Metabolomics and lipidomics seek to achieve comprehensive profiling of metabolites and lipids in biological and plant systems. However, the wide diversity in their physicochemical properties, such as polarity and optical activity, poses considerable challenges to conventional one-dimensional liquid chromatography (1DLC). Multidimensional liquid chromatography (MDLC) addresses these limitations by combining orthogonal separation mechanisms, offering a powerful approach for resolving complex metabolome and lipidome. This review outlines the fundamental configurations of MDLC—including heart-cutting, stop-flow, and comprehensive modes—along with key modulation techniques such as loop-based and trapping-column-based interfaces. It further summarizes applications in metabolomics and lipidomics focusing on biological and plant samples over the past decade. Owing to its high sensitivity and rich information, mass spectrometry operated in both untargeted and targeted modes has become the dominant detection platform coupled with MDLC. Overall, the integration of MDLC with MS has enhanced sensitivity and separation resolution for targeted analytes and entire sample compositions, while significantly expanding the coverage of metabolites and lipids across diverse polarity ranges.

Acrylamide (AA) is a neurotoxic and potentially carcinogenic compound whose detection in processed foods and food additives has raised significant public health concerns, underscoring the urgent need for sensitive yet straightforward analytical methods. Conventional analytical approaches based on GC or HPLC have limited applicability in case of AA due to its high polarity and low molecular weight. Therefore, various derivatization strategies have been explored; however, challenges remain in terms of selectivity. Herein, we report a novel HPLC-FLD method for AA analysis using 5-(dimethylamino)-N-(2-sulfanylethyl)naphthalene-1-sulfonamide (DNS-SH) as a fluorescent derivatization reagent. DNS-SH was selected to provide moderate hydrophobicity for reversed-phase retention, long-wavelength fluorescence distinct from that of food components, and a reactive thiol group for the selective derivatization of AA. The AA-DNS derivative was structurally confirmed by LC-HRMS, 1D-NMR, and 2D-NMR. The developed HPLC-FLD method exhibited excellent linearity (r² > 0.9999), reproducibility (RSD < 2.76 %), and a low LOQ of 0.1 µg/mL. Its applicability was validated using an “Amino Acid-Monosaccharide Reaction Product,” where AA was detected at 0.1 µg/g in spiked samples. This approach provides a reliable tool for monitoring AA in food additives and potentially broader food matrices.

This study proposes a novel analytical method for the sensitive and rapid detection of residual solvents in pharmaceutical formulations. In this method, a powdered tablet is dissolved and treated via purge and trap extraction using a functional polytetrafluoroethylene (PTFE) membrane and a needle-type extraction device. Specifically, the powdered sample and sodium chloride were added to 5 mL of pure water in a glass solid-phase extraction cartridge, followed by stirring until the dissolution of the solids. Subsequently, 100 mL of headspace gas was collected using a needle-type extraction device packed with Carbopack X and carbon molecular sieve as adsorbents. During the active sampling, a purging gas was introduced into the solution from the bottom of the cartridge through a functional PTFE membrane, which retained the solution while allowing the purging gas to pass. The typical sample collection time was 10 min. Following the sample collection, the extraction needle was directly inserted into a heated gas chromatographic injection port, and the extracted analytes were thermally desorbed and analyzed using a flame ionization detector. The limits of detections for benzene, carbon tetrachloride, 1,2-dichloroethane, 1,1-dichloroethylene, and 1,1,1-trichloroethane (Class 1 compounds) were 0.005, 0.5, 0.005, 0.01, and 0.01 w/w ppm, respectively. The recovery of the target compounds did not depend on the formulation, and the proposed method demonstrated sufficient recovery and sensitivity for hydrophobic analytes, whereby allowing simple and rapid sample preparation.

Ion mobility spectrometry (IMS) has become an analytical technique for rapid chiral discrimination, offering significant potential to accelerate various studies such as drug discovery, clinical diagnostics, and bioimaging. This review highlights recent advancements in chiral discrimination using IMS, based on studies published from 2006 to 2024. The methods are categorized into three main approaches: (1) direct separation in a chiral environment, (2) non-covalent complexation, and (3) diastereomeric separation using a covalently bonded chiral tag (chiral derivatization). For each approach, landmark studies are discussed with emphasis on critical notes such as selection of the additive and the ion form for efficient separation. Subsequently, representative applications of chiral derivatization to asymmetric catalyst development, rapid bioanalysis, and biomolecular imaging are explained in detail. The enantioselective imaging of chiral metabolites is more challenging than others but attracts growing interest from biologists and analytical chemists, making the description regarding on-tissue charged chiral derivatization, which improves ionization efficiency and separation of analyte, particularly relevant. Independent studies have demonstrated that IMS, under optimized conditions, can achieve performance comparable to high-performance liquid chromatography (HPLC) in determining enantiomeric excess. Considering higher throughput and simplicity of IMS over HPLC, further instrumental and/or chemical advancements would make IMS a more versatile technique for chiral discrimination.

Photostability and chiral stability of d-chlorpheniramine maleate (d-CPM, S (+)-form), which is an old but clinically useful antihistamine, were investigated by HPLC with an ovomucoid (OVM) or a β-cyclodextrin (β-CD) derivative immobilized chiral columns. Enantiomers of chlorpheniramine maleate (dl-CPM) were successfully separated by an OVM column with resolution (Rs) 3.2 within 10 min under the reversed-phase mode. A β-CD immobilized column was also useful for the separation of enantiomers. Using validated chiral HPLC methods, photostability and chiral inversion study of d-CPM drug substances, its various formulations (syrups, tablets, injections, and powders) under UV irradiation and heating stress conditions were performed in addition to its quality evaluation. As a result of quality evaluation, large content (1-6%) of the minor enantiomer, l-CPM was detected in both d-CPM drug substances and its formulations. Especially ca. 6% l-CPM, which was the largest among tested formulations, was observed in injections. This may be due to its production process (probably sterilization by heat). In photostability studies, maleic acid (cis-form) in d-CPM was converted to fumaric acid (trans-form) in all formulations including the solid type and aqueous solutions of d-CPM, although chiral inversion was not observed under UV irradiation. On the other hand, under the heating conditions, chiral inversion of d-CPM was observed in all formulations and aqueous solutions of d-CPM with an increase of heating temperature and heating time, in addition to cis-trans rearrangement of maleic acid. An increase of l-CPM (chiral inversion) was also observed in d-CPM syrups, powders and tablets stored at the room temperature for one year, showing d-CPM is relatively easily chiral inverted by heat.

Momilactones, allelochemicals from rice plants, exhibit diverse effects, including functioning as crop-friendly pesticides, and possessing antifungal and anticancer properties. The present study aimed to optimize the isolation of momilactones A (MA) and B (MB) using high-speed countercurrent chromatography (HSCCC), prioritizing the minimization of organic solvent waste and utilizing discarded rice husk. By employing techniques such as solvent reuse and HSCCC, we successfully isolated the highly purified fractions of MA and MB (>95% purity, as verified by HPLC) from rice husk. The optimized two-phase solvent system (n-hexane/ethyl acetate/methanol/water, 7/3/5/5, v/v/v/v) was specifically chosen for its efficiency in HSCCC, applied to extracts from 2 kg of rice husk. The separation was performed using the lower mobile phase of the two-phase solvent system at a flow rate of 1.0 mL/min after rotating the multilayer coiled column at the revolution speed of 1000 rpm. The retention of the stationary phase was observed to be 77%. The purity of isolated MA (3.0 mg) and MB (2.0 mg) was determined to be 99.0% and 98.2% via LC-UV chromatogram, respectively. Significantly, the organic solvents used in the extraction and HSCCC processes were redistilled and recycled, leading to an over 80% reduction in solvent consumption. This study demonstrates the feasibility of a sustainable, economical, and effective chromatographic method for isolating allelochemicals from waste plant materials.

Due to the recent discoveries of several D-amino acids in mammals and also by the elucidation of their physiological functions and/or availabilities as diagnostic markers, the enantioselective analysis of intrinsic amino acids has been the focus of attention. Based on the determination of amino acids with chiral discrimination, the clarification of their distributions, regulation mechanisms, physiological functions and the diagnostic/therapeutic values of a wide variety of D-amino acids is expected. Although various analytical techniques have been reported, the improvement of the selectivity is still in demand especially for the accurate determination of trace levels of D-amino acids in complicated biological matrices. One of the straightforward approaches to address the selectivity issue is the multi-dimensional LC analysis in which several separation modes are combined. In the present review, the two-dimensional (2D) LC methods in combination with the detection using MS/MS are introduced and the applications to various biological samples are summarized. The 2D LC-MS/MS methods were designed by integrating a reversed-phase purification (first dimension), an enantioselective separation (second dimension) and the detection of specific precursor/product ion pairs of analytes using MS/MS. Using these methods, the precise and simultaneous determination of D-alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), leucine (Leu), proline (Pro) and serine (Ser) in human plasma and urine was carried out. The in vivo regulation of D-Ala, Leu, Pro, Ser and tryptophan (Trp) amounts by D-amino acid oxidase (DAO) was clarified by analyzing their concentrations in the plasma and urine of DAO deficient mice. For the accurate analysis of D-amino acid residues in proteins, the hydrolysis using deuterium chloride/deuterium oxide and 2D LC-MS/MS determination were combined, and the stereoinversion of the Ala, Asp, Glu, Pro and Ser residues was evaluated in several proteins.

The success of the phosphoproteome analysis depends on the separation efficiency of phosphorylated peptides in ion-pair reversed-phase liquid chromatography coupled with tandem mass spectrometry. Here, we report that the phosphorylated peptides were less susceptible to the column temperature than the unphosphorylated peptides when trifluoroacetic acid (TFA) was used as an ion-pair reagent. This trend was partially reversed when TFA was replaced by acetic acid, and the retention order of phosphorylated and unphosphorylated peptide pairs was also partially reversed. Our results indicate that the retention behavior of phosphorylated peptides is relatively predictable in the TFA system, which has long been used in ion-pair liquid chromatographic analysis of peptides, but in the acetic acid system, which is compatible with mass spectrometry, the retention behavior is more diverse, and further data accumulation is needed to quantitatively understand the retention behavior.

Nonaqueous capillary zone electrophoresis (NACZE) utilizing cationic reagents as an electrophoresis promoter has the potential for separating water-insoluble synthetic polymers and that with tetrahydrofuran based electrophoretic media containing cationic reagents has been reported. In this study, NACZE using hexafluoroisopropyl alcohol (HFIP)-based media was developed for separating polyesters and polyamides, which were insoluble in tetrahydrofuran. Nonaqueous media of HFIP containing 400 mM cetyltrimethylammonium chloride (CTAC) and 50 mM trihexyl(tetradecyl)phosphonium chloride (P66614Cl) were used, and it was found that hydrophobic P66614 cation enhanced the electrophoresis of both polyesters and polyamides compared with cetyltrimethylammonium cation. The media containing CTAC resulted in partially separated poly(ethylene terephthalate)/poly(hexamethylene terephthalate) and poly(ethylene terephthalate)/poly(ethylene naphthalate), but P66614Cl failed to separate them. By contrast, nylon 6I and nylon 6/6I were separated with the P66614Cl media, whereas they were co-eluted in the media with CTAC.

γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter having anti-anxiety, stress-reducing and sleep-enhancing effects. The blood GABA concentration has the potential to become a diagnostic index for depression and schizophrenia. In this study, we developed a method for the quantification of GABA in human serum by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) combined with derivatization using a pair of reagents, 4-diethylaminobenzoic acid N-succinimidyl ester (DEABAS) and its deuterated isotopologue (dDEABAS). The deproteinized serum samples were derivatized with DEABAS, to which the dDEABAS-derivatized standard GABA of known amount was then added. The DEABAS-derivatization enhanced not only the ESI-MS/MS sensitivity but also the LC retention, which enabled the separation of GABA from the interfering isomers. Satisfactory assay precision and accuracy were also acquired by adding dDEABAS-derivatized GABA, which served as the internal standard and worked well for correcting the matrix effect and instrument drift. The developed method had satisfactory applicability to the real sample analysis because it was successfully used to quantify the serum GABA of the healthy subjects.