Label-Free Composition Analysis of Supramolecular Polymer-Nanoparticle Hydrogels by Reversed-Phase Liquid Chromatography Coupled with a Charged Aerosol Detector.

Supramolecular hydrogels formed through polymer-nanoparticle interactions are promising biocompatible materials for translational medicines. This class of hydrogels exhibits shear-thinning behavior and rapid recovery of mechanical properties, providing desirable attributes for formulating sprayable and injectable therapeutics. Characterization of hydrogel composition and loading of encapsulated drugs is critical to achieving the desired rheological behavior as well as tunable in vitro and in vivo payload release kinetics. However, quantitation of hydrogel composition is challenging due to material complexity, heterogeneity, high molecular weight, and the lack of chromophores. Here, we present a label-free approach to simultaneously determine hydrogel polymeric components and encapsulated payloads by coupling a reversed phase liquid chromatographic method with a charged aerosol detector (RPLC-CAD). The hydrogel studied consists of modified hydroxypropylmethylcellulose, self-assembled PEG-b-PLA nanoparticles, and a therapeutic compound, bimatoprost. The three components were resolved and quantitated using the RPLC-CAD method with a C4 stationary phase. The method demonstrated robust performance, applicability to alternative cargos (i.e., proteins) and was suitable for composition analysis as well as for evaluating in vitro release of cargos from the hydrogel. Moreover, this method can be used to monitor polymer degradation and material stability, which can be further elucidated by coupling the RPLC method with (1) a multi-angle light scattering detector (RPLC-MALS) or (2) high resolution mass spectrometry (RPLC-MS) and a Fourier-transform based deconvolution algorithm. We envision that this analytical strategy could be generalized to characterize critical quality attributes of other classes of supramolecular hydrogels, establish structure-property relationships, and provide rational design guidance in hydrogel drug product development.

In-silico method development and optimization of on-line comprehensive two-dimensional liquid chromatography via a shortcut model.

Comprehensive two-dimensional liquid chromatography (LCxLC) represents a valuable alternative to conventional single column, or one-dimensional, liquid chromatography (1D-LC) for resolving multiple components in a complex mixture in a short time. However, developing LCxLC methods with trial-and-error experiments is challenging and time-consuming, which is why the technique is not dominant despite its significant potential. This work presents a novel shortcut model to in-silico predicting retention time and peak width within an RPLCxRPLC separation system (i.e., LCxLC systems that use reversed-phase columns (RPLC) in both separation dimensions). Our computationally effective model uses the hydrophobic-subtraction model (HSM) to predict retention and considers limitations due to the sample volume, undersampling and the maximum pressure drop. The shortcut model is used in a two-step strategy for sample-dependent optimization of RPLCxRPLC separation systems. In the first step, the Kendall's correlation coefficient of all possible combinations of available columns is evaluated, and the best column pair is selected accordingly. In the second step, the optimal values of design variables, flow rate, pH and sample loop volume, are obtained via multi-objective stochastic optimization. The strategy is applied to method development for the separation of 8, 12 and 16 component mixtures. It is shown that the proposed strategy provides an easy way to accelerate method development for full-comprehensive 2D-LC systems as it does not require any experimental campaign and an entire optimization run can take less than two minutes.

Octanol/water partition coefficients estimated using retention times in reverse-phase liquid chromatography and calculated in silico as one of the determinant factors for pharmacokinetic parameter estimations of general chemical substances.

The octanol/water partition coefficient P (logP) is a hydrophobicity index and is one of the determining factors for the pharmacokinetics of orally administered substances because it influences membrane permeability. To illustrate the wide-ranging variety of compounds in the chemical space, a two-dimensional data plot consisting of 25 blocks was previously proposed based on a substance's in silico chemical descriptors. The logP values of approximately 200 diverse chemicals (test plus reference compounds covering all 25 blocks of the chemical space) were estimated experimentally using retention times in reverse-phase liquid chromatography; these values were compared with those of authentic reference compounds with established logP values (available for 17 of 60 reference substances in the Organization for Economic Co-operation and Development Test Guideline 117). The logP values of 140 of 165 chemicals successfully estimated using four different mobile phase conditions (pH 2, 4, 7, and 10 for molecular forms) correlated significantly with those calculated using the in silico packages ChemDraw and ACD/Percepta (r > 0.72). Although substances that neighbored authentic compounds in the chemical space had precisely correlated logP values estimated experimentally and in silico, some compounds that were more distant from authentic substances showed lower logP values than those estimated in silico. These results indicate that additional authentic reference materials with wider ranging chemical diversity and their logP values from reverse-phase liquid chromatography should be included in the international test guidance to promote simple and reliable estimation of octanol/water partition coefficients, which are important determinant factors for the pharmacokinetics of general chemicals.

Optimization of elution conditions and comparison of emerging biocompatible columns on the resolving power and detection sensitivity of oligonucleotides by ion-pairing reversed-phase liquid chromatography mass spectrometry.

A generic performance comparison strategy has been developed to evaluate the impact of mobile-phase additives (ion-pairing agent / counter ion systems), distinct stationary phases on resulting resolving power, and MS detectability of oligonucleotides and their critical impurities in gradient IP-RPLC. Stationary-phase considerations included particle type (core-shell vs. fully porous particles), particle diameter, and pore size. Separations were carried out at 60°C to optimize mass transfer (C-term). The incorporation of an active column preheater mitigated thermal mismatches, leading to narrower peaks and overcoming peak splitting. Acetonitrile as organic modifier outweighed methanol in terms of peak-capacity generation and yielded a 30% lower back pressure. Performance screening experiments were conducted varying ion-pairing agents and counter ions, while adjusting gradient span achieved an equivalent effective retention window. Hexafluoromethylisopropanol yielded superior chromatographic resolution, whereas hexafluoroisopropanol yielded significantly higher MS detection sensitivity. The 1.7 µm core-shell particle columns with 100 Å pores provided maximum resolving power for small (15-35 mers) oligonucleotides. Sub-min analysis for 15-35 polyT ladders was achieved operating a 50 mm long column at the kinetic performance limits. High-resolution separations between a 21-mer modified RNA sequence oligonucleotides and its related (shortmer and phosphodiester) impurities and complementary strand were obtained using a coupled column set-up with a total length of 450 mm.

Determination of lipophilic marine biotoxins (azaspiracids, brevetoxins, and okadaic acid group) and domoic acid in mussels by solid-phase extraction and reversed-phase liquid chromatography with tandem mass spectrometry.

An accurate and efficient method was developed for the determination of azaspiracid shellfish toxins (azaspiracids-1, -2, and -3), neurotoxic shellfish toxins (brevetoxins-2 and -3), diarrhetic shellfish toxins (okadaic acid and dinophysistoxins-1 and -2), and the amnesic shellfish toxin (domoic acid) in mussels (Mytilus galloprovincialis). Lipophilic marine biotoxins (azaspiracids, brevetoxins, and okadaic acid group) were extracted with 0.5 % acetic acid in methanol under heating at 60°C to improve the extraction efficiency of okadaic acid group toxins and then cleaned up with a C18 solid-phase extraction cartridge. Domoic acid was extracted with 50 % aqueous methanol and then cleaned up with a graphitized carbon solid-phase extraction cartridge. Lipophilic marine biotoxins and domoic acid were quantified by reversed-phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The developed method had insignificant matrix effects for the nine analytes and good recoveries in the range of 79.0 % to 97.6 % at three spiking levels for all analytes except brevetoxin-2 (43.8-49.8 %). The developed method was further validated by analyzing mussel tissue certified reference materials, and good agreement was observed between certified and determined values.

Identification and quantification of chain-pairing variants or mispaired species of asymmetric monovalent bispecific IgG1 monoclonal antibody format using reverse-phase polyphenyl chromatography coupled electrospray ionization mass spectrometry.

Developing a knob-into-hole asymmetric bispecific IgG1 monoclonal antibody (mAb) poses manufacturing challenges due to the expression of chain pairing variants, also called mispaired species, in the desired product. The incorrect pairing of light and heavy chains could result in heterogeneous mispaired species of homodimers, heterodimers, light chain swapping, and low molecular weight species (LMWS). Standard chromatography, capillary electrophoretic, or spectroscopic methods poorly resolve these from the main variants. Here, we report a highly sensitive reverse-phase polyphenyl ultra-high-performance liquid chromatography (RP-UHPLC) method to accurately measure mispaired species of Duet mAb format, an asymmetric IgG1 bispecific mAb, for both process development and quality control analytical tests. Coupled with electrospray ionization mass spectrometry (ESI-MS), it enabled direct online characterization of mispaired species. This single direct assay detected diverse mispaired IgG-like species and LMWS. The method resolved eight disulfide bonds dissociated LMWS and three mispaired LMWS. It also resolved three different types of IgG-like mispaired species, including two homodimers and one heterodimer. The characterization and quantification simultaneously enabled the cell line selection that produces a lesser heterogeneity and lower levels of mispaired species with the desired correctly paired product. The biological activity assessment of samples with increased levels of these species quantified by the method exhibited a linear decline in potency with increasing levels of mispaired species in the desired product. We also demonstrated the utility of the technique for testing in-process intermediate materials to determine and assess downstream purification process capability in removing diverse mispaired IgG-like species and LMWS to a certain level during the downstream purification process. Our investigation demonstrates that adopting this method was vital in developing asymmetric bispecific mAb from the initial stage of cell line development to manufacturing process development. Therefore, this tool could be used in the control strategy to monitor and control mispaired species during manufacturing, thus improving the quality control of the final product.

Two-dimensional liquid chromatography with reversed phase in both dimensions: A review.

Two-dimensional liquid chromatography (2D-LC), and in particular comprehensive two-dimensional liquid chromatography (LC×LC), offers increased peak capacity, resolution and selectivity compared to one-dimensional liquid chromatography. It is commonly accepted that the technique produces the best results when the separation mechanisms in the two dimensions are completely orthogonal; however, the use of similar separation mechanisms in both dimensions has been gaining popularity as it helps avoid difficulties related to mobile phase incompatibility and poor column efficiency. The remarkable advantages of using reversed phase in both dimensions (RPLC×RPLC) over other separation mechanisms made it a promising technique in the separation of complex samples. This review discusses some physical and practical considerations in method development for 2D-LC involving the use of RP in both dimensions. In addition, an extensive overview is presented of different applications that relied on RPLC×RPLC and 2D-LC with reversed phase column combinations to separate components of complex samples in different fields including food analysis, natural product analysis, environmental analysis, proteomics, lipidomics and metabolomics.

Mechanistic studies to understand peak tailing due to sulfinic acid- and carboxylic acid-silanophilic interactions in reversed-phase liquid chromatography.

Silanophilic interactions are a primary contributor to peak tailing of acidic pharmaceutical compounds, thus a thorough understanding is especially important for reversed-phase liquid chromatography (RPLC) method development. Herein, a sulfinic acid compound that exhibited severe peak tailing in RPLC with acidic mobile phases was carefully studied. Results indicated that the neutral protonated form of the sulfinic acid is involved in the strong interaction that leads to peak tailing, but that tailing can be mitigated with a blocking effect achieved through use of acetic acid modifier in the mobile phase. Peak tailing was also observed with other structurally-similar sulfinic acids and carboxylic acids but was, in general, less severe with the latter. The Hydrophobic Subtraction Model (HSM) was applied to six commercial C18 columns that exhibited different tailing behaviors for the sulfinic acid compound in attempts to identify key sites of interaction within the stationary phase. A combination of heated acid column wash experiments and density functional theory (DFT) calculations indicate that the differential interactions of the acids with vicinal silanol pairs in the stationary phase play a major role in peak tailing.

Simultaneous Determination of Enantiomeric Purity and Organic Impurities of Dexketoprofen Using Reversed-Phase Liquid Chromatography-Enhancing Enantioselectivity through Hysteretic Behavior and Temperature-Dependent Enantiomer Elution Order Reversal on Polysaccharide Chiral Stationary Phases.

A reversed-phase high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of the potential impurities of dexketoprofen, including the distomer R-ketoprofen. After screening the separation capability of four polysaccharide columns (Lux Amylose-1, Lux Amylose-2, Lux Cellulose-1 and Lux Cellulose-2) in polar organic and in reversed-phase modes, appropriate enantioseparation was observed only on the Lux Amylose-2 column in an acidified acetonitrile/water mixture. A detailed investigation of the mobile phase composition and temperature for enantio- and chemoselectivity showed many unexpected observations. It was observed that both the resolution and the enantiomer elution order can be fine-tuned by varying the temperature and mobile phase composition. Moreover, hysteresis of the retention times and enantioselectivity was also observed in reversed-phase mode using methanol/water mixtures on amylose-type columns. This could indicate that the three-dimensional structure of the amylose column can change by transitioning from a polar organic to a reversed-phase mode, which affects the enantioseparation process. Temperature-dependent enantiomer elution order and rare enthalpic/entropic controlled enantioseparation in the operative temperature range were also observed in reversed-phase mode. To find the best methodological conditions for the determination of dexketoprofen impurities, a full factorial optimization design was performed. Using the optimized parameters (Lux Amylose-2 column with water/acetonitrile/acetic acid 50/50/0.1 (v/v/v) at a 1 mL/min flow rate at 20 °C), baseline separations were achieved between all compounds within 15 min. Our newly developed HPLC method was validated according to the current guidelines, and its application was tested on commercially available pharmaceutical formulations. According to the authors' knowledge, this is the first study to report hysteretic behavior on polysaccharide columns in reversed-phase mode.

Detection of 26 Drugs of Abuse and Metabolites in Quantitative Dried Blood Spots by Liquid Chromatography-Mass Spectrometry.

A method was developed for the determination of 26 drugs of abuse from different classes, including illicit drugs in quantitative dried blood spots (qDBSs), with the aim to provide a convenient method for drug testing by using only 10 µL of capillary blood. A satisfactory limit of quantification (LOQ) of 2.5 ng/mL for 9 of the compounds and 5 ng/mL for 17 of the compounds and a limit of detection (LOD) of 0.75 ng/mL for 9 of the compounds and 1.5 ng/mL for 17 of the compounds were achieved for all analytes. Reversed-phase liquid chromatography was applied on a C18 column coupled to MS, providing selective detections with both +ESI and -ESI modes. Extraction from the qDBS was performed using AcN-MeOH, 1:1 (v/v), with recovery ranging from 84.6% to 106%, while no significant effect of the hematocrit was observed. The studied drugs of abuse were found to be stable over five days under three different storage conditions (at ambient temperature 21 °C, at -20 °C, and at 35 °C), thus offering a highly attractive approach for drug screening by minimally invasive sampling for individuals that could find application in forensic toxicology analysis.

Ion Quantitation in Liposomal Products Using RP-HPLC with Charged Aerosol Detection.

Ion concentration in liposomal drugs is important for drug stability and drug release profile. However, quantifying ion concentration in liposomal drugs is challenging due to the absence of chromophores or fluorophores of ions and the efficiency of their release from the liposome structure. To address these issues, a method based on reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a charged aerosol detector (CAD) has been developed to determine total, internal, and external ions in drug-loaded liposomal products. In this protocol, we focused on the quantitation of ammonium and sulfate ions in liposomal products, using generic PEGylated liposomal doxorubicin as an example. This method can be extended to calcium, acetate, and other ions in different liposomal formulations with slight modifications.

Preparation and investigation of two-component silica-modified hydrophobic layer for minimizing retention loss of reversed-phase chromatographic columns using fully aqueous mobile phases.

Chromatographers often employ fully aqueous mobile phases to retain highly polar compounds in reversed-phase liquid chromatography (RPLC). However, when the flow rate is interrupted, either accidentally or intentionally, a substantial loss in retention occurs due to the spontaneous dewetting of water from the hydrophobic surface of conventional RPLC-C18 particles. Previous studies have shown that maintaining a low C18 surface coverage (approximately 1.5 µmol/m2) can mitigate water dewetting by increasing chain disorder, facilitating the intercalation of water clusters between the C18-bonded chains, and keeping the mesopores wetted. In this research, we explore the potential and additional benefits of using two-component surface bonding materials (C8/C18 and PhenylHexyl (PhHx)/C18) at a constant and low total surface coverage of 1.51 ± 0.15 µmol/m2. We synthesized seven one- and two-component modified silica particles with a volume average particle size of 5.22 µm and an average mesopore size of 104 Å. The surface coverage was increased from 0 to 0.54, 1.00, and to 1.66 µmol2 for C8 chains and from 0 to 0.52, 0.70, and to 1.65 µmol2 for PhHx ligands. To prevent interactions between water and any unreacted silanols, all seven derivatized particles were heavily endcapped with trimethylsilane (TMS) reagent. The fraction of the surface area remaining in contact with water was determined by measuring the retention times of weakly (thiourea) and strongly (thymine) retained compounds at intervals of 1, 2, 4, 8, 16, 32, and 64 minutes following the cessation of flow. Two distinct column temperatures, 24°C and 60°C, were employed in the experiments. Retention losses were found to be minimized in the presence of a small quantity of C8 chains (less than 40% of the total surface coverage). Additionally, it is essential to consider substantial fractions of PhHx chains, as long as the presence of the PhHx ligand does not significantly impact retention and selectivity. Combining mixed RPLC bondings with a low total surface coverage of approximately 1.5 µmol/m2 emerges as a viable solution for further minimizing retention loss in standard C18-bonded RPLC columns, particularly within the surface coverage range of 2.5-3.0 µmol/m2.

Mass Spectrometry-Based Metabolomics Multi-platform for Alzheimer's Disease Research.

The integration of complementary analytical platforms is nowadays the most common strategy for comprehensive metabolomics analysis of complex biological systems. In this chapter, we describe methods and tips for the application of a mass spectrometry multi-platform in Alzheimer's disease research, based on the combination of direct mass spectrometry and orthogonal hyphenated approaches, namely, reversed-phase ultrahigh-performance liquid chromatography and gas chromatography. These procedures have been optimized for the analysis of multiple biological samples from human patients and transgenic animal models, including blood serum, various brain regions (e.g., hippocampus, cortex, cerebellum, striatum, olfactory bulbs), and other peripheral organs (e.g., liver, kidney, spleen, thymus).

Resolving phytosterols in microalgae using offline two-dimensional reversed phase liquid chromatography-supercritical fluid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Sterols are unsaponifiable lipids resulting from plant metabolism that exhibit interesting bioactive properties. Microalgae are a major source of specific phytosterols, most of which are still not fully characterized. The similarity in sterol structures and the existence of positional isomers make the separation of phytosterols challenging. A method was developed based on an offline two-dimensional (2D) system, reversed-phase liquid chromatography (RPLC)-supercritical fluid chromatography (SFC)/quadrupole time-of-flight (Q-ToF) mass spectrometry, for the identification of sterols in microalgae. Subsequent positive-mode MS/MS was used to confirm the identified phytosterols. The 2D chromatogram exhibited a pattern related to the positions of the double bonds, which were confirmed by standard injection, enabling structural elucidation. The analysis of the unsaponifiable fraction of two algae, namely Scenedesmus obliquus, a freshwater microalgae, and Padina pavonica, a marine macroalgae, highlighted the ability of the method to distinguish a large number of sterol isomers.

Design and evaluation of semicarbazide-embeddedd stationary phases for liquid chromatography.

Semicarbazide, as a derivative of urea, constitutes a great variety of functional molecules for different needs. Herein, novel stationary phases with an incorporated semicarbazide group were proposed. Using aliphatic (docosanoyl, C22) and aromatic (benzoyl, Bz) hydrazides, the semicarbazide-embedded ligands were synthesized before chemical modification of the silica gel, allowing for an accurate interpretation of the chromatographic properties of the corresponding packings. The new stationary phases were water-wettable, due to the presence of highly polar groups. In particular, Bz-semicarbazide (Bz-SCD) stationary phase was sufficiently hydrophilic to run in hydrophilic interaction (HILIC) mode, whilst the C22 (C22-SCD) equivalent, in spite of its reversed-phase nature, was markedly less hydrophobic than the referenced polar-embedded ones. The versatility of C22-SCD was demonstrated with a large selection of analytes, including geometric isomers and standard mixtures of polycyclic aromatic hydrocarbons, sulfonamides, sulfonylurea, substituted ureas, pyridines and carbamates, fat-soluble colorants, antifungal metabolites, angiotensin II receptor blockers and calcium channel blockers.

Facile preparation of embedded polar group-containing pentafluorophenyl stationary phases for highly selective separations of diverse analytes.

Pentafluorophenyl (PFP) stationary phase is one of the most important phases after the C18 phase in terms of its applications. Three embedded polar groups (EPG)-containing stationary phases were newly synthesized to act the EPGs as additional interaction sites. The silica surface was initially modified with (3-aminopropyl)trimethoxysilane (APS). The APS-modified silicas were coupled with 2,3,4,5,6-pentafluorobenzoic acid, 2,3,4,5,6-pentafluorophenylacetic acid, and 2,3,4,5,6-pentafluoro-anilino(oxo)acetic acid to obtain Sil-PFP-BA, Sil-PFP-AA, and Sil-PFP-AN phases, respectively. The new phases were characterized by elemental analysis, ATR-FTIR, scanning electron microscopy (SEM), and thermogravimetric analysis (TGA). The phases were evaluated with the Tanaka and Neue tests in reversed-phase liquid chromatography (RPLC). In addition, they were characterized as hydrophilic phases by the Tanaka test protocol used in hydrophilic interaction chromatography (HILIC) separation mode. The Sil-PFP-AA phase showed the highest molecular shape selectivity in RPLC, while Sil-PFP-AN achieved the highest separability in HILIC compared to the commercial PFP reference column. The Sil-PFP-AA phase was successfully applied for the analysis of capsaicinoids from real samples of fresh chili peppers (Capsicum spp.) in RPLC and the Sil-PFP-AN phase for vitamin C (ascorbic acid) in HILIC.

Development, validation and application of an ion-pair reversed-phase liquid chromatography-tandem mass spectrometry method for the quantification of nusinersen.

Background: The fully phosphorothioate-modified oligonucleotide (OGN) nusinersen has low ionization efficiency in the negative ion mode, resulting in a low mass spectrometry response. There have been no relevant reports on developing a LC-MS method for the determination of nusinersen by optimizing mobile phase composition. Materials & methods: Mobile phase additives comprised of 15 mM triethylamine/25 mM 1,1,1,3,3,3-hexafluoro-2-propanol with a pH of 9.6. Nusinersen was extracted from plasma using Oasis® HLB solid-phase extraction (Waters, MA, USA). Results & conclusion: By adjusting the pH of the mobile phase to 9.6 by optimizing the type and concentration of ion-pair reagents, a high mass spectrometry response was obtained. The developed method was applied to nusinersen and met the requirements for the pharmacokinetic study of nusinersen in rabbits.

Automated high-throughput buffer exchange platform enhances rapid flow analysis of antibody drug conjugates by high resolution mass spectrometry.

Antibody drug conjugates (ADCs) are an increasingly important therapeutic class of molecules for the treatment of cancer. Average drug-to-antibody ratio (DAR) and drug-load distribution are critical quality attributes of ADCs with the potential to impact efficacy and toxicity of the molecule and need to be analytically characterized and understood. Several platform methods including hydrophobic interaction chromatography (HIC) and native size-exclusion chromatography-mass spectrometry (nSEC-MS) have been developed for that purpose; however, each presents some limitations. In this work, we assessed a new sample preparation and buffer exchange platform coupled with high-resolution mass spectrometry for characterizing the drug-load and distribution of several cysteine-linked ADCs conjugated with a variety of chemotypes. Several criteria were evaluated during the optimization of the buffer exchange-mass spectrometry system performance and the data generated with the system were compared with results from nSEC-MS and HIC. The results indicated that the platform enables automated and high throughput quantitative DAR characterization for antibody-drug conjugates with high reproducibility and offers several key advantages over existing approaches that are used for chemotype-agnostic ADC characterization.

Identification of oxygenated triacylglycerols in pistachio nuts' lipids by reverse-phase high-resolution-liquid chromatography-ESI mass spectrometry.

Hydroxy- and peroxy-triacylglycerols are common products of lipid peroxidation formed during oil storage or heating or as enzymatic oxidation product of arachidonic acid as signaling molecules in mammals. In this study, oxygenated triacylglycerides (TAG) were identified in pistachio oil based on reverse phase(RP), high-performance liquid chromatography coupled with electrospray ionization and mass spectrometry (HPLC- ESI -MS). 20 novel lipid plant metabolites, classified based on their fragment spectra into a hydroxy (TAG-OH), an epoxy (TAG-O), and hydroperoxide (TAG-OOH) groups. We believe that this class of compounds has been for the first time observed as genuine secondary plant metabolites in a natural source in this case pistachio lipids of dietary relevance.

Temperature-responsive comprehensive two-dimensional liquid chromatography coupled to high resolution mass spectrometry for the elucidation of the oxidative degradation processes of chemicals of environmental concern.

This study explores the possibilities offered by temperature-responsive liquid chromatography (TRLC) based comprehensive 2-dimensional liquid chromatography in combination with reversed-phase liquid chromatography (RPLC) for the analysis of degradation products formed upon oxidative treatment of persistent organic pollutants, in this case exemplified through carbamazepine (CBZ). The TRLC×RPLC combination offers the possibility to overcome peak overlap and incomplete separation encountered in 1D approaches, while the transfer of the purely aqueous mobile phase leads to refocusing of all analytes on the second dimension column. Consequently, this allows for about method-development free and hence, easier LC×LC. The study focuses on the oxidative degradation of CBZ, a compound of environmental concern due to its persistence in water bodies. The TRLC×RPLC combination effectively separates and identifies CBZ and its degradation products, while offering improved selectivity over the individual TRLC or RPLC separations. This allows gathering more understanding of the degradation cascade and allows real-time monitoring of the appearance and disappearance of various degradation products. The compatibility with high-resolution mass spectrometry is last shown, enabling identification of 21 CBZ-related products, nine of which were not previously reported in CBZ degradation studies. The approach's simplicity, optimization-free aspects, and ease of use make it a promising tool for the analysis of degradation pathways in environmental contaminants.