In this report, C70-fullerene (C70), which has aspheric structure and greater π electrons compared to C60-fullerene (C60), was effectively immobilized onto the surface of a silica-monolithic capillary, and then the retention characteristics for polycyclic aromatic hydrocarbons (PAHs) due to π-π interaction were evaluated in liquid chromatography (LC). For the immobilization of C70, a C70 derivative via thermal reactive agent, perfluorophenylazide (PFPA) and N-hydroxysuccinimide (NHS) was synthesized. In comparison with a typical thermal reaction in an oil bath and a microwave, the latter reaction provided higher yield. Then, both the C70 and C60 monoliths were evaluated by LC with a variety of PAHs as the solutes. The results suggested that the specific interactions, such as dipole and induced dipole interactions were contributed between the bonded C70 and PAHs. Absorption spectra of PAHs with C60 or C70 and a computer simulation also supported these interactions. Furthermore, the comparison of the separations for PAHs and brominated benzenes with the C70 column and a porous graphite carbon packed column suggested that the interactions based on the polarization of the molecules contributed the separations.
The circadian profiles of D-alanine (Ala) amounts in human serum and urine have been clarified using a two-dimensional HPLC system combining a microbore reversed-phase C18 column and a Pirkle-type enantioselective column. The two-dimensional HPLC system enables the determination of trace amounts of D-Ala in the clinical samples without severe interference from intrinsic substances in the human physiological fluids. In the volunteers having normal sleep-awake patterns, the amounts of D-Ala in the serum were high in the late evening and low in the morning. On the other hand, in the volunteers having the reversed sleep-awake patterns, the amounts of D-Ala in the serum were high in the morning and low in the nighttime. The similar circadian changes of D-Ala amounts were observed in the urine. These 24-h profiles of D-Ala are almost the same as those observed in rats and mice, suggesting that D-Ala has fundamental physiological functions related to rest-active conditions in mammals, and could also be useful as the biomarker for sleep-awake profiles.
The aim of this study was to investigate the applicability of a new technique using in-source collision-induced dissociation (CID) for improving quantitative linear range of various compounds in liquid chromatography-tandem mass spectrometry (LC/MS/MS). To determine whether the linear range shift due to in-source CID occurs under various MS conditions, we investigated the quantitative linear ranges of reserpine, indomethacin, and furosemide in both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in positive- and negative-ion detection modes. We observed 3–30-fold linear range shifts upon changing the declustering potential to adjust in-source CID in positive- and negative-ion detection modes in both ESI and APCI. These results indicated that this new technique could be applied for arbitrary adjustment of linear range regardless of ionization process and polarities in LC/MS/MS. Therefore, this technique could be applied to simultaneous quantification, in biological fluids, of compounds with quite different sensitivities under various MS conditions.
The formation of D-amino acid residues in proteins is considered as one of the deterioration processes, and the determination of these D-amino acid residues is highly expected for the screening of new biomarkers under various disease conditions. In the present study, a two-dimensional (2D) HPLC-MS/MS system following the hydrolysis with deuterium chloride (2HCl/2H2O) and derivatization of amino acids with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) has been designed/developed and applied to the analysis of proteins stored under various conditions. As the target, 5 major D-amino acid residues (Ala, Asp, Glu, Pro and Ser) were selected. The analytical procedure was validated using a model peptide, NH2-Gly-Pro-Glu-Ala-Asp-Ser-Gly-OH, and the obtained calibration lines of %D for the 5 target amino acids were linear with correlation coefficients greater than 0.998. The RSD values for the intra-day precision and inter-day precision were lower than 5%. In most of the proteins tested, the amounts of the D-Ser and D-Asp residues increased during storage, and the highest value (14%, D-Ser) was observed in ovalbumin (OVA) after storage at pH 9.5 for 4 weeks.
Sodium dehydroacetate (SDA) was extracted from cosmetic powders using a Na2CO3-methanol-H2O system. After the extract was neutralized, the resulting dehydroacetic acid (DAA) reacted with 4-nitrophenylhydrazine∙HCl (4-NPH∙HCl) to give the corresponding hydrazone, which was determined by an HPLC-VIS (400 nm) system. Using 4-NPH∙HCl in great excess was suitable for detecting the presence of DAA, which almost reacted with the hydrazine to yield the corresponding hydrazone. As a result, it was determined that contaminants in the cosmetic powder did not interfere with analysis of the hydrazone. Importantly, the molar absorptivity of the hydrazone (1.29 × 104 at 400 nm) is five times that of DAA (2.57 × 103 at 230 nm). Accordingly, detection of the hydrazone at 400 nm in a new analytical method might have fivefold higher sensitivity compared with that of DAA at 230 nm in the standard method.
Reagent-release hydrogels containing an acid or a base solution were prepared and employed as ion suppliers in isoelectric focusing using short capillaries. In conventional isoelectric focusing techniques using microfluidic channels, complicated liquid manipulations are required, which can decrease analytical performance such as the reproducibility of a focusing position. To develop a rapid and sensitive assay based on microfluidic isoelectric focusing, we previously prepared reagent-release capillaries retaining ampholytes by physical adsorption on the inner surface of a short capillary, which were then applied to isoelectric focusing. In this study, reagent-release hydrogels containing an acid or a base solution were developed for isoelectric focusing using short capillaries in place of the acid and base solutions used in conventional microfluidic isoelectric focusing. The prepared reagent-release hydrogels showed some advantages for isoelectric focusing, e.g., high durability for at least five weeks of storage, easy handling as compared to the conventional solutions, and suppression of hydrodynamic flow in capillaries, which often decreases the reproducibility in capillary isoelectric focusing. The migration behavior of ions from the hydrogels into the capillary and the formation of a pH gradient in isoelectric focusing using the reagent-release hydrogels were investigated, which confirmed that the prepared reagent-release hydrogels could form a similar pH gradient to that obtained with conventional capillary isoelectric focusing using the solutions.