Miniaturization is a key technological approach to realize high performance separation systems that have several important features required in modern analytical methods. A smaller scale of the stationary phase synthesis enables a systematic analysis of the retention behavior and also a more efficient development of novel stationary phases in liquid chromatography. Development of microscale sample preparation technique has been regarded as a valuable supporting approach to the development of microscale separation methods. Effective on-line coupling of the microscale sample preparation and separation allows an efficient and rapid analysis without a loss of sensitivity for most of the analytical separations. In this review, development of the miniaturized separation techniques and the applications have been described from the view point of the authors' research group, including the development of novel stationary phase, microscale sample preparation techniques for liquid and gas sample matrices.

We developed a rapid and simple UHPLC-UV method for quantitative determination of erlotinib and its metabolite, OSI-420, in order to monitor their serum levels in patients with non-small cell lung cancer (NSCLC). Erlotinib and OSI-420 were extracted from 100 μL of human serum by liquid–liquid extraction using t-butyl methyl ether. The analytes were separated on Inertsil ODS-3 (100 mm × 2.1 mm I.D., 2 μm) as an analytical column using 20 mM potassium phosphate buffer (pH 2.5)/acetonitrile (74:26, v/v) as the mobile phase at a flow rate of 0.6 mL/min, and monitored at a UV wavelength of 345 nm. This method covered a linear concentration range of 6–6000 ng/mL for erlotinib and 6–2000 ng/mL for OSI-420, respectively (r > 0.999). The intra- and inter-day precisions of the analysis were < 6.8%, and the accuracy was ± 7.4%. This method has been successfully applied to measure erlotinib and OSI-420 in the serum of an NSCLC patient.

Single-walled carbon nanotube (SWCNT) was dispersed in an aqueous surfactant solution, and the dispersion of CNT was analyzed by capillary electrophoresis (CE). The dispersion was evaluated through a broad peak of the electropherograms,while the aggregation of the CNT was attributed to the shot signals in the electropherograms. Water-soluble nonionic polymer was also added in the separation buffer to examine the dispersion in the surfactant solution as well as to control the migration behavior. Polyethylene glycol, polyvinyl alcohol, and polyvinylpyrrolidone were examined. The electrophoretic mobility of the broad peak got smaller with increasing concentrations of the polymer examined. The theoretical number of plates of the broad peak was improved by the addition of the polymer. Moderately broad peak was preferable for the dispersion of the SWCNT in the surfactant solution.

A novel derivatization method for the selective and sensitive liquid chromatography (LC)-tandem mass spectrometric (MS/MS) analysis of polyamines (putrescine, cadaverine, spermidine, and spermine) was developed. In this study, to utilize the specific affinity between perfluoroalkyl compounds, called ‘fluorous’ affinity, two to four amino groups in each polyamine molecule were transformed with a relatively short perfluoroalkyl reagent, N-succinimidyl 4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluorononanoate to achieve the corresponding diperfluoroalkyl- to tetraperfluoroalkyl-derivatives. Compared with the non-fluorous compounds, the fluorophilicity of the multi-fluorous derivatives was sufficient for realizing strong retention on an LC column with a perfluoroalkyl-modified stationary phase. Furthermore, sensitive analysis of these derivatives could be performed using the multiple reaction monitoring mode in positive electrospray ionization-MS/MS. The limits of detection of the polyamines were in the range of 0.31–1.4 nM. The method was validated using human plasma samples. Although the recoveries from spiked human plasma after ultrafiltration were in the range of 66.4–103%, the derivatives could be determined without interference from matrix effects because of their selective retention on the column, which excluded non-fluorous biological matrix components, such as phospholipids. Therefore, this sensitive and selective analysis method was useful for the determination of trace amounts of polyamines in human plasma.

Temperature-responsive chromatography, in which the characteristics of the stationary phase can be controlled by varying the column temperature with only an aqueous eluent, was applied to the analysis of psychoactive drugs. Temperature-responsive poly(N-isopropylacrylamide) (PNIPAAm)-based copolymers were synthesized with n-butyl methacrylate (BMA) and N-acryloyl L-phenylalanine methyl ester (Phe-OMe) as a comonomer. These polymers were grafted onto aminopropyl silica and used as the stationary phase. Seven psychoactive drugs could be separated simultaneously by the column, including BMA. Among the seven drugs, for triazolam, which has a triazole ring in its structure, the order of elution changed on changing the column temperature. The phenomenon could be explained by NH–π interactions between the NH groups of PNIPAAm and π electrons of the triazole ring. The conformational changes of PNIPAAm altered the degree of exposure of the NH groups and would affect the elution order of the analytes. To enhance the molecular recognition ability for the triazole ring, the column containing Phe-OMe was used. Noticeable changes in the retention factors occurred due to the additional π–π interactions between the phenyl moieties of Phe-OMe and the triazole ring. The effects of the π–π interactions were also altered by the changes in column temperature. This chromatographic system, which needs no toxic organic solvent, offers an effective way to determine the causes of addiction in medical institutions.

The separation of neutral and water-insoluble linear synthetic polymers in non-aqueous capillary zone electrophoresis using the hydrophobic phosphonium-based ionic liquid trihexyl(tetradecyl)phosphonium chloride (P₆₆₆₁₄Cl) were investigated using polystyrene, polymethylmethacrylates, polybutadiene, and polycarbonate as test analytes. The solubility of P₆₆₆₁₄Cl in tetrahydrofuran was above 500 mM, which was significantly higher than that of the general ionic surfactants used in our previous studies. The separation of the four polymers was achieved using a binary mixture of tetrahydrofuran/methanol or tetrahydrofuran/acetonitrile containing P₆₆₆₁₄Cl as the electrophoretic medium. It was confirmed that an increase in hydrophobicity or bulkiness of the cationic reagent improved the polymer separation. The interaction between polycarbonate with P66614Cl was reduced by the addition of acetonitrile, and enhanced by the methanol addition.

An analytical method for quantifying branched-chain keto acids (BCKAs) - namely α-keto-isovaleric acid (KIV), α-keto-isocaproic acid (KIC), and α-keto-β-methylvaleric acid (KMV) - in cell extracts was developed and applied to a human chronic myelogenous leukemia cell line. Reversed-phase liquid chromatography was conducted for separation of the BCKAs after derivatization into fluorescent quinoxalines by reaction with o-phenylenediamine. The calibration curve was linear over the range from 0.5 to 50 μM. The precision values in intra- and inter-day assays were less than 3.1% and 5.6% (n = 4), respectively. Intracellular concentrations of KIV, KIC, and KMV in K562 cells were 47.6 ± 10.2, 312.4 ± 40.6, and 282.4 ± 71.6 pmol/1 x 10⁶ cells, respectively (n = 5 for each BCKAs). Furthermore, this method was applied to study the effect of gabapentin - an inhibitor of branched-chain amino acid aminotransferase 1 - on intracellular BCKA levels.

In this study, the application of a new type of monolithic silica column to the analysis of amino acids in human plasma samples has been investigated. The monolithic silica column (3.0 × 150 mm, MonoTower C18) provided good separation of the amino acids derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Although the separation ability of this column was not much improved in comparison with the previous monolithic silica columns, it was similar with that of a 3 μm particle packed column. The column with length 150 mm had the same separation property as a tandem connection of columns of lengths 50 mm and 100 mm. After 1000 injections of human plasma samples, the peak widths for the NBD-amino acids were increased by only 8%. Thus, the newly developed monolithic silica column can be potentially applied for faster analysis and/or more efficient separation of biological compounds.