High-performance liquid chromatography (HPLC) and electrophoresis are liquid phase separation methods and are frequently used in various research fields. Although their origins are sufficiently old, the development of novel separation methods based on HPLC and electrophoresis are still required for both fundamental and applicational functions. In this paper, we introduced our fundamental studies of the development of novel analytical methods based on HPLC and electrophoresis toward enhancing separation efficiency. The contents can be categorized as follows: (1) enhancement of separation in pressurized flow-driven capillary electrochromatography, (2) orthogonal electrochromatography for simultaneous two-dimensional separation, (3) development of new stationary phases/columns in HPLC, (4) development of low and ultra-low-temperature HPLC using liquefied gas mobile phase, (5) development of nonaqueous capillary electrophoresis for analyzing water-insoluble synthetic polymers, and (6) development of frequency division multiplex HPLC-MS.

Photo-exposure has a crucial effect on both the quality and quantity of photosensitive pharmaceuticals due to the decrease of their contents or the expression of adverse effects derived from generated photoproducts. The photostability test guidelines (ICH-Q1B, Photostability testing of new drug substances and products) have been established for the photostability evaluation in the new drug application, and obtained results from these tests are required for the elucidation of the final packaging and the dosage form. However, there is no information in the interview form about the photostability of medicines that are taken out from a press through package (PTP) sheet and their dosage forms are changed for the purpose of the improvement of medication compliance of patients. It is an important issue to clarify the photostability of medicines that are crushed or suspended and determine the potential photoproducts for safe use. Herein, in this review, results of the evaluation of the photodegradability of photosensitive active pharmaceutical ingredients (APIs) and their formulations, the determination of several photoproducts generated by ultraviolet-light (UV) irradiation and the development of photostabilization strategy for them based on the addition of several photostabilizers are summarized.

Three different types of analytical methods for D,L-amino acid were developed. First, a method for simultaneous analysis of D,L-amino acids in food was constructed by combining pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), UHPLC separation, and UV and circular dichroism (CD) detection. Using this method, D,L-amino acids in food samples was determined at a very short time of 5.5 minutes. Second, a pre-column derivatization LC/MS method (direct method) for D-alanine, D-serine, and D-aspartic acid and L-isomers in biological samples was developed. In this method, D,L-amino acids were derivatized with an achiral reagent, p-N,N,N-trimethylammonioanilyl N′-hydroxysuccinimidyl carbamate iodide (TAHS), separated with a chiral column, and detected using mass spectrometry (MS). Third, a pre-column derivatization LC/MS method (indirect method) for highly sensitive and selective analysis of 20 D,L-amino acids using a chiral derivatization reagent, (R)-4-nitrophenyl-N-[2-(diethylamino)-6,6-dimethyl-[1,1-biphenyl]-2-yl] carbamate hydrochloride ((R)-BiAC), was developed. The unique structure of the (R)-BiAC reagent, which has a biaryl axially chiral structure, allowed for higher resolution and a high sensitive analysis of the 20 D,L-amino acids within 12 minutes. This method has been applied to various D-amino acid research studies, and it can be applied to a wide range of analyses from food to biological samples. Lastly, an LC/MS method was established that simultaneously analyzes tryptophan and 15 of its metabolites, which is involved in neurotransmission and immunity in vivo.

Equipment-free detection devices are attractive for point-of-care analysis, especially in resource-limited regions. Distancebased detection is the measurement of color change along the detection path related to the concentration of analytes. Hydrogels are three-dimensional crosslinked polymer networks with tunable properties and may be suitable for distance-based sensors. However, only small ions have been used as the targets in the previous studies. In this work, we demonstrated the possibility of using hydrogel as the distance-based measurement of large biomolecules such as proteins. The hydrogel was prepared with poly(ethylene glycol) diacrylate and a fluorescein derivative as the fluorescent detection probe. The fluorescent alteration in the presence of a target protein, trypsin, can be observed in both intensity-based and distance-based detection. It was found that the effect of the concentration of a fluorescent reagent was not significantly impacted in distance-based detection, while it is essential in intensity-based detection. The relationship between the distance change and the trypsin concentration in the range of 0.5 – 5 mM can be observed. This result showed the possibility of using hydrogel for distance-based detection of large biomolecules.

Helium is an inactive gas that has been used as a carrier gas in gas chromatography (GC). Due to a recent helium shortage of last several years, a method transfer from GC to high-performance liquid chromatography (HPLC) has been increasingly considered. Here, camphor and menthol, which are used in many pharmaceutical products and have been widely analyzed by GC, were determined by reversed-phase liquid chromatography (LC) coupled with electrospray ionization single quadrupole mass spectrometry (ESI/MS). An excellent linearity was confirmed in the concentration range of 0.5–500 µg/mL for camphor and 1–1000 µg/mL for menthol. Diluted, commercially available eye drops were analyzed with the developed LC-MS method, where both of these compounds were successfully quantitated with a good precision as well as excellent accuracy. The developed LC-MS method can be applied as a promising alternative to the analysis of similar classes of volatile compounds in GC.

A B6DAO^-/- mouse strain lacking D-amino acid oxidase (DAO) activity with the genetic background of C57BL strain, frequently used to produce disease model mice in the current medical/life science areas, was newly developed. In the plasma of the control C57BL/6J and mutant B6DAO^-/- mice, five D-amino acids widely found in mammals were determined using a highly-selective two-dimensional LC-MS/MS system. As the targets, D-alanine (Ala), D-aspartic acid (Asp), D-leucine (Leu), D-proline (Pro) and D-serine (Ser) were selected. All five chiral amino acids were clearly observed, and the D-enantiomers of the neutral amino acids (Ala, Leu, Pro and Ser) significantly increased in the mutant B6DAO^-/- mice (3.69-36.91 nmol/mL) compared to those in the control C57BL/6J mice (0.24-2.03 nmol/mL), whereas the amounts of D-Asp were almost equal between the two strains. The obtained values are consistent with those in previous reports using ddY/DAO^-/- mice having spontaneous DAO deficiency and the control ddY/DAO^+/+ mice. These results indicated that the newly-developed B6DAO^-/- strain is practically useful to clarify the controlling mechanism of intrinsic D-amino acids that are expected as new bio-functional molecules and/or biomarkers in mammals.