Immobilized enzyme microreactors for analysis of tryptic peptides in ß-casein and ß-lactoglobulin.

In this study, our primary objective was to develop an effective analytical method for studying trypsin-digested peptides of two proteins commonly found in cow's milk: ß-casein (ßCN) and ß-lactoglobulin (ßLG). To achieve this, we employed two distinct approaches: traditional in-gel protein digestion and protein digestion using immobilized enzyme microreactors (µ-IMER). Both methods utilized ZipTip pipette tips filled with C18 reverse phase media for sample concentration. The µ-IMER was fabricated through a multi-step process that included preconditioning the capillary, modifying its surface, synthesizing a monolithic support, and further surface modification. Its performance was evaluated under HPLC chromatography conditions using a small-molecule trypsin substrate (BAEE). Hydrolysates from both digestion methods were analyzed using MALDI-TOF MS. Our findings indicate that the µ-IMER method demonstrated superior sequence coverage for oxidized molecules in ßCN (33 ± 1.5%) and ßLG (65 ± 3%) compared to classical in-gel digestion (20 ± 2% for ßCN; 49 ± 2% for ßLG). The use of ZipTips further improved sequence coverage in both classical in-gel digestion (26 ± 1% for ßCN; 60 ± 4% for ßLG) and µ-IMER (41 ± 3% for ßCN; 80 ± 5% for ßLG). Additionally, phosphorylations were identified. For ßCN, no phosphorylation was detected using classical digestion, but the use of ZipTips showed a value of 27 ± 4%. With µ-IMER and µ-IMER-ZipTip, the values increased to 30 ± 2% and 33 ± 1%, respectively. For ßLG, the use of ZipTip enabled the detection of a higher percentage of modified peptides in both classical (79 ± 2%) and µ-IMER (79 ± 4%) digestions. By providing a comprehensive comparison of traditional in-gel digestion and µ-IMER methods, this study offers valuable insights into the advantages and limitations of each approach, particularly in the context of complex biological samples. The findings set a new benchmark in protein digestion and analysis, highlighting the potential of µ-IMER systems for enhanced sequence coverage and post-translational modification detection.

Cholesterol Stationary Phase in the Separation and Identification of siRNA Impurities by Two-Dimensional Liquid Chromatography-Mass Spectrometry.

The aim of this research was to develop a simple and efficient ion-pair reagent-free chromatographic method for the separation and qualitative determination of oligonucleotide impurities, exemplified by synthesis of raw products of the two single strands of patisiran siRNA. The stationary phases with mixed hydrophobic/hydrophilic properties (cholesterol and alkylamide) were firstly used for this purpose with reversed-phased high-performance liquid chromatography. Several different chromatographic parameters were tested for their impact on impurities separation: type, concentration, pH of salt, as well as organic solvent type in the mobile phase. The pH was the most influential factor on the separation and signal intensities in mass spectrometry detection. Finally, the optimized method included the application of cholesterol stationary phase, with mobile phase containing 20 mM ammonium formate (pH 6.5) and methanol. It allowed good separation and the identification of most impurities within 25 min. Since not all closely related impurities could be fully resolved from the main peak in this oligonucleotide impurity profiling, two-dimensional liquid chromatography was used for peak purity determination of the target oligonucleotides. The Ethylene Bridged Hybrid (BEH) Amide column in hydrophilic interaction liquid chromatography was applied in the second dimension, allowing additional separation of three closely related impurities.

Optimization of the packing process of microcolumns with the embedded phosphodiester stationary phases.

The development of new home-made stationary phases involves their packaging procedure and is crucial to obtain satisfactory working parameters. The parameter that illustrates the quality of the packed bed is its efficiency measured as the height equivalent to the theoretical plate. According to the Van Deemetr's equation, it depends on three factors, but only one of them, eddy diffusion, does not depend on the linear flow velocity. Therefore, in order to obtain it as low as possible, it is necessary to focus on a good filling of the column. Among many parameters affecting the quality of column packing, in our work we have focused on the choice of slurry solvent. Novel stationary phases with an embedded phosphodiester group were investigated. The suspensions in 16 solvents and solvent mixtures were studied for their stability, aggregation, sedimentation, and viscosity comparison. The efficiency of the packed microcolumns and its comparison was determined by chromatographic analyses using a polar (thymidine) and a nonpolar compound (naphthalene). The results obtained led to the conclusion that for these stationary phases, the best slurry solvent is the one that aggregates the phase while maintaining stability and having high viscosity.

CE-DAD-MS/MS in the simultaneous determination and identification of selected antibiotic drugs and their metabolites in human urine samples.

In this study, a new analytical method was developed and validated for the simultaneous analysis of antibiotic drugs (amoxicillin, cefotaxime, ciprofloxacin, clindamycin, linezolid, metronidazole) and their metabolites (amoxycilloic acid, amoxicillin diketopiperazine, 3-desacetyl cefotaxime lactone, clindamycin sulfoxide, ciprofloxacin piperazinyl-N4-sulfate, linezolid N-oxide, metronidazole-OH) in human urine. Capillary electrophoresis (CE) along with the tandem mass spectrometry (MS/MS) was used to determine and identify all analytes. Appropriate conditions for MS/MS measurements along with the use of the central composite design were optimized. The effects of different analytical conditions (the composition, the concentration, and the pH value of the background electrolyte, the time and pressure of the injection, the capillary temperature and influence of the organic modifier) on the migration and separation of antibiotic drugs and metabolites were examined using the CE-DAD. The analytical procedure was linear for concentrations ranging from 20 to 1000 ng/mL, with determination coefficients higher than 0.99 for all the analytes. The validated analytical procedure was then applied to the measurement of antibiotic drugs and their metabolites in human urine samples.

Synthesis and application of stationary phase for DNA-affinity chromatographic analysis of unmodified and antisense oligonucleotide.

The goal of the research was the synthesis and application of an oligonucleotide immobilized stationary phase for the analysis of unmodified and antisense oligonucleotides. The method for attaching these molecules to aminopropyl silica modified with pentanedioic acid was developed. Each step of the synthesis was carefully controlled with the application of spectroscopic, elemental, and chromatographic analyses. The oligonucleotide-based stationary phase was applied for the retention studies. Unmodified oligonucleotides of different complementarity to the molecule attached as a stationary phase, as well as antisense oligonucleotides, were tested. The comparative study upon complex optimization of oligonucleotide analysis in different liquid chromatography modes was performed. Results have shown that this stationary phase may be applied for oligonucleotide analysis in hydrophilic interaction liquid chromatography and ion exchange chromatography, but no unique sequence-based selectivity was obtained. Contrary results were observed for affinity chromatography, which allowed for specific separation of the complementary strands based on hydrogen bonding and stacking interactions, where the temperature was the main factor influencing the selectivity of the separation. Furthermore, the oligonucleotide-based stationary phase may be applied for comparative antisense oligonucleotide hybridization studies to a specific RNA sequence. All of the results have shown that affinity chromatography with oligonucleotide-based stationary phases is a powerful technique for the specific base recognition of polynucleotides.

Analysis of Natural Dyes from Historical Objects by High Performance Liquid Chromatography and Electromigration Techniques.

Based on material published between 1989 and 2018 in this paper high performance liquid chromatography and electromigration techniques used in studies of natural dyes that can be found in historical objects are rewieved. Different aspects of analysis have been discussed: the stationary and mobile phase, the choice of sample solvent, methods of extraction and detection, including sensitivity parameters, such as LOD and/or LOQ. The discussed dyes have been divided into three categories (a) red antraquinone dyes along with dyes extracted from bark and tree juices, (b) yellow flavonoid dyes and saffron and (c) blue indigoid dyes. The main markers (chromophores or auxochromes) typical for each dye source were presented which allows to identify specific species of source plants and animals. The first part of the study involved the analysis of most critical findings when it comes to HPLC or represented an significant analytical approach. The second part of the study is focused on different aspects of electromigration techniques application in analysis of natural dyes with special attention paid on such parameters as the running buffer/mobile phase composition and sample solvent. Detection methods along with LOD and LOQ comparison in HPLC and electromigration techniques were also discussed. Methods of sample preparation, such as hydrolysis and extraction, used in HPLC and electromigration techniques were also briefly discussed.

Preparation of an improved hydrophilic monolith to make trypsin-immobilized microreactors.

In the present work the preparation of capillary-based microreactors with immobilized trypsin was investigated. The monolithic support was synthesized from 2-hydroxyethyl methacrylate (HEMA) as a functional monomer and N,N'-methylenebis(acrylamide) (MBA) as a hydrophilic crosslinker. Two monomers contents in the polymerization mixture (27% and 35%) at the ratio of HEMA:MBA=3:2 were tested. The results indicated that the heated mixture of the above monomers and the porogen consisting of 40% 1-decanol, 40% 2-propanol and 20% water was the most appropriate polymerization mixture for preparation of HEMA-MBA copolymer. The elaborated novel procedure of HEMA-MBA hydrophilic monolith preparation allowed for the introduction of higher monomers content compared to earlier literature data. The obtained monolith exhibited lower adsorption of BSA in comparison with the commonly used glycidyl methacrylate-co-ethylene dimethacrylate (GMA-EDMA) material. In the second step, the covalent enzyme attachment on the hydroxyl groups via 1,1'-carbonyldiimidazole activation was performed. Two immobilization techniques were tested. The first approach involved direct trypsin attachment to the hydroxyl groups of two-carbons HEMA chain (microreactor T1). In the other strategy, 5-amino-1-pentanol was employed to form a twelve-atom spacer arm (microreactor TS1). The prepared microreactor s activities and specificities were evaluated using low molecular weight trypsin substrate (Nα-benzoyl-l-arginine ethyl ester (BAEE)) and bovine serum albumin (BSA). The chromatographic analysis of the eluates from T1 and TS1 microreactors indicated that the microreactor T1 showed higher activity toward the BAEE than the microreactor TS1. However, the BSA digestion and subsequent MALDI-TOF MS (Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis of the collected eluates resulted in a sequence coverage of 43.9% and 35.7% in the case of TS1 and T1 microreactors, respectively.

Application of a cholesterol stationary phase in the analysis of phosphorothioate oligonucleotides by means of ion pair chromatography coupled with tandem mass spectrometry.

The main aim of this study was the investigation of the influence of several ion pair reagents towards both the retention and the mass spectrometry sensitivity of phosphorothioate oligonucleotides. A cholesterol stationary phase was applied for the first time in the analysis of this group of compounds. The mobile phase composition was modified by changing the concentration and the type of amines and acetates or 1,1,1,3,3,3-hexafluoroisopropanol. It has been shown that the increase of amines concentration results in the retention factor increase for each oligonucleotide, on each adsorbent. The only exception was the mobile phase composed of triethylamine and 1,1,1,3,3,3-hexafluoroisopropanol. This is a consequence of interactions taking place between a cholesterol molecule and an alcohol. This effect was convenient when the mass spectrometry detection was applied, since it allowed an increase in the sensitivity. Moreover, optimization of the mobile phase composition and its impact on the efficiency of ionization process and on the sensitivity in mass spectrometry were also presented. The optimization of this new method, based on cholesterol stationary phase coupled with mass spectrometry detection, was finally applied for the determination of phosphorothioate oligonucleotides impurity in a real sample.

Preparation and evaluation of dual-enzyme microreactor with co-immobilized trypsin and chymotrypsin.

The preparation of capillary microfluidic reactor with co-immobilized trypsin and chymotrypsin with the use of a low-cost commercially available enzymatic reagent (containing these proteases) as well as the evaluation of its usefulness in proteomic research were presented. The monolithic copolymer synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) was used as a support. Firstly, the polymerization conditions were optimized and the monolithic bed was synthesized in the fused silica capillary modified with 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS). The polymer containing epoxy groups was then modified with 1,6-diaminohexane, followed by the attachment of glutaraldehyde and immobilization of enzymes. The efficiency of the prepared monolithic Immobilized Enzyme Microreactor (µ-IMER) with regard to trypsin activity was evaluated using the low-molecular mass compound (Nα-benzoyl-l-arginine ethyl ester, BAEE). The activities of both enzymes were investigated using a macromolecular protein (human transferrin, Tf) as a substrate. In the case of BAEE, the reaction product was separated from the substrate using the capillary liquid chromatography and the efficiency of the reaction was determined by the peak area of the substrate. The hydrolysis products of transferrin were analyzed with MALDI-TOF which allows for the verification of the prepared enzymatic system applicability in the field of proteomic research.

Cholesterol-based polymeric monolithic columns for capillary liquid chromatography. Part II.

This paper presents a second part of our research devoted to cholesterol-based polymeric monolithic stationary phase. The obtained capillary columns were successfully used for separations of alkylbenzenes, steroid hormones and polycyclic aromatic hydrocarbons during isocratic or gradient elutions. The columns showed excellent thermal stability. Increasing the temperature resulted in decrease of the retention factors and peak widths, but selectivity and efficient separations have been retained in the studied temperature range of 20 to100°C. Additionally, the van't Hoff model presented a non-linear relationship of lnk versus 1/T plots, which is likely the result of liquid crystal properties of cholesterol. The studied capillary monolithic columns showed extremely fast re-equilibration after gradient elution and found to be stable under such conditions as: fast flow rate, high acetonitrile content in the mobile phase (no swelling) and high temperature.

Cholesterol-based polymeric monolithic columns for capillary liquid chromatography.

A novel, cholesterol-based polymeric monolithic stationary phase for capillary liquid chromatography, was prepared by thermally initiated in-situ polymerization. Cholesteryl methacrylate (CholMA) was used as a functional monomer and trimethylolpropane trimethacrylate (TRIM) was a cross-linker, while azobisisobutyronitrile (AIBN) was an initiator. Isooctane and toluene were chosen as "poor" and "good" solvent, respectively, as constituents of the porogen solvent. Isocratic elutions of alkylbenzenes and separation of the testing mixture of o-terphenyl and triphenylene were conducted for all of the monoliths to assess their hydrophobicity and planar selectivity characteristic for cholesterol-based stationary phases. The synthesized columns demonstrated efficiency exceeding N=10,000 plates and a plate height of ca. H=30 µm. Column preparation was found to be highly reproducible; the relative standard deviation (RSD) values (n=3) for day-to-day and column-to-column were less than 4.08 and 2.02%, respectively, based on retention factor of alkylbenzenes.

Monolithic molecularly imprinted polymeric capillary columns for isolation of aflatoxins.

Monolithic molecularly imprinted polymers extraction columns have been prepared in fused-silica capillaries by UV or thermal polymerization in a two-step process. First, a poly-(trimethylolpropane trimethacrylate) (polyTRIM) core monolith was synthesized either by UV or thermal polymerization. Then it was grafted with the mixture of methacrylic acid (MAA) as a functional monomer, ethylene dimethacrylate (EDMA) as a cross-linking agent, 5,7-dimethoxycoumarin (DMC) as an aflatoxin-mimicking template, toluene as a porogen solvent and 2,2-azobis-(2-methylpropionitrile) (AIBN) as an initiator of the polymerization reaction. Different thermal condition of the photografting and different concentrations of the grafting mixture were tested during polymerization. The extraction capillary columns were evaluated in the terms of their hydrodynamic and chromatographic properties. Retention coefficients for aflatoxin B1 and DMC were used for assessment of the selectivity and imprinting factor. The obtained results indicate that the temperature of photografting and concentration of the grafting mixture are key parameters that determine the quality of the prepared MIPs. From the MIP columns characterized by the highest permeability the column of the highest imprinting factor was applied for isolation of aflatoxins B1, B2, G1 and G2 from the model aqueous sample followed by on-line chromatographic separation. The process was performed using a micro-MISPE-microLC-LIF system of a novel design, which allowed for detection of the eluates from the sample preparation part as well as from the chromatographic separation.

Preparation of Monolithic Capillary Chromatographic Columns Using Supercritical Fluid as a Porogen Solvent.

Monolithic polymeric beds were synthesized in fused silica capillaries using either trimethylolpropane trimethacrylate (TRIM) or a mixture of butyl methacrylate (BMA) with ethylene glycol dimethacrylate (EDMA) as monomers. Carbon dioxide at temperature and pressure conditions above its critical values was used as a porogen solvent. The purpose of using the supercritical carbon dioxide was to have the possibility of changing the solvation power (and thus the porosity of the resulting monolith) of the porogen by pressure and temperature changes instead of changing the porogen composition. The experiments were performed using a special setup consisting of a stainless steel high-pressure reactor to which the fused silica capillary was connected. The synthesized monoliths underwent liquid chromatographic evaluation. The polyTRIM capillary monoliths were characterized by different permeability, which depended on the pressure of the synthesis. BMA/EDMA columns were applied for separation of alkylbenzenes and a model mixture of proteins.

Supramolecular recognition of estrogens via molecularly imprinted polymers.

The isolation and preconcentration of estrogens from new types of biological samples (acellular and protein-free simulated body fluid) by molecularly imprinted solid-phase extraction has been described. In this technique, supramolecular receptors, namely molecularly imprinted polymers (MIPs) are used as a sorbent material. The recognition sites of MIPs were prepared by non-covalent multiple interactions and formed with the target 17beta-estradiol as a template molecule. High-performance liquid chromatography with spectroscopic UV, selective, and a sensitive electrochemical CoulArray detector was used for the determination of 17beta-estradiol, estrone, and estriol in simulated body fluid which mimicked human plasma.

Effect of zeta potential value on bacterial behavior during electrophoretic separation.

The aggregation and/or adhesion of bacterial cells is a serious disadvantage of electrophoretic separations. In this study, physicochemical surface characteristics of bacteria were measured to establish their role in bacterial adhesion and aggregation on the basis of electrophoretic behavior of different clinical strains of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria. The number and the shape of peaks obtained on the electropherograms were connected with the zeta potential measurements and in-line microscope observation using specially designed CE fluorescence stereomicroscope setup. These results suggest that the lower the zeta potential, the higher the number of smaller peaks detected. The direct microscopic observation of electrophoretic movement proved the presence of many small aggregates originating from individual or clustered bacterial cells. On the other hand, lower zeta potential was also observed for dead bacterial cells, which suggested that some of the peaks can be attributed to viable cells while the other to the dead ones.

Differentiation of Staphylococcus aureus strains by CE, zeta potential and coagulase gene polymorphism.

Staphylococcus aureus is a common cause of infection in both hospitals and the community, and it is becoming increasingly virulent and resistant to antibiotics. Possibilities of fast, sensitive and cheap determination of these pathogenic bacteria are extremely important in antimicrobial therapy. In the present study, CE with chemically modified capillary and zeta potential measurements were used for differentiation of three different clinical strains of S. aureus. The data presented in this contribution suggested that electrophoretic behavior and the values of zeta potential should be very useful in distinguishing between closely related strains, which exhibited coagulase gene/protein polymorphism. Understanding the differences between S. aureus strains could help to improve our knowledge about S. aureus pathogenecity and to monitor for and respond to emergence of more virulent strains.

Migration of bacteria through a monolith.

The separation of bacteria by electromigration techniques was a subject of several of our previous papers. This contribution presents the results of investigation of the porosity of the monolithic bed and migration of Staphylococcus aureus cells through it. The gigaporous monolith was thermally synthesized using glycidyl methacrylate, triethylene glycol dimethacrylate and trimethylolpropane trimethacrylate as the monomers in the presence of porogen solvent containing 1-decanol, polyethylene glycol and 2-methoxyethanol. The porous properties were evaluated by inverse size-exclusion chromatography (ISEC) using a wide range of polystyrene standards of different molecular weights. The results have shown, that large pores (ca. 300 nm) dominate in the monolithic bed structure, however much larger flow-through pores must also be present as ca. 1 microm sized S. aureus bacteria were able to migrate through the bed.

Effect of temperature during photopolymerization of capillary monolithic columns.

Polymeric monolithic capillary columns were synthesized using butyl methacrylate (BMA), ethylene glycol dimethacrylate (EDMA), and 2-acrylamido-2-methylpropanesulfonic acid (AMPS) as monomers and 1,4-butanediol, 1-propanol, and water as a porogen mixture. The synthesis was performed over a wide temperature range from -15 degrees C to +70 degrees C using UV radiation to trigger the polymerization process initiated by benzoin methyl ether (BME). The columns exhibited different hydrodynamic properties (permeability) as well as efficiency. The most efficient and the most permeable columns were those polymerized at low temperatures (-15 degrees C to +10 degrees C). The columns photopolymerized at higher temperatures (T > or = 30 degrees C) were completely useless because of low permeability or efficiency. The presented results indicate that temperature can be another factor which allows control of porosity during the preparation of monoliths by photopolymerization.

EOF in monolithic poly(styrene-co-divinylbenzene) capillary columns.

In this paper, we present the result of our study on generation of EOF on uncharged poly(styrene-co-divinylbenzene)-based monolithic capillary columns. Three types of neutral monoliths were used: unmodified poly(styrene-co-divinylbenzene) and two modified with octadecyl chains: using grafting process or Friedel-Crafts reaction. The electroosmotic mobilities (mu(EOF)) versus pH profiles are compared, as well as molecular modeling using HyperChem software was employed to describe the observed phenomena. The mu(EOF) was almost independent of pH when pH was increased from 2.5 to 9.5 during the consecutive experiments; however, it decreased significantly when pH was decreasing between these values. The modeling has shown that the buffer molecules can be attracted by the neutral polystyrene surface only in the presence of water molecules, thus creating a negatively charged layer.