BUNSEKI KAGAKU Abstracts

We have developed a novel chromatography system, an environmental-responsive chromatography, based on a multifunctional polymer. We designed a temperature-responsive copolymer with N-isopropylacrylamide and various monomers. This copolymer undergoes a reversible phase transition from a hydrophilic to a hydrophobic microstructure when triggered by a change of temperature. Also, with this chromatographic system is possible to separate the analytes by using only an aqueous solution or water as the mobile phase. Temperature-responsive chromatography could prove to be highly useful for bioactive molecules, such as steroid, amino acid and peptide separation. There are some advantages not to need an organic solvent, compared to traditional reversed-phase chromatography. For example, we achieved a successful separation of enzymes without a loss of bioactivity, and for environmental use as a new green technology, and for reducing the cost of handling and disposing of a mobile phase. Applications to bio-separation in various fields, such as medical and pharmaceutical sciences, are expected in the future.

Silicone polymer-coated packing materials were developed by coating silica with a silicone polymer, and modifying it with vinyl compounds in the presence of hexachloroplatinic acid. This silicone polymer coating indicated that a homogeneous silicone polymer film of 7 Å in thickness was formed and that it was formed even inside small micropores. This silicone polymer-coated silicas showed both a high resolution of chemically bonded silicas and an alkaline resistance of an organic porous polymer. Polymer-coated C18 packings optimized in the balance of the polymer coating and the introduction of C18 groups showed a high symmetrical peak shape for amitriptyline, a strong basic compound, under neutral conditions obtained in different organic modifiers, methanol and acetonitrile. The silicone polymer-coated packing materials showed good reproducibility in a comparison with conventional chemically bonded silicas of different surface activity.

The retention behavior of the reversed phase was evaluated under 100% aqueous conditions. It is commonly said that reversed phases, such as C18 (ODS) and C8, show a decrease in the retention time under 100% aqueous conditions. It was found that a 100% aqueous mobile phase was expelled from the pores of the packing materials, so that the stationary phase in contact with the mobile phase decreased and the retention time decreased. Some parameter, such as the pore size, length and ligand density of the alkyl group of the stationary phase, the amount of residual silanol groups of the stationary phase, salt and the ion-pair reagent concentration in the mobile phase, temperature and back pressure of the column, were shown to influence the decrease in retention. Furthermore, the wettability between the C18 stationary phase and water as a mobile phase was analyzed. It was concluded that the retention behavior could be explained by capillarity, and reversed-phase separation could be carried out under 100% aqueous conditions, even if a mobile phase can not wet the stationary phase. Finally, these phenomena were applied to reversed-phase separation using the C18 stationary phase and the mobile phase with less than 70% methanol and more than 30% water.

A high-performance liquid-chromatographic detection method was developed for a highly selective determination of flame retardants, such as PBBs and PBDEs. The method is based on on-line dehalogenation by irradiation with UV light of the column effluent. The response of a conductivity detector was proportional to the concentration of the analyte. However the numbers of bromide liberated from PBDEs were almost unity regardless of the UV irradiation time. The detection limit of DecaBDE was 3 ng for a 3 : 1 S/N ratio, following about 0.55 min irradiation with a 15 W low-pressure mercury lamp. The calibration curves from peak areas of PBDEs were linear with high correlation coefficients of more than 0.996 at the range over 3 to 100 ng. A recovery test from a laboratory fortified with PBDEs polyester resin showed good results for PBDEs, ranging from 87.3 to 109%. The proposed detection system permits a simple and rapid screening of brominated flame retardants using a small sample mass.

We developed a device comprising a spin column packed with ion exchange type (SCX and SAX) monolithic silica for extracting ionic compounds from biological samples. The methods involving the use of these spin column are not useful for the extraction of ionic analytes, but are highly reproducible for the analysis in serum and urine. This spin column enabled sample preparation in less than 10 min. Handling such as sample loading, washing, and elution of analytes, was exhibited by the centrifugation of a spin column. In addition, many samples could be processed at the same time. This method has many advantages : easy operation, low volume of the extraction solvent, and without evaporation. This spin column has potential as a new tool for the routine extraction of ionic compounds in biological materials.

The charged aerosol detector (CAD) has recently become a new alternative detection system in HPLC. This detection approach was applied in a new HPLC method for quantification. We tried to use CAD for the quantification of impurities in a 17β-estradiol reagent. This detector could not analyze a high-concentration sample because of a baseline noise. The component of 17β-estradiol was removed by using a valve-switching device. Quantification of impurities in 17β-estradiol reagent was determined by a calibration curve of 17β-estradiol (USP). The results of quantification used by CAD with the calibration curve of 17β-estradiol were compared with those used by UV with the calibration curve of each standard of impurity. There was correspondence between the results of quantification using CAD and UV. By this result, we confirmed that CAD is useful in analyzing some impurities in the 17β-estradiol reagent.

High-speed Protein A-HPLC analysis was developed for the quantification of human monoclonal antibody (hMab). A perfusion type of Protein A HPLC column (POROS 50A ; 4.6 mm i.d. × 5 cm ; particle size, 50 μm) was used, and the chromatographic conditions, such as the flow rate, salt concentration in the buffer solution, and equilibration time were examined. HPLC was performed at a flow rate of 5 mL min⁻¹ using stepwise elution from 50 mM sodium phosphate that contained 300 mM sodium chloride (pH 7.0) to the same buffer (pH 2.8), which was selected to reduce the leading of elution peaks of hMabs and the carry-over of hMabs. To minimize the time per analysis, we deleted a re-equilibration step from the method program, because the necessary volume of the equilibration buffer flowed in the column while the sample was injected by an autosampler. This enabled us to analyze 1 sample within 2 to 2.5 min (including the time required for sample injection). A column lifetime study not only showed that this deletion had no effect on repeated quantification, but also demonstrated that more than 3000 analyses can be performed with one column. We report here on the fast quantification method with a long column lifetime. This high-throughput quantification method is very useful for the process development of monoclonal antibody pharmaceuticals.

We have focused on skin as an information source of the cholesterol level from a living body. In this study, skin components were extracted by placing a palm in contact with an organic solvent. The resulting solution was analyzed by using high-performance liquid chromatography. Some important parameters, such as the extracting time, area and solvent, in extracting skin components were investigated. Cholesterol, cholesterol oxide and suqualene were extracted from skin by using ethanol as an extracting solvent. The concentration of skin cholesterol was higher along with an increase in the extracting time, and because saturated after 60 s. A plastic cup (diameter ; 4.2 cm ; extract area : 12.8 cm²) was appropriate for extracting skin cholesterol to discriminate individual difference and easiness in handling. The concentration of skin cholesterol correlated with the amount of blood cholesterol measured by a medical institution, which suggests that this method has potential usefulness for non-invasive cholesterol measurements.

An HPLC method with a charged aerosol detector (CAD) was optimized and validated for the quantification of six carbohydrates (xylose, fructose, glucose, sucrose, lactose, maltose) and sugar alcohol (erythritol) in drinks. Chromatographic separation was achieved using gradient elution with a mixture of water-methanol (7 : 3) and acetonitril. Carbohydrates were cleaned up using a strong cation exchange cartridge (Bond Elut SCX, Varian). Although an interfering peak appeared without any clean up, it was confirmed that interfering substance had be removed by a treatment using this cartridge and its peak had disappeared. The determinations were performed in the linear range of 5∼10000 μg/mL for six carbohydrates and sugar alcohol, the correlation coefficients (R) were 0.998∼1.000, the relative standard deviations (RSDs) ranged over 0.4∼4.5% (spiked level at 500 μg/mL, n = 5), recoveries ranged between 84∼113% (spiked level at 500 μg/mL, n = 3), and the limits of quantification were 0.4∼19.3 μg/mL. In addition, we performed analysis of carbohydrates with CAD, an evaporative light scattering detector (ELSD) and a refractive index detector (RID) to compare and investigate the sensitivity and analysis precision in the each optimum condition. As for the CAD, high sensitivity and good analysis precision were provided. Because it is not necessary to optimize the conditions, CAD is the simplest and easiest device in these detectors. It became clear that CAD was the most suitable to perform correct quantitative analysis of carbohydrates in drinks.

An ultra high-speed liquid chromatograph (UHLC) offers a reduction in the analysis time compared with conventional HPLC by using a column with finer particles and an apparatus resistant to high pressure. Although postcolumn methods are excellent techniques for the analysis of samples containing contaminants, such as foodstuffs, the postcolumn method has not been applied to UHLC because of problems associated with diffusion of the target peak and the reactivity. A postcolumn method using UHLC has not been reported due to some problems such as diffusion of the sample in a reaction part. We successfully applied a postcolumn method using UHLC through optimization of the instrumental system and the analytical conditions with Ascentis Express RP-Amide and LaChromUltra C18-AQ columns. This afforded the high-speed analysis of 10 organic acids ; tartaric acid, formic acid, malic acid, lactic acid, acetic acid, pyroglutamic acid, citric acid, succinic acid, propionic acid and fumaric acid ; within 4 min with good separation and reproducibility. Their limits of detection (S/N = 3) were between 9 to 29 mg/L. The results of measurements of food samples by the postcolumn method using UHLC were similar to those obtained by the postcolumn method using HPLC. Therefore, it can be said that effective measurements are possible using the postcolumn method by UHLC, and that this system is particularly useful for materials that contain large amounts of contaminants, such as foodstuffs.

The standard method for determining gaseous aldehydes involves the collection of the aldehydes on a 2,4-dinitrophenyl hydrazine (DNPH)-coated silica gel cartridge, followed by solvent extraction of the cartridge and analysis of the aldehyde derivatives using high-performance liquid chromatography (HPLC). However, acid catalysis is required for a fast and quantitative derivatization reaction, and the DNPH derivatives of α,β-unsaturated aldehydes, such as acrolein, disappear during storage of the acidified DNPH-coated cartridges. In this paper, we describe a new method that involves the collection of acrolein and other lower aldehydes on a non-acidified DNPH-coated silica-gel cartridge. Using this method, we were able to carry out highly sensitive simultaneous sampling, as indicated by the quantitative collection of acrolein and other lower aldehydes on the cartridge. The acrolein hydrazone was stable on the cartridge at room temperature, and the gaseous acrolein concentrations measured in a test atmosphere were in good agreement with those obtained by means of the traditional DNPH-solution method. In addition, the new method was tested on an ambient air sample. The operational procedure for the non-acidified DNPH-coated cartridge is nearly the same as that for the standard DNPH/HPLC method. This new method will permit the determination of acrolein, formaldehyde, and acetaldehyde in atmospheric environmental applications.