Screening of stationary phase selectivities for global lipid profiling by ultrahigh performance supercritical fluid chromatography

Publication date: 4 May 2018
Source:Journal of Chromatography A, Volume 1548
Author(s): Said Al Hamimi, Margareta Sandahl, Marina Armeni, Charlotta Turner, Peter Spégel
The performance of seven sub-2-μm particle packed columns (2-picolylamine, 2-PIC; charged surface hybrid fluoro-phenyl, CSH-FP; high strength silica C18 SB, HSS-C18; diethylamine, DEA; 1-aminoanthracene, 1-AA; high density diol and ethylene bridged hybrid; BEH) was examined for lipid separation in ultra-high performance supercritical fluid chromatography (UHPSFC) coupled to quadrupole time-of-flight mass spectrometry. Based on the results of the column screening a method for profiling of multiple lipid species from the major lipid classes was developed.Stationary phases containing β-hydroxy amines, i.e. 1-AA, DEA and 2-PIC, yielded strong retention and poor peak shapes of zwitterionic lipids with primary amine groups, such as phosphatidylserines, phosphatidylethanolamines and its lyso forms. The BEH and HSS-C18 columns showed strong retention of polar and nonpolar lipids, respectively. The Diol column retained the majority of major lipid classes and also produced symmetric peaks. In addition, this column also produced the highest resolution within and between major lipid classes. An injection solvent composed of methanol:chloroform (1:2, v:v) and the addition of 20 mM ammonium formate in the mobile phase improved chromatographic separation and mass spectrometry detection in comparison to ammonium acetate or absence of additive. Finally, chromatographic and mass spectrometric parameters were optimized for the Diol column using a design of experiments approach. The separation mechanism on the Diol column depended on the lipid functionality and the length and degree of unsaturation of the acyl groups. The developed method could resolve 18 lipid classes and multiple lipids within each class, from blood serum and brain tissue in 11 min.

Publication date: 4 May 2018
Source:Journal of Chromatography A, Volume 1548

Author(s): Said Al Hamimi, Margareta Sandahl, Marina Armeni, Charlotta Turner, Peter Spégel

The performance of seven sub-2-μm particle packed columns (2-picolylamine, 2-PIC; charged surface hybrid fluoro-phenyl, CSH-FP; high strength silica C18 SB, HSS-C18; diethylamine, DEA; 1-aminoanthracene, 1-AA; high density diol and ethylene bridged hybrid; BEH) was examined for lipid separation in ultra-high performance supercritical fluid chromatography (UHPSFC) coupled to quadrupole time-of-flight mass spectrometry. Based on the results of the column screening a method for profiling of multiple lipid species from the major lipid classes was developed. Stationary phases containing β-hydroxy amines, i.e. 1-AA, DEA and 2-PIC, yielded strong retention and poor peak shapes of zwitterionic lipids with primary amine groups, such as phosphatidylserines, phosphatidylethanolamines and its lyso forms. The BEH and HSS-C18 columns showed strong retention of polar and nonpolar lipids, respectively. The Diol column retained the majority of major lipid classes and also produced symmetric peaks. In addition, this column also produced the highest resolution within and between major lipid classes. An injection solvent composed of methanol:chloroform (1:2, v:v) and the addition of 20 mM ammonium formate in the mobile phase improved chromatographic separation and mass spectrometry detection in comparison to ammonium acetate or absence of additive. Finally, chromatographic and mass spectrometric parameters were optimized for the Diol column using a design of experiments approach. The separation mechanism on the Diol column depended on the lipid functionality and the length and degree of unsaturation of the acyl groups. The developed method could resolve 18 lipid classes and multiple lipids within each class, from blood serum and brain tissue in 11 min.





Evaluation of flavour profiles in e-cigarette refill solutions using gas chromatography–tandem mass spectrometry

Publication date: 27 April 2018
Source:Journal of Chromatography A, Volume 1547
Author(s): Justyna Aszyk, Paweł Kubica, Mateusz Kacper Woźniak, Jacek Namieśnik, Andrzej Wasik, Agata Kot-Wasik
Many flavour compounds that are present in e-liquids for e-cigarettes are responsible for specific tastes and smoking sensations for users. Data concerning content and specific types of flavours is often limited and unknown to users. The aim of the research was to define and compare flavour profiles of e-liquids with the same group taste from different manufacturers. Gas chromatography coupled with tandem mass spectrometry (GC–MS/MS) was used to separate and identify 90 popular compounds (98, including isomers) of interest. The developed method was validated in terms of accuracy (88–113%) for three spiking levels and the intra-day (0.2–13%) and inter-day precision (1–10%). Limits of quantitation were in the range of 10–816 ng/mL, while the matrix effects for 80% of the compounds were at negligible levels. The proposed method is rapid, simple and reliable and uses a green and modern GC–MS/MS technique. Twenty-five samples of five different flavours (tobacco, strawberry, cherry, menthol and apple) from five different producers were analysed, and the determined compounds were categorized and differentiated. The approach proposed in this study allowed for the evaluation of which compounds/group of compounds are responsible for taste and to distinguish common flavour compounds among the investigated brands for each flavour. Furthermore, the presented research can be considered in future toxicological studies.

Graphical abstract

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Publication date: 27 April 2018
Source:Journal of Chromatography A, Volume 1547

Author(s): Justyna Aszyk, Paweł Kubica, Mateusz Kacper Woźniak, Jacek Namieśnik, Andrzej Wasik, Agata Kot-Wasik

Many flavour compounds that are present in e-liquids for e-cigarettes are responsible for specific tastes and smoking sensations for users. Data concerning content and specific types of flavours is often limited and unknown to users. The aim of the research was to define and compare flavour profiles of e-liquids with the same group taste from different manufacturers. Gas chromatography coupled with tandem mass spectrometry (GC–MS/MS) was used to separate and identify 90 popular compounds (98, including isomers) of interest. The developed method was validated in terms of accuracy (88–113%) for three spiking levels and the intra-day (0.2–13%) and inter-day precision (1–10%). Limits of quantitation were in the range of 10–816 ng/mL, while the matrix effects for 80% of the compounds were at negligible levels. The proposed method is rapid, simple and reliable and uses a green and modern GC–MS/MS technique. Twenty-five samples of five different flavours (tobacco, strawberry, cherry, menthol and apple) from five different producers were analysed, and the determined compounds were categorized and differentiated. The approach proposed in this study allowed for the evaluation of which compounds/group of compounds are responsible for taste and to distinguish common flavour compounds among the investigated brands for each flavour. Furthermore, the presented research can be considered in future toxicological studies.

Graphical abstract

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Co-Al bimetallic hydroxide nanocomposites coating for online in-tube solid-phase microextraction

Publication date: 18 May 2018
Source:Journal of Chromatography A, Volume 1550
Author(s): Xiuqin Wang, Juanjuan Feng, Yu Tian, Chuannan Luo, Min Sun
The Co-Al bimetallic hydroxide nanocomposites were synthesized and used as in-tube solid-phase microextraction coating. The nanocomposites-coated fibers were packed in a PEEK tube and tested for the extraction of eight polycyclic aromatic hydrocarbons (PAHs) coupled with high performance liquid chromatography. Several parameters affecting the PAHs extraction including the sampling volume, sampling flow rate, methanol content and desorption time were investigated. Under the optimized conditions, the extraction tube with nanocomposites coating exhibited remarkable extraction performance towards PAH targets. An online analysis method was achieved with the limits of detection less than 0.02 μg L−1 and the linearity in the range from 0.03 to 15 μg L−1. The recoveries of the method for two water samples with spiking levels of 1 and 10 μg L−1 ranged from 83 to 121%. Extraction repeatability and preparation repeatability were less than 4.0% and 10.9%, respectively.

Publication date: 18 May 2018
Source:Journal of Chromatography A, Volume 1550

Author(s): Xiuqin Wang, Juanjuan Feng, Yu Tian, Chuannan Luo, Min Sun

The Co-Al bimetallic hydroxide nanocomposites were synthesized and used as in-tube solid-phase microextraction coating. The nanocomposites-coated fibers were packed in a PEEK tube and tested for the extraction of eight polycyclic aromatic hydrocarbons (PAHs) coupled with high performance liquid chromatography. Several parameters affecting the PAHs extraction including the sampling volume, sampling flow rate, methanol content and desorption time were investigated. Under the optimized conditions, the extraction tube with nanocomposites coating exhibited remarkable extraction performance towards PAH targets. An online analysis method was achieved with the limits of detection less than 0.02 μg L−1 and the linearity in the range from 0.03 to 15 μg L−1. The recoveries of the method for two water samples with spiking levels of 1 and 10 μg L−1 ranged from 83 to 121%. Extraction repeatability and preparation repeatability were less than 4.0% and 10.9%, respectively.





Preparation of polymer monolithic column functionalized by arsonic acid groups for mixed-mode capillary liquid chromatography

Publication date: 27 April 2018
Source:Journal of Chromatography A, Volume 1547
Author(s): Zhang-Na Qin, Qiong-Wei Yu, Ren-Qi Wang, Yu-Qi Feng
A mixed-mode polymer monolithic column functionalized by arsonic acid groups was prepared by single-step in situ copolymerization of monomers p-methacryloylaminophenylarsonic acid (p-MAPHA) and pentaerythritol triacrylate (PETA). The prepared poly(p-MAPHA-co-PETA) monolithic column has a homogeneous monolithic structure with good permeability and mechanical stability. Zeta potential measurements reveal that the monolithic stationary phase holds a negative surface charge when the mobile phase resides in the pH range of 3.0–8.0. The retention mechanisms of prepared monolithic column are explored by the separation of selected polycyclic aromatic hydrocarbons (PAHs), nucleosides, and three basic compounds. The results indicate that the column functions in three different separation modes associated with reversed-phase chromatography based on hydrophobic interaction, hydrophilic interaction chromatography, and cation-exchange chromatography. The column efficiency of prepared monolithic column is estimated to be 70,000 and 76,000 theoretical plates/m for thiourea and naphthalene, respectively, at a linear flow velocity of 0.85 mm/s using acetonitrile/H2O (85/15, v/v) as the mobile phase. Furthermore, an analysis of the retention factors obtained for the PAHs indicates that the prepared monolithic column exhibits good reproducibility with relative standard deviations of 2.9%, 4.0%, and 4.7% based on run-to-run injections, column-to-column preparation, and batch-to-batch preparation, respectively. Finally, we investigate the separation performance of the proposed monolithic column for select phenols, sulfonamides, nucleobases and nucleosides.

Publication date: 27 April 2018
Source:Journal of Chromatography A, Volume 1547

Author(s): Zhang-Na Qin, Qiong-Wei Yu, Ren-Qi Wang, Yu-Qi Feng

A mixed-mode polymer monolithic column functionalized by arsonic acid groups was prepared by single-step in situ copolymerization of monomers p-methacryloylaminophenylarsonic acid (p-MAPHA) and pentaerythritol triacrylate (PETA). The prepared poly(p-MAPHA-co-PETA) monolithic column has a homogeneous monolithic structure with good permeability and mechanical stability. Zeta potential measurements reveal that the monolithic stationary phase holds a negative surface charge when the mobile phase resides in the pH range of 3.0–8.0. The retention mechanisms of prepared monolithic column are explored by the separation of selected polycyclic aromatic hydrocarbons (PAHs), nucleosides, and three basic compounds. The results indicate that the column functions in three different separation modes associated with reversed-phase chromatography based on hydrophobic interaction, hydrophilic interaction chromatography, and cation-exchange chromatography. The column efficiency of prepared monolithic column is estimated to be 70,000 and 76,000 theoretical plates/m for thiourea and naphthalene, respectively, at a linear flow velocity of 0.85 mm/s using acetonitrile/H2O (85/15, v/v) as the mobile phase. Furthermore, an analysis of the retention factors obtained for the PAHs indicates that the prepared monolithic column exhibits good reproducibility with relative standard deviations of 2.9%, 4.0%, and 4.7% based on run-to-run injections, column-to-column preparation, and batch-to-batch preparation, respectively. Finally, we investigate the separation performance of the proposed monolithic column for select phenols, sulfonamides, nucleobases and nucleosides.





Development of a micro-solid-phase extraction molecularly imprinted polymer technique for synthetic cannabinoids assessment in urine followed by liquid chromatography–tandem mass spectrometry

Publication date: 18 May 2018
Source:Journal of Chromatography A, Volume 1550
Author(s): Juan Sánchez-González, Sara Odoardi, Ana María Bermejo, Pilar Bermejo-Barrera, Francesco Saverio Romolo, Antonio Moreda-Piñeiro, Sabina Strano-Rossi
Several molecularly imprinted polymers (MIPs) have been synthesized for the first time using various synthetic cannabinoids (JWH007, JWH015 and JWH098) as template molecules. Ethylene dimethacrylate (EDMA) was used as a functional monomer for all cases. Similarly, divinylbenzene (DVB) and 2,2′-azobisisobutyronitrile (AIBN) were used as cross-linker and initiator, respectively. The prepared MIPs have been fully characterized and evaluated as new selective adsorbents for micro-solid phase extraction (μ-SPE) of synthetic cannabinoids in urine. The developed MIP-μ-SPE devices consisted of a polypropylene (PP) porous membrane containing the adsorbent (novel porous membrane protected micro-solid phase extraction based on a cone-shaped device) for operating in batch mode, which allowed a fast and integrated extraction-cleanup procedure. High performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was used for quantifying the analytes after MIP-μ-SPE. The best performances were obtained for MIPs prepared from JWH015 as a template. Optimum loading conditions were found to be urine pH of 5.0 and adsorption time of 8.0 min under mechanical (orbital-horizontal) stirring at 100 rpm. The composition of the eluting solution consisted of 75:20:5 heptane/2-propanol/ammonium hydroxide. The elution was assisted by ultrasounds (37 kHz, 325 W) for 8.0 min. In addition, studies regarding selectivity have also been addressed for several drugs of abuse under optimized loading/adsorption conditions. Validation of the method showed good precision and analytical recovery by intra-day and inter-day assays (RSD values lower than 7 and 10% for intra-day and inter-day precision, and within the 83–100% range for intra-day and inter-day analytical recovery).

Publication date: 18 May 2018
Source:Journal of Chromatography A, Volume 1550

Author(s): Juan Sánchez-González, Sara Odoardi, Ana María Bermejo, Pilar Bermejo-Barrera, Francesco Saverio Romolo, Antonio Moreda-Piñeiro, Sabina Strano-Rossi

Several molecularly imprinted polymers (MIPs) have been synthesized for the first time using various synthetic cannabinoids (JWH007, JWH015 and JWH098) as template molecules. Ethylene dimethacrylate (EDMA) was used as a functional monomer for all cases. Similarly, divinylbenzene (DVB) and 2,2′-azobisisobutyronitrile (AIBN) were used as cross-linker and initiator, respectively. The prepared MIPs have been fully characterized and evaluated as new selective adsorbents for micro-solid phase extraction (μ-SPE) of synthetic cannabinoids in urine. The developed MIP-μ-SPE devices consisted of a polypropylene (PP) porous membrane containing the adsorbent (novel porous membrane protected micro-solid phase extraction based on a cone-shaped device) for operating in batch mode, which allowed a fast and integrated extraction-cleanup procedure. High performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was used for quantifying the analytes after MIP-μ-SPE. The best performances were obtained for MIPs prepared from JWH015 as a template. Optimum loading conditions were found to be urine pH of 5.0 and adsorption time of 8.0 min under mechanical (orbital-horizontal) stirring at 100 rpm. The composition of the eluting solution consisted of 75:20:5 heptane/2-propanol/ammonium hydroxide. The elution was assisted by ultrasounds (37 kHz, 325 W) for 8.0 min. In addition, studies regarding selectivity have also been addressed for several drugs of abuse under optimized loading/adsorption conditions. Validation of the method showed good precision and analytical recovery by intra-day and inter-day assays (RSD values lower than 7 and 10% for intra-day and inter-day precision, and within the 83–100% range for intra-day and inter-day analytical recovery).





Reversible Concanavalin A (Con A) ligands immobilization on metal chelated macroporous cellulose monolith and its selective adsorption for glycoproteins

Publication date: 4 May 2018
Source:Journal of Chromatography A, Volume 1548
Author(s): Kaifeng Du, Shunmin Dan
The present work deals with the development of novel affinity monolith with reversible protein ligands for protein chromatography. As for the formation of reversible ligands, Concanavalin A (Con A) is chelated with Cu(II)-iminodiacetic acid (IDA) immobilized macroporous cellulose monolith (MCM) for glycoprotein adsorption. The reversible immobilization is realized by Cu ions, which bridge affinity ligands and support by strong chelation interaction. The fabrication process of reversible Con A immobilized adsorbent is studied, especially with regards to the effect of synthesis conditions on the ligands immobilization. The adsorption behavior is then evaluated to elucidate the potential of Con A-Cu(II)-IDA-MCM for protein chromatography. It reveals that the static adsorption capacity and dissociation constant of glucose oxidase (GOD) on Con A-Cu(II)-IDA-MCM are determined to be 17.4 ± 0.6 mg mL−1 and 0.055 ± 0.011 mg mL−1 by Langmuir model. With frontal analysis, the dynamic binding capacity of GOD at 10% breakthrough point is about 11.4 ± 1.0 mg mL−1 and changes less with an increase of flow velocity from 0.2 to 1.0 mL min−1. Moreover, Con A-Cu(II)-IDA-MCM displays weak nonspecific adsorption for the impurities and is able to successfully enrich glycoprotein ovalbumin (OVA) from diluted chicken egg white. In addition, Con A-Cu(II)-IDA-MCM exhibits excellent stability by the repeated adsorption/desorption operations. By taking these advantages of high adsorption capacity, excellent specificity and structure stability, the prepared affinity adsorbent of Con A-Cu(II)-IDA-MCM has great potential for high performance protein chromatography.

Publication date: 4 May 2018
Source:Journal of Chromatography A, Volume 1548

Author(s): Kaifeng Du, Shunmin Dan

The present work deals with the development of novel affinity monolith with reversible protein ligands for protein chromatography. As for the formation of reversible ligands, Concanavalin A (Con A) is chelated with Cu(II)-iminodiacetic acid (IDA) immobilized macroporous cellulose monolith (MCM) for glycoprotein adsorption. The reversible immobilization is realized by Cu ions, which bridge affinity ligands and support by strong chelation interaction. The fabrication process of reversible Con A immobilized adsorbent is studied, especially with regards to the effect of synthesis conditions on the ligands immobilization. The adsorption behavior is then evaluated to elucidate the potential of Con A-Cu(II)-IDA-MCM for protein chromatography. It reveals that the static adsorption capacity and dissociation constant of glucose oxidase (GOD) on Con A-Cu(II)-IDA-MCM are determined to be 17.4 ± 0.6 mg mL−1 and 0.055 ± 0.011 mg mL−1 by Langmuir model. With frontal analysis, the dynamic binding capacity of GOD at 10% breakthrough point is about 11.4 ± 1.0 mg mL−1 and changes less with an increase of flow velocity from 0.2 to 1.0 mL min−1. Moreover, Con A-Cu(II)-IDA-MCM displays weak nonspecific adsorption for the impurities and is able to successfully enrich glycoprotein ovalbumin (OVA) from diluted chicken egg white. In addition, Con A-Cu(II)-IDA-MCM exhibits excellent stability by the repeated adsorption/desorption operations. By taking these advantages of high adsorption capacity, excellent specificity and structure stability, the prepared affinity adsorbent of Con A-Cu(II)-IDA-MCM has great potential for high performance protein chromatography.





O-carboxymethyl chitosan Schiff base complexes as affinity ligands for immobilized metal-ion affinity chromatography of lysozyme

Publication date: 18 May 2018
Source:Journal of Chromatography A, Volume 1550
Author(s): Ömür Acet, Talat Baran, Demet Erdönmez, Neşe Hayat Aksoy, İhsan Alacabey, Ayfer Menteş, Mehmet Odabaşi
We synthesized Ni2+-attached O-Carboxymethyl chitosan Schiff base complexes embedded composite cryogels (Ni2+-O-CMCS-CCs) by means of polymerization of gel-forming precursors at subzero temperatures. Prepared affinity cryogel showed excellent adsorption performance for lysozyme selected as model protein to test adsorption parameters, demonstrating an adsorption capacity of 244.6 mg/g (15.3 mg/g for Ni2+ minus O-CMCS-CCs), with fast adsorption equilibrium within 30 min and good reversibility. The performance of Ni2+-O-CMCS-CCs for lysozyme was also evaluated by SDS-PAGE, and a purification efficiency of 86.9% with 89.5% purification yield was determined. The swelling test, FT-IR, and SEM analysis were carried out for the characterization of Ni2+-O-CMCS-CCs. At the end of 35 adsorption-desorption cycles, there was no significant change in the adsorption capacity.

Publication date: 18 May 2018
Source:Journal of Chromatography A, Volume 1550

Author(s): Ömür Acet, Talat Baran, Demet Erdönmez, Neşe Hayat Aksoy, İhsan Alacabey, Ayfer Menteş, Mehmet Odabaşi

We synthesized Ni2+-attached O-Carboxymethyl chitosan Schiff base complexes embedded composite cryogels (Ni2+-O-CMCS-CCs) by means of polymerization of gel-forming precursors at subzero temperatures. Prepared affinity cryogel showed excellent adsorption performance for lysozyme selected as model protein to test adsorption parameters, demonstrating an adsorption capacity of 244.6 mg/g (15.3 mg/g for Ni2+ minus O-CMCS-CCs), with fast adsorption equilibrium within 30 min and good reversibility. The performance of Ni2+-O-CMCS-CCs for lysozyme was also evaluated by SDS-PAGE, and a purification efficiency of 86.9% with 89.5% purification yield was determined. The swelling test, FT-IR, and SEM analysis were carried out for the characterization of Ni2+-O-CMCS-CCs. At the end of 35 adsorption-desorption cycles, there was no significant change in the adsorption capacity.





Construction of chiral ligand exchange capillary electrochromatography for d,l-amino acids enantioseparation and its application in glutaminase kinetics study

Publication date: 4 May 2018
Source:Journal of Chromatography A, Volume 1548
Author(s): Liping Zhao, Juan Qiao, Ke Zhang, Dan Li, Hongyi Zhang, Li Qi
A chiral ligand exchange capillary electrochromatography (CLE-CEC) protocol was designed and implemented for d,l-amino acids enantioseparation with poly(maleic anhydride-styrene-methacryloyl-l-arginine methyl ester) as the coating. The block copolymer was synthesized through the reversible addition fragmentation chain transfer reaction. In the constructed CLE-CEC system, poly (methacryloyl-l-arginine methyl ester) moiety of the block copolymer played the role as the immobilized chiral ligand and Zn (II) was used as the central ion. Key factors, including pH of buffer solution, ratio of Zn (II) to ligands, the mass ratio of monomers in the block copolymer, which affect the enantioresolution were investigated. Comparing with the bare capillary, the CLE-CEC enantioresolution was enhanced greatly with the coating one. 5 Pairs of d,l-amino acids enantiomers obtained baseline separation with 5 pairs partly separated. The mechanism of enhancement enantioresolution of the developed CLE-CEC system was explored briefly. Further, good linearities were achieved in the range of 25.0 μM–5.0 mM for quantitative analysis of d-glutamine (r2 = 0.997) and l-glutamine (r2 = 0.991). Moreover, the proposed CLE-CEC assay was successfully applied in the kinetics study of glutaminase by using l-glutamine as the substrate.

Publication date: 4 May 2018
Source:Journal of Chromatography A, Volume 1548

Author(s): Liping Zhao, Juan Qiao, Ke Zhang, Dan Li, Hongyi Zhang, Li Qi

A chiral ligand exchange capillary electrochromatography (CLE-CEC) protocol was designed and implemented for d,l-amino acids enantioseparation with poly(maleic anhydride-styrene-methacryloyl-l-arginine methyl ester) as the coating. The block copolymer was synthesized through the reversible addition fragmentation chain transfer reaction. In the constructed CLE-CEC system, poly (methacryloyl-l-arginine methyl ester) moiety of the block copolymer played the role as the immobilized chiral ligand and Zn (II) was used as the central ion. Key factors, including pH of buffer solution, ratio of Zn (II) to ligands, the mass ratio of monomers in the block copolymer, which affect the enantioresolution were investigated. Comparing with the bare capillary, the CLE-CEC enantioresolution was enhanced greatly with the coating one. 5 Pairs of d,l-amino acids enantiomers obtained baseline separation with 5 pairs partly separated. The mechanism of enhancement enantioresolution of the developed CLE-CEC system was explored briefly. Further, good linearities were achieved in the range of 25.0 μM–5.0 mM for quantitative analysis of d-glutamine (r2 = 0.997) and l-glutamine (r2 = 0.991). Moreover, the proposed CLE-CEC assay was successfully applied in the kinetics study of glutaminase by using l-glutamine as the substrate.





Characterization of new polymer-grafted protein cation exchangers developed by partial neutralization of carboxyl groups derivatized by modification of poly(ethylenimine)-Sepharose with succinic anhydride

Publication date: 18 May 2018
Source:Journal of Chromatography A, Volume 1550
Author(s): Yangyang Zhao, Xiaoyan Dong, Linling Yu, Yang Liu, Yan Sun
Previously, we have studied protein adsorption and chromatographic behaviors on poly(ethylenimine) (PEI)-grafted Sepharose FF anion-exchange resins, and found that protein uptake rates increased greatly when PEI grafting density reached over a critical ionic capacity (cIC) due to the occurrence of the “chain delivery” effect. Moreover, by partial charge neutralization of starting resin FF-PEI-L740 (IC = 740 mmol/L, larger than the cIC) with sodium acetate to FF-PEI-R440, it exhibited a three-fold increase in uptake rate over FF-PEI-L740. In this work, to take the advantages of PEI and extend the applications of the PEI-grafted resins in cation-exchange chromatography, a series of cation exchangers of five different ICs were developed. First, the charged of FF-PEI-L740 was reversed from positive to negative by reaction with excess succinic anhydride, which created a cation-exchanger with an IC of 970 mmol/L (FF-FEI-C970). FF-PEI-C970 was further modified with ethanolamine for partial charge neutralizations, leading to the preparation of four charge-reduced cation exchangers with IC values (in mmol/L) of 780, 630, 560 and 430, which were denoted as FF-PEI-CR780, −CR630 −CR560 and −CR430, respectively. Protein adsorption and chromatographic behaviors were investigated using lysozyme (Lys) as the model protein. It was found that, the resins of high and moderate IC values (IC ≥ 560 mmol/L) afforded adsorption capacities up to over 230 mg/mL. Besides, the uptake rate, represented by the effective pore diffusivity (De/D0), exhibited significant increase from 0.067 (FF-PEI-C970 and FF-PEI-CR780) to 0.343 (FF-PEI-CR630 and FF-PEI-CR560) and then to 1.035 (FF-PEI-CR430) with decreasing IC. It was considered that decreasing IC led to the decreased protein binding sites (binding strength), which encouraged the occurrence of the “chain delivery” effect. Moreover, the resins of high and moderate IC values, particularly, the resins of moderate IC values (FF-PEI-CR630 and FF-PEI-CR560), presented both high adsorption capacities and uptake kinetics at 0–100 mmol/L NaCl. Besides, dynamic binding capacity achieved 150 mg/mL for the resins of moderate IC values at 0 mmol/L NaCl concentration, and afforded >110 mg/mL for the resin of high IC values at 0–100 mmol/L NaCl concentration. The results proved the excellent IEC performance of the PEI-derived cation exchangers.

Publication date: 18 May 2018
Source:Journal of Chromatography A, Volume 1550

Author(s): Yangyang Zhao, Xiaoyan Dong, Linling Yu, Yang Liu, Yan Sun

Previously, we have studied protein adsorption and chromatographic behaviors on poly(ethylenimine) (PEI)-grafted Sepharose FF anion-exchange resins, and found that protein uptake rates increased greatly when PEI grafting density reached over a critical ionic capacity (cIC) due to the occurrence of the “chain delivery” effect. Moreover, by partial charge neutralization of starting resin FF-PEI-L740 (IC = 740 mmol/L, larger than the cIC) with sodium acetate to FF-PEI-R440, it exhibited a three-fold increase in uptake rate over FF-PEI-L740. In this work, to take the advantages of PEI and extend the applications of the PEI-grafted resins in cation-exchange chromatography, a series of cation exchangers of five different ICs were developed. First, the charged of FF-PEI-L740 was reversed from positive to negative by reaction with excess succinic anhydride, which created a cation-exchanger with an IC of 970 mmol/L (FF-FEI-C970). FF-PEI-C970 was further modified with ethanolamine for partial charge neutralizations, leading to the preparation of four charge-reduced cation exchangers with IC values (in mmol/L) of 780, 630, 560 and 430, which were denoted as FF-PEI-CR780, −CR630 −CR560 and −CR430, respectively. Protein adsorption and chromatographic behaviors were investigated using lysozyme (Lys) as the model protein. It was found that, the resins of high and moderate IC values (IC ≥ 560 mmol/L) afforded adsorption capacities up to over 230 mg/mL. Besides, the uptake rate, represented by the effective pore diffusivity (D e/ D 0), exhibited significant increase from 0.067 (FF-PEI-C970 and FF-PEI-CR780) to 0.343 (FF-PEI-CR630 and FF-PEI-CR560) and then to 1.035 (FF-PEI-CR430) with decreasing IC. It was considered that decreasing IC led to the decreased protein binding sites (binding strength), which encouraged the occurrence of the “chain delivery” effect. Moreover, the resins of high and moderate IC values, particularly, the resins of moderate IC values (FF-PEI-CR630 and FF-PEI-CR560), presented both high adsorption capacities and uptake kinetics at 0–100 mmol/L NaCl. Besides, dynamic binding capacity achieved 150 mg/mL for the resins of moderate IC values at 0 mmol/L NaCl concentration, and afforded >110 mg/mL for the resin of high IC values at 0–100 mmol/L NaCl concentration. The results proved the excellent IEC performance of the PEI-derived cation exchangers.





Gradient elution behavior of proteins in hydrophobic interaction chromatography with U-shaped retention factor curves

Publication date: 27 April 2018 Source:Journal of Chromatography A, Volume 1547 Author(s): Arch Creasy, Joseph Lomino, Gregory Barker, Anurag Khetan, Giorgio Carta Protein retention in hydrophobic interaction chromatography i…

Publication date: 27 April 2018
Source:Journal of Chromatography A, Volume 1547

Author(s): Arch Creasy, Joseph Lomino, Gregory Barker, Anurag Khetan, Giorgio Carta

Protein retention in hydrophobic interaction chromatography is described by the solvophobic theory as a function of the kosmostropic salt concentration. In general, an increase in salt concentration drives protein partitioning to the hydrophobic surface while a decrease reduces it. In some cases, however, protein retention also increases at low salt concentrations resulting in a U-shaped retention factor curve. During gradient elution the salt concentration is gradually decreased from a high value thereby reducing the retention factor and increasing the protein chromatographic velocity. For these conditions, a steep gradient can overtake the protein in the column, causing it to rebind. Two dynamic models, one based on the local equilibrium theory and the other based on the linear driving force approximation, are presented. We show that the normalized gradient slope determines whether the protein elutes in the gradient, partially elutes, or is trapped in the column. Experimental results are presented for two different monoclonal antibodies and for lysozyme on Capto Phenyl (High Sub) resin. One of the mAbs and lysozyme exhibit U-shaped retention factor curves and for each, we determine the critical gradient slope beyond which 100% recovery is no longer possible. Elution with a reverse gradient is also demonstrated at low salt concentrations for these proteins. Understanding this behavior has implications in the design of gradient elution since the gradient slope impacts protein recovery.





Integrated strategy for identifying minor components in complex samples combining mass defect, diagnostic ions and neutral loss information based on ultra-performance liquid chromatography-high resolution mass spectrometry platform: Folium Artemisiae Argyi as a case study

Publication date: 18 May 2018 Source:Journal of Chromatography A, Volume 1550 Author(s): Dabing Ren, Lu Ran, Chong Yang, Meilin Xu, Lunzhao Yi Ultra-performance liquid chromatography coupled to high-resolution mass spectromet…

Publication date: 18 May 2018
Source:Journal of Chromatography A, Volume 1550

Author(s): Dabing Ren, Lu Ran, Chong Yang, Meilin Xu, Lunzhao Yi

Ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS) has been used as a powerful tool to profile chemicals in traditional Chinese medicines. However, identification of potentially bioactive compounds is still a challenging work because of the large amount of information contained in the raw UPLC-HRMS data. Especially the ubiquitous matrix interference makes it more difficult to characterize the minor components. Therefore, rapid recognition and efficient extraction of the corresponding parent ions is critically important for identifying the attractive compounds in complex samples. Herein, we propose an integrated filtering strategy to remove un-related or interference MS1 ions from the raw UPLC-HRMS data, which helps to retain the MS features of the target components and expose the compounds of interest as effective as possible. The proposed strategy is based on the use of a combination of different filtering methods, including nitrogen rule, mass defect, and neutral loss/diagnostic fragment ions filtering. The strategy was validated by rapid screening and identification of 16 methoxylated flavonoids and 55 chlorogenic acids analogues from the raw UPLC-HRMS dataset of Folium Artemisiae Argyi. Particularly, successful detection of several minor components indicated that the integrated strategy has obvious advantages over individual filtering methods, and it can be used as a promising method for screening and identifying compounds from complex samples, such as herbal medicines.





Graphene-coated polystyrene-divinylbenzene dispersive solid-phase extraction coupled with supercritical fluid chromatography for the rapid determination of 10 allergenic disperse dyes in industrial wastewater samples

Publication date: 18 May 2018
Source:Journal of Chromatography A, Volume 1550
Author(s): Chaoyan Lou, Can Wu, Kai Zhang, Dandan Guo, Lei Jiang, Yang Lu, Yan Zhu
Allergenic disperse dyes are a group of environmental contaminants, which are toxic and mutagenic to human beings. In this work, a method of dispersive solid-phase extraction (d-SPE) using graphene-coated polystyrene-divinylbenzene (G@PS-DVB) microspheres coupled with supercritical fluid chromatography (SFC) was proposed for the rapid determination of 10 allergenic disperse dyes in industrial wastewater samples. G@PS-DVB microspheres were synthesized by coating graphene (G) sheets onto polystyrene-divinylbenzene (PS-DVB) polymers. Such novel sorbents were employed in d-SPE for the purification and concentration of allergenic disperse dyes in wastewater samples prior to the determination by SFC with UV detection. To achieve the maximum extraction efficiency for the target dyes, several parameters influencing d-SPE process such as sorbent dosage, extraction time, desorption conditions were investigated. SFC conditions including stationary phase, modifier composition and percentage, column temperature, backpressure and flow rate were optimized to well separate the allergenic disperse dyes. Under the optimum conditions, satisfactory linear relationship (R ≥ 0.9989) was observed with the concentration of dyes ranging from 0.02 to 10.0 μg/mL. The limits of detection (LOD, S/N = 3) for the ten dyes were in the range of 1.1–15.6 ng/mL. Recoveries for the spiked samples were between 89.1% and 99.7% with relative standard deviations (RSD) lower than 10.5% in all cases. The proposed method is time-saving, green, precise and repeatable for the analysis of the target dyes. Furthermore, the application of G@PS-DVB based d-SPE process can be potentially expanded to isolate and concentrate other aromatic compounds in various matrices and supercritical fluid chromatography methodology featuring rapidity, accuracy and green will be an ideal candidate for the analysis of these compounds.

Publication date: 18 May 2018
Source:Journal of Chromatography A, Volume 1550

Author(s): Chaoyan Lou, Can Wu, Kai Zhang, Dandan Guo, Lei Jiang, Yang Lu, Yan Zhu

Allergenic disperse dyes are a group of environmental contaminants, which are toxic and mutagenic to human beings. In this work, a method of dispersive solid-phase extraction (d-SPE) using graphene-coated polystyrene-divinylbenzene (G@PS-DVB) microspheres coupled with supercritical fluid chromatography (SFC) was proposed for the rapid determination of 10 allergenic disperse dyes in industrial wastewater samples. G@PS-DVB microspheres were synthesized by coating graphene (G) sheets onto polystyrene-divinylbenzene (PS-DVB) polymers. Such novel sorbents were employed in d-SPE for the purification and concentration of allergenic disperse dyes in wastewater samples prior to the determination by SFC with UV detection. To achieve the maximum extraction efficiency for the target dyes, several parameters influencing d-SPE process such as sorbent dosage, extraction time, desorption conditions were investigated. SFC conditions including stationary phase, modifier composition and percentage, column temperature, backpressure and flow rate were optimized to well separate the allergenic disperse dyes. Under the optimum conditions, satisfactory linear relationship (R ≥ 0.9989) was observed with the concentration of dyes ranging from 0.02 to 10.0 μg/mL. The limits of detection (LOD, S/N = 3) for the ten dyes were in the range of 1.1–15.6 ng/mL. Recoveries for the spiked samples were between 89.1% and 99.7% with relative standard deviations (RSD) lower than 10.5% in all cases. The proposed method is time-saving, green, precise and repeatable for the analysis of the target dyes. Furthermore, the application of G@PS-DVB based d-SPE process can be potentially expanded to isolate and concentrate other aromatic compounds in various matrices and supercritical fluid chromatography methodology featuring rapidity, accuracy and green will be an ideal candidate for the analysis of these compounds.





Liquid chromatography – high resolution mass spectrometry method for monitoring of 17 mycotoxins in human plasma for exposure studies

Publication date: 4 May 2018
Source:Journal of Chromatography A, Volume 1548
Author(s): Irina Slobodchikova, Dajana Vuckovic
Mycotoxins are secondary metabolites produced by filamentous fungi. Primary route of human exposure to mycotoxins is the intake of the contaminated food. Minimizing mycotoxin exposure is important for population health, as their chronic toxic effects have been associated with kidney and liver diseases, some types of cancer and immunosuppression. The objective of this work was to develop and validate a multi-class mycotoxin method suitable for exposure monitoring of mycotoxins in human plasma. A sensitive liquid chromatography – mass spectrometry method was developed for 17 mycotoxins: nivalenol (NIV), deoxynivalenol, fusarenon X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, T-2 toxin, HT-2 toxin, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, zearalenone, α-zearalenol (α-ZOL), β-zearalenol, zearalanone, α-zeranol and, and β-zeranol. The method relies on three-step liquid–liquid extraction with ethyl acetate to eliminate the need for immunoaffinity extraction and minimize ionization matrix effects. Chromatographic separation of mycotoxins, including all isomers, was achieved with pentafluorophenyl column and water/methanol mobile phase. Mycotoxin detection and quantitation were performed using high-resolution mass spectrometry on LTQ Velos Orbitrap, in both positive and negative electrospray ionization (ESI(+) and (ESI(−)). The use of 0.02% acetic acid as mobile phase additive for ESI(−) resulted in significant increase in ionization efficiency ranging from 1.7 to 26 times for mycotoxins that ionize better in ESI(−). The optimized method was validated according to FDA guidance procedures. LOQs of all mycotoxins ranged from 0.1 to 0.5 ng/ml, except NIV which resulted in LOQ of 3 ng/ml because of low extraction recovery of this highly polar mycotoxin. Mean intra-day accuracy ranged from 85.8% to 116.4%, and intra-day precision (n = 6) ranged from 1.6% to 12.5% RSD for all mycotoxins except α-ZOL where mean accuracy ranged from 72.9% to 97.2%. Inter-day accuracy and precision were 85.6%–111.5% and 2.7–15.6% RSD respectively, showing good analytical performance of the method for biomonitoring.

Publication date: 4 May 2018
Source:Journal of Chromatography A, Volume 1548

Author(s): Irina Slobodchikova, Dajana Vuckovic

Mycotoxins are secondary metabolites produced by filamentous fungi. Primary route of human exposure to mycotoxins is the intake of the contaminated food. Minimizing mycotoxin exposure is important for population health, as their chronic toxic effects have been associated with kidney and liver diseases, some types of cancer and immunosuppression. The objective of this work was to develop and validate a multi-class mycotoxin method suitable for exposure monitoring of mycotoxins in human plasma. A sensitive liquid chromatography – mass spectrometry method was developed for 17 mycotoxins: nivalenol (NIV), deoxynivalenol, fusarenon X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, T-2 toxin, HT-2 toxin, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, zearalenone, α-zearalenol (α-ZOL), β-zearalenol, zearalanone, α-zeranol and, and β-zeranol. The method relies on three-step liquid–liquid extraction with ethyl acetate to eliminate the need for immunoaffinity extraction and minimize ionization matrix effects. Chromatographic separation of mycotoxins, including all isomers, was achieved with pentafluorophenyl column and water/methanol mobile phase. Mycotoxin detection and quantitation were performed using high-resolution mass spectrometry on LTQ Velos Orbitrap, in both positive and negative electrospray ionization (ESI(+) and (ESI(−)). The use of 0.02% acetic acid as mobile phase additive for ESI(−) resulted in significant increase in ionization efficiency ranging from 1.7 to 26 times for mycotoxins that ionize better in ESI(−). The optimized method was validated according to FDA guidance procedures. LOQs of all mycotoxins ranged from 0.1 to 0.5 ng/ml, except NIV which resulted in LOQ of 3 ng/ml because of low extraction recovery of this highly polar mycotoxin. Mean intra-day accuracy ranged from 85.8% to 116.4%, and intra-day precision (n = 6) ranged from 1.6% to 12.5% RSD for all mycotoxins except α-ZOL where mean accuracy ranged from 72.9% to 97.2%. Inter-day accuracy and precision were 85.6%–111.5% and 2.7–15.6% RSD respectively, showing good analytical performance of the method for biomonitoring.





A screening method for cardiovascular active compounds in marine algae

Publication date: 18 May 2018 Source:Journal of Chromatography A, Volume 1550 Author(s): S. Agatonovic-Kustrin, E. Kustrin, M.J. Angove, D.W. Morton The interaction of bioactive compounds from ethanolic extracts of selected mar…

Publication date: 18 May 2018
Source:Journal of Chromatography A, Volume 1550

Author(s): S. Agatonovic-Kustrin, E. Kustrin, M.J. Angove, D.W. Morton

The interaction of bioactive compounds from ethanolic extracts of selected marine algae samples, separated on chromatographic plates, with nitric/nitrous acid was investigated. The nature of bioactive compounds in the marine algae extracts was characterised using UV absorption spectra before and after reaction with diluted nitric acid, and from the characteristic colour reaction after derivatization with anisaldehyde. It was found that diterpenes from Dictyota dichotoma, an edible brown algae, and sterols from green algae Caulerpa brachypus, bind nitric oxide and may act as a nitric oxide carrier. Although the carotenoid fucoxanthin, found in all brown marine algae also binds nitric oxide, the bonds between nitrogen and the fucoxanthin molecule are much stronger. Further studies are required to evaluate the effects of diterpenes from Dictyota dichotoma and sterols from green algae Caulerpa brachypus to see if they have beneficial cardiovascular effects. The method reported here should prove useful in screening large numbers of algae species for compounds with cardiovascular activity.





Flow variation as a factor determining repeatability of the internal standard-based qualitative and quantitative analyses by capillary electrophoresis

Publication date: 4 May 2018
Source:Journal of Chromatography A, Volume 1548
Author(s): Paweł Mateusz Nowak, Michał Woźniakiewicz, Paweł Kościelniak
The use of migration times and peak areas referred to another sample component − internal standard, brings many benefits in improving reliability of capillary electrophoresis. However, it is quite commonly overlooked that despite relative migration time and peak area ratio are more stable than the absolute values upon alteration in the flow rate, some shift should always be expected. The present work offers a new look at this analytically-important issue. We have derived a simple model allowing to estimate the magnitude of error for the selected pair of molecules of known mobilities upon the given flow alteration. Then, we have confronted the theoretical predictions with the experimental results obtained for the model sample separated in various flow conditions reached by the external pressure manipulation, including several internal standards of different mobilities. A good agreement has been obtained, pointing out that the magnitude of error may be large even for the seemingly “good” internal standards. Several potentially useful means have been tested to address this issue: the use of electrophoretic mobilities and electrophoretic mobility ratios instead migration times in the qualitative analysis, and performing time-correction of peak area ratios, or alternatively, transformation of electropherograms from the time-related scale into the electrophoretic mobility-related scale in the quantitative analysis. We have also considered some additional factors. The results may be of interest for all users dealing with the development and optimization of analytical methods using capillary electrophoresis.

Publication date: 4 May 2018
Source:Journal of Chromatography A, Volume 1548

Author(s): Paweł Mateusz Nowak, Michał Woźniakiewicz, Paweł Kościelniak

The use of migration times and peak areas referred to another sample component − internal standard, brings many benefits in improving reliability of capillary electrophoresis. However, it is quite commonly overlooked that despite relative migration time and peak area ratio are more stable than the absolute values upon alteration in the flow rate, some shift should always be expected. The present work offers a new look at this analytically-important issue. We have derived a simple model allowing to estimate the magnitude of error for the selected pair of molecules of known mobilities upon the given flow alteration. Then, we have confronted the theoretical predictions with the experimental results obtained for the model sample separated in various flow conditions reached by the external pressure manipulation, including several internal standards of different mobilities. A good agreement has been obtained, pointing out that the magnitude of error may be large even for the seemingly “good” internal standards. Several potentially useful means have been tested to address this issue: the use of electrophoretic mobilities and electrophoretic mobility ratios instead migration times in the qualitative analysis, and performing time-correction of peak area ratios, or alternatively, transformation of electropherograms from the time-related scale into the electrophoretic mobility-related scale in the quantitative analysis. We have also considered some additional factors. The results may be of interest for all users dealing with the development and optimization of analytical methods using capillary electrophoresis.





Gold nanoparticles dispersion stability under dynamic coating conditions in capillary zone electrophoresis

Publication date: 18 May 2018 Source:Journal of Chromatography A, Volume 1550 Author(s): Szymon Dziomba, Krzesimir Ciura, Paulina Kocialkowska, Adam Prahl, Bartosz Wielgomas Capillary zone electrophoresis (CZE) of unmodified …

Publication date: 18 May 2018
Source:Journal of Chromatography A, Volume 1550

Author(s): Szymon Dziomba, Krzesimir Ciura, Paulina Kocialkowska, Adam Prahl, Bartosz Wielgomas

Capillary zone electrophoresis (CZE) of unmodified gold nanoparticles (Au NPs) was investigated in terms of dispersion stability in a presence of buffering counter-ions in background electrolyte (BGE). Capillary length, migration time and electric field strength were identified among factors influencing particles CZE. Moreover, BGE electrolysis was found to significantly affect analyses repeatability. The adsorption of NPs to capillary wall was recognized as the main problem. It was shown that this inconvenience can be overcome by the application of relatively big counter-ions. According to this observation, steric stabilization of NPs suspension by BGE components during CZE was hypothesized. In result, repeatable CZE of bare Au NPs under dynamic coating conditions was shown.





Fabrication of a high selectivity magnetic solid phase extraction adsorbent based on β-cyclodextrin and application for recognition of plant growth regulators

Publication date: 27 April 2018
Source:Journal of Chromatography A, Volume 1547
Author(s): Jiuyan Chen, Shurui Cao, Ming Zhu, Cunxian Xi, Lei Zhang, Xianliang Li, Guomin Wang, Yuantao Zhou, Zhiqiong Chen
An adsorbent, consisting of silica-coated Fe3O4 grafted graphene oxide and β-cyclodextrin (Fe3O4@SiO2/GO/β-CD), which possessed the merits of antioxidation, superparamagnetism, high surface area, high supramolecular recognition and environment friendly, was successfully fabricated. Considering the synergy between β-CD and graphene oxide in adsorption mechanism, the synthesized adsorbent could grasp compounds especially with aromatic structures through π-π interaction, hydrophobic interaction and host-guest inclusion complex forming. Based on the advantages, a magnetic solid phase extraction (MSPE) method for 9 PGRs using Fe3O4@SiO2/GO/β-CD as adsorbents was developed in this study. The characterizations of Fe3O4@SiO2/GO/β-CD were performed on Fourier transform infrared spectrometry (FT-IR), X-ray photoelectron spectroscopy (XPS), CHNS/O elemental analyzer, scanning electron microscopy (SEM) and vibrating sample magnetometry (VSM). Under the optimal MSPE condition, the Fe3O4@SiO2/GO/β-CD exhibited selectivity capability toward 9 PGRs when compared with Fe3O4@SiO2/GO. Meanwhile, the selectivity capability of Fe3O4@SiO2/GO/β-CD was higher than that of Fe3O4@SiO2/GO/α-CD except for 4-FPA. When the developed MSPE procedure was coupled with ultra-high performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry (UHPLC-QTrap-MS/MS) to quantitative analysis of 9 PGRs, linearities ranging from 2 to 50 μg/kg were achieved for 9 PGRs with the correlation coefficients (r2) in the range of 0.9975-0.9999. The limits of detection (LODs) for 9 analytes varied from 0.04 to 0.29 μg/kg. Finally, the proposed technique was applied to analyze PGRs residues in mutiple vegetable samples.

Publication date: 27 April 2018
Source:Journal of Chromatography A, Volume 1547

Author(s): Jiuyan Chen, Shurui Cao, Ming Zhu, Cunxian Xi, Lei Zhang, Xianliang Li, Guomin Wang, Yuantao Zhou, Zhiqiong Chen

An adsorbent, consisting of silica-coated Fe3O4 grafted graphene oxide and β-cyclodextrin (Fe3O4@SiO2/GO/β-CD), which possessed the merits of antioxidation, superparamagnetism, high surface area, high supramolecular recognition and environment friendly, was successfully fabricated. Considering the synergy between β-CD and graphene oxide in adsorption mechanism, the synthesized adsorbent could grasp compounds especially with aromatic structures through π-π interaction, hydrophobic interaction and host-guest inclusion complex forming. Based on the advantages, a magnetic solid phase extraction (MSPE) method for 9 PGRs using Fe3O4@SiO2/GO/β-CD as adsorbents was developed in this study. The characterizations of Fe3O4@SiO2/GO/β-CD were performed on Fourier transform infrared spectrometry (FT-IR), X-ray photoelectron spectroscopy (XPS), CHNS/O elemental analyzer, scanning electron microscopy (SEM) and vibrating sample magnetometry (VSM). Under the optimal MSPE condition, the Fe3O4@SiO2/GO/β-CD exhibited selectivity capability toward 9 PGRs when compared with Fe3O4@SiO2/GO. Meanwhile, the selectivity capability of Fe3O4@SiO2/GO/β-CD was higher than that of Fe3O4@SiO2/GO/α-CD except for 4-FPA. When the developed MSPE procedure was coupled with ultra-high performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry (UHPLC-QTrap-MS/MS) to quantitative analysis of 9 PGRs, linearities ranging from 2 to 50 μg/kg were achieved for 9 PGRs with the correlation coefficients (r2) in the range of 0.9975-0.9999. The limits of detection (LODs) for 9 analytes varied from 0.04 to 0.29 μg/kg. Finally, the proposed technique was applied to analyze PGRs residues in mutiple vegetable samples.





Enantioselective determination of aspartate and glutamate in biological samples by ultrasonic-assisted derivatization coupled with capillary electrophoresis and linked to Alzheimer’s disease progression

Publication date: 18 May 2018
Source:Journal of Chromatography A, Volume 1550
Author(s): Ya-Hui Hsieh, Fang-Yi Liao, Yuan-Han Yang, Jing-Ru Weng, Su-Hwei Chen, Chia-Hsien Feng
A simple cyclodextrin-mediated capillary zone electrophoresis method equipped with a laser-induced fluorescence detector was developed for chiral analysis of the excitatory amino acids aspartate (Asp) and glutamate (Glu). Plasma and cerebrospinal fluid (CSF) samples were pretreated with centrifugal filter devices before analysis to remove high-molecular-weight proteins (molecular weight cut-off: 3000) and then derivatized using 10 mM 6-carboxyfluorescein N-hydroxysuccinimide ester in DMSO under sonication at 25 °C for 2 h. After the derivatization reaction, reacted samples were diluted 100-fold with 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein hydrochloride (DTAF) solution and then hydrodynamically subjected to capillary electrophoresis (0.5 psi for 5 s, injection volume 8.27 nL). The separation buffer consisted of 50 mM borate buffer (pH 9.0) with 6 mM γ-CD and 0.1% polyvinylpyrrolidone, and the separation voltage was set at 20 kV. In the linearity calculations for the determination of d/l-Asp and d/l-Glu in plasma and CSF, a standard addition method was utilized to spike solutions with 0–20.0 μg mL−1l-Glu and 0–2.0 μg mL−1d-Glu and d/l-Asp to construct calibration curves. Correlation coefficients were above 0.998 for every analyte. The limits of detection (S/N = 3) for d/l-Asp and d/l-Glu standard solutions were 0.85–0.96 μg mL−1. The proposed method was applied successfully to determine d/l-Asp and d/l-Glu concentrations in the plasma and CSF samples of 26 patients with Alzheimer’s disease (AD), and the association between these concentrations and disease severity was investigated. Statistical analysis showed a moderately negative correlation (r = −0.158) between plasma l-Asp concentration and AD severity.

Publication date: 18 May 2018
Source:Journal of Chromatography A, Volume 1550

Author(s): Ya-Hui Hsieh, Fang-Yi Liao, Yuan-Han Yang, Jing-Ru Weng, Su-Hwei Chen, Chia-Hsien Feng

A simple cyclodextrin-mediated capillary zone electrophoresis method equipped with a laser-induced fluorescence detector was developed for chiral analysis of the excitatory amino acids aspartate (Asp) and glutamate (Glu). Plasma and cerebrospinal fluid (CSF) samples were pretreated with centrifugal filter devices before analysis to remove high-molecular-weight proteins (molecular weight cut-off: 3000) and then derivatized using 10 mM 6-carboxyfluorescein N-hydroxysuccinimide ester in DMSO under sonication at 25 °C for 2 h. After the derivatization reaction, reacted samples were diluted 100-fold with 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein hydrochloride (DTAF) solution and then hydrodynamically subjected to capillary electrophoresis (0.5 psi for 5 s, injection volume 8.27 nL). The separation buffer consisted of 50 mM borate buffer (pH 9.0) with 6 mM γ-CD and 0.1% polyvinylpyrrolidone, and the separation voltage was set at 20 kV. In the linearity calculations for the determination of d/l-Asp and d/l-Glu in plasma and CSF, a standard addition method was utilized to spike solutions with 0–20.0 μg mL−1 l-Glu and 0–2.0 μg mL−1 d-Glu and d/l-Asp to construct calibration curves. Correlation coefficients were above 0.998 for every analyte. The limits of detection (S/N = 3) for d/l-Asp and d/l-Glu standard solutions were 0.85–0.96 μg mL−1. The proposed method was applied successfully to determine d/l-Asp and d/l-Glu concentrations in the plasma and CSF samples of 26 patients with Alzheimer’s disease (AD), and the association between these concentrations and disease severity was investigated. Statistical analysis showed a moderately negative correlation (r = −0.158) between plasma l-Asp concentration and AD severity.





In-line Fourier-transform infrared spectroscopy as a versatile process analytical technology for preparative protein chromatography

Publication date: 27 April 2018
Source:Journal of Chromatography A, Volume 1547
Author(s): Steffen Großhans, Matthias Rüdt, Adrian Sanden, Nina Brestrich, Josefine Morgenstern, Stefan Heissler, Jürgen Hubbuch
Fourier-transform infrared spectroscopy (FTIR) is a well-established spectroscopic method in the analysis of small molecules and protein secondary structure. However, FTIR is not commonly applied for in-line monitoring of protein chromatography. Here, the potential of in-line FTIR as a process analytical technology (PAT) in downstream processing was investigated in three case studies addressing the limits of currently applied spectroscopic PAT methods. A first case study exploited the secondary structural differences of monoclonal antibodies (mAbs) and lysozyme to selectively quantify the two proteins with partial least squares regression (PLS) giving root mean square errors of cross validation (RMSECV) of 2.42 g/l and 1.67 g/l, respectively. The corresponding Q2 values are 0.92 and, respectively, 0.99, indicating robust models in the calibration range. Second, a process separating lysozyme and PEGylated lysozyme species was monitored giving an estimate of the PEGylation degree of currently eluting species with RMSECV of 2.35 g/l for lysozyme and 1.24 g/l for PEG with Q2 of 0.96 and 0.94, respectively. Finally, Triton X-100 was added to a feed of lysozyme as a typical process-related impurity. It was shown that the species could be selectively quantified from the FTIR 3D field without PLS calibration. In summary, the proposed PAT tool has the potential to be used as a versatile option for monitoring protein chromatography. It may help to achieve a more complete implementation of the PAT initiative by mitigating limitations of currently used techniques.

Graphical abstract

image

Publication date: 27 April 2018
Source:Journal of Chromatography A, Volume 1547

Author(s): Steffen Großhans, Matthias Rüdt, Adrian Sanden, Nina Brestrich, Josefine Morgenstern, Stefan Heissler, Jürgen Hubbuch

Fourier-transform infrared spectroscopy (FTIR) is a well-established spectroscopic method in the analysis of small molecules and protein secondary structure. However, FTIR is not commonly applied for in-line monitoring of protein chromatography. Here, the potential of in-line FTIR as a process analytical technology (PAT) in downstream processing was investigated in three case studies addressing the limits of currently applied spectroscopic PAT methods. A first case study exploited the secondary structural differences of monoclonal antibodies (mAbs) and lysozyme to selectively quantify the two proteins with partial least squares regression (PLS) giving root mean square errors of cross validation (RMSECV) of 2.42 g/l and 1.67 g/l, respectively. The corresponding Q 2 values are 0.92 and, respectively, 0.99, indicating robust models in the calibration range. Second, a process separating lysozyme and PEGylated lysozyme species was monitored giving an estimate of the PEGylation degree of currently eluting species with RMSECV of 2.35 g/l for lysozyme and 1.24 g/l for PEG with Q 2 of 0.96 and 0.94, respectively. Finally, Triton X-100 was added to a feed of lysozyme as a typical process-related impurity. It was shown that the species could be selectively quantified from the FTIR 3D field without PLS calibration. In summary, the proposed PAT tool has the potential to be used as a versatile option for monitoring protein chromatography. It may help to achieve a more complete implementation of the PAT initiative by mitigating limitations of currently used techniques.

Graphical abstract

image