Quantitative method of active 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 containing active sulfur molecular species (hereinafter simply referred to as "active sulfur") in a sample, and more particularly to a quantitative determination method utilizing chromatography-mass spectrometry. [Background technology]

[0002] Active sulfur is a general term for highly reactive sulfur compounds, such as cysteine ​​persulfide, in which an excess (usually two or more) sulfur atoms are added to the thiol (SH) group of cysteine. Active sulfur is present in large quantities in various organs and blood in the body, and is known to function as a major antioxidant that exhibits the ability to eliminate active oxygen in the body. For this reason, active sulfur is expected to be applied in various fields related to medicine, such as preventing human aging and developing diagnostic, preventive, and therapeutic drugs for various diseases and illnesses, including cancer, in which oxidative stress is involved.

[0003] For these reasons, there has been a demand for a method for quantifying active sulfur in living organisms. However, active sulfur, especially reduced active sulfur, is a highly reactive compound and is easily decomposed during pretreatment and analysis, making accurate quantification difficult.

[0004] Known methods for quantifying active sulfur, including reduced active sulfur, include a method using a liquid chromatograph-tandem mass spectrometer (LC-MS / MS), as described in, for example, Non-Patent Documents 1 and 2. In this method, reduced active sulfur is converted into a stable derivative using an electrophilic alkylating agent, and then the active sulfur is selectively detected by multiple reaction monitoring (MRM) measurement using LC-MS / MS. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Tomoaki Ida and 15 others, "Reactive cysteine ​​persulfides and S-polythiolation regulate oxidative stress and redox signaling", Proc. Natl. Acad. Sci. USA, May 27, 2014, Vol.111, No.21, pp.7606-7611 (Internet)<URL:https: / / doi.org / 10.1073%2Fpnas.1321232111> ) [Non-Patent Document 2] Hide Ihara and 2 others, "New developments in research on reactive sulfur", Biochemistry, Vol. 91, No. 3, pp. 388-398, published June 25, 2019 Summary of the Invention [Problem to be solved by the invention]

[0006] Generally, to perform quantification using the above-mentioned methods, a standard substance containing the target component at a known concentration is required. However, for chemically unstable active sulfur such as cysteine ​​persulfide, standard substances are not generally provided. Therefore, it is practically difficult for general users to perform quantitative analysis of active sulfur using such methods.

[0007] The present invention has been made to solve these problems, and its main object is to provide a quantitative determination method that can easily and precisely quantify various sulfur compounds, including chemically unstable active sulfur, using standard substances that are generally easily available. [Means for solving the problem]

[0008] A first aspect of the method for quantifying active sulfur according to the present invention, which has been made to solve the above problems, is a method for quantifying sulfur compounds containing active sulfur in a sample using a chromatography mass spectrometer, and the method quantifies sulfur compounds contained in at least one group to be analyzed, selected from 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 of measuring a standard substance having a known concentration of a basic compound, which is cystine, cysteine, oxidized glutathione, or reduced glutathione, contained in the group to be analyzed, by a chromatography mass spectrometer; a quantitative reference information acquisition step of obtaining quantitative reference information for quantifying the basic compounds and other active sulfur compounds contained in the group to be analyzed based on the measurement results obtained in the standard substance measurement step, on the assumption that the signal intensities of the multiple sulfur compounds contained in the same group will have a predetermined relationship when the concentrations of the multiple sulfur compounds are the same; a sample measurement step of measuring each sulfur compound contained in the group to be analyzed in a sample by a chromatography mass spectrometer, the measurement being performed under analysis conditions previously determined for each sulfur compound such that, when the concentrations of multiple sulfur compounds contained in the same group are the same, the signal intensities of the multiple sulfur compounds have the predetermined relationship; a quantification step of quantifying each sulfur compound contained in the group to be analyzed using the measurement results obtained in the sample measurement step and the quantitative reference information; has.

[0009] A second 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 chromatography mass spectrometer, the method being for quantifying a plurality of sulfur compounds contained in at least one group to be analyzed, selected from 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, the group containing reduced glutathione and related active sulfur, the group containing sulfuric acid and related inorganic sulfur compounds, and the group containing by-products generated from sulfur compounds by a derivatization process for stabilizing active sulfur, A pretreatment step of carrying out a derivatization treatment on the specimen using a predetermined derivatization reagent to stabilize active sulfur; a sample measurement step of measuring each sulfur compound contained in the group to be analyzed in the sample pretreated by the pretreatment step using a chromatographic mass spectrometer, the measurement being performed under analytical conditions previously determined for each sulfur compound such that when the concentrations of multiple sulfur compounds contained in the same group are the same, the signal intensities of the multiple sulfur compounds have a predetermined relationship; A quantification step of relatively quantifying a plurality of sulfur compounds contained in the group to be analyzed based on the measurement results obtained in the sample measurement step; has.

[0010] A third aspect of the method for quantifying active sulfur according to the present invention is a method for quantifying a sulfur compound containing active sulfur contained in a sample using a chromatography mass spectrometer, comprising the steps of: a standard substance measurement step in which a plurality of sulfur compounds having the same structure except for the chain bonds of sulfur atoms but different numbers of sulfur atoms constituting the chain bonds are grouped together, and 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 smallest number of sulfur atoms constituting the chain bonds is measured by a chromatographic mass spectrometer; a quantitative reference information acquisition step of obtaining quantitative reference information for quantifying the sulfur compounds contained in the same group based on the measurement results obtained in the standard substance measurement step, under the assumption that the signal intensities of the sulfur compounds contained in the same group will have a predetermined relationship when the concentrations of the sulfur compounds are the same; a sample measurement step of measuring a target sulfur compound in a sample using a chromatography mass spectrometer under analysis conditions that are predefined for each sulfur compound such that, when the concentrations of a plurality of sulfur compounds included in the same group are the same, the signal intensities of the plurality of sulfur compounds have the predetermined relationship; a quantification step of quantifying a target sulfur compound using the measurement result obtained by the sample measurement step and the quantitative reference information; has. Effect of the Invention

[0011] According to the first and third aspects of the method for quantifying active sulfur of the present invention, various sulfur compounds containing active sulfur, which are chemically unstable and for which standard substances are difficult to obtain, can be quantified simply and with sufficient accuracy by using standard substances of chemically stable sulfur compounds that are generally easily available.

[0012] In addition, according to the second aspect of the method for quantifying active sulfur of the present invention, even if there is a large difference in concentration between multiple sulfur compounds included in the same group, including unstable active sulfur, it is possible to accurately perform relative quantification of such multiple sulfur compounds without using standard substances for the sulfur compounds. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram of an example of an analysis system for carrying out the method for quantifying active sulfur according to the present invention. [Diagram 2] 2 is a flowchart showing an outline of the procedure for quantitatively analyzing sulfur compounds containing active sulfur using the analysis system shown in FIG. 1. [Diagram 3] A list of the 17 sulfur compounds that are the targets of the sulfur compound analysis method. [Figure 4] FIG. 1 shows an example of derivatization to stabilize active sulfur. [Diagram 5] Figure showing an example of a measured chromatogram of 17 types of sulfur compounds in a sample. [Figure 6] FIG. 2 is a graph showing an example of a comparison of the signal intensity of each sulfur compound before and after adjustment of MS analysis conditions. [Figure 7] FIG. 1 shows an example of chromatograms of cysteine ​​and cysteine ​​persulfide before and after adjustment of MS analysis conditions. [Figure 8] FIG. 1 shows an example of a comparison of the relative signal intensities of sulfur compounds using an actual sample (plasma sample) after adjusting the MS analysis conditions. [Figure 9] FIG. 1 shows an example of a comparison of the relative signal intensities of sulfur compounds using real samples (cells) after adjusting the MS analysis conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0015] [Sulfur compounds to be analyzed] The main purpose of the quantitative determination method according to this embodiment is to determine the amount of sulfur compounds containing active sulfur present in a specimen such as human blood (plasma) or cells taken from an organ, but the specimen is not limited to these as long as it is a biological sample.

[0016] FIG. 3 shows 17 types of sulfur compounds that are targets of quantitative analysis in this quantitative method. As shown on the right side of Figure 3, the 17 types of sulfur compounds are divided into six groups. Of these, the four important active sulfurs are the cysteine ​​group, the cystine group, the reduced glutathione group, and the oxidized glutathione group. Of 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 not marked with an asterisk. The active sulfurs contained in the cysteine ​​group and the reduced glutathione group have highly reactive thiol groups (-SH), and are non-stable active sulfurs that are particularly poor in chemical stability.

[0017] The other two groups are the sulfate group and the by-reactant group. The sulfur compounds in the above four groups are organic compounds, whereas the sulfur compounds in the sulfate group are inorganic compounds, in this example, sulfate and thiosulfate. On the other hand, the sulfur compounds in the by-reactant group are not compounds originally contained in the specimen, but are sulfur compounds derived during the pretreatment process described below. Generally, sulfur compounds that can be contained in the sulfate group, such as sulfate and thiosulfate, are not active sulfur, but are important compounds as sulfur sources in the metabolism in vivo, and therefore can be considered as sulfur compounds equivalent to active sulfur.

[0018] As can be seen from FIG. 3, in this example, the sulfur compounds belonging to the same group in the above four groups are compounds that differ only in the number of sulfur atoms constituting the chain bonds of sulfur atoms (...-SS-...), and the structure (composition) of the other parts is the same. For example, the three sulfur compounds belonging to the cysteine ​​group have 1 to 3 sulfur atoms in the chain bonds. Since there are also sulfur compounds with the number of sulfur atoms being 4 or more, such compounds may be added to the same group. The same applies to other groups. In addition, even if the structures other than the chain bonds of sulfur atoms are not completely identical, it may be possible to include them in the same group. In addition, for the side reactant group, the binding site where the reaction occurs is not necessarily uniquely determined, so various side reactants may be generated. Therefore, the side reactant group may include multiple sulfur compounds with different structures even if the number of sulfur atoms in the chain bonds is the same.

[0019] Furthermore, among the above four groups, sulfur compounds for which standard substances are readily available are compounds in which the number of sulfur atoms constituting chain bonds is 1 or the smallest (specifically, 2) in the group. Thus, regardless of the number of sulfur compounds contained in a group and regardless of the structure of the sulfur compounds contained in a group, a compound in which the number of sulfur atoms constituting chain bonds is 1 or the smallest among the multiple sulfur compounds in the group may be suitable as a standard substance.

[0020] [Pretreatment for stabilizing active sulfur] The unstable active sulfur described above is difficult to analyze as it is. Therefore, in order to stabilize the active sulfur, a pretreatment is carried out by derivatization. Specifically, β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM) is used as the derivatization reagent.

[0021] FIG. 4 is a diagram showing a derivatization reaction using HPE-IAM. HPE-IAM selectively reacts with SH groups contained in reduced active sulfur to derivatize the active sulfur. The side reaction to polysulfide chains is suppressed by the action of OH groups contained in the derivative, making it chemically stable. In FIG. 3 and FIGS. 5, 6, 8, and 9 described below, the abbreviation "-HPE" in the compound name indicates that it is a derivative using HPE-IAM. Although such derivatization is essentially necessary as a pretreatment for the measurement of reduced active sulfur, it is not necessary to use HPE-IAM as a reagent.

[0022] [Quantitative principle of active sulfur] In the quantitative determination method of the present embodiment, a liquid chromatograph-triple quadrupole mass spectrometer (LC-MS / MS) is used to comprehensively measure the 17 types of sulfur compounds contained in a sample. In LC-MS / MS, the 17 types of sulfur compounds and other various impurities in the sample are roughly separated in time by a liquid chromatograph in the front stage, and the 17 types of sulfur compounds are selectively detected in a triple quadrupole mass spectrometer in the rear stage, and an ion intensity signal corresponding to the amount of each compound is obtained.

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

[0024] When quantifying components in a sample using LC-MS / MS or the like, the most common method is the absolute quantification method using an external standard, in which a calibration curve is prepared in advance using a standard substance with a known concentration, and the unknown concentration is obtained from the peak area value, etc., by referring to the calibration curve. However, as described above, it is generally difficult to obtain a standard substance for active sulfur. Therefore, here, we carry out relative quantification, in which a standard substance is a commonly available sulfur compound as a standard, and the concentration ratio between multiple sulfur compounds is obtained, and pseudo absolute quantification, in which a calibration curve prepared using a standard substance of one type of sulfur compound is also used to calculate the absolute concentration of active sulfur related to that sulfur compound.

[0025] However, even for sulfur compounds in the same group, there is a large difference in the signal intensity (typically the peak area value in a chromatogram) obtained for the same concentration. Therefore, we consider adjusting the MS analysis conditions for each group so that the signal intensities for the multiple sulfur compounds in one group are roughly uniform.

[0026] There are several parameters that affect the signal intensity in LC-MS / MS. For example, there are usually multiple MRM transitions for selectively detecting a certain compound, and the sensitivity differs depending on the MRM transition. In addition, changing the collision energy during collision-induced dissociation (CID) for MS / MS analysis changes the manner of ion dissociation, so the signal intensity in a specific MRM transition changes. In many cases, the signal intensities for multiple sulfur compounds can be roughly matched by appropriately selecting an MRM transition for each sulfur compound and appropriately adjusting the collision energy value.

[0027] However, when the signal intensity is adjusted by adjusting the MRM transition and collision energy value, the signal intensity is adjusted to the lowest one among multiple sulfur compounds, which may result in a low detection sensitivity. Therefore, in order to avoid the detection sensitivity becoming too low, a method is adopted in which the detection sensitivity is increased and the signal intensity is adjusted for some sulfur compounds by lowering the mass resolution. In addition, in a triple quadrupole mass spectrometer, the mass resolution can be adjusted by adjusting the m / z value width of the ions passing through the front and rear quadrupole mass filters.

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

[0029] Figure 6 shows an example of the signal intensities and their signal intensity ratios (A) before parameter adjustment and the signal intensities and their signal intensity ratios (B) after parameter adjustment for all 17 types of sulfur compounds mentioned above. As with Figure 7, this is also the measurement result when a standard substance of the same concentration (a specially synthesized standard substance for active sulfur) was used by LC / MS / MS.

[0030] As can be seen from Figure 6(A), the two sulfur compounds, CysSSSCys and HSO3-HPE, have particularly low signal intensities in each group. Therefore, if the collision energy is adjusted to lower the signal intensities of other sulfur compounds to match these sulfur compounds, the overall detection sensitivity will be too low. Therefore, for these two sulfur compounds, the mass resolution is adjusted (actually lowered) to increase the signal intensity, and for the other sulfur compounds, the collision energy value is adjusted to lower the signal intensity, so that the signal intensities for the same concentration in each group are uniform. As a result, as shown on the right side of Figure 6(B), the signal intensity after adjustment is within the range of approximately 20 to 60% of the maximum signal intensity before adjustment, and a significant decrease in detection sensitivity is avoided. In addition, the variation in signal intensity within each group is within 10%.

[0031] However, the results in Figure 6 are the results of measuring a standard substance that does not contain impurities, and since actual specimens are affected by various impurities other than sulfur compounds, it is necessary to evaluate these effects as well. Therefore, using plasma and cells as real samples, we performed an addition recovery test for each sulfur compound under the parameters after adjusting as shown in Figure 6(B) and calculated the recovery rate. The results are shown in Figures 8 and 9. In both the case of plasma and cells, it was confirmed that by diluting the sample appropriately, the relative intensity difference for almost all sulfur compounds could be kept within the range of approximately ±20%.

[0032] As described above, for the above 17 types of sulfur compounds, the signal intensities of the multiple sulfur compounds included in the same group can be made almost uniform by appropriately selecting the MRM transitions and appropriately adjusting the collision energy value and the mass resolution. That is, when the concentrations of the multiple sulfur compounds included in the same group are the same, the signal intensities of the multiple sulfur compounds can be made to have a predetermined relationship. Since this relationship holds for any concentration, the relative quantification of the multiple sulfur compounds included in each group can be performed with high accuracy and a wide dynamic range. In addition, if the measurement results for the standard substances of the sulfur compounds that are chemically stable and easily available are obtained for each group, it is possible to obtain quantitative reference information for absolute quantification of all the sulfur compounds in the group from the results.

[0033] In the quantitative determination method of this embodiment, a manufacturer that provides a system and method file for performing quantitative determination uses standard substances of the above 17 types of sulfur compounds to examine, for each sulfur compound, MRM transitions, collision energy values, and mass resolutions that will give approximately the same signal intensity for the same concentration of sulfur compounds in the same group. Then, based on the results, analytical conditions (LC analytical conditions and MS analytical conditions) including individual MS analytical conditions for each sulfur compound are determined, and a method file for performing LC / MS / MS analysis under the analytical conditions is created.

[0034] [Example of analysis system configuration] An example of an analysis system used for the measurement will be described. Fig. 1 is a schematic diagram of an example of the analysis system. This analysis system is an LC-MS / MS system including a measurement unit including 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 a mobile phase (solvent) is stored, a liquid delivery pump 12 that draws in and delivers the mobile phase from the mobile phase container 11, an injector 13 that injects a sample into the mobile phase, and a column 14 that separates multiple components contained in the sample in the time direction. Although not shown, an autosampler is usually connected to the injector 13 in order to sequentially analyze multiple specimens.

[0036] The mass spectrometer 2 is a triple quadrupole mass spectrometer, and includes 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 of which is evacuated by a vacuum pump (not shown). The ionization chamber 201 is provided with an ESI sprayer 21 that performs ionization by an ElectroSpray Ionization (ESI) method, and the ionization chamber 201 and the next-stage first intermediate vacuum chamber 202 are connected by a desolvation tube 22. An ion guide 23 that focuses and transports ions is disposed in the first intermediate vacuum chamber 202, and the first intermediate vacuum chamber 202 and the next-stage second intermediate vacuum chamber 203 are connected through a small hole formed at the top of a skimmer 24. A multipole ion guide 25 that focuses and transports ions is also disposed in the second intermediate vacuum chamber 203.

[0037] In the high vacuum chamber 204, a pre-quadrupole mass filter 26, a collision cell 27, a post-quadrupole mass filter 28, and an ion detector 29 are arranged along the ion flow. A quadrupole ion guide is arranged inside the collision cell 27. The pre-quadrupole mass filter 26 and the post-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 from the outside into the collision cell 27, and the collision cell 27 has the function of dissociating the introduced ions 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 the data, and includes, as functional blocks, a data collection 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 that includes information indicating analysis conditions dedicated to sulfur metabolite quantification stored in an internal storage unit. The central control unit 5 mainly executes overall control of each unit and user interface through the input unit 6, display unit 7, etc. As described above, the sulfur compound analysis method 41 includes individual MS analysis conditions for each sulfur compound.

[0039] In general, the data processing unit 3, analysis control unit 4, and central control unit 5 are actually personal computers or computers called higher performance workstations, and the functions of the above-mentioned functional blocks can be realized by running dedicated software (computer programs) preinstalled on the computer. In other words, the sulfur compound analysis method 41 is also a kind of program that provides parameters for analysis and procedures for data processing.

[0040] [Outline of measurement operation] In the above analytical system, MRM measurements are repeatedly performed for a predetermined MRM transition within a predetermined measurement time range in the measurement section during the analysis of sulfur compounds. The measurement operation including this MRM measurement will be briefly described below.

[0041] In the liquid chromatograph 1, the liquid delivery pump 12 draws in the mobile phase from the mobile phase container 11 and delivers it to the injector 13 at a substantially constant flow rate. The injector 13 injects a predetermined amount of sample (specimen) into the mobile phase at a predetermined timing. The sample rides along with the flow of the mobile phase and is introduced into the column 14, and various components in the sample are separated in the time direction and eluted while passing through the column 14. The eluate discharged from the outlet of the column 14 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 in the process of the solvent in the droplets evaporating, the compound molecules in the sample are ionized.

[0042] The generated ions are sent to a first intermediate vacuum chamber 202 via a desolvation tube 22, and further to a high vacuum chamber 204 via an ion guide 23, a small hole in a skimmer 24, and a multipole ion guide 25. Ions derived from the sample components are introduced into a pre-quadrupole mass filter 26, and only ions having a predetermined m / z value corresponding to the voltage applied to the electrodes constituting the pre-quadrupole mass filter 26 selectively pass through as precursor ions. The precursor ions entering the collision cell 27 come into contact with a CID gas and are dissociated, generating various product ions.

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

[0044] The analysis control unit 4 controls the mass spectrometer 2 so that voltages corresponding to the target MRM transition are applied to the electrodes of the front-stage quadrupole mass filter 26 and the rear-stage quadrupole mass filter 28. In addition, DC voltages applied to each unit, including the ion transport optical system (not shown), are set so that ions that have passed through the front-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 ion intensity of ions corresponding to a specific MRM transition among ions derived from various components contained in the sample, in other words, product ions having a specific m / z generated by dissociation of a precursor ion having a specific m / z.

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

[0046] [Procedure for quantitative analysis of sulfur compounds] Fig. 2 is a flow chart showing an example of a schematic procedure for quantitative analysis of sulfur compounds. Note that the following explanation is an example of quantifying all of the 17 types of sulfur compounds shown in Fig. 3, but it is not essential to quantify all of them, and it is possible to quantify at least one active sulfur contained in one group.

[0047] First, the user who wants 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 according to the sulfur compound analysis method 41 (step S1). That is, for the cysteine group, cysteine is measured; for the cystine group, cystine is measured; for the reduced glutathione group, reduced glutathione is measured; for the oxidized glutathione group, oxidized glutathione is measured; and for the sulfate group, a standard substance of a basic compound that is either sulfuric acid or thiosulfuric acid is measured. Note that for the side reaction product group, generally available standard substances usually do not exist, so measurement of the standard substance is not performed.

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

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

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

[0051] When the basic compounds of each group are measured using the above-mentioned analysis system, the chromatogram data acquired by repeating the MRM measurements is stored in the data collection unit 31. The quantitative calculation unit 32 then analyzes the chromatogram data to detect peaks corresponding to each basic compound, calculates the area values ​​of the peaks, and obtains quantitative reference information indicating 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 in a mathematical formula. When creating a calibration curve, it is desirable to use standard substances with multiple levels of concentration. The quantitative reference information may also be a combination of a certain concentration value and an area value. Although this quantitative reference information is for the basic compound, the parameters are adjusted here so that the signal intensities of the basic compound and active sulfur for the same concentration for each group are roughly the same, so that the quantitative reference information is used as it is as quantitative reference information for active sulfur.

[0053] Thereafter, the user performs the above-mentioned pretreatment of derivatization on the reduced active sulfur in the target specimen to stabilize the active sulfur (step S3). The user then places the pretreated specimen in the measurement unit and issues an instruction to start measurement via the input unit 6. Upon receiving the instruction, the analysis control unit 4 executes LC / MS / MS analysis of the specimen according to the sulfur compound analysis method (step S4). The analysis conditions at this time are the same as those used in the measurement of the standard substance in step S1.

[0054] The chromatogram data obtained by this measurement is temporarily stored in the data collecting unit 31. In the mass spectrometer 2, MRM measurement is performed on each sulfur compound in the sample according to the above-mentioned MS analysis conditions and adjusted parameters, so that, 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, the quantitative values ​​of the sulfur compounds, that is, the concentration values ​​as absolute quantitative values ​​or the concentration ratios as relative quantitative values, are calculated based on the area values ​​corresponding to the sulfur compounds contained in one group and the quantitative reference information of 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 contained in a 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 a sample is obtained from the area value of cysteine ​​in a sample and quantitative reference information, and the relative concentration of cysteine ​​persulfide in a sample, that is, the relative quantitative value, is obtained from the area value of cysteine ​​persulfide in a sample and quantitative reference information.In addition, when performing absolute quantification of multiple sulfur compounds contained in a cysteine ​​group, a calibration curve showing the relationship between the concentration of cysteine ​​and the area value is obtained as quantitative reference information.Therefore, the absolute concentration value of cysteine ​​in a sample is obtained from the area value of cysteine ​​in a sample and quantitative reference information, and the approximate absolute concentration value of cysteine ​​persulfide is obtained from the area value of cysteine ​​persulfide in a sample and quantitative reference information.The same applies to sulfur compounds contained in other groups. For the by-reactant group, there is no quantitative reference information based on the measurement results of a standard substance. However, if the concentrations of the multiple sulfur compounds contained in the group are the same, the area values ​​should be approximately the same, so relative quantification can be performed based on the ratio of the area values ​​of each sulfur compound.

[0057] As described above, according to the quantitative determination method of the present embodiment, it is possible to easily determine the amount of active sulfur, for which it is generally difficult to obtain a standard substance.

[0058] [Variations] The numerical values ​​shown in the above analysis conditions are merely examples, and may vary depending on the model of the apparatus used, etc. Furthermore, the systems and procedures used in the above explanation are merely examples, and it goes without saying that they are not limited to those described here.

[0059] In the above embodiment, the active sulfur contained in the four groups of cysteine, cystine, reduced glutathione, and oxidized glutathione is the target of the quantification, but it is natural that the quantification can be performed by the same method for various other types of active sulfur. For example, mercaptopyruvate and coenzyme A are reduced active sulfurs having a thiol group, and there are multiple sulfur compounds in which one or more sulfur atoms are bonded in a chain between the thiol group and other structures, as in cysteine. On the other hand, cystathionine is an oxidized active sulfur similar to cystine, and there are multiple sulfur compounds in which the same partial structures are connected via a chain of sulfur atoms and the number of sulfur atoms is different. Even in such sulfur compounds, multiple sulfur compounds in which the number of sulfur atoms in the chain bond is different and the other structures are substantially the same are grouped, and a compound in which the number of sulfur atoms in the chain bond is one or the smallest among the sulfur compounds in the group is used as a standard substance, thereby making it possible to quantify multiple sulfur compounds in the group.

[0060] [Various aspects] It will be apparent to those skilled in the art that the above-described exemplary embodiments are illustrative of the following aspects.

[0061] (Item 1) One 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 chromatography mass spectrometer, and the method quantifies sulfur compounds contained in at least one group to be analyzed, selected from 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, comprising: A standard substance measurement step of measuring a standard substance having a known concentration of a basic compound, which is cystine, cysteine, oxidized glutathione, or reduced glutathione, contained in the group to be analyzed, by a chromatography mass spectrometer; a quantitative reference information acquisition step of obtaining quantitative reference information for quantifying the basic compounds and other active sulfur compounds contained in the group to be analyzed based on the measurement results obtained in the standard substance measurement step, on the assumption that the signal intensities of the multiple sulfur compounds contained in the same group will have a predetermined relationship when the concentrations of the multiple sulfur compounds are the same; a sample measurement step of measuring each sulfur compound contained in the group to be analyzed in a sample by a chromatography mass spectrometer, the measurement being performed under analysis conditions previously determined for each sulfur compound such that, when the concentrations of multiple sulfur compounds contained in the same group are the same, the signal intensities of the multiple sulfur compounds have the predetermined relationship; a quantification step of quantifying each sulfur compound contained in the group to be analyzed using the measurement results obtained in the sample measurement step and the quantitative reference information; has.

[0062] According to the method for quantifying active sulfur described in paragraph 1, various sulfur compounds containing active sulfur, which are chemically unstable and for which standard substances are difficult to obtain, can be quantified easily and with sufficient accuracy by using a standard substance of a chemically stable sulfur compound that is generally easily available. Note that the "active sulfur related thereto" as used herein typically refers to a sulfur compound that is different from the sulfur compound, i.e., cystine, only in the number of sulfur atoms (an integer of 1 or more) in the chain bond of sulfur atoms and has the same structure as the sulfur compound. However, the structures of the sulfur compounds in the same group do not have to be completely the same as long as they are substantially the same.

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

[0064] (Item 2) In the method for quantifying active sulfur according to item 1, the quantifying step may involve determining a ratio of concentrations of a plurality of sulfur compounds contained in each group.

[0065] According to the method for quantifying active sulfur described in paragraph 2, it is possible to perform relative quantification of sulfur compounds contained in each group. Although there may be a large difference in concentration of sulfur compounds contained in a sample even within the same group, when measuring a sample, for example, when the concentrations of multiple sulfur compounds contained in the same group are the same, the measurement is performed under analysis conditions previously determined for each sulfur compound so that the ratio of the measurement results of the multiple sulfur compounds falls within a predetermined allowable range. Therefore, signal saturation of a component with a high concentration and, conversely, detection omission of a component with a low concentration are unlikely to occur, and each component can be quantified over a wide dynamic range.

[0066] (Item 3) In the method for quantifying active sulfur described in item 1, the quantitative reference information acquisition step can include obtaining a calibration curve showing the relationship between concentration and signal intensity as the quantitative reference information, and the quantification step can include performing absolute quantification by an external standard method using the quantitative reference information.

[0067] According to the method for quantifying active sulfur described in paragraph 3, for example, a pseudo external standard method using a readily available standard substance such as cysteine ​​can be used to determine the approximate absolute concentration value of cysteine ​​persulfide, for which it is difficult to obtain a standard substance.

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

[0069] Here, the collision energy can be adjusted by a DC voltage applied to the entrance of the collision cell in the triple quadrupole mass spectrometer and the ion transport optical system (including the front-stage quadrupole mass filter) located ahead of the entrance. In addition, the mass resolution is determined by the m / z width of the ions passing through the two (front and rear) quadrupole mass filters in the triple quadrupole mass spectrometer, and therefore can be adjusted by the voltage applied to the electrodes that constitute the quadrupole mass filters.

[0070] According to the method for quantifying active sulfur described in item 4, not only the MRM transition and collision energy, which have a large effect on the signal intensity of ions, but also the mass resolution can be adjusted as necessary, so that even if there is a large difference in the signal intensities of multiple sulfur compounds contained in one group for the same component concentration under the same analytical conditions, it is possible to adjust the signal intensities so that they are roughly the same. This allows the active sulfur contained in the above-mentioned four groups to be quantified satisfactorily.

[0071] (Item 5) 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 chromatography mass spectrometer, the method being a method for quantifying a plurality of sulfur compounds contained in at least one group to be analyzed, selected from 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, the group containing reduced glutathione and related active sulfur, the group containing sulfuric acid and related inorganic sulfur compounds, and the group containing by-products generated from sulfur compounds by a derivatization process that stabilizes active sulfur, A pretreatment step of carrying out a derivatization treatment on the specimen using a predetermined derivatization reagent to stabilize active sulfur; a sample measurement step of measuring each sulfur compound contained in the group to be analyzed in the sample pretreated by the pretreatment step using a chromatographic mass spectrometer, the measurement being performed under analytical conditions previously determined for each sulfur compound such that when the concentrations of multiple sulfur compounds contained in the same group are the same, the signal intensities of the multiple sulfur compounds have a predetermined relationship; A quantification step of relatively quantifying a plurality of sulfur compounds contained in the group to be analyzed based on the measurement results obtained in the sample measurement step; has.

[0072] (Item 6) In the method for quantifying active sulfur described in item 5, the analytical conditions predefined for each sulfur compound may be defined so that the signal intensities of the multiple sulfur compounds are approximately the same.

[0073] In the method for quantifying active sulfur described in paragraph 6, when a sample contains multiple sulfur compounds in a certain group at the same concentration, the signal intensities of the multiple sulfur compounds are approximately the same. Therefore, the ratio of the signal intensities of the multiple sulfur compounds represents the relative concentration ratio, and relative quantification is possible. Even if the signal intensities of the multiple sulfur compounds in the same group are not the same but have a known predetermined relationship (ratio), it is clear that the relative ratio of the concentrations can be calculated by utilizing the known predetermined relationship.

[0074] Thus, according to the method for quantifying active sulfur described in paragraphs 5 and 6, it is possible to perform relative quantification of multiple sulfur compounds with high accuracy and a wide dynamic range within each group without performing measurements on standard substances of sulfur compounds.

[0075] (Item 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 contained in a sample using a chromatography mass spectrometer, comprising the steps of: a standard substance measurement step in which a plurality of sulfur compounds having the same structure except for the chain bonds of sulfur atoms but different numbers of sulfur atoms constituting the chain bonds are grouped together, and 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 smallest number of sulfur atoms constituting the chain bonds is measured by a chromatographic mass spectrometer; a quantitative reference information acquisition step of obtaining quantitative reference information for quantifying the sulfur compounds contained in the same group based on the measurement results obtained in the standard substance measurement step, under the assumption that the signal intensities of the sulfur compounds contained in the same group will have a predetermined relationship when the concentrations of the sulfur compounds are the same; a sample measurement step of measuring a target sulfur compound in a sample using a chromatography mass spectrometer under analysis conditions that are predefined for each sulfur compound such that, when the concentrations of a plurality of sulfur compounds included in the same group are the same, the signal intensities of the plurality of sulfur compounds have the predetermined relationship; a quantification step of quantifying a target sulfur compound using the measurement result obtained by the sample measurement step and the quantitative reference information; has.

[0076] According to the method for quantifying active sulfur described in item 7, not only the sulfur compounds related to cystine, cysteine, oxidized glutathione, and reduced glutathione described above, but also a wide range of sulfur compounds containing active sulfur can be quantified easily and with sufficient accuracy by using standard substances of chemically stable sulfur compounds that are generally easily available. [Explanation of symbols]

[0077] 1. Liquid chromatograph 11...Mobile phase container 12...Liquid delivery pump 13...Injector 14…Column 2...Mass spectrometer 201…Ionization chamber 202…First intermediate vacuum chamber 203…Second intermediate vacuum chamber 204…High vacuum chamber 21…ESI Spray 22...Desolvation tube 23…Ion Guide 24…Skimmer 25...Multipole ion guide 26...Pre-quadrupole mass filter 27…Collision cell 28...Post-quadrupole mass filter 29...Ion detector 3. Data processing section 31…Data collection section 32...Quantitative calculation section 33…Quantitative reference information storage unit 4. Analysis control section 41…Active Sulfur Analysis Method 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 chromatography mass spectrometer, the method quantifying sulfur compounds contained in at least one group to be analyzed, selected from 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, comprising: A standard substance measurement step of measuring a standard substance having a known concentration of a basic compound, which is cystine, cysteine, oxidized glutathione, or reduced glutathione, contained in the group to be analyzed, by a chromatography mass spectrometer; a quantitative reference information acquisition step of obtaining quantitative reference information for quantifying the basic compounds and other active sulfur compounds contained in the group to be analyzed based on the measurement results obtained in the standard substance measurement step, on the assumption that the signal intensities of the multiple sulfur compounds contained in the same group will have a predetermined relationship when the concentrations of the multiple sulfur compounds are the same; a sample measurement step of measuring each sulfur compound contained in the group to be analyzed in a sample by a chromatography mass spectrometer, the measurement being performed under analysis conditions previously determined for each sulfur compound such that, when the concentrations of multiple sulfur compounds contained in the same group are the same, the signal intensities of the multiple sulfur compounds have the predetermined relationship; a quantification step of quantifying each sulfur compound contained in the group to be analyzed using the measurement results obtained in the sample measurement step and the quantitative reference information; The method for quantifying active sulfur comprises the steps of:

2. The method for quantifying active sulfur according to claim 1 , wherein the quantitative determination step determines a ratio of concentrations of the plurality of sulfur compounds contained in each group.

3. The method for quantifying active 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 quantification step, absolute quantification is performed by an external standard method using the quantitative reference information.

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

5. A method for quantifying sulfur compounds containing active sulfur in a sample using a chromatography mass spectrometer, the method comprising the steps of: quantifying a plurality of sulfur compounds contained in at least one group to be analyzed, the group being 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-products generated from sulfur compounds by a derivatization process for stabilizing active sulfur, the method comprising the steps of: A pretreatment step of carrying out a derivatization treatment of the specimen using a predetermined derivatization reagent to stabilize active sulfur; a sample measurement step of measuring each sulfur compound contained in the group to be analyzed in the sample pretreated by the pretreatment step using a chromatographic mass spectrometer, the measurement being performed under analytical conditions previously determined for each sulfur compound such that when the concentrations of multiple sulfur compounds contained in the same group are the same, the signal intensities of the multiple sulfur compounds have a predetermined relationship; A quantification step of relatively quantifying a plurality of sulfur compounds contained in the group to be analyzed based on the measurement results obtained in the sample measurement step; The method for quantifying active sulfur comprises the steps of:

6. 6. The method for quantifying active sulfur according to claim 5, wherein the analytical conditions predefined for each sulfur compound are determined so that the signal intensities of a plurality of sulfur compounds are approximately the same.

7. A method for quantifying a sulfur compound containing active sulfur contained in a sample using a chromatography mass spectrometer, comprising the steps of: a standard substance measurement step in which a plurality of sulfur compounds having the same structure except for the chain bonds of sulfur atoms but different numbers of sulfur atoms constituting the chain bonds are grouped together, and 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 bonds is measured by a chromatographic mass spectrometer; a quantitative reference information acquisition step of obtaining quantitative reference information for quantifying the sulfur compounds contained in the same group based on the measurement results obtained in the standard substance measurement step, under the assumption that the signal intensities of the sulfur compounds contained in the same group will have a predetermined relationship when the concentrations of the sulfur compounds are the same; a sample measurement step of measuring a target sulfur compound in a sample using a chromatography mass spectrometer under analysis conditions that are predefined for each sulfur compound such that, when the concentrations of a plurality of sulfur compounds included in the same group are the same, the signal intensities of the plurality of sulfur compounds have the predetermined relationship; a quantification step of quantifying a target sulfur compound using the measurement result obtained by the sample measurement step and the quantitative reference information; The method for quantifying active sulfur comprises the steps of:

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