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.

The retention of ethane in cryogenic high performance liquid chromatography (HPLC) at -196 °C was investigated by comparing with the retentions in ambient temperature gas chromatography (GC) at 50 °C (analyte: n-pentane) and ambient temperature HPLC at 25 °C (analyte: acenaphthene) using commercially available linear (trimethyl, dimethyl-n-butyl, dimethyl-n-octyl, and dimethyl-n-octadecyl silica) and laboratory-made branched alkyl chain (dimethylisopropyl and dimethylthexyl silica) stationary phases. For this investigation, the densities of the functional and end capping groups were evaluated using the carbon and hydrogen contents in the packing materials, as measured by CHNS analysis. To evaluate retention in each stationary phase and separation mode, the retention factor per single functional group and the retention per single carbon unit (CH₂ or CH₃) were calculated. A detailed study on the retention behavior revealed that the retention behavior observed with linear alkyl chain stationary phases in cryogenic HPLC was similar to that in ambient temperature GC, but not ambient temperature HPLC. The stationary phase of the branched thexyl group resulted in a weak interaction for the test analytes in HPLC_-196°C and HPLC_25°C. By contrast, the stationary phase of the isopropyl group did not suppress the analyte retention in either liquid chromatography.

A simple and accurate analytical method was developed for determining carbon tetrachloride (CCl₄) at the ppm level in wastewater from the manufacturing process as part of process monitoring and control. In this method, CCl₄ was extracted from wastewater using toluene and analyzed by gas chromatography (GC) equipped with a flame ionization detector (FID) and an OVI-G43 capillary column (film thickness: 3 μm; inner diameter: 0.53 mm; and length: 30 m）, with nitrogen as the carrier gas. The proposed method was validated in terms of its specificity, linearity, accuracy, and repeatability. The regression curve demonstrated excellent linearity (r = 1.000, r² = 1.000) over a concentration range of 0.5–10 ppm. The mean recovery at concentrations between 0.5 and 5 ppm was 90 %, and the relative standard deviation for repeatability was 2.0 %. The limit of quantification was 0.5 ppm based on the linearity and accuracy results. The proposed method requires only simple liquid–liquid extraction, a versatile GC-FID system, and a sustainable carrier gas (nitrogen), making it highly suitable for the analysis of CCl₄ at manufacturing sites.

Several years have passed since the dynamics of d-amino acids (AAs) in the living body of mammals have drawn attention as potential biomarkers of several diseases. So far, researchers have developed an enantiomer-separable method using liquid chromatography and mass spectrometry (LC-MS). Using these excellent methods for dl-AA analysis, much knowledge was obtained about d-AAs. In our previous study, (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine-2-carboxylate (DMT-(S)-Pro-OSu: DMT(S)), which was a chiral derivatization reagent for LC-MS analysis, showed high sensitivity that enabled detection of dl-AAs at the low concentration level of biological samples. However, DMT(S) required five different elution conditions and two columns for 19 dl-AA analysis with resolution (Rs) > 1.5. Herein, we report the development of novel derivatization reagents, which have a modified structure based on DMT(S) to enhance enantiomeric separation. A comparison of the separation efficiency of developed reagents and DMT(S) was higher for DMT(S) and the isopropoxy-modified reagent (i.e., DiPT(S)). In addition, a multiple regression model for the prediction of Rs was also developed. Our binary mobile phase system prediction model showed high accuracy with only five condition test data sets. The resulting analytical method, using parallel derivatization (DMT(S) and DiPT(S)) and an elution condition predicted by the developed model, enabled simultaneous analysis of 19 dl-AAs in 45 min LC run by stepwise elution. These techniques contribute to the efficient and speedy development of an analysis method with chiral derivatization for dl-AAs and other chiral metabolites.

Chiral stability and photostability of bepotastine besylate (BB, S (+)-form) and levocetirizine dihydrochloride (LCTZ, R (-)-form), both are clinically useful antihistamines, were investigated by HPLC with an ovomucoid (OVM) or a β-cyclodextrin (β-CD) immobilized enantioselective columns under the reversed-phase mode. Enantiomers of BB were successfully separated by an OVM column with resolution (Rs) 3.6 within 7 min and a β-CD column within 13 min with Rs 2.7. Optical purity testing of BB has been included in the monograph of BB in the Japanese Pharmacopoeia 18^th edition (JP18), where a β-CD column is used. Enantiomers eluted within 20 min with Rs 2.4 by the JP18 method. Through changing buffer pH of the mobile phase in the official JP method, faster enantiopurity evaluation of BB was achieved without loss of Rs value. Enantiomers of LCTZ, i.e., cetirizine (CTZ) were also successfully separated by HPLC with an OVM column with Rs 1.8 within 10 min. Using validated chiral HPLC methods, photostability and chiral inversion studies of BB orally disintegrated (OD) tablets and its suspensions, and LCTZ tablets and syrups under UV irradiation and the heating stress conditions were performed in addition to its quality evaluation. In photostability studies, formulations of both BB and LCTZ were stable for UV irradiation. Under heat stress testing, chiral inversion of BB was observed in OD tablets (70 ºC, 1 week, 0.5 % R(-)-form) and its suspensions (70 ºC, 1 week, 6 % R (-)-form). On the other hand, chiral inversion of LCTZ formulations by heat (70 ºC, 80 ºC, 1 week) was not observed, although chemical structure of LCTZ is very similar to BB. In our previous chiral inversion study of d-chlorpheniramine maleate (d-CPM, S (+)-form), d-CPM was quite easily chiral inverted under the heating conditions. From these chiral inversion studies of d-CPM, BB and LCTZ, chiral inversion mechanism is proposed by comparing three chemical structures. Imine-enamine tautomerism seems to be a key step for chiral inversion of these antihistamines.

This study introduces F-trap pyrene Ox, a reagent peak-free pre-column fluorescence derivatization reagent for sensitive analysis of amines and amino acids. By oxidizing the thioether moiety of previously reported F-trap pyrene to sulfone, we significantly enhanced reactivity toward amino groups, achieving complete derivatization in 10 min at 90 °C. The reagent incorporates a fluorophilic perfluoroalkyl tag that enables selective removal of excess unreacted reagent through fluorous solid-phase extraction (F-SPE) with 75 % aqueous acetonitrile as eluent, achieving >99.4 % removal rate while maintaining excellent recovery. Method validation for aliphatic amines demonstrated good linearity (r² > 0.995) over 50 fmol to 50 pmol, excellent precision (intra-day RSD ≤ 6.7 %), and detection limits 4-times lower than F-trap pyrene. Additionally, we developed an online column-switching F-SPE system that automates the removal process. The method successfully analyzed 13 aliphatic amines and 16 amino acids without interference from reagent peaks, offering significant advantages for sensitive analysis of biologically active amines in complex matrices.

Fatty acids (FAs) are carboxylic acid with carbon chain and classified according to the length of carbon chain and the number of double bonds. The source of FAs in a human body is the absorption from intake foods, the release from several lipid classes by hydrolysis and the de novo lipogenesis including the condensation of acetyl-CoA. They have multiple roles including energy sources, precursor compounds for the synthesis of cellular lipids, regulators of gene expression and signal mediators. In recent years, the functions of FAs have attracted a great deal of attention, and it is important to analyze them accurately by FA species. To quantify levels of FA species accurately without derivatization and also to develop the method of simultaneous analysis of FA and several lipid classes, in this study, authors carried out the optimization of MS conditions of 10 FA species including the combination of a precursor ion and a product ion systematically. We clarified that both carbon chain length and the number of double bonds affect the ionization efficiency of FA species, resulting in the difference of slope values obtained from calibration curves. These differences are useful as correction parameters, which enable to quantify FA species in the internal standardized method accurately without the use of deuterium-labeled internal standards. Furthermore, the developed method was applied to FA profiling of 8 mammalian sera. Total FA levels of mouse serum was the highest compared with other mammalians, suggesting higher activity of FA synthesis and/or lipolysis of mouse.

The surface area of gold nanoparticles (AuNPs) was estimated through the adsorption of cysteine onto the AuNPs. The AuNPs used were synthesized by a solution plasma process and were polycrystalline. Capillary zone electrophoresis (CZE) was employed to monitor the cysteine adsorption. An AuNP dispersed solution with cysteine adsorption was directly introduced into the CZE system, and a broad peak was obtained in the electropherograms with the dispersed AuNPs. Shot signal for the aggregated AuNPs was not detected in the electropherograms, indicating that the dispersion of the AuNPs was maintained even after the cysteine adsorption. The effective electrophoretic mobility of the AuNPs was reduced by the adsorption of cysteine to the AuNP surface. Any peak attributed to the cysteine was not detected at low concentrations of the cysteine, and the added cysteine was mostly adsorbed onto the AuNPs. An additional peak attributed to the residual cysteine was detected at higher concentrations of cysteine from 100 μmol L⁻¹, and a linear relationship was obtained between the concentration of cysteine and the peak area of the cysteine with an x-intercept at 86 μmol L⁻¹. This concentration represents the saturation point of surface adsorption onto the AuNPs. The surface area of the AuNP dispersed solution was estimated from the adsorption amount of cysteine and the cross-section area of a cysteine molecule, resulting in an estimated surface area of 0.078 m² mL⁻¹. The estimated surface area was also verified through the adsorption of glutathione onto the AuNP.