This review summarizes our recent works concerned on the analysis of glycoprotein glycans. Glycoprotein glycans are highly complicated and have variations that number into the thousands. Therefore the method for their analyses should be carefully chosen according to the complexity and quantity of glycans in samples. To shorten the time for the preparation of labeled glycans from glycoprotein, we proposed two methods: PNGase F/NBD-F method enables the preparation of fluorescently labeled glycans from glycoproteins about 2 hr. A combination of a neutrally coated capillary and borate buffer eliminates the purification steps for the removal of excess reagents from the glycan samples for capillary electrophoresis. Partial filling affinity capillary electrophoresis using glycan-recognizing proteins such as lectins, glycosidases and immunoglobulins, was used for the structural analysis of glycans. The method was applied to the specific detection of NeuGc and α-Gal containing glycans known as the heterogenic antigens. Sensitivity of the detection of glycoprotein glycans separated by microchip-based electrophoresis was enhanced by in situ fabricated sufonate-type polyacrylamide gel. This method enhances the sensitivity by a factor of 105. In situ fabrication of lectin impregnated gels enables specific entrapment and analysis of glycans. Conversion reaction from fluorescently labeled glycans (1-amino-1-alditols) to free oligosaccharides will be helpful for the functional analysis of glycans.
This review summarizes the possibility to diagnosing the chronic diseases and application of determination of metabolites of the noninvasive human nails. A target derivatization UPLC-ESI-MS method is a powerful tool for the analysis of bioactive compounds with high sensitivity and selectivity. We developed target derivatization UPLC-ESI-MS methods to analyze the biological trace components in human fingernail. Moreover, we have used these methods to detect the metabolites in human fingernails, such as DL-amino acids, polyamines and Advanced Glycation End products (AGEs). Through detecting the biological trace components in the real samples from human, we tested the possibility to use these new methods to diagnosing the chronic diseases, i.e., diabetes, lung cancer. Some applications utilizing these target derivatization methods for the analyses determination of metabolites in the human fingernails are also described in this paper.
We synthesized 3-(4-sulfo-1,8-naphthalimido)propyl-modified silica (SNAIP) for capillary electrochromatography (CEC). The unique structure of SNAIP contributed to the retention by three interactions including hydrophobic, electrostatic and π-π interactions and the acceleration of electroosmotic flow at an acidic condition. The CEC employing SNAIP was successively applied to the rapid separations of several drugs, peptides and polar compounds. Also, several examples for applying CEC to real sample analyses were demonstrated. Furthermore, with the idea to use adamantane as a shield to reduce the peak tailing of charged solutes in CEC, adamantyl (ADM)-functionalized polymer monolith by a single-step copolymerization with the monomer containing ADM structure and a cross-linker was presented. Three chiral stationary phases with different phenylalanine (Phe) peptide lengths, Phe4, Phe8, Phe12, were prepared to study the effect of peptide length on enantioseparation in reversed-phase HPLC. The highest resolution was observed for the selector with intermediate peptide length (i.e., Phe8). The side chain of amino acids was also found to play a role for the separation performance of the chiral stationary phases (CSPs). The IR spectra suggested that the Phe peptides immobilized on the CSPs were assumed to be mainly in the α-helical state. Also, it was found that the conformation strongly contributed to the chiral recognition of the CSPs by thermodynamic study.
A narrow-bore packed column (packed-capillary column) that designed for a direct installation in conventional capillary gas chromatographic (GC) system had been developed. In this column, particulate sorbents that employed in typical GC packed columns were packed into a stainless-steel capillary of 1.0 mm i.d. By attaching a pair of stainless-steel capillaries of 1.27 mm o.d. at both ends of the column, the packed-capillary column can be directly installed into the conventional capillary GC system. In this article, fundamental of the packed-capillary column developed by our research group is mainly reviewed, along with the various applications, such as column switching analysis and evaluation of catalytic property based on the determinations of CO and CO2.
D-Amino acids are now increasingly recognized as physiologically functional molecules and biomarkers in mammals. However, the amounts of D-amino acids are extremely low in most cases, thus highly sensitive and selective analytical methods are practically essential. In the present review article, enantioselective two-dimensional high-performance liquid chromatographic (2D-HPLC) methods are introduced and their applications to the determination of D-amino acids in mammalian samples are described. For the sensitive determination, amino acid enantiomers are derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and are determined by a fluorescence detector. The 2D-HPLC system consists of the reversed-phase non-enantioselective, but chemoselective separation of NBD-amino acids in the first dimension, and the sequential chiral separation in the second dimension. By using the system, neuroactive D-amino acids (D-Ser and D-Ala) were determined in the brain of various mammalian species and the regulation of their intrinsic amounts are also demonstrated. The physiological and diagnostic meanings of the trace amounts of D-Ser are also discussed. The 2D-HPLC systems for the acidic D-amino acids (D-Asp and D-Glu), proline analogues (D-Pro, trans- and cis-4-hydroxy-D-Pro) and hydrophilic D-amino acids (D-Arg, D-Asn, D-Asp, D-Gln, D-Glu, D-His, D-Ser, D-allo-Thr and D-Thr) are also shown.
Two separated peaks were detected when a small volume (approximately less than 0.5 µL) of one compound was injected into a HPLC system using an autosampler equipped with a 6-port valve. The origin of the two peaks was found to be injection by an autosampler that sucked the sample solution twice during the injection of each sample. First, the autosampler sucked in a sample solution to fill the needle, and then, after the rotation of the 6-port valve, the autosampler sucked in the sample solution again to fill the injection loop. Because the twice suck generated two separated sample zones in a 6-port valve and these sample zones were injected into the column separately, two separated peaks were observed. The increase in the sample volume induced disappearance of second peak and only one peak was observed. And also the same phenomenon was observed with a manual injector instead of the autosampler even when small volume of sample was injected.