Method for providing information relating to urinary metabolite contained in urine sample, and method for evaluating neuroblastoma using said method for providing information

Abstract

The present invention addresses the problem of providing a method for providing information relating to one or more urinary metabolites contained in a urine sample, with reduced variation in elution time when an aqueous mobile phase is used. The problem can be solved by a method for providing information relating to one or more urinary metabolites contained in a urine sample, the method comprising: a step for separating the one or more urinary metabolites contained in a urine sample with use of a reversed phase column having an adamantyl group; and a step for providing information relating to the types of the one or more separated urinary metabolites. The types of the one or more urinary metabolites provided in the information providing step include a 3-methoxytyramine sulfate conjugate and / or vanillactate, and the provided information is used to evaluate neuroblastoma.

Description

Information providing method for providing information on urinary metabolites contained in a urine sample and method for evaluating neuroblastoma using said information providing method

[0001] The disclosure of the present application relates to an information providing method for providing information on urinary metabolites contained in a urine sample, and a method for evaluating neuroblastoma using the information provided by the information providing method.

[0002] Childhood cancer is the leading cause of death among children, yet countermeasures remain inadequate. It has been shown that the earlier the stage of most childhood cancers, the better the treatment outcomes, and early detection directly impacts prognosis. However, early diagnosis is extremely difficult, and cancers are often discovered after infiltration into other organs or distant metastasis, making treatment difficult. Furthermore, when surgical resection is difficult or there is a possibility of residual tumor after surgical resection, chemotherapy or radiation therapy is often performed. For tumors for which no useful tumor markers have been identified, there is no effective means of assessing treatment efficacy other than diagnostic imaging. Examples of such tumors include neuroblastoma, Wilms' tumor, rhabdomyosarcoma, osteosarcoma, and Ewing's sarcoma family tumors. While imaging tests such as computed tomography (CT) and positron emission tomography (PET) are effective for assessing residual tumors, when these tests are inconclusive, biopsy or resection of the residual tumor mass is necessary, resulting in significant invasive procedures. On the other hand, even if residual tumors can be evaluated using PET scans, many cases of "pediatric cancer" require continued imaging diagnosis over the long term, and there is debate about the carcinogenicity of these tests themselves.

[0003] Methods and devices for detecting catecholamine metabolites (vanillylmandelic acid (VMA) and homovanillic acid (HVA)) in urine, for example, for cancer screening of neuroblastoma, a type of childhood cancer, have been reported (e.g., Patent Documents 1 to 3).

[0004] Furthermore, Patent Document 4 describes that metabolites in the urine of pediatric cancer patients and those in the urine of healthy children of the same age were comprehensively analyzed using a liquid chromatography mass spectrometer (LC / MS) to search for tumor markers, and that multiple metabolites that change between healthy children and pediatric cancer patients were identified, and that a group of urinary tumor markers that are promising for evaluating pediatric cancer were identified based on multivariate analysis.

[0005] Japanese Patent Laid-Open No. 5-113438 Japanese Patent Publication No. 7-92456 Japanese Patent Publication No. 6-58364 Japanese Patent Laid-Open No. 2019-168319

[0006] Patent Document 4 describes that a large number of urinary metabolites can be tumor markers for childhood cancer, particularly neuroblastoma. Incidentally, when actually conducting clinical tests using urine samples, it is preferable to have as high reproducibility as possible. However, the invention described in Patent Document 4 is an invention that searches for urinary metabolites that can be tumor markers by combining various analyses, and does not disclose anything about reproducibility.

[0007] The present invention has been disclosed to solve the above-mentioned problems. As a result of extensive research, the present inventors have found that when the same separation column is used to repeatedly separate urinary metabolites contained in a urine sample, the following problems occur: (1) The ODS column (octadecylsilyl group (C １８ H ３７ The following new findings were found: (1) when a column packed with a stationary phase modified with methyl methyl acrylate (Si) is used, variations in the elution time of urinary metabolites may occur in each separation run when an aqueous mobile phase is used; (2) when a reversed-phase column having an adamantyl group is used, variations in the elution time of urinary metabolites in each separation run are reduced compared to an ODS column, even when an aqueous mobile phase is used; and (3) information on 3-methoxytyramine sulfate conjugates and / or vanyl lactate as types of urinary metabolites can be used to evaluate neuroblastoma.

[0008] That is, the purpose of the disclosure in the present application is to provide an information providing method (hereinafter, sometimes simply referred to as "information providing method") that provides information on urinary metabolites contained in a urine sample with little variation in elution time when an aqueous mobile phase is used, and a method for evaluating neuroblastoma (hereinafter, sometimes simply referred to as "evaluation method") using the information providing method.

[0009] The disclosure of the present application relates to an information providing method for providing information on urinary metabolites contained in a urine sample and a method for evaluating neuroblastoma using the information providing method, as described below.

[0010] (1) A method for providing information about urinary metabolites contained in a urine sample, the method comprising: a separation step of separating urinary metabolites contained in the urine sample using a reverse-phase column having an adamantyl group; and an information providing step of providing information about the types of the separated urinary metabolites, wherein the types of urinary metabolites provided in the information providing step include 3-methoxytyramine sulfate conjugates and / or vanyl lactate, and the provided information is used for evaluating neuroblastoma. (2) The method for providing information according to (1), wherein the types of urinary metabolites further include at least one selected from the group consisting of vanillylmandelic acid, homovanillic acid, 3-methoxytyramine, and 3-methoxytyrosine. (3) The information on the 3-methoxytyramine sulfate conjugate provided in the information providing step includes quantitative information obtained using a triple quadrupole mass spectrometer, and when quantifying by the internal standard method using the triple quadrupole mass spectrometer, a measurement specimen and positive ion with m / z 248.1 and an internal standard and positive ion with m / z 252.1 are used as precursor ions, and a measurement specimen and positive ion with m / z 151.0 and an internal standard and positive ion with m / z 155.0 are used as product ions. (4) The information providing method according to any one of (1) to (3), wherein the information on vanyl lactate provided in the information providing step includes quantitative information obtained using a triple quadrupole mass spectrometer, and when quantifying by an internal standard method using the triple quadrupole mass spectrometer, the measurement specimen and negative ions having m / z 211.1 and the internal standard and negative ions having m / z 214.1 are used as precursor ions, and the measurement specimen and negative ions having m / z 150.0 and the internal standard and negative ions having m / z 152.0 are used as product ions. (5) A method for evaluating neuroblastoma, wherein the evaluation method evaluates neuroblastoma in a subject using information provided by the information providing method according to any one of (1) to (4). (6) The evaluation method according to (5), wherein the types of urinary metabolites include the 3-methoxytyramine sulfate conjugate, and when evaluating neuroblastoma, the histological malignancy according to the International Classification of Neuroblastoma is evaluated as being high or low according to the International Classification of Neuroblastoma.(7) The evaluation method according to (5), wherein the types of urinary metabolites include vanyl lactate, and when evaluating neuroblastoma, whether or not the patient is at high risk according to the International Neuroblastoma Risk Classification System is evaluated.

[0011] The information providing method disclosed in the present application (liquid chromatography mass spectrometer using a reversed-phase column having an adamantyl group) reduces the variation in elution times of separated urinary metabolites compared to an ODS column when repeatedly separating urinary metabolites contained in a urine sample using the same separation column, even when an aqueous mobile phase is used. In clinical tests, urinary metabolites are repeatedly separated using the same separation column. By implementing the information providing method disclosed in the present application, the variation in elution times of the same type of urinary metabolite is reduced, thereby reducing the variation in the information provided about urinary metabolites. Furthermore, the information providing method disclosed in the present application can separate 3-methoxytyramine sulfate conjugates and / or vanyl lactate, which can be used to evaluate neuroblastoma, in a single separation step.

[0012] FIG. 1A is a graph showing overlaid mass chromatograms obtained by injecting the same sample three times using an ADME column with an adamantyl ethyl group in Example 1. The samples were 3-methoxytyramine sulfate conjugate (MTS) and 3-methoxytyramine (3MT). A mass chromatogram is a chart in which the ion intensity of a specific mass number is extracted and plotted against time on the horizontal axis. FIG. 1B is a graph showing overlaid mass chromatograms (3MT, MTS) obtained by injecting the same sample three times using an ODS column in Comparative Example 1. FIG. 2 is a graph showing the results of separation of six sample mixtures (MTR, VMA, VLA, and HVA) using an ADME column in Example 2. FIG. 3A is a graph showing that when 3-methoxytyramine sulfate conjugate (MTS) is used as a tumor marker, it is possible to accurately distinguish cases with poor prognosis and high histological malignancy. FIG. 3B is a graph showing that when vanyl lactate (VLA) is used as a tumor marker, high-risk cases with poor prognosis can be identified with high accuracy.

[0013] The information providing method and evaluation method disclosed in this application will be described in detail below.

[0014] (Embodiment of Information Providing Method) First, an information providing method according to an embodiment will be described. The information providing method includes a separation step of separating urinary metabolites contained in a urine sample using a reversed-phase column having an adamantyl group, and an information providing step of providing information on the types of the separated urinary metabolites.

[0015] The reversed-phase column having an adamantyl group contains adamantane, which is a cage-shaped C10 molecule represented by the following formula (1) in which 10 carbon atoms are arranged in the same manner as in the diamond structure.

[0016] The reversed-phase column having an adamantyl group is not particularly limited as long as the adamantyl group is used as the functional group. For example, an ADME column having an adamantylethyl group as the functional group can be obtained by, but is not limited to, filling a column with a packing material in which adamantane and a silica gel substrate are bonded together using an ethyl group as a spacer. Packing materials having an adamantylethyl group as the functional group and columns containing such packing materials can be prepared, for example, with reference to the method described in Japanese Patent No. 6199140. Alternatively, a commercially available ADME column may be used. Examples of commercially available products include ADME-HR from Osaka Soda Co., Ltd.

[0017] The information provision method uses urine as a sample. Compared to substances in the blood, urinary metabolites are less susceptible to enzymes and are structurally stable, making them highly useful as tumor markers. Urine is also a convenient sample, as it can be easily collected even from children.

[0018] The information provided by implementing the information provision method can be used to evaluate neuroblastoma. Neuroblastoma is a type of childhood cancer that originates in the sympathetic ganglia of the trunk (the axial part of the body excluding the limbs) or the adrenal medulla. Approximately 65% ​​of cases occur in the abdomen, and half of those originate in the adrenal medulla. Neuroblastoma is not caused by any specific factors such as genetics or lifestyle, and the cause of its development is often unknown. Therefore, there is a need to discover suitable major markers for evaluating neuroblastoma.

[0019] As shown in the Examples and Comparative Examples below, when a urine sample is repeatedly separated using a reversed-phase column having the same adamantyl group, the variation in elution times of urinary metabolites is reduced compared to an ODS column when an aqueous mobile phase is used. In other words, the information provision method disclosed in this application can be said to be a selective invention that achieves significant benefits by combining a target marker with a reversed-phase column having an adamantyl group, among many other samples and separation columns. In particular, the newly discovered marker, 3-methoxytyramine sulfate conjugate (MTS), is highly hydrophilic, and an aqueous mobile phase is preferable for its separation, making this method particularly effective. Therefore, the information about the separated urinary metabolites (including not only the type but also the elution times during repeated separation) can be said to be novel information that cannot be obtained using conventional ODS columns.

[0020] As mentioned above, the new marker, 3-methoxytyramine sulfate conjugate (MTS), is highly hydrophilic. Therefore, a 100% aqueous mobile phase is preferably used for its separation. However, as long as the effects disclosed herein are obtained, the aqueous mobile phase may contain, for example, a few percent of organic substances other than water. The non-water substance is not particularly limited as long as it is a substance commonly used in the relevant technical field, and examples include alcohols such as methanol and 2-propanol, acetic acid, formic acid, trifluoroacetic acid, organic solvents, etc. In this specification, the term "aqueous mobile phase" preferably refers to a 100% aqueous mobile phase, but is not limited to a 100% aqueous mobile phase. A 100% aqueous mobile phase may also be referred to as a 100% aqueous mobile phase that does not contain organic substances. As described in the Examples below, it is preferable to use an aqueous mobile phase for the separation when separating 3-methoxytyramine sulfate conjugate (MTS), which is highly hydrophilic. However, when separating multiple urinary metabolites in a single analysis, separation may be performed under gradient conditions using an organic solvent.

[0021] A urine sample refers to urine collected from a subject, and samples obtained by treating the urine (for example, urine to which a preservative such as toluene, xylene, or hydrochloric acid has been added).

[0022] The subjects are children, specifically children aged 0 to 15. However, the subjects are not limited to humans, and may include other mammals, such as primates (monkeys, chimpanzees, etc.), livestock animals (cows, horses, pigs, sheep, etc.), pet animals (dogs, cats, etc.), and laboratory animals (mice, rats, rabbits, etc.).

[0023] The types of urinary metabolites provided by the information provision method disclosed in the present application include at least 3-methoxytyramine sulfate conjugate (MTS) represented by the following formula (2) and / or vanyl lactate (VLA) represented by the following formula (3), for use in evaluating neuroblastoma.

[0024] As shown in the Examples below, the 3-methoxytyramine sulfate conjugate represented by formula (2) is a novel marker newly discovered by the present inventors as a biomarker capable of evaluating whether or not a tumor has a high histological malignancy according to the International Neuroblastoma Pathological Classification. As shown in the Examples below, vanyl lactate represented by formula (3) is a novel marker newly discovered by the present inventors as a biomarker capable of evaluating whether or not a tumor has a high risk according to the International Neuroblastoma Risk Classification. Therefore, the information provided by carrying out the information providing method includes at least 3-methoxytyramine sulfate conjugate and / or vanyl lactate.

[0025] The types of urinary metabolites provided may further include at least one selected from the group consisting of vanillylmandelic acid (VMA), homovanillic acid (HVA), 3-methoxytyramine (3MT), and 3-methoxytyrosine (MTR). Vanillylmandelic acid, homovanillic acid, 3-methoxytyramine, and 3-methoxytyrosine (hereinafter, sometimes referred to as "four types of urinary metabolites") are known markers already described in Patent Document 1. However, as shown in the Examples below, it has been confirmed that when urinary metabolites are separated using an ADME column, the four types of urinary metabolites can be simultaneously separated along with the 3-methoxytyramine sulfate conjugate and vanyl lactate. Therefore, by providing information on at least one of the four types of urinary metabolites in addition to the novel markers 3-methoxytyramine sulfate conjugate and / or vanyl lactate in a single separation operation, useful information for evaluating neuroblastoma can be efficiently provided. It should be noted that Patent Document 1 describes many tumor markers other than the four types mentioned above, and the description of Patent Document 1 is incorporated herein by reference.

[0026] The information providing step is not particularly limited as long as it can provide information about the types of urinary metabolites separated in the separation step. Without limitation, the urinary metabolites separated in the separation step may be analyzed using a mass spectrometer (MS). The information providing step may include quantitative information about the types of urinary metabolites in addition to the information about the types.

[0027] Optionally, a triple quadrupole mass spectrometer may be used as a mass spectrometer for analyzing urinary metabolites. This triple quadrupole mass spectrometer consists of two quadrupole mass spectrometers (Q) connected in series (Q-q-Q) with a collision chamber (q) in between. Triple quadrupole mass spectrometers are generally used in conjunction with a chromatograph as a pretreatment device. A system connected to a liquid chromatograph is called LC-MS / MS. Using a mass spectrometer as a chromatograph detector provides high selectivity through mass separation, enabling highly sensitive quantitative analysis. Mass spectrometers have higher selectivity than other chromatographic detectors, and the selected ion detection (SIM) method of a single quadrupole mass spectrometer often provides sufficient sensitive analysis. However, the mass selectivity of a quadrupole is approximately 0.7 Da, and compounds with a mass difference smaller than this cannot be separated. Therefore, blood, urine, biological tissue, etc. may contain matrices with masses close to those of the analyte. Of course, quantitative analysis is possible if pretreatment and chromatographic separation are possible, but this requires time to consider the conditions.

[0028] The triple quadrupole mass spectrometer is an analytical method to avoid the influence of interfering substance ions with similar mass differences. In a triple quadrupole mass spectrometer, the precursor ion to be analyzed is selected in the first quadrupole mass spectrometer, the ion is fragmented in the collision cell, and the product ions, which depend on the molecular structure, are detected in the third quadrupole mass spectrometer, thereby increasing selectivity and enabling highly accurate quantitative analysis.

[0029] In addition, the use of stable isotope-labeled compounds is effective for quantifying markers using this method. For example, stable isotope-labeled compounds (internal standards) generated by substituting some of the intramolecular hydrogen atoms with deuterium generally have the same physicochemical properties as the original compounds. Therefore, while their behavior in LC is identical, the mass-to-charge ratio (m / z) information obtained by mass spectrometry differs between the internal standard and the original compounds, making them distinguishable by mass spectrometry. This method employs an internal standard method. In this method, the internal standard is added in equal amounts to a calibration curve sample (sample with known concentration) and a measurement sample (sample with unknown concentration). The concentration of the target compound in the sample can be estimated by comparing the sensitivity ratios of the internal standard to the target compound in the calibration curve sample and the measurement sample. Additionally, this method can offset various irregular changes that the analytical sample undergoes during analysis, potentially improving analytical accuracy.

[0030] (Embodiment of Neuroblastoma Evaluation Method) Next, a neuroblastoma evaluation method according to an embodiment will be described. In the evaluation method, neuroblastoma in a subject is evaluated using information provided by the information providing method according to the embodiment described above. The provided information may include at least 3-methoxytyramine sulfate conjugate and / or vanyl lactate, which are novel tumor markers for neuroblastoma. If necessary, the information may also include at least one or more compounds selected from the group consisting of vanillylmandelic acid, homovanillic acid, 3-methoxytyramine, and 3-methoxytyrosine. Furthermore, if necessary, the information may also include a tumor marker described in Patent Document 1. Neuroblastoma can be evaluated using the information on the urinary metabolites described above.

[0031] The information provision method and evaluation method using the provided information disclosed in this application achieve the following effects. (1) When urinary metabolites are repeatedly separated using a reversed-phase column with the same adamantyl group, the elution time variation of the same type of urinary metabolite is reduced when using an aqueous mobile phase compared to an ODS column. Therefore, assuming actual clinical tests in which the same column is repeatedly used, the variation in the provided information is reduced. Maintaining the initial performance of the column for a longer period is important for obtaining accurate analytical results and reducing costs. There are several analytical conditions for extending the life of a column, one of which is the mobile phase. In a mobile phase with a high water content, hydrophobic substances dissolved in the sample or mobile phase are retained and accumulated by the modification groups of the packing material. This results in column deterioration such as changes in retention time, abnormal peak leading, tailing, and peak splitting. Since this application separates urinary metabolites contained in hydrophilic urine, the mobile phase begins with an aqueous one, and therefore, particular attention must be paid to the column deterioration described above. As shown in the Examples and Comparative Examples described below, when an ODS column was used, the elution times of MTS and 3MT varied. On the other hand, when an ADME column, a reversed-phase column with an adamantyl group, was used, the elution times of MTS and 3MT were reduced. Therefore, from the perspective of separating urinary metabolites contained in urine samples, the use of a reversed-phase column with an adamantyl group is of significant significance. (2) When information on 3-methoxytyramine sulfate conjugates is provided by the information provision method, it is possible to evaluate whether the histological malignancy is high according to the International Neuroblastoma Pathological Classification. (3) When information on vanyl lactate is provided by the information provision method, it is possible to evaluate whether the patient is at high risk according to the International Neuroblastoma Risk Classification. (4) When a reversed-phase column with an adamantyl group is used, it is possible to separate and quantify the new tumor markers 3-methoxytyramine sulfate conjugates and vanyl lactate, as well as the conventional tumor markers vanillylmandelic acid, homovanillic acid, 3-methoxytyramine, and 3-methoxytyrosine, in a single separation step. Therefore, a variety of information regarding the pathological condition can be provided at once.(5) When ionized by the ionization method (electrospray method) used in LC / MS-MS, the 3-methoxytyramine sulfate conjugate may undergo deconjugation. That is, the 3-methoxytyramine sulfate conjugate undergoes structural transformation to 3-methoxytyramine (hereinafter, sometimes referred to as the "deconjugate (3-methoxytyramine)"). In this case, if the 3-methoxytyramine sulfate conjugate (in other words, the components detected as the "3-methoxytyramine sulfate conjugate" and the "deconjugate (3-methoxytyramine)" in LC / MS-MS) cannot be separated from the 3-methoxytyramine originally contained in the urine sample, the amount of 3-methoxytyramine originally contained in the urine sample may be overestimated. When a reversed-phase column having an adamantyl group is used, the 3-methoxytyramine sulfate conjugate and the 3-methoxytyramine originally contained in the urine sample are clearly separated, and this problem does not occur.

[0032] The following examples are provided to specifically explain the embodiments disclosed in the present application, but these examples are merely for the purpose of explaining the embodiments and are not intended to limit or restrict the scope of the invention disclosed in the present application.

[0033] [Sample Separation] <Example 1 and Comparative Example 1> (1) Sample: 3-Methoxytyramine sulfate conjugate (MTS: 3-Methoxy Tyramine Sulfate manufactured by TLC Pharmaceutical Standards). Concentration adjusted to 1 μg / mL. 3-Methoxytyramine (3MT: 3-Methoxytyramine hydrochloride 1.0 mg / mL in methanol (as free base) manufactured by Merck). Concentration adjusted to 1 μg / mL. Solvent: Water (to create an aqueous solvent simulating a urine sample). (2) Measurement Conditions: An ADME column was used in Example 1, and an ODS (C18) column was used in Comparative Example 1. The measurement conditions were as follows:

[0034] Instrument: Vanquish (Thermo) Analytical column: ADME column: CAPCELL PAK ADME HR (2.1 mm I.D. x 100 mm, particle size 2 μm), Osaka Soda C18 column: Myghtysil RP-18 GP (2.0 mm I.D. x 100 mm, particle size 3 μm), Kanto Chemical Mobile phase: A: water / acetic acid (1000:2, v / v) B: methanol Run time: 15 min Gradient: ・Column temperature: 50℃ ・Autosampler temperature: 5℃ ・Injection volume: 1μL ・Instrument: Orbitrap Q executive (thermo)・Mode: Full MS ・Polarity: positive ・Scan Range: m / z 100-800 ・Resolution: 70000

[0035] The chromatogram drawing conditions are as follows: Software: Xcalibur ver. 4.1 (Thermo) 3MT: 168.10191 (C ９ H １３ NO ２ ) ・MTS:248.05872(C ９ H １３ NO ５ S) ・Tolerance: 5ppm

[0036] Figure 1A shows a graph overlaid with MS chromatograms obtained when the same sample was injected three times using an ADME column. Figure 1B shows a graph overlaid with MS chromatograms obtained when the same sample was injected three times using an ODS column. As is clear from Figures 1A and 1B, when a sample was separated three times using the same ADME column, the variability in the elution times of MTS and 3MT was confirmed to be reduced compared to an ODS column. In particular, MTS is a novel tumor marker disclosed in this application. Therefore, assuming an actual clinical test in which the same column is used repeatedly, the ADME column reduces the variability in the information obtained even with repeated use. Furthermore, as shown in Table 1, an aqueous mobile phase was used up to 10.4 min. However, as shown in Figure 1, it was confirmed that the novel marker MTS and 3MT originally contained in the sample eluted by 4 min. In other words, it was confirmed that when separating only the novel marker MTS and 3MT originally contained in the sample, separation was possible using only an aqueous mobile phase.

[0037] Example 2 In addition to the samples used in Example 1, 3-methoxytyramine sulfate conjugate (MTS) and 3-methoxytyramine (3MT), the following samples were added and mixed: 3-methoxytyrosine (MTR: Toronto Research Chemicals), vanillylmandelic acid (VMA: Tokyo Chemical Industry), vanyl lactate (VLA: Toronto Research Chemicals), and homovanillic acid (HVA: Tokyo Chemical Industry). The resulting solution was further diluted with phosphate buffer to prepare a standard solution of the concentration to be used. An internal standard solution was also prepared in the same manner. Finally, the standard solution, internal standard solution, and water were mixed, and the supernatant after centrifugation was used as the mixed sample. Sample separation was then performed using the same procedure as in Example 1, except that the mixed sample was used.

[0038] Taking into account the urinary concentrations of the markers, the concentrations of the standard substances were set as follows: 3MT: 1000 ng / mL MTS, VLA: 5000 ng / mL MTR: 10000 ng / mL HVA: 50000 ng / mL VMA: 100000 ng / mL

[0039] The results are shown in Figure 2. As is clear from Figure 2, the use of an ADME column confirmed that the novel tumor markers 3-methoxytyramine sulfate conjugate (MTS) and vanyl lactate (VLA), as well as the conventional tumor markers vanillylmandelic acid (VMA), homovanillic acid (HVA), 3-methoxytyramine (3MT), and 3-methoxytyrosine (MTR), can be separated and quantified in a single separation step. Furthermore, as shown in Figure 2, two peaks were observed for 3MT, one at around 2.8 min (see the arrows in Figure 2; the lower peak of MTS) and the other at around 3.8 min. The peak at around 2.8 min represents the structural transformation of MTS into 3MT (elimination of the sulfate conjugate) upon introduction into the MS (when ionized by an ionization method called electrospray), and the other represents the peak of 3MT contained in the mixed sample.

[0040] The conditions for detecting the six compounds using the triple quadrupole mass spectrometer are shown in Table 2 below. In Table 2, IS stands for internal standard. As can be seen from Table 1, an internal standard substituted with deuterium is used, so the m / z detected for the product ion of the internal standard is larger than that of the standard. However, in the case of VMA and VMA-IS, the product ions have the exact same structure, with no deuterium moiety, and therefore have the same m / z.

[0041] The information provision method disclosed in the present application has been confirmed to clearly separate the deconjugate (3-methoxytyramine) resulting from structural transformation of 3-methoxytyramine sulfate conjugate (MTS) from 3-methoxytyramine (3MT). Therefore, the quantification of 3-methoxytyramine (3MT) originally contained in a sample is not affected by the deconjugate (3-methoxytyramine), thereby achieving accurate quantification of the 3-methoxytyramine (3MT) originally contained in the sample. Furthermore, the deconjugate (3-methoxytyramine) observed around 2.8 min in Figure 2 was structurally transformed when introduced into the MS (during ionization), and is a component originally contained in the sample as 3-methoxytyramine sulfate conjugate (MTS).

[0042] [On the Usefulness of Novel Tumor Markers, 3-Methoxytyramine Sulfate (MTS) and Vanillactate (VLA)] Example 3 In Example 3, a total of 295 participants participated, including 227 control subjects and 68 pediatric neuroblastoma patients. Comprehensive analyses were performed on 110 urine samples from 110 control participants and 18 pretreatment urine samples from 18 pediatric patients with newly diagnosed neuroblastoma. Approximately 1,500 metabolites were detected in the urine samples from control subjects and neuroblastoma patients. Based on random forest classification for neuroblastoma screening, the ranking of catecholamine metabolites according to their importance among all metabolites was as follows: HVA (1st), MTS (2nd), VMA (3rd), and VLA (4th). Therefore, VLA and MTS were selected as novel urinary markers for neuroblastoma.

[0043] Next, quantitative analysis was performed on 41 pretreatment urine samples from 41 pediatric patients with newly diagnosed neuroblastoma, and the results are shown in Figure 3. The quantitative analysis was performed using the same procedures as in Example 2, except for different samples. Of the 41 cases, the values ​​of the novel marker candidate MTS and the currently used clinical HVA+VMA were compared in 20 and 9 cases with high and low histological malignancy grades according to the International Neuroblastoma Pathological Classification (INPC). As shown in Figure 3A, ROC analysis revealed that MTS (AUC: 0.950) could more accurately identify cases with high histological malignancy grades and poor prognosis than the combination of HVA and VMA (AUC: 0.711).

[0044] Next, we compared the values ​​of the novel marker candidate VLA with the currently used clinical HVA+VMA in 22 high-risk and 10 low-risk cases according to the International Neuroblastoma Risk Classification (INRG pretreatment risk) of the 41 cases. As shown in Figure 3B, the ROC analysis revealed that VLA (AUC: 0.900) could more accurately identify high-risk cases with poor prognosis than the conventional combination of HVA and VMA (AUC: 0.645).

[0045] The ROC (Receiver Operating Characteristic) curve is a statistical method for analyzing the usefulness of a diagnostic method. It is a curve in which the sensitivity and specificity when a specific cutoff value is set are plotted on the vertical axis and the horizontal axis (1 - specificity), respectively, and connected by a broken line. It is quantified by the area under the curve (AUC). If the diagnostic method has high discriminatory ability, the AUC value will approach 1.

[0046] The information providing method disclosed in the present application enables evaluation of neuroblastoma. Therefore, the information providing method and neuroblastoma evaluation method disclosed in the present application are useful in the medical industry.

Claims

1. An information providing method for providing information regarding urinary metabolites contained in a urine sample, said information providing method comprising: a separation step of separating urinary metabolites contained in the urine sample using a reversed-phase column having an adamantyl group; and an information providing step of providing information regarding the types of urinary metabolites separated, wherein the types of urinary metabolites provided in the information providing step include 3-methoxytyramine sulfate conjugates and / or vanyl lactate, and the provided information is used in the evaluation of neuroblastoma.

2. The information provision method according to claim 1, wherein the types of urinary metabolites further include at least one selected from the group consisting of vanillylmandelic acid, homovanillic acid, 3-methoxytyramine, and 3-methoxytyrosine.

3. The information provision method according to claim 1, wherein the information on the 3-methoxytyramine sulfate conjugate provided in the information provision step includes quantitative information obtained using a triple quadrupole mass spectrometer, and when quantifying by the internal standard method using the triple quadrupole mass spectrometer, a measurement specimen and positive ion with m / z 248.1 and an internal standard and positive ion with m / z 252.1 are used as precursor ions, and a measurement specimen and positive ion with m / z 151.0 and an internal standard and positive ion with m / z 155.0 are used as product ions.

4. The information provision method according to claim 1, wherein the information on vanyl lactate provided in the information provision step includes quantitative information obtained using a triple quadrupole mass spectrometer, and when quantifying by the internal standard method using the triple quadrupole mass spectrometer, a measurement specimen and negative ion with m / z 211.1 and an internal standard and negative ion with m / z 214.1 are used as precursor ions, and a measurement specimen and negative ion with m / z 150.0 and an internal standard and negative ion with m / z 152.0 are used as product ions.

5. A method for evaluating neuroblastoma, comprising evaluating neuroblastoma in a subject using information provided by the information providing method according to any one of claims 1 to 4.

6. The evaluation method according to claim 5, wherein the types of urinary metabolites include 3-methoxytyramine sulfate conjugates, and when evaluating neuroblastoma, whether or not the histological malignancy is high according to the International Neuroblastoma Pathological Classification is evaluated.

7. The method of claim 5, wherein the types of urinary metabolites include vanyl lactate, and when evaluating neuroblastoma, whether or not the patient is at high risk according to the International Neuroblastoma Risk Classification System is evaluated.

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