Mixed-mode Liquid Chromatography Research Articles

Preparation of dicationic ionic liquid modified silica stationary phase for mixed-mode liquid chromatography and its application for food additive detection

BackgroundFood safety has become an essential aspect of public concern and there are lots of detection means. Liquid chromatography plays a dominating role in food safety inspection because of its high separation efficiency and reproducibility. However, with the increasing complexity of real samples and monitoring requirements, conventional single-mode chromatography would require frequent column replacement and cannot separate different kinds of analytes on a single column simultaneously, which is costly and time-consuming. There is a great need for fabricating mixed-mode stationary phases and validating the feasibility of employing mixed-mode stationary phases for food safety inspection. ResultsThis work fabricated multifunctional stationary phases for liquid chromatography to determine diverse food additives under the mixed mode of RPLC/HILIC/IEC. Two dicationic ionic liquid silanes were synthesized and bonded onto the silica gel surface. The functionalized silica was characterized by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and elemental analysis. Both columns provide satisfactory separation performance towards 6 hydrophilic nucleosides, 4 hydrophobic polycyclic aromatic hydrocarbons, and 5 anions. Great repeatability of retention (RSD <0.1 %) and column efficiency (100330 plate/m) were obtained. Thermomechanical analysis and linear solvation energy relationship investigated the retention mechanism. Finally, the better in two prepared columns was employed to separate and determine the contents of NO2− and NO3− in vegetables(highest 4906 mg kg−1 NO3− in spinach), preservatives in bottled beverages (180.8 mg kg−1 sodium benzoate in soft drink), and melamine in milk with satisfactory performance and recovery rates ranging from 96.4 % to 105.6 %. SignificanceThis work developed a novel scheme for preparing mixed-mode stationary phases by dicationic ionic liquid which provides great separation selectivity. Most importantly, this work proved the superiority of employing mixed-mode stationary phases for food safety inspection, which might avoid high-cost and frequent changes of columns and chromatography systems in the near future.

Preparation and post-modified of dicationic ionic liquid stationary phase and their application in mixed-mode liquid chromatography

This work prepared two dicationic ionic liquid stationary phases for mixed-mode chromatography via thioe-ene click reaction, one of which was post-modified by p-tolyl isocyanate. The successful synthesis of two stationary phases was demonstrated by several characterizations. The separation of a wide range of substances, including nucleosides and bases, alkylbenzene, polycyclic aromatic hydrocarbons, anilines, phenols, inorganic anions and organic acids, shows that homemade stationary phases can not only be applie to RPLC/HILIC/IEC mixed-mode chromatography, but also has better separation selectivity than commercial column. Therefore, the post-modification of p-tolyl isocyanate can greatly improve the separation effect in RPLC mode, which proves that this modification method is of research significance. The retention mechanisms of two stationary phases were investigated by varying the chromatographic conditions and linear solvation energy relationships (LSER). In addition, the stationary phase is able to detect preservatives in foods quickly and accurately. In actual sample testing, the limit of detection ≤ 0.074mg·L−1 and the inter-day precision was not more than 3.33 %.

An ionic liquid assisted hydrogel functionalized silica stationary phase for mixed-mode liquid chromatography

An ionic liquid assisted hydrogel modified silica was synthesized using a one-pot polymerization and physical coating technique and subsequently applied to mixed-mode liquid chromatography. Analytical techniques, including Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and elemental analysis, etc., confirmed the successful prepared of this innovative stationary phase. The unique combination of amide, long alkyl chain, and imidazole ring in the hydrogel coating enables the stationary phase to function effectively in hydrophilic/reversed-phase/ion exchange liquid chromatography. Notably, the stationary phase exhibited superior separation performance owing to the synergistic effect of the ionic liquid and hydrogel. This was particularly evident when analyzing various analytes such as organic acids, nucleosides/bases, polycyclic aromatic hydrocarbons (PAHs) and anions. Furthermore, under our operating conditions, an excellent column efficiency of 53,642.9 plates/m was achieved for theobromine. In summary, we have proposed a straightforward strategy to enhance the separation performance of hydrogel coatings in liquid chromatography, thereby broadening the potential applications of hydrogels in the field of separation.

Sample preparation for suspect screening of persistent, mobile and toxic substances and their phase II metabolites in human urine by mixed-mode liquid chromatography

Persistent, mobile and toxic substances have drawn attention nowadays due to their particular properties, but they are overlooked in human monitorization works, limiting the knowledge of the human exposome. In that sense, human urine is an interesting matrix since not only parent compounds are eliminated, but also their phase II metabolites that could act as biomarkers. In this work, 11 sample preparation procedures involving preconcentration were tested to ensure maximum analytical coverage in human urine using mixed-mode liquid chromatography coupled with high-resolution tandem mass spectrometry. The optimized procedure consisted of a combination of solid-phase extraction and salt-assisted liquid-liquid extraction and it was employed for suspect screening. Additionally, a non-discriminatory dilute-and-shoot approach was also evaluated. After evaluating the workflow in terms of limits of identification and type II errors (i.e., false negatives), a pooled urine sample was analysed. From a list of 1450 suspects and in-silico simulated 1568 phase II metabolites (i.e. sulphates, glucuronides, and glycines), 44 and 14 substances were annotated, respectively. Most of the screened suspects were diverse industrial chemicals, but biocides, natural products and pharmaceuticals were also detected. Lastly, the complementarity of the sample preparation procedures, columns, and analysis conditions was assessed. As a result, dilute-and-shoot and the Acclaim Trinity P1 column at pH = 3 (positive ionization) and pH = 7 (negative ionization) allowed the maximum coverage since almost 70 % of the total suspects could be screened using those conditions.

Fabrication of polyethyleneimine grafted maleopimaric acid glycidyl methacrylate ester stationary phase for mixed-mode liquid chromatography

Due to their advantages of low green toxicity, diverse sources, renewable nature, and excellent biocompatibility, natural products have been extensively utilized in the exploration and development of novel stationary phase materials for liquid chromatography. In this study, we fabricated a rosin-based stationary phase material for mixed-mode chromatography by utilizing maleopimaric acid glycidyl methacrylate ester (MPAG) and polyethyleneimine (PEI) as ligands. Initially, silica was functionalized with MPAG through thiol-ene click reaction, followed by the grafting of PEI via an imidization reaction to produce Sil-MPAG-PEI material. This material exhibited both anion exchange and hydrophilic interaction properties due to the presence of PEI, allowing for the switching of its retention mode from reversed-phase mode to a mixed-mode incorporating reversed-phase, hydrophilic interaction and ion-exchange chromatography (RPLC/HILIC/IEC) under different mobile phase conditions. Consequently, Sil-MPAG-PEI demonstrated the ability to provide various interactions, including hydrophobic, π-π, hydrogen bond, hydrophilic, ion exchange, and electrostatic interactions. These interactions enabled selective separation among compounds with different polarities, such as nucleosides/bases, benzoic acids, flavonoids, alkylbenzenes, phenols, anilines, and polycyclic aromatic hydrocarbons (PAHs). Furthermore, Sil-MPAG-PEI demonstrated excellent chromatographic stability, as evidenced by the low relative standard deviations (RSDs) of the retention time for interday precision and interday precision were 0.042 %-0.275 % (n = 10) and 0.159 %-0.932 % (n = 7). The column was also successfully employed for the quantitative determination of evodiamine and rutaecarpine in an extract solution of Evodiae fructus, achieving concentrations of 9.102 and 3.256 mg/g, respectively. These results demonstrate the potential of Sil-MPAG-PEI in the analysis of complexsamples. Briefly, this study investigated and confirmed the potential application of rosin derivatives in mixed-mode liquid chromatography separations has been investigated and verified. The exceptional separation ability of Sil-MPAG-PEI indicates that it is a promising separation material for chromatographic separation.

Preparation of ionic liquid stationary phase copolymerized with anion and cation for mixed-mode liquid chromatography

Ionic liquids (ILs) are a promising material to develop stationary phases for mixed-mode chromatography (MMC). In order to investigate the effect of anion type and polymerization method of ionic liquid, this work prepared and compared two stationary phases (Sil-IM-Br and Sil-IM-SAG) based on the polymeric ionic liquid, one of which copolymerized cation and anion. Two stationary phases were characterized and provided satisfactory retention repeatability (RSD < 0.3 %) and column efficiency (84600 plates/m). Chromatographic conditions (ACN content, salt concentration, and pH) were varied to reveal the retention characters and interactions between stationary phases and solutes. The results showed that both two developed stationary phases can operate under the mixed mode of RPLC/HILIC/IEC, but diverse selectivity was provided. So, the retention mechanisms were studied by linear solvation energy relationship equation (LSER) and thermodynamic research. Two stationary phases showed totally different retention towards carboxylic acids, which resulted from the effect of anion bonded. Lastly, the developed stationary phases were applied to separate alkylbenzenes and polycyclic aromatic hydrocarbons (PAHs) under RPLC. Furthermore, 7 carboxylic acids and 7 nucleosides and bases and complex samples were successfully separated under HILIC mode, Sil-IM-Br and Sil-IM-SAG provide better performances and different selectivity when compared with commercial mixed-mode columns. This work proves that cationic and anionic copolymerization provides a new method for stationary phase design, and better selectivity can be obtained by changing the anions.

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.

Dilute-and-shoot coupled to mixed mode liquid chromatography-tandem mass spectrometry for the analysis of persistent and mobile organic compounds in human urine

In this work, a comprehensive method for the simultaneous determination of 33 diverse persistent and mobile organic compounds (PMOCs) in human urine was developed by dilute-and-shoot (DS) followed by mixed-mode liquid chromatography coupled with tandem mass spectrometry (MMLC-MS/MS). In the sample preparation step, DS was chosen since it allowed the quantification of all targets in comparison to lyophilization. For the chromatographic separation, Acclaim Trinity P1 and P2 trimodal columns provided greater capacity for retaining PMOCs than reverse phase and hydrophilic interaction liquid chromatography. Therefore, DS was validated at 5 and 50 ng/mL in urine with both mixed mode columns at pH = 3 and 7. Regarding figures of merit, linear calibration curves (r2 > 0.999) built between instrumental quantification limits (mostly below 5 ng/mL) and 500 ng/mL were achieved. Despite only 60% of the targets were recovered at 5 ng/mL because of the dilution, all PMOCs were quantified at 50 ng/mL. Using surrogate correction, apparent recoveries in the 70–130% range were obtained for 91% of the targets. To analyse human urine samples, the Acclaim Trinity P1 column at pH = 3 and 7 was selected as a consensus between analytical coverage (i.e. 94% of the targets) and chromatographic runs. In a pooled urine sample, industrial chemicals (acrylamide and bisphenol S), biocides and their metabolites (2-methyl-4-isothiazolin-3-one, dimethyl phosphate, 6-chloropyridine-3-carboxylic acid, and ammonium glufosinate) and an artificial sweetener (aspartame) were determined at ng/mL levels. The outcomes of this work showed that humans are also exposed to PMOCs due to their persistence and mobility, and therefore, further human risk assessment is needed.

The secret of reversed-phase/weak cation exchange retention mechanisms in mixed-mode liquid chromatography applied for small drug molecule analysis

Resolving complex sample mixtures by liquid chromatography in a single run is challenging. The so-called mixed-mode liquid chromatography (MMLC) which combines several retention mechanisms within a single column, can provide resource-efficient separation of solutes of diverse nature. The Acclaim Mixed-Mode WCX-1 column, encompassing hydrophobic and weak cation exchange interactions, was employed for the analysis of small drug molecules. The stationary phase's interaction abilities were assessed by analysing molecules of different ionisation potentials. Mixed Quantitative Structure-Retention Relationship (QSRR) models were developed for revealing significant experimental parameters (EPs) and molecular features governing molecular retention. According to the plan of Face-Centred Central Composite Design, EPs (column temperature, acetonitrile content, pH and buffer concentration of aqueous mobile phase) variations were included in QSRR modelling. QSRRs were developed upon the whole data set (global model) and upon discrete parts, related to similarly ionized analytes (local models) by applying gradient boosted trees as a regression tool. Root mean squared errors of prediction for global and local QSRR models for cations, anions and neutrals were respectively 0.131; 0.105; 0.102 and 0.042 with the coefficient of determination 0.947; 0.872; 0.954 and 0.996, indicating satisfactory performances of all models, with slightly better accuracy of local ones. The research showed that influences of EPs were dependant on the molecule's ionisation potential. The molecular descriptors highlighted by models pointed out that electrostatic and hydrophobic interactions and hydrogen bonds participate in the retention process. The molecule's conformation significance was evaluated along with the topological relationship between the interaction centres, explicitly determined for each molecular species through local models. All models showed good molecular retention predictability thus showing potential for facilitating the method development.

Preparation of spherical silica hydroxyl-functionalized covalent organic polymer composites for mixed-mode liquid chromatography.

Covalent organic polymers are an emerging class of amorphous microporous materials that have raised increasing concerns in analytical chemistry due to their unique structural and surface chemical properties. However, the application of covalent organic polymers as mixed-mode stationary phases in chromatographic separations has rarely been reported. Herein, novel spherical silica hydroxyl-functionalized covalent organic polymer composites were successfully prepared via a layer-by-layer approach. The structure and morphology of the materials were carefully characterized by elemental analysis, Fourier-transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, X-ray diffraction, thermogravimetric analysis, Brunauer-Emmett-Teller, and contact angle measurements. Baseline separations of various alkylbenzenes, polycyclic aromatic hydrocarbons, and nucleosides and bases were achieved on the prepared stationary phase under reversed-phase/hydrophilic interaction mode. The column efficiencies of 23 853 and 36 580 plates/m were obtained for butylbenzene and uracil, respectively, and the relative standard deviation of the retention time for continuous injections was less than 1.38% (n=10), suggesting satisfactory column efficiency and repeatability. Additionally, this novel stationary phase realized the complete separation of the endocrine-disrupting chemicals in river water. This work affords a new route for synthesizing covalent organic polymers-based mixed-mode stationary phase and further reveals their great potential in chromatographic separation.

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.

Comprehensive profiling of Sanguisorba officinalis using offline two-dimensional mixed-mode liquid chromatography × reversed-phase liquid chromatography, tandem high-resolution mass spectrometry, and molecular network.

The profiling of natural products is important in modern biological sciences and new drug development. However, the separation and characterization of complex herbal extracts are significantly challenging for researchers in the biochemical field. Herein, an offline two-dimensional mixed-mode liquid chromatography × reversed-phase liquid chromatography system is developed. Our system exhibits high orthogonality and is composed of a newly prepared stationary phase in the first dimension and a traditional C18 phase in the second dimension, and is operated in combination with a high-resolution mass spectrometry and molecular network. Sanguisorba officinalis L. is studied using the proposed method owing to its bioactivity. With the aid of orthogonal separation, the ionization of the individual components is improved. The number of detected compounds and separated peaks are significantly increased when one-dimensional liquid chromatography is upgraded to two-dimensional liquid chromatography. In addition, 270 compounds (127 of which are tentatively characterized as new compounds, and further confirmation is needed) are successfully characterized based on their fragmentation patterns under the guidance of molecular network, while only 95 compounds are characterized using one-dimensional liquid chromatography and high-resolution mass spectrometry. The results indicate that the developed offline two-dimensional mixed-mode liquid chromatography × reversed-phase liquid chromatography, tandem high-resolution mass spectrometry, and molecular network method are effective for profiling complex samples.

Analysis of hydrophilic per- and polyfluorinated sulfonates including trifluoromethanesulfonate using solid phase extraction and mixed-mode liquid chromatography-tandem mass spectrometry

Ultra-hydrophilic per- and polyfluorinated sulfonates (PFSA) are increasingly scrutinized in recent years due to their ubiquitous occurrence, persistence, and aqueous mobility in the environment, yet analysis remains a challenge. This study developed methods for the analysis of trifluoromethanesulfonate, perfluorobutanesulfonate, 10-camphorsulfonate, and a di-fluorinated sulfonate utilizing mixed-mode liquid chromatography, where all analytes were adequately retained and separated. Chromatography and electrospray ionization parameters were optimized; instrumental limits of quantification for the anionic target analytes were in the range of 4.3 - 16.1 ng L−1. Solid phase extraction (SPE) methods were developed using Oasis WAX cartridges; SPE recoveries for the analytes ranged from 86% to 125%. Salinity and total organic carbon both impaired the SPE performance to different extents, depending on the respective analyte. Utilizing widely accessible instrumentation and materials, this is a single method to simultaneously analyze conceivably the most hydrophilic PFAS chemical, i.e., trifluoromethanesulfonate, and moderately hydrophobic PFSAs.

Preparation of hydrogel nanocomposite functionalized silica microspheres and its application in mixed-mode liquid chromatography

Hydrogel is a kind of three-dimensional network structure polymer that can absorb water and swell in water. It has been widely used in many fields due to its flexible functionality. We proposed the design strategy of dual-network hydrogel assisted by a metal-organic-framework (MOF) and modified them on the surface of silica (with average particle diameter of 5 μm and average pore diameter of 76 Å). On the basis of effectively avoiding shortcomings such as osmotic pressure caused by swelling, abundant mesh types of composite material also improves the separation selectivity of the stationary phase. A variety of analytes such as nucleosides/bases, antibiotics, organic acids, carbohydrates, alkylbenzenes, polycyclic aromatic hydrocarbons, pesticides and anions can be selectively separated. The research on the retention behavior and the interaction mechanism proves that the column can be used in mixed mode liquid chromatography. By comparing with the optimized chromatographic conditions of commercial HILIC column and C18 column, this new type of stationary phase also has some significant advantages in the selective separation of mixed analytes. This new stationary phase also has excellent acid/base stability. The intraday relative standard deviation of their retention time under acidic conditions is 0.05%-0.26% (n = 10), and the intraday relative standard deviation under basic conditions is 0.11–0.14% (n = 10). After optimizing the chromatographic conditions, the efficiency of this new type of chromatographic column can reach 90,300 plates/m (sucrose). In short, a new strategy for applying hydrogel to liquid chromatography with high selectivity and chromatographic separation performance is proposed.

Mesoporous nanomaterial-assisted hydrogel double network composite for mixed-mode liquid chromatography.

By introducing functional groups such as quaternary amine groups, sulfonic acid groups, triazine groups, and other mespore nanomaterials into the hydrogel, better separation effect of some organic framework materials has been obtained. Due to a reasonable design and preparation strategy, the hydrogel composite-modified silica can be used in the selective separation of various analytes such as pesticides, alkylbenzenes, polycyclic aromatic hydrocarbons, nucleosides/bases, benzoic acids, antibiotics, and carbohydrates. Through the exploration of chromatographic retention behavior, it is proved that the column can be used in mixed-mode liquid chromatography. The intra-day relative standard deviation for retention time of this new stationary phase is 0.12-0.16% (n = 10), and the inter-day relative standard deviation is less than 0.39% (n = 5). This new stationary phase can also be used for separation in complex samples. The limit of detection (LOD) for chlorotoluron in farm irrigation water is 0.21µg/L and the linear range is 2-250µg/L. After optimizing the chromatographic conditions, the highest efficiency of the hydrogel column in RPLC and HILIC modes has reached 32,400 plates/m (chlorobenzuron) and 41,300 plates/m (galactose). This new type of hydrogel composite is a porous network material with flexible functional design and simple preparation method and its application has been expanded in liquid chromatography separation successfully. The hydrogel composed of triallyl cyanate cross-linking agent and 3-(2-(methacryloyloxy) ethyl) dimethylamine) propane-1-sulfonate (SBMA) monomer which were co-modified on the surface of mesoporous silica with MOF-919 for separation in mixed-mode liquid chromatography.

Simultaneous quantitation of inorganic ions in oligonucleotides using mixed-mode liquid chromatography coupled with a charged aerosol detector

The amounts of inorganic ions (e.g. sodium, potassium, magnesium, calcium, chloride, and phosphate) in intrathecally administered oligonucleotide drug products need to be controlled in order to meet the final target formulation composition. During process development, these ions are measured to ensure the solution composition does not change during purification or drug product compounding and to ensure the final drug product targets are achieved. A quantitative method for ion analysis in antisense oligonucleotide (ASO) drugs was developed by mixed-mode anion exchange/cation exchange-hydrophilic interaction liquid chromatography (AEX/CEX-HILIC) with a charged aerosol detector (CAD). This thirteen-minute method measures monovalent and multivalent cations and anions simultaneously. LC separation conditions, CAD parameters, and sample preparation were judiciously optimized to ensure that the method is specific, accurate, precise, linear, robust, and rugged. With this LC-CAD approach, ion analysis for oligonucleotide drugs can be performed in most analytical labs to support drug development.

Non-conjugated flexible network for the functional design of silica-based stationary phase for mixed-mode liquid chromatography

The functionalized non-conjugated organic network modified silica microspheres are proposed as the stationary phase of liquid chromatography, which can effectively avoid some defects of organic framework materials in liquid chromatography. Due to the co-existing of pyridine ring, carbonyl group, amide group and triazine ring in the non-conjugated flexible organic network on the silica surface, the developed mixed-mode stationary phase exhibits multiple interactions between the stationary phase and the analytes during the separation process. A variety of nucleoside bases, organic acids, antibiotics, pesticides, alkylbenzenes, polycyclic aromatic hydrocarbons and sulfonamides achieved ideal resolution and flexible selectivity in separation. Compared with the commercial chromatographic columns under their optimized chromatographic conditions, it shows better performance for the separation of complex analytes. The influence of chromatographic conditions on retention behavior indicates that the column's multiple retention mechanisms make it suitable for mixed-mode liquid chromatography. The stationary phase prepared by the new design strategy also has excellent chromatographic reproducibility, repeatability and stability with the intraday RSD of 0.09%–0.12% (n = 10) and the interday RSD of 0.37%–1.64% (n = 5) for the retention time. The separation results of actual samples also prove its potential in the analysis of complex samples. In short, we designed and prepared the non-conjugated flexible network modified silica stationary phase material for liquid chromatography that is different from organic framework materials. Its excellent separation ability shows that we have successfully reported a new kind of liquid chromatography packing with functional design and facile preparation method.

Combination of different chromatographic and sampling modes for high-resolution mass spectrometric screening of organic microcontaminants in water.

This study explores the combination of two sampling strategies (polar organic compounds integrative sampler (POCIS) vs. spot sampling) and four chromatographic retention modes (reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), mixed-mode liquid chromatography (MMLC) and supercritical fluid chromatography (SFC)) for high-resolution mass spectrometry (HRMS) screening of organic pollutants in water samples. To this end, a suspect screening approach, using iterative data-dependent tandem mass spectrometry (MS/MS) driven by a library of 3227 chemicals (including pharmaceuticals, pesticides, drugs of abuse, human metabolites, industrial chemicals and other pollutants), was employed. Results show that POCIS can afford a larger number of positive identifications as compared to spot sampling. On the other hand, the best suited retention mechanisms, in terms of identified analytes, are SFC, and followed by RPLC, MMLC and HILIC. However, the best combination (POCIS + SFC) would only allow the identification of 67% of the detected analytes. Thus, the combination of the two sampling strategies, spot and passive sampling, with two orthogonal retention mechanisms, RPLC and SFC, is proposed in order to maximize the number of analytes detected (89%). This strategy was applied to different surface water (river and estuary) samples from Galicia (NW Spain). A total of 155 compounds were detected at a confidence level 2a, from which the major class was pharmaceuticals (61%).

Modern Trends in Mixed-Mode Liquid Chromatography (LC) Columns

Commercialization of columns that provide multiple modes of chromatographic separations have recently been on the rise. For example, combinations of retention modes, such as ion-exchange and reversed-phase, often enable the separation of complex mixtures of analytes not possible using single-mode columns. In this work, recent trends in what is often referred to as “mixed-mode” phase are investigated. Particular attention is paid to recent fundamental research, stationary phase development and design, and areas of application.

Simultaneous quantification of polysorbate 20 and poloxamer 188 in biopharmaceutical formulations using evaporative light scattering detection

Polysorbates and Poloxamer 188 constitute the most common surfactants used in biopharmaceutical formulations owing to their excellent protein-stabilizing properties and good safety profiles. In recent years, however, a vast number of reports concerning potential risk factors closely related with their applications, such as the accumulation of degradation products, their inherent heterogeneity and adsorption effects of proteins at silicon/oil interfaces have drawn the focus to potential alternatives. Apart from tedious efforts to evaluate new excipient candidates, the use of mixed formulations leveraging combinations of well-established surfactants appears to be a promising approach to eliminate or, at least, minimize and postpone adverse effects associated with the single compounds. Due to the similar molecular properties of non-ionic surfactants, however, baseline separation of these mixtures, which is mandatory for their reliable quantification, poses a great challenge to analytical scientists. For this purpose, the present work describes the development of a robust mixed-mode liquid chromatography method coupled to evaporative light scattering detection (mixed-mode LC-ELSD) for simultaneous determination of the (intact) Polysorbate 20 and Poloxamer 188 content in biopharmaceutical formulations containing monoclonal antibodies. Extensive qualification and validation studies, comprising the evaluation of method specificity, robustness, linearity, accuracy and precision according to ICH guidelines, demonstrated its suitability for quality control studies. A case study on the storage stability of a formulated antibody was conducted to underline the method’s practical utility. Finally, the versatility of the developed approach was successfully tested by quantifying Polysorbate 20-related surfactants, such as Polysorbate 80 and super-refined Polysorbate.