In the field of liquid chromatography (LC) analysis, derivatization is a method of converting an analyte into a chemical structure that is advantageous for analysis. In particular, many derivatization methods have been reported that aim to improve the response to detectors, such as fluorescence derivatization. In this study, the derivatization methods were developed not only to improve the response to the detector, but also to improve the selectivity and quantification compared to the conventional method by utilizing the special physical properties of the derivatization reagent. First, the study focused on the specific affinity (fluorous) between perfluoroalkyl groups, and a fluorous derivatization method was developed to chemically modify the target compounds. Next, for the protein, we developed a derivatization method for isotope labeling and high-sensitivity derivatization of compounds to different functional groups, and a derivatization method that enables evaluation of small structural changes in protein. The developed derivatization methods have been applied to LC-Fluorescence detection (LC-FL) and LC-Mass spectrometry (LC-MS).

Homochirality of amino acid residues of protein is essential for life. For a long time, it was believed that D-amino acids were excluded from living systems, and that proteins consisted only of L-amino acids. However, recent developments in analytical techniques have led to the discovery of D-amino acids in living organisms, such as peptides and proteins. Many D-amino acids are aspartates, which are located in various sites within metabolically inactive tissues. In order to identify such aspartate residues in protein, a combination of complicated methods and instruments has been previously applied. In contrast, our laboratory has recently developed a rapid, easy and comprehensive method to identify D-Asp and β-Asp in tissues using LC-MS/MS, thus using mass spectrometry as a “mass detection device”. A total of four samples from the same tissue and proteins, generated in two steps of enzymatic digestion, are used for LC-MS/MS. The identification of each iso-Asp containing peptide peak is performed by the disappearance of the chromatogram due to the alteration of molecular weight by each enzymatic digestion. By using this methodology, it is possible to identify and distinguish each D-Asp and β-Asp in protein without a set of complicated methods and instruments.

Recently, digital electrophoresis has attracted attention as a separation and preconcentration method based on the passage or non-passage of sample molecules through various functional hydrogels. However, the complex fabrication of the device filled with various hydrogels has been a major issue. In this study, an easy-to-fabricate capillary device for digital electrophoresis is proposed and developed by the successive introduction of prepolymer solutions. Digital molecular sieving electrophoresis using polyacrylamide hydrogels with different densities was employed for a fundamental characterization of the developed device. As a result, the correlation between the total concentration of acrylamide species in the prepolymer solution (%T) and the molecular weights of the passing or non-passing proteins was clarified. The evaluation of the concentration efficiency showed that the sensitivity increased up to about 180 times. In addition, digital molecular sieve electrophoresis using the device successively plugged with 5, 15, and 25 %T polyacrylamide hydrogels achieved simultaneous separation and preconcentration of proteins with different molecular weights. These results demonstrate high-resolution with high-sensitivity digital electrophoresis using the simply fabricated device.

8-Aminopyrene-1,3,6-trisulfonate (APTS) is one of the most frequently used reagent in capillary electrophoresis for glycans separation. Three sulfonate groups in APTS generate fast electrophoretic mobilities of derivatized glycans, therefore very suitable for CE-LIF applications. However, these groups also make separation with partition chromatography difficult. A novel column for hydrophilic interaction liquid chromatography (HILIC) with DCpak PTZ was examined for the separation of APTS-labeled glycans derived from glycoproteins. This separation mode has enhanced capability for the size resolution of neutral and acidic oligosaccharides. We utilized this column for the fractionation of APTS-labeled glycans from human transferrin and examined their migration times with CE by comigration with a mixture of glycoprotein glycans. We also utilized this method for identification of high-mannose type glycans of HepG2 cell with CE by comigration with a mixture of bovine ribonuclease B derived glycans. The use of HILIC mode of DCpak PTZ column promise for the separation and preparation of glycoprotein-derived glycans labeled with APTS.

We developed a trap column based on a monolithic silica for nanoflow LC to analyze intact proteins dissolved in buffered solutions containing surfactants by high-precision LC and LC/MS. The C4 modification onto the silica surface as an adsorption layer was examined for the separation of proteins from each surfactant. The use of the trap column of the C4 modified silica provided good separations of proteins from the surfactant (CHAPS) under an isocratic condition. We also analyzed proteins dissolved in high concentration solubilizer (10 mmol L⁻¹ CHAPS, 1 mmol L⁻¹ EDTA · 2Na, 6 mol L⁻¹ guanidine buffer) by LC and LC/MS. The obtained MS data provided the proper molecular weight with deconvolution of MS spectra. The monolithic silica trap column provides high linearity of the calibration curve toward low concentration proteins without any adsorption loss. Thus, the monolithic silica column works well in the analysis of intact proteins by LC and LC/MC.

In this study, a new stationary phase modified with a peptide containing 2-aminoisobutyric acid (Aib, an unnatural amino acid) was prepared, and its separation behavior in HPLC was investigated. Peptide Aib 3mer (L-Leu-L-Leu-Aib) and peptide Aib 6mer (L-Leu-L-Leu-Aib-L-Leu-L-Leu-Aib) were introduced into aminopropylsilyl silica gel (APS) and packed into a stainless-steel column. The effect of organic solvent content in the mobile phase on the retention of hydrophobic compounds was investigated for the prepared columns. The results showed a typical reversed-phase mode separation behavior, in which retention decreased with increasing organic solvent content. Next, the effect of organic solvent content in the mobile phase on the retention of hydrophilic compounds was investigated. As a result, the separation behavior in the region where the organic solvent content in the mobile phase is high follows the HILIC mode, where the retention of hydrophilic compounds increases with increasing organic solvent content. All of this demonstrates the versatility of the prepared new stationary phase and its efficiency in separating both hydrophobic and hydrophilic compounds.

Here, we evaluated the effects of anticoagulants and storage conditions on the measurement of plasma cortisol and 6β-hydroxycortisol (6β-OHF) by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC–MS/MS), using stable isotope-labeled compounds as internal standards for calculating plasma or serum 6β-OHF/cortisol concentration ratios. Blood samples were obtained from five healthy subjects and divided into tubes containing three anticoagulants: sodium heparin, serum separation gel, and EDTA-2 potassium. There was no statistically significant difference in cortisol and 6β-OHF concentrations and 6β-OHF/cortisol ratios in the samples obtained from the three anticoagulants measured on the day of blood sampling. Serum samples obtained from serum separation gel were stored at 4°C for 4 days then at –20°C for 3 days, or at 4°C for 7 days. Whole blood samples with EDTA were stored at 4°C for 7 days. The cortisol and 6β-OHF concentrations in serum samples after 7 days of storage under two storage conditions were slightly higher and lower, respectively, than the value on day 1. The concentration of 6β-OHF stored as whole blood was higher than that of the serum storage sample due to the high blood/plasma ratio of 6β-OHF. We compared 6β-OHF/cortisol ratios with different anticoagulants and storage conditions. The change in the ratio was within 15 % in the serum sample, but –4.9 % ∼ 17.7 % in the whole blood sample. Although the storage condition affected the 6β-OHF/cortisol ratio, it ded not affect the subject's ratio order. Therefore, blood samples with any anticoagulant and under any storage conditions can be used; however, it is more desirable to use serum samples stored at –20°C than whole blood samples.

Glycans have important functions in living organisms. In the analysis of glycans, fluorescence derivatization is followed by separation analysis using high-performance liquid chromatography. It is necessary to remove excess unreacted derivatization reagent. In this study, we investigated the selective extraction of 2-aminobenzamide labelled glycans using a monolithic silica disk-packed spin column, MonoSpin column, in order to simplify this pretreatment. MonoSpin Amide and MonoSpin NH2 columns, which have amide and amino groups as functional groups, respectively, showed the same efficiency as the conventional solid-phase extraction method in the removal of 2-AB and the recovery of labeled glycan samples. The MonoSpin column is expected to be applied to high-throughput glycan sample preparation since it can be easily pretreated only by centrifugation.