Three-dimensional ionic liquid-ferrite functionalized graphene oxide nanocomposite for pipette-tip solid phase extraction of 16 polycyclic aromatic hydrocarbons in human blood sample J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-21 Yun Zhang, Yong-Gang Zhao, Wei-Sheng Chen, He-Li Cheng, Xiu-Qiong Zeng, Yan Zhu
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitously found in the environment and have been proved to be prospectively associated with the risk of cancer. In this study, a simple method based on pipette-tip solid phase extraction (PT-SPE) and gas chromatography-mass spectrometry (GC-MS) has been firstly developed for the determination of 16 PAHs in human whole blood. Three-dimensional ionic liquid-ferrite functionalized graphene oxide nanocomposite (3D-IL-Fe3O4-GO) was used as sorbent in PT-SPE. Compared with conventional SPE method, the PT-SPE method was solvent-saving (1.0 mL), reusable (at least 10 times) and required less blood sample (200 μL). Affecting parameters on extraction efficiency were investigated and optimized. Under the optimized conditions, a good linearity was obtained and the recoveries of 16 PAHs at three spiked levels ranged from 85.0% to 115%. The limits of quantification (LOQs) were in the range of 0.007-0.013 μg/L. Furthermore, the developed method was successfully applied to the analysis of 16 PAHs in 14 human blood samples. The results showed that the predominant PAHs in human whole blood was low-molecular-weight PAHs, with the rank order phenanthrene (PHE)> naphthalene (NAP)> fluorene (FLU)> fluoranthene (FLT)> pyrene (PYR). Because of its simplicity, accuracy and reliability, the PT-SPE method combined with GC-MS demonstrated the applicability for clinical analysis and provided more information for PAHs exposure studies.
Utility of a high coverage phenyl-bonding and wide-pore superficially porous particle for the analysis of monoclonal antibodies and related products J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-21 Balázs Bobály, Matthew Lauber, Alain Beck, Davy Guillarme, Szabolcs Fekete
A wide-pore silica-based superficially porous material with a high coverage phenyl bonding was evaluated for the analysis of monoclonal antibodies and antibody-drug conjugates. This new material is based on 2.7 μm particles having a shell thickness of 0.40 μm and average pore size of approximately 450 Å. Various important features of this reversed phase column technology were explored, including kinetic performance for large biomolecules (i.e. speed of analysis, efficiency and peak capacity), recovery of proteins, selectivity for resolving modifications, and the possibility to reduce the amount of trifluoroacetic acid in the mobile phase. A systematic comparison was also performed with other existing modern wide-pore phases possessing differences in structure/morphology and chemistry. If all these figures of merit are considered, it is clear that this phenyl bonded wide-pore superficially porous stationary phase is one of the most promising materials to have been developed in recent years. Indeed, it offers kinetic performance comparable to the most efficient wide-pore SPP column on the market. In terms of protein recovery, this new phase was found to be superior to silica-based and silica-hybrid C4 bonded materials, particularly with separations performed at sub-80 °C temperature. Under such conditions, it in fact shows recoveries that are quite similar to a divinyl benzene (DVB) polymer-based material. More importantly, due to its unique, high coverage phenyl bonding, it offers additional steric effects and potentially even π-π interactions that yield advantageous selectivity for mAb sub-unit peaks and ADC species as compared to commonly used C4 or C18 bonded phases. Last but not least, mobile phases consisting of only 0.02–0.05% trifluoroacetic acid can be successfully used with this column, without significant loss in recovery and peak capacity.
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 J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-21 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.
QSRR modeling for the chromatographic retention behavior of some β-lactam antibiotics using forward and firefly variable selection algorithms coupled with multiple linear regression J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-21 Marwa A. Fouad, Enas H. Tolba, Manal A. El-Shal, Ahmed M. El Kerdawy
The justified continuous emerging of new β-lactam antibiotics provokes the need for developing suitable analytical methods that accelerate and facilitate their analysis. A face central composite experimental design was adopted using different levels of phosphate buffer pH, acetonitrile percentage at zero time and after 15 min in a gradient program to obtain the optimum chromatographic conditions for the elution of 31 β-lactam antibiotics. Retention factors were used as the target property to build two QSRR models utilizing the conventional forward selection and the advanced nature-inspired firefly algorithm for descriptor selection, coupled with multiple linear regression. The obtained models showed high performance in both internal and external validation indicating their robustness and predictive ability. Williams-Hotelling test and student’s t-test showed that there is no statistical significant difference between the models’ results. Y-randomization validation showed that the obtained models are due to significant correlation between the selected molecular descriptors and the analytes’ chromatographic retention. These results indicate that the generated FS-MLR and FFA-MLR models are showing comparable quality on both the training and validation levels. They also gave comparable information about the molecular features that influence the retention behavior of β-lactams under the current chromatographic conditions. We can conclude that in some cases simple conventional feature selection algorithm can be used to generate robust and predictive models comparable to that are generated using advanced ones.
Validation and application of analytical method for glyphosate and glufosinate in foods by liquid chromatography-tandem mass spectrometry J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-20 Yang Liao, Jean-Marie Berthion, Isabelle Colet, Mathilde Merlo, Alexandre Nougadère, Renwei Hu
A reliable and sensitive method was developed for simultaneous determination of glyphosate and glufosinate in various food products by liquid chromatography-tandem mass spectrometry. Based on extraction, derivatization with 9-fluorenylmethylchloroformate and purification on solid phase extraction column, quantification was done by using isotopic-labeled analytes as internal standard and calibration in matrix. Good selectivity and sensitivity were achieved with a limit of quantification of 5 μg/kg. The recoveries of these two pesticides ranged from 91% to 114% with inter-day and relative standard deviation of 3.8 to 6.1% in five matrices of cereal group spiked at 5, 10, and 20 μg/kg. An accuracy profile was performed for method validation, demonstrating the accuracy and precision of the method for the studied food groups. The verification results in expanded food groups indicated extensive applicability for the analysis of glyphosate and glufosinate. Finally, the developed method was applied to analyze 136 food samples including milk-based baby foods from the French Agency for Food, Environmental and Occupational Health & Safety. Glyphosate residues were detected in two breakfast cereal samples (6.0 and 34 μg/kg). Glufosinate residues were found in a sample of boiled potatoes (9.8 μg/kg). No residues were detected in the other samples, including milk-based baby foods with limits of detection ranging from 1 to 2 μg/kg. The method has been applied for routine national monitoring of glyphosate and glufosinate in various foods.
High temperature multidimensional gas chromatographic approach for improved separation of triacylglycerols in olive oil J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-19 Habtewold D. Waktola, Chadin Kulsing, Yada Nolvachai, Philip J. Marriott
Heart-cut multidimensional gas chromatographic (H/C MDGC) methods under suitable flow and high temperature (T) program conditions were developed to separate olive oil triacylglycerols (TAGs). Different column sets were selected for further evaluation, each with relatively short non-polar first dimension (1D) and mid-polar second dimension (2D) columns of high T limits (350 °C). The 1D separation displayed three major groups of peaks in an area ratio of approximately 5:33:62 (of increasing retention), using flame ionisation detection (FID). Four groups of minor peaks, with 2 of them located between the major peaks, were also detected. The H/C fractions of the minor peaks, and sub-sampled regions across the major peaks eluting from the 1D outlet, were cryotrapped at the 2D inlet. The trapped TAGs then underwent temperature programmed 2D separation. Each of the ‘H/C’ zones generally gave 2–5 – and in some cases more – separated peaks of TAGs on the 2D column, under suitable flow condition and phase polarity that resulted in improved separation. Six sub-sampled H/Cs from various regions of the individual peaks from the 1D column were simultaneously trapped and released to 2D, resulting in apparently more than 22 individual TAG peaks. According to their different retention times, different TAGs were revealed within each of the 3 major groups, using H/C sub-sampling. A comprehensive sampling strategy that covers most of the 1D peaks further revealed the presence of more TAGs in the olive oil sample. This tandem column strategy was able to resolve more components than that usually observed on a single column.
Gold nanoparticles dispersion stability under dynamic coating conditions in capillary zone electrophoresis ☆ J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-19 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.
Development of a fast and simple gas chromatographic protocol based on the combined use of alkyl chloroformate and solid phase microextraction for the assay of polyamines in human urine J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-19 Attilio Naccarato, Rosangela Elliani, Brunella Cavaliere, Giovanni Sindona, Antonio Tagarelli
Polyamines are aliphatic amines with low molecular weight that are widely recognized as one of the most important cancer biomarkers for early diagnosis and treatment. The goal of the work herein presented is the development of a rapid and simple method for the quantification of free polyamines (i.e., putrescine, cadaverine, spermidine, spermine) and N-monoacetylated polyamines (i.e., N1-Acetylspermidine, N8-Acetylspermidine, and N1-Acetylspermine) in human urine. A preliminary derivatization with propyl chloroformate combined with the use of solid phase microextraction (SPME) allowed for an easy and automatable protocol involving minimal sample handling and no consumption of organic solvents. The affinity of the analytes toward five commercial SPME coatings was evaluated in univariate mode, and the best result in terms of analyte extraction was achieved using the divinylbenzene/carboxen/polydimethylsiloxane fiber. The variables affecting the performance of SPME analysis were optimized by the multivariate approach of experimental design and, in particular, using a central composite design (CCD). The optimal working conditions in terms of response values are the following: extraction temperature 40 °C, extraction time of 15 min and no addition of NaCl. Analyses were carried out by gas chromatography-triple quadrupole mass spectrometry (GC-QqQ-MS) in selected reaction monitoring (SRM) acquisition mode. The developed method was validated according to the guidelines issued by the Food and Drug Administration (FDA). The satisfactory performances reached in terms of linearity, sensitivity (LOQs between 0.01 and 0.1 μg/mL), matrix effect (68–121%), accuracy, and precision (inter-day values between −24% and +16% and in the range 3.3–28.4%, respectively) make the proposed protocol suitable to be adopted for quantification of these important biomarkers in urine samples.
O-Carboxymethyl Chitosan Schiff Base Complexes as Affinity Ligands for Immobilized Metal-ion Affinity Chromatography of Lysozyme J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-19 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.
Supercritical fluid chromatography versus high performance liquid chromatography for enantiomeric and diastereoisomeric separations on coated polysaccharides-based stationary phases: application to dihydropyridone derivatives J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-19 Vanessa Hoguet, Julie Charton, Paul-Emile Hecquet, Chahinaze Lakhmi, Emmanuelle Lipka
For analytical applications, SFC has always remained in the shadow of LC. Analytical enantioseparation of eight dihydropyridone derivatives, was run in both High Performance Liquid Chromatography and Supercritical Fluid Chromatography. Four polysaccharide based chiral stationary phases namely amylose and cellulose tris(3, 5-dimethylphenylcarbamate), amylose tris((S)-α-phenylethylcarbamate) and cellulose tris(4-methylbenzoate) with four mobile phases consisted of either n-hexane/ethanol or propan-2-ol (80:20 v:v) or carbon dioxide/ethanol or propan-2-ol (80:20 v:v) mixtures were investigated under same operatory conditions (temperature and flow-rate). The elution strength, enantioselectivity and resolution were compared in the two methodologies. For these compounds, for most of the conditions, HPLC afforded shorter retention times and a higher resolution than SFC. HPLC appears particularly suitable for the separation of the compounds bearing two chiral centers. For instance compound 7 was baseline resolved on OD-H CSP under n-Hex/EtOH 80/20, with resolution values equal to 2.98, 1.55, 4.52, between the four stereoisomers in less than 17 minutes, whereas in SFC, this latter is not fully separated in 23 minutes under similar eluting conditions. After analytical screenings, the best conditions were transposed to semi-preparative scale.
Poly(butylene terephthalate) based novel achiral stationary phase investigated under supercritical fluid chromatography conditions J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-17 Kanji Nagai, Tohru Shibata, Satoshi Shinkura, Atsushi Ohnishi
Poly(butylene terephthalate) based novel stationary phase (SP), composed of planar aromatic phenyl group together with ester group monomer units, was designed for supercritical fluid chromatography (SFC) use. As expected from its structure, this phase shows planarity recognition of isomeric aromatics and closely similar compounds. Interestingly, for most analytes, the retention behavior of this SP is significantly distinct from that of the 2-ethylpyridine based SPs which is among the most well-known SFC dedicated phases. Although the poly(butylene terephthalate) is coated on silica gel, the performance of the column did not change by using extended range modifiers such as THF, dichloromethane or ethyl acetate and column robustness was confirmed by cycle durability testing.
Multi-class multi-residue analysis of veterinary drugs in meat using enhanced matrix removal lipid cleanup and liquid chromatography-tandem mass spectrometry J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-17 Limian Zhao, Derick Lucas, David Long, Bruce Richter, Joan Stevens
This study presents the development and validation of a quantitation method for the analysis of multi-class, multi-residue veterinary drugs s using lipid removal cleanup cartridges, enhanced matrix removal lipid (EMR-Lipid), for different meat matrices by liquid chromatography tandem mass spectrometry detection. Meat samples were extracted using a two-step solid-liquid extraction followed by pass-through sample cleanup. The method was optimized based on the buffer and solvent composition, solvent additive additions, and EMR-Lipid cartridge cleanup. The developed method was then validated in five meat matrices, porcine muscle, bovine muscle, bovine liver, bovine kidney and chicken liver to evaluate the method performance characteristics, such as absolute recoveries and precision at three spiking levels, calibration curve linearity, limit of quantitation (LOQ) and matrix effect. The results showed that >90% of veterinary drug analytes achieved satisfactory recovery results of 60–120%. Over 97% analytes achieved excellent reproducibility results (relative standard deviation (RSD) < 20%), and the LOQs were 1–5 μg/kg in the evaluated meat matrices. The matrix co-extractive removal efficiency by weight provided by EMR-lipid cartridge cleanup was 42–58% in samples. The post column infusion study showed that the matrix ion suppression was reduced for samples with the EMR-Lipid cartridge cleanup. The reduced matrix ion suppression effect was also confirmed with <15% frequency of compounds with significant quantitative ion suppression (>30%) for all tested veterinary drugs in all of meat matrices. The results showed that the two-step solid-liquid extraction provide efficient extraction for the entire spectrum of veterinary drugs, including the difficult classes such as tetracyclines, beta-lactams etc. EMR-Lipid cartridges after extraction provided efficient sample cleanup with easy streamlined protocol and minimal impacts on analytes recovery, improving method reliability and consistency.
Preparative concentration of nucleic acids fragments by capillary isotachophoretic analyzer ☆ J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-15 Vladimíra Datinská, Ivona Voráčová, Jan Berka, František Foret
Sample preparation plays an important role in the DNA analysis workflow. Real samples often include a complex matrix, such as blood and other bodily fluids, or exogenous impurities, e.g., from the scene of crime. Most of the common nucleic acids isolation techniques are based on extractions; however, isotachophoretic focusing has recently attracted some interest for its simplicity and potential for very high enrichment factors and ease of automation. Here, we report on the use of a commercial isotachophoretic instrument for optimization of DNA focusing and preparative fraction collection. In order to achieve a high recovery and enrichment, experimental factors including electric current, sample amount and matrix were investigated experimentally as well as by computer simulation. The sample of a DNA ladder was injected in 30 μl volume and after ITP focusing the DNA zone was recovered using an on-column micropreparative collection valve. The DNA content in the collected sample was verified by fluorescence spectrometry and chip capillary electrophoresis with fluorescence detection.
Liquid Chromatography – High Resolution Mass Spectrometry Method for Monitoring of 17 Mycotoxins in Human Plasma for Exposure Studies J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-15 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-zeranoland, 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% to 111.5% and 2.7 to 15.6% RSD respectively, showing good analytical performance of the method for biomonitoring.
Feasibility study for high-resolution multi-component separation of protein mixture using a cation-exchange cuboid packed-bed device J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-15 Guoqiang Chen, Alisha Gerrior, Raja Ghosh
In recent papers we have discussed the optimization of design and operating conditions for cuboid packed-bed device for chromatographic separations. The efficiency metrics used in these studies included the number of theoretical plates per unit bed height as well as attributes of flow-through and eluted peaks. These studies were carried out using equivalent columns as benchmarks. The cuboid packed-bed devices consistently outperformed the columns in terms of the above metrics. The current study examines how well, or indeed if at all these superior efficiency metrics translate to superiority in multi-component protein separation. Cation exchange resin was examined in the current study using appropriate multi-component model protein system which was chosen with close isoelectric points to make the separation challenging. Effects of operating and experimental parameters such as flow rate, loop size and linear gradient length on separation performance were systematically investigated. Separation metrics examined included peak width, tailing factor, asymmetry factor and resolution of separated protein peaks. The results obtained showed that the cation exchange cuboid packed-bed device significantly outperformed its equivalent commercial column (e.g., the number of theoretical plates per unit bed height was 8636/m for the cuboid packed-bed device as opposed to 1480/m for the column at a flow rate of 0.5 mL/min. The difference in efficiency was particularly high at lower flow rate and when shorter gradients were employed. The results suggest that the cuboid packed-bed devices could potentially have promising application in preparative separations such as biopharmaceutical purifications.
Construction of chiral ligand exchange capillary electrochromatography for D,L-amino acids enantioseparation and its application in glutaminase kinetics study ☆ J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-15 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 to 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.
Reversible Concanavalin A (Con A) ligands immobilization on metal chelated macroporous cellulose monolith and its selective adsorption for glycoproteins J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-15 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.
Enzyme assay for d-amino acid oxidase using optically gated capillary electrophoresis-laser induced fluorescence detection ☆ J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-14 Ning Zhang, Miaomiao Tian, Xin Liu, Li Yang
Because d-amino acids (AAs) play an essential role in the regulation of many processes in living cells, detection of D-AAs and assay of d-amino acid oxidase (DAAO) activity are of vital importance in bioanalytical science. However, the reliability and accuracy of DAAO assays could be interfered due to the facts that DAAO presents broad substrate activity towards different D-AAs and there could be abundant L-AA enantiomers in biological samples. In this study we presented the first application of optically gated capillary electrophoresis with LIF detection (OGCE-LIF) for efficient assay of DAAO activity. High repeatability of the OGCE-LIF assay of amino acids (AAs) was achieved with relative standard deviation (RSD) (n = 15) less than 1.5% and 2.7% for migration time and peak height, respectively. Under the optimal OGCE-LIF conditions, five pairs of D/L-AA enantiomers were efficiently separated in less than 1 min with low limit of detection of 1.3 μM. Enzymatic assays of DAAO were successfully achieved by detection the substrate consumption with OGCE-LIF, for either single or mixed AA substrates. Kinetic analysis of the parallel oxidation reactions of two different substrates was performed, which was in good agreement with the experimental results. Our study indicates OGCE-LIF can perform rapid and efficient separation of mixed pairs of AA enantiomers and is a promising method for quantitatively assaying DAAO catalyzed reaction with the presence of L-AA enantiomers in the sample. Our study would pave the way for accurate determination of D-AAs and DAAO enzymes in complicated biological samples.
Screening of Stationary Phase Selectivities for Global Lipid Profiling by Ultrahigh Performance Supercritical Fluid Chromatography J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-14 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 minutes.
Flow variation as a factor determining repeatability of the internal standard-based qualitative and quantitative analyses by capillary electrophoresis J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-14 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.
Development of an analytical method for urocanic acid isomers in fish based on reactive extraction cleanup and chaotropic chromatography techniques J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-14 Jian-Jun Zhong, Ningbo Liao, Mingfeng He, Yunfeng Pu, Donghong Liu
Urocanic acid (UA), existing in trans- or cis-isoform, is of fairly recent interest to food researchers because of its potential public health hazards of scombrotoxicity and immunotoxicity, as well as associating with fish spoilage. This work is among the first efforts to study the analytical chemistry of UA in fish. With 0.6 M perchloric acid UA was extracted, and co-extracted fish matrix components were efficiently removed through a reactive extraction of UA. The optimum conditions for the reactive extraction, which allowed an 80% recovery of UA, were sample pH adjustment to 9, twice extractions with 32% (w/w) di (2-ethylhexyl) phosphate in hexanol, and a back-extraction with 0.1 M hydrochloric acid at 1:1 phase ratio. A chaotropic hexafluorophosphate salt was added to acidic water-acetonitrile mobile phases to improve the reversed-phase chromatography of UA, which otherwise was poorly retained. Optimum separation conditions were obtained for fish samples and enabled a fast (10 min), convenient-to-use chromatography that clearly outperforms cumbersome legacy ion-pair chromatography. Intended for routine use in our laboratory, the proposed method passed an in-house validation test for linearity, matrix effect (on reactive extraction), accuracy, precision, and detectability.
Systematic profiling and comparison of the lipidomes from Panax ginseng, P. quinquefolius, and P. notoginseng by ultrahigh performance supercritical fluid chromatography/high-resolution mass spectrometry and ion mobility-derived collision cross section measurement J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-14 Xiaojian Shi, Wenzhi Yang, Shi Qiu, Jinjun Hou, Wanying Wu, Dean Guo
Lipidomics currently is still confronted with challenges from chromatographic separation and lipids identification. Here we report a lipidomics platform by integrating ultrahigh performance supercritical fluid chromatography/quadrupole time-of-flight mass spectrometry (UHPSFC/QTOF-MS) and collision cross section (CCS) measurement using ion mobility spectroscopy/time-of-flight mass spectrometry (IMS/QTOF-MS), aiming to enhance the profiling performance and identification reliability of lipids. The lipidomes extracted from three congeneric Panax species (P. ginseng, P. quinquefolius, and P. notoginseng) by methyl tert-butyl ether are comprehensively profiled and compared by use of this platform. A potent UHPSFC/QTOF-MS approach was developed on a 1.7-μm particles packed Torus 2-PIC column using CH3OH (in CO2) as a modifier and CH3OH/0.2 mM ammonium acetate as the makeup liquid, enabling well resolution of six lipid subclasses by both positive and negative MSE modes. In contrast to the reversed-phase chromatography, “normal-phase” like elution order and better resolution of polar lipids and some lipid isomers were achieved by UHPSFC separation. Pattern recognition chemometric analysis of 60 batches of Ginseng samples ultimately unveiled 24 lipid markers, of which triacylglycerols were the most important. Aside from the automated MS database searching against HMDB and LIPID MAPS, the application of CCS retrieval or CCS prediction improved lipid identification by reducing the possible hits. In conclusion, this integral platform can significantly improve the chromatographic separation and the reliability of lipids identification in lipidomics studies. It is the first report that systematically compares the lipidomic difference of three reputable Panax species, providing useful information for their quality control in addition to ginsenoside analysis.
Liquid chromatography with alkylammonium formate ionic liquid mobile phases and fluorescence detection J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-14 Neil D. Danielson, Fotouh R. Mansour, Ling Zhou, Casey V. Connell, Erin M. Dotlich, Justin N. Gibler, Blake E. Norman, Shau Grossman, Wenjun Wei, Ying Zhang
Fluorescence detection of various pharmaceuticals and the amino acid tryptophan (low molecular weight organic compounds) as well as the enzyme lactate dehydrogenase LDH (high molar mass compound) has been studied in aqueous solutions using alkyl ammonium formate ionic liquids as the primary solvent component. It was expected that the high viscosity of such ionic liquid-water mixtures would enhance fluorescence. Pharmaceuticals such as riboflavin and naproxen showed no such enhancement in the presence of ethylammonium formate (EAF) or isopropylammonium formate (IPAF) but the fluorescence of warfarin was substantially enhanced. However, this improved fluorescence using alkylammonium formates did not seem to be general for other coumarin compounds. Enhancement of tryptophan fluorescence was also seen for both EAF and IPAF. During the reversed phase elution of LDH on a polymeric HPLC column, remarkable enhancements in LDH peak intensity and activity were observed by incorporating 6% PEG 8000 in the organic mobile phase that contained either acetonitrile or IPAF. Using higher concentrations of PEG 8000 is not recommended, not only because of the high viscosity, but also because the stabilizing effect of PEG 8000 is gradually reduced at higher concentrations.
Development of a partitioned liquid-liquid extraction- dispersive solid phase extraction procedure followed by liquid chromatography-tandem mass spectrometry for analysis of 3-monochloropropane-1,2-diol diesters in edible oils ☆ J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-13 J.A. Custodio-Mendoza, R.A. Lorenzo, I.M. Valente, P.J. Almeida, M.A. Lage, J.A. Rodrigues, A.M. Carro
A fast and effective method using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach which includes partitioned liquid-liquid extraction (PLLE) and dispersive solid phase extraction (dSPE) clean-up step for the determination of seven 3-monochloropropane-1,2-diol (3-MCPD) fatty acid diesters in vegetable oils is developed and validated according to the Food and Drug Administration (FDA) guidelines. Due to the complexity of the matrices, combination of silica based sorbents (Silica Strong Anion Exchange (Si-SAX), Supel™ QuE Z-Sep+ (Z-Sep+) and Primary Secondary Amine (PSA) were tested for lipid removal. The effect of several experimental factors on the efficiency of the extraction procedure was studied by a screening design 3422//16 and a response surface Doehlert design. The separation and determination was carried out by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The method provided suitable linearity (r2 > 0.9960), precision (relative standard deviation (RSD) lower than 10%) and accuracy, in terms of recovery. The limits of detection (LOD) and limits of quantification (LOQ) ranged from 10 to 20 μg kg −1 and from 25 to 50 μg kg−1, respectively. The recoveries at three spiking levels of 100, 250, and 500 μg kg−1 were over the range of 71.4–122.9% with RSD lower than 13%. The method was successfully applied in edible oils and fatty food samples. The results provide valuable information to assess the risk of exposure to these foodborne contaminants.
Characterization of plant polysaccharides from dendrobium officinale by multiple chromatographic and mass spectrometric techniques ☆ J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-13 Huiying Ma, Keke Zhang, Qing Jiang, Diya Dai, Hongli Li, Wentao Bi, David Da Yong Chen
Plant polysaccharides have numerous medicinal functions. Due to the differences in their origins, regions of production, and cultivation conditions, the quality and the functions of polysaccharides can vary significantly. They are macromolecules with large molecular weight (MW) and complex structure, and pose great challenge for the analytical technology used. Taking Dendrobium officinale (DO) from various origins and locations as model samples. In this investigation, mechanochemical extraction method was used to successfully extract polysaccharides from DO using water as solvent, the process is simple, fast (40 s) and with high yield. The MWs of the intact saccharides from calibration curve and light scattering measurement were determined and compared after separation with size exclusion chromatography (SEC). The large polysaccharide was acid hydrolyzed to oligosaccharides and the products were efficiently separated and identified using liquid chromatography coupled to a high resolution tandem mass spectrometry (LC–MS2). Obvious differences were observed among LC–MS2 chromatograms of digested products, and the chemical structures for the products were proposed based on accurate mass values. More importantly, isomeric digested carbohydrate compounds were explored and characterized. All the chromatographic and mass spectrometric results in this study provided a multi-dimensional characterization, fingerprint analysis, and molecular structure level assessment of plant polysaccharides.
Facile synthesis of hierarchical porous carbon from crude biomass for high-performance solid-phase microextraction J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-13 Hu Cheng, Yang Song, Fang Wang, Rongting Ji, Xiaona Li, Yuhao Fu, Xinglun Yang, Yongrong Bian, Xin Jiang
Porous materials have great prospective applications for solid-phase microextraction (SPME) technology because of their large specific surface area and pore volume. In this study, a hierarchical porous carbon (HPC) was synthesized by simple hydrothermal reaction and potassium hydroxide (KOH) activation of crude biomass and found to be an efficient adsorbent for SPME of organic pollutants. Results show that the as-prepared HPC has a partly graphitic amorphous-like structure with ultrahigh specific surface area (2551 m2/g), high pore volume (1.53 cm3/g), good pore size distribution (PSD) (mesopore/micropore ratio of 68%), and great thermal stability (>450 °C). When we utilized it as SPME fiber coating, the extraction capacities for chlorobenzenes (CBs), polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and phthalates (PAEs) were, respectively, 1.13–39.46, 2.40–7.78, 1.34–36.02, and 1.50–1.83 times higher than those of a commercial polydimethylsiloxane (PDMS) fiber. Under the optimized extraction conditions, an analytical method for CBs with low detection limits (0.01–0.24 ng/L), good repeatability (1.00%–4.93% for intra-day, 1.11%–6.94% for inter-day), and great reproducibility (1.48%–8.91%, n = 3) was developed. Moreover, we evaluated the practicality of the developed method for environmental water sample and obtained satisfactory recoveries (86.21%–104.34%). The findings provide a novel and promising HPC from crude biomass using a low-cost and facile synthetic route for SPME applications.
Method development and optimization for the determination of benzene, toluene, ethylbenzene and xylenes in water at trace levels by static headspace extraction coupled to gas chromatography–barrier ionization discharge detection J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-13 Raffaella Pascale, Giuliana Bianco, Stefania Calace, Salvatore Masi, Ignazio M. Mancini, Giuseppina Mazzone, Donatella Caniani
Benzene, toluene, ethylbenzene, and xylenes, more commonly named BTEX, represent one of the most ubiquitous and hazardous groups of atmospheric pollutants. The goal of our research was the trace quantification of BTEX in water by using a new simple, low-cost, and accurate method, based on headspace (HS) extraction and gas chromatography (GC) coupled to barrier ionization discharge detector (BID). This water application dealt with simple matrices without protein, fat, or humic material that adsorb target analytes, thus the external standard calibration was suitable to quantify each compound. The validation steps included the study of linearity, detection and quantification limits, and accuracy. LODs and LOQs varied from 0.159 to 1.845 μg/L and from 0.202 to 2.452 μg/L, respectively. The recovery was between 0.74 ± 0.13 and 1.15 ± 0.09; relative standard deviations (% RDSs) were less than 12.81% (n = 5) and 14.84% (n = 10). Also, GC performance was evaluated in term of efficiency, peak tailing and resolution. Preliminary results from practical applications to analyses of real samples are presented. The results indicate that static HS coupled to GC–BID is a successful method for BTEX analysis in water samples at the μg/L levels, provided that hydrocarbons interference occur at similar concentration levels. GC-BID may become a routine reference method alongside the official analytical techniques for quality control purposes of contaminated waters. Moreover, the new method is amenable to automation by using commercial HS units.
A monolithic column based on covalent cross-linked polymer gels for online extraction and analysis of trace aflatoxins in food sample J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-12 Tianfu Wei, Zhengyi Chen, Gongke Li, Zhuomin Zhang
Aflatoxins are highly toxic mycotoxin contamination, which pose serious food safety incidents. It is very important to precisely and rapidly determine trace aflatoxins in food. In this study, we designed porous monolithic column based on covalent cross-linked polymer gels for online extraction and analysis of trace aflatoxins in food samples with complicated matrices coupled with high-performance liquid chromatography-ultraviolet detector (HPLC-UV). The prepared monolithic column showed excellent enrichment performance due to its good permeability, good reproducibility and long life span. The study of adsorption mechanism suggested that the excellent enrichment performance of this monolithic column was attributed to the multiple effect of π-π stacking interaction, hydrophobic effect and steric effect. When the online analytical method was applied for the determine of trace aflatoxins in real food samples, aflatoxins G1 and aflatoxins B1 could be actually found in one positive bean sauce sample and quantified to be 32.8 and 26.4 μg/kg, respectively. Aflatoxins G1 in one bean sample could be also found and quantified to be 25.9 μg/kg. The low detection limits of the developed method were achieved in range of 0.08-0.2 μg/kg. And the recoveries for spiked samples were in range from 76.1 to 113% with RSDs of 1.1-9.6%. The developed method was proved to be a promising method for online enrichment and analysis of trace aflatoxins in complicated food samples.
Reversed phase ion-pair chromatographic separation of sugar alcohols by complexation with molybdate ion J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-12 Tomoko Kemmei, Shuji Kodama, Atsushi Yamamoto, Yoshinori Inoue, Kazuichi Hayakawa
In this study, we developed a simple and sensitive reversed phase ion-pair chromatographic method for the analysis of C4-C6 sugar alcohols. The method is based on the on-line complexation of sugar alcohols with molybdate ion. The resulting dinuclear anionic complexes can be separated on a reversed-phase C18 column using tetrabutylammonium chloride as an ion-pairing reagent. The mobile phase (pH 3.1) consisted of 0.1 mM disodium molybdate, 1 mM hydrochloric acid and 0.4 mM tetrabutylammonium chloride − 10% v/v methanol. By complexing with molybdate ion, sugar alcohols can be detected by their UV absorption at 247 nm with high resolution and sensitivity. The quantification limits of the examined sugar alcohols calculated at S/N = 10 were 0.1 mM for erythritol and xylitol and 0.01 mM for arabitol, sorbitol, mannitol and dulcitol. The detector response was linear over three orders of magnitude of sugar alcohol concentration. The proposed method was successfully applied to measure sugar alcohols in health drinks, eyedrops and mouthwashes.
Multilevel characterization of marine microbial biodegradation potentiality by means of flow-modulated comprehensive two-dimensional gas chromatography combined with a triple quadrupole mass spectrometer J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-12 Mariosimone Zoccali, Simone Cappello, Luigi Mondello
The present research is focused on the use of a triple quadrupole mass spectrometer (QqQ MS) coupled with flow modulated comprehensive two-dimensional gas chromatography (FM GC × GC) for a multilevel elucidation of biodegradation potentiality of natural marine microbial populations during a bioremediation (biostimulation) treatment. The crude oil used for the evaluation of the bioremediation process, namely Dansk Blend Pier E1, represents a very complex sample. Hence, in order to understand the metabolic activity of microbial populations during the bioremediation process, a GC × GC system was used. The high separation power has allowed a detailed characterization of the different chemical families; moreover, thanks to the high acquisition frequency of the QqQ MS spectrometer, both full scan and multiple reaction monitoring (MRM) data were acquired in the same run. By using this system, both qualitative analysis of untargeted hydrocarbons mixture (crude oil) and qualitative analysis of biomarker compounds, present in low amount and often hindered under the bulk of the sample (i.e. adamantanes, diamantanes, steranes and hopanes), were performed simultaneously. The bioremediation capability of biostimulated bacteria was evaluated at four (T4), eight (T8) and fourteen (T14) days. Progressive degradation of linear, branched, and aromatic hydrocarbons, adamantanes, and diamantanes has been showed, whereas, results underline the lack of any kind of activity against steranes, and hopanes.
Quantitative determination of major oxidation products in edible oils by direct NP-HPLC-DAD analysis J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-11 Joaquín Velasco, Arturo Morales, M. Victoria Ruiz-Méndez, Gloria Márquez-Ruiz
The objective of the present study was to explore the possibilities of the direct analysis of vegetable oils by normal-phase HPLC-DAD to evaluate the amounts of the main oxidation products of triacylglycerols containing linoleate, i.e. hydroperoxy-, keto- and hydroxy-dienes. A follow-up of oxidation at 40 °C of trilinolein, used as a simplified model lipid system, high-linoleic sunflower oil and high-oleic sunflower oil was performed to evaluate samples with different fatty acid compositions and different oxidation levels. The results showed that the HPLC-DAD method proposed allows for determining the concentrations of mono-hydroperoxydienes in edible oils without applying any isolation or derivatization step. The method was found to be direct, sensitive (LOQ 0.06 mmol/kg oil), precise (CV ≤ 5%) and also accurate, with 99% of analyte recovery. It also enabled the estimation of the minor amounts of ketodienes, but not those of hydroxydienes, which presented wide chromatographic bands and coeluted with a number of different minor oxidation compounds.
Chemical Modification of Protein A Chromatography Ligands with Polyethylene Glycol. I: Effects on IgG Adsorption Equilibrium, Kinetics, and Transport J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-09 Justin Weinberg, Shaojie Zhang, Gillian Crews, Giorgio Carta, Todd Przybycien
Chemical modification of Protein A (ProA) chromatography ligands with polyethylene glycol (PEGylation) has been proposed as a strategy to increase the process selectivity and resin robustness by providing the ligand with a steric repulsion barrier against non-specific binding. This article comprises a comprehensive study of IgG adsorption and transport in Repligen CaptivA PriMAB resin with PEGylated ProA ligands that are modified using 5.2 and 21.5 kDa PEG chains. We studied the impact of PEG chain molecular weight of the PEG as well as the extent of PEGylation for the 5.2 kDa PEG modification. In all cases, PEGylation of ProA ligands decreases the resin average pore size, particle porosity, and static binding capacity for IgG proportional to the volume of conjugated PEG in the resin. Resin batch uptake experiments conducted in bulk via a stirred-tank system and with individual resin particles under confocal laser scanning microscopy suggests that PEGylation introduces heterogeneity into IgG binding kinetics: a fraction of the IgG binding sites are transformed from typical fast association kinetic behavior to slow kinetic behavior. pH gradient elution experiments of an IgG molecule on the modified resins show an increase in IgG elution pH for all modified resins, implying a decrease in IgG-ProA binding affinity on modification. Despite losses in static binding capacity for all resins with PEGylated ligands, the loss of 10% dynamic binding capacity (DBC10%) ranged more broadly from almost 0 to 47% depending on the PEG molecular weight and the extent of PEGylation. Minimal losses in DBC10% were observed with a low extent of PEGylation with a smaller molecular weight PEG, while higher losses were observed at higher extents of PEGylation and with higher molecular weight PEG due to decreased static binding capacity and increased mass transfer resistance. This work provides insight into the practical implications for resin performance if PEGylation is considered as a strategy for selectivity enhancement in affinity chromatography with macromolecular ligands.
Gradient elution behavior of proteins in hydrophobic interaction chromatography with U-shaped retention factor curves J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-09 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 where 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.
A simple and highly sensitive on-line column extraction liquid chromatography-tandem mass spectrometry method for the determination of protein-unbound tacrolimus in human plasma samples J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-07 Heike Bittersohl, Björn Schniedewind, Uwe Christians, Peter B. Luppa
Therapeutic drug monitoring (TDM) of the immunosuppressive drug tacrolimus is essential to avoid side effects and rejection of the allograft after transplantation. In the blood circulation, tacrolimus is largely located inside erythrocytes or bound to plasma proteins and less than 0.1% is protein-unbound (free). One basic principle of clinical pharmacology is that only free drug is pharmacologically active and monitoring this portion has the potential to better reflect the drug effect than conventional measurements of total tacrolimus in whole blood. To address this, a highly sensitive and straightforward on-line liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed, validated and applied to patient plasma samples. The sample preparation included ultracentrifugation and addition of the stable isotope labeled drug analogue D2,13C-tacrolimus, followed by on-line sample extraction and measurement using a Sciex QTRAP® 6500 in the multiple reaction monitoring mode. Due to very low concentrations of protein-unbound tacrolimus, it was important to develop a highly sensitive, precise and accurate assay. Here, we first report the efficient formation of tacrolimus lithium adduct ions, which greatly increased assay sensitivity. A lower limit of quantification (LLOQ) of 1 pg/mL (10 fg on column) was achieved and the assay was linear between 1 and 200 pg/mL. There was no carry-over detected. The inaccuracy ranged from −9.8 to 7.4% and the greatest imprecision was 7.5%. The matrix factor was found to be smaller than 1.1%. In summary, this method represents a suitable tool to investigate the potential clinical value of free tacrolimus monitoring in organ transplant recipients.
Salt-dependent elution of uncharged aromatic solutes in ion-exchange chromatography J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-06 Atsushi Hirano, Kazuki Iwashita, Shun Sakuraba, Kentaro Shiraki, Tsutomu Arakawa, Tomoshi Kameda
Ion-exchange chromatography and multimodal ion-exchange chromatography are widely used for the separation of small molecules, peptides and proteins. Salts generally attenuate the electrostatic interactions between charged moieties of solutes and those of resins through electrostatic screening. However, little is known about how salts affect the interaction between the uncharged moieties of the solutes, such as aromatic moieties, and the charged moieties of the resins. In this study, we used alkyl gallates as model aromatic solutes to investigate the interaction mechanism of aromatic moieties with multimodal and conventional ion-exchange resins. Interestingly, alkyl gallates retained by these resins were readily eluted from the columns by the addition of 0.01–1 M NaCl, even though the alkyl gallates used contained no charged group. Molecular dynamics (MD) simulations were performed to understand the mechanism of these interactions. The MD simulation with a conventional force field showed that 1 M NaCl enhances the binding of an alkyl gallate molecule to the ligand, which contradicts the experimental results. Thus, we modified the force field to express a cation–π interaction between sodium ions and aromatic moieties, which successfully reproduced the experimental results at 1 M, suggesting that the cation–π interaction between sodium ions and aromatic moieties plays a crucial role in reducing the binding affinity of alkyl gallates for the ligands. These results provide new information indicating that aromatic moieties, including the aromatic residues of proteins and nucleobases of nucleic acids, favorably interact with multimodal and conventional ion-exchange resins and that cations, such as sodium ions, contribute to attenuating the binding of aromatic moieties to the ligands.
QuEChERS and ultra-high performance liquid chromatography-tandem mass spectrometry method for the determination of parabens and ultraviolet filters in human milk samples J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-06 F. Vela-Soria, L.M. Iribarne-Durán, V. Mustieles, I. Jiménez-Díaz, M.F. Fernández, N. Olea
Concerns are growing about human exposure to endocrine disrupting chemicals (EDCs), especially during developmental stages. Parabens (PBs) and ultraviolet filters (UVFs) are prevalent EDCs widely used as additives in cosmetics and personal care products (PCPs). The objective of this study was to develop a method to determine four PBs and ten UVFs in human milk using QuEChERS treatment and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Multivariate strategies were applied to optimize experimental parameters. Limits of quantification ranged from 0.1 to 0.2 ng mL−1 and inter-day variability (evaluated as relative standard deviation) from 6% to 13%. The method was validated using matrix-matched standard calibration followed by a recovery assay with spiked samples. Recovery percentages ranged from 87% to 112%. The method was satisfactorily applied to assess target compounds in human milk samples from 15 donors. This analytical procedure can provide information on newborn exposure to these EDCs.
Preparation and evaluation of a hydrophilic interaction and cation-exchange chromatography stationary phase modified with 2-methacryloyloxyethyl phosphorylcholine J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-06 Caifeng Xiong, Jie Yuan, Zhiying Wang, Siyao Wang, Chenchen Yuan, Lili Wang
In this work, 2-methacryloyloxyethyl phosphorylcholine (MPC) was used as a ligand to prepare a novel mixed-mode chromatography (MMC) stationary phase by the thiol-ene click reaction onto silica (MPC-silica). It was found that this MPC-silica showed the retention characteristics of hydrophilic interaction chromatography (HILIC) and weak cation exchange chromatography (WCX) under suitable mobile phase conditions. In detail, acidic and basic hydrophilic compounds and puerarin from pueraria were separated quickly with HILIC mode. Meanwhile, six standard proteins were allowed to reach baseline separation in WCX mode, and protein separation from egg white was also achieved with this mode. In addition, reduced/denatured lysozyme could be refolded with the MPC-silica column. In the meantime, the MPC-silica has been applied for refolding with simultaneous purification of recombinant human Delta-like1-RGD (rhDll1-RGD) expressed in Escherichia coli. The results show that the mass recovery and purity of rhDll1-RGD could reach 63.4% and 97% by one step, respectively. Furthermore, the reporter assay results demonstrated that refolded with simultaneously purified rhDll1-RGD could efficiently activate the signalling pathway in a dose-dependent manner. In general, this MPC-silica has good resolution and selectivity in the separation of polar compounds and protein samples in different high-performance liquid chromatography (HPLC) modes, and it successfully achieved refolding with simultaneous purification of denatured protein.
Determination of finasteride and its metabolite in urine by dispersive liquid-liquid microextraction combined with field-enhanced sample stacking and sweeping J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-05 Chun-Hsien Chen, Yu-Ying Chao, Yi-Hui Lin, Yen-Ling Chen
The on-line preconcentration technique of field-enhanced sample stacking and sweeping (FESS-sweeping) are combined with dispersive liquid-liquid microextraction (DLLME) to monitor the concentrations of finasteride, which is used in the treatment of androgenetic alopecia, and its metabolite, finasteride carboxylic acid (M3), in urine samples. DLLME is used to concentrate and eliminate the interferences of urine samples and uses chloroform as an extracting solvent and acetonitrile as a disperser solvent. A high conductivity buffer (HCB) was introduced into capillary and then sample plug (90.7% capillary length) was injected into capillary. After applying voltage, the sodium dodecyl sulfate (SDS) swept the analytes from the low conductivity sample solution into HCB. The analytes were concentrated on the field-enhanced sample stacking boundary. The limit of detection for the analytes is 20 ng mL−1. The sensitivity enrichment of finasteride and M3 are 362-fold and 480-fold, respectively, compared with the conventional MEKC method. The on-line preconcentration technique of field-enhanced sample stacking and sweeping possess good selectivity because the endogenous steroid did not interfere the detection of finasteride and M3. The analytical technique is applied to investigate the concentrations in urine samples from patients who have been administered finasteride for the treatment of androgenetic alopecia; the amount of M3 detected in 12 h was 72.69 ± 4.18 μg.
In-line Fourier-Transform Infrared Spectroscopy as a Versatile Process Analytical Technology for Preparative Protein Chromatography J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-05 Steffen Großhans, Matthias Rüdt, Adrian Sanden, Nina Brestrich, Josefine Morgenstern, Stefan Heissler, Jürgen Hubbuch
Evaluation of flavour profiles in e-cigarette refill solutions using gas chromatography-tandem mass spectrometry J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-05 Justyna Aszyk, Paweł Kubica, Mateusz Kacper Woźniak, Jacek Namieśnik, Andrzej Wasik, Agata Kot-Wasik
Preparation of polymer monolithic column functionalized by arsonic acid groups for mixed-mode capillary liquid chromatography J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-05 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.
Fabrication of a high selectivity magnetic solid phase extraction adsorbent based on β-cyclodextrin and application for recognition of plant growth regulators J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-04 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 2–50 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.
One-pot preparation of magnetic carbon adsorbent derived from pomelo peel for magnetic solid-phase extraction of pollutants in environmental waters J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-03 Youfang Huang, Jinghe Peng, Xiaojia Huang
In this work, magnetic carbon material derived from pomelo peels (MCMPs) was conveniently fabricated utilizing one-pot synthesis method and employed as adsorbent of magnetic solid-phase extraction (MSPE). Several characterized measures including infrared spectroscopy, scanning electron microscopy, transmission electron microscopy and vibrating sample magnetometer were used to investigate the morphology, spectroscopic and magnetic properties of prepared adsorbent. Apolar parabens and polar fluoroquinolones (FQs) were used to investigate the extraction performance of MCMPs. Under the optimized extraction conditions, the MCMPs displayed satisfactory extraction performance for target analytes. At the same time, the MCMPs/MSPE was combined with HPLC-DAD for the sensitive determination of parabens and FQs in real-life water samples. Results showed that the limits of detection (S/N = 3) for parabens and FQs were in the ranges of 0.011–0.053 μg/L and 0.012–0.46 μg/L, respectively. The spiked recoveries were in the range of 76.6–116% for parabens and 80.2-114% for FQs with good repeatability (relative standard deviations less than 10%). In comparison to reported methods, the developed MCMPs/MSPE-HPLC-DAD showed some merits including low-cost, simplicity, satisfactory sensitivity and green non-pollution.
A non-targeted metabolomic approach to identify food markers to support discrimination between organic and conventional tomato crops J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-03 María Jesús Martínez Bueno, Francisco José Díaz-Galiano, Łukasz Rajski, Víctor Cutillas, Amadeo R. Fernández-Alba
In the last decade, the consumption trend of organic food has increased dramatically worldwide. However, the lack of reliable chemical markers to discriminate between organic and conventional products makes this market susceptible to food fraud in products labeled as “organic”. Metabolomic fingerprinting approach has been demonstrated as the best option for a full characterization of metabolome occurring in plants, since their pattern may reflect the impact of both endogenous and exogenous factors. In the present study, advanced technologies based on high performance liquid chromatography-high-resolution accurate mass spectrometry (HPLC-HRAMS) has been used for marker search in organic and conventional tomatoes grown in greenhouse under controlled agronomic conditions. The screening of unknown compounds comprised the retrospective analysis of all tomato samples throughout the studied period and data processing using databases (mzCloud, ChemSpider and PubChem). In addition, stable nitrogen isotope analysis (δ15N) was assessed as a possible indicator to support discrimination between both production systems using crop/fertilizer correlations. Pesticide residue analyses were also applied as a well-established way to evaluate the organic production. Finally, the evaluation by combined chemometric analysis of high-resolution accurate mass spectrometry (HRAMS) and δ15N data provided a robust classification model in accordance with the agricultural practices. Principal component analysis (PCA) showed a sample clustering according to farming systems and significant differences in the sample profile was observed for six bioactive components (L-tyrosyl-L-isoleucyl-L-threonyl-L-threonine, trilobatin, phloridzin, tomatine, phloretin and echinenone).
Dispersive liquid-liquid microextraction and gas chromatography accurate mass spectrometry for extraction and non-targeted profiling of volatile and semi-volatile compounds in grape marc distillates J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-03 Ariel Fontana, Isaac Rodríguez, Rafael Cela
The suitability of dispersive liquid-liquid microextraction (DLLME) and gas chromatography accurate mass spectrometry (GC-MS), based on a time-of-flight (TOF) MS analyzer and using electron ionization (EI), for the characterization of volatile and semi-volatile profiles of grape marc distillates (grappa) are evaluated. DLLME conditions are optimized with a selection of compounds, from different chemical families, present in the distillate spirit. Under final working conditions, 2.5 mL of sample and 0.5 mL of organic solvents are consumed in the sample preparation process. The absolute extraction efficiencies ranged from 30 to 100%, depending on the compound. For the same sample volume, DLLME provided higher responses than solid-phase microextraction (SPME) for most of the model compounds. The GC-EI-TOF-MS records of grappa samples were processed using a data mining non-targeted search algorithm. In this way, chromatographic peaks and accurate EI-MS spectra of sample components were linked. The identities of more than 140 of these components are proposed from comparison of their accurate spectra with those in a low resolution EI-MS database, accurate masses of most intense fragment ions of known structure, and available chromatographic retention index. The use of chromatographic and spectral data, associated to the set of components mined from different grappa samples, for multivariate analysis purposes is also illustrated in the study.
A two-step purification strategy using calmodulin as an affinity tag J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-02 Lianyun Lin, Chen Liu, Bidhan Chandra Nayak, Weiyi He, Minsheng You, Zhiguang Yuchi
Calmodulin (CaM) is a Ca2+-binding protein that plays an important role in cellular Ca2+-signaling. CaM interacts with diverse downstream target proteins and regulates their functions in a Ca2+-dependent manner. CaM changes its conformation and hydrophobicity upon [Ca2+] change and consequently changes its interaction with CaM-binding domains from the targets. Based on these special properties of CaM, it was used as an affinity tag to develop a novel purification strategy by using it for two sequential orthogonal purification steps: 1) an affinity purification step, in which CaM-tag interacts with an immobilized CaM-binding domain; and 2) a hydrophobic interaction chromatography step, during which CaM binds to a phenyl sepharose column. In both steps, the CaM-tagged protein binds in the presence of Ca2+ and unbinds in the presence of ethylenediaminetetraacetic acid (EDTA). An optional third step can be added to remove the CaM-tag if necessary. We used green fluorescent protein (GFP) as a test protein to demonstrate the effectiveness of the method. High yield and high purity of GFP with proper function was obtained using this novel strategy. We believe that this method can be applied to a wide range of protein targets for structural and functional studies.
A highly selective dispersive liquid–liquid microextraction approach based on the unique fluorous affinity for the extraction and detection of per- and polyfluoroalkyl substances coupled with high performance liquid chromatography tandem–mass spectrometry J. Chromatogr. A (IF 3.981) Pub Date : 2018-03-02 Juan Wang, Yali Shi, Yaqi Cai
In the present study, a highly selective fluorous affinity-based dispersive liquid–liquid microextraction (DLLME) technique was developed for the extraction and analysis of per- and polyfluoroalkyl substances (PFASs) followed by high performance liquid chromatography tandem–mass spectrometry. Perfluoro-tert-butanol with multiple C-F bonds was chosen as the extraction solvent, which was injected into the aqueous samples with a dispersive solvent (acetonitrile) in a 120:800 (μL, v/v) mixture for PFASs enrichment. The fluorous affinity-based extraction mechanism was confirmed by the significantly higher extraction recoveries for PFASs containing multiple fluorine atoms than those for compounds with fewer or no fluorine atoms. The extraction recoveries of medium and long-chain PFASs (CF2 > 5) exceeded 70%, except perfluoroheptanoic acid, while those of short-chain PFASs were lower than 50%, implying that the proposed DLLME may not be suitable for their extraction due to weak fluorous affinity. This highly fluoroselective DLLME technique can greatly decrease the matrix effect that occurs in mass spectrometry detection when applied to the analysis of urine samples. Under the optimum conditions, the relative recoveries of PFASs with CF2 > 5 ranged from 80.6-121.4% for tap water, river water and urine samples spiked with concentrations of 10, 50 and 100 ng/L. The method limits of quantification for PFASs in water and urine samples were in the range of 0.6–8.7 ng/L. Furthermore, comparable concentrations of PFASs were obtained via DLLME and solid-phase extraction, confirming that the developed DLLME technique is a promising method for the extraction of PFASs in real samples.
Sensitive determination of brassinosteroids by solid phase boronate affinity labeling coupled with liquid chromatography-tandem mass spectrometry J. Chromatogr. A (IF 3.981) Pub Date : 2018-02-28 Xiao-Tong Luo, Bao-Dong Cai, Lei Yu, Jun Ding, Yu-Qi Feng
Brassinosteroids (BRs) are regarded as the sixth plant hormone that is widely distributed in the plant kingdom. Sensitive quantification of BRs will be greatly benefit to illuminate the detail mechanisms about how BRs play crucial role in plant developmental processes such as cell division, cell expansion, cytodifferentiation, seed germination, vegetative growth and resisting biological or abiotic stress. In the current study, we developed a method for rapid and sensitive determination of endogenous BRs in plant tissues by combining LC–MS and a novel sample preparation strategy, in which the plant tissue extract was supplied to solid phase boronate affinity labeling and extraction, followed by desorption and salt-induced phase transition extraction for further purification. Under the optimized conditions, good linearity was obtained for 6 BR with correlation coefficients (r) ranging from 0.9988 to 0.9999. The limits of detection (LODs, S/N = 3) ranged from 1.4 to 2.8 pg mL−1. The recoveries were between 93.4% and 116.2% with the relative standard deviations (RSDs) ranging from 2.8% to 15.8%. Finally, the developed method was successfully applied to the analysis of 6 endogenous BR in various plant tissues including 20 mg FW Oryza sativa shoot, 10 mg FW Oryza sativa root, 20 mg FW Arabidopsis thaliana shoot, 4 Arabidopsis thaliana flowers (2.8 mg) and one Brassica napus stamen (3.0 mg) with concentration ranging from 0.26 to 157.28 ng g−1 FW.
A method for analysis of marker persistent organic pollutants in low-volume plasma and serum samples using 96-well plate solid phase extraction J. Chromatogr. A (IF 3.981) Pub Date : 2018-02-27 Jordan Stubleski, Petr Kukucka, Samira Salihovic, P. Monica Lind, Lars Lind, Anna Kärrman
The objective of this study was to develop and validate a 96-well plate solid phase extraction method for analysis of 23 lipophilic persistent organic pollutants (POPs) in low-volume plasma and serum samples which is applicable for biomonitoring and epidemiological studies. The analysis of selected markers for internal exposure: 16 polychlorinated biphenyls (PCBs), 5 organochlorine pesticides (OCPs), octachlorinated dibenzo-p-dioxin (OCDD), and polybrominated diphenylether 47 (BDE 47) was evaluated by comparing two SPE sorbents and GC-HRMS or GC-MS/MS detection. The final method extracted 23 POPs from 150 μL of serum and plasma using a 96-well extraction plate containing 60 mg Oasis HLB sorbent per well prior to GC-HRMS magnetic sector analysis. The extraction method was applied to 40 plasma samples collected for an epidemiological study. The recovery of selected POPs ranged from 31% to 63% (n = 48), and detection limits ranged from 2.2 to 45 pg/mL for PCBs, 4.2 to 167 pg/mL for OCPs, 7.8 pg/mL for OCDD and 6.1 pg/mL for BDE 47. This method showed good precision with relative standard deviations of selected POP concentrations in quality control samples (n = 48) ranging from 11% to 25%. The trueness was determined with standard reference material serum (n = 48) and the deviation from certified values ranged from 1 to 27%. Of the 23 POPs analyzed, 18 were detected in 43% to 100% of plasma samples collected for the epidemiological study. The method showed good robustness with low inter-well plate variation (11–31%) determined by twelve 96-well plate extractions, and can extract 96 samples, including quality controls and procedural blanks in 2–3 days. Comparison with GC-MS/MS analysis showed that similar concentrations (within 0.5% to 30%) of most POPs could be obtained with GC-APCI-MS/MS. Larger deviations were observed for PCB 194 (60%) and trans-nonachlor (43%). The developed method produces accurate concentrations of low-level marker POPs in plasma and serum, providing a suitable high-throughput sample preparation procedure for biomonitoring and epidemiological studies involving large sample size and limited sample volume. GC-HRMS was chosen over GC-MS/MS, however the latter showed promising results, and could be used as an alternative to GC-HRMS analysis for most POPs.
Magnetic molecularly imprinted polymers for the selective determination of cocaine by ion mobility spectrometry ☆ J. Chromatogr. A (IF 3.981) Pub Date : 2018-02-27 Aitor Sorribes-Soriano, Francesc Albert Esteve-Turrillas, Sergio Armenta, Ana Montoya, José Manuel Herrero-Martínez, Miguel de la Guardia
Magnetic molecularly imprinted polymers (MMIPs) were prepared for cocaine recognition by bulk polymerization in the presence of magnetic nanoparticles (MNPs). Two reagents (polyethylene glycol (PEG) and 3-(trimethoxysilyl)propyl methacrylate (V)) were used for MNPs modification. MMIPs were characterized and compared in terms of loading capacity, reusability, accuracy and precision for the extraction of cocaine from saliva samples. It was observed that V-MMIPs gave higher physical stability than PEG-MMIPs. Thus, V-MMIP were used for the analysis of cocaine users saliva. The developed procedure based on ion mobility spectrometry (IMS) provided limits of detection and quantification of 4 and 14 μg L−1, respectively, and recoveries in cocaine free saliva samples spiked at 80, 270 and 560 μg L−1 ranging from 80 to 99%. Results found by the proposed method were statistically comparable to those obtained by two reference procedures; a lateral flow immunoassay and an ultra-high performance liquid chromatography coupled with tandem mass spectrometry. Therefore, MMIP-IMS can be considered as a fast, selective and sensitive alternative to reference methods with affordable cost avoiding the requirement of skilled operator.
Development of a large volume injection method using a programmed temperature vaporization injector – Gas chromatography hyphenated to ICP-MS for the simultaneous determination of mercury, tin and lead species at ultra-trace levels in natural waters J. Chromatogr. A (IF 3.981) Pub Date : 2018-02-27 J. Terán-Baamonde, S. Bouchet, E. Tessier, D. Amouroux
The current EU legislation lays down Environmental Quality Standards (EQS) for 45 priority substances in surface waters; among them levels for (organo)metallic species of Hg, Sn and Pb are set between ng L−1 (for Hg and Sn) and μg L−1 (for Pb). To date, only a few analytical methods can reach these very restrictive limits and there is thus a need for comprehensive methods able to analyze these species down to these levels in natural waters. The aim of this work was to develop an online automated pre-concentration method using large volume injections with a Programmed Temperature Vaporization (PTV) injector fitted with a sorbent packed liner coupled to GC-ICP-MS to further improve the detection limits associated to this well-established method. The influence of several parameters such as the PTV transfer temperature and time, carrier gas flow rate and amount of packing material was investigated. Finally, the maximum volume injected through single or multiple injection modes was optimized to obtain the best compromise between chromatographic resolution and sensitivity. After optimization, very satisfactory results in terms of absolute and methodological detection limits were achieved, down to the pg L−1 level for all species studied. The potential of the method was exemplified by determining the concentrations of organometallic compounds in unpolluted river waters samples from the Adour river basin (SW France) and results were compared with conventional (splitless) GC-ICP-MS. The strength of this analytical method lies in the low detection limits reached for the simultaneous analysis of a wide group of organometallic compounds, and the potential to transfer this method to other gas chromatographic applications with inherent lower sensitivity.
Separation of enantiomers of selected chiral sulfoxides with cellulose tris(4-chloro-3-methylphenylcarbamate)-based chiral columns in high-performance liquid chromatography with very high separation factor J. Chromatogr. A (IF 3.981) Pub Date : 2018-02-26 Tamar Khatiashvili, Rusudan Kakava, Iza Matarashvili, Hadi Tabani, Chiara Fanali, Alessandro Volonterio, Tivadar Farkas, Bezhan Chankvetadze
The present study reports successful separations of enantiomers of selected chiral sulfoxides with very high separation factor in high-performance liquid chromatography by using chiral columns prepared with the chiral selector cellulose tris(4-chloro-3-methylphenylcarbamate). High separation factors were observed in polar organic, as well as in hydrocarbon-alcohol-type mobile phases. The key structural components of the solute for obtaining high chiral recognition are discussed as well as thermodynamic quantities of analyte adsorption on the chiral stationary phase were determined. Experiment aimed at the enantioselective extraction of racemates from solution are also described.
Sensitive method for glycosaminoglycan analysis of tissue sections J. Chromatogr. A (IF 3.981) Pub Date : 2018-02-25 Lilla Turiák, Gábor Tóth, Oliver Ozohanics, Ágnes Révész, András Ács, Károly Vékey, Joseph Zaia, László Drahos
A simple, isocratic HPLC method based on HILIC-WAX separation, has been developed for analyzing sulfated disaccharides of glycosaminoglycans (GAGs). To our best knowledge, this is the first successful attempt using this special phase in nano-HPLC–MS analysis. Mass spectrometry was based on negative ionization, improving both sensitivity and specificity. Detection limit for most sulfated disaccharides were approximately 1 fmol; quantitation limits 10 fmol. The method was applied for glycosaminoglycan profiling of tissue samples, using surface digestion protocols. This novel combination provides sufficient sensitivity for GAG disaccharide analysis, which was first performed using prostate cancer tissue microarrays. Preliminary results show that GAG analysis may be useful for identifying cancer related changes in small amounts of tissue samples (ca. 10 μg).
Heparin/Heparan Sulfate Analysis by Covalently Modified Reverse Polarity Capillary Zone Electrophoresis-Mass Spectrometry J. Chromatogr. A (IF 3.981) Pub Date : 2018-02-24 Patience Sanderson, Morgan Stickney, Franklin E. Leach III, Qiangwei Xia, Yanlei Yu, Fuming Zhang, Robert J. Linhardt, I. Jonathan Amster
Reverse polarity capillary zone electrophoresis coupled to negative ion mode mass spectrometry (CZE-MS) is shown to be an effective and sensitive tool for the analysis of glycosaminoglycan mixtures. Covalent modification of the inner wall of the separation capillary with neutral or cationic reagents produces a stable and durable surface that provides reproducible separations. By combining CZE-MS with a cation-coated capillary and a sheath flow interface, a rapid and reliable method has been developed for the analysis of sulfated oligosaccharides from dp4 to dp12. Several different mixtures have been separated and detected by mass spectrometry. The mixtures were selected to test the capability of this approach to resolve subtle differences in structure, such as sulfation position and epimeric variation of the uronic acid. The system was applied to a complex mixture of heparin/heparan sulfate oligosaccharides varying in chain length from dp3 to dp12 and more than 80 molecular compositions were identified by accurate mass measurement.
Separating large microscale particles by exploiting charge differences with dielectrophoresis J. Chromatogr. A (IF 3.981) Pub Date : 2018-02-24 Danielle V. Polniak, Eric Goodrich, Nicole Hill, Blanca H. Lapizco-Encinas
Dielectrophoresis (DEP), the migration of particles due to polarization effects under the influence of a nonuniform electric field, was employed for characterizing the behavior and achieving the separation of larger (diameter > 5 μm) microparticles by exploiting differences in electrical charge. Usually, electrophoresis (EP) is the method of choice for separating particles based on differences in electrical charge; however, larger particles, which have low electrophoretic mobilities, cannot be easily separated with EP-based techniques. This study presents an alternative for the characterization, assessment, and separation of larger microparticles, where charge differences are exploited with DEP instead of EP. Polystyrene microparticles with sizes varying from 5 to 10 μm were characterized employing microdevices for insulator-based dielectrophoresis (iDEP). Particles within an iDEP microchannel were exposed simultaneously to DEP, EP, and electroosmotic (EO) forces. The electrokinetic behavior of four distinct types of microparticles was carefully characterized by means of velocimetry and dielectrophoretic capture assessments. As a final step, a dielectropherogram separation of two distinct types of 10 μm particles was devised by first characterizing the particles and then performing the separation. The two types of 10 μm particles were eluted from the iDEP device as two separate peaks of enriched particles in less than 80 seconds. It was demonstrated that particles with the same size, shape, surface functionalization, and made from the same bulk material can be separated with iDEP by exploiting slight differences in the magnitude of particle charge. The results from this study open the possibility for iDEP to be used as a technique for the assessment and separation of biological cells that have very similar characteristics (shape, size, similar make-up), but slight variance in surface electrical charge.
Synthesis of high generation thermo-sensitive dendrimers for extraction of rivaroxaban from human fluid and pharmaceutic samples J. Chromatogr. A (IF 3.981) Pub Date : 2018-02-24 Negin Parham, Homayon Ahmad Panahi, Alireza Feizbakhsh, Elham Moniri
In this present study, poly (N-isopropylacrylamide) as a thermo-sensitive agent was grafted onto magnetic nanoparticles, then ethylenediamine and methylmethacrylate were used to synthesize the first generation of poly amidoamine (PAMAM) dendrimers successively and the process continued alternatively until the ten generations of dendrimers. The synthesized nanocomposite was investigated using Fourier transform infrared spectrometry, thermalgravimetry analysis, X-ray diffractometry, elemental analysis and vibrating-sample magnetometer. The particle size and morphology were characterized using dynamic light scattering, field emission scanning electron microscopy and transmission electron microscopy. Batch experiments were conducted to investigate the parameters affecting adsorption and desorption of rivaroxaban by synthesized nanocomposite. The maximum sorption of rivaroxaban by the synthesized nanocomposite was obtained at pH of 8. The resulting grafted magnetic nanoparticle dendrimers were applied for extraction of rivaroxaban from biologic human liquids and medicinal samples. The specifications of rivaroxaban sorbed by a magnetic nanoparticle dendrimer showed good accessibility and high capacity of the active sites within the dendrimers. Urine and drug matrix extraction recoveries of more than 92.5 and 99.8 were obtained, respectively.
Chemical Modification of Protein A Chromatography Ligands with Polyethylene Glycol. II: Effects on Resin Robustness and Process Selectivity J. Chromatogr. A (IF 3.981) Pub Date : 2018-02-24 Justin Weinberg, Shaojie Zhang, Allison Kirkby, Enosh Shachar, Giorgio Carta, Todd Przybycien
We have proposed chemical modification of Protein A (ProA) chromatography ligands with polyethylene glycol (PEGylation) as a strategy to increase the resin selectivity and robustness by providing the ligand with a steric repulsion barrier against non-specific binding. Here, we report on robustness and selectivity benefits for Repligen CaptivA PriMAB resin with ligands modified with 5.2 kDa and 21.5 kDa PEG chains, respectively. PEGylation of ProA ligands allowed the resin to retain a higher percentage of static binding capacity relative to the unmodified resin upon digestion with chymotrypsin, a representative serine protease. The level of protection against digestion was independent of the PEG molecular weight or modification extent for the PEGylation chemistry used. Additionally, PEGylation of the ligands was found to decrease the level of non-specific binding of fluorescently labeled bovine serum albumin (BSA) aggregates to the surface of the resin particles as visualized via confocal laser scanning microscopy (CLSM). The level of aggregate binding decreased as the PEG molecular weight increased, but increasing the extent of modification with 5.2 kDa PEG chains had no effect. Further examination of resin particles via CLSM confirmed that the PEG chains on the modified ligands were capable of blocking the “hitchhiking” association of BSA, a mock contaminant, to an adsorbed mAb that is prone to BSA binding. Ligands modified with 21.5 kDa PEG chains were effective at blocking the association, while ligands modified with 5.2 kDa PEG chains were not. Finally, ligands with 21.5 kDa PEG chains increased the selectivity of the resin against host cell proteins (HCPs) produced by Chinese Hamster Ovary (CHO) cells by up to 37% during purification of a monoclonal antibody (mAb) from harvested cell culture fluid (HCCF) using a standard ProA chromatography protocol. The combined work suggests that PEGylating ProA chromatography media is a viable pathway for increasing both resin lifetime and host cell impurity clearance in downstream bioprocessing.
Optimized anion exchange column isolation of zirconium-89 (89Zr) from yttrium cyclotron target: Method development and implementation on an automated fluidic platform J. Chromatogr. A (IF 3.981) Pub Date : 2018-02-24 Matthew J. O’Hara, Nathaniel J. Murray, Jennifer C. Carter, Samuel S. Morrison
Zirconium-89 (89Zr), produced by the (p, n) reaction from naturally monoisotopic yttrium (natY), is a promising positron emitting isotope for immunoPET imaging. Its long half-life of 78.4 h is sufficient for evaluating slow physiological processes. A prototype automated fluidic system, coupled to on-line and in-line detectors, has been constructed to facilitate development of new 89Zr purification methodologies. The highly reproducible reagent delivery platform and near-real time monitoring of column effluents allows for efficient method optimization. The separation of Zr from dissolved Y metal targets was evaluated using several anion exchange resins. Each resin was evaluated against its ability to quantitatively capture Zr from a load solution high in dissolved Y. The most appropriate anion exchange resin for this application was identified, and the separation method was optimized. The method is capable of a high Y decontamination factor (>105) and has been shown to remove Fe, an abundant contaminant in Y foils, from the 89Zr elution fraction. Finally, the method was evaluated using cyclotron bombarded Y foil targets; the method was shown to achieve >95% recovery of the 89Zr present in the foils. The anion exchange column method described here is intended to be the first 89Zr isolation stage in a dual-column purification process.
EVALUATION OF SUPERCIRTICAL FLUID CHROMATOGRAPHY COUPLED TO TANDEM MASS SPECTROMETRY FOR PESTICIDE RESIDUES IN FOOD J. Chromatogr. A (IF 3.981) Pub Date : 2018-02-23 Víctor Cutillas, María Martínez Galera, Łukasz Rajski, Amadeo R. Fernández-Alba
Supercritical fluid chromatography coupled to triple quadrupole mass spectrometry has been evaluated for pesticide residues in food. In order to check its advantages and limitations it was developed a method to identify and quantify 164 pesticides in three different matrices (tomato, orange and leek). A carbon dioxide gradient with methanol (containing 1 mM ammonium formate) was used allowing a flow rate of 1.5 mL/min that made the total run time of 12 min without any problem of overpressure. Addition of a post column flow 150 μL/min of Methanol with ammonium formate/formic acid was necessary to improve the ionization. The matrix effect study revealed that the percentages of pesticides with irrelevant matrix effect (suppression lower than 20%) was 99% in tomato, 87% in orange and 62% in leek, whereas significant suppression (higher than 50%) was not found in tomato and only 1% of the compounds in orange and 3% in leek.These results compare favorably with that typically obtained in LC-MS/MS. The absence of water in the mobile phase, also provided some important advantages regarding LC-MS/MS as (i) higher retention of polar compounds in the column, which elute with high sensitivity and good peak shape and (ii) a general increase of the sensitivity of the analysis, consequence of the high ionization and ion extraction efficiency. Pesticides evaluated were identified following the SANTE/11813/2017. At the spiking concentration of 5 μg/kg, 98% of the pesticides were identified in tomato, 98% in orange and 94% in leek, whereas for the concentration of 10 μg/kg all the compounds were identified in tomato and only spiromesifen was not identified in orange and leek. At the concentration of 20 μg/kg, spiromesifen was also identified in these two matrices. The linearity and reproducibility of the method were evaluated with results which guarantee high quality in the analytical measurements. Even though only 2 μL of final extract were injected, the sensitivity of the SFC method was enough to achieve stringent LOQs.Real samples, including 6 different fruits and vegetables, were analyzed by the SFC-MS/MS proposed method, the results being similar to those obtained by LC-MS/MS. The method was also applied to a proficiency test of fipronil in eggs with good results in all the cases. Carbon dioxide as mobile phase with methanol as modifier can represent a good alternative to LC-MS/MS with reduction of matrix effects and shorter run times.
A miniaturized sorbent phase-based extraction device in the form of syringe filter holder using molecularly imprinted polymer as sorbent and its application to extract benzophenones J. Chromatogr. A (IF 3.981) Pub Date : 2018-02-22 Xiao Sun, Xin-yue Jiao, Jing Li, Li Xu
The molecularly imprinted polymer using 2,4-dihydroxybenzophenone as the template (DHBP-MIP) was synthesized via sacrificial support method. The DHBP-MIP was demonstrated to possess good adsorption capacity and selectivity towards benzophenones. Moreover, a miniaturized sorbent phase-based extraction device in the form of syringe filter holder using DHBP-MIP as the sorbent was proposed, and named as μ-SPE-SFH-MIP device. The μ-SPE-SFH-MIP device consisted of a reusable syringe filter holder, flexible amount of sorbent and a sub-microporous membrane, which could be conveniently connected to different driving forces, like syringe pump, solid-phase extraction device and peristaltic pump. It was successfully applied to extract five benzophenones from swimming pool water and human urine samples, combined with high performance liquid chromatograph-UV analysis, with detection limits of 0.12-0.68 μg L−1 and 0.21-1.05 μg L−1, respectively. The accuracy and precision of the analytes were in the range of 89.27%-113.35% for swimming pool water samples and 90.65%-108.00% for urine samples, with all the relative standard deviation values below 7.89%. The μ-SPE-SFH-MIP device was portable, cost-effective operational convenient and suitable for the small-size samples; moreover, with the MIP as the sorbent, it afforded additional high selectivity and sensitivity.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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