Chiral amino acid analysis, especially the determination of trace levels of D-enantiomers, is currently gathering attention in a variety of research areas including the food/clinical sciences. These D-amino acids had long been believed to be absent in the higher animals. However, by the advances of analytical technologies, some of the D-enantiomers are found in mammals including humans and increasingly recognized as novel physiologically-active substances and/or biomarkers. For the determination of these D-amino acids and related compounds in real world samples, utilization of sensitive and selective methods is essential and multi-dimensional HPLC is one of the straightforward approaches. In the present review, two/three-dimensional HPLC methods and biological/medical applications focusing on our current studies are summarized.

Niemann–Pick disease type C (NPC) is an autosomal recessive disorder that features progressive neurodegeneration. Because NPC patients have mutations in NPC1 or NPC2 cholesterol transporting proteins, failures in cholesterol and other lipid trafficking occur. NPC patients represent a wide clinical spectrum in terms of age of onset and type of symptoms. Because conventional diagnostic methods are time-consuming and complicated, biomarker tests have attracted increased attention. In this review, recently reported biomarkers for NPC are discussed in detail. For example, oxysterols and some cholenoic acid conjugates, metabolized from excess cholesterol in NPC cells, can be detected in urine or plasma. In addition, two types of lysosphingolipids, sphingosylphosphorylcholine and glycosylsphingosine, are present at increased concentrations in NPC patients. A more recently discovered biomarker is N-palmitoyl-O-phosphorylcholine, previously called “lysosphingomyelin-509.” These biomarkers are helpful for the early diagnosis of NPC and may contribute to a good prognosis for NPC patients.

Microfluidic device is capable of reducing amount of sample consumption, shortening the analysis time, and miniaturizing instruments. Many applications of microfluidic devices have been developed not only in chemical analysis but also in medical diagnosis, and the food and agricultural industries. Electrophoresis or chromatography in a microfluidic device has improved the speed, reproducibility and separation resolution. Some of them have been integrated with complex experimental functionality on a small substrate, of which concept is known as micro total analysis systems. To build up a system, microfluidic components that carry out an experimental functionality in a microfluidic channel and novel technology to support the developments of microfluidic components are indispensable. In this review, three-dimensional (3D) fabrication by thiol-ene quick reaction, sample injection with an inkjet ejector, and molecular detection based on electroosmosis are focused briefly. The 3D fabrication realizes to make various 3D microstructures that conventional fabrication method cannot make without specific equipment. The sample injection by using inkjet technology can apply tiny amount of sample solution into multiple microfluidic channel on some precise spots, which leads to rapid analysis with high accuracy. The electroosmosis-based molecular detection indicates a possibility to develop a new portable device without any peripherals for detection or pretreatment for specimen labeling. Abstract Microfluidic device is capable of reducing amount of sample consumption, shortening the analysis time, and miniaturizing instruments. Many applications of microfluidic devices have been developed not only in chemical analysis but also in medical diagnosis, and the food and agricultural industries. Electrophoresis or chromatography in a microfluidic device has improved the speed, reproducibility and separation resolution. Some of them have been integrated with complex experimental functionality on a small substrate, of which concept is known as micro total analysis systems. To build up a system, microfluidic components that carry out an experimental functionality in a microfluidic channel and novel technology to support the developments of microfluidic components are indispensable. In this review, three-dimensional (3D) fabrication by thiol-ene quick reaction, sample injection with an inkjet ejector, and molecular detection based on electroosmosis are focused briefly. The 3D fabrication realizes to make various 3D microstructures that conventional fabrication method cannot make without specific equipment. The sample injection by using inkjet technology can apply tiny amount of sample solution into multiple microfluidic channel on some precise spots, which leads to rapid analysis with high accuracy. The electroosmosis-based molecular detection indicates a possibility to develop a new portable device without any peripherals for detection or pretreatment for specimen labeling.

An enantioselective analytical method for the determination of synephrine, an ingredient in health food, by liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) was developed. By derivatizing synephrine with FMOC and using TCI Chiral MB-S as the chiral column, FMOC-synephrine enantiomers were well separated. The electrospray ionization (ESI) negative mode was adopted for TOF-MS measurement. Good linearity (r > 0.999) was obtained in the concentration range of 0.5 to 50 μg/mL. The limit of detection (LOD, S/N = 3) and the limit of quantification (LOQ, S/N > 10) were 0.25 μg/mL and 0.5 μg/mL, respectively. The intra-day and inter-day accuracy of synephrine enantiomers at the LOQ level (0.5 μg/mL), the intermediate concentration level (10 μg/mL), and the high concentration level (50 μg/mL) ranged from 90.0 to 107.4%. The intra-day and inter-day precisions were ≤ 9.24% and ≤ 10.63%, respectively. As a result of analyzing synephrine-containing health foods using this method, approximately half of the products showed high optical purity with 80–100% enantiomeric excess of the l-isomer. In contrast, nearly 20% of the products contained racemate with 0–50% enantiomeric excess, and variations in optical purity were observed for the products. It was speculated that the l-isomer could be converted into the d-isomer during the manufacturing process.

Quantification of four extracellular matrices commonly found in foods with functional claims was conducted using capillary zone electrophoresis with direct ultraviolet (UV) absorbance detection at 200 nm. The four polymeric compounds, namely, proteoglycan, chondroitin sulfate, hyaluronic acid, and collagen, were separated with 100 mM borate buffer (pH 10.0) using capillaries coated with poly-N,N-dimethylacrylamide on the inner wall, which almost completely suppressed the electroosmotic flow to give reproducible migration times for the analytes. The addition of 5 mM sodium dodecyl sulfate to the running solutions improved the shapes of the peaks and the linearity of the calibration curves. Some commercial products, including the extracellular matrices under investigation, were analyzed with the method developed.

To determine the fate and behavior of pharmaceuticals in the aquatic environment, in this study, we investigated the photo-conversion of phenytoin (PH), which is one of the antiepilepsy drug, induced by ultraviolet light (UV) irradiation in the aqueous media. The degradation of PH and the generation of its photoproducts were monitored by means of high-performance liquid chromatography (HPLC). Based on the fragmentation patterns in electrospray ionization time-of-flight mass spectrometry (ESI-TOF/MS/MS) and coupling patterns in nuclear magnetic resonance (NMR) spectrum, the main photoproduct of PH was determined as benzophenone. Evaluation of toxicity by means of luminescent bacteria test (ISO11348) indicated that a PH solution was nontoxic, whereas a UV-irradiated PH solution and a benzophenone solution were toxic (EC₅₀: 19.46 mg/L and 24.40 mg/L). It was shown that benzophenone has a contribution to the increase of ecotoxicity of PH solution for luminescent bacteria after UV irradiation. This is the first report of the photo-conversion of PH to benzophenone induced by UV irradiation in the absence of photo-catalysts. These results indicate that the importance of evaluating not only parent compounds but also their photoproducts for the risk assessment of pharmaceuticals in the aquatic environment.

A pillar array column with a perfectly ordered internal structure has a higher separation efficiency than a particle-packed column used in conventional high-performance liquid chromatography. However, applying the pillar array column technology to quantitative analysis is challenging as the injection volume of the sample varies for each injection. This occurs because the sample volumes are in the order of nanoliters and the sample is injected manually. In this study, an automated sample injection system was developed to solve this problem. The system was composed of two pumps (one for the sample and other for the mobile phase), a six-way valve that can be controlled by a PC, and an autosampler. The peak height deviation, which is more than 20% in the conventional manual injection system, was improved to 1.2% and 0.4% for the two coumarin dye samples. This result indicated that the automated sample injection method developed in this study could be applied to pillar array columns to allow for quantitative analysis.