Pharmaceuticals are essential for the medical treatment of various diseases. From the herbal medicines (crude drugs), synthetic medicines to biopharmaceuticals, to assure its efficacy and safety, the production procedures and the quality control methods of pharmaceuticals are important. These are strictly regulated by Good Manufacturing Practice (GMP) and various guidelines. The key is to develop the “Specifications and testing methods” of the pharmaceutical scientifically and rationally according to the production process. There are many testing items such as identity tests, purity tests, assay, etc. in “Specifications and testing methods”. Among them, assay and purity test are core testing items. Optical purity evaluation is required for the single enantiomer pharmaceutical. Chromatographic methods and its detection methods are useful for the purpose. High-performance liquid chromatography (HPLC), thin layer chromatography (TLC) and gas chromatography (GC) are commonly used because the target analyses are almost a mixture. In this review, results of the development of the quality evaluation methods of relatively low molecular pharmaceuticals and herbal medicines by HPLC and capillary electrophoresis (CE) we have been investigated are summarized.

Focused metabolomics facilitates the understanding of the current phenotype of living organisms, involves highly practical methods for investigation, and allows for the acquisition of highly quantitative data for important endogenous metabolites. The authors developed novel precolumn derivatization reagents for the analysis of amino acids, which are particularly important endogenous metabolites, through precolumn liquid chromatography/tandem mass spectrometry (LC/MS/MS). One of the reagents facilitated sub-femtomole-to-attomole-scale amino acid detection. We also developed an automated precolumn derivatization amino acid analyzer using another reagent for liquid chromatography/mass spectrometry (LC/MS). With this device, nearly 40 amino acids can be analyzed within a short time (<10 min), compared with the time (~2 h) taken by a conventional automated postcolumn derivatization amino acid analyzer. For highly accurate and precise analysis of free amino acid concentration in human plasma, we validated all steps, from blood collection to measurement, by using the proposed analyzer and developed a test procedure for studying free amino acid metabolomics in human plasma. While calculating the reference intervals of plasma amino acid concentrations in healthy individuals, it was found that the balance of amino acid concentrations in plasma is useful for risk screening of diseases including cancer, diabetes, and myocardial infarction. This functional-group-targeted analysis through LC/MS for amino acid was also effective for dipeptide, tripeptide, and D, L-amino acid analyses and amino acid detection in tissue by imaging mass spectrometry.

Microchip electrophoresis (ME) has emerged as a promising tool for rapid analysis of various sample types and it is one of the most important modules of miniaturized total analysis systems. Electrokinetic effects provide both efficient transport and separation of sample components in microfluidic channels. Moreover, miniaturization of devices drastically reduces reagent consumption, significantly decreases analysis time, and allows for easier parallel screening and automation. However, most microfluidic systems are insensitive, which is a serious problem. In order to increase the sensitivity of ME, several on-line pretreatment applications such as specific and non-specific preconcentration, extraction, and derivatization have been proposed. Here, we review various specific on-line preconcentration methods utilizing in situ photopolymerized polyacrylamide-based preconcentrator gels. These techniques allowed for highly sensitive specific detection of various sample types by ME.

A method was developed for the specific entrapment and separation of thrombin aptamers using a thrombin-immobilized polyacrylamide gel fabricated at the channel crossing point of a microfluidic electrophoresis chip. The channel intersection of the poly(methyl methacrylate) (PMMA) microchip was filled with a solution comprising thrombin, acrylamide, N,N-methylene-bis-acrylamide, and 2,2’-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], which functioned as a photocatalytic initiator. In situ polymerization at the channel crossing point was performed by irradiation with an LED laser beam. The fabricated thrombin-immobilized gel (100 ×100 × 30 µm) contained approximately 40 fmol of thrombin and therefore could entrap thrombin aptamers at the femtomolar level. The electrophoretically trapped thrombin aptamers were released from the gel by switching the voltage, which delivered high concentrations of phosphate ions in a background electrolyte. The broad sample band eluted from the gel was effectively reconcentrated at the boundary of a pH junction generated by sodium ions delivered from the outlet reservoir. The reconcentrated sample components were then separated and fluorometrically detected at the end of the separation channel. Under the optimized conditions, the thrombin aptamers were concentrated by a factor of 1,000-fold, and the peak resolution was comparable to that obtained by pinched injection. This method was successfully utilized to preconcentrate and analyze thrombin aptamers.

The residual status of clenbuterol in various edible parts of livestock products was investigated using the LC-MS/MS and LC-MS/MS/MS methods. The target food products were the edible tissues of pig and cattle, and clenbuterol was extracted using acetonitrile containing anhydrous sodium sulfate and sodium chloride. Following the dispersive solid-phase extraction using C18 particles and purification with an anion-exchange solid-phase extraction cartridge, the resultant test sample solutions were subjected to the LC analysis. A C18 column was used as the analytical column, and the peak of clenbuterol was eluted at 4.8 min with the gradient elution from 5% acetonitrile to 99% acetonitrile in 10 min. As a result of the LC-MS/MS analysis, interfering peaks were detected around the retention time of clenbuterol. By using the LC-MS/MS/MS analysis, which was developed as a quantitative analysis method that reduces the influence of contaminants, most of the unknown peaks were removed, and it was confirmed that the food samples obtained in Kanagawa prefecture did not contain clenbuterol.

Two-steps acid dissociation constants of L-ascorbic acid (AA) were determined through the changes in the effective electrophoretic mobility in capillary zone electrophoresis. Although ascorbic acid is oxidatively degradable in an aqueous solution, especially in alkaline conditions, the effective electrophoretic mobility of AA was successfully measured in the pH range between 1.87 and 11.97. In the analysis of the first-step acid dissociation constant (pK_a1) at weakly acidic pH conditions, a coated capillary with 1,3-propanesultone and a pressure-assist were utilized to detect anionic AA. In the analysis of the second-step acid dissociation constant (pK_a2) at weakly alkaline pH conditions, AA was successfully detected at the pH range up to 11.97 with the help of Cu catalyst to remove the dissolved oxygen in the separation buffer. Acid dissociation constants were independently determined as pK_a1 = 4.15±0.01 and pK_a2 = 12.07±0.04 by non-linear least-squares analyses. AA did not fully dissociate at the weakly alkaline pH range, and the effective electrophoretic mobility of the dianion form of AA was extrapolated for the analysis.

In this paper we describe a highly sensitive and selective LC method for the determination of 5-hydroxyindoles (serotonin, N-acetylserotonin, 5-hydroxyindole-3-acetamide, 5-hydroxytryptophan, 5-hydroxyindole-3-acetic acid, and 5-hydroxytryptophol) through post-column electrochemical derivatization and fluorescence detection. These 5-hydroxyindoles can be separated within 40 min by reversed-phase liquid chromatography using 250 mM acetate buffer/acetonitrile [pH 6.5; 95:5 (v/v)] under conditions of isocratic elution; then they were subjected to electrochemical oxidation with benzylamine to produce their corresponding fluorescent derivatives. We detected these derivatives spectrofluorometrically at 480 nm upon excitation at 345 nm. The detection limits (S/N = 3) of these 5-hydroxyindoles were in the range 3.2–180 fmol per 20-µL injection.

The retention behaviors of various low-molecular-weight compounds in low-temperature HPLC with an ODS (octadecyl silica) column and liquid CO₂ mobile phase containing methanol or acetonitrile as modifiers were investigated. The addition of methanol/acetonitrile reduced the retention of xanthone, which was dominated by the liquid CO₂ content, independent of the modifier. Additionally, the retention of polycyclic aromatic hydrocarbons (PAHs) was reduced by the addition of methanol/acetonitrile. Because acetonitrile resulted in a rapid reduction in the retention compared with methanol, it was suggested that the CH- interactions have an important role in the retention of PAHs in the C18 stationary phase. Notably, the retention of alkylbenzenes were only slightly affected by the addition of methanol/acetonitrile. Acetonitrile slightly increased the retention of alkylbenzenes with longer alkyl chains than those with shorter alkyl chains. It is suggested that the interaction of the phenyl moiety in alkylbenzenes with C18 chains was reduced by the increase in the acetonitrile content, and enhanced in the case of the alkyl chain moieties. The retentions of alkyl benzenes were approximately compensated, when the alkyl chain length was relatively short.

Dansyl-DL-amino acids were separated by tube radial distribution chromatography (TRDC) comprising a commercially available HPLC system, where open-tubular capillary tubes manufactured for capillary GC were used as the separation column. Initially, the separation performance of GC capillary tubes in TRDC was assessed by separating a model analyte mix of 1-naphthol and 2,6-naphthalenedisulfonic acid by using water/acetonitrile/ethyl acetate solutions (3:8:4 volume ratio, organic solvent-rich and 4:3:1 volume ratio, water-rich) as the eluent and high-polarity and non-polarity capillary tubes. Next, enantiomer separation of dansyl-DL-methionine and dansyl-DL-valine was examined using a water/acetonitrile/ethyl acetate solution (8:2:1 volume ratio, water-rich) containing 1.0 mM -cyclodextrin and non-polarity capillary tubing. The D- and L-enantiomers were separated and detected in this order through interaction between amino acids and -cyclodextrin by tube radial distribution flow in the TRDC system.