Reversed-phase Liquid Chromatography Mode Research Articles

One-pot synthesis of a poly(styrene-acrylic acid) copolymer-modified silica stationary phase and its applications in mixed-mode liquid chromatography

As modified ligands with a wide range of sources, abundant functional groups, and good biocompatibility, polymers have been widely used in the development of silica-based chromatographic stationary phases. In this study, a poly(styrene-acrylic acid) copolymer-modified silica stationary phase (SiO2@P(St-b-AA)) was prepared via one-pot free-radical polymerization. In this stationary phase, styrene and acrylic acid were used as functional repeating units for polymerization and vinyltrimethoxylsilane (VTMS) was used as a silane coupling agent to link the copolymer and silica. Various characterization methods, such as Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), scanning electron microscopy (SEM), N2 adsorption-desorption analysis, and Zeta potential analysis, confirmed the successful preparation of the SiO2@P(St-b-AA) stationary phase, which had a well-maintained uniform spherical and mesoporous structure. The retention mechanisms and separation performance of the SiO2@P(St-b-AA) stationary phase in multiple separation modes were then evaluated. Hydrophobic and hydrophilic analytes as well as ionic compounds were selected as probes for different separation modes, and changes in the retention of the analytes under various chromatographic conditions, including different methanol or acetonitrile contents and buffer pH values, were investigated. In reversed-phase liquid chromatography (RPLC) mode, the retention factors of alkyl benzenes and polycyclic aromatic hydrocarbons (PAHs) on the stationary phase decreased with increasing methanol content in the mobile phase. This finding could be attributed to the hydrophobic and π-π interactions between the benzene ring and analytes. The retention changes of alkyl benzenes and PAHs revealed that the SiO2@P(St-b-AA) stationary phase, similar to the C18 stationary phase, exhibited a typical reversed-phase retention behavior. In hydrophilic interaction liquid chromatography (HILIC) mode, as the acetonitrile content increased, the retention factors of hydrophilic analytes gradually increased, and a typical hydrophilic interaction retention mechanism was inferred. In addition to hydrophilic interaction, the stationary phase also demonstrated hydrogen-bonding and electrostatic interactions with the analytes. Compared with the C18 and Amide stationary phases prepared by our groups, the SiO2@P(St-b-AA) stationary phase exhibited excellent separation performance for the model analytes in the RPLC and HILIC modes. Owing to the presence of charged carboxylic acid groups in the SiO2@P(St-b-AA) stationary phase, exploring its retention mechanism in ionic exchange chromatography (IEC) mode is of great importance. The effect of the mobile phase pH on the retention time of organic bases and acids was further studied to explore the electrostatic interaction between the stationary phase and charged analytes. The results revealed that the stationary phase has weak cation exchange ability toward organic bases and electrostatically repels organic acids. Moreover, the retention of organic bases and acids on the stationary phase was influenced by the analyte structure and mobile phase. Thus, the SiO2@P(St-b-AA) stationary phase could provide multiple interactions, as demonstrated by the separation modes described above. The SiO2@P(St-b-AA) stationary phase showed excellent performance and reproducibility in the separation of mixed samples with different polar components, indicating that it has promising application potential in mixed-mode liquid chromatography. Further investigation of the proposed method confirmed its repeatability and stability. In summary, this study not only described a novel stationary phase that could be used in RPLC, HILIC, and IEC modes but also presented a facile "one-pot" preparation approach that could provide a new route for the development of novel polymer-modified silica stationary phases.

Ionic liquid-functionalized dendrimer grafted silica for mixed-mode chromatographic separation and online solid-phase extraction

The efficient sample pretreatment and high-performance chromatographic separation are essential for an accurate analysis of the complex sample. Finding a material that can apply as both sorbent for sample preparation and stationary phase for chromatography is greatly important for analytical chemistry. With a large number of active sites and designable properties, the dendrimers displayed potential performance for sample preparation and chromatographic separation. In order to improve its selectivity restricted by the limited type of functional groups (amide, ethylidene, amino groups), ionic liquid (1,1′-(1,4-butyricdiyl)bis(1-aminopropyl imidazolium)dibromide) were introduced to functionalize dendrimers. With porous silica as the base, ionic liquid-functionalized dendrimer grafted silica (SiO2@G1.5-IL) was obtained and characterized by X-ray photoelectron spectroscopy, elemental analyzer, as well as specific surface and porosity analyzer. The material was used as stationary phase for mixed-mode chromatography and sorbent for SPE, respectively. Under reversed-phase liquid chromatography (RPLC), it successfully separated some hydrophobic analytes (five polycyclic aromatic hydrocarbons (PAHs), two bisphenols, diphenylpropane, and diphenylmethane), by utilizing hydrophobic, π-stacking and hydrogen bonding interactions. Based on the hydrophilic effect, it could separate hydrophilic analytes (nucleosides and alkaloids), which coincident with hydrophilic interaction liquid chromatography (HILIC). And it exhibited better separation capacity than NH2@SiO2 and SiO2@G1.5 under the above two modes. Furthermore, the thermodynamic parameters (ΔH, ΔS, and ΔG) of the above types of analytes on the SiO2@G1.5-IL column were also investigated in detail, revealing the controlling factors during the chromatographic separation. And the relative standard deviations (RSDs, 0.03–2.95%, n = 10) of the repeatability of chromatographic separation were satisfactory. After that, SiO2@G1.5-IL-based SPE column was online combined with high-performance liquid chromatography to investigate the extraction selectivity for three types of hydrophobic organic pollutants (PAHs, estrogens, and bisphenols), and compared to SiO2 and C18 columns. According to their extraction performance, PAHs were selected as the target analytes, and the online analytical method was established. The limits of detection were as low as 0.001 μg L-1 due to the high enrichment factor reach to 3869, limits of quantity were 0.003–0.17 μg L-1, linear ranges were from 0.003 to 15.0 µg L-1, the adsorption capacity was 16.10 μg g−1, as well as RSDs were 0.5–3.7%, 0.6–6.7%, and 4.2–9.0% for intraday, interday tests and reproducibility. Finally, it was applied to analyze the environmental water samples (tap water, snow water, and rainwater), and satisfactory results were obtained (spiked recoveries were 86.8%-120.9%). Therefore, SiO2@G1.5-IL was a powerful material that can be used for the chromatographic separation of both hydrophobic and hydrophilic samples by RPLC or HILIC mode, as well as the efficient online extraction of hydrophobic targets prior to C18 column-based high-performance liquid chromatography.

Prediction of peptides retention behavior in reversed-phase liquid chromatography based on their hydrophobicity.

Hydrophobicity is an important physicochemical property of peptides and proteins. It is responsible for their conformational changes, stability, as well as various chemical intramolecular and intermolecular interactions. Enormous efforts have been invested to study the extent of hydrophobicity and how it could influence various biological processes, in addition to its crucial role in the separation and purification endeavor as well. Here, we have reviewed various studies that were carried out to determine the hydrophobicity starting from (i) simple amino acids solubility behavior, (ii) experimental approach that was undertaken in the reversed-phase liquid chromatography mode, and ending with (iii) some examples of more advanced computational and machine learning models.

Ultra-Fast Middle-Up Reversed Phase Liquid Chromatography Analysis of Complex Bispecific Antibodies Obtained in Less Than One Minute.

This work illustrates the benefits and limitations of using ultra-short reversed phase liquid chromatography (RPLC) columns for the characterization of various complex bispecific antibodies after prolonged thermal stress at the middle-up level of analysis. First, we have demonstrated that alternative organic modifiers, such as isopropanol, can be used in RPLC mode without generating excessive pressure, thanks to the prototype 10 × 2.1 mm, 2.7 µm particle column. However, compared to acetonitrile, the selectivity was not improved, at least for the selected biopharmaceutical products. Importantly, very fast separations (sub-1 min) of high quality were systematically obtained for the different samples when using a spectroscopic detector, but a severe loss of performance was observed with mass spectrometry (MS) detection due to dispersion effects. Based on these results, there is a clear need to improve the interfacing between LC and MS (shorter/thinner tubing) to mitigate band broadening.

A chiral porous organic polymer COP-1 used as stationary phase for HPLC enantioseparation under normal-phase and reversed-phase conditions.

A spherical chiral porous organic polymer (POPs) COP-1 is synthesized by the Friedel-Crafts alkylation reaction of Boc-3-(4-biphenyl)-L-alanine (BBLA) and 4,4'-bis(chloromethyl)-1,1'-biphenyl (BCMBP), which was used as a novel chiral stationary phase (CSPs) for mixed-mode high-performance liquid chromatography (HPLC) enantioseparation. The racemic compounds were resolved in normal-phase liquid chromatography (NPLC) using n-hexane/isopropanol as mobile phase and reversed-phase liquid chromatography (RPLC) using methanol/water as mobile phase. The COP-1-packed column exhibited excellent separation performance toward various racemic compounds including alcohols, amines, ketones, esters, epoxy compounds, organic acids, and amino acids in NPLC and RPLC modes. The effects of analyte mass and column temperature on the separation efficiency of racemic compounds were investigated. In addition, the chiral resolution ability of the COP-1-packed column not only can be complementary in RPLC/NPLC modes but also exhibit a good chiral recognition complementarity with Chiralpak AD-H column and chiral porous organic cage (POC) NC1-R column. The relative standard deviations (RSD) (n = 5) of the retention time, resolution value, and peak area by repeated separation of 1-(4-chiorophenyl)ethanol are all below 3.0%. The COP-1 column shows high column efficiency (e.g., 17,320 plates m-1 for 1-(4-chlorophenyl)ethanol on COP-1 column in NPLC), high enantioselectivity, and good reproducibility toward various racemates. This work demonstrates that chiral POPs microspheres are promising chiral materials for HPLC enantioseparation.

Sub/supercritical fluid chromatography versus liquid chromatography for peptide analysis

The aim of this study was to evaluate the potential of ultra-high performance supercritical fluid chromatography (UHPSFC) for peptide analysis by comparing its analytical performance to several chromatographic approaches based on reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC) and mixed-mode liquid chromatography. First, the retention behavior of synthetic peptides with 3 to 30 amino acids and different isoelectric points (acid, neutral, and basic) was evaluated. For all the tested conditions (13 peptides in 8 conditions), only 4 results were not exploitable (not retained or not eluted), confirming that all the tested chromatographic conditions can be successfully applied when analyzing a wide range of diverse peptides. Average tailing factor were quite comparable across all chromatographic modes, while the best peak capacity values were obtained under mixed-mode LC conditions. Selectivity for each chromatographic mode was also evaluated for six closely related peptides having minor modifications on their structures. The LC-based chromatographic modes confirmed their superior selectivity over UHPSFC. By contrast, when analyzing short peptides (di- or tripetides), UHPSFC was the only technique allowing to simultaneously separate highly polar and less polar peptides within the same run confirming its unique versatility. In addition, the sensitivity of each chromatographic approach was accessed by for two representative peptides by both UV and MS detection. With UV detection, limit of detection (LOD) values were comparable among the different chromatographic modes, ranging from 0.5 to 2 µg mL−1. However, major differences were found when employing MS detection (LOD values ranged from 0.05 to 5 µg mL−1). The best results were obtained under HILIC conditions, followed by SFC, and finally mixed-mode LC and RPLC modes.

Preparation and evaluation of two silica-based hydrophilic-hydrophobic and acid-base balanced stationary phases via in-situ surface polymerization

Two new hydrophilic-hydrophobic and acid-base balanced liquid chromatographic stationary phases were obtained by using two long-chain monomers including undecylenic acid (UA) and oleic acid (OA) combined with short-chain 4-vinyl pyridine (Py) in-situ polymerized on silica microspheres surfaces, respectively. These two stationary phases can be used for reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) modes. Hydrophilic, hydrophobic, acidic and basic analytes can be separated on two new stationary phases in RPLC or HILIC mode. Compared with commercial hydrophobic C18 columns and other HILIC columns, two new stationary phases have excellent selectivity and separation performance in the separation of 9 polycyclic aromatic hydrocarbons, 10 aromatic acids, 10 anilines and 5 oxazolidinones, respectively. This novel polymer-functionalized silica design strategy has opened up new ideas for the future of mixed-mode stationary phases with multiple functions and specificity.

Comparison of chromatographic performance of co-grafted silica using octadecene respectively with vinylpyrrolidone, vinylimidazole and vinylpyridine

Three reversed-phase liquid chromatography (RPLC) stationary phases were obtained by using long-chain 1-octadecene (OD) co-grafted with three short-chain monomers, including N-vinylpyrrolidone (NVP), 1-vinylimidazole (VIm) and 4-vinylpyridine (VPy), respectively (noted as Sil@ODNVP, Sil@ODVIm and Sil@ODVPy). Peak broadening phenomenon in RPLC mode which resulted by short-chain was examined carefully. Compared with Sil@ODNVP, both of Sil@ODVIm and Sil@ODVPy had smaller peak width and higher column efficiency in the separation of 10 polycyclic aromatic hydrocarbons (PAHs), 7 alkyl benzenes, 7 aromatic acids, 7 aromatic esters and 9 phenols. In addition, VPy has the strongest ion exchange capacity than other two short-chains. In this case, we can see that VPy and VIm maybe more suitable to be used as functional monomeric modifiers of new chromatographic stationary phases.

Preparation of two amide-bonded stationary phases and comparative evaluation under mixed-mode chromatography.

In this work, the conventional reactions were used to functionalize the silica surface with amide and hydrocarbon chain groups affording two different mixed-mode stationary phases (Sil-amide-C11 and Sil-C12-amide). The prepared stationary phases were analyzed by elemental analysis and thermogravimetric analysis. The retention of benzene, phenol, pyridine, and aniline was investigated and compared with synthesized and commercial columns, and this led to prove the existence of different interactions on the synthesized stationary phases. The mixed-mode stationary phases showed multiple interactions, and different chromatography modes were found under distinct chromatographic conditions. According to the type of amide group (either free or within the hydrocarbon chain), different interactions can be made on the columns. The alkylbenzenes and polycyclic aromatic hydrocarbons, as nonpolar hydrocarbons, were chromatographed under reversed-phase liquid chromatography modes, in which amide groups on the silica could efficiently separate polar analytes under hydrophilic interaction liquid chromatography mode in both prepared stationary phases. The performance of the columns was compared by the separation of the carboxylic acid group and biological samples. The bonding method and the type of amide group showed different interactions leading to different separation and performance.

Preparation and evaluation a mixed-mode stationary phase with C18 and 2-methylindole for HPLC.

A modified C18 column (Silpr-2MI-C18) was prepared using 2-methylindole and C18 reagent. The extent of C18 hydrocarbon chain, conjugative rings and anion exchange site provided multiple retention mechanisms, including reversed-phase liquid chromatography (RPLC), π-π interaction, hydrophilic interaction liquid chromatography (HILIC) and anion exchange chromatography (AEC). The separation of protected amino acids was investigated on the commercial C18 and Silpr-2MI-C18 columns, while the chromatographic conditions, including methanol content and pH of the mobile phase, were studied. The separation arrangement of the hydrophilic amino acids was different on the Silpr-2MI-C18 column compared to the commercial C18 column under RPLC mode. Furthermore, these amino acids were separated on the Silpr-2MI-C18 column under HILIC mode. The modified C18 column was employed to separate amino acids, alkylbenzenes and polycyclic aromatic hydrocarbons under RPLC mode and inorganic anion under AEC mode. The results confirm that this new stationary phase of RPLC/HILIC/AEC has multiple interactions with different analytes. Effective retention of biological samples was found on the Silpr-2MI-C18 column by comparing the results obtained from the commercial C18 column.

Preparation of brazilein from Caesalpinia sappan by high performance countercurrent chromatography

Brazilein is among the main chemical constituents of Caesalpinia sappan. It has diverse pharmacological activities. Modern pharmacological studies have shown that the compound has antitumor, anti-inflammatory, antibacterial, antioxidant, immunomodulatory, and other pharmacological activities. Brazilein is often used as a stain in various industries. The separation of brazilein by traditional column chromatography will not only result in contamination of the chromatographic column materials, but also lead to loss of the active ingredient. Countercurrent chromatography is an advanced liquid-liquid chromatographic separation technique. It has been widely used for natural product separation and isolation as it offers several advantages, such as low solvent consumption, a highly selective solvent system, and high recoveries. Typical countercurrent chromatography techniques include centrifugal partition chromatography (CPC), high-speed countercurrent chromatography (HSCCC), and high performance countercurrent chromatography (HPCCC). It is well known that choosing a suitable solvent system is vital in countercurrent separation. Therefore, two methods were introduced for choosing a suitable solvent system. One is the generally useful estimation of solvent systems (GUESS) method, which employs thin-layer chromatography (TLC) to identify a suitable solvent system with minimal labor for the rapid purification of target compounds, and another is the Shake-Flash method. The solvent system could be determined by observing the distribution of the sample in the upper and lower phases. Two kinds of solvent systems were screened using the TLC-GUESS and Shake-Flash methods, and tested through the analysis mode of the HPCCC instrument. The results showed that chloroform-methanol-water (4:3:2, v/v/v) was the optimal solvent system for HPCCC separation. A total of 15.2 mg of brazilein and 5.7 mg of caesappanin C were obtained from an ethyl acetate extract with high purities (95.6% and 89.0%, analyzed by HPLC) in one step using the preparation mode of HPCCC, the reversed-phase liquid chromatography mode with the apparatus rotated at 1600 r/min, a flow rate of 10 mL/min, separation temperature of 25℃, and detection wavelength of 285 nm. Their structures were determined by spectroscopic and spectrometric analyses. Brazilein stained the solid packing material in the column and was difficult to elute. The results showed that the use of HPCCC for the separation of brazilein can not only prevent the loss of target active ingredients in Caesalpinia sappan, but also shorten the separation and purification times and improve the operating efficiency. Therefore, HPCCC can be used for the separation and preparation of other pigment compounds in Caesalpinia sappan and other dye plants.

Preparation of reversed-phase/hydrophilic chromatographic stationary phase based on dialdehyde microcrystalline cellulose-functionalized C18

A novel reversed/hydrophilic chromatographic stationary phase based on dialdehyde microcrystalline cellulose (DMCC)-functionalized C18 was prepared by covalent bonding between the amino groups of octadecylamine with the aldehyde groups of DMCC, which was used in reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) modes. The prepared DMCC-functionalized C18 modified silica (C18-DMCC/SiO2) stationary phase exhibited good hydrophobic selectivity and aromatic selectivity by separating alkylbenzenes and polycyclic aromatic hydrocarbons (PAHs) in the RPLC mode. Polar compounds, including anilines, phenols, and glycosides were chosen as analytes to evaluate the polar selectivity of this column in the RPLC mode, and the evaluation results were satisfactory compared with the commercial C18 column. Nucleobases were used for evaluating the hydrophilic interaction liquid chromatography performance of the C18-DMCC/SiO2 column. By investigating the impact of organic solvent content on the retention, it could be found that this new stationary phase had the typical characteristics of reversed-phase/hydrophilic chromatography.

Retention of stevioside polar compounds on a sulfonic acid-functionalized stationary phase

To explore the retention and separation of stevioside polar compounds on a sulfonic acid-functionalized cation exchange column, the effects of different organic solvent-water mobile phases on the retention behavior of polar rebaudioside A (RA) and its analogues on the column were investigated over a wide range of organic solvent contents. The obtained U-shape curves hinted that the retention of the compounds on the same column transitioned from a reversed-phase liquid chromatography (RPLC) mode to a hydrophilic interaction liquid chromatography (HILIC) mode when the water-rich state in the mobile phases changed to an organic solvent-rich state. Under the RPLC mode, no separation of RA from its analogues was observed. The HILIC mode was beneficial to the retention and separation of RA and its analogues. Compared with polar protic solvents, aprotic solvents were more conducive to the retention and separation of the polar compounds based on the HILIC mode in organic solvent-rich mobile phases. Three models were used to evaluate and discuss the HILIC retention and separation of the compounds on the column. In the aprotic solvent-rich mobile phase, the HILIC retention of RA and its analogues was effectively described by a mixed-mode model; in the polar proton solvent-rich mobile phase, the retention of analytes was best described by an linear solvation strength (LSS) model. The content and composition of the organic solvent in the mobile phase were determined to be important influencing factors that regulated the retention time for the RA and its analogues, and even the separation mechanism for HILIC. The present work provides a theoretical basis for guiding one to prepare high-purity RA from its analogues by predicting the retention time.

Chromatographic behavior and characterization of polydisperse surfactants using Ultra-High-Performance Liquid Chromatography hyphenated to High-Resolution Mass Spectrometry

Surfactants are widely used in various petrochemical applications. Thus, it is essential to have highly efficient analytical tools to monitor the different classes of surfactants commonly used. Three among the four known classes of surfactants were studied in the present work: anionic (i.e. alkylbenzene sulfonate, alkyl ether carboxylic acid), nonionic (i.e. alkylcyclohexyl alcohol ethoxylated, alkyl alcohol ethoxylated) and cationic (i.e. alkyl trimethylammonium). Thanks to high-resolution mass spectrometry (HRMS), a useful mass list was created including 119 m/z values (error in mass around 5 ppm). This list was the foundation of a HRMS database, which, for the sake of simplicity, will be further denoted only “database”. To avoid ion competition and streamline attribution of structural formulas for isobar molecules, a suitable chromatographic method was used before MS. The retention behavior of six surfactants (trimethyloctadecylammonium bromide, myristyltrimethylammonium bromide, Brij®C10, Triton X-100 reduced, glycolic acid ethoxylate lauryl ether, 4-dodecylbenzenesulfonic acid) was evaluated under three separation modes: reversed-phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC) and mixed-mode chromatography (combination of anion-exchange and reversed-phase mechanisms). In RPLC mode, six columns were tested including C4, C18, C30, polar-embedded C18, PFP and phenyl chemistries. Two HILIC columns were also tested including bare silica and urea chemistries. An anion-exchange combined with RPLC mechanism was investigated as mixed-mode mechanism. Using ammonium formate at 10 mM as buffer provided the best signal in HRMS. In liquid chromatography, acid conditions (pH 3.5) were preferred, to avoid peak tailing due to residual silanols. The mixed-mode separation mode clearly appears as the best compromise for the characterization of the three surfactants classes. Nevertheless, the orthogonality observed for the separations obtained in HILIC and RPLC modes offers some possibilities for further multidimensional separations.

Determination of tracheal cytotoxin in pertussis and diphtheria tetanus acellular pertussis vaccines using liquid chromatography-tandem mass spectrometry

Tracheal cytotoxin (TCT) is a toxic glycopeptide, which contribute to the adverse effects of pertussis toxin (PT) and related vaccines. Although pharmacopeias limit the amount of TCT in PT product, there is no recommended TCT determination method in any pharmacopeia. In this study, a liquid chromatography-tandem mass spectrometry method was developed to determine TCT. Chromatographic conditions, including column-type and mobile-phase composition, were optimized. According to the literature reports, both reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) can provide a good retention for TCT. A large amount of organic solvent is usually used for protein precipitation, which may affect the RPLC mode, leading to peak distortion, while such effects were not observed in HILIC mode. Thus, HILIC mode was used to analyze TCT in this study. The developed method had a wide linear range (5.76-369 ng/L), good precision (no more than 3.9%), satisfied recoveries in various matrices (96.4%-102.5%). The limit of quantification (LOQ) of the developed method was 1279 times lower than the one required by Chinese Pharmacopeia, wherein the required amount of TCT should be less than 2 pmol per dose. The developed method was used to detect TCT in pertussis vaccine (acellular component), pertussis vaccine (acellular, co-purified), co-purified diphtheria tetanus pertussis vaccine, and component diphtheria tetanus acellular pertussis vaccine. As a result, TCT was not detected in any of the selected samples indicating the safety of these vaccines and PT products.

Preparation and evaluation of a reversed-phase/hydrophilic interaction/ion-exchange mixed-mode chromatographic stationary phase functionalized with dopamine-based dendrimers

In this work, a novel dendritic stationary phase was synthesized by the repeated grafting of 1,4-butanediol diglycidyl ether (BDDE) and dopamine (DA) on the surface of silica for performing mixed-mode high-performance liquid chromatography (MHPLC). Elemental analysis (EA), thermogravimetric analysis (TGA) and Fourier transform infrared spectrometry (FT-IR) showed the successful preparation of the dendritic stationary phase. The prepared stationary phase showed the retention mechanisms of reversed-phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC) and ion-exchange chromatography (IEC) under different mobile phase conditions. In detail, alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs) and hydrophobic positional isomers were separated successfully in the RPLC mode. The baseline separation of nucleobases, nucleosides and flavonoids was achieved under HILIC mode, respectively. Meanwhile, some acidic and basic analytes were used to evaluate the IEC mode. The effects of different chromatographic conditions, such as acetonitrile content, salt concentration and pH in the mobile phase, on the different chromatographic modes were also investigated. In addition, the application of the mixed-mode dendritic stationary phase was demonstrated by the analysis of traditional Chinese medicine (TCM), including Carthamus tinctorius L. and Abelmoschus manihot (Linn.) Medicus. Interestingly, the stationary phase also has the ability for the capture and separation of boric acids. These meaningful applications confirmed that the mixed-mode dendritic stationary phase can be potentially applied in the analysis of complex samples.

Voltammetric and electrogeneration approaches for the assessment of the oxidative drug metabolism.

In this paper, electrochemical (EC) methods have been proposed to evaluate the oxidative behavior of drugs as rapid, simple, and cheap strategies to predict some metabolic features. Various commercial drugs belonging to different therapeutic families have been assayed to deal with a wide variety of biotransformations and to cover different metabolism extents. First, differential pulse voltammetry has been applied to evaluate the oxidative behavior of drugs. Voltammetric assays have demonstrated to be highly efficient to predict the metabolism extent from the current intensity data. The second objective of this work has been the comparison of metabolite profiles from both EC and in vitro methods based on liver microsome assays. The resulting samples have been analyzed by reversed-phase liquid chromatography mode using a core-shell column and UV detection. Chromatographic methods have been established for each particular drug and its metabolites using 0.1% (v/v) formic acid aqueous solution and methanol (MeOH) as the components of the mobile phase. Drug oxidation products from both EC- and microsome-based methodologies have been compared in terms of variety and percentage from the corresponding chromatographic profiles. In general, most of the metabolites occurring in vitro have also been reproduced in the EC runs. Besides, it has been found that compositional profiles from EC experiments are dependent on experimental variables such as pH and potential. In general, acid (pH2) and basic (pH10) conditions and too high potentials can contribute to the generation of oxidation artifacts which differ from metabolites while milder potentials and neutral pH values may reproduce more accurately the microsome patterns. The proposed methodology is suitable for a first study of the oxidative behavior of molecules that can be related to relevant metabolic properties. The obtained information could be of great interest to prioritize or discard compounds, as a first screening, on the research of drug candidates.

Hypercrosslinked cholesterol-based polystyrene monolithic capillary columns

Cholesteryl methacrylate has been used to obtain hypercrosslinked poly(cholesteryl methacrylate-co-4-methylstyrene-co-vinylbenzyl chloride-co-divinylbenzene) monolithic stationary phase for capillary liquid chromatography. Different contents of cholesteryl methacrylate and toluene in polymerization mixture have been tested to obtain chromatographic performance. The best column has been prepared using 7% of cholesteryl methacrylate and 1% of toluene in polymerization mixture. Such a monolithic material exhibited a surface area of 452m2/g (BET). The column efficiency of about 63 000 plates/m (H=15μm) was achieved for benzene. The polystyrene column with cholesteryl methacrylate providing an additional functional group gave the highest efficiency compared to known poly(4-methylstyrene-co-vinylbenzyl chloride-co-divinylbenzene) and poly(cholesteryl methacrylate-co-trimethylolpropane trimethacrylate) monolithic columns. All of the above capillary columns were used for the separations of small molecules, such as alkylbenzenes, o-terphenyl/triphenylene, parabens, and proteins in reversed-phase liquid chromatography mode. The relative standard deviation of day-to-day reproducibility of hypercrosslinked poly(cholesteryl methacrylate-co-4-methylstyrene-co-vinylbenzyl chloride-co-divinylbenzene) monolithic columns were between 12.39-15.95% for retention factors and 10–24% for plate heights, respectively.

Separation of substrates and closely related glucuronide metabolites using various chromatographic modes

The aim of this study was to assess the retention and selectivity of a cocktail of 10 substrates of uridine diphosphate glucuronosyltransferase enzymes (UGTs) and their respective glucuronides using four chromatographic approaches. For this purpose, seven different stationary phases were employed in reversed phase liquid chromatography (RPLC), two in hydrophilic interaction liquid chromatography (HILIC), one in aqueous normal phase chromatography (ANPC) and four in subcritical fluid chromatography (SFC). Highly orthogonal separations were achieved with these chromatographic modes. Hydrophobic interactions mainly governed the retention of the substrates and their polar glucuronides in RPLC despite the use of different chemical stationary phase bonding, involving additional possible interactions. In ANPC, atypical separations and poor peak shapes were observed with the selected compounds. In HILIC and SFC conditions, the metabolites were more retained than the substrates because of the polarity increase related to the glucuronic acid moiety. For the latter, a very high proportion of organic solvent (up to 80%) was required to elute the glucuronides that often displayed poor peak shapes. Finally, the selectivity of nine chromatographic systems was compared for the separation of isomeric and diastereoisomeric compounds. The stationary phases used in RPLC mode were more selective towards the two positional isomers of morphine glucuronides since they possess distinct lipophilicity. HILIC and SFC columns were found to be promising for the separation of a critical diastereoisomers pair, namely epitestosterone-glucuronide and testosterone-glucuronide.

A novel two-dimensional liquid-chromatography method for online prediction of the toxicity of transformation products of benzophenones after water chlorination.

Benzophenone-type UV filters (BPs) are ubiquitous in the environment. Transformation products (TPs) of BPs with suspected toxicity are likely to be produced during disinfection of water by chlorination. To quickly predict the toxicity of TPs, in this study, a novel two-dimensional liquid-chromatography (2D-LC) method was established in which the objective of the first dimension was to separate the multiple components of the BPs sample after chlorination, using a reversed-phase liquid-chromatography mode. A biochromatographic system, i.e. bio-partitioning micellar chromatography with the polyoxyethylene (23) lauryl ether aqueous solution as the mobile phase, served as the second dimension to predict the toxicity of the fraction from the first dimension on the basis of the quantitative retention-activity relationships (QRARs) model. Six BPs, namely 2,4-dihydroxybenzophenone, oxybenzone, 4-hydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid, 2,2'-dihydroxy-4,4'-dimethoxybenzophenone and 2,2'-dihydroxy-4-methoxybenzophenone, were the target analytes subjected to chlorination. The products of these BPs after chlorination were directly injected to the 2D-LC system for analysis. The results indicated that most TPs may be less toxic than their parent chemicals, but some may be more toxic, and that intestinal toxicity of TPs may be more obvious than blood toxicity. The proposed method is time-saving, high-throughput, and reliable, and has great potential for predicting toxicity or bioactivity of unknown and/or known components in a complex sample. Graphical Abstract The scheme for the 2D-LC online prediction of toxicity of the transformation products of benzophenone-type UV filters after chlorination.