We applied the deep eutectic solvent (DES) extraction for analyzing capecitabine (Cape), 5’-deoxy-5-fluorocytidine (DFCR), 5’-deoxy-5-fluorouridine (DFUR), and 5-fluorouracil (5-FU) in human plasma with LC–UV method. In this method, the DES, which was prepared by mixing trioctylmethylammonium chloride and pentafluorophenol in a 1:2 molar ratio, was used as extraction solvent for target analytes in the sample solution. The target analytes extracted by DES was then injected into a reversed-phase LC–UV system and were detected with ultraviolet at 270 nm for 5-FU, DFCR, and DFUR, and at 240 nm for Cape. Under the optimized conditions, this extraction resulted in enrichment factors for Cape, DFCR, DFUR, and 5-FU, ranging from 1.4 to 5.6. The determination coefficients of the calibration curves were r² > 0.9952 in the range of 0.05–10 µg/mL. The limits of detection and quantification ranged from 1.9 to 4.4 and from 6.4 to 14.7 ng/mL, respectively. The relative standard deviations of the peak areas obtained during intra-day determinations were within 9.8 %. The proposed method was further validated by the analysis of spiked human plasma samples.

In this study, we developed and validated a novel method of simultaneously measuring serum concentrations of linezolid (LZD) and its major metabolites, PNU-142300 and PNU-142586, in human serum using high-performance liquid chromatography coupled with an ultraviolet detector (HPLC-UV) for pharmacokinetic–pharmacodynamic analysis. LZD is an antibacterial agent, and its major metabolites are suspected to cause severe thrombocytopenia. To elucidate the relationship between the blood concentrations of these substances and platelet counts, an analytical method is needed that can simultaneously quantify LZD, PNU-142300, and PNU-142586 and be applied to clinical samples. We investigated an analytical method that shows high linearity at concentrations of 0.1–50 μg/mL (LZD and PNU-142300) and 0.2–50 μg/mL (PNU-142586), which covers the practical concentration range of clinical samples predicted through pharmacokinetic simulation. The intra- and inter-assay accuracies ranged above 88 % for LZD, PNU-142300, and PNU-142586. Furthermore, the precision of all intra- and inter- assays was less than 15 %. This is the first report on the simultaneous measurement of LZD concentrations and those of its major metabolites in human serum using an HPLC-UV system with a neutral isocratic mobile phase.

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.

In this review, applications of synthetic polymer materials with fibrous shape in chromatographic field were described, especially focusing on the approach by the author's group. Introducing synthetic polymer materials with good heat-resistance, chemical-resistance, and mechanical strength as separation/extraction media in chromatographic techniques, novel stationary phases and sample preparation media have been developed. Taking advantage of the physicochemical properties, synthetic fibrous materials packed into a short capillary could be employed as both of the separation and extraction media in chromatography. Filament-packed columns consisted of longitudinal fibrous materials were successfully developed in gas chromatography as well as liquid chromatography, and a unique retention behavior for aromatic compounds was confirmed on the fibrous polymer phase. In applying synthetic polymer filaments as extraction media, sample preparation devices with novel functionalities were developed in addition to the conventional concentration performance based on a trap-and-release process because fibrous shape allows flexible arrangement within capillary. These results also suggest a future possibility of the fine synthetic fibrous materials in microscale separation science.

Protein phosphorylation is a crucial regulatory mechanism in cellular signaling, yet comprehensive analysis of the phosphoproteome is challenging due to its extensive dynamic range and complexity. This review explores the chromatographic behavior of phosphopeptides, and the strategies developed to tackle the difficulties in detecting the low-abundance phosphoproteome, particularly focusing on motif-targeting phosphoproteomics that utilize protein kinase substrate specificity. The review highlights how in vitro kinase reactions facilitate the selective enrichment of specific subsets of the phosphoproteome, thereby reducing sample complexity and improving the detection of low-abundance species, such as tyrosine phosphorylation. Two complementary approaches are discussed: motif-targeting phosphoproteomics, which involves performing kinase reactions on target samples to create enriched phosphopeptide populations for direct analysis, and motif-centric phosphoproteomics, where kinase-generated phosphopeptides are isobarically labeled and used as carrier channels to enhance signals from low-abundance species. Additionally, recent advancements in phosphopeptide chromatography, including mobile phase optimization and temperature-dependent retention behavior, are examined. Applications in tyrosine phosphoproteome analysis and kinase inhibitor profiling illustrate the effectiveness of integrating biochemical selectivity with chromatographic separation for pathway-focused phosphoproteomics, even from limited sample amounts.

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.

Microchip electrophoresis (MCE) have attracted considerable attention as high-performance electro-driven separation methods and applied to various research fields. To improve concentration sensitivity in MCE, we have developed and modified online sample preconcentration techniques for a sample solution introduced into a whole separation channel. By employing such enrichment approaches, we investigated the development of highly-sensitive electrophoretic microchips that eliminate the need for complex voltage-programs in the sample injection processes. In the present focusing review, the contents can be categorized as follows: (1) application of large volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) to MCE, (2) development of a novel channel modification method for controlling EOF, (3) sensitivity enhancements by combining LVSEP with field-amplified sample injection (FASI), (4) sensitivity enhancements by coupling LVSEP with transient isotachophoresis (tITP), and (5) sensitivity enhancements by integrating large-volume dual preconcentration by isotachophoresis and stacking (LDIS) with FASI.

In-capillary continuous enzyme assay in electrophoretically mediated microanalysis was simulated using MATLAB software, employing the hydrolysis of p-nitrophenylphosphate (NPP) with alkaline phosphatase (ALP) as a model system. In the simulation, a short plug of NPP substrate solution was injected into a separation capillary, while the separation buffer contained the enzyme ALP. The migration of NPP in the capillary was modeled based on its apparent electrophoretic mobility, and enzymatic hydrolysis of NPP occurred continuously within the NPP zone at a defined reaction rate. A product of p-nitrophenol (NP) was continuously generated at the substrate zone, and it was immediately resolved from the substrate zone with its apparent electrophoretic mobility. Electrophoretic migration and enzymatic reaction were iterated every second, resulting in a simulated plateau response of NP in the continuous enzyme assay. Electropherograms exhibiting the plateau response were reproduced by adjusting several parameters, including instrumental conditions (capillary lengths, injected plug length, applied voltage), reaction conditions (reagent concentrations, reaction rate), and electrophoretic mobility of both the substrate and the product. The height of the plateau response reflected the Michaelis-Menten constant and the reaction kinetics, and the simulation results showed good agreement with previously reported experimental data. At low substrate concentrations, the plateau response transitioned to a slope profile, indicating significant substrate depletion. Additional parameter sets were explored to achieve the plateau response. This study demonstrates that MATLAB-based simulation is a helpful tool for estimating the conditions to achieve a plateau response in electrophoretically mediated microanalysis.

Various betaines (amino acid derivatives possessing a quaternary ammonium group) are present in various foods and have significant roles in health maintenance and enhancement for humans. Although rice bran (RB) fermented with Aspergillus oryzae (A. oryzae) is expected to be a foodstuff rich in the bioactive betaines, its betaine contents have not been elucidated. To address this concern, a hydrophilic interaction liquid chromatography (HILIC)/electrospray ionization-tandem mass spectrometry method was developed and validated for the determination of the five bioactive betaines, i.e., γ-butyrobetaine, L-carnitine, L-stachydrine, L-ergothioneine and L-hercynine, in the A. oryzae-fermented RB products. These betaines were satisfactorily retained and separated on an InertSustain Amide column with an acetonitrile-rich mobile phase. A solvent extraction-based pretreatment, HILIC separation and internal standard calibrations provided the precise (relative standard deviations: ≤ 5.6 %) and accurate (analytical recovery rates: 97.1–100.6 %) measured values. No significant matrix effect was observed. By using this method, we demonstrated that the A. oryzae-fermentation significantly increased the contents of all five betaines in the RB. The A. oryzae-fermented RB products had compatible or greater betaine contents compared to the plant-based foods that have been reported to be rich in betaines.

I This study employs ethylene glycol (EG) as a solvent for dissolving 2,4-dinitrophenylhydrazine (DNPH) in the collection and derivatization of gaseous formaldehyde using a miniaturized liquid absorption cartridge comprising a polypropylene cartridge and a functional polytetrafluoroethylene (PTFE) membrane, which permits the passage of gas and blocks the flow of liquid. DNPH dissolved in 400 μL EG was used as the absorption solvent in the cartridge. The gas sample was collected at 50 mL/min through the PTFE membrane, and the analyte formaldehyde was derivatized and collected in the solvent. After air collection, the absorption solution was heated at 80 °C for 5 min to enhance the derivatization reaction. Thereafter, the absorption solvent was injected into a high-performance liquid chromatography system for the determination of formaldehyde. EG has a considerably higher viscosity than acetonitrile, which enables a high flow rate during sample gas collection. Additionally, the loss of absorption solvent during sample collection and heating due to solvent evaporation is prevented. The limit of detection of the proposed method is 2.5 ng/L, and the relative standard deviation of the peak area for the standard formaldehyde gas sample (10 ng/L) is less than 7 %.

Denopamine (DP, R(-)-1-(p-hydroxyphenyl)-2-((3,4-dimethoxyphenethyl)amino)ethanol), which has been developed as a single enantiomer from its pharmacological aspect, is a clinically useful cardiotonic drug. From our previous photostability and chiral stability studies of DP, chiral inversion of DP was observed in aqueous solutions of DP drug substances and suspensions of DP tablets under the heat stress conditions dependent on the heating temperature and the storage period. In this paper, chiral inversion mechanism of DP was investigated employing phenylephrine hydrochloride (HCl) (R (-)-form) and ephedrine HCl (1R, 2S (-)-form) as samples for comparison. These drugs have the same phenylethylamine structure and the relationship between asymmetric C-atom and N-atom is similar. As a result, phenylephrine HCl and ephedrine HCl were found to be stable for heat stress. No chiral inversion was observed in both drugs. The difference between DP and phenylephrine HCl is the bond position of the hydroxy group in the phenyl group, and the difference between DP and ephedrine HCl is the existence of the hydroxy group in the phenyl group. From this study, an important step for the chiral inversion of DP was speculated as the formation of the stable benzyl cation in DP due to its hydroxy group at para-position of the phenyl group.