Osamu Shirota

Separation of fluorescent oligosaccharide derivatives by microcolumn techniques based on electrophoresis and liquid chromatography

Various aldose oligosaccharides can be quantitatively derivatized into primary amines for subsequent reaction with fluorogenic reagents, such as 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde or 3-benzoyl-2-naphthaldehyde. Capillary electrophoresis (CE) and microcolumn liquid chromatography (LC), coupled with laser-induced fluorescence detection, were evaluated as a means of separating complex oligosaccharide mixtures. Whereas microcolumn LC and open-tubular CE appear confined in their utility to relatively small oligosaccharides, unprecedented results were obtained with polyacrylamide gel-filled capillaries on hydrolyzed malto-oligosaccharides and enzymatically degraded samples of chondroitin sulfate and hyaluronic acid.

Direct determination of bisphenol A and nonylphenol in river water by column-switching semi-microcolumn liquid chromatography/electrospray mass spectrometry

A rapid and sensitive analytical method based on column-switching semi-microcolumn high-performance liquid chromatography (HPLC) with electrospray mass spectrometry was developed for determining trace levels of bisphenol A (2,2-bis(4-hydroxyphenyl)propane) and nonylphenol (4-nonylphenol) in river water. An aliquot of sample solution was directly injected into the precolumn packed with Capcellpak MF-Ph for sample cleanup and enrichment. The compounds of interest were then transferred to a C-18 analytical column for main separation through a change in flow path by a programmed switching valve. Bisphenol A, nonylphenol, and interfering substances were satisfactorily separated with a simple gradient elution complete within 35 min. Detection of their deprotonated molecules was conducted in negative ion mode. A reduced flow rate (100 &mgr;L/min) optimized for the narrow bore column was found advantageous in obtaining a high degree of sensitivity during electrospray detection. The influence of carrier additives on sensitivity was also examined. This method produced detection limits of 0.5 ng/mL for bisphenol A and 10 ng/mL for nonylphenol (signal-to-noise ratio 3). Calibration curves were observed in the range of 2.5-50 ng/mL (r(2) = 0.999) for bisphenol A and 50-500 ng/mL (r(2) = 0.998) for nonylphenol. Recoveries of the compounds from spiked distilled water and river water were 99.7-138.5% with a relative standard deviation (RSD) ranging from 2.2-9.7%. Copyright 1999 John Wiley & Sons, Ltd.

Synthesis and characterization of polymer-coated mixed functional stationary phases with several different hydrophobic groups for direct analysis of biological samples by liquid chromatography

Three types of polymer-coated mixed-functional (PCMF) silica column-packing materials for the direct analysis of biological liquids have been synthesized. These packing materials all have polyoxyethylene groups as their hydrophilic part but differ in their hydrophobic part (methyl groups for Me-POE, phenyl groups for Ph-POE, and octyl groups for Oc-POE). Retention characteristics with respect to several drug molecules, and protein recovery, were studied for each of the three types with different amounts of hydrophobic groups attached. Of the PCMFs prepared, Oc-POE showed the greatest overall retention, and Me-POE displayed the greatest protein recovery.

Synthesis and characterization of a polymer-coated C18 stationary phase with high carbon content for liquid chromatography

Abstract High-purity porous silica with a large surface area (particle diameter 5 μm, mean pore diameter 8.4 nm, surface area 350 m2/g) was coated with a homogeneous silicone polymer film, which was thereafter modified with octadecyl groups to be used as a stationary phase for reversed-phase chromatography. The stationary phase had a high carbon content (18%), and showed a relatively large overall retention for various analytes among other stationary phases available in the market. It also showed preferable characteristics in separation of basic compounds, such as pyridine, and hinokitiol, a substance that easily coordinates with metals, and also had a high durability under an alkaline mobile phase.

Direct determination of pindolol enantiomers in human serum by column-switching LC-MS/MS using a phenylcarbamate-β-cyclodextrin chiral column

A direct analytical method of pindolol enantiomers in body fluids was developed by means of column-switching semi-microcolumn liquid chromatography/tandem mass spectrometry (LC-MS/MS). A pre-column packed with a silica-based cation-exchanger was used for on-line sample cleanup. Subsequent enantioseparation was conducted with a phenylcarbamate-beta-cyclodextrin (ph-beta-CD) bonded semi-micro chiral column (2.0 mm inner diameter (i.d.)). A 25-microl aliquot of serum/urine samples was directly injected into the system after simple filtration with a membrane filter. Separated enantiomers were monitored with positive electrospray ionization (ESI) and selected reaction monitoring (SRM). R(+)- and S(-)-pindolol in serum and urine were determined separately within 16 min at a resolution factor of 1.9. The detection limits at a signal-to-noise (S/N) ratio of 5 were 0.13 ng ml(-1) for both enantiomers. The linearity of the method was in the range of 0.25-100 ng ml(-1) with regression coefficient greater than 0.997. Recoveries from spiked serum and urine samples, estimated by the external standard method, were between 94.8 and 117.6% with a relative standard deviation (RSD) ranging from 2.1 to 18%.

High-sensitivity analysis of sialyl-oligosaccharide glycosylation sites in glycoproteins by miniaturized tryptic digestion and microcolumn liquid chromatography

A highly sensitive analytical procedure was developed to assess the site of glycosylation in a model glycoprotein, bovine fetuin. First, sample cleavage with immobilized trypsin and the peptide map development are accomplished by microcolumn liquid chromatography. Second, the sialic acids content is measured fluorometrically using their precolumn conversion to quinoxaline derivatives. A unique preconcentration system was developed to secure sensitivity of the second measurement.

Analysis of ibuprofen metabolites by semi-microcolumn liquid chromatography with ultraviolet absorption and pulsed amperometric detectors☆

Semi-microcolumn (1.5 mm i.d.) liquid chromatography (LC) system with ultraviolet (UV) and pulsed amperometric detector (PAD) was constructed for analysis of ibuprofen metabolites in human urine. PAD was connected in series with the UV detector, and an alkaline solution was post-column added to a mobile phase after the UV detector. By a gradient elution, five ibuprofen metabolites were detected with UV detection from 1 microliter of human urine at 3.5 h after the administration, and information concerning their glucuronation was simultaneously obtained by PAD response.