Method for quantifying activated sulfur

Abstract

To simply quantify active sulfur that hardly acquires a standard substance with sufficient accuracy.SOLUTION: A quantitative method includes a standard substance measurement step (S1) for measuring a standard substance of a known concentration of a basic compound of cysteine or the like contained in an analysis object group by an LC-MS / MS, a quantitative reference information acquisition step (S2) for acquiring quantitative reference information for quantifying the basic compound and active sulfur other than it on the basis of the measurement result under the premise that signal intensity of a plurality of sulfur compounds has a prescribed relation in the case that the concentrations of the plurality of sulfur compounds contained in the same group are the same, a sample measurement step (S4) for measuring each sulfur compound in a sample by the LC-MS / MS under the analysis condition predetermined to make the signal intensity of the plurality of sulfur compounds the prescribed relation in the case that the concentrations of the plurality of sulfur compounds contained in the same group are the same, and a quantitative step (S5) for quantifying each sulfur compound by using the measurement result and the quantitative reference information.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a method for quantifying sulfur compounds including reactive sulfur molecular species (hereinafter simply referred to as "reactive sulfur") in a specimen, and more particularly to a quantification method using chromatographic mass spectrometry.

Background Art

[0002] Reactive sulfur is a general term for highly reactive sulfur compounds, typified by cysteine persulfide, which is formed when an excessive number (usually two or more) of sulfur atoms are added to the thiol (SH) group of cysteine. Reactive sulfur exists abundantly in various organs and blood in vivo, and is known to function as a major antioxidant that exerts reactive oxygen species scavenging ability in vivo. Therefore, reactive sulfur is expected to be applied to various medical-related fields such as prevention of human aging, diagnosis of various diseases and disorders including cancer in which oxidative stress is involved, and development of preventive and therapeutic drugs.

[0003] For these reasons, there has been a conventional demand for establishing a method for quantifying reactive sulfur in vivo. However, since reactive sulfur, especially reduced reactive sulfur, is a particularly highly reactive compound, it is easily decomposed during pretreatment and analysis, making accurate quantification difficult.

[0004] containing reduced reactive sulfur Sulfur compounds As a quantification method, for example, a method using a liquid chromatography-tandem mass spectrometer (LC-MS / MS) described in Non-Patent Documents 1 and 2 is known. In this method, reduced reactive sulfur is converted into a stable derivative using an electrophilic alkylating agent, and then reactive sulfur is selectively detected by multiple reaction monitoring (MRM) measurement using LC-MS / MS.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

[0006] Generally, quantitative analysis using the methods described above requires a standard substance containing the target component at a known concentration. However, standard substances for chemically unstable active sulfur, such as cysteine ​​persulfide, are not generally available. Therefore, it is practically difficult for the average user to perform quantitative analysis of active sulfur using these methods.

[0007] This invention was made to solve these problems, and its main objective is to provide a quantitative method that can easily and accurately quantify various sulfur compounds, including chemically unstable activated sulfur, using readily available standard substances. [Means for solving the problem]

[0008] A first aspect of the method for quantifying active sulfur according to the present invention, which was made to solve the above problems, is a method for quantifying sulfur compounds containing active sulfur in a sample using a chromatograph-mass spectrometer, and is a method for quantifying sulfur compounds that are the target of analysis in at least one of the following groups: a group containing cystine and related active sulfur, a group containing cysteine ​​and related active sulfur, a group containing oxidized glutathione and related active sulfur, and a group containing reduced glutathione and related active sulfur. A standard substance measurement step involves measuring known concentrations of standard substances containing the basic compounds cystine, cysteine, oxidized glutathione, or reduced glutathione, which are included in the group to be analyzed, using a chromatographic mass spectrometer. Based on the premise that the signal intensities of multiple sulfur compounds in the same group have a predetermined relationship when their concentrations are the same, a quantitative reference information acquisition step is performed to obtain quantitative reference information for quantifying the basic compounds and other active sulfur compounds in the group to be analyzed, based on the measurement results obtained in the standard substance measurement step. A sample measurement step comprising measuring each sulfur compound included in the group to be analyzed in the sample using a chromatographic mass spectrometer, wherein the measurement is performed under predetermined analytical conditions for each sulfur compound so that when the concentrations of multiple sulfur compounds included in the same group are the same, the signal intensities of the multiple sulfur compounds have a predetermined relationship; A quantitative step is performed to quantify each sulfur compound included in the group to be analyzed, using the measurement results obtained in the sample measurement step and the quantitative reference information. It holds.

[0010] Furthermore, the method for quantifying activated sulfur according to the present invention 2 The embodiment is a method for quantifying sulfur compounds containing active sulfur in a sample using a chromatographic mass spectrometer, A standard substance measurement step involves grouping multiple sulfur compounds that have the same structure other than the chain bond of sulfur atoms but differ in the number of sulfur atoms constituting the chain bond, and measuring a standard substance of a known concentration of a sulfur compound that is included in the same group as the target sulfur compound to be quantified and has one or the minimum number of sulfur atoms constituting the chain bond, using a chromatographic mass spectrometer. Based on the premise that the signal intensities of multiple sulfur compounds in the same group have a predetermined relationship when their concentrations are the same, a quantitative reference information acquisition step is performed to obtain quantitative reference information for quantifying the sulfur compounds in the group, based on the measurement results obtained in the standard substance measurement step. A sample measurement step in which a chromatograph-mass spectrometer is used to measure the target sulfur compound in a sample under predetermined analytical conditions for each sulfur compound, such that when the concentrations of multiple sulfur compounds in the same group are the same, the signal intensities of the multiple sulfur compounds have the predetermined relationship. A quantitative step is performed to quantify the target sulfur compound using the measurement results obtained in the sample measurement step and the quantitative reference information. It holds. [Effects of the Invention]

[0011] The first and 2 According to this embodiment, various sulfur compounds, including active sulfur which is chemically unstable and for which standard materials are difficult to obtain, can be easily and accurately quantified using standard materials of chemically stable sulfur compounds that are generally readily available. [Brief explanation of the drawing]

[0013] [Figure 1] A schematic diagram of an example of an analytical system for carrying out the method for quantifying activated sulfur according to the present invention. [Figure 2] A flowchart showing the general procedure for quantitative analysis of sulfur compounds, including active sulfur, using the analytical system shown in Figure 1. [Figure 3]A figure showing a list of 17 sulfur compounds that are analysis targets in the sulfur compound analysis method. [Figure 4] A figure showing an example of derivatization for stabilizing active sulfur. [Figure 5] A figure showing an actual measurement example of chromatograms of 17 sulfur compounds in a sample. [Figure 6] A figure showing an example of comparison between signal intensity before adjustment of MS analysis conditions and signal intensity after adjustment for each sulfur compound. [Figure 7] A figure showing an example of chromatograms of cysteine and cysteine persulfide before and after adjustment of MS analysis conditions. [Figure 8] A figure showing an example of comparison of relative signal intensity of each sulfur compound using an actual sample (the sample is plasma) after adjustment of MS analysis conditions. [Figure 9] A figure showing an example of comparison of relative signal intensity of each sulfur compound using an actual sample (the sample is cells) after adjustment of MS analysis conditions. MODE FOR CARRYING OUT THE INVENTION

[0014] Hereinafter, a method for quantifying active sulfur, which is one embodiment of the present invention, will be described with reference to the accompanying drawings.

[0015] [Sulfur compounds to be analyzed] The quantification method according to this embodiment is mainly intended to quantify sulfur compounds including active sulfur present in samples such as cells collected from human blood (plasma) or organs. However, the sample is not limited to these as long as it is a biological sample.

[0016] Figure 3 is a diagram showing 17 sulfur compounds that are targets of quantitative analysis in the present quantification method. As shown on the right side of Figure 3, the 17 types of sulfur compounds can be divided into 6 groups. Of these, the four groups that are important as active sulfur are the cysteine ​​group, the cystine group, the reduced glutathione group, and the oxidized glutathione group. In these four groups, the sulfur compounds marked with an asterisk in Figure 3 are chemically stable sulfur compounds (i.e., not active sulfur in the general narrow sense), and standard substances with known concentrations are generally available. Standard substances are not generally available for the other sulfur compounds that are not marked with an asterisk. The active sulfur contained in the cysteine ​​group and the reduced glutathione group has a highly reactive thiol group (-SH) and is particularly unstable active sulfur with poor chemical stability.

[0017] Furthermore, the other two groups besides the four mentioned above are the sulfuric acid group and the by-reactant group. While the sulfur compounds in the four groups mentioned above are organic compounds, the sulfur compounds in the sulfuric acid group are inorganic compounds, specifically sulfuric acid and thiosulfuric acid in this example. On the other hand, the sulfur compounds in the by-reactant group are not compounds originally present in the sample, but rather sulfur compounds that are generated as a byproduct during the pretreatment process described later. Generally speaking, although sulfur compounds that may be included in the sulfuric acid group, such as sulfuric acid and thiosulfuric acid, are not active sulfur compounds, they can be considered as sulfur compounds similar to active sulfur because they are important sulfur sources in metabolism within the body.

[0018] As can be seen from Figure 3, in this example, multiple sulfur compounds belonging to the same group in the four groups mentioned above are compounds in which only the number of sulfur atoms constituting the chain bond (...-SS-...) of sulfur atoms differs, and the structure (composition) of the rest of the compound is identical. For example, the three sulfur compounds belonging to the cysteine ​​group have 1 to 3 sulfur atoms in the chain bond. Since there are also sulfur compounds with 4 or more sulfur atoms, such compounds can also be added to the same group. The same applies to the other groups. Furthermore, even if the structure other than the chain bond of sulfur atoms is not completely identical, it may still be possible to include them in the same group. In addition, for the by-reactant group, the bonding site where the reaction occurs is not necessarily uniquely determined, so various by-reactants may be produced. Therefore, the by-reactant group may include multiple sulfur compounds with different structures, even if they have the same number of sulfur atoms in the chain bond.

[0019] Furthermore, among the four groups mentioned above, sulfur compounds for which standard materials are readily available are those in which the number of sulfur atoms constituting the chain bond is 1 or the minimum number within the group (specifically, 2). Thus, regardless of the number of sulfur compounds included in the group, and regardless of the structure of the sulfur compounds included in the group, a compound in which the number of sulfur atoms constituting the chain bond is 1 or the minimum number within the group may be suitable as a standard material.

[0020] [Pretreatment for stabilizing activated sulfur] The aforementioned unstable active sulfur is difficult to analyze directly. Therefore, a pretreatment by derivatization is performed to stabilize the active sulfur. Specifically, β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM) is used as the derivatization reagent.

[0021] Figure 4 shows the derivatization reaction using HPE-IAM. HPE-IAM selectively reacts with the SH group contained in reduced active sulfur, derivatizing the active sulfur. The OH group contained in the derivative suppresses side reactions to the polysulfide chain, making it chemically stable. In Figure 3 and Figures 5, 6, 8, and 9 described later, "-HPE" in the abbreviation of the compound name indicates that it is a derivative by HPE-IAM. Although such derivatization is practically essential as a pretreatment for measurement of reduced active sulfur, the use of HPE-IAM as a reagent is not essential.

[0022] [Quantitative Principle of Active Sulfur] In the quantitative method of this embodiment, a liquid chromatograph-triple quadrupole mass spectrometer (LC-MS / MS) is used to comprehensively measure the 17 types of sulfur compounds contained in the sample. In the LC-MS / MS, the liquid chromatograph in the preceding stage separates the 17 types of sulfur compounds and various other impurities in the sample roughly over time, and the triple quadrupole mass spectrometer in the subsequent stage selectively detects each of the 17 types of sulfur compounds and obtains an ionic intensity signal corresponding to their amount.

[0023] In a triple quadrupole mass spectrometer, MRM (Multiple Reaction Mass Spectrometer) measurements are performed targeting multiple reaction transitions, which are pairs of specific precursor and product ions with corresponding mass-to-charge ratios (m / z), within a predetermined measurement time range near the retention time for each of the 17 sulfur compounds. This yields data that constitutes an extracted ion chromatogram (hereinafter simply referred to as a chromatogram) for each of the 17 sulfur compounds. If a certain sulfur compound is present in the sample, a peak will appear in the chromatogram corresponding to that sulfur compound. Since the area (or height) of this peak depends on the amount or concentration of that sulfur compound, quantitative values ​​such as content and concentration can be determined based on the area or height value.

[0024] When quantifying components in a sample using LC-MS / MS, the most common method is absolute quantification using an external standard, where a calibration curve is created beforehand using a standard substance of known concentration, and the unknown concentration is determined from the peak area value, etc., by referring to the calibration curve. However, as mentioned above, standard substances for active sulfur are generally difficult to obtain. Therefore, here we will perform relative quantification, which uses sulfur compounds for which standard substances are generally available as a reference and determines the ratio of concentrations among multiple sulfur compounds, and pseudo-absolute quantification, which reuses a calibration curve created using a standard substance of one type of sulfur compound to calculate the absolute concentration of active sulfur related to that sulfur compound.

[0025] However, even within the same group of sulfur compounds, there are significant differences in the signal intensity (typically the peak area value in the chromatogram) obtained for the same concentration. Therefore, we will consider adjusting the MS analysis conditions for each group so that the signal intensity for multiple sulfur compounds contained within that group is roughly the same.

[0026] Several parameters affect signal intensity in LC-MS / MS. For example, while multiple MRM transitions are usually available for selectively detecting a particular compound, their sensitivity differs. Furthermore, changing the collision energy during collision-induced dissociation (CID) for MS / MS analysis alters the ion dissociation pattern, thus changing the signal intensity in a specific MRM transition. In many cases, by appropriately selecting the MRM transition for each sulfur compound and adjusting the collision energy value accordingly, the signal intensity for multiple sulfur compounds can be roughly standardized.

[0027] However, when equalizing signal intensity by adjusting MRM transition and collision energy values, the signal intensity will be matched to the one with the lowest signal intensity among multiple sulfur compounds, which may result in a lower detection sensitivity. Therefore, to avoid the detection sensitivity becoming too low, for some sulfur compounds, a method is adopted to increase the detection sensitivity and equalize the signal intensity by lowering the mass resolution. In a triple quadrupole mass spectrometer, the mass resolution can be adjusted by adjusting the m / z range of ions passing through the pre- and post-quadrupole mass filters.

[0028] Figure 7 shows examples of chromatograms of cysteine ​​and cysteine ​​persulfide (two types) before and after adjustment of the MS analysis conditions. These are measurement results obtained by LC / MS / MS with standard substances of the same concentration (a specially synthesized standard substance for cysteine ​​persulfide). Figure 7(A) shows the chromatograms obtained for each compound under MS analysis conditions that maximize signal intensity, while Figure 7(B) shows the chromatograms obtained under MS analysis conditions after adjustment to equalize signal intensity. In this case, since the difference in signal intensity is relatively small before adjustment, it can be seen that the signal intensity can be roughly equalized by adjusting only the collision energy value.

[0029] Figure 6 shows an example of the signal intensity and signal intensity ratio (A) before parameter adjustment, and the signal intensity and signal intensity ratio (B) after parameter adjustment, for all 17 sulfur compounds mentioned above. Similar to Figure 7, these are measurement results obtained by LC / MS / MS using standard substances of the same concentration (specially synthesized standard substances for active sulfur).

[0030] As can be seen in Figure 6(A), the two sulfur compounds CysSSSSCys and HSO3-HPE have particularly low signal intensities within each group. Therefore, if the collision energy and other parameters are adjusted to lower the signal intensity of the other sulfur compounds to match these two, the overall detection sensitivity becomes too low. To address this, the signal intensity of these two sulfur compounds is increased by adjusting (actually decreasing) the mass resolution, while the signal intensity of the other sulfur compounds is decreased by adjusting the collision energy value, so that the signal intensity for the same concentration is consistent within each group. As a result, as shown on the right side of Figure 6(B), the adjusted signal intensity is within approximately 20-60% of the maximum signal intensity before adjustment, thus avoiding a significant decrease in detection sensitivity. Furthermore, the variation in signal intensity within each group is kept within 10%.

[0031] However, the results in Figure 6 are based on measurements of standard substances free of impurities. Actual samples are affected by various impurities other than sulfur compounds, so it is necessary to evaluate their effects as well. Therefore, plasma and cells were used as actual samples, and the addition and recovery tests of each sulfur compound were performed under the parameters adjusted as shown in Figure 6(B), and the recovery rates were determined. The results are shown in Figures 8 and 9. In both the case of plasma and cells, it was confirmed that by appropriately diluting the samples, the relative intensity difference for almost all sulfur compounds could be kept within a range of approximately ±20%.

[0032] As described above, for the 17 types of sulfur compounds listed, by appropriately selecting the MRM transition and adjusting the collision energy value and mass resolution, the signal intensity for multiple sulfur compounds in the same group can be made nearly identical. That is, when the concentrations of multiple sulfur compounds in the same group are the same, the signal intensity of these multiple sulfur compounds can be made to have a predetermined relationship. Since this relationship holds for any concentration, relative quantification of multiple sulfur compounds in a group can be performed accurately and with a wide dynamic range for each group. Furthermore, if measurement results for a standard substance of the sulfur compound that is chemically stable and readily available can be obtained for each group, it is possible to obtain quantitative reference information for the absolute quantification of all sulfur compounds in the group from these results.

[0033] In the quantitative analysis method of this embodiment, the manufacturer providing the system and method file for quantitative analysis investigates the MRM transition, collision energy value, and mass resolution for each sulfur compound, using standard substances of the 17 sulfur compounds listed above, such that the signal intensity for the same concentration of sulfur compounds in the same group is approximately the same. Based on these results, analytical conditions (LC analysis conditions and MS analysis conditions) including individual MS analysis conditions for each sulfur compound are determined, and a method file is created for performing LC / MS / MS analysis under these analytical conditions.

[0034] [Example of an analysis system configuration] An example of an analytical system used for measurement is described below. Figure 1 is a schematic diagram of an example of the analytical system. This analytical system is an LC-MS / MS system that includes a measurement unit containing a liquid chromatograph (LC) 1 and a mass spectrometer 2, a data processing unit 3, an analysis control unit 4, a central control unit 5, an input unit 6, and a display unit 7.

[0035] The liquid chromatograph 1 includes a mobile phase container 11 in which the mobile phase (solvent) is stored, a liquid delivery pump 12 for aspirating and supplying the mobile phase from the mobile phase container 11, an injector 13 for injecting the sample into the mobile phase, and a column 14 for separating multiple components contained in the sample over time. Although not shown in the diagram, an autosampler is usually connected to the injector 13 to sequentially analyze multiple samples.

[0036] The mass spectrometer 2 is a triple quadrupole mass spectrometer and comprises an ionization chamber 201 maintained at approximately atmospheric pressure, and a first intermediate vacuum chamber 202, a second intermediate vacuum chamber 203, and a high vacuum chamber 204, each evacuated by a vacuum pump (not shown). The ionization chamber 201 is equipped with an ESI spray 21 for ionization by the electrospray ionization (ESI) method, and the ionization chamber 201 and the next stage, the first intermediate vacuum chamber 202, are connected by a desolvation tube 22. An ion guide 23 for transporting ions while focusing them is located in the first intermediate vacuum chamber 202, and the first intermediate vacuum chamber 202 and the next stage, the second intermediate vacuum chamber 203, are connected through a small hole formed at the top of a skimmer 24. A multipole ion guide 25 for transporting ions while focusing them is also located in the second intermediate vacuum chamber 203.

[0037] Inside the high-vacuum chamber 204, a pre-stage quadrupole mass filter 26, a collision cell 27, a post-stage quadrupole mass filter 28, and an ion detector 29 are arranged along the flow of ions. A quadrupole-type ion guide is placed inside the collision cell 27. The pre-stage quadrupole mass filter 26 and the post-stage quadrupole mass filter 28 each have the function of selectively passing ions having a predetermined m / z. An inert collision-induced dissociation (CID) gas such as argon is introduced into the collision cell 27 from the outside, and the introduced ions are dissociated by contacting them with the CID gas to generate product ions.

[0038] The data processing unit 3 receives detection data from the ion detector 29 and performs processing based on that data. Its functional blocks include a data acquisition unit 31, a quantitative calculation unit 32, and a quantitative reference information storage unit 33. The analysis control unit 4 controls the operation of the liquid chromatograph 1 and the mass spectrometer 2 according to a sulfur compound analysis method (method file) 41, which contains information indicating analysis conditions specifically for the quantitative determination of sulfur metabolites and is stored in an internal storage unit. The central control unit 5 primarily performs overall control of each unit and provides a user interface through the input unit 6 and the display unit 7. As described above, the sulfur compound analysis method 41 includes individual MS analysis conditions for each sulfur compound.

[0039] Generally, the data processing unit 3, the analysis control unit 4, and the central control unit 5 are actually personal computers or more powerful workstations, and the functions of each of these functional blocks can be realized by running dedicated software (computer programs) pre-installed on these computers. In other words, the sulfur compound analysis method 41 is also a type of program that provides parameters for analysis and procedures for data processing.

[0040] [Overview of measurement operation] In the analysis system described above, when analyzing sulfur compounds, the measurement unit repeatedly performs MRM measurements for predetermined MRM transitions within a predetermined measurement time range. The measurement operation, including this MRM measurement, will be explained in general terms.

[0041] In the liquid chromatograph 1, the liquid delivery pump 12 draws the mobile phase from the mobile phase container 11 and sends it to the injector 13 at a nearly constant flow rate. The injector 13 injects a predetermined amount of sample into the mobile phase at a predetermined timing. The sample is introduced into the column 14 along with the flow of the mobile phase, and the various components in the sample are separated and eluted over time as they pass through the column 14. The eluate exiting the column 14 outlet reaches the ESI spray 21 of the mass spectrometer 2. In the ESI spray 21, the sample is sprayed into the ionization chamber 201 as fine charged droplets. The charged droplets come into contact with residual gas molecules and split, and as the solvent in the droplets vaporizes, the compound molecules in the sample are ionized.

[0042] The generated ions are sent to the first intermediate vacuum chamber 202 via the desolvation tube 22, and further sent to the high vacuum chamber 204 via the ion guide 23, the pores of the skimmer 24, and the multi-pole ion guide 25. Ions originating from the sample components are introduced into the pre-stage quadrupole mass filter 26, and only ions with a predetermined m / z corresponding to the voltage applied to the electrodes constituting the pre-stage quadrupole mass filter 26 selectively pass through as precursor ions. Precursor ions incident on the collision cell 27 are dissociated upon contact with the CID gas, generating various product ions.

[0043] The various product ions generated are introduced into a downstream quadrupole mass filter 28. Only product ions with a predetermined m / z corresponding to the voltage applied to the electrodes constituting the downstream quadrupole mass filter 28 selectively pass through and reach the ion detector 29. The ion detector 29 generates a detection signal corresponding to the amount of incident ions, and the digitized detection data, converted by an analog-to-digital converter (not shown), is input to the data processing unit 3.

[0044] The analysis control unit 4 controls the mass spectrometer 2 so that a voltage corresponding to the desired MRM transition is applied to the electrodes of the pre-stage quadrupole mass filter 26 and the post-stage quadrupole mass filter 28, respectively. It also sets the DC voltage applied to each part, including the ion transport optical system (not shown), so that ions passing through the pre-stage quadrupole mass filter 26 enter the collision cell 27 with a predetermined collision energy. This allows for the acquisition of detection data indicating the ionic intensity of product ions with a specific m / z, generated by the dissociation of precursor ions with a specific m / z, among the ions originating from various components in the sample, corresponding to a specific MRM transition.

[0045] In the mass spectrometer 2, precursor ions are dissociated by CID within the collision cell 27, but the manner of dissociation differs depending on the kinetic energy, i.e., collision energy, of the precursor ions. The collision energy is determined by the DC potential difference between the inlet end of the collision cell 27 and its preceding stage (in Figure 1, this is the preceding quadrupole mass filter 26, but it may also be an ion optical element such as another ion lens), and therefore the collision energy is usually expressed by this potential difference. Accordingly, the collision energy can be adjusted by applying a DC bias voltage to either the preceding quadrupole mass filter 26 or the preceding ion optical element, or both.

[0046] [Procedure for quantitative analysis of sulfur compounds] Figure 2 is a flowchart illustrating an example of a general procedure for the quantitative analysis of sulfur compounds. Note that the following explanation is an example of quantifying all 17 sulfur compounds shown in Figure 3; however, quantifying all of them is not mandatory, and it is sufficient to quantify at least one active sulfur compound within one group.

[0047] First, a user who intends to perform quantification prepares standard substances of known concentrations of chemically stable sulfur compounds (basic compounds) for which standard substances are available in each group shown in FIG. 3. Then, each standard substance is measured using the analysis system shown in FIG. 1 in accordance with the sulfur compound analysis method 41 (step S1). That is, for the cysteine group, cysteine is used; for the cystine group, cystine is used; for the reduced glutathione group, reduced glutathione is used; for the oxidized glutathione group, oxidized glutathione is used; and for the sulfate group, a standard substance of a basic compound which is either sulfuric acid or thiosulfuric acid is measured. Note that, for the side reaction product group, generally no commercially available standard substance exists, so measurement for a standard substance is not performed.

[0048] An example of main LC analysis conditions and MS analysis conditions for measuring the 17 types of sulfur compounds described above is as follows. However, it is assumed herein that an LCMS-8060NX manufactured by Shimadzu Corporation is used as the measurement unit. Further, MRM transitions for each sulfur compound are as described in FIG. 8 and FIG. 9. <LC analysis conditions> ·Column type: PFPP column ·Gradient conditions: Mobile phase A (formate-Water) and mobile phase B (formate-Metha n nol) gradient elution used ·Mobile phase flow rate: 0.3 mL / min ·Sample injection volume: 2 μL ·Column temperature: 40 °C

[0049] ·Nebulizer gas flow rate: 3.0 L / min ·Drying gas flow rate: 10.0 L / min ·Heating gas flow rate: 10.0 L / min ·Desolvation line temperature: 250 °C ·Heat block (in ionization chamber) temperature: 400 °C ·Ionization mode: IonFocus ESI

[0050] The 17 sulfur compounds listed above can generally be separated over time under the LC analysis conditions described above. Figure 5 is an example of a chromatogram obtained by measuring an actual sample using LC-MS / MS under the above conditions. In Figure 5, some sulfur compounds overlap over time, but these overlapping compounds can be separated by MRM measurement.

[0051] When measurements are performed on the basic compounds of each group using the analysis system described above, the chromatogram data obtained by repeating the MRM measurement is stored in the data acquisition unit 31. Subsequently, the quantitative calculation unit 32 analyzes the chromatogram data to detect peaks corresponding to each basic compound, calculates the area value of those peaks, and obtains quantitative reference information showing the relationship between the known concentration and area value for each group (step S2). The obtained quantitative reference information is stored in the quantitative reference information storage unit 33.

[0052] This quantitative reference information can be, for example, a calibration curve that expresses the relationship between concentration and area value using a mathematical formula. When creating a calibration curve, it is desirable to use standard substances at multiple concentration levels. Alternatively, the quantitative reference information may be a combination of a single concentration value and an area value. Furthermore, although this quantitative reference information is for the basic compound, here, the parameters are adjusted so that the signal intensity is approximately the same for the basic compound and activated sulfur at the same concentration for each group, so the above quantitative reference information can be used directly as quantitative reference information for activated sulfur.

[0053] Next, the user performs the derivatization pretreatment described above on the reduced active sulfur in the target sample to stabilize the active sulfur (step S3). Then, the user sets the pretreated sample in the measurement unit and instructs the input unit 6 to start the measurement. Upon receiving the instruction, the analysis control unit 4 performs LC / MS / MS analysis on the sample according to the sulfur compound analysis method (step S4). The analysis conditions at this time are the same as those used for measurement of the standard substance in step S1.

[0054] The chromatogram data obtained by this measurement is temporarily stored in the data acquisition unit 31. In the mass spectrometer 2, MRM measurement is performed for each sulfur compound in the sample according to the MS analysis conditions and adjusted parameters described above. For example, if the sample contains cysteine ​​(CysS-HPE) and cysteine ​​persulfide (CysSS-HPE) at the same concentration, the peaks on the chromatogram corresponding to these sulfur compounds will have approximately the same area value.

[0055] The quantitative calculation unit 32 analyzes the chromatogram data and calculates the area value of the peak corresponding to each sulfur compound. Then, for each group, it calculates the quantitative value of multiple sulfur compounds, that is, the concentration value as an absolute quantitative value, or the concentration ratio as a relative quantitative value, based on the area values ​​corresponding to the multiple sulfur compounds contained in one group and the quantitative reference information for each group read from the quantitative reference information storage unit 33 (step S5). The quantitative values ​​thus obtained are displayed on the display unit 7.

[0056] For example, when performing relative quantification of multiple sulfur compounds included in the cysteine ​​group, the area value relative to a specified concentration of cysteine ​​is obtained as quantitative reference information. Therefore, the concentration value of cysteine ​​in the sample can be determined from the area value of cysteine ​​in the sample and the quantitative reference information, and the relative concentration, i.e., the relative quantitative value, of cysteine ​​persulfide can be determined from the area value of cysteine ​​persulfide in the sample and the quantitative reference information. Furthermore, when performing absolute quantification of multiple sulfur compounds included in the cysteine ​​group, a calibration curve showing the relationship between the concentration and area value of cysteine ​​is obtained as quantitative reference information. Therefore, the absolute concentration value of cysteine ​​in the sample can be determined from the area value of cysteine ​​in the sample and the quantitative reference information, and the approximate absolute concentration value of cysteine ​​persulfide can be determined from the area value of cysteine ​​persulfide in the sample and the quantitative reference information. The same applies to sulfur compounds included in other groups. For the by-reactant group, there is no quantitative reference information based on the measurement results of standard substances. However, if the concentrations of the multiple sulfur compounds included in the group are the same, the area values ​​should be approximately the same. Therefore, relative quantification can be performed based on the ratio of the area values ​​of each sulfur compound.

[0057] As described above, the quantitative method of this embodiment allows for the easy quantification of activated sulfur, for which standard substances are generally difficult to obtain.

[0058] [Differentiation] The numerical values ​​shown in the above analysis conditions are merely examples and may naturally vary depending on the type of equipment used. Furthermore, the systems and procedures used in the above explanation are merely examples and are not limited to those described herein.

[0059] Furthermore, while the above embodiment focused on quantifying active sulfur compounds belonging to four groups—cysteine, cystine, reduced glutathione, and oxidized glutathione—it is naturally possible to quantify various other types of active sulfur compounds using a similar method. For example, mercaptopyruvic acid and coenzyme A are reduced active sulfur compounds having a thiol group, and like cysteine, there are multiple sulfur compounds in which one or more sulfur atoms are linked in a chain between the thiol group and the other structures. On the other hand, cystathionine is an oxidized active sulfur compound similar to cystine, and there are multiple sulfur compounds in which the same substructure is linked via a chain of sulfur atoms, but with a different number of sulfur atoms. Even with these sulfur compounds, multiple sulfur compounds that differ in the number of sulfur atoms in the chain links but have substantially the same other structures can be grouped together, and a compound in that group with one or the minimum number of sulfur atoms in the chain links can be used as a standard substance to quantify the multiple sulfur compounds in that group.

[0060] [Various forms] It will be obvious to those skilled in the art that the exemplary embodiments described above are specific examples of the following embodiments.

[0061] (Section 1) One embodiment of the method for quantifying active sulfur according to the present invention is a method for quantifying sulfur compounds containing active sulfur in a sample using a chromatograph-mass spectrometer, and is a method for quantifying sulfur compounds that are the target of analysis in at least one of the following groups: the group containing cystine and related active sulfur, the group containing cysteine ​​and related active sulfur, the group containing oxidized glutathione and related active sulfur, and the group containing reduced glutathione and related active sulfur. A standard substance measurement step involves measuring known concentrations of standard substances containing basic compounds, such as cystine, cysteine, oxidized glutathione, or reduced glutathione, which are included in the group to be analyzed, using a chromatographic mass spectrometer. Based on the premise that the signal intensities of multiple sulfur compounds in the same group have a predetermined relationship when their concentrations are the same, a quantitative reference information acquisition step is performed to obtain quantitative reference information for quantifying the basic compounds and other active sulfur compounds in the group to be analyzed, based on the measurement results obtained in the standard substance measurement step. A sample measurement step comprising measuring each sulfur compound included in the group to be analyzed in the sample using a chromatographic mass spectrometer, wherein the measurement is performed under predetermined analytical conditions for each sulfur compound so that when the concentrations of multiple sulfur compounds included in the same group are the same, the signal intensities of the multiple sulfur compounds have a predetermined relationship; A quantitative step is performed to quantify each sulfur compound included in the group to be analyzed, using the measurement results obtained in the sample measurement step and the quantitative reference information. It holds.

[0062] According to the method for quantifying active sulfur described in paragraph 1, various sulfur compounds containing active sulfur, which is chemically unstable and for which standard materials are difficult to obtain, can be quantified simply and with sufficient accuracy by using chemically stable sulfur compound standard materials that are generally readily available. Here, "related active sulfur" typically refers to a sulfur compound that differs from that, i.e., a certain sulfur compound such as cystine, only in the number of sulfur atoms in the chain bond of sulfur atoms (an integer of 1 or more), and whose other structures are the same. However, for multiple sulfur compounds included in the same group, the other structures do not need to be exactly the same; they only need to be substantially the same.

[0063] Furthermore, since the active sulfur contained in the cysteine ​​and reduced glutathione groups is particularly unstable, it is desirable to perform a stabilization pretreatment using a reagent that selectively reacts with thiol groups (typically HPE-IAM mentioned above) prior to measurement.

[0064] (Section 2) In the method for determining active sulfur described in Section 1, the determination step may involve determining the ratio of concentrations of multiple sulfur compounds contained in each group.

[0065] According to the method for quantifying active sulfur described in paragraph 2, relative quantification of sulfur compounds contained in each group can be performed. Even within the same group, there may be large concentration differences among the sulfur compounds contained in the sample. However, when measuring the sample, for example, if the concentrations of multiple sulfur compounds contained in the same group are the same, the measurement is performed under predetermined analytical conditions for each sulfur compound so that the ratio of the measurement results of these multiple sulfur compounds falls within a predetermined acceptable range. This reduces the likelihood of signal saturation of high-concentration components or, conversely, failure to detect low-concentration components, allowing for quantification of each component within a wide dynamic range.

[0066] (3) In the method for determining active sulfur described in paragraph 1, the quantitative reference information acquisition step may involve obtaining a calibration curve showing the relationship between concentration and signal intensity as quantitative reference information, and the determination step may involve performing absolute determination using an external standard method with respect to the quantitative reference information.

[0067] According to the method for quantifying active sulfur described in paragraph 3, for example, the approximate absolute concentration of cysteine ​​persulfide, for which standard substances are difficult to obtain, can be determined by a pseudo-external standard method using readily available standard substances such as cysteine.

[0068] (Section 4) In the method for determining active sulfur described in Section 1, the chromatograph-mass spectrometer is a liquid chromatograph-triple quadrupole mass spectrometer, and the analytical conditions adjusted for each sulfur compound in the sample measurement step may include multiple reaction monitoring (MRM) transitions, collision energy, and mass resolution.

[0069] Here, the collision energy can be adjusted by the DC voltage applied to the ion transport optical system (including the pre-quadrupole mass filter) located at the entrance of the collision cell and further ahead in the triple quadrupole mass spectrometer. Furthermore, the mass resolution is determined by the m / z width of the ions passing through the two (pre- and post-quadrupole) quadrupole mass filters in the triple quadrupole mass spectrometer, and can therefore be adjusted by the voltage applied to the electrodes constituting these quadrupole mass filters.

[0070] According to the method for quantifying active sulfur described in Section 4, not only the MRM transition and collision energy, which greatly affect the signal intensity of ions, but also the mass resolution can be adjusted as needed. Therefore, even if there are large differences in the signal intensity of multiple sulfur compounds contained in one group for the same component concentration under the same analytical conditions, it is possible to adjust them so that their signal intensities are roughly the same. This allows for good quantification of active sulfur contained in the four groups mentioned above.

[0071] (Section 5) Another embodiment of the method for quantifying active sulfur according to the present invention is a method for quantifying sulfur compounds containing active sulfur in a sample using a chromatographic mass spectrometer, wherein the method quantifies a plurality of sulfur compounds that are the target of analysis in at least one of the following groups: a group containing cystine and related active sulfur, a group containing cysteine ​​and related active sulfur, a group containing oxidized glutathione and related active sulfur, a group containing reduced glutathione and related active sulfur, a group containing sulfuric acid and related inorganic sulfur compounds, and a group containing by-reactants produced from sulfur compounds by a derivatization treatment that stabilizes active sulfur, A pretreatment step in which a derivatization treatment is performed on the sample using a predetermined derivatization reagent to stabilize active sulfur, A sample measurement step comprising measuring each sulfur compound included in the group to be analyzed in the sample pretreated by the above pretreatment step using a chromatographic mass spectrometer, wherein the measurement is performed under predetermined analytical conditions for each sulfur compound so that when the concentrations of multiple sulfur compounds included in the same group are the same, the signal intensities of the multiple sulfur compounds have a predetermined relationship, A quantitative step is performed to relatively quantify multiple sulfur compounds included in the group to be analyzed, based on the measurement results obtained in the sample measurement step, It holds.

[0072] (Item 6) In the method for determining active sulfur described in Item 5, the analytical conditions predetermined for each sulfur compound may be set such that the signal intensities of multiple sulfur compounds are substantially the same.

[0073] In the method for quantifying active sulfur described in Section 6, if multiple sulfur compounds belonging to a certain group are present in the sample at the same concentration, the signal intensities of these multiple sulfur compounds will be approximately identical. Therefore, the ratio of the signal intensities of multiple sulfur compounds represents the ratio of their relative concentrations, making relative quantification possible. Furthermore, even if the signal intensities of multiple sulfur compounds belonging to the same group are not identical when their concentrations are the same, and instead follow a known predetermined relationship (ratio), it is clear that the relative ratio of concentrations can be calculated using this known predetermined relationship.

[0074] Thus, according to the quantitative determination methods for active sulfur described in paragraphs 5 and 6, it is possible to perform highly accurate and wide-dynamic-range relative quantification of multiple sulfur compounds within each group without performing measurements against standard substances of sulfur compounds.

[0075] (Section 7) Another aspect of the method for quantifying active sulfur according to the present invention is a method for quantifying sulfur compounds containing active sulfur in a sample using a chromatographic mass spectrometer, A standard substance measurement step involves grouping multiple sulfur compounds that have the same structure other than the chain bond of sulfur atoms but differ in the number of sulfur atoms constituting the chain bond, and measuring a standard substance of a known concentration of a sulfur compound that is included in the same group as the target sulfur compound to be quantified and has one or the minimum number of sulfur atoms constituting the chain bond, using a chromatographic mass spectrometer. Based on the premise that the signal intensities of multiple sulfur compounds in the same group have a predetermined relationship when their concentrations are the same, a quantitative reference information acquisition step is performed to obtain quantitative reference information for quantifying the sulfur compounds in the group, based on the measurement results obtained in the standard substance measurement step. A sample measurement step in which a chromatograph-mass spectrometer is used to measure the target sulfur compound in a sample under predetermined analytical conditions for each sulfur compound, such that when the concentrations of multiple sulfur compounds in the same group are the same, the signal intensities of the multiple sulfur compounds have the predetermined relationship. A quantitative step is performed to quantify the target sulfur compound using the measurement results obtained in the sample measurement step and the quantitative reference information. It holds.

[0076] According to the method for quantifying active sulfur described in Section 7, the quantification of not only sulfur compounds related to cystine, cysteine, oxidized glutathione, and reduced glutathione, as mentioned above, but also a wide range of sulfur compounds including active sulfur, can be easily and accurately performed using readily available, chemically stable sulfur compound standards. [Explanation of symbols]

[0077] 1…Liquid Chromatography 11...Mobile phase container 12…Liquid transfer pump 13…Injector 14... Columns 2...Mass spectrometer 201... Ionization Room 202...First Intermediate Vacuum Chamber 203...Second Intermediate Vacuum Chamber 204…High vacuum chamber 21…ESI spray 22…Desolvation tube 23…Aeon Guide 24... Skimmer 25…Multipole type ion guide 26…Pre-stage quadrupole mass filter 27...Collision cell 28... Quadrupole mass filter in the later stage 29…Ion detector 3…Data Processing Unit 31…Data Collection Department 32...Quantitative calculation section 33…Quantitative reference information storage unit 4…Analysis and Control Section 41… Sulfur compounds Analysis Methods 5…Central Control Unit 6...Input section 7...Display section

Claims

1. A method for quantifying sulfur compounds containing active sulfur in a sample using a chromatographic mass spectrometer, wherein the method quantifies sulfur compounds belonging to at least one of the following groups that are the target of analysis: a group containing cystine and related active sulfur, a group containing cysteine ​​and related active sulfur, a group containing oxidized glutathione and related active sulfur, and a group containing reduced glutathione and related active sulfur. A standard substance measurement step involves measuring known concentrations of standard substances containing basic compounds, such as cystine, cysteine, oxidized glutathione, or reduced glutathione, which are included in the group to be analyzed, using a chromatographic mass spectrometer. Based on the premise that the signal intensities of multiple sulfur compounds in the same group have a predetermined relationship when their concentrations are the same, a quantitative reference information acquisition step is performed to obtain quantitative reference information for quantifying the basic compounds and other active sulfur compounds in the group to be analyzed, based on the measurement results obtained in the standard substance measurement step. A sample measurement step comprising measuring each sulfur compound included in the group to be analyzed in the sample using a chromatographic mass spectrometer, wherein the measurement is performed under predetermined analytical conditions for each sulfur compound so that when the concentrations of multiple sulfur compounds included in the same group are the same, the signal intensities of the multiple sulfur compounds have a predetermined relationship; A quantitative step is performed to quantify each sulfur compound included in the group to be analyzed, using the measurement results obtained in the sample measurement step and the quantitative reference information. A method for quantifying active sulfur, comprising the characteristics of a method for quantifying active sulfur.

2. The method for quantifying active sulfur according to claim 1, wherein the quantitative step involves determining the ratio of concentrations of multiple sulfur compounds contained in each group.

3. The method for quantifying activated sulfur according to claim 1, wherein in the quantitative reference information acquisition step, a calibration curve showing the relationship between concentration and signal intensity is obtained as the quantitative reference information, and in the quantitative step, absolute quantification is performed using an external standard method with respect to the quantitative reference information.

4. The chromatograph-mass spectrometer is a liquid chromatograph-triple quadrupole mass spectrometer, and the analytical conditions adjusted for each sulfur compound in the sample measurement step include multiple reaction monitoring transitions, collision energy, and mass resolution, as described in claim 1, for the determination of active sulfur.

5. A method for quantifying sulfur compounds containing active sulfur in a sample using a chromatographic mass spectrometer, A standard substance measurement step involves grouping multiple sulfur compounds that have the same structure other than the chain bond of sulfur atoms but differ in the number of sulfur atoms constituting the chain bond, and measuring a standard substance of a known concentration of a sulfur compound that is included in the same group as the target sulfur compound to be quantified and has one or the minimum number of sulfur atoms constituting the chain bond, using a chromatographic mass spectrometer. Based on the premise that the signal intensities of multiple sulfur compounds in the same group have a predetermined relationship when their concentrations are the same, a quantitative reference information acquisition step is performed to obtain quantitative reference information for quantifying the sulfur compounds in the group, based on the measurement results obtained in the standard substance measurement step. A sample measurement step in which a chromatograph-mass spectrometer is used to measure the target sulfur compound in a sample under predetermined analytical conditions for each sulfur compound, such that when the concentrations of multiple sulfur compounds in the same group are the same, the signal intensities of the multiple sulfur compounds have the predetermined relationship. A quantitative step is performed to quantify the target sulfur compound using the measurement results obtained in the sample measurement step and the quantitative reference information. A method for quantifying active sulfur, comprising the characteristics of a method for quantifying active sulfur.

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