Chemistry: analytical and immunological testing – Oxygen containing – Hydroxyl containing
Reexamination Certificate
1999-12-10
2001-12-25
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Oxygen containing
Hydroxyl containing
C436S056000, C436S140000, C436S172000, C436S169000, C436S082000
Reexamination Certificate
active
06333199
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of analyzing bisphenols and polyphenols; and, in particular, to a method of analyzing a bisphenol and/or a polyphenol having at least two phenolic hydroxyl groups contained in a test sample.
2. Related Background Art
Bisphenols are used in large amounts as materials (stabilizer, plasticizer, and the like) for polycarbonate resins, phenol resins, and the like in familiar everyday goods and the like. It has recently been reported that bisphenols leak into the environment from such everyday goods or wastes thereof, so that attention is being paid to their distribution in the environment, their influences on the ecosystem, their toxicity to humans, and so forth. It has also been indicated that bisphenols may be one of exogenous endocrine disrupting chemicals, but there are still many unclear points concerning their actions on organisms and their mechanism. One of the reasons behind this unclearness is attributable to the imperfection in the method of analyzing bisphenols. Conventionally, gas chromatography/mass spectrometry (GC/MS) method, liquid chromatography/mass spectrometry (LC/MS) method, and LC/fluorescence detection method have been used in general as the method of analyzing bisphenols.
SUMMARY OF THE INVENTION
As a result of repeated diligent studies, the inventors have found that the following problems exist in the above-mentioned conventional analyzing methods.
1) Though the GC/MS and LC/MS methods have a high selectivity, their preprocessing operations are complicated, and their apparatus cost and running cost are so high that it tends to be difficult for them to be used for the routine analysis of a number of test samples (specimens).
2) The LC/fluorescence detection method is a method in which the phenolic hydroxyl groups of bisphenols are fluorescence-labeled with a fluorescent reagent such as dansyl chloride or the like, and their fluorescence is detected, whereby both of its apparatus cost and running cost are relatively inexpensive. In this method, however, many monophenols and amines are also fluorescence-labeled. As a result, background fluorescence enhances, the selectivity for bisphenols is lower, and a sufficient sensitivity may not be obtained, whereby it is not suitable for analyzing bisphenols in the environment.
3) While simple kits for analyzing bisphenol A (e.g., ELISA Kit manufactured by Takeda Chemical Industries, Ltd.) are commercially available, they include many compounds which generate a crossing reaction, whereby they are only applicable to limited samples and are problematic in that their preprocessing is complicated.
In view of such circumstances, it is an object of the present invention to provide a simple method of analyzing bisphenols and polyphenols, which can analyze bisphenols and/or polyphenols with a high sensitivity and a high selectivity without necessitating complicated preprocessing.
The inventors have repeated diligent studies in order to achieve the above-mentioned object, and have found that, when a labeling reagent having a pyrene group is caused to act on and label at least two phenolic hydroxyl groups in a molecule of a bisphenol or a polyphenol, and thus labeled compound is optically excited, an intramolecular excimer is formed, so that fluorescence having a wavelength specific to this excimer is quantitatively emitted, whereby the present invention is accomplished.
Namely, the method of analyzing bisphenols and polyphenols in accordance with the present invention is a method of analyzing a polyphenol and/or a bisphenol having at least two phenolic hydroxyl groups contained in a test sample, the method comprising a step of reacting a bisphenol and/or a polyphenol with a labeling reagent having a pyrene group, so as to generate a fluorescent derivative; a step of irradiating the fluorescent derivative with excitation light and detecting fluorescence emitted from the fluorescent derivative; a step of calculating an amount of the bisphenol and/or polyphenol in the test sample according to thus detected fluorescence intensity and a relationship between a known concentration of a bisphenol and/or a polyphenol contained in a standard sample and fluorescence intensity.
In such a method of analyzing bisphenols and polyphenols, the labeling reagent substitutes for hydrogen atoms in the phenolic hydroxyl groups in a molecule of a bisphenol and/or a polyphenol, thereby generating a fluorescent derivative labeled with pyrene groups. When this fluorescent derivative is irradiated with excitation light, the pyrene groups within the molecule are optically excited, and thus excited pyrene groups are dimerized within the molecule, whereby an intramolecular excimer is formed. The intramolecular excimer emits fluorescence having a wavelength different from that of the fluorescence (monomer fluorescence) emitted by the pyrene groups alone upon optical excitation. According to the resulting fluorescence intensity and a relationship (calibration curve or working curve) between the concentration and fluorescence intensity determined concerning a standard sample containing a known concentration of bisphenols and/or polyphenols, or upon addition of the standard sample, for example, the quantity of the bisphenol and/or polyphenol in the test sample is determined.
In the step of detecting fluorescence, it is preferred that fluorescence having a wavelength of 450 to 530 nm be detected. Such a fluorescence wavelength (450 to 530 nm) includes a wavelength region of the fluorescence emitted from the above-mentioned intramolecular excimer of the fluorescent derivative and is longer than the wavelength of the monomer fluorescence emitted from the pyrene group monomer such as one in the labeling reagent or the like. Therefore, the selectivity for bisphenols or polyphenols is enhanced, so as to enable high-sensitivity analysis.
Preferably, as the test sample, one containing an oxyphenyl group at a terminal within its molecule is used, whereby the present invention becomes quite effective. More preferably, a bisphenol is at least one selected from the group consisting of 2,2-bis(4-hydroxyphenyl) propane, 2,2-bis(4-hydroxyphenyl) butane, 4,4′-ethylidene bisphenol, bis(4-hydroxyphenyl) methane, 4,4′-(1-phenylethylidene) bisphenol, and 4,4′-(1,4-phenylene-di-isopropylidene) bisphenol.
Preferably, the method further comprises a step of adding an amine having a polar group, more preferably an amidosulfonic acid, aliphatic amino acid, or amino sugar to the test sample in which the fluorescent derivative is generated. As a consequence, the excess of labeling reagent, which remains within the test sample while being unreacted with a bisphenol or a polyphenol or after hydrolysis, is decomposed. When the separation of components in the test sample by high-performance liquid chromatography (hereinafter referred to as “HPLC”) is concurrently used, decomposition products tend to be eluted faster than the labeling reagent itself. Here, the above-mentioned amine is preferably at least one selected from the group consisting of taurine, arginine, &ggr;-aminolactic acid, glucosamine, and mannosamine in particular.
If the labeling reagent having a pyrene group is at least one selected from the group consisting of 4-(1-pyrene) butanoyl chloride, 4-(1-pyrene) pentanoyl chloride, and 4-(1-pyrene) hexanoyl chloride, it will be useful since the efficiency of labeling reaction of bisphenols and/or polyphenols is remarkably enhanced.
REFERENCES:
Boileau et al. “End-to-End Cyclization Dynamics of a Pyrene End-Capped (Bisphenol A-diethylene glycol) Polycarbonate”, Polym. Prep. (Am. Chem. Soc.), 1988, v.29 (1), pp. 509-510.*
Boileau et al. “End-to-End Cyclization of a Pyrene End-Capped Polycbisphenol AF-diethylene glycol carbonate)”, Macromolecules, 1989, v. 22, pp. 215-220.*
Duhamel et al. “End-to-End Cyclization of a Pyrene End-Capped Poly(bisphenol AF-diethylene glycol carbonate) in Solution”, Eur. Polym. J., 1994, v. 30, No. 1, pp. 129-134.*
Wilken et al. “End-Group Dyn
Nohta Hitoshi
Yamaguchi Masatoshi
Yoshida Hideyuki
Gakh Meline
Laboratory of Molecular Biophotonics
Leydig , Voit & Mayer, Ltd.
Warden Jill
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