d-Amino acid residues have been observed in aged proteins in various diseases, and the correlations between the formation of d-form residues and functional changes are of increasing interest. As the factors causing the epimerization of amino acids residues, external stresses, such as high temperature and alkaline conditions, have been reported, and there is a pressing need to clarify the production of d-form residues in proteins exposed to various stress conditions. In the present study, d-amino acid residues of alanine (Ala), aspartic acid (Asp) and serine (Ser) were determined in proteins stored under high temperature and/or basic pH conditions. The protein samples were hydrolyzed, then the obtained amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, and analyzed by using a two-dimensional chiral LC-MS/MS system equipped with Singularity RP18 (first dimension) and Singularity CSP-013S (second dimension) columns. As a result, the d-Ser residue (3.5 %) was detected in ovalbumin (OVA) stored at 37 ºC and pH 7.4 for 4 weeks (physiological conditions), while relatively large amounts of d-Asp (5.4 %) and d-Ser (9.4 %) residues were observed in the same protein stored at 60 ºC and pH 7.4 for 4 weeks. In OVA stored at 60 ºC and pH 9.6 for 4 weeks, large amounts of d-Asp and d-Ser residues (21.9 % and 37.8 %) were determined, while a non-negligible amount of d-Ala residues was observed (2.9 %). Also, in human serum albumin stored at 60 ºC and pH 9.6 for 4 weeks, the d-forms of all target amino acid residues were observed and the %d values were 2.3 for Ala, 15.3 for Asp and 33.5 for Ser. These results indicated that epimerization of various amino acid residues occurs (not only for Asp and Ser) by the external stress and further detailed studies are expected.

This study reports a new analytical method for the musty odor compounds 2-methylisoborneol and geosmin in water samples using a high-flow-rate purge-and-trap extraction with a solid-phase extraction (SPE)–type collection device, followed by analysis using gas chromatography–mass spectrometry (GC–MS). For the sample preparation, 500 mL of water sample and sodium chloride were placed in a glass vial. An SPE device packed with styrene–divinylbenzene polymer particles was attached to the vial cap, and the headspace gas was collected at a flow rate of 1 L/min for 30 min. During the gas sampling, clean air was introduced into the sample solution through a purge gas supply tube, while the musty odor compounds were purged from the water sample and extracted onto the adsorbent. The extracted compounds were eluted with 1.0 mL of dichloromethane, and the eluent was concentrated to 0.1 mL via nitrogen blowdown. The analytes were measured by GC–MS after injection of the eluent. The limit of detection for the analytes were 0.5 ng/L. The spike recoveries of the analytes from tap and river water ranged from 94 to 105%. Finally, the applicability of the method was investigated for determining the musty odor compounds 2,4,6-trichloroanisole and 2,4,6-tribromoanisole in water samples.

An acid dissociation constant of hydrolyzable p-nitrophenyl phosphate (NPP), pK_a2, was determined in this study through its effective electrophoretic mobility in capillary zone electrophoresis (CZE). NPP could undergo hydrolysis in an aqueous solution to produce p-nitrophenol (NP), and a reliable value for its pK_a2 has not been reported previously. When NPP solutions were freshly prepared each day and analyzed by CZE on the same day, only a peak assigned to NPP was detected in the electropherograms, with no detectable peak corresponding to NP. The effective electrophoretic mobility of anionic NPP increased with pH, and analysis of the mobility change yielded a second acid dissociation constant of pK_a2 = 5.46±0.02. The pK_a2 value was also determined by spectrophotometric titration without interference from hydrolyzed NP, giving pK_a2 = 5.53±0.08. These two pK_a2 values were in good agreement, demonstrating that the pK_a2 of NPP was successfully determined in this study.

In order to evaluate the possibility of using sodium p-fluorobenzenesulfinate (NaFBS) as a probe for magnetic resonance imaging with fluorine (¹⁹F-MRI) in diagnosis of melanoma, ion chromatography (IC) and capillary-zone electrophoresis (CZE) were applied to determination of fluoride ion (F^-) in samples for melanoma and control cells cultured in the corresponding medium containing NaFBS at various concentrations. Under the conditions employed, a peak for F^- was detected and identified on the chromatograms and electropherograms, and the F^- level determined by IC and CZE was elevated depending on increase in the amount of NaFBS added to media, especially in melanoma cells. As the accelerated accumulation of F in melanoma cells was caused possibly due to enhanced generation of 6-fluorobenzenesulfonyldopa by the reaction of NaFBS with dopaquinone as an intermediate of the tyrosinase reaction, the results of IC and CZE analyses suggested that NaFBS may be a potential candidate ¹⁹F-MRI probe for diagnosing melanoma with the highly elevated tyrosinase activity.

The Pirkle-type chiral stationary phases for enantioselective high-performance liquid chromatographic (HPLC) separation of all the proteinogenic amino acids plus allo-forms (allo-isoleucine and allo-threonine) were designed and developed. The developed three columns having N-(3,5-dinitrophenylaminocarbonyl)-l-amino acids at their chiral recognition sites successfully separated the enantiomers of all the proteinogenic amino acids and allo-forms individually. The obtained separation factors of the enantiomers were 1.08–1.42, 1.09–1.52 and 1.14–1.55 for the Singularity CSP-001S, 016S and 017S columns, respectively. The elution orders of the enantiomers were ideal for all the amino acids using these three columns; d-enantiomers (normally minor isomers in biological matrices) eluted faster than the predominant l-forms indicating that these columns are helpful for the determination of trace amounts of the d-forms. The separation profiles of 3 chiral amino acids (alanine and 2 model interfering amino acids) revealed that these newly designed columns have different separation properties. The accurate analysis of trace amounts of d-amino acids in complicated biological samples is often disturbed by the co-elution of numerous interfering substances. Therefore, the newly designed stationary phases would contribute to cross-checking the quantitative values of chiral amino acids besides their comprehensive determination in real-world matrices.

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.