JP2013137326A - Analyzing method and analyzer for test object - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of analyzing a plurality of sulfur containing compounds such as cysteine and cystine included in a measurement sample simultaneously in a short time.SOLUTION: There is provided an analyzing method for two or more kinds of test objects including a step (1) of making the measurement sample flow to a flow passage together with a moving phase; a step (2) of making a moving phase which does not include the measurement sample flow to a flow passage P2 and a flow passage P3; and a step (3) of acquiring chromatograms of two or more kinds of test objects exiting from analysis columns through the steps (1) and (2) (where the flow passage passes through a pre-processing colum forward and then passes through the analysis column to reach a detector, the flow passage P2 passes through the analysis column to reach the detector without passing through the pre-processing column, and the flow passage P3 passes through the pre-processing column backward and passes neither the analysis column nor the detector to be discharged). The step (1) ends after the two or more kinds of test objects pass through the pre-processing column in the step (1), and then the step (2) starts.

Description

  The present invention relates to a method for analyzing a test object and an analysis apparatus, and more particularly to a method for analyzing a sulfur-containing compound contained in a measurement sample, particularly a substance having a thiol group such as cysteine and cystine, or a disulfide bond. The present invention is composed of multiple components such as sugars, electrolytes, amino acids, etc. like infusion preparations and biological samples, and is suitable for highly sensitive and highly accurate analysis in viscous measurement samples.

  It is known that cysteine, which is one of the active ingredients such as infusion preparations, is less stable than other amino acids and is changed to cystine during storage and the content is reduced. Therefore, accurate quantification of cysteine, cystine, etc. in the preparation is important for quality control.

  As a conventional standard analysis method for L-cysteine, L-cysteine is reacted with 4,4′-dithiodipyridine (4-PDS) at a maximum absorption wavelength of 324 nm derived from 4-thiopyridone as a reaction product. An ultraviolet-visible absorbance measurement method (4-PDS method) for measuring absorbance can be mentioned. However, the 4-PDS method is an inefficient test method that requires a long analysis operation and is complicated.

  A conventional standard analysis method for L-cystine is an amino acid analysis method, and includes a post-column derivatization method using a derivatization reagent that selectively reacts with an amino group. The post-column derivatization method is a method in which an amino acid is separated by cation exchange chromatography, a derivatization reagent (ninhydrin) is fed, reacted with the amino acid, and analyzed with a visible absorbance detector. The post-column derivatization method is an inefficient analysis method because the analysis time per sample is as long as about 134 minutes.

  Moreover, the conventional analysis method described above cannot simultaneously analyze L-cysteine and L-cystine.

On the other hand, sulfur-containing amino acids, peptides, and proteins are known to play an important role in physiological activities of biological systems. The thiol group (—SH) is more active than hydroxyl groups (—OH) such as alcohols, and easily forms a dimer through a disulfide bond (—S—S—) due to a change in pH or an enzyme. Or other molecules with thiol groups.
Biological systems skillfully utilize the reactivity of thiol groups to maintain life while performing biosynthesis and metabolism.
There are many known amino acids such as cysteine-cystine, homocysteine-homocystine, glutathione-glutathione disulfide, and thiamine thiol type-thiamin disulfide known as vitamin B1. (See Non-Patent Documents 1 and 2).

For samples with complex compositions, such as biological samples, pretreatment with a fluorescent derivatization reagent that selectively reacts with thiol groups, separation by liquid chromatography, and fluorescence detection or using carbon electrodes A method of directly detecting a thiol group with an electrochemical detector is generally used.
In the derivatization method (pre-column derivatization method), it is necessary to adjust the sample to neutral to weak alkaline during the derivatization reaction. Under these conditions, thiol groups tend to form disulfide bonds, and many samples can be accurately obtained. It is very difficult to process.
As described later, an electrochemical detector using a carbon electrode has poor stability, and it is very difficult to perform measurement with high accuracy.

  The usefulness of the -S-S- bond is unquestionably given an example. For example, allicin, which is known as an active ingredient of garlic, is disulfatide, but alliamines produced by the combination of allicin and thiol-type thiamine are moderately fat-soluble and thus improve intestinal absorption. Even in health foods and pharmaceuticals, it is valuable as a method for absorbing the active ingredients in the body. In the body, these compounds are reduced by enzymes. It is a so-called pro-drug usage. Also, in plants, it has been found that these thiols play an important role in the production of active ingredients by biosynthesis (actual ingredients can be produced in the middle of various circuits → metabolic pathways). In recent years there have been interesting reports. Low molecular thiols have an extremely high fragrance threshold and are important in determining good fragrance. For example, furanthiol, which is famous as an incense of strawberry, mercaptohexanol, which is important as an incense of grapefruit, determines an incense that may be in the order of ppb to ppt. In wine, products containing mercaptohexanol have high added value, and it has become important to manage their production. In grapes, mercaptohexanol is produced in the form of a precursor that is -SS-linked to cysteine. By measuring these before harvesting, the best time can be determined (see Non-Patent Document 3).

  These current measurement methods are as follows. Grape juice is poured into a reaction column on which an enzyme that cleaves the -S-S- bond is immobilized, mercaptohexanol is recovered with an organic solvent, and then concentrated in a nitrogen stream or the like to obtain GC / An extremely expensive preprocessing operation for MS and an expensive analyzer are required.

The electrochemical detector is often used after the UV-visible, the differential refractive index, and the fluorescence detector, and is characterized by high selectivity and high sensitivity. For this reason, application to the biochemical field and the environmental field, which includes a large amount of contaminants and has a very small amount of the target component, is being advanced. The main advantages of this electrochemical detector are as follows: (1) It is possible to analyze trace components that require a sensitivity of 4 orders of magnitude or more than the UV-visible light detector generally used in liquid chromatography. (2 ) From the reaction principle, it is highly selective because only the oxidizing or reducing substance reacts electrochemically.
Many compounds form reaction products that form an inert film on the working electrode surface, which causes the detector response to change over time. Among them, phenol is known as a very bad film-forming substance, and in an electrochemical detector using a carbon electrode (graphite, glassy carbon, etc.) as a working electrode, the response continuously decreases even after only a few hours of operation. In that case, the cell must be disassembled and the working electrode surface must be polished and renewed. It takes several hours to reassemble and equilibrate until trace components begin to be analyzed. The fact that the response changes continuously or requires elaborate maintenance is limited in spite of various advantages due to the fact that it is cumbersome and very expensive for ordinary users. ing.
In addition, since a high applied voltage cannot be applied to a glassy carbon electrode, it is difficult to stably detect a disulfide bond having a high reaction potential.

  On the other hand, a diamond electrode using conductive diamond imparted with conductivity by doping boron into the working electrode has been widely proposed. The conductive diamond electrode has a wide potential window in an aqueous solution, that is, a wide potential region in which electrolysis of water does not occur, and can perform a stable reaction by applying a high applied voltage. Furthermore, the width of the potential window is determined by the overpotential for hydrogen generation and oxygen generation on the electrode surface, but these reactions are generally multistage multielectron transfer reactions via a reaction intermediate that is weakly adsorbed on the electrode surface. It is. The conductive diamond surface has only a negligible site for such intermediates to be adsorbed, and therefore, a high sensitivity analysis with a low background current value and a low noise level is possible.

Conductive diamond electrodes are used for various types of detection.
Some of the present inventors use a conductive diamond electrode for the detector flow cell of liquid chromatography, and enable detection by applying a high potential to a hardly reactive substance (Patent Document 1). This method, in combination with a coulometric cell, improves the separation by removing electrochemically active contaminants and trace substances and converting them to another substance.
Fujishima et al. (Patent Document 2) utilizes the property that a diamond electrode is specifically sensitive to hydrogen peroxide, and is applied to the concentration detection of a substance by an enzyme reaction.
Furthermore, Nagaoka et al. (Patent Document 3) provide a flow-type amino acid analyzer with a detection unit that uses a conductive diamond electrode that has been subjected to mirror surface treatment and subjected to oxygen termination. Oxygen termination is performed using plasma in an oxygen-saturated atmosphere, but this method cannot be performed while the mobile phase is flowing with the electrode installed and the system is assembled, and is offline before the start of detection. It is necessary to do it in. For this reason, when performing multi-sample and continuous repeated analysis, the electrode surface state is inevitably changed, and stable analysis is impossible.

International Publication No. 01/67089 Pamphlet JP 2003-121410 A JP 2005-69692 A

Biomed. Chromatogr. 3,166-172 (1989) Cancer Res. 61, 4365-4370 (2001) Tominaga Takatoshi: Kiroiro Incense, Fragrance Journal

  An object of the present invention is to provide an efficient analysis method for an object to be detected contained in a measurement sample and an analysis apparatus therefor. Preferably, an object of the present invention is to provide a method capable of simultaneously analyzing a plurality of sulfur-containing compounds such as cysteine and cystine contained in a measurement sample in a short time. More preferably, it is composed of multiple components such as sugars, electrolytes, and amino acids such as infusion preparations and biological samples, and can analyze sulfur-containing compounds such as cysteine and cystine in a viscous sample with high accuracy in a short time. It is an object to provide a method.

The present inventors diligently studied to solve the above problems, and completed the present invention. The present invention includes the following matters.
[1] A method for analyzing a test object contained in a measurement sample,
A step (1) of flowing the measurement sample together with the mobile phase to the following flow path P1;
A step (2) of flowing a mobile phase not containing a measurement sample in the following flow paths P2 and P3, and
(3) obtaining a chromatogram of the test object from the analytical column in steps (1) and (2) by an electrochemical detector having a diamond electrode as a working electrode (however, The flow path P1 is a flow path that passes through the pretreatment column in the forward direction and then passes through the analysis column to the detector, and the flow path P2 does not pass through the pretreatment column and flows to the detector through the analysis column. The flow path P3 is a flow path that passes through the pretreatment column in the reverse direction and is discharged without passing through the analysis column and without passing through the detector.
The said analysis method which complete | finishes a process (1) and starts a process (2) after the said test object passes a pre-processing column in a process (1).
[2] The analysis method according to [1], wherein a contaminant that requires a significantly longer passage time than the test object contained in the measurement sample does not pass through the analysis column.
[3] The analysis method according to [1] or [2], wherein the test object is a sulfur-containing compound.
[4] The analysis method according to [3], wherein the sulfur-containing compound is a compound having a thiol group or a disulfide bond.
[5] The analysis method according to [3], wherein the sulfur-containing compound is a sulfur-containing amino acid.
[6] The analysis method according to [3], wherein the sulfur-containing compound is N-acetylcysteine.
[7] The analysis method according to [3], wherein the sulfur-containing compound is homocysteine or glutathione.
[8] The analysis method according to the above [1] or [2], wherein the test object is two or more sulfur-containing compounds.
[9] The analysis method according to [8], wherein the two or more sulfur-containing compounds are cysteine and cystine.
[10] The analysis method according to [1] or [2] above, wherein the test object is two or more amino acids.
[11] The analysis method according to any one of [1] to [10], wherein the mobile phase contains water and an electrolyte.
[12] The analysis method according to any one of [1] to [10], wherein the mobile phase contains water, an electrolyte, and an ion pair compound, and the pH is 1 to 3.
[13] The analysis method according to [12], wherein the ion pair compound is at least one selected from the group consisting of alkyl sulfonates and alkyl sulfates.
[14] The analysis method according to any one of [1] to [13], wherein the analysis column is any one of a reverse phase column, a normal phase column, and an ion exchange column.
[15] The analysis method according to [14], wherein the analysis column is a reverse phase column.
[16] The analysis method according to any one of [1] to [15] above, wherein the diamond electrode is a conductive diamond electrode.
[17] The analysis method according to [16], wherein the conductive diamond electrode is an electrode subjected to oxidative electropolishing.
[18] The analysis method according to [16], wherein the conductive diamond electrode is an electrode that has been subjected to oxidative electropolishing with a mobile phase flowing.
[19] The analysis method according to any one of [1] to [18], wherein the measurement sample is any one of an infusion preparation, a dialysis agent, a fermentation broth, and a biological sample.
[20] The analysis method according to [1], wherein steps (1), (2), and (3) are started after step (3) is completed, and continuous repeated analysis is performed.
[21] A method for simultaneously detecting two or more sulfur-containing compounds contained in a measurement sample, wherein the analysis method according to any one of [1] to [20] is used.
[22] An apparatus for analyzing a test object contained in a measurement sample,
A flow path switching device capable of reversibly switching the direction of the flow path;
A sample introduction flow path for flowing the mobile phase together with the measurement sample to the flow path switching device;
A mobile phase introduction flow path for flowing the mobile phase alone to the flow path switching device;
A flow path that returns from the flow path switching device and returns to the flow path switching device again through the pretreatment column;
A flow path from the flow path switching device to the electrochemical detector having a diamond electrode as a working electrode capable of acquiring a chromatogram of the test object through the analysis column;
A discharge path from the flow path switching device to the outside of the device without passing through the pretreatment column or the analysis column,
The flow path switching device is configured to be switched to the following states (A) and (B).
(A) A state in which a flow path P1 is formed from the flow path for sample introduction to the pretreatment column in the forward direction and then to the detector through the analysis column,
(B) The pretreatment column passes through the pretreatment column in the reverse direction from the flow path for sample introduction, does not pass through the pretreatment column, passes through the analysis column, and reaches the detector. The state which comprises the flow path P3 which leads to a path.
[23] The apparatus according to [22], wherein the test object is a sulfur-containing compound.
[24] The apparatus according to [23], wherein the sulfur-containing compound is a compound having a thiol group or a disulfide bond.
[25] The apparatus according to [23], wherein the sulfur-containing compound is a sulfur-containing amino acid.
[26] The apparatus according to [23], wherein the sulfur-containing compound is N-acetylcysteine.
[27] The apparatus according to [23], wherein the sulfur-containing compound is homocysteine or glutathione.
[28] The apparatus according to [22], wherein the test object is two or more sulfur-containing compounds.
[29] The apparatus according to [28], wherein the two or more sulfur-containing compounds are cysteine and cystine.
[30] The apparatus according to [22], wherein the test object is two or more amino acids.
[31] The apparatus according to any one of [22] to [30], wherein the analytical column is any one of a reverse phase column, a normal phase column, or an ion exchange column.
[32] The apparatus according to [31], wherein the analytical column is a reverse phase column.
[33] The apparatus according to any one of [22] to [32], wherein the diamond electrode is a conductive diamond electrode.
[34] The apparatus according to [33], wherein the conductive diamond electrode is an electrode subjected to oxidative electropolishing.
[35] The apparatus according to [33], wherein the conductive diamond electrode is an electrode that has been subjected to oxidative electropolishing with a mobile phase flowing.
[36] The apparatus according to any one of [22] to [36], wherein the measurement sample is any one of an infusion preparation, a dialysis agent, a fermentation broth, and a biological sample.

  According to the present invention, highly accurate and stable measurement can be performed by using a diamond electrode type electrochemical detector having a high selectivity and a low noise level and a column switching method. In other words, contaminants that may be contained in the sample solution and cause the measurement time to increase remain in the vicinity of the inlet of the pretreatment column in step (1), and the mobile phase is reversed in step (2). By flowing, this contaminant can be removed quickly without going through the analytical column, so that the next measurement can be started immediately, and as a result, the measurement time can be greatly shortened. Therefore, continuous analysis of many samples is possible.

  In one aspect of the present invention, by using reverse phase chromatography using an ion-pair compound, a plurality of sulfur-containing substances that are suitable for testing from viscous samples containing multiple components such as infusion preparations and dialysis agents Compounds, particularly cysteine and cystine, can be clearly separated in a short time and analyzed with high accuracy. In the present invention, the separation method is not limited to reverse phase analysis using an ion pair compound, and can be used in ordinary reverse phase, normal phase, and ion exchange methods, and does not depend on the separation mechanism.

  In one aspect of the present invention, all of the sample and standard solution preparations, mobile phase, etc. are carried out under acidic conditions, and are simple operations that are not easily affected by oxidation and the formation of new disulfide bonds. In particular, a plurality of sulfur-containing compound components can be clearly separated in a short time and analyzed with high accuracy.

  In one aspect of the present invention, by using online regeneration, a plurality of sulfur-containing compound components can be clearly separated in a short time stably with high fastness, and can be analyzed accurately and simultaneously.

  According to the present invention, it is expected to provide an analysis method as a standard test method or quality test method for pharmaceutical products for application.

  According to one aspect of the present invention, two sulfur-containing amino acids, particularly cysteine and cystine, contained in a dialysis agent and the like can be clearly separated in a short time, and can be analyzed accurately and simultaneously.

In one aspect of the present invention, stable measurement can be performed without changing the system by performing an oxidative electropolishing treatment of a conductive diamond electrode while flowing a mobile phase.
Table 1 summarizes the performance of the present invention and existing analytical methods in cysteine and cystine analysis.

FIG. 1 schematically shows the flow path P1. FIG. 2 schematically shows the flow path P2 and the flow path P3. FIG. 3 shows a chromatogram obtained by analysis of a standard solution. Example 1 FIG. 4 shows a chromatogram of cysteine without oxidative electropolishing. Example 1 FIG. 5 shows a chromatogram of cysteine with oxidative electropolishing. Example 1 FIG. 6 shows a chromatogram obtained by analysis of the infusion preparation. Example 1 FIG. 7 shows a chromatogram obtained by analysis of a standard solution. (Example 2) FIG. 8 shows a chromatogram obtained by analysis of the infusion preparation. (Example 2) FIG. 9 shows a chromatogram obtained by analysis of a standard solution. (Example 3) FIG. 10 shows a chromatogram obtained by analysis of a standard solution (Example 4). FIG. 11 shows a chromatogram of a mouse plasma sample. Example 4 FIG. 12 shows a chromatogram of a sample obtained by adding a standard solution to mouse plasma. Example 4 FIG. 13 shows an enlarged view of FIG. Example 4 FIG. 14 shows an enlarged view of FIG. Example 4 FIG. 15 shows a chromatogram obtained by continuously measuring a rat plasma sample. (Example 5) FIG. 16 shows a chromatogram obtained by analysis of a standard solution. Example 1 FIG. 17 shows a chromatogram obtained by analysis of red wine. (Example 7) FIG. 18 shows a chromatogram of 3-mercapto-1-hexanol. (Example 8) FIG. 19 shows a chromatogram of furanthiol. Example 9 FIG. 20 shows a chromatogram in HILIC mode. (Example 10)

  Hereinafter, the analysis method of the test object contained in the measurement sample according to the present invention may be simply referred to as the method of the present invention. Hereinafter, the case where the test object is “cysteine and cystine” will be mainly described. However, in the present invention, the test object is not particularly limited.

  The measurement sample of the present invention is not particularly limited as long as it is a sample to be analyzed, and specific examples include infusion preparations, dialysis agents, fermentation solutions, biological samples (plasma / tissue) and the like. The analysis of the test object includes confirming the absence of the test object when it does not exist. Therefore, it is not necessary that the test object is included in the measurement sample. Preferably, the test object is two or more amino acids. The amino acids in the present invention include not only natural amino acids but also non-natural amino acids, amino acid derivatives, and amino acids, and sulfur-containing amino acids are preferred. For example, homocysteine, sulfocysteine, N-acetylcysteine (for example, N-acetyl-L-cysteine) and the like are included. The two or more sulfur-containing amino acids are preferably cysteine and cystine. Cysteine is a kind of amino acid and has a thiol group in the side chain. Cystine is a kind of amino acid and has a structure in which two molecules of cysteine are connected via a disulfide (—S—S—) bond generated by oxidation of a thiol group (—SH).

In the present invention, the test object may be a sulfur-containing compound. A sulfur-containing compound is a compound having a sulfur atom in its chemical structure. For example, a compound having a sulfur-containing functional group such as a thiol group, a disulfide bond, a sulfide bond, a sulfoxide bond, a sulfone bond, sulfinic acid, sulfonic acid, and sulfuric acid. And a compound having a thiol group or a disulfide bond is preferable. The sulfur-containing compound is preferably a sulfur-containing amino acid. Cysteine and cystine described above are specific examples of sulfur-containing amino acids. Specific examples of sulfur-containing amino acids include methionine, N-acetylcysteine (for example, N-acetyl-L-cysteine), cysteine sulfinic acid, homozygote. Examples include cysteine sulfinic acid, homocysteine, and homocystin. As a sulfur-containing compound that is not a sulfur-containing amino acid, glutathione (including reduced glutathione and oxidized glutathione) that is a sulfur-containing peptide, other 3-mercapto-1-hexanol, furanthiol, and the like are assumed.
As the sulfur-containing compound, cysteine, cystine, N-acetylcysteine, homocysteine, and glutathione are preferable.
Only one type of sulfur-containing compound may be the test subject, or two or more types may be the test subject. As will be described later, since the analysis method of the present invention can simultaneously analyze a plurality of substances, an embodiment in which two or more sulfur-containing compounds are to be tested is preferable.

  The analysis method of the test object contained in the measurement sample, particularly two or more sulfur-containing compounds (especially cysteine and cystine) is a concept that broadly encompasses finding the state of the test object in the measurement sample, The analysis may be a qualitative analysis or a quantitative analysis. Further, the method of the present invention may be used as a pharmaceutical standard test method or a quality control test method in a factory. The method of the present invention may be a method for finding out whether or not a test object is included in a measurement sample, that is, a detection method. The method of the present invention may be a method for measuring at least one concentration of cysteine and cystine in a measurement sample.

According to one embodiment of the present invention, in a pretreatment column suitable for cysteine and cystine, the effect is great in a measurement sample including a contaminant that requires a significantly longer passage time than cysteine and cystine. That is, the contaminant remains in the vicinity of the inlet of the pretreatment column in step (1), and is quickly removed without passing through the analytical column by flowing the mobile phase in the reverse direction in step (2). Examples of such contaminants include electrochemically active amino acids such as tyrosine, tryptophan, histidine, and methionine.
Note that contaminants such as sugar (such as glucose) and electrolytes (such as sodium chloride, sodium lactate, calcium gluconate, magnesium sulfate, and zinc sulfate) contained in the sample are not detected by the method of the present invention.

In the method of the present invention, a technique of liquid chromatography can be used as appropriate. For example, reverse phase column liquid chromatography using a mobile phase containing water and an electrolyte can be employed. Hereinafter, although the aspect using a reverse phase column is demonstrated, it cannot be overemphasized that this invention is not limited to this.
A general acidic mobile phase may be used as the mobile phase. In the present invention, the pH of the mobile phase is preferably 1 to 3. When the pH of the mobile phase is within the above range, there is an advantage that the analysis can be performed with high accuracy, stability and robustness in a short time. The electrolyte contained in the mobile phase is not particularly limited. Dipotassium hydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, sodium citrate, sodium acetate, Examples thereof include sodium carbonate.

An example of a suitable mobile phase is a mixture of the following components A and B.
Here, the A component is a phosphate buffer (pH 1 to 3) composed of 10 to 100 mM sodium dihydrogen phosphate and 1 to 10 mM sodium octanesulfonate, and the B component is acetonitrile. The weight ratio (A / B) between the component A and the component B is preferably 90.0 / 10.0 to 99.0 / 1.0.

  According to the knowledge of the present inventors, the analysis accuracy of cysteine and cystine is improved by including an ion pair compound in the mobile phase. In an analysis in a reverse phase system using an ODS column or the like, an ionic substance such as an acidic substance or a basic substance is in a dissociated state depending on the conditions of the mobile phase and cannot be sufficiently held in the column. In such a case, a method of holding the compound by adding an ionic substance having a charge opposite to that of the target component to the mobile phase is called ion pair chromatography, and the ionic substance to be added is called an ion pair compound. The target component and the ion pair compound form an ion pair in the mobile phase, so that the influence of the charge is reduced, and the target component and the ion pair compound are easily held in the stationary phase. In addition, by holding the ion pair compound in the stationary phase, it is expected to have a pseudo ion exchange effect and to retain the target component in the stationary phase. When an acidic mobile phase is used, since the amino group of the amino acid is ionized, an acidic ion pair compound such as an alkyl sulfonate or an alkyl sulfate is used. The ion pair compound is preferably selected from the group consisting of alkyl sulfonates and alkyl sulfates.

  The number of carbon atoms in the alkyl moiety of the alkyl sulfonate is preferably 5-12, more preferably 5-8. The “salt” in the alkyl sulfonate is preferably an alkali metal salt, and among them, a sodium salt is preferable. More specific examples of the alkyl sulfonate include sodium octane sulfonate, sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, sodium dodecane sulfonate, and the like.

  The number of carbon atoms in the alkyl moiety of the alkyl sulfate is preferably 5-12, more preferably 5-8. The “salt” in the alkyl sulfate is preferably an alkali metal salt, and a sodium salt is particularly preferable. More specific examples of the alkyl sulfate include sodium dodecyl sulfate.

  FIG. 1 schematically shows the flow path P1. However, the present invention is not limited to the illustrated embodiment. In the step (1) of the present invention, the mobile phase and the measurement sample are passed through the flow path P1. The flow path P1 is a flow path that passes through the pretreatment column 1 in the forward direction and then passes through the analysis column 2 to the detector 3. The pretreatment column 1 can be selected from those in which a plurality of sulfur-containing compounds (such as cysteine and cystine) can pass in about 4 to 15 minutes. For example, commercially available reverse phase columns include inert sill ODS-3 (manufactured by GL Science), Develosil ODS-UG (manufactured by Nomura Chemical), YMC-PackODS series (manufactured by YMC), CAPCELL PAK C18 (manufactured by Shiseido). ZORBAX Eclipse XDB (manufactured by Agilent) and the like.

  When the flow path P1 is configured in the analysis apparatus of the present invention, the flow path switching apparatus 6 that can reversibly switch the direction of the flow path is used to connect the sample introduction flow path to the flow path switching apparatus 6. Then, the flow is switched from the flow path switching device 6 to the flow path that returns to the flow path switching device 6 through the pretreatment column 1 and is further returned from the flow path switching device 6 to the test column 2 through the analysis column 2. The piping and the flow path switching device 6 may be arranged so as to reach the detector 3 that can acquire the chromatogram of the object. Here, the channel switching device 6 is typically one or more valves, and the sample introduction channel generally drives a mobile phase supply source (tank or the like) 51 and the flow of the mobile phase. A liquid feeding device (pump or the like) 41 and an injector 7 into which a measurement sample is to be introduced are provided.

  The pretreatment column 1 acts as a stationary phase in liquid chromatography. The mobile phase normally flows through the analytical column 2 in one direction. In the present invention, the flow direction in the analytical column when flowing the mobile phase to the pretreatment column 1 in the flow path P1 used in the step (1) is defined as “forward direction”, and the opposite direction is defined as “reverse direction”. Is defined. For convenience of explanation, the inlet 11 and outlet 12 of the pretreatment column 1 are defined as follows. That is, when the flow direction is “forward”, the liquid enters the column 1 from the “inlet” 11 of the pretreatment column 1 and exits from the “outlet” 12 of the pretreatment column. Therefore, when the flow direction is “reverse direction”, the mobile phase enters the column from the “exit” 12 of the pretreatment column 1 and exits from the “inlet” 11 of the pretreatment column.

  In the present invention, by passing the measurement sample together with the mobile phase through the pretreatment column 1, the solution containing the test object preferably reaches the analysis column 2 after preferably removing at least a part of the contaminants. Analytical column 2 also acts as a stationary phase in liquid chromatography and can separate, for example, cysteine and cystine. As long as the test object (a sulfur-containing compound such as cysteine or cystine) can be separated, the material and shape of the analysis column 2 are not particularly limited, and the same type of column as the pretreatment column 1 may be used.

The present invention is not limited to reverse phase chromatography. For example, analysis can also be performed by normal phase chromatography or ion exchange chromatography.
An example of a suitable mobile phase for normal phase chromatography is the following mixture of A and B components. Here, A component consists of 10-100 mM acetic acid and sodium acetate (pH 3-5), and B component is acetonitrile. The volume ratio (A / B) of the A component and the B component is preferably 40.0 / 60.0 to 5.0 / 95.0. As the normal phase column to be used, a HILIC (Hydrophilic Interaction Chromatography) column that can use an aqueous mobile phase is preferably used. Examples of commercially available HILIC columns include ZIC-HILIC (manufactured by Nomura Chemical Co., Ltd.).
An example of a suitable mobile phase for ion exchange chromatography is 0.1 to 0.3 M sodium citrate buffer. Examples of commercially available ion exchange columns include Shodex CXpak manufactured by Showa Denko KK.

  The detector 3 is a device that receives a chromatogram signal based on the presence of an object to be examined, performs calculations as necessary, and transmits information to a display means (not shown) such as a chart. The type of detector 3 is an electrochemical detector having a diamond electrode as a working electrode. The electrochemical detector is a device that has a working electrode and a reference electrode, and detects a change in current due to an oxidation reaction on the surface of the working electrode when a predetermined voltage, preferably 1200 to 2000 mV, is applied to the working electrode. . Preferably, the working electrode is a conductive diamond electrode. As the reference electrode, a general silver / silver chloride electrode or the like can be used.

  The conductive diamond electrode is an electrode made of diamond that exhibits conductivity, such as a semiconductor or metal, to which impurities of Group 3 or Group 5 are added. It is known that a conductive diamond electrode can be manufactured by a CVD method or the like, and can be obtained by appropriately taking into account known documents such as Patent Document 2. Use of a conductive diamond electrode has the advantage of being able to perform simultaneous analysis with high sensitivity, in a short period of time, with high accuracy, without fluctuations in daily stability, and with robustness. It is preferable to use on-line regeneration for electrode processing, in which case the robustness of the system is increased and the analysis accuracy is improved. Furthermore, in order to enhance the robustness, good results can be obtained if the surface of the electrode is highly oxygen-terminated so that the ratio of oxygen termination to hydrogen termination on the electrode does not gradually change. As a method for producing the oxygen termination, a method known per se in the art can be adopted without any particular limitation. For example, as described in Akira Fujishima et. Al. Edited by Diamond Chemistry, 218 (2005) Elsevier. 1. Flow oxygen gas at high temperature. Boil in a sulfuric acid-nitric acid mixture; 3. irradiation with oxygen plasma; 4. Treatment with an oxidizing agent Oxidation electrolytic polishing is preferably employed. Among these, the electrolytic electrolytic polishing method that can be executed online while feeding the mobile phase in the same manner as the analysis state without destroying the configuration of the analysis system is highly reproducible and useful for the present invention.

  The liquid feeding means for the mobile phase and the measurement sample is not particularly limited, and the mobile phase can be sent from the mobile phase supply source 51 using a general pump 41 or the like. In the embodiment of FIG. 1, the measurement sample is put into the flow path P1 in the injector 7. What is necessary is just to set the flow volume per time suitably according to the capability etc. of a column, Preferably it is 0.2-1.0 mL / min.

  In the present invention, after a plurality of sulfur-containing compounds (such as cysteine and cystine) pass through the pretreatment column 1 in the step (1), the step (1) is finished and the step (2) is started. Since the time required for a plurality of sulfur-containing compounds to pass through the pretreatment column 1 is roughly determined by the combination of the mobile phase, the flow rate, and the stationary phase, the required time may be measured in advance, or the pretreatment column 1 Monitoring may be performed by providing a detection means (not shown) in the vicinity of the outlet 12. In the step (2), the flow path P2 and the flow path P3 are used.

  FIG. 2 schematically shows the flow path P2 and the flow path P3. However, the present invention is not limited to the illustrated embodiment. In the step (2) of the present invention, a mobile phase not containing a measurement sample is passed through the flow path P2 and the flow path P3. The direction of the flow path P2 is indicated by a black arrow in FIG. The flow path P <b> 2 is a flow path that reaches the detector 3 through the analysis column 2 without passing through the pretreatment column 1. The analysis column 2 and the detector 3 are the same as those used in the flow path P1. By flowing the mobile phase through the flow path P2, a plurality of sulfur-containing compounds to be tested (cysteine, cystine, etc.) remaining in the analytical column 2 or the previous pipe in step (1) are chromatographed in the analytical column 2. Analytical work can be continued for the graphic processing. By not allowing the measurement sample to flow through the flow path P2 in the step (2), there is no entry of contaminants into the analysis column 2, so that the analysis accuracy is unlikely to decrease.

  The direction of the flow path P3 is indicated by a white arrow in FIG. The flow path P3 is a flow path that passes through the pretreatment column 1 in the reverse direction and is discharged without passing through the analysis column 2 and without passing through the detector 3. The pretreatment column 1 is the same as that used in the flow path P1. Among the contaminants remaining in the pretreatment column 1 in the step (1), those having a long retention time remain in the vicinity of the inlet 11 of the pretreatment column 1. Conventionally, in order to remove such contaminants having a long holding time, it has been necessary to keep the mobile phase flowing through the pretreatment column 1 for a long time. According to the present invention, the mobile phase flowing through the flow path P3 is Since it flows from the outlet 12 of the processing column 1 to the inlet 11, foreign substances remaining in the vicinity of the inlet 11 can be removed in a relatively short time. Therefore, the measurement for the next measurement sample can be started in a short time, and the measurement can be speeded up. Furthermore, in a preferred embodiment of the present invention, steps (1), (2) and (3) are started in the second analysis immediately after completion of step (3) in the first analysis, and this is continuously and repeatedly analyzed. Is to do.

  When the flow path P2 is configured in the analyzer of the present invention, the flow from the sample introduction flow path is immediately passed through the analysis column 2 to the flow path to the detector 3 by switching the flow path switching device 6. You should guide it. Thus, when the flow direction is determined by the flow path switching device 6, it is important that the flow path P3 is configured at the same time. In this case, the flow path P3 guides the flow entering the flow path switching device 6 from the mobile phase introduction flow path to the outlet side 12 of the pretreatment column 1, passes the pretreatment column in the reverse direction, and again passes the flow path. The flow path is determined so as to enter the switching device 6 and then toward the discharge path.

  The mobile phases passed in step (1) and step (2) are preferably all of the same type. In that case, in addition to simple operation, the possibility that the retention behavior of the substance in the columns 1 and 2 due to the change in the type of mobile phase will be reduced. In the drawing, two mobile phase supply sources 51 and 52 are shown for convenience, but the mobile phase may be supplied to the flow paths P1 to P3 from the same supply source. In the step (1) and the step (2), the switching of the flow paths P1 to P3 can be easily realized by combining a flow path switching device (valve or the like) 6 and piping as shown in the figure.

When the flow paths P2 and P3 are configured, the test object is guided to the diamond electrode type electrochemical detector after being separated by the analysis column 2, and selectively detected by the electrochemical activity specific to the substance. Contaminants whose passage time is substantially the same as that of the test object are also introduced to the analytical column 2, but are not detected because they do not show activity in the diamond electrode type electrochemical detector. On the other hand, contaminants that require a significantly longer passage time than the test object flow from the pretreatment column 1 in the reverse direction and are discharged. Therefore, by adopting this configuration, it becomes possible to simultaneously detect a plurality of sulfur-containing compounds having electrochemical activity without the influence of foreign substances.
The present invention enables a highly accurate and stable measurement method for a plurality of compounds in a short time by using a diamond electrode type electrochemical detector having a high selectivity and a low noise level and a column switching method.

  EXAMPLES Hereinafter, although this invention is demonstrated in more detail using an Example, these examples do not limit this invention at all.

(Mobile phase)
An acidic (pH 2.2) buffer solution was prepared by adding a predetermined amount of phosphoric acid to a 50 mmol / L sodium dihydrogen phosphate solution and a 5 mmol / L sodium octanesulfonate solution. A mobile phase was obtained by adding 2.5 parts by weight of acetonitrile to 97.5 parts by weight of this buffer solution.

(Standard solution)
L-cysteine for quantification dried for 3 hours with a desiccator (reduced pressure, phosphorus pentoxide) was dissolved in the mobile phase. Separately, L-cystine for quantification dried at 105 ° C. for 3 hours is dissolved in a small amount of 1 mol / L hydrochloric acid test solution, and water is added to make a predetermined amount. Next, a predetermined amount of each of the L-cysteine solution and the L-cystine solution was weighed, and the mobile phase was added, mixed and diluted to obtain a standard solution.

(Analysis conditions)
In this example, an analysis system schematically represented in FIGS. 1 and 2 was constructed and analyzed. Detailed conditions are as follows.
Analytical column: Inertosyl ODS-3 (manufactured by GL Sciences Inc.)
150 mm x 3.0 mm I.D. D. dp = 3 μm
Pretreatment column: Inertosyl ODS-3 (manufactured by GL Sciences Inc.)
33 mm x 3.0 mm I.D. D. dp = 3 μm
Column temperature: 40 ° C
Detector: Electrochemical detector, conductive diamond electrode 1600 mV
Reference electrode, Ag / AgCl
Flow rate: 0.4 mL / min
Injection volume: 10 μL

(Oxidation electrolytic polishing of diamond electrode)
Mobile phase conditions: acetonitrile / 0.1% phosphoric acid = 50/50
Flow rate: 0.4 mL / min
Applied voltage: 5000 mV
Treatment time: allowed to stand for 24 hours Oxidation electropolishing of the diamond electrode was carried out under the above conditions with a mobile phase flowing in a state where a system used for actual measurement was constructed. This condition is an example that was effective for cysteine. For various compounds, the mobile phase was allowed to flow at a rate almost the same as the measurement condition under strongly acidic conditions, and was allowed to stand for 6 hours or longer at a high voltage of 2000 mV or higher. It is preferable to complete the polishing.

(Analysis of standard solution)
Using the standard solution described above, the applied voltage of the working electrode of the detector 3 was set to 1600 mV in the measurement system schematically shown in FIGS. 1 and 2, and the current change was detected. At this time, a region surrounded by a dotted line in FIGS. 1 and 2, that is, a region including the pretreatment column 1, the analysis column 2, and the valve 6 was kept constant at 40 ° C. As a result of this operation, it was confirmed that simultaneous analysis of L-cysteine and L-cystine was possible. FIG. 3 is a chromatogram obtained by analysis of this standard solution.

  If the diamond electrode was not subjected to oxidative electropolishing, the cysteine peak area value decreased to about 66% after 10 hours from the initial analysis. However, when oxidative electropolishing was performed, the area of 96% The value and the area value of the initial state were maintained.

  FIG. 4 is a chromatogram obtained by performing the same analysis after 5 hours and 10 hours without performing the oxidative electrolytic polishing treatment, and Table 2 shows the area values.

  FIG. 5 is a chromatogram obtained by performing the same analysis after 5 hours and 10 hours after the oxidation electropolishing treatment, and Table 3 shows the area values.

  It was confirmed that oxidative electropolishing while flowing the mobile phase is sufficiently effective for this system.

(Analysis of infusion preparations)
An infusion preparation (sugar / electrolyte / amino acid solution “Twinpal (registered trademark)” manufactured by Ajinomoto Co., Inc.) was diluted with the above mobile phase to obtain a sample solution. When this sample solution was analyzed in the same manner as the standard solution, it was confirmed that specific analysis of L-cysteine and L-cystine was possible. FIG. 6 is a chromatogram obtained by analysis of this infusion preparation.

  The composition of this infusion preparation is as shown in Table 4 (I layer (sugar / electrolyte solution)) and Table 5 (II layer (amino acid solution)). In the analysis, a sample obtained by mixing the II layer or the I layer and the II layer was used. FIG. 6 shows a chromatogram when the layer II is used as a sample.

  In the case of this infusion preparation, there are two volumes of 500 mL capacity (I layer: 350 mL, II layer: 150 mL) and 1000 mL capacity (I layer: 700 mL, II layer: 300 mL), and 2 volumes were used as samples.

In the analysis method of the present invention, accuracy and concurrence accuracy are also good. L-cysteine; recovery rate: 101%, relative standard deviation: 0.9%, L-cystine; recovery rate: 100%, relative standard deviation: 0. 7% (all performed with n = 9).
In particular, in this embodiment, the column switching method shown in FIGS. 1 and 2 plays an important role. Although the electrochemical detector using a diamond electrode is a highly selective device, in addition to cysteine and cystine, amino acids such as methionine, tyrosine, histidine, and phenylalanine are also detected. These amino acids are components that are also contained a lot in infusion preparations and biological samples. Without the column switching method, these amino acids elute between 45 and 280 minutes (FIG. 16). For this reason, the analysis time per sample becomes very long, and it cannot be actually used for the measurement of the actual sample.

(Standard solution)
N-acetyl-L-cysteine for quantification dried at 80 ° C. for 3 hours was dissolved in the mobile phase. Next, a predetermined amount of this N-acetyl-L-cysteine solution was weighed and diluted by adding a mobile phase to obtain a standard solution. The mobile phase was the same as in Example 1.

(Analysis conditions)
In this example, an analysis system schematically represented in FIGS. 1 and 2 was constructed and analyzed. Detailed conditions are the same as in the first embodiment.

(Analysis of standard solution)
As a result of performing the same operation as in Example 1 in the measurement system schematically shown in FIGS. 1 and 2 using the standard solution described above, simultaneous analysis of N-acetyl-L-cysteine is possible. Was confirmed. FIG. 7 is a chromatogram obtained by analysis of this standard solution.

(Analysis of infusion preparations)
An infusion preparation (amino acid / electrolyte solution “commercially available vitamin B1, sugar / electrolyte / amino acid solution”) was diluted with the above mobile phase to obtain a sample solution. When this sample solution was analyzed in the same manner as the standard solution, it was confirmed that a specific analysis of N-acetyl-L-cysteine was possible. FIG. 8 is a chromatogram obtained by analysis of this infusion preparation.

  The composition of this infusion preparation is as shown in Table 6 (upper chamber fluid (amino acid / electrolyte solution)) and Table 7 (lower chamber fluid (vitamin B1, sugar / electrolyte solution)). In the analysis, a sample obtained by mixing the upper chamber solution or the upper chamber solution and the lower chamber solution was used. FIG. 8 shows a chromatogram when the upper chamber solution is used as a sample.

  In the case of this infusion preparation, there are two volumes of 500 mL (upper chamber fluid: 150 mL, lower chamber fluid: 350 mL) and 1000 mL (upper chamber fluid: 300 mL, lower chamber fluid: 700 mL), and two volumes were used as samples.

(Standard solution)
L-cysteine sulfinic acid, L-homocysteine sulfinic acid, DL-homocysteine and reduced glutathione dried for 3 hours in a desiccator (reduced pressure, phosphorus pentoxide) were dissolved in the mobile phase to obtain a mixed solution. Similarly, dry L-homocystine is dissolved in a small amount of 1 mol / L hydrochloric acid test solution, and water is added to obtain a predetermined amount to obtain an L-homocystine solution. Next, a predetermined amount of each of the mixed solution and the L-homocystine solution was weighed, mixed and diluted by adding a mobile phase to obtain a standard solution of sulfur-containing amino acid / sulfur-containing compound. The mobile phase was the same as in Example 1.

(Analysis conditions)
In this example, an analysis system schematically represented in FIGS. 1 and 2 was constructed and analyzed. Detailed conditions are the same as in the first embodiment.

(Analysis of standard solution)
As a result of performing the same operation as in Example 1 in the measurement system schematically shown in FIGS. 1 and 2 using the standard solution described above, it is possible to simultaneously analyze the standard solutions of sulfur-containing amino acids and sulfur-containing compounds. It was confirmed that. FIG. 9 is a chromatogram obtained by analysis of this standard solution.
This method is considered to be an effective method for searching for degradation products of amino acids such as L-cysteine. In FIG. 9, the peak with a retention time of 2.1 minutes is derived from L-cysteine sulfinic acid, and the peak at 2.5 minutes is derived from L-homocysteine sulfinic acid.

(Standard solution)
Cysteine, cystine, reduced glutathione and homocysteine were weighed in predetermined amounts and dissolved using dilute hydrochloric acid. Then, water was added and diluted sequentially to obtain a standard solution containing 6 μmol / L cysteine, 15 μmol / L cystine, 3 μmol / L reduced glutathione and 6 μmol / L homocysteine.

(Preparation of plasma sample)
A trichloroacetic acid solution was added to plasma of mice, rats, dogs, humans, etc., and protein components were removed by centrifugation. The centrifugal supernatant was diluted with water to obtain a plasma sample.

(Analysis conditions)
In this example, an analysis system schematically represented in FIGS. 1 and 2 was constructed and analyzed. Detailed conditions are as follows.
Analytical column: Inertosyl ODS-3 (manufactured by GL Sciences Inc.)
100 mm × 3.0 mm I.V. D. dp = 3 μm
Pretreatment column: Inertosyl ODS-3 (manufactured by GL Sciences Inc.)
10 mm × 3.0 mm I.V. D. dp = 5 μm
Column temperature: 50 ° C
Mobile phase: A mixture of 98.5 parts by weight of a buffer containing 1-sodium heptanesulfonate (20 mM) and phosphoric acid (25 mM) and 1.5 parts by weight of acetonitrile Detector: Electrochemical detector, conductive Diamond electrode 1600mV
Reference electrode, Ag / AgCl
Flow rate: 0.8mL / min
Injection volume: 10 μL
Column switching time 2.1 min
Detection: ECD 20.0 min 1600mV
20.1 min 4000mV
21.0 minutes 1600mV
After 20.1 minutes after the test object was detected and the analysis was completed, dirt accumulated on the electrode surface was removed by applying a high voltage, and electrochemically regenerated.

(Analysis of standard solution)
Using the standard solution described above, the applied voltage of the working electrode of the detector 3 was set to 1600 mV in the measurement system schematically shown in FIGS. 1 and 2, and the current change was detected. At this time, a region surrounded by a dotted line in FIGS. 1 and 2, that is, a region including the pretreatment column 1, the analysis column 2, and the valve 6 was kept constant at 50 ° C. As a result of this operation, it was confirmed that simultaneous analysis of cysteine, cystine, reduced glutathione and homocysteine was possible.
FIG. 10 is a chromatogram obtained by analyzing 6 μmol / L of cysteine, 3 μmol / L of reduced glutathione, 6 μmol / L of homocysteine, and 15 μmol / L of cystine in a standard solution, and FIG. FIG.

(Analysis of plasma samples)
An analysis of the plasma sample subjected to the above pretreatment was performed in the same manner as the standard solution. As a result, it was confirmed that specific analysis of cysteine, cystine, reduced glutathione, and homocysteine in plasma was possible. . FIG. 11 is a chromatogram obtained by analysis of a mouse plasma sample.

(Analysis of plasma-added sample)
FIG. 12 shows the analysis results obtained by adding 6 μmol / L standard solution, 3 μmol / L reduced glutathione, 6 μmol / L homocysteine, and 15 μmol / L cystine to plasma (FIG. 11). FIG. 14 is an enlarged view of the homocysteine elution portion of FIG.

In FIG. 10, only added cysteine, reduced glutathione, homocysteine, and cystine are detected from the standard solution analysis. In FIG. 11, cysteine, reduced glutathione, and cystine are detected as components contained in plasma.
Since homocysteine is not detected in plasma, the concentration of homocysteine added to plasma in FIG. 12 is 6 μmol / L, the same concentration as in FIG. The homocysteine peak obtained in FIG. 12 and the homocysteine peak in FIG. 10 are almost the same, and it was confirmed from FIGS. 13 and 14 that the height of the peak and the noise were comparable. From the above results, it was found that the same sensitivity as in the standard solution analysis can be obtained in the analysis of plasma containing contaminants in the matrix.

Actual biological samples such as plasma contain various contaminants, and some of them show electrochemical activity. However, in the prior art, only the standard product analysis data that does not contain contaminants is presented at present.
Selectivity adjustment that combines electrochemical selectivity and separation techniques such as column switching is essential when applying this system to materials containing complex matrices. In fact, the detection limit in pharmaceutical and food analysis is defined as 3σ in the background, and even if the diamond electrode electrochemical detector has excellent detection ability, it cannot achieve the adjustment of selectivity that can be separated from contaminants. As long as the sensitivity does not increase.
Thus, the plasma blank sample, which is a complex matrix sample and contains contaminants, has the same sensitivity as the standard solution analysis. This system combines electrochemical selectivity and column switching techniques. Is achieved for the first time.

  Regarding this method, as a result of confirming quantitativeness using rat plasma, favorable results such as linearity, simultaneous reproducibility, daily reproducibility, stability (autosampler 4 ° C.), accuracy, and the like were obtained. The quantification ranges were cysteine, homocysteine: 6 to 120 μM, reduced glutathione: 3 to 60 μM, and cystine: 15 to 300 μM.

(Preparation of plasma sample with standard solution added)
Protein is denatured by adding 20 μL of water and 80 μL of 10% trichloroacetic acid to 100 μL of rat plasma. A plasma sample was prepared by adding 10 μL of a cysteine / cystine mixed sample and 890 μL of purified water to 100 μL of the centrifuged supernatant and stirring.
Continuous analysis was performed using this plasma sample.

(Analysis conditions)
In the measurement system schematically shown in FIGS. 1 and 2, the applied voltage of the working electrode of the detector 3 was set to 1600 mV, and the current change was detected.
Detailed conditions are the same as in the first embodiment. The analysis cycle was 20 minutes per analysis.

(Continuous measurement of plasma-added sample)
FIG. 15 shows chromatograms at the initial stage, the 500th time, and the 1000th time in 1000 continuous analyses.
In the initial 500 times and 1000 times of chromatograms, the peak intensities of cysteine and cystine do not change and the baseline is stable.
Table 8 shows the retention time and the variation rate (CV%) of the retention time and the area value in the 9th continuous analysis of the 21st to 29th times, 481 to 489th time, and 961 to 969th time among 1000 continuous analysis. . Good results were obtained with both cysteine and cystine having a retention rate variation of 1% or less and an area value CV value of at most about 3.9%.

The peak area values after 7 hours (20th time), 160 hours (480th time), and 320 hours (960th time) after 1000 times of continuous analysis in Tables 9 and 10 were picked up. The decrease rate was shown. Even after 320 hours, the rate of decrease was as small as 5% or less, confirming the stability of the system.
From the above, it was shown that a highly accurate quantitative analysis can be performed in a short time using a plasma sample, which is a complex matrix containing a very large amount of contaminants, as a sample.
For this purpose, the column switching method was used to efficiently remove contaminants, shortening the analysis time and preventing electrode deterioration, and using conductive diamond electrodes, carbon is an alkylsulfonic acid that imposes a load on the electrodes. It is considered that the salt can be used at a sufficient concentration, and that the electrode surface can be cleaned by oxidative electropolishing by applying a high applied voltage, and that the cleaning can be performed at each injection, which contributes greatly. This enables stable analysis.

  On the other hand, as a comparative control of data stability, a standard solution using a glassy carbon electrode was analyzed. Since the glassy carbon electrode cannot apply a high applied voltage, cystine having a high reaction potential cannot be detected and only cysteine is used.

(Analysis conditions)
Analysis column: Inertsil ODS-3 (manufactured by GL Sciences) 3 μm 3.0 × 150 mm
Mobile phase: 100 mM phosphate buffer-5 mM sodium octane sulfonate (pH 2.2) / methanol = 95/5
Flow rate: 0.8mL / min
Column temperature: 40 ° C
Detection: Electrochemical detector Glassy carbon electrode Applied voltage: 500 mV

(Analysis of standard solution using glassy carbon electrode)
Tables 11 to 13 show the change in the area value when the standard solution was adjusted to three concentrations and the day passed.
In the cysteine analysis using a glassy carbon electrode, the area value changed with the passage of time, and since the area value had already decreased by 20% or more in 96 hours, it was confirmed that the stability was lacking.
From this, the result was conspicuous how stable the analysis system constructed with the diamond electrode was despite the harsh conditions in which the sample contained a lot of contaminants.

(Analysis of fermented red wine)
As an analysis example of the fermentation broth, red wine and its forced oxidation product were analyzed using the system of the present invention.
In the measurement system schematically shown in FIGS. 1 and 2, the applied voltage of the working electrode of the detector 3 was set to 1600 mV, and the current change was detected. Detailed conditions are the same as those in the fourth embodiment.

(Oxidation conditions and sample pretreatment conditions)
About 1/3 amount of red wine was put into a screw vial, capped, and then subjected to ultrasonic waves for 30 minutes. Thereafter, untreated red wine and oxidized red wine were deproteinized with 10% trichloroacetic acid, and the centrifugal supernatant was diluted and measured with the system of the present invention.

(result of analysis)
FIG. 17 shows the result. By using the system of the present invention, it has become possible to measure a fermentation broth containing many components in a short time with good reproducibility. When the two chromatograms are compared, the same separation and intensity are obtained for signals up to about 10 minutes. On the other hand, the peak intensity greatly fluctuates for signals of 10 to 11 minutes and 14 to 15 minutes. These compounds are signals indicating the oxidation state of the sample, and can be analyzed accurately and in a short time by using the system of the present invention.

(Analysis of standard solution)
3-mercapto-1-hexanol, which has a thiol group in its structure and is known as a grapefruit fragrance, was forcibly oxidized, and the oxidized form was analyzed by the system of the present invention.

Analysis column: Inertsil ODS-3 (manufactured by GL Sciences) 3 μM 2.1 × 150 mm
Mobile phase: 50 mM phosphate buffer (pH 2.8) / acetonitrile = 50/50
Flow rate: 0.2 mL / min
Column temperature: 40 ° C
Detection: Electrochemical detector Voltage applied to conductive diamond electrode: 1600 mV

(Oxidation conditions for 3-mercapto-1-hexanol)
A water-dimethylformamide mixed solution (1: 1) was added to 10 μL of 3-mercapto-1-hexanol, and the mixture was sonicated for 30 minutes and then allowed to stand at room temperature for 12 hours. This was diluted 6-fold with water and used as an oxidized sample of 3-mercapto-1-hexanol for analysis.

(result of analysis)
FIG. 18 is a chromatogram obtained by analysis of an oxidized sample of 3-mercapto-1-hexanol.
When 3-mercapto-1-hexanol was oxidized, 2-mercapto-1-hexanol was detected in 5 minutes, and two peaks considered to be 3-mercapto-1-hexanol dimer were detected in 10.5 minutes. .
It was confirmed that a monomer and a dimer of a compound having a thiol group can be analyzed simultaneously.

(Analysis of standard solution)
Furanthiol, which has a thiol group in its structure and is known as strawberry odor, was forcibly oxidized together with cysteine, and the oxidized form was analyzed by the system of the present invention.

(Analysis conditions)
Analysis column: Inertsil ODS-3 (manufactured by GL Sciences) 3 μM 2.1 × 150 mm
Mobile phase: 50 mM phosphate buffer (pH 2.8) / acetonitrile = 50/50
Flow rate: 0.2 mL / min
Column temperature: 40 ° C
Detection: Electrochemical detector Voltage applied to conductive diamond electrode: 1600 mV

(Furanthiol and cysteine oxidation conditions)
Cysteine 10 mg / mL (water: dissolved in dimethylformamide = 1: 1) and furanthiol 10 μL are mixed, sonicated for 30 minutes, and then allowed to stand at room temperature for 12 hours, and analyzed as a furanthiol-cysteine mixed oxidation sample Used for.
Cysteine 10 mg / mL (dissolved in water: dimethylformamide = 1: 1) was oxidized in the same manner as above, diluted 10-fold with dimethylformamide, and used as an oxidized sample of cysteine for analysis.
10 μL of furanthiol was oxidized and used for analysis as described above.

(result of analysis)
FIG. 19 is a chromatogram obtained by the analysis of the above-mentioned furanthiol-cysteine mixed oxidation sample, furanthiol, and cysteine.
When only furanthiol was oxidized, a furanthiol dimer peak was detected at 7.5 minutes and at 24.5 minutes, and could be identified by mass spectrum.
When furanthiol and cysteine are mixed and oxidized, a cysteine peak and several peaks that were not seen in the oxidation of furanthiol appear, and by mixing cysteine, furanthiol and cysteine react to form an oxidant. It was thought that it was. It was confirmed that an electrochemical detector using a diamond electrode can measure not only thiol homodimers but also oxidants formed between different thiols.

  As an example of a separation mode other than ion pair chromatography, analysis in HILIC mode was performed. FIG. 20 shows chromatograms of cysteine, cystine, reduced glutathione, and oxidized glutathione.

(Analysis conditions)
Analytical column: ZIC-HILIC 4.6 × 250 mm
Mobile phase: acetonitrile / 50 mM acetic acid + 5 mM ammonium acetate = 70/30
Column oven temperature: 40 ° C. Set flow rate: 1.0 ml / min
Injection volume: 10 μl
Detection: ECD 30 min 1600 mV
31 min 4000 mV
31 min 1600 mV
When comparing the mobile phase in HILIC mode with the mobile phase in ion pair chromatography (Examples 1 and 4), the buffer type, salt strength, presence / absence of ion pair reagent, and organic solvent concentration are greatly different. Recognize. Even under such different mobile phase conditions, cysteine, cystine, reduced glutathione, and oxidized glutathione were detected with no trouble by the electrochemical detector with diamond electrode (FIG. 20). From the above, in this analysis system, it is considered possible to analyze sulfur-containing amino acids and sulfur-containing compounds without depending on the separation mode.

This application is based on Japanese Patent Application No. 2006-166556 filed in Japan, the contents of which are incorporated in full herein.
While the invention has been presented or described with reference to preferred embodiments thereof, various changes in form and detail may be made herein without departing from the scope of the invention as encompassed by the appended claims. It will be appreciated by those skilled in the art. All patents, patent publications and other publications shown or referenced herein are incorporated by reference in their entirety.

DESCRIPTION OF SYMBOLS 1 Pretreatment column 11 Inlet 12 Outlet 2 Analytical column 3 Detector 41, 42 Pump 51, 52 Mobile phase supply source 6 Valve 7 Injector

Claims (36)

An analysis method for a test object contained in a measurement sample,
A step (1) of flowing the measurement sample together with the mobile phase to the following flow path P1;
A step (2) of flowing a mobile phase not containing a measurement sample in the following flow paths P2 and P3, and
(3) obtaining a chromatogram of the test object from the analytical column in steps (1) and (2) by an electrochemical detector having a diamond electrode as a working electrode (however, The flow path P1 is a flow path that passes through the pretreatment column in the forward direction and then passes through the analysis column to the detector, and the flow path P2 does not pass through the pretreatment column and flows to the detector through the analysis column. The flow path P3 is a flow path that passes through the pretreatment column in the reverse direction and is discharged without passing through the analysis column and without passing through the detector.
The said analysis method which complete | finishes a process (1) and starts a process (2) after the said test object passes a pre-processing column in a process (1).   The analysis method according to claim 1, wherein a contaminant that requires a significantly longer passage time than the test object contained in the measurement sample does not pass through the analysis column.   The analysis method according to claim 1 or 2, wherein the test object is a sulfur-containing compound.   The analysis method according to claim 3, wherein the sulfur-containing compound is a compound having a thiol group or a disulfide bond.   The analysis method according to claim 3, wherein the sulfur-containing compound is a sulfur-containing amino acid.   The analysis method according to claim 3, wherein the sulfur-containing compound is N-acetylcysteine.   The analysis method according to claim 3, wherein the sulfur-containing compound is homocysteine or glutathione.   The analysis method according to claim 1 or 2, wherein the test object is two or more sulfur-containing compounds.   The analysis method according to claim 8, wherein the two or more sulfur-containing compounds are cysteine and cystine.   The analysis method according to claim 1 or 2, wherein the test object is two or more amino acids.   The analysis method according to any one of claims 1 to 10, wherein the mobile phase contains water and an electrolyte.   The analysis method according to any one of claims 1 to 10, wherein the mobile phase contains water, an electrolyte, and an ion pair compound, and has a pH of 1 to 3.   The analysis method according to claim 12, wherein the ion pair compound is at least one selected from the group consisting of alkyl sulfonates and alkyl sulfates.   The analytical method according to any one of claims 1 to 13, wherein the analytical column is any one of a reverse phase column, a normal phase column, and an ion exchange column.   The analysis method according to claim 14, wherein the analysis column is a reverse phase column.   The analysis method according to claim 1, wherein the diamond electrode is a conductive diamond electrode.   The analysis method according to claim 16, wherein the conductive diamond electrode is an electrode subjected to oxidative electropolishing.   The analysis method according to claim 16, wherein the conductive diamond electrode is an electrode that has been subjected to oxidative electropolishing with a mobile phase flowing.   The analysis method according to any one of claims 1 to 18, wherein the measurement sample is any one of an infusion preparation, a dialysis agent, a fermentation broth, and a biological sample.   The analysis method according to claim 1, wherein after the step (3) is completed, the steps (1), (2) and (3) are started to perform continuous repeated analysis.   A method for simultaneously detecting two or more sulfur-containing compounds contained in a measurement sample, wherein the analysis method according to any one of claims 1 to 20 is used. An analysis device for a test object contained in a measurement sample,
A flow path switching device capable of reversibly switching the direction of the flow path;
A sample introduction flow path for flowing the mobile phase together with the measurement sample to the flow path switching device;
A mobile phase introduction flow path for flowing the mobile phase alone to the flow path switching device;
A flow path that returns from the flow path switching device and returns to the flow path switching device again through the pretreatment column;
A flow path from the flow path switching device to the electrochemical detector having a diamond electrode as a working electrode capable of acquiring a chromatogram of the test object through the analysis column;
A discharge path that exits the flow path switching device and goes out of the apparatus without passing through the pretreatment column or the analysis column, and the flow path switching device switches between the following states (A) and (B) An apparatus for analyzing a test object, configured to be able to
(A) A state in which a flow path P1 is formed from the flow path for sample introduction to the pretreatment column in the forward direction and then to the detector through the analysis column,
(B) The pretreatment column passes through the pretreatment column in the reverse direction from the flow path for sample introduction, does not pass through the pretreatment column, passes through the analysis column, and reaches the detector. The state which comprises the flow path P3 which leads to a path.   The apparatus according to claim 22, wherein the test object is a sulfur-containing compound.   The apparatus according to claim 23, wherein the sulfur-containing compound is a compound having a thiol group or a disulfide bond.   The apparatus of claim 23, wherein the sulfur-containing compound is a sulfur-containing amino acid.   The apparatus of claim 23, wherein the sulfur-containing compound is N-acetylcysteine.   The apparatus of claim 23, wherein the sulfur-containing compound is homocysteine or glutathione.   The apparatus according to claim 22, wherein the test object is two or more sulfur-containing compounds.   The apparatus of claim 28, wherein the two or more sulfur-containing compounds are cysteine and cystine.   The apparatus according to claim 22, wherein the test object is two or more amino acids.   The apparatus according to any one of claims 22 to 30, wherein the analytical column is any one of a reverse phase column, a normal phase column, and an ion exchange column.   32. The apparatus of claim 31, wherein the analytical column is a reverse phase column.   33. Apparatus according to any one of claims 22 to 32, wherein the diamond electrode is a conductive diamond electrode.   34. The apparatus of claim 33, wherein the conductive diamond electrode is an oxidatively electropolished electrode.   34. The apparatus of claim 33, wherein the conductive diamond electrode is an electrode that has been oxidatively electropolished with the mobile phase flowing.   37. The apparatus according to any one of claims 22 to 36, wherein the measurement sample is any one of an infusion preparation, a dialysis agent, a fermentation broth, and a biological sample.

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