-Ming Liu, Xun Liao
Flowers of Rosa chinensis are widely used in traditional Chinese medicine as well as in food industry. Flavonoid glycosides are believed to be the major components in R. chinensis that are responsible for its antioxidant activities. In this work, a liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for analysis of flavonoid glycosides presented in ethyl acetate extract of dried R. chinensis flowers. Twelve flavonoid glycosides were separated and detected. By comparing the retention times, UV spectra, and tandem MS fragments with those of respective authentic compounds, eight flavonoid glycosides were unequivocally identified. Although the other four were also identified as flavonoid glycosides, the glycosylation positions could not be determined due to lack of authentic compounds. Fortunately, the glycosylation effects were clearly observed in the 13C NMR spectrum of the extract. The detailed structural information was, therefore, obtained to identify the four flavonoid glycosides as quercetin-3-O-d-glucoside, quercetin-3-O-d-xyloside, kaempferol-3-O-d-xyloside and quercetin-3-O-d-(6″-coumaroyl)-galactoside. These flavonoid glycosides were detected and identified for the first time in this botanic material. This work reports on the first use of 13C NMR of a mixture to enhance a rapid HPLC–MS/MS analysis. The proposed analytical protocol was validated with a mixture of authentic flavonoid glycosides.
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