Liquid chromatography (LC) is an essential separation and analytical technique in the pharmaceutical sciences, but despite advances in LC separation technology and mass spectrometer performance, there are still many analytes and applications that require higher selectivity and sensitivity. We have developed new LC-based analytical methods for optically active substances and therapeutic monoclonal antibodies (mAbs) for use in pharmaceutical sciences by introducing unique technologies. In this review, the author presents our LC related research achievements on (1) dress-up chiral columns: new removable chiral stationary phases for enantioseparation of optically active substances, (2) development of a derivatization-LC analytical method for chiral compounds using the condensing agent DMT-MM as an enantioseparation enhancer, (3) sensitive and comprehensive LC-MS/MS analyses of chiral pharmaceuticals and their hepatic metabolites using ovomucoid column, (4) development of methods for intact bioanalysis of therapeutic mAbs, (5) development of LC-based bioanalytical methods using anti-idiotype DNA aptamers as capture ligands, (6) development of a versatile DNA aptamer acquisition method for therapeutic mAbs using LC.

Due to the recent discoveries of several D-amino acids in mammals and also by the elucidation of their physiological functions and/or availabilities as diagnostic markers, the enantioselective analysis of intrinsic amino acids has been the focus of attention. Based on the determination of amino acids with chiral discrimination, the clarification of their distributions, regulation mechanisms, physiological functions and the diagnostic/therapeutic values of a wide variety of D-amino acids is expected. Although various analytical techniques have been reported, the improvement of the selectivity is still in demand especially for the accurate determination of trace levels of D-amino acids in complicated biological matrices. One of the straightforward approaches to address the selectivity issue is the multi-dimensional LC analysis in which several separation modes are combined. In the present review, the two-dimensional (2D) LC methods in combination with the detection using MS/MS are introduced and the applications to various biological samples are summarized. The 2D LC-MS/MS methods were designed by integrating a reversed-phase purification (first dimension), an enantioselective separation (second dimension) and the detection of specific precursor/product ion pairs of analytes using MS/MS. Using these methods, the precise and simultaneous determination of D-alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), leucine (Leu), proline (Pro) and serine (Ser) in human plasma and urine was carried out. The in vivo regulation of D-Ala, Leu, Pro, Ser and tryptophan (Trp) amounts by D-amino acid oxidase (DAO) was clarified by analyzing their concentrations in the plasma and urine of DAO deficient mice. For the accurate analysis of D-amino acid residues in proteins, the hydrolysis using deuterium chloride/deuterium oxide and 2D LC-MS/MS determination were combined, and the stereoinversion of the Ala, Asp, Glu, Pro and Ser residues was evaluated in several proteins.

Aqueous solutions of platinum nanoparticles (PtNP) and silver nanoparticles (AgNP) were prepared by a solution plasma process in the presence of hydrogen peroxide. Both nanoparticle solutions did not show visible-range photo-absorption by surface plasmon resonance, and capillary zone electrophoresis (CZE) was utilized for the characterization of the nanoparticles. An anionic broad peak was detected with the PtNP by the CZE analysis, whereas both an anionic and an electrically neutral peaks were detected with the AgNP. Serious shot signals attributed to the agglomerate of the nanoparticles were not detected with either the PtNP or the AgNP, and the nanoparticles were stably dispersed in an aqueous solution for at least a few tens of week. Dispersion stability of the nanoparticles was also evaluated in salt solutions, as well as with ethanol co-solvent.

Wound exudate is produced during the healing process and plays a crucial role in natural defense and repair mechanisms. Proper wound management is necessary by monitoring exudate levels. Amino acid concentrations in wound exudates are altered under wound conditions during the wound-healing process. Thus, analysis of amino acids in wound exudates is useful for determining wound conditions. In this study, we developed a validated analytical method for amino acids in rat wound exudates by high-performance liquid chromatography (HPLC)-fluorescence detection using the fluorescence derivatization reagent, 2,3-naphthalenedicarboxaldehyde (NDA). Chromatographic separation of 18 NDA-amino acids and 5-aminovaleric acid (internal standard) was achieved within 100 min on an octadecylsilyl column under gradient elution using an eluent consisting of water, acetonitrile, and trifluoroacetic acid. The calibration curves were linear (0.25–25 pmol/injection) and exhibited high correlation coefficients (r2 > 0.998) for each amino acid. The developed method was applied to a wound exudate sample and 18 amino acids were quantified. The amino acid concentrations in the wound exudate samples were 24.2–1694 µM. This method was successfully applied to amino acids quantification in wound exudates, showing its potential for clinical application.

In this study, a novel cryogenic high-performance liquid chromatography (HPLC) system, where the separation temperature can be controlled from -196 to -50 °C, was developed using liquefied propane as the mobile phase. Pyrene retention in a bare silica-gel column increased as the column temperature decreased, ranging from -130 to -50 °C. The linear van’t Hoff plot for pyrene (r² = 0.9926) suggested that a variation in the retention mechanism for pyrene did not occur in this temperature range. The separation of pyrene and pyrene-d₁₀ was enhanced by the decrease in the separation temperature in the bare silica-gel column, that is, the H/D selectivity increased with a decrease in temperature. The H/D pyrene was not separated in an ODS column even though the cryogenic HPLC at -90 °C. This suggested that hydrogen bonding between H/D pyrene and hydroxy groups in the mobile phase chemicals is essential for H/D separation in reversed-phase HPLC.