Elution Behavior Research Articles

Atomistic Details of Peptide Reversed-Phase Liquid Chromatography from Molecular Dynamics Simulations

Peptide separations by reversed-phase liquid chromatography (RPLC) are an integral part of bottom-up proteomics. These separations typically employ C18 columns with water/acetonitrile gradient elution in the presence of formic acid. Despite the widespread use of such workflows, the exact nature of peptide interactions with the stationary and mobile phases is poorly understood. Here, we employ microsecond molecular dynamics (MD) simulations to uncover details of peptide RPLC. We examined two tryptic peptides, a hydrophobic and a hydrophilic species, in a slit pore lined with C18 chains that were grafted onto SiO2 support. Our simulations explored peptide trapping, followed by desorption and elution. Trapping in an aqueous mobile phase was initiated by C18 contacts with Lys butyl moieties. This was followed by extensive anchoring of nonpolar side chains (Leu/Ile/Val) in the C18 layer. Exposure to water/acetonitrile triggered peptide desorption in a stepwise fashion; charged sites close to the termini were the first to lift off, followed by the other residues. During water/acetonitrile elution, both peptides preferentially resided close to the pore center. The hydrophilic peptide exhibited no contacts with the stationary phase under these conditions. In contrast, the hydrophobic species underwent multiple transient Leu/Ile/Val binding interactions with C18 chains. These nonpolar interactions represent the foundation of differential peptide retention, in agreement with the experimental elution behavior of the two peptides. Extensive peptide/formate ion pairing was observed in water/acetonitrile, particularly at N-terminal sites. Overall, this work uncovers an unprecedented level of RPLC molecular details, paving the way for MD simulations as a future tool for improving retention prediction algorithms and for the design of novel column materials.

Development of Centrifugal Partition Chromatography for the Purification of Antibody-Drug Conjugates.

Monoclonal antibody-drug conjugates (ADCs) are an expanding therapeutic class of biomolecules for which relatively few analytical and preparative separation options exist. Purification of ADCs with a specific drug antibody ratio is even more challenging. We report the first application of countercurrent separation (CCS) to this problem. An ADC mimic was successfully chromatographed using an aqueous two-phase system (ATPS) consisting of PEG 1000/sodium citrate pH 7.5/water, 17.75/17.75/64.50 (w/w/w). Notably, different partition coefficients (K) in this ATPS for the ADC mimic (0.09 < K < 0.16) and its monoclonal antibody backbone, IgG (0.16 < K < 0.27), were observed using CCS. Differential elution behavior of such high-molecular-weight biomolecules, 146,441 vs. ∼150,000 Da, using CCS has no precedent. The results provide a proof of concept for further exploration of the application of ATPSs and CCS to the separation of ADCs.

Factors Influencing the Prediction Accuracy of Model Peptides in Reversed-Phase Liquid Chromatography

Hydrophobicity is an important physicochemical property of peptides in solution. As well as being strongly associated with peptide stability and aggregation, hydrophobicity governs the solution based chromatographic separation processes, specifically reversed-phase liquid chromatography (RPLC). In addition, hydrophobicity is a major physicochemical property of peptides in comparison to H-bonding, electrostatic, and aromatic properties in intermolecular interactions. However, a wide range of molecular factors can influence peptide hydrophobicity, with accurate predictions depending on specific peptide amino acid compositions, structure, and conformation. It is noticeable that peptide composition, the position of the amino acid, and its neighbouring groups play a crucial role in the elution process. In light of this, the same amino acid behaved differently depending on its position and neighbouring amino acid in the peptide chain. Extra attention should be paid to the denaturation process during the course of elution, as it has been shown to complicate and alter the elution pattern. This paper reports on the key peptide properties that can alter hydrophobicity and, consequently, the RPLC elution behaviour of the peptides, and it will conclude by proposing improved prediction algorithms for peptide elution in RPLC.

Elution Behavior of Carbohydrates for Core-Shell Ion-Exchange Resin St-60 with Different Degrees of Cross-Linking in the Porous Shell

Elution Behavior of Carbohydrates for Core-Shell Ion-Exchange Resin St-60 with Different Degrees of Cross-Linking in the Porous Shell

Performance of global retention models in the optimisation of the chromatographic separation (I): Simple multi-analyte samples

Conventional retention models lead to accurate descriptions of the elution behaviour from the fitting of data for single solutes or from a set of solutes, one by one. However, the simultaneous fitting of several solutes through a regression process that separates the contributions of column and solvent from those of each solute is also possible. The result is a global retention model constituted by a set of equations with some common parameters (those associated with column and solvent), whereas others, specific to each solute, differ for each equation. This work explores the possibilities, advantages, and limitations of global models when they are applied to the optimisation of chromatographic resolution. A set constituted by 13 drugs (diuretics and β-blockers) and a training experimental design of seven multi-linear gradients are considered. Since standards for all compounds were available, the optimisation based on global models could be compared with the conventional optimisation, which is based on individual models. In their current state, global models do not predict changes in elution order, but they do allow for incorporating additional solutes (e.g., new analytes or matrix peaks) with only one new experiment. This possibility is explored by extending the model for the 13 analytes to include 26 peaks associated with a contamination in the injector. The combination of individual and global models allows an optimisation where the effects of matrix peaks on the separation of analytes can be integrated.

Anti-Langmuir elution behavior of a bispecific monoclonal antibody in cation exchange chromatography: Mechanistic modeling using a pH-dependent Self-Association Steric Mass Action isotherm

The objective of this scientific work was to model and simulate the complex anti-Langmuir elution behavior of a bispecific monoclonal antibody (bsAb) under high loading conditions on the strong cation exchange resin POROS™ XS. The bsAb exhibited anti-Langmuirian elution behavior as a consequence of self-association expressed both in uncommon retentions and peak shapes highly atypical for antibodies. The widely applied Steric Mass Action (SMA) model was unsuitable here because it can only describe Langmuirian elution behavior and is not able to describe protein-protein interactions in the form of self-association. For this reason, a Self-Association SMA (SAS-SMA) model was applied, which was extended by two activity coefficients for the salt and protein in solution. This model is able to describe protein-protein interactions in the form of self-dimerization and thus can describe anti-Langmuir elution behavior. Linear gradient elution (LGE) experiments were carried out to obtain a broad dataset ranging from pH 4.5 to 7.3 and from 50 to 375 mmol/L Na+ for model parameter determination. High loading LGE experiments were conducted with an increasing load from 0.5 up to 75.0 mgbsAb/mLresin. Thereby, pH-dependent empirical correlations for the activity coefficient of the solute protein, for the equilibrium constant of the self-dimerization process and for the shielding factor could be set up and ultimately incorporated into the SAS-SMA model. This pH-dependent SAS-SMA model was thus able to simulate anti-Langmuir behavior over extended ranges of pH, counterion concentration, and column loading. The model was confirmed by experimental verification of simulated linear pH gradient elutions up to a load of 75.0 mgbsAb/mLresin.

Two peak elution behavior of a monoclonal antibody in cation exchange chromatography as a screening tool for excipients

Aggregation of proteins is a critical quality attribute and a major concern during the purification of therapeutic proteins, like monoclonal antibodies. In-solution experiments applying different stress scenarios, e.g., mechanical, or physical stresses, can determine the overall conformational stability of the protein to enhance drug product shelf-life. Several groups have reported surface-induced unfolding and aggregation of monoclonal antibodies and their derivatives during cation exchange chromatography, which results in a two-peak elution behavior of the protein and its species. We have investigated universal influencing factors, like temperature and hold time, on this phenomenon. The formation of the second peak is a kinetic process, which is strongly influenced by temperature during the hold time. However, our main focus was the application of excipients and their influence on the two-peak elution behavior. We compared the on-column screening results with results obtained through a “traditional” in-solution screening using nanoDSF. Mostly, stabilizing excipients, like Sucrose, show their stabilizing abilities in both systems, but some discrepancies, e.g., using Arginine, between the two orthogonal techniques show the potential of the on-column screening system to lead to unexpected results, which would not necessarily be visible in in-solution experiments.

Degradation pathways and impurity profiling of the anticancer drug apalutamide by HPLC and LC-MS/MS and separation of impurities using Design of Experiments.

Apalutamide, an androgen receptor inhibitor, is used to treat prostate cancer. A stability-indicating high-performance liquid chromatography method was developed for the estimation of assay and organic impurities of apalutamide in drug substance and in tablet dosages using Design of Experiments. The chromatographic separation was achieved within 30 min using Atlantis dC18 , 100 × 4.6mm, 3.0 μm and the binary gradient program (10 mm KH2 PO4 , pH3.5; acetonitrile). The detection wavelength, flow rate, column temperature and injection volume used were 270 nm, 1.0 ml/min, 45°C and 10 μl, respectively. The interaction of independent variables (pH, column temperature and flow rate) and their influences on HPLC parameters were studied using a central composite design, and then the peak separation and elution behaviors between apalutamide and its seven impurities were determined. The method validation was performed for linearity, detection limit, quantitation limit, accuracy, precision and robustness as per the International Conference on Harmonization. A high-quality recovery with good precision (91.7-106.0%) and correlations (r2 > 0.997) within a linear range of 0.12-2.24 μg/ml (0.05-0.3%, w/w) were achieved consistently for assay and organic impurities of apalutamide. The stability-indicating characteristics of the proposed method were assessed through forced degradation and mass balance studies. An effort was made to figure out the chemical structures of newly formed degradation products (DP1-DP5) using LC-MS/MS.

Elution behavior of mercury in desulfurization gypsum produced in a coal-fired power plant

Batch and semi-batch elution tests were conducted to determine the elution pattern and dissolvable amount of mercury in desulfurization gypsum produced in a coal-fired power plant. Two gypsum samples obtained from double-contact-flow-type desulfurization equipment were used. Chemical forms of mercury in gypsum were identified by temperature programmed desorption–cold vapor atomic absorption spectroscopy. The elution behavior of mercury varied significantly with the testing method. In the batch elution test, an industrial standard method, mercury eluted into the water rapidly and was then adsorbed by gypsum. This behavior was kinetically simulated with the sequential elution and adsorption of mercury, showing the maximum elution peak. In contrast, in the semi-batch method, the adsorption phenomenon was negligible, and the ultimate amount of eluted mercury could be determined. Thus, the semi-batch method is more accurate for evaluating mercury elution from desulfurization gypsum.

Analysis of complex drugs by comprehensive two-dimensional gas chromatography and high-resolution mass spectrometry: detailed chemical description of the active pharmaceutical ingredient sodium bituminosulfonate and its process intermediates

The European pharmacopeia provides analytical methods for the chemical characterization of active pharmaceutical ingredients (APIs). However, the complexity of some APIs exceeds the limitations of the currently prevailing physicochemical methods. Sodium bituminosulfonate (SBS) is described by the collection of key parameters of generalizing criteria such as dry matter, sulfur and sodium content, and neutrality, but techniques to unravel the complexity on a molecular level are lacking. We present a study based on online derivatization with tetramethylammonium hydroxide in combination with comprehensive two-dimensional gas chromatography coupled to an electron ionization high-resolution time-of-flight mass spectrometer (GC × GC-HR-ToF–MS) for the chemical description of SBS as well as its process intermediates. The application of GC × GC allowed the comprehensive description of the chemical components in the API and the process intermediates for the first time. Furthermore, it was possible to classify peaks regarding their elemental and structural composition based on accurate mass information, elution behavior, and mass fragmentation pattern. This work demonstrates not only the general applicability, advantages but also limitations of GC × GC for the characterization of APIs for complex drugs.Graphical

Effect of a micronization method on the particle-size distribution and eluted phosphate-ion concentration for methane fermentation residue sludge

Micronized methane fermentation residue sludge can be used as a liquid fertilizer because the micronized solid particles can be directly sprayed. Thus, the objective of this study was to clarify the effect of the micronization method on the particle-size distribution and eluted phosphate-ion concentration of sludge micronized by ultrasonication or ball milling. Although the average particle size in the sludge micronized by ball milling was ~15 µm smaller than that micronized by ultrasonication, many finer particles (<10 µm) were observed in the ultrasonication-micronized sludge. Compared with untreated sludge, the concentration of the eluted phosphate ions of ultrasonication-micronized sludge was almost 15% higher, whereas that of the ball-milled sludge changed insignificantly. These results indicate that the particle sizes in the ball-milled and ultrasonication-micronized sludge were different, resulting in different phosphate ion elution behavior. Thus, the particle size and phosphate ion concentration can be controlled by changing and/or combining micronization techniques and optimizing their operating conditions. Effect of a micronization method on the particle-size distribution and eluted phosphate-ion concentration for methane fermentation residue sludgeAll authorsTakamasa Mori , Yui Komiya & Mitsuyasu Yabehttps://doi.org/10.1080/23311916.2022.2138869Published online:27 October 2022Display full size

Radiation stability of two extraction chromatography resin materials containing substituted diglycolamide ligands in an ionic liquid

The radiation stability of two extraction chromatography resins containing diglycolamide ligands viz. TPDGA (N,N,N’,N’-tetra-n-pentyl diglycolamide) and TODGA (N,N,N’,N’-tetra-n-octyl diglycolamide) in a room temperature ionic liquid (C4mim.NTf2) was studied by exposing to gamma rays from a 60Co source. The resins were irradiated to varying gamma ray dose up to 1000 kGy where both the dry resins and wet resins (containing a fixed amount nitric acid in contact) were used. The performance assessment of the resins was done by physicochemical characterization as well as uptake studies; both by batch as well as column methods. The physicochemical characterization of the resins was done using FTIR analysis while the surface morphology of the resins was carried out by scanning electron microscopy. The uptake of the metal ions, typically that of Am(III) and Eu(III), representing the trivalent actinides and lanthanides was studied by batch method. The dry resins and those in contact with nitric acid yielded nearly identical results suggesting minimal effect of the radiolytic products of nitric acid on the resin performance. There was negligible change in the Kd values up to an absorbed dose of 300 kGy beyond which there was sharp decrease. However, the Kd values were still quite large even after an absorbed dose of 1000 kGy. The column performance of the irradiated resins was also assessed by the uptake and elution of Eu(III) ion and though loading was affected, the elution behavior was found to have insignificant effect.

Loading Glyphosate in Attapulgite and Sodium Alginate Hydrogels to Construct pH-Responsive Controlled Release Microsphere for Enhanced Soil Sustained Release

Herein, we constructed the microsphere SAH-Gly by loading glyphosate (Gly) with dopamine-modified attapulgite (DA-Gly) and capsuling in sodium alginate hydrogels. The Gly releasing rate and the hydrogel swelling rate were manifestly increased to the highest of 60 and 26% within 70 and 24 h in alkaline solution (pH = 8.5), respectively, while only 20% Gly releasing rate and less than 15% hydrogel swelling rate were observed in both acidic (pH = 5.5) and neutral solutions (pH = 7.0). The abundant carboxyl groups in SAH-Gly provided the swelling characters through H+ ionization. Both DA-Gly and SAH-Gly enhanced the stability of Gly to ultraviolet rays and temperature variability. The sustained release of Gly from SAH-Gly resulted in its partial absorption in soils. DA-Gly and SAH-Gly released maximum Gly molecular concentrations after 28 and 32 h, respectively, and then degraded completely in 200 h in soil. First-order kinetics showed that the half-life (DT50) of the Gly residue in soil was 65.89 h, while the DT50 values of DA-Gly and SAH-Gly were 31.18 and 66.27 h, respectively, with the following order: DA-Gly < Gly < SAH-Gly. In addition, leaching experiments depicted that SAH-Gly displayed a weaker elution behavior and longer elution time in the upper soil ring of the column. The sustained release behavior of Gly from SAH-Gly in soil could decelerate the degradation, adsorption, and leaching characters of Gly molecular, helping reduce the loss of active Gly and enhancing its sustainable agricultural practice in response to environmental factors.

Effect of parameters on retention behavior of ovalbumin and immunoglobulin E in asymmetrical flow field‐flow fractionation

Asymmetrical flow field-flow fractionation is a rising useful technique to separate and characterize macromolecules. Elution behaviors of allergen protein ovalbumin and immunoglobulin E were studied here. Effects of flow rates, kinds of carrier solutions, and injection amounts were investigated in the desire to experiment and verify the effect of every factor and to validate asymmetrical flow field-flow fractionation theory. Results suggested that cross-flow is the most important factor among all the factors that have been studied. Too-low focus flow (0.5 ml/min) and outflow (0.4 ml/min) are harmful for protein separation and characterization. Smaller size ovalbumin (with a monomer is 11.4 nm) is more prone to be influenced compared with the bigger analyte immunoglobulin E (with a monomer diameter is 32.1 nm) by flow rates. The adopted types of carrier fluids did not change the retention behavior of the two proteins. Still, protein aggregation and membrane adsorption should be paid more attention to under different carrier fluids.

Synthesis and application of chloroacetamides in pyridinium based ionic liquid for high temperature extraction of uranyl ion: A novel and 'green' approach for extractive mass transfer at elevated temperature

Two different chloroacetamides viz 2-chloro-N-methyl-N-phenylacetamide (MACA) and 2-chloro-N-(3-methylpyridin-2-yl)acetamide (AMPCA) have been synthesized and applied for the extraction of uranyl ions from acidic feed. 1-Ethyl-2-methylpyridinium bis(trifluoromethylsulfonyl)imide ionic liquid (IL) has been used at elevated temperature for liquid–liquid biphasic mass transfer. An effort has also been made to study the mechanism of extraction, kinetics, thermodynamics, speciation, radiolytic stability and elution behavior of UO22+. The investigation revealed the predominance of ‘cation exchange’ mechanism. The D values in 2-chloro-N-methyl-N-phenylacetamide (MACA) were found to be more than that of 2-chloro-N-(3-methylpyridin-2-yl)acetamide (AMPCA). The species were found to be[(UO2(NO3)(MACA)2]+ and [(UO2(NO3)(AMPCA)2]+. Back extraction studies using aqueous solution of oxalic acid and sodium carbonate revealed that, the stripping of uranyl ion from AMPCA in ionic liquid was better than that of MACA. However, MACA exhibited better radiolytic stability as compared to AMPCA in ionic liquid. To understand the complexation of UO22+ with MACA and AMPCA, theoretical calculations were carried out.

Pilot study comparison of regenerable and emerging single-use anion exchange resins for treatment of groundwater contaminated by per- and polyfluoroalkyl substances (PFASs)

This study reports the results of an 8-month pilot study comparing both regenerable and emerging single-use anion exchange resins (AERs) for treatment of per- and polyfluoroalkyl substances (PFASs) at a source zone impacted by historical use of aqueous film-forming foam (AFFF). Two regenerable (Purolite A860 and A520E) and three single-use (Purolite PFA694E, Calgon CalRes 2301, and Dowex PSR2+) AERs were tested in parallel, collecting effluent samples after treatment for 30-sec and 2-min total empty bed contact time (EBCT). Results demonstrate that single-use AERs significantly outperform regenerable resins, particularly for treatment of long-chain perfluoroalkyl carboxylic acids (PFCAs) and perfluoroalkyl sulfonic acids (PFSAs). No detectable concentrations of ≥C7 PFCAs or PFSAs were observed within 150,000 bed volumes (BVs) after treatment with the single-use resins (2-min EBCT). Analysis of effluent samples following 30-sec EBCT treatment shows that even the shortest-chain PFSAs do not reach 50% breakthrough within the first 350,000 BVs, though differences in removal of short-chain PFCAs was less dramatic. The regenerable polyacrylic A860 resin performed very poorly compared to all polystyrene resins, with >90% breakthrough of all PFASs occurring within 10,000 BVs. The greater affinity of polystyrene resins is attributed to increased hydrophobic interactions in addition to electrostatic ion exchange. Analysis of breakthrough profiles reveals empirical correlation with ion exchange affinity coefficients (logKex) measured in batch experiments. Postmortem analysis of PFASs extracted from spent resins revealed chromatographic elution behavior and competition among PFASs for adsorption to the resins. PFSAs and long-chain PFCAs were preferentially adsorbed to earlier sections in the AER columns, whereas short-chain PFCAs were competitively displaced towards the later sections of the columns and into the effluent, consistent with effluent concentrations of the latter structures exceeding influent values. These results provide insights into the mechanisms that govern PFAS adsorption to AERs in real multisolute groundwater matrices and support findings from other diverse sites regarding PFAS affinity, elution behavior, and competition for exchange sites.

Do Self-adhesive Resin Composites Release More Monomers? A Comparative High-performance Liquid Chromatographic Analysis.

To comparatively evaluate the elution of residual monomers (bis-GMA, bis-EMA, TEG-DMA, and HEMA) from two self-adhesive flowable resin composites, a giomer, and a nano-flowable resin composite over five different time intervals, using high-performance liquid chromatography (HPLC). Four flowable resin composites were investigated (Vertise Flow, Constic, Beautifil Flow Plus F03, and Filtek Z350 XT). Immediately after polymerization, each sample was immersed in 75% ethanol/water solution and stored in amber-colored bottles at room temperature. HPLC analysis was performed at predefined time intervals: 1 h, 24 h, 4 days, 8 days and 16 days. The extraction solution was changed after each analysis. Data were analyzed with repeated-measures ANOVA and one-way ANOVA with Tukey's post-hoc test at p < 0.05. The highest mean concentration of residual monomers was eluted from Beautifil, followed by Filtek, and both were significantly higher (p < 0.05) than the mean concentration of eluates from self-adhesive resin composites (Vertise Flow and Constic). Vertise Flow released significantly higher concentrations of HEMA than all the other tested materials. At 1 h post-immersion, 52.2% of monomers were eluted, and continued to elute at a reduced rate throughout the study duration. TEG-DMA was the fastest monomer to leach out, while bis-GMA exhibited significantly higher total mean concentration. The elution rate was significantly dependent on the molecular weight of the eluted monomers. No specific elution behavior can be attributed to self-adhesive RBCs. Elution of residual monomers is dependent on each material's composition, resin matrix characteristics, and the monomer's molecular weight.

Integrated process model for the prediction of biopharmaceutical manufacturing chromatography and adjustment steps

A fundamental process understanding of an entire downstream process is essential for achieving and maintaining the high-quality standards demanded for biopharmaceutical drugs. A holistic process model based on mechanistic insights could support process development by identifying dependencies between process parameters and critical quality attributes across unit operations to design a holistic control strategy. In this study, state-of-the-art mechanistic models were calibrated and validated as digital representations of a biopharmaceutical manufacturing process. The polishing ion exchange chromatography steps (Q Sepharose FF, Poros 50 HS) were described by a transport-dispersive model combined with a colloidal particle adsorption model. The elution behavior of four size variants was analyzed and included in the model. Titration curves of pH adjustments were simulated using a mean-field approach considering interactions between the protein of interest and other ions in solution. By including adjustment steps the important process control inputs ionic strength, dilution, and pH were integrated. The final process model was capable to predict online and offline data at manufacturing scale. Process variations at manufacturing scale of 94 runs were adequately reproduced by the model. Furthermore, the process robustness against a 20% input variation of concentration, size variant and ion composition, volume, and pH could be confirmed with the model. The presented model demonstrates the potential of the integrated approach for predicting manufacturing process performance across scales and operating units.

Influence of ligand density variations on the two peak elution behavior of a monoclonal antibody in cation exchange chromatography

Cation exchange chromatography, as part of the monoclonal antibody purification train, is known as a mild polishing technique. However, in the last couple of years, more and more publications have shown unusual elution behavior, resulting from e.g. on-column (reversible) unfolding and aggregation of the predominantly mAb molecules. The stability of the investigated protein seems to play a significant role in this phenomenon. We have used a glycosylated IgG1 antibody as a model protein and investigated several influencing factors, including pH value and ligand density variations of three prototype Fractogel® cation exchange resins. Ligand density, pH and salt concentration are the main contributing factors in the Donnan effect, i.e. distribution of ions, between resin pore volume and bulk volume. This leads to a significantly lower pH value the protein is subjected to during the on-column hold time and therefore influences the conformational stability of our protein. Nano-DSF and kinetic SEC measurements show that the protein is destabilized at low pH values, but also, that the binding to the CEX resin and the elution with increasing salt concentration is responsible for the resulting two-peak elution behavior and partially reversible unfolding and aggregation.

Chromatography columns packed with thermoresponsive-cationic-polymer-modified beads for therapeutic drug monitoring

Therapeutic drug monitoring, which is used to determine appropriate drug doses, is critical in pharmacological therapy. In this study, we developed thermoresponsive chromatography columns with various cationic properties for effective therapeutic drug monitoring. Thermoresponsive cationic copolymer poly(N-isopropylacrylamide-co–n-butyl methacrylate-co–N,N-dimethylaminopropyl acrylamide) (P(NIPAAm-co-BMA-co-DMAPAAm))-modified silica beads, which were used as the chromatographic stationary phase, were prepared by modifying the radical initiator of the silica beads, followed by radical polymerization. Characterization of the prepared silica beads demonstrated that thermoresponsive polymers with various cationic properties successfully modified the beads. The elution behavior of several steroids in the prepared bead-packed columns at various temperatures indicated that the optimal column operating temperature was 30 °C. Appropriate measurement conditions for 13 drugs were investigated by varying the cationic properties of the columns and the pH of the mobile phase. Drug concentrations in serum samples were determined using the developed columns and mobile phases with a suitable pH. Voriconazole concentrations in human serum samples were determined using the developed columns with all-aqueous mobile phases. We anticipate that the developed chromatography columns can be used for therapeutic drug monitoring because drug concentrations can be measured using all-aqueous mobile phases that are suitable in clinical settings.