Aloe species plural (Aloe spp.) is a perennial succulent plant having various pharmacological activities such as laxative, antibacterial and anti-inflammatory effects, etc. Among more than 400 species are known, A. arborescens and A. vera are mainly used in foods and cosmetics. Health food products prepared from Aloe spp. may naturally contain their components at high concentrations, while some of them lack the ingredient display. Aloe components are bioactive so that a simple separation method for each component is required to prepare at high purity. In the present study, the systematic separation method of the ingredients containing in commercially available crude aloin was developed using countercurrent chromatography based on liquid-liquid partitioning. Aloesin and aloenin were separated using an organic-aqueous two-phase solvent system composed of ethyl acetate/1-butanol/water (3 : 2 : 5, v/v) with the lower mobile phase. And then, after collected and concentrated the upper stationary phase, aloe-emodin and other anthraquinones containing barbaloin and isobarbaloin were next separated using n-hexane/ethyl acetate/methanol/water (7 : 3 : 7 : 3, v/v) with the lower mobile phase. Furthermore, barbaloin and isobarbaloin were successfully separated using ethyl acetate/1-butanol/water (45 : 5 : 50, v/v) with the lower mobile phase. The overall results suggest that five components can be systematically separated from crude aloin at high purity using a simple preparation procedure with countercurrent chromatography by selecting suitable two-phase solvent systems.

L-Carnitine (CRN) and acetyl-L-carnitine (AcCRN) play several important roles, primarily energy production, and the ingestion of sufficient amounts of them is essential for human health. Rice bran (RB) fermented with Aspergillus oryzae (A. oryzae) is expected to be a CRN- and AcCRN-rich plant-based foodstuff. To prove this concept, a liquid chromatography (LC)/electrospray ionization-tandem mass spectrometry method was developed for measuring the CRN and AcCRN contents in the A. oryzae-fermented RB product. The derivatization using 1-aminonaphtharene was employed to enhance the LC retention of the analytes, which led to negligible matrix effects, satisfactory precision (all intra- and inter-assay relative standard deviations, ≤ 8.7%) and accuracy (all analytical recoveries, 98.2–102.7%). The CRN and AcCRN contents in the RB product after the fermentation for 46 h were 101 ± 8 and 23.8 ± 10.2 µg/g (mean ± standard deviation, 5 lots), which were tens and hundred times greater than those in the unfermented RB material (1.43 ± 0.09 and 0.0519 ± 0.0041 µg/g, respectively). Thus, the A. oryzae-fermentation was an effective approach for enriching the RB-based material with CRN and AcCRN, and the resultant product could be one of the most CRN- and AcCRN-abundant plant-based foodstuffs.

For the highly-selective analysis of lactic acid (LA), 2-hydroxybutyric acid (2HB), 3-hydroxybutyric acid (3HB) and malic acid (MA) enantiomers in human physiological fluids, a three-dimensional (3D) HPLC method following the fluorescence derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole has been designed and developed. The 3D-HPLC system was composed of reversed-phase, mixed-mode and enantioselective columns, and the conditions of all the dimensions were thoroughly investigated. In the first and second dimensions, the target hydroxy acids were separated from most of the interfering substances in the clinical samples. In the third dimension, the enantiomers were separated with resolution values higher than 3.47. The present method was validated by checking the calibration curves, precision and accuracy using standard solutions and human samples, and satisfactory results were acquired. Using the 3D-HPLC system, D-LA, L-LA, D-3HB, L-3HB and L-2HB were determined in the human plasma and urine (MA enantiomers were not determined). The concentrations of the detected enantiomers in the human plasma were 6.2, 660, 34.0, 1.0 and 19.0 µM, and those in the human urine were 13.4, 88.3, 4.5, 3.7 and 4.5 µM, respectively.

Direct enantiomer separation of denopamine (DP, active form: R (-)-form), which is an optically active and an orally administrable cardiotonic agent, was investigated by HPLC with β-cyclodextrin (β-CD) immobilized chiral stationary phases (CSPs). Successful enantiomer separation under the reversed-phase mode was achieved by a β-CD having a phenyl moiety immobilized CSP with large resolution within 10 min. Optical purity testing and assay of tablets by the HPLC method developed were validated for specificity, linearity, and recovery. Validation results indicate the method is accurate and has a good linearity and sensitivity. Limit of detection and limit of quantitation of the minor enantiomer were 0.01% and 0.03%, respectively. Assay of DP tablets 5 mg by the internal standard method was also successfully achieved within 10 min. Further, photostability of DP was evaluated by this chiral HPLC method and the reversed-phase HPLC with an ODS column. Chemical structures of two photoproducts generated in an aqueous media were determined by LC-MS/MS, showing these were p-hydroxybenzaldehyde and 3,4-dimethoxyphenethylamine. Other than these two main photoproducts, low level (0.17%) of the minor enantiomer was detected.

We have developed a comprehensive method for analyzing gene expression levels by combining the fluorous-fluorescence hybrid probes, oligonucleic acids modified with a perfluoroalkyl tag and fluorescent probes, with the fluorous liquid chromatography (LC) – fluorescence detection. When Taq polymerase chain reaction (PCR) was performed using this probe, fluorescent mononucleotides were released according to the amount of template gene targeted. Because each probe differs in the linker length of the fluorescent probe and the type of nucleobase, multiple gene expression levels can be analyzed by LC as the amount of fluorescent mononucleotides produced. The excess unreacted probe, which did not undergo the Taq PCR reaction, was selectively removed by adsorption in the fluorous LC column introduced as a pretreatment column. The method established in this study allows simultaneous LC quantification of three housekeeping genes. In addition, LC separation conditions for the simultaneous analysis of nine gene expression levels were established, and comprehensive detection of cytokine genes in lipopolysaccharide-stimulated leukocytes was successfully achieved.

A simple and sensitive method for determining flavor compounds in foamable liquid samples was developed using the purge-and-trap method with the addition of glass wool and a functional porous polytetrafluoroethylene (PTFE) membrane. Flavor compounds purged from sample solutions were trapped by a needle-type extraction device. The functional PTFE membrane, which retained the aqueous sample while allowing purge gas to pass, was fixed on a glass solid-phase extraction cartridge. Hence, purge gas was continuously introduced into the sample solution from the bottom of the cartridge. To trap the foam, 0.4 g of glass wool was fixed in the sample headspace, and headspace gas was collected using a needle-type extraction device placed on the upper side of the wool. During the purge process, although foam climbed through the cartridge, it was adsorbed on the glass wool. However, the flavor compounds were not adsorbed on the inactive glass wool surface as confirmed in this study. This method does not require the dilution of sample solutions or the adding of antifoam agents to sample solutions. This simple and inexpensive construction enables the sensitive detection of flavor compounds.

A new analytical method for the quantification of volatile polycyclic aromatic hydrocarbons (PAHs) in soil samples using a needle-type extraction device was developed. Naphthalene (Nap), acenaphthylene (Acy), acenaphthene (Ace), and fluorene (Flu) were investigated as analytes. Samples were prepared by placing 10 g of soil in a glass cartridge and immersing the cartridge in a 75°C water bath. The needle-type extraction device packed with Carbopack X and carbon molecular sieve was inserted into the glass cartridge, and 300 mL of gaseous sample was collected. During gas sampling, N₂ was introduced into the cartridge through the tip of the cartridge. After collecting the sample, the extraction needle was inserted into a heated gas chromatographic (GC) injection port, and the extracted PAHs were thermally desorbed followed by the GC separation on a fused-silica capillary column. The limits of quantification for Nap, Acy, Ace, and Flu were 36, 36, 15, and 36 ng through flame ionization detector and 3.6, 7.5, 7.5, and 36 ng through mass spectrometry, respectively.

To improve the concentration sensitivity of cationic analytes in microchip electrophoresis, on-line sample preconcentration by a large-volume sample stacking with an electroosmotic flow pump (LVSEP) was performed using non-aqueous background solutions (BGSs) in cationic polymer-coated microchannels. In the LVSEP analyses using dimethyl sulfoxide-based BGS, however, poly(vinyl alcohol)/poly(allylamine) mixture-coated microchannel gave insufficient enrichments and broader peaks for cationic histamine. By employing a methanolic BGS containing (hydroxypropyl)methyl cellulose, on the other hand, good enrichments of histamine were achieved by LVSEP with the sensitive enhancement factor of 67. To our knowledge, this is first time that LVSEP analyses of cationic analytes were successfully realized in non-aqueous media.

A novel core-shell ion-exchange resin (St-70) having a core-shell monomer weight ratio of 30:70 was prepared for use in highperformance liquid chromatography (HPLC), and the effect of the degree of cross-linking (10–55%) of the porous shell on the separation of carbohydrates was examined. A mixture of inositol, glucose, fructose, and sucrose was separated under alkaline conditions (100 and 150 mmol/L NaOH) at flow rates of 0.3–0.7 mL/min using St-70, which exhibited a degree of cross-linking of 10, 40, and 55%. The retention time of carbohydrates decreased with increasing of the degree of cross-linking in the porous shell. The theoretical plate number (N) of glucose, fructose, and sucrose decreased with decreasing the degree of cross-linking when 100 mmol/L NaOH eluent was used at flow rates of 0.3 and 0.5 mL/min.