When analyzing trace components in vivo, it is important to truly capture the target molecules, and even if advanced separation analysis methods are used, it is necessary to perform sufficient pretreatment. In recent years, chromatography and mass spectrometry have become commonly used for the analysis of biological components. Rapid development in both of these technologies has made it possible to capture previously undocumented phenomena. We have comprehensively analyzed cholesterol metabolites, including bile acids and their conjugated metabolites, in vivo, using both technologies. In addition, we have applied these techniques not only to small molecules in vivo but also to the analysis of biopolymers interacting with them. These results have contributed to the development of drug therapy, diagnosis, and other medical procedures, and we introduce some of these examples in this paper.
Several D-amino acids have been identified in higher animals including humans, and are now increasingly recognized as biologically active substances and/or biomarkers. Therefore, the presence of related compounds, such as dipeptides, containing D-amino acids in biological matrices is one of the recent topics of increasing interest. In the present study, on-line multi-dimensional HPLC systems combining a narrowbore reversed-phase column (KSAARP, 1.5 mm i.d. x 500 mm), a weak anion-exchange column (KSAAAX, 1.5 mm i.d. x 250 mm) and a Pirkle-type enantioselective column (KSAACSP-001S or R, 1.5 mm i.d. x 250 mm) have been designed for the chiral analysis of Ser-containing dipeptides in fermented products. As target analytes, Ser-Gly and Gly-Ser were selected. For the sensitive analysis, the dipeptides were pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to form the NBD-dipeptides, which were detected upon their fluorescence. During the selective multi-dimensional analysis, the target dipeptides were separated from other substances by the reversed-phase and anion-exchange columns as their D/L scalemic mixtures and automatically introduced to the enantioselective column, where the D,L-forms were separated with selectivity / separation factor (α) and resolution (Rs) values higher than 1.07 and 0.71, respectively. The present 3D-HPLC system was well applicable to the analysis of Japanese traditional amber rice vinegar where relatively large amounts of the L-dipeptides and trace amount of D-Ser-Gly were observed.
An on-line coupled comprehensive two-dimensional liquid chromatography (2D-LC) system was developed for the separation of triglycerides (TGs) in this work. Taking advantage of two-dimensional separation on the basis of two separation columns with different selectivities, where an silver-ion column and an octylsilica column were employed as the columns in the 1st and 2nd dimensions, respectively, all the TGs in perilla and tuna oils were well separated. Introducing the systematic qualitative analysis in gas chromatography (GC), it was possible to make the assignments for all the fatty acid compositions in the TGs that were fractionated after the comprehensive 2D-LC separations. In the subsequent GC analysis, 23 and 76 types of TGs were determined for perilla and tuna oils, respectively. The results clearly showed an excellent performance of the two-dimensional separation system for the separation of TGs in oils and fats, suggesting a future applications to the high performance separation of other types of fish oils with the on-line coupled comprehensive 2D-LC system developed in this work.
A needle-type sample preparation device packed cyclohexanone-coated fibers as an extraction medium has been developed for the gas chromatographic (GC) analysis of volatile amines in air samples. For a simultaneous derivatization/extraction of monoethanolamine (MEA), a fiber-packed needle was employed, where a bundle of heat resistant filaments, Zylon, poly(p-phenylene-2,6-benzobisoxazole) was packed longitudinally into the specially-designed needle. To the needle-type device, cyclohexanone was loaded in the preconditioning process. Gaseous MEA was sampled by a vacuum air sampler connected to the needle, and a simultaneous derivatization/extraction was carried out therein. Desorption of MEA derivative was successfully made at a heated GC injection port without using any desorption solvent. Introducing the simultaneous derivatization with cyclohexanone, the sensitivity was significantly improved, allowing a more sensitive detection of volatile amines. Taking advantage of the specially-designed needle, the needle was able to be stored for several days at room temperature after the sampling, where the derivatives of the volatile amines were stably trapped on the surface of the Zylon filaments in the needle.
A highly-sensitive two-dimensional high-performance liquid chromatographic (2D-HPLC) system with narrowbore reversed-phase and microbore enantioselective columns has been developed for the determination of alanine (Ala) and serine (Ser) enantiomers. The amounts of D-amino acids in biological samples are extremely low in most cases and improvement in the sensitivity is still required. For a highly-sensitive analysis, a narrowbore reversed-phase column, KSAARP (1.5 mm x 500 mm, having a wider ID than the microbore column) was used in the first dimension, and a microbore enantioselective column, KSAACSP-001S (1.0 mm x 250 mm, having a narrower ID than the frequently reported enantioselective columns) was used in the second dimension. The amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and detected by the fluorescence detectors. The 2D-HPLC system was validated using a mouse cerebrum in addition to the standard amino acids, and satisfactory calibration lines and precision results were obtained. In the mouse cerebrum, a large amount of D-Ser and a small amount of D-Ala were clearly observed. These results indicated that the present method is applicable to the determination of the trace amounts of D-amino acids in tissue samples.
A fine fibrous polyimide (PI) material was introduced as a stationary phase in packed capillary gas chromatography (GC). Packed longitudinally into a capillary, the bundle of the PI filaments exhibited retentivity for a set of test analytes, showing that the PI-packed column was able to work as a stationary phase in GC. On the basis of a systematic consideration of the retention factors for homologous alkanes, it was confirmed that a good linear relationship between the carbon number and the logarithmic retention factor for alkanes at a constant column temperature. Linear van't Hoff plots were also obtained for alkanes, where logarithmic retention factors were plotted against the reciprocal absolute column temperatures. On the basis of the tendency in the retention factors of homologous alkane test analytes, the PI stationary phase has a trend for the retention behavior similar to that of conventional GC stationary phases widely employed, although a certain contribution of polar functional groups in the PI stationary phase to the retention of alcohols was observed. Taking advantage of the good heat-resistance of the PI material, an applicability to temperature-programmed GC separations was also suggested for the fibrous PI stationary phase.
A novel polymer-based particles was introduced as a stationary phase in liquid chromatography (LC). Spherical particles of a copolymer, poly(benzoguanamine-co-formaldehyde) (BF), was packed into a conventional blank LC column and the retention behavior of polycyclic aromatic compounds (PACs) was evaluated in reversed-phase LC. The trend for the retention was compared with that obtained on commercially-available octadecylsilica (ODS) and phenylbutylsilica (PBS) phases. In terms of the trend on the retentivity for PACs, the BF phase showed a similar behavior as that on the ODS phase, although the selectivity to some critical pairs was somewhat different from that on the ODS phase. The results suggest that the BF phase has a retentivity basically on the basis of hydrophobic interaction between the stationary phase ligands and the solute molecule, however, at the same time, the BF phase has a different selectivity from the PBS phase for the recognition of PACs molecules.On the basis of the comparison with the PBS phase, the unique selectivity of the BF phase can be attributed to the restricted moving of the phenyl ligands.