JP2010048568A - Method and apparatus for simultaneously and continuously analyzing thiol compound - Google Patents

Abstract

A method for simultaneous and continuous analysis of thiol compounds capable of simultaneously and simply analyzing oxidized thiols and reduced thiols of thiol compounds, and an analysis apparatus usable for the same.
SOLUTION: After separating oxidized thiols and reduced thiols of a thiol compound, the oxidized thiols are reduced to be converted to reduced thiols, and derivatized with a fluorescent derivatizing reagent such as pyrene. By analyzing the excimer fluorescence emitted from the derivative, the oxidized thiols and the reduced thiols of the thiol compound can be simultaneously and continuously analyzed.
[Selection] Figure 1

Description

  The present invention relates to a method for simultaneous and continuous analysis of thiol compounds in a sample and a simultaneous and continuous analysis apparatus used therefor, and more specifically, simultaneous and continuous oxidation and reduction thiols of thiol compounds contained in a sample. The present invention relates to an analysis method and a simultaneous continuous analysis apparatus used therefor.

  In recent years, high-performance liquid chromatography (HPLC) has been used in the fields of medicine, environment, food, etc., and HPLC detection is performed based on detecting the physical and chemical properties of the substance to be measured. Electrochemistry, mass spectrometry (MS), fluorescence, chemiluminescence, etc. are used as detectors. If the target chemical does not respond to such a detector or does not respond sufficiently, detection by HPLC may be impossible or insufficient as it is. In such a case, it is possible to enable detection by HPLC by converting the target substance into a substance showing a strong response to the detector by a chemical reaction such as so-called derivatization. Among the derivatization methods, the fluorescence derivatization method is widely used, and many fluorescent derivatization reagents necessary for this purpose have been developed (Non-patent Document 1).

  Some fluorescent derivatization reagents have a reaction site for labeling the target substance and a fluorescent site (fluorophore) involved in fluorescence emission in the same molecule. For example, pyrene, acridine, anthracene, coumarin, cyanine, dansyl, fluorescein, rhodamine and the like are known (for example, see Non-Patent Documents 1-4). When using these fluorescent derivatizing reagents to detect trace components in a sample, an excessive amount of fluorescent derivatizing reagent may be added to derivatize a large amount of coexisting substances present in the sample. In many cases, the fluorescent reagent added excessively in this way may interfere with the analysis of the substance to be measured. Attempts have also been made to solve these problems, not only improving pretreatment methods and separation techniques, but also developing fluorescent derivatization methods using non-fluorescent reagents (Non-Patent Documents 5-7), Attempts have been made to develop a derivatization method (Non-Patent Documents 8-10) in which a special fluorescence phenomenon (fluorescence polarization, time-resolved fluorescence, fluorescence transfer energy transfer, etc.) is introduced.

  The present inventors have developed a derivatization method in which excimer fluorescence, which is one of such special fluorescence phenomena, is introduced, and multi-functional test substances are labeled at multiple points with a pyrene reagent. Fluorescence detection in the excimer fluorescence region (440-540 nm) in the longer wavelength range than the region (monomer fluorescence: 360-420 nm) enables selective analysis of specific compounds with multiple functional groups . As a result, the present inventors developed a method for analyzing polyfunctional compounds such as polyamines, polyphenols, and polycarboxylic acids, and simple and highly selective quantification of bioactive substances, pharmaceuticals, agricultural chemicals, environmental hormones, and the like. (For example, refer nonpatent literature 11).

  On the other hand, thiol compounds include polythiols having a plurality of thiol structures (sulfhydryl groups; —SH) in the molecule, and monothiols having one thiol structure. Various forms of thiol compounds exist in vivo as cysteines and glutathione. In addition, since monothiols and polythiols coexist in an oxidized form and a reduced form and have mutually different functions, it is necessary to measure them simultaneously.

  In general, the reduced thiol compound can be analyzed by derivatization with various fluorescent reagents and then separation by high performance liquid chromatography (HPLC). At this time, when the pyrene reagent is used, the detection wavelength of polythiols is 450-500 nm, that is, excimer fluorescence, whereas the detection wavelength of monothiols is significantly different from 360-420 nm, that is, monomer fluorescence. By detecting excimer fluorescence, polythiols can be selectively detected without being disturbed by monothiols. Among such thiol compounds, oxidized thiols can be detected by excimer fluorescence or monomer fluorescence by reducing with a reducing agent before derivatization.

Since polythiols can be detected with high sensitivity and high selectivity by the fluorescence prelabel analysis method, this fluorescence prelabel analysis method has been widely used in recent years. However, since the analytical methods reported so far can only measure the total amount of oxidized and reduced thiols, or only reduced thiols, the amount of oxidized thiols can be measured using oxidized and reduced thiols. The amount of reduced thiols was subtracted from the total amount. At present, it can be said that there is no method for easily and simultaneously measuring the oxidized and reduced forms of polythiols. Theoretically, if LC-MS analysis is used, it is thought that measurement can be performed as it is even in a state where oxidized thiols and reduced thiols coexist. However, such methodological papers have been known so far. As far as it does not exist. Therefore, development of a method capable of easily and simultaneously measuring oxidized and reduced polythiols has been awaited.
Masatoshi Yamaguchi, Hitoshi Noda: Bunkeki, 291 (3), 204 (1999) Y. Ohkura, M. Kai, H. Nohta: J. Chromatogr. B., 659, 85 (1994) M. Yamaguchi, J. Ishida: "Modern derivatization methods for separation sciences", T. Toyo'oka (Ed.), P. 99 (1999), (John Wiley and Sons Ltd. New York) K. Fukushima, N. Usui, Y. Santa, K. Imai: J. Pharm. Biomed. Anal., 30, 1655 (2003) S. Uchiyama, et al .: Anal. Chem., 73, 2165 (2001) K. Gyimesi-Forras, et al .: J. Chromatogr. A, 1083, 80 (2005) T. Yoshitake, et al .: Biomed. Chromatogr., 20, 267 (2006) K. Abe, et al .: BUNSEKI KAGAKU, 44, 555 (1995) K. Matsumoto, et al .: J. Chromatogr. B, 773, 135 (2002) M. Yoshitake, et al .: Anal. Chem., 78, 920 (2005) H. Yoshida, et al .: BUNSEKI KAGAKU, 55 (4), 213-221 (2006)

  Therefore, the present inventors have combined the above excimer fluorescence derivatization method with an HPLC-online reduction method that combines the HPLC-online reduction method capable of simultaneously and selectively analyzing oxidized and reduced thiol compounds. -It has been found that the excimer fluorescence derivatization analysis method can enable simultaneous and selective analysis of oxidized thiols and reduced thiols of a thiol compound contained in a sample. It came to be completed.

  Therefore, the present invention is a method for simultaneous and continuous analysis of thiol compounds. First, a thiol compound in a sample is separated into oxidized thiols and reduced thiols, and the oxidized thiols are reduced using a reducing agent. After the reduction, the reduced thiols originally present and the reduced thiols converted by converting the oxidized thiols can be labeled with pyrene for fluorescence detection. It is a method for simultaneous analysis of thiol compounds based on the revolutionary HPLC-on-line reduction-fluorescence derivatization analysis method that enables simultaneous and selective analysis of oxidized and reduced thiol compounds simply by injection. .

  Therefore, the present invention provides a simultaneous and continuous analysis of thiol compounds that can simultaneously and selectively analyze oxidized thiols and reduced thiols of thiol compounds contained in a sample by a method for simultaneous and continuous analysis of thiol compounds. It aims to provide a method.

  In addition, the present invention can apply an HPLC-online reduction-fluorescence derivatization analysis method that can simultaneously and selectively analyze oxidized and reduced thiol compounds. HPLC-online reduction-fluorescence derivatization analysis An object is to provide a system for simultaneous and continuous analysis of thiol compounds.

  In order to achieve the above object, the present invention is based on the HPLC-on-line reduction-fluorescence derivatization analysis method for thiol compounds capable of simultaneously and selectively analyzing oxidized and reduced thiol compounds. It is to provide a continuous analysis method.

  More specifically, the present invention relates to a method for simultaneous and continuous analysis of thiol compounds in a sample by high performance liquid chromatography (HPLC), wherein the thiol compounds are separated into oxidized thiols and first reduced thiols. A separation step of reducing the separated oxidized thiols with a reducing agent to convert them into second reduced thiols; and derivatizing the first and second reduced thiols with a fluorescent derivatization reagent. And a method for simultaneous analysis of thiol compounds comprising continuously analyzing oxidized thiols and reduced thiols in the sample by the simultaneous continuous analysis method.

  As a preferred embodiment, the present invention further includes a derivatization step of derivatizing only the first reduced thiol by not reducing the oxidized thiols separated in the separation step. Provide a continuous analysis method.

  In a preferred embodiment of the present invention, the reducing agent is, for example, tripropylphosphine, tri-n-butylphosphine, tri-t-butylphosphine, di-t-butylmethylphosphine, di-t-butylneopentylphosphine. , Trialkylphosphines such as tris (2-carboxyethyl) phosphine, tricycloalkylphosphines such as tricyclohexylphosphine and tricyclopentylphosphine, triphenylphosphine, tri (4-chlorophenyl) phosphine, tri (o- or p- Triarylphosphines such as tolyl) phosphine and diphenyl (o-tolyl) phosphine or tri (alkyl and / or cycloalkyl and / or aryl) phosphines such as t-butyldiphenylphosphine and cyclohexyldiphenylphosphine The present invention provides a method for simultaneous and continuous analysis of thiol compounds, characterized by being phosphines such as thiones.

  According to a preferred embodiment of the present invention, the fluorescent derivatization reagent is a pyrene, anthracene, acridine, coumarin, cyanine, dansyl, fluorescein, rhodamine, indacene, oxoquinoxaline or benzoxazi Provided is a method for simultaneous and continuous analysis of thiol compounds consisting of azoles.

  As another form of the present invention, an HPLC-online reduction-fluorescence derivatization analysis method that can simultaneously and selectively analyze oxidized and reduced thiol compounds can be applied. An apparatus for simultaneous and continuous analysis of thiol compounds based on a fluorescence derivatization analysis system is provided.

  As a preferred embodiment of the present invention, there is provided a simultaneous and continuous analysis apparatus for thiol compounds by high performance liquid chromatography (HPLC) for simultaneously analyzing thiols in a sample into oxidized thiols and reduced thiols, A separation unit that separates the thiols in the sample into oxidized thiols and first reduced thiols; a reducing unit that reduces the separated oxidized thiols into second reduced thiols; There is provided a simultaneous and continuous analysis apparatus for a thiol compound, comprising: a derivatization unit for derivatizing each second reduced thiol with a fluorescent derivatization reagent; and a fluorescence measurement unit.

  In a preferred embodiment of the present invention, a simultaneous thiol compound simultaneous analysis apparatus combines the separation unit and the fluorescence derivatization unit, and includes a first flow path including the reduction unit; A thiol compound simultaneous analysis apparatus further comprising: a second flow path not containing the reducing part, wherein the part is coupled to the fluorescent derivatization part.

  As a preferred embodiment of the present invention, the simultaneous thiol compound simultaneous analysis apparatus provides a thiol compound simultaneous continuous analysis apparatus in which the first flow path is further provided with a reduction column that constitutes the reduction unit.

  In a preferred embodiment of the present invention, the reduction column is provided with a trialkylphosphine such as tripropylphosphine or tri-n-butylphosphine, a tricycloalkylphosphine such as tricyclohexylphosphine, or a trialkylphosphine such as triphenylphosphine. An apparatus for simultaneous and continuous analysis of thiol compounds containing a reducing agent comprising phosphines such as reel phosphines or tri (alkyl and / or cycloalkyl and / or aryl) phosphines is provided.

  According to a preferred embodiment of the present invention, the fluorescent derivatization reagent is a pyrene, anthracene, acridine, coumarin, cyanine, dansyl, fluorescein, rhodamine, indacene, oxoquinoxaline or benzoxazi An apparatus for simultaneous and continuous analysis of thiol compounds that are azoles is provided.

  Furthermore, the present invention provides, as another form thereof, a method for simultaneous and continuous analysis of the thiol compound using the simultaneous and continuous analyzer for the thiol compound, wherein the oxidized thiols and the first reduced form separated by the separation unit The thiols are allowed to flow through the first flow path, the separated oxidized thiols are converted into second reduced thiols by the reducing section provided in the first flow path, and then the fluorescent derivatization section is converted to a fluorescent derivatization section. The fluorescence measuring unit measures the first reduced thiols and the second reduced thiols, and if necessary, the oxidized thiols and the first reduced thiols separated by the separating unit in the second flow path. The present invention provides a method for simultaneous and continuous analysis of thiol compounds, which comprises measuring only the first reduced thiols by fluorinating and measuring without fluorination.

  The simultaneous method of the thiol compound according to the present invention is such that the thiol compound contained in the sample can be simultaneously and selectively analyzed into oxidized thiols and reduced thiols simply by injecting the sample into the HPLC. A phased HPLC-on-line reduction-excimer fluorescence derivatization analysis method as well as a simultaneous continuous analysis system applicable to this method. In addition, this invention can be expected to increase the selectivity and sensitivity of the analysis of oxidized thiols and reduced thiols.

  The method according to the present invention is a method for simultaneous analysis of thiol compounds in a sample by high performance liquid chromatography (HPLC), wherein the thiol compound is separated into oxidized thiols and first reduced thiols. A reduction step of reducing the separated oxidized thiols with a reducing agent to convert them into second reduced thiols; and a derivatization step of derivatizing the first and second reduced thiols with a fluorescent derivatizing reagent. And a simultaneous continuous analysis method for a thiol compound capable of continuously analyzing oxidized thiols and reduced thiols in the sample. According to the simultaneous continuous analysis method of the present invention, it is possible to analyze a thiol compound contained in a sample simultaneously and selectively into oxidized thiols and reduced thiols simply by injecting the sample into HPLC. It is a continuous analysis method of a typical thiol compound.

  In the present invention, first, a sample is injected into a separation column of high performance liquid chromatography (HPLC) to separate a thiol compound contained in the sample into oxidized thiols and reduced thiols. The sample that can be used in the present invention is not particularly limited, and any sample containing a thiol compound in the sample may be used. As the thiol compound, oxidized thiols such as lipoic acid and thiooctamide A sample in which reduced thiols are mixed is preferable. Examples of such samples include pharmaceutical preparations containing α-lipoic acid (thioctic acid) such as thioctic acid injection, pharmaceutical preparations containing thiooctamide such as thioctic acid amide, various supplements containing lipoic acid, and the like. And biological samples such as urine and blood, such as α-lipoic acid and thiooctamide, when taking or supplements. The present invention can also be applied to measurement of samples containing peptide hormones such as cysteine-containing peptides such as oxytocin, vasotocin and vasopressin.

  Depending on the separation, the order in which the oxidized thiols and the reduced thiols elute varies depending on the structure of the analyte and the nature of the separation column, and thus cannot be specified. However, in this invention, since the eluate containing oxidized thiols and reduced thiols can be continuously sent to the reduction process, it is necessary to consider the elution order of oxidized thiols and reduced thiols. There is no.

  The eluate eluted from the separation column of high performance liquid chromatography (HPLC) is sent to the reduction process where the reducing agent is continuously placed, and only the separated oxidized thiols in the eluate are reduced by the reducing agent. And converted to the corresponding reduced thiols. That is, the S—S bond (disulfide bond) of the oxidized thiol is reduced by the reducing agent and converted into —SH group (sulfhydryl group).

  If the oxidized thiols in the sample are reduced and converted to reduced thiols before separation, the total amount of oxidized thiols and reduced thiols can be measured. It should be noted that individual quantification with type thiols is not possible. Therefore, it is necessary to convert oxidized thiols to reduced forms after separation from reduced thiols.

  Examples of the reducing agent that can be used in the present invention include reducing agents such as phosphines, but are not limited thereto, and any reducing agent that can reduce oxidized thiols is used. be able to. Examples of such phosphines include tripropylphosphine, tri-n-butylphosphine, tri-t-butylphosphine, di-t-butylmethylphosphine, di-t-butylneopentylphosphine, and tris (2-carboxyethyl). Trialkylphosphines such as phosphine, tricycloalkylphosphines such as tricyclohexylphosphine and tricyclopentylphosphine, triphenylphosphine, tri (4-chlorophenyl) phosphine, tri (o- or p-tolyl) phosphine, diphenyl (o- Triarylphosphine such as (tolyl) phosphine or tri (alkyl and / or cycloalkyl and / or aryl) phosphine such as t-butyldiphenylphosphine and cyclohexyldiphenylphosphine. it can. Of these, trialkylphosphines are preferable, and tri-n-butylphosphine is more preferable.

  As described above, the reduced thiols are passed through the reduction step, and the reduced thiols contained in the sample from the beginning (hereinafter also referred to as “first reduced thiols”), This means that reduced thiols produced by reducing oxidized thiols in the reduction step (hereinafter also referred to as “second reduced thiols”) are included. In addition, since this invention can analyze a thiol compound, this invention can be applied even when the reduced thiols are not contained in the sample from the beginning. Therefore, the present invention can be applied even when it is unclear whether the sample contains oxidized thiols or reduced thiols.

  The sample that has passed through the reduction process as described above is subsequently sent to the derivatization process. In the derivatization step, both the first reduced thiols and the second reduced thiols in the sample are derivatized.

  Various types of derivatization methods can be used in the present invention. Examples of the derivatization method include a fluorescence derivatization method using a non-fluorescent reagent as described above (for example, see Non-Patent Documents 5-7), a special fluorescence phenomenon (fluorescence polarization, time-resolved fluorescence, And derivatization methods (for example, see Non-Patent Documents 8-11) and the like in which fluorescence resonance energy transfer, intramolecular excimer fluorescence, and the like are introduced.

  Excimer fluorescence, for example, when aromatic fluorescent molecules with relatively long fluorescence lifetimes, such as pyrenes and anthracenes, are present in close proximity to each other, do not form a stable dimer in the ground state, but one molecule When excited light is absorbed and becomes an excited state, it associates with other ground state molecules to form an excited dimer (excimer), which is a light emission phenomenon from this excimer. This excimer fluorescence includes intermolecular excimer fluorescence generated between two fluorescent molecules and intramolecular excimer fluorescence generated between two luminophores existing in the same molecule (Non-patent Document 11). .

  In this invention, by introducing the excimer fluorescence phenomenon into the derivatization analysis, a derivative in which one luminophore is introduced per molecule of the target substance, which has been difficult by the conventional derivatization method, and a plurality of derivatives introduced. It is preferred to use an intramolecular excimer fluorescence derivatization method that can be distinguished spectroscopically. By this method, a fluorescent derivatization reagent having a plurality of pyrene structures and the like is introduced into a measurement target substance having a plurality of reaction sites, and excimer fluorescence emitted from the derivatized measurement target substance is detected. Selective analysis of compounds can be done. In other words, this makes it possible to selectively analyze target compounds without being affected by measurement reagents that have only a single reaction site coexisting in a large amount in a fluorescent reagent or sample that exists in excess in the reaction solution. it can.

  That is, the fluorescent derivatization reagent that can be used in the present invention can reduce the sulfhydryl group (SH) of the reduced thiols of the thiol compound, absorb the excitation light, enter the excited state, and emit excimer fluorescence. Any aromatic fluorescent molecule can be used as long as it can bind to the sulfhydryl group (SH) of the reduced thiols. Examples of such aromatic fluorescent molecules include pyrenes, anthracenes, acridines, coumarins, cyanines, dansyls, fluoresceins, rhodamines, indacenes, oxoquinoxalines, and benzoxadiazoles. . Accordingly, examples of fluorescent derivatization reagents that can be used in the present invention include N- (1-pyrenyl) maleimide (NPM), N- [4- (5,6-dimethoxy-2-phthalimidyl) phenyl] maleimide (DPM), N- [ 4- (6-Dimethylamino-2-benzofuranyl) phenyl] maleimide (DBPM), N- [4- (6-Methylamino-2-benzofuranyl) phenyl] maleimide (MBPM), N- (9-acridinyl) maleimide (NAM), N- [ 4- (2-Benzoxazolyl) phenyl] maleimide (BPM), 4- (1-pyrene) butanoyl chloride (PBC), 4- (1-pyrene) butanoic acid N-hydroxysuccinimidyl ester (PSE), 4 , 4-difluoro-4-borer 3α, 4α-diazers-indacene (BODIPY) -N- (2-aminoethyl) maleimide, 6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl chloride (DMEQ), 7-fluorobe And Nzu-2-oxa-1,3-diazole-4-sulfonamide (ABD-F). Of these fluorescent derivatization reagents, pyrenes are preferred, and N- (1-pyrenyl) maleimide (NPM) is more preferred.

  In this invention, excimer fluorescence is generated by derivatizing the thiol compound as described above and irradiating the intrinsic excitation light of the derivatization reagent used, so that the thiol in the sample can be analyzed by analyzing the fluorescence. The oxidized and reduced thiols of the compound can be analyzed simultaneously and continuously. In the case of N- (1-pyrenyl) maleimide (NPM) as a typical excimer fluorescence-expressing compound, when excitation light with an excitation wavelength of 345 nm is irradiated, normal fluorescence from the monomer has a fluorescence maximum near 375 nm. Excimer fluorescence shifts long wavelength around 485 nm. As a result, the oxidized and reduced thiols in the sample can be analyzed simultaneously and continuously for both monothiols and polythiols (see FIG. 1).

  Here, referring to FIG. 2, simultaneous use of a simultaneous continuous analyzer (HPLC) of oxidized thiols and reduced thiols based on the HPLC-online reduction-excimer fluorescence derivatization system according to the present invention. A continuous analysis method will be described.

  As shown in FIG. 2, the mobile phase 1 performs measurement of a measurement sample by HPLC smoothly and quickly, and also has effects such as promoting a derivatization reaction, adjusting pH, preventing oxidation, and reducing interference by metals. For example, it is preferable to use an aqueous methanol solution of tris (hydroxymethyl) aminomethane and EDTA as the mobile phase. A sample as an object to be measured is injected by the injector 4 into the mobile phase 1 pumped into the HPLC apparatus from the container 2 containing the mobile phase 1 by the pump 3, and sent to the separation column 5. Note that the composition condition of the mobile phase 1 is preferably adjusted as appropriate depending on the type of the measurement sample. The HPLC separation column 5 separates the thiol compound in the sample into oxidized thiols and reduced thiols (first reduced thiols) (separation step). The eluate eluted from the separation column 5 is sequentially sent to the reduction column 8 through the flow path 7 by the injector 6. The reducing column 8 is filled with the above reducing agent, and the oxidized thiols contained in the eluate are reduced (reducing step) to be converted into reduced thiols (second reduced thiols) to be It is sent to the derivatization process. However, since the first reduced thiols in the eluate are not reduced by the reducing agent, they pass through the reduction column 8 as they are and are sent to the next derivatization step.

  On the other hand, a part of the eluate containing oxidized thiols and reduced thiols separated by the separation column 5 can be sent to a flow channel 9 different from the flow channel 7 as necessary. . Since no reduction column is disposed in the flow path 9, the oxidized thiols in the eluate pass through without being reduced and are sent to the next derivatization step. That is, the eluate that has passed through the flow path 9 contains oxidized thiols and reduced thiols (first reduced thiols) of the thiol compound in the sample.

  In the next derivatization step, the fluorescent reagent solution 10 pumped by the pump 12 is injected from the container 11 containing the fluorescent reagent solution 10 into the sample sent from the flow path 7 or 9. The fluorescent reagent solution 10 can be prepared by mixing a suitable solvent with a fluorescent derivatizing reagent such as pyrenes. The mixed solution of the sample thus prepared and the fluorescent derivatization reagent of the fluorescent reagent solution 10 is sent to the reaction tube 13. The reactor is not limited to the reaction tube, and may have any shape as long as it meets the purpose. In this reaction tube 13, the reduced thiols in the sample react with the fluorescent derivatization reagent in the fluorescent reagent solution 10 to derivatize the reduced thiols. This derivatization reaction is performed in the fluorescent reagent solution 10, but the reaction temperature and reaction time can be appropriately changed depending on the type of thiol compound in the sample, the fluorescent derivatization reagent used, and the like.

  On the other hand, the oxidized thiols in the sample sent from the flow path 9 are not derivatized with the fluorescent derivatization reagent, and therefore remain in the measurement sample as they are. However, the reduced thiols (first reduced thiols) present in the sample are naturally derivatized with a fluorescent derivatization reagent in the derivatization step.

  After completion of the derivatization process, the measurement sample is sent to a fluorescence measurement process including a fluorescence detector 14 and an integrator 15, and irradiated with excitation light having a predetermined excitation wavelength, so that the derivatized reduced thiols are qualitatively Quantitative analysis is possible. The fluorescence measurement method can be carried out by a method commonly used in the technical field.

  That is, as shown in FIG. 1, according to the present invention, the reduced polythiols (first reduced thiols) in the sample are derivatized with a fluorescent derivatization reagent and the excimer fluorescence is measured. Can be analyzed. On the other hand, oxidized polythiols in a sample are analyzed by reducing them once to reducing polythiols (second reduced thiols) with a reducing agent, derivatizing with a fluorescent derivatizing reagent, and measuring the excimer fluorescence. can do. In addition, monothiols in a sample can be analyzed by derivatizing with a fluorescent derivatization reagent and measuring the monomer fluorescence. Therefore, the polythiols and monothiols in the sample can be separated and analyzed by the difference in fluorescence.

  In contrast, oxidized thiols and reduced thiols in a sample can be analyzed by converting oxidized thiols to reduced thiols, derivatizing them, and measuring their excimer fluorescence. In addition, as described above, the oxidized thiols that were sent through another flow path different from the flow path in which the reduction column was arranged were similarly treated, and thus were originally included in the sample. Only the reduced thiols in the original sample can be quantitatively analyzed without being disturbed by oxidized thiols. Moreover, the method for simultaneous and continuous analysis of thiol compounds according to the present invention has the great advantage that it can be carried out simply by injecting a sample into the apparatus.

  DL-thioctic acid (α-lipoic acid) and DL-6,8-thiooctamide (pharmaceuticals), which are polythiols showing oxidized and reduced forms, were used as measurement samples. Alpha lipoic acid functions as a coenzyme for various enzymes in the human body and has recently been approved as a food for specified health use. N- (1-pyrenyl) maleimide (NPM) was used as a pyrene fluorescent derivatization reagent.

Specific HPLC conditions when the above compound was analyzed by an online reduction post-label HPLC system (see FIG. 2) are as follows.
HPLC conditions Separation column: XBridge ^TM C ₁₈ (inner diameter 3.0 mm × length 15 cm, particle size 5 [mu] m, manufactured by Waters Co.)
Reduction column: XBridge ^TM C ₁₈ (inner diameter 2.1 mm x length 3 cm, particle size 5 μm, manufactured by Waters) holding tri-n-butylphosphine injected from an injector Mobile phase: 10 mM Tris (hydroxy 35% (v / v) methanol (0.4 mL / min) in which (methyl) aminomethane and 5 mM EDTA · 2Na are dissolved
Derivatization solution: 50 μM NPM (prepared with acetonitrile) (flow rate 0.4 mL / min)
Fluorescence detection: excitation wavelength 345 nm, excimer fluorescence 485 nm

  The reducing column was prepared by injecting an optimum concentration of tri-n-butylphosphine (reducing agent) from the injector 6 in FIG. After injecting the reducing agent, the mobile phase was fed for about 30 minutes. Note that no reduction in reducing power was observed even during 48 hours of continuous operation. However, when a solvent having a strong dissolution power was fed, peeling of the injected reducing agent from the column was observed. These facts show that the prepared reduction column has sufficient durability, and the column can be easily washed, initialized, and refilled with the reducing agent.

  For routine analysis, not only the mobile phase solution but also the stability of the post-column derivatization reagent (fluorescent reagent) solution is an indispensable factor. It was confirmed that the NPM solution prepared with acetonitrile remained stable for more than a week and continuously provided good data with no variation when stored in the dark with the outside air removed.

  A 5 μL mixture of lipoic acid and thiooctamide, which is the analyte, was injected from the injector 4 into the online reduction post-label HPLC system, analyzed under the above HPLC conditions, and analyzed at the excimer fluorescence wavelength. The analysis result is shown by a chromatogram (FIG. 3).

  As shown in FIG. 3, when the oxidized / reduced mixed solution of lipoic acid and thiooctamide is analyzed under the condition of passing through the reducing column (channel 7), lipoic acid (oxidized) → lipoic acid (reduced) → thiooctamide They were separated and detected in the order of (oxidized type) → thiooctamide (reduced type). On the other hand, when each of the oxidized form and reduced form of lipoic acid and thiooctamide was analyzed under conditions that did not pass through the reducing column (channel 9), only the reduced form gave a peak. These results indicated that the structure of the oxidized / reduced structure can be easily confirmed by simply switching the flow path. In FIG. 3, the left two chromatograms are data not passing through the reduction column.

  To create a calibration curve for lipoic acid and thiooctamide, prepare oxidized and reduced samples of lipoic acid and thiooctamide to be stepped to 0.5 mM, 1 mM, 2.5 mM, 5 mM, and 10 mM, respectively. 5 μL was injected into an online reduction post-label HPLC system, processed in the same manner as in Example 1, and analyzed at the excimer fluorescence wavelength. A calibration curve and a detection limit value obtained by analysis under the above conditions are shown in FIG. From this result, it became clear that any compound can be measured with extremely high sensitivity and high selectivity by a simple operation of sample injection.

  The oxidized cysteine-containing peptides vasopressin, vasotocin and oxytocin were analyzed using an HPLC system in the same manner as in Example 1. The analysis result is shown by a chromatogram (FIG. 5). When the cysteine-containing peptide was analyzed under the condition of passing through the reduction column (channel 7), each peptide was detected. On the other hand, each peptide did not give a peak under the condition not passing through the reduction column (flow path 9). From these results, it was confirmed that it can be applied not only to lipoic acid-related compounds but also to measurement of cysteine-containing peptides. In addition, the left chromatogram in FIG. 5 is data not passing through the reduction column.

  This invention can be applied to any thiol compound by simply injecting the oxidized / reduced thiols of thiol compounds in biological samples such as urine and blood such as pharmaceuticals and supplements into the HPLC system. Since compounds can also be measured with extremely high sensitivity and high selectivity, they can be usefully used particularly in the inspection department in the field of pharmaceuticals and supplements.

Explanatory drawing which shows the concept of the on-line reduction-excimer fluorescence derivatization method of a thiol compound. 1 shows an HPLC-online reduction-excimer fluorescence derivatization system. The figure which shows the chromatogram obtained from the lipoic acid and thiooctamide liquid mixture (Example 1). The figure which shows the detection limit and calibration curve about lipoic acid and thiooctamide (Example 2). The figure which shows the chromatogram obtained from the cysteine containing peptide liquid mixture (Example 3).

Claims (14)

A method for simultaneous and continuous analysis of thiol compounds in a sample by high performance liquid chromatography (HPLC),
A separation step of separating the thiol compound into oxidized thiols and first reduced thiols;
A reduction step of reducing the separated oxidized thiols with a reducing agent to convert them into second reduced thiols;
A derivatization step of derivatizing the first and second reduced thiols with a fluorescent derivatization reagent;
A method for simultaneous and continuous analysis of thiol compounds, characterized in that oxidized thiols and reduced thiols in the sample are continuously analyzed by the simultaneous and continuous analysis method.   The method for simultaneous continuous analysis of thiol compounds according to claim 1, further comprising a derivatization step of derivatizing only the first reduced thiols without reducing the oxidized thiols separated in the separation step. A method for simultaneous and continuous analysis of thiol compounds.   3. The method for simultaneous and continuous analysis of thiol compounds according to claim 1 or 2, wherein the reducing agent is a phosphine.   4. The method for simultaneous analysis of thiol compounds according to claim 3, wherein the phosphine is a trialkyl phosphine, tricycloalkyl phosphine, triaryl phosphine or tri (alkyl and / or cycloalkyl and / or aryl) phosphine. A method for simultaneous and continuous analysis of thiol compounds.   The method for simultaneous analysis of thiol compounds according to any one of claims 1 to 4, wherein the fluorescent derivatization reagent comprises pyrenes, anthracenes, acridines, coumarins, cyanines, dansyls, fluoresceins, A method for simultaneous and continuous analysis of thiol compounds, which is rhodamines, indacenes, oxoquinoxalines or benzoxadiazoles. An apparatus for simultaneous and continuous analysis of thiol compounds by high performance liquid chromatography (HPLC) for simultaneous and continuous analysis of thiols in a sample into oxidized thiols and reduced thiols,
A separation unit for separating thiols in the sample into oxidized thiols and first reduced thiols;
A reducing unit that reduces the separated oxidized thiols to second reduced thiols;
A derivatization unit for derivatizing each of the first and second reduced thiols with a fluorescent derivatization reagent;
A fluorescence measuring unit;
An apparatus for simultaneous and continuous analysis of thiol compounds, comprising: The simultaneous analysis apparatus for thiol compounds according to claim 6,
A first flow path that combines the separation unit and the fluorescent derivatization unit and includes the reduction unit;
A second flow path that combines the separation unit and the fluorescent derivatization unit and does not include the reduction unit, if necessary;
The thiol compound simultaneous analysis apparatus characterized by further including.   The simultaneous continuous analyzer according to claim 6 or 7, wherein the first flow path is provided with a reducing column that constitutes the reducing unit.   9. The simultaneous and continuous analysis apparatus for thiol compounds according to claim 8, wherein the reducing column is filled with a reducing agent.   The simultaneous and continuous analysis apparatus for thiol compounds according to claim 9, wherein the reducing agent is a phosphine.   The simultaneous thiol compound analysis apparatus according to claim 9 or 10, wherein the phosphine is a trialkylphosphine, a tricycloalkylphosphine, a triarylphosphine, or tri (alkyl and / or cycloalkyl and / or aryl). An apparatus for simultaneous and continuous analysis of thiol compounds characterized by being a phosphine.   The thiol compound simultaneous analysis apparatus according to claim 6, wherein the fluorescent derivatization reagent is pyrenes, anthracenes, acridines, coumarins, cyanines, dansyls, fluoresceins, rhodamines, indacenes, oxo An apparatus for simultaneous and continuous analysis of thiol compounds, which is a quinoxaline or a benzoxadiazole.   The implementation of the method for simultaneous and continuous analysis of thiol compounds according to any one of claims 1 to 5 using the simultaneous and continuous analysis apparatus for thiol compounds according to any one of claims 6 to 12. The sample containing the oxidized thiols and the first reduced thiols separated in step 1 is sent to the first channel, and the separated oxidized thiols are second reduced by the reducing unit provided in the first channel. After conversion to thiols, the first and second reduced thiols are sent to the fluorescent derivatization section, derivatized with a fluorescent derivatization reagent, and measured by the fluorescence measurement section simultaneously. Analysis method.   14. The simultaneous continuous analysis method for a thiol compound according to claim 13, wherein, if necessary, the oxidized thiols and the first reduced thiols separated in the separation section are flowed through the second flow path and are not reduced. A method for simultaneous and continuous analysis of thiol compounds, characterized in that only the first reduced thiols are measured by fluorescence measurement.