Hisami Matsunaga

Preparation of magnetic molecularly imprinted polymers for bisphenol A and its analogues and their application to the assay of bisphenol A in river water

Magnetic molecularly imprinted polymers (M-MIPs) for bisphenol A (BPA) and its structural analogues have been prepared by a multi-step swelling and polymerization method using uniformly-sized magnetic particles as a shape template. Binding experiments and Scatchard analyses revealed that two classes of binding sites were formed on M-MIP(BPA). The retention and molecular-recognition properties of M-MIPs for BPA and its structural analogues were evaluated using a mixture of phosphate buffer and acetonitrile or a mixture of water and acetonitrile as a mobile phase by LC. M-MIPs for BPA and [²H₁₆]BPA (BPA-d₁₆), M-MIP(BPA) and M-MIP(BPA-d16), showed almost the same imprinting factors for BPA, while M-MIP for bisphenol B (2,2-bis(4-hydroxyphenyl)butane, BPB), M-MIP(BPB), gave the moderate imprinting factor for BPA. Furthermore, M-MIP(BPB) was applied for the selective extraction and determination of BPA in environmental water samples. The concentration of BPA in river water samples was determined to be 68 ng/L.

Molecularly imprinted polymers for simultaneous determination of antiepileptics in river water samples by liquid chromatography-tandem mass spectrometry.

A restricted access media-molecularly imprinted polymer (RAM-MIP) for cyclobarbital has been developed for selective extraction of antiepileptics in river water samples. The RAM-MIP was prepared using 4-vinylpyridine and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multi-step swelling and polymerization method followed by a surface modification technique. The RAM-MIP for cyclobarbital showed molecular recognition abilities for phenobarbital, amobarbital and phenytoin as well as cyclobarbital. Thus, selective analysis of antiepileptics in river water samples was attained with RAM-MIP extraction followed by column-switching liquid chromatography-tandem mass spectrometry. The concentrations of phenobarbital and phenytoin in river water samples were about 15 and 4 ng/L, respectively, while that of amobarbital was below the limit of quantitation.

Simultaneous determination of non-steroidal anti-inflammatory drugs in river water samples by liquid chromatography–tandem mass spectrometry using molecularly imprinted polymers as a pretreatment column

A restricted access media-molecularly imprinted polymer (RAM-MIP) for flufenamic acid has been developed for the simultaneous determination of non-steroidal anti-inflammatory drugs (NSAIDs) in river water samples. The RAM-MIP was prepared using 4-vinylpyridine and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multi-step swelling and polymerization method followed by a surface modification technique. The RAM-MIP for flufenamic acid showed excellent molecular recognition abilities for flufenamic acid and mefenamic acid, and moderate molecular recognition abilities for indomethacin, etodolac and ketoprofen. The simultaneous determination of NSAIDs (mefenamic acid, indomethacin, etodolac and ketoprofen) in river water samples was carried out by LC-MS/MS using the RAM-MIP for flufenamic acid as a pretreatment column. The concentrations of mefenamic acid, indomethacin and etodolac in river water samples were determined to be 0.4, 0.7 and 0.3ng/L, respectively, while ketoprofen was below the limit of quantitation.

Molecularly imprinted polymer for chlorogenic acid by modified precipitation polymerization and its application to extraction of chlorogenic acid from Eucommia ulmodies leaves.

Molecularly imprinted polymers (MIPs) for chlorogenic acid (CGA) were prepared by modified precipitation polymerization using methacrylic acid as a functional monomer, divinylbenzene as a crosslinker and methanol or dimethylsulfoxide as a co-solvent. The prepared MIPs were microspheres with a narrow particle size distribution. Binding experiments and Scatchard analyses revealed that two classes of binding sites, high and low affinity sites, were formed on the MIP. The retention and molecular-recognition properties of the prepared MIP were evaluated using a mixture of water and acetonitrile as a mobile phase in hydrophilic interaction chromatography. With an increase of acetonitrile content, the retention factor of CGA was increased on the MIP. In addition to shape recognition, hydrophilic interactions seem to work for the recognition of CGA on the MIP. The MIP had a specific molecular-recognition ability for CGA, while other related compounds, such as caffeic acid, gallic acid, protocatechuic acid and vanillic acid, could not be recognized by the MIP. Furthermore, the MIP for CGA was successfully applied for extraction of CGA in the leaves of Eucommia ulmodies.

Protein domain of chicken α1-acid glycoprotein is responsible for chiral recognition

Abstract Ovoglycoprotein from chicken egg whites (OGCHI) has been used as a chiral selector to separate drug enantiomers. However, neither the amino acid sequence of OGCHI nor the responsible part for the chiral recognition (protein domain or sugar moiety) has yet to be determined. First, we isolated a cDNA clone encoding OGCHI, and clarified the amino acid sequence of OGCHI, which consists of 203 amino acids including a predictable signal peptide of 20 amino acids. The mature OGCHI shows 31–32% identities to rabbit and human α1-acid glycoproteins (α1-AGPs). Thus, OGCHI should be the chicken α1-AGP. Second, the recombinant chicken α1-AGP was prepared by the Escherichia coli expression system, and its chiral recognition ability was confirmed by capillary electrophoresis. Since proteins expressed in E. coli are not modified by any sugar moieties, this result shows that the protein domain of the chicken α1-AGP is responsible for the chiral recognition.

Preparation of molecularly imprinted polymers for organophosphates and their application to the recognition of organophosphorus compounds and phosphopeptides.

Monodisperse molecularly imprinted polymers (MIPs) for diphenyl phosphate (DPP) and 1-naphthyl phosphate (1-NapP) have been prepared by a multi-step swelling and polymerization method using 4-vinylpyridine as a functional monomer, glycerol dimethacrylate as a crosslinker and cyclohexanol or 1-hexanol as a porogen. The retention and molecular-recognition properties of these MIPs for organophosphorus compounds were evaluated by HPLC using a mixture of phosphate buffer and acetonitrile as an eluent. In addition to shape recognition, hydrogen bonding and hydrophobic interactions could play an important role in the retention and molecular recognition of DPP and 1-NapP. Furthermore, the MIPs were applied to the separation of adenosine and adenosine phosphates (AMP, ADP and ATP). These phosphates were retained on the MIPs according to the number of phosphate groups in the molecule and were well separated from one another. Hydrogen bonding and hydrophobic interactions seemed to affect the retention and recognition of adenosine phosphates in low acetonitrile content, while hydrophilic interactions affected these properties in high acetonitrile content. Finally, the MIPs were applied to the trapping of phosphopeptides. The MIPs non-selectively trapped phosphopeptides, which have phosphorylated tyrosine, serine or threonine in the sequences, and successfully trapped four phosphopeptides in tryptic digests of bovine α-casein.

Molecularly imprinted polymer for caffeic acid by precipitation polymerization and its application to extraction of caffeic acid and chlorogenic acid from Eucommia ulmodies leaves

Molecularly imprinted polymers (MIPs) for caffeic acid (CA) were prepared using 4-vinylpyridine and methacrylamide (MAM) as functional monomers, divinylbenzene as a crosslinker and acetonitrile-toluene (3:1, v/v) as a porogen by precipitation polymerization. The use of MAM as the co-monomer resulted in the formation of microsphere MIPs and non-imprinted polymers (NIPs) with ca. 3- and 5-μm particle diameters, respectively. Binding experiments and Scatchard analyses revealed that the binding capacity and affinity of the MIP to CA are higher than those of the NIP. The retention and molecular-recognition properties of the prepared MIPs were evaluated using water-acetonitrile and sodium phosphate buffer-acetonitrile as mobile phases in hydrophilic interaction chromatography (HILIC) and reversed-phase chromatography, respectively. In HILIC mode, the MIP showed higher molecular-recognition ability for CA than in reversed-phase mode. In addition to shape recognition, hydrophilic interactions seem to work for the recognition of CA on the MIP in HILIC mode, while hydrogen bonding and hydrophobic interactions seem to work for the recognition of CA in reversed-phase mode. The MIP had a specific molecular-recognition ability for CA in HILIC mode, while other structurally related compounds, such as chlorogenic acid （CGA), gallic acid, protocatechuic acid and vanillic acid, could not be recognized by the MIP. Furthermore, the MIP was successfully applied for extraction of CA and CGA in the leaves of Eucommia ulmodies in HILIC mode.

Separation of basic drug enantiomers by capillary electrophoresis using methylated glucuronyl glucosyl β-cyclodextrin as a chiral selector

Separations of basic drug enantiomers by capillary electrophoresis have been investigated using methylated glucuronyl glucosyl β-cyclodextrin (Me GUG β-CD) as the chiral selector in the background electrolyte. Methylation was performed by reaction with methyl iodide, barium oxide, and barium hydroxide in dimethylformamide, varying the reaction temperature and time. The resulting Me GUG β-CD derivatives differed in their degree of substitution and were characterized using matrix-assisted laser desorption ionization time-of-flight mass spectra. Among the Me GUG β-CD derivatives prepared, the one with a degree of substitution of 2.8 showed the highest resolution abilities for basic drug enantiomers. Chiral resolution of 16 basic drugs was attained using the Me GUG β-CD derivative (10 mM solution in 40 mM sodium phosphate buffer at pH 3.5). The chiral recognition abilities of Me GUG β-CD were compared with those of GUG β-CD. Me GUG β-CD showed higher resolution for bupivacaine, clorprenaline, isoprenaline, pindolol, salbutamol, and terbutaline than GUG β-CD, while GUG β-CD showed higher resolution for atenolol, chlorpheniramine, dimethindene, homochlorcyclizine, ketamine, piperoxan, promethazine, propranolol, trimetoquinol, and verapamil than Me GUG β-CD. Thus, Me GUG β-CD and GUG β-CD can be complementarily used for the resolution of basic drug enantiomers.

Separation of basic drug enantiomers by capillary electrophoresis using ovoglycoprotein as a chiral selector: comparison of chiral resolution ability of ovoglycoprotein and completely deglycosylated ovoglycoprotein.

Separations of basic drug enantiomers by capillary electrophoresis (CE) using ovoglycoprotein (OGCHI) as a chiral selector are described. The effects of running buffer pH and 2‐propanol content on the migration times and resolution of basic drug enantiomers were examined using a linear polyacrylamide‐coated capillary. High resolution of basic drug enantiomers was attained using a mixture of 50 mM sodium phosphate buffer (pH 4.5–6.0) and 2‐propanol (5–30%) including 50 νM OGCHI. It was found that ionic and hydrophobic interactions could work for the recognition of basic drug enantiomers. Further, we compared the chiral resolution ability of OGCHI with that of completely deglycosylated OGCHI (cd‐OGCHI) using them as chiral selectors in CE. OGCHI showed higher resolution for basic drug enantiomers tested than cd‐OGCHI. The results suggest that the chiral recognition site(s) for OGCHI exists on the protein domain of OGCHI.

Preparation of molecularly imprinted polymers for strychnine by precipitation polymerization and multistep swelling and polymerization and their application for the selective extraction of strychnine from nux‐vomica extract powder

Monodisperse molecularly imprinted polymers for strychnine were prepared by precipitation polymerization and multistep swelling and polymerization, respectively. In precipitation polymerization, methacrylic acid and divinylbenzene were used as a functional monomer and crosslinker, respectively, while in multistep swelling and polymerization, methacrylic acid and ethylene glycol dimethacrylate were used as a functional monomer and crosslinker, respectively. The retention and molecular recognition properties of the molecularly imprinted polymers prepared by both methods for strychnine were evaluated using a mixture of sodium phosphate buffer and acetonitrile as a mobile phase by liquid chromatography. In addition to shape recognition, ionic and hydrophobic interactions could affect the retention of strychnine in low acetonitrile content. Furthermore, molecularly imprinted polymers prepared by both methods could selectively recognize strychnine among solutes tested. The retention factors and imprinting factors of strychnine on the molecularly imprinted polymer prepared by precipitation polymerization were 220 and 58, respectively, using 20 mM sodium phosphate buffer (pH 6.0)/acetonitrile (50:50, v/v) as a mobile phase, and those on the molecularly imprinted polymer prepared by multistep swelling and polymerization were 73 and 4.5. These results indicate that precipitation polymerization is suitable for the preparation of a molecularly imprinted polymer for strychnine. Furthermore, the molecularly imprinted polymer could be successfully applied for selective extraction of strychnine in nux-vomica extract powder.