In recent years, innovative pharmaceuticals such as nanotechnology-based pharmaceuticals (nanomedicines) and mid-sized pharmaceuticals have been developed. In particular, nanomedicines have contributed significantly to the rapid dissemination of COVID-19 vaccines and may continue to be an essential formulation technology for the development of vaccines against various infectious diseases and cancers. Because chromatography is a major analytical technique for pharmaceuticals, it is important to develop a high-performance separation method for innovative pharmaceuticals with unconventional characteristics using this technique. We have promoted research focused on the development of high-performance separation and analysis methods for nanomedicines and mid-sized pharmaceuticals using chromatography. In this review, the author presents our related research achievements on: (1) the development of a size separation method for nanoparticles using monolithic capillary columns, (2) the development of a size separation method for liposome formulations using monolithic capillary LC, (3) size exclusion chromatography (SEC)-multi-angle light scattering (MALS) detector for block copolymer micelle characterization, (4) development of a highly sensitive method for measuring concentrations of the anticancer drug doxorubicin and its metabolites in biological samples, (5) development of a high-resolution analytical method for cyclosporine.

In high performance liquid chromatography (HPLC), packing materials based on porous silica gel are commonly used. Among them, ODS (OctaDecylSilyl)-modified silica gel, in which the silica-based material is chemically modified with octadecylsilyl groups, is the most widely used reverse-phase column due to its versatility in separating (bio)chemical compounds through hydrophobic interactions. The retention can be controlled by adjusting the ratio of organic solvents and aqueous solutions (typically with a pH range of 2–8) in the mobile phase. This principle is widely recognized and applied by chromatography researchers and practitioners. In contrast we have developed a novel concept of chromatography by surface-modifying the base of packing material with functional polymers. This review focuses on temperature- and pH-responsive chromatography using temperature- and pH-responsive polymers among stimuli-responsive polymers, as well as alkali-resistant column packing materials, which are developed by modifying the surface of porous calcium carbonate particles inspired by biominerals with amphiphilic polymers. First, the principles of elution control in 100% aqueous solutions through external stimuli (temperature and pH) in temperature- and pH-responsive chromatography, as well as its application to the separation of small molecules and proteins are discussed. Subsequently, it introduces the development of novel packing materials by electrostatically adsorbing poly(maleic acid-alt-1-octadecene) (PMAcO) onto the surface of synthetic porous calcium carbonate microspheres and eggshell powder, and their application to the separation and preparative purification of basic compounds using alkali mobile phases.

Ion mobility spectrometry (IMS) has become an analytical technique for rapid chiral discrimination, offering significant potential to accelerate various studies such as drug discovery, clinical diagnostics, and bioimaging. This review highlights recent advancements in chiral discrimination using IMS, based on studies published from 2006 to 2024. The methods are categorized into three main approaches: (1) direct separation in a chiral environment, (2) non-covalent complexation, and (3) diastereomeric separation using a covalently bonded chiral tag (chiral derivatization). For each approach, landmark studies are discussed with emphasis on critical notes such as selection of the additive and the ion form for efficient separation. Subsequently, representative applications of chiral derivatization to asymmetric catalyst development, rapid bioanalysis, and biomolecular imaging are explained in detail. The enantioselective imaging of chiral metabolites is more challenging than others but attracts growing interest from biologists and analytical chemists, making the description regarding on-tissue charged chiral derivatization, which improves ionization efficiency and separation of analyte, particularly relevant. Independent studies have demonstrated that IMS, under optimized conditions, can achieve performance comparable to high-performance liquid chromatography (HPLC) in determining enantiomeric excess. Considering higher throughput and simplicity of IMS over HPLC, further instrumental and/or chemical advancements would make IMS a more versatile technique for chiral discrimination.

Photostability and chiral stability of d-chlorpheniramine maleate (d-CPM, S (+)-form), which is an old but clinically useful antihistamine, were investigated by HPLC with an ovomucoid (OVM) or a β-cyclodextrin (β-CD) derivative immobilized chiral columns. Enantiomers of chlorpheniramine maleate (dl-CPM) were successfully separated by an OVM column with resolution (Rs) 3.2 within 10 min under the reversed-phase mode. A β-CD immobilized column was also useful for the separation of enantiomers. Using validated chiral HPLC methods, photostability and chiral inversion study of d-CPM drug substances, its various formulations (syrups, tablets, injections, and powders) under UV irradiation and heating stress conditions were performed in addition to its quality evaluation. As a result of quality evaluation, large content (1-6%) of the minor enantiomer, l-CPM was detected in both d-CPM drug substances and its formulations. Especially ca. 6% l-CPM, which was the largest among tested formulations, was observed in injections. This may be due to its production process (probably sterilization by heat). In photostability studies, maleic acid (cis-form) in d-CPM was converted to fumaric acid (trans-form) in all formulations including the solid type and aqueous solutions of d-CPM, although chiral inversion was not observed under UV irradiation. On the other hand, under the heating conditions, chiral inversion of d-CPM was observed in all formulations and aqueous solutions of d-CPM with an increase of heating temperature and heating time, in addition to cis-trans rearrangement of maleic acid. An increase of l-CPM (chiral inversion) was also observed in d-CPM syrups, powders and tablets stored at the room temperature for one year, showing d-CPM is relatively easily chiral inverted by heat.

A method was developed for the on-line derivatization of polysaccharides with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) using a twisted nylon monofilament mold microchip. We embedded a twisted nylon monofilament in a polydimethylsiloxane (PDMS) solution to fabricate a twisted channel. We utilized a polystyrene case with 2 holes and inserted twisted nylon monofilament sutures through these holes. The PDMS prepolymer was poured onto the case and heated. After cooling the PDMS, we peeled the PDMS from the case and cut the PDMS to generate a twisted microchannel and other reservoirs. The two PDMS layers were then exposed to oxygen plasma. Under this manufacturing condition, the two sutures intertwine to form microchip microchannels, resulting in a non-uniform inner diameter and efficient solution mixing. We used maltopentaose as a model sample to optimize the flow rate, reaction temperature, and number of times the sample passed through the microchannel. These conditions were also used for on-line fluorescent labeling of isomaltooligosaccharides. By repeatedly pumping liquid through this twisted microchannel, the reductive amination of saccharides could be achieved in a short period of time.

Amino acids play essential roles in mammals, and their analysis in biological samples can provide valuable physiological insights. Recently, interest in health management has expanded, encompassing humans and companion animals. This study aimed to develop a high-performance liquid chromatography-fluorescence method to analyze plasma amino acids in cats—the most popular companion animals. Unlike humans, cats cannot synthesize taurine, an essential biomolecule for their health that must be acquired through external dietary sources. We developed a method for the simultaneous analysis of taurine and amino acids. 4-Fluoro-7-nitro-2, 1, 3-benzoxadiazole was used as a fluorescence derivatization reagent owing to its advantages, such as the formation of stable derivatives and high reaction efficiency. The separation conditions were optimized, and 16 amino acids, including taurine, were successfully separated. The developed method demonstrated good validation data in amino acid standards and cat plasma samples and was applied to assess cat plasma samples obtained from healthy cats and those with a history of ureterolithiasis. The plasma concentrations of glutamic acid, taurine, and citrulline were significantly elevated in cats with a history of ureterolithiasis. The analytical method developed in this study could be valuable for exploring the relationship between plasma amino acids and cat health.