Method of quantizing polyamines and collection kit

Abstract

To accurately quantize polyamines.SOLUTION: A method of quantizing polyamines includes: a measurement step of acquiring the measured quantity of polyamines and the measured quantity of an internal standard substance in a sample for measurement including a biological sample collected from a living body, a predetermined amount of internal standard substance, and a chelating agent; and a normalization step of correcting the measured quantity of polyamines with the measured quantity of the internal standard substance to calculate normalized concentration. The internal standard substance is 1,6-diaminohexane.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for quantifying polyamines and a collection kit thereof. [Background technology]

[0002] In recent years, methods have been developed to use substances contained in biological samples such as saliva, urine, or blood as markers to determine whether or not a person has a corresponding target disease based on the concentration of the marker in the biological sample. Such methods can easily determine the possibility of a disease using biological samples that can be easily collected, and the search for useful markers for each disease is ongoing.

[0003] Polyamines have been noted to be related to the concentration in a biological sample and cell proliferation, and are attracting attention as tumor markers. For example, Patent Document 1 proposes a method for testing breast cancer in which a saliva sample collected from a subject is analyzed to quantify polyamines and determine whether or not the subject has breast cancer.

[0004] Furthermore, Non-Patent Document 1 discloses a method for quantitatively determining the concentration of polyamines in a sample by mass spectrometry using 1,6-diaminohexane as an internal standard substance. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2017-20979 A [Non-patent literature]

[0006] [Non-Patent Document 1] Haruhito Tsutsui et. al., High-throughput LC-MS / MS based simultaneous determination of polyamines including N-acetylated forms in human saliva and the diagnostic approach to breast cancer patients, Analytical Chemistry, 85(24) p.11835-11842, 2013 Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have found that there is room for improvement in the quantitative accuracy of conventional methods for quantifying polyamines.

[0008] An object of one aspect of the present invention is to provide a method capable of quantifying polyamines with high accuracy. [Means for solving the problem]

[0009] In order to solve the above-mentioned problems, a method for quantifying polyamines according to one embodiment of the present invention includes a measurement step of obtaining measured amounts of polyamines and measured amounts of the internal standard in a measurement sample containing a biological sample collected from a living organism, a predetermined amount of an internal standard, and a chelating agent, and a standardization step of correcting the measured amounts of polyamines with the measured amount of the internal standard to calculate a standardized concentration, wherein the internal standard is 1,6-diaminohexane. Effect of the Invention

[0010] According to one aspect of the present invention, a method capable of quantifying polyamines with high accuracy can be realized. [Brief description of the drawings]

[0011] [Figure 1]FIG. 1 shows some of the results of a storage stability test on saliva samples to which EDTA-2Na was added in order to quantify polyamines. [Diagram 2] FIG. 1 shows some of the results of a storage stability test on saliva samples to which EDTA-2Na was added in order to quantify polyamines. [Diagram 3] FIG. 1 shows the results of plotting the measured amounts of spermine at each concentration before standardization with an internal standard against the concentration of EDTA-2Na. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] An embodiment of the present invention will be described below. In this specification, the symbol "to" means a range including both ends of the symbol.

[0013] A method for quantifying polyamines according to one embodiment of the present invention includes a measurement step and a standardization step, and may further include a calibration curve generation step.

[0014] [Measurement process] The measuring step is a step of obtaining the measured amounts of polyamines and the measured amount of the internal standard in a measurement sample containing a biological sample collected from a living body, a predetermined amount of the internal standard, and a chelating agent.

[0015] (Living organisms and biological samples) In one embodiment of the present invention, the measurement sample includes a biological sample taken from a living body. The living body is not particularly limited, and may be, for example, a human, a non-human mammal, or an animal other than a mammal. Non-human mammals include pet animals, livestock, and laboratory animals, and more specifically, dogs, cats, rabbits, monkeys, chimpanzees, horses, cows, sheep, goats, pigs, mice, and rats.

[0016] The biological sample collected from such a living body may be, for example, a liquid or solid biological sample. Examples of the liquid biological sample include saliva, urine, tears, blood, lymph, tissue fluid, ascites, and digestive tract secretions. Examples of the solid biological sample include oral tissue, digestive tract tissue, and diseased tissue. When using a solid biological sample, it may be dissolved in a suitable dissolving solution and included in the measurement sample. The biological sample is preferably a liquid biological sample from the viewpoint of ease of handling and quantitative accuracy, and is preferably saliva or urine from the viewpoint of non-invasiveness and easy collection.

[0017] (internal standard substance) In one embodiment of the present invention, the measurement sample contains 1,6-diaminohexane as an internal standard. When the amount of polyamines to be measured is obtained, for example, by mass spectrometry, it is required that the internal standard is not contained in the biological sample to be measured, is eluted at a position close to the polyamines to be measured, and is separable from the peaks of components other than polyamines. In addition, when the amount of polyamines to be measured is obtained, for example, by a method of derivatizing polyamines and performing mass spectrometry (Non-cited Document 1, etc.), it is required that the internal standard is also derivatized in the same manner as the polyamines. Since 1,6-diaminohexane satisfies these conditions, it can be suitably used as an internal standard.

[0018] The predetermined amount of the internal standard in the measurement sample is not particularly limited, but is preferably an amount that can be stably detected by mass spectrometry or the like. The predetermined amount of the internal standard is preferably the same concentration as the concentration of the internal standard in the calibration curve sample in the calibration curve generation step described below. Here, the same concentration does not need to be exactly the same concentration, but is a concept that allows for difficult-to-avoid errors due to handling errors, measurement errors, and the like.

[0019] The predetermined amount of the internal standard is, for example, 10 to 2000 μmol / m 3 may be 40 to 500 μmol / m 3is preferably 100 to 250 μmol / m 3 It is more preferable that:

[0020] (chelating agent) In one embodiment of the present invention, the measurement sample contains a chelating agent. The present inventors have found that a chelating agent improves the quantitative accuracy of polyamines, which is a new finding in the present invention. Specifically, the present inventors have found that when 1,6-diaminohexane is used as an internal standard, the measured amount of 1,6-diaminohexane is likely to vary (for example, the coefficient of variation is likely to be large). The present inventors have also found that when a sample containing 1,6-diaminohexane also contains a chelating agent, the variation in the measured amount of 1,6-diaminohexane can be reduced.

[0021] Variations in the measured amount of the internal standard can have a significant adverse effect on the quantitative accuracy of all polyamines. According to one embodiment of the present invention, the variability in the measured amount of the internal standard can be reduced, thereby improving the quantitative accuracy of polyamines.

[0022] As described above, one embodiment of the present invention has the advantageous effect that, by including a chelating agent in a measurement sample, the variability in the measured amount of an internal standard substance is reduced, thereby improving the quantitative accuracy of polyamines.

[0023] Improving the quantitative accuracy of polyamines is expected to reduce false negatives and false positives in disease testing, making it possible to accurately predict which patients need treatment. Such effects will also contribute to achieving Goal 3 of the United Nations' Sustainable Development Goals (SDGs), "Ensure healthy lives and promote well-being for all."

[0024] The chelating agent is not particularly limited as long as it is a substance that reacts with metal ions to form a complex. Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA), hydroxyethylethylenediaminetriacetic acid (HeEDTA), di(ortho-hydroxybenzyl)-ethylenediaminediacetic acid (HBED), diethylenetriaminepentaacetic acid (DTPA), porphyrin, citric acid, etidronic acid, ethylenediaminedisuccinic acid, edetic acid gluconic acid, glutamic acid diacetic acid, diethylenetriaminepentaacetic acid, hydroxyethanediphosphonic acid, hydroxyethanediphosphonic acid, phytic acid, pentetic acid, and metaphosphoric acid, and may be metal salts thereof. Examples of the metal salt include sodium salts and potassium salts. Among them, the chelating agent is preferably ethylenediaminetetraacetic acid or a metal salt thereof. The chelating agent may be one of these, or a combination of two or more of them.

[0025] The concentration of the chelating agent in the measurement sample is, for example, 0.1 to 500 mol / m 3 may be 0.2 to 250 mol / m 3 is preferably 0.5 to 160 mol / m 3 More preferably, the ratio is 1 to 120 mol / m 3 It is more preferable that:

[0026] The method for preparing the measurement sample is not particularly limited, but for example, an internal standard may be added to a mixture containing a biological sample and a chelating agent. Alternatively, a biological sample may be mixed with a mixture of the internal standard and the chelating agent, a mixture of the biological sample and the internal standard and the chelating agent may be mixed, or a biological sample may be mixed with the internal standard and the chelating agent.

[0027] The present inventors have found that the chelating agent does not impair the storage stability of polyamines in a biological sample. Therefore, the biological sample and the chelating agent may be mixed at any timing. For example, the chelating agent may be contained in a container for collecting the biological sample, the biological sample and the chelating agent may be mixed immediately after the biological sample is collected, or the biological sample and the chelating agent may be mixed after the biological sample is stored or transported and before the measurement of polyamines. When the biological sample is saliva or the like that may contain microorganisms, the chelating agent is expected to have an effect of reducing the activity of the microorganisms. Therefore, it is preferable that the biological sample and the chelating agent are mixed early, such as before the storage or transport of the biological sample.

[0028] (Polyamines) The polyamines are not particularly limited as long as they are aliphatic compounds containing multiple amino groups, and may be, for example, polyamines or polyamine precursors. The number of amino groups that the polyamines have may be two, three, four, or five or more. The number of hydrocarbons that the polyamines have may be 2 to 30, 2 to 25, 2 to 20, or 3 to 15. The polyamines may have a substituent other than the amino group and the hydrocarbon group, and such a substituent may be, for example, a substituent containing an oxygen atom such as an oxo group, a hydroxyl group, or a carboxyl group. The polyamines to be measured may be one type or two or more types.

[0029] Specifically, the polyamines may include at least one selected from the group consisting of spermine, N1-acetylspermine, spermidine, N1-acetylspermidine, N8-acetylspermidine, cadaverine, N1N12-diacetylspermine, N1N8-diacetylspermidine, putrescine, N1-acetylputrescine, and diaminopropane.

[0030] (Measurement quantity) The method for obtaining the measured amount of polyamines and the measured amount of internal standard is not particularly limited. The method is preferably mass spectrometry from the viewpoint of accuracy. Examples of mass spectrometry include ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS / MS), liquid chromatography tandem mass spectrometry (LC-MS / MS), liquid chromatography mass spectrometry (LC-MS), gas chromatography mass spectrometry (GC-MS), inductively coupled plasma mass spectrometry (ICP-MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS). The method for obtaining the measured amount of polyamines and the measured amount of internal standard may be a combination of these mass spectrometry methods, or may be a method other than mass spectrometry, such as X-ray spectrometry or emission spectrometry.

[0031] From the viewpoint of improving the measurement accuracy, polyamines and internal standard substances may be subjected to deproteinization treatment with an organic solvent or the like and derivatization with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) or the like as described in Non-Patent Document 1, before being subjected to mass spectrometry.

[0032] The measured amount of polyamines and the measured amount of the internal standard may be the peak area of ​​each peak representing the polyamines or the internal standard when the measured amount is obtained by mass spectrometry. The range of the peak area in the chromatogram may be set by the analysis software or manually.

[0033] [Standardization process] The standardization step is a step of calculating a standardized concentration by correcting the measured amount of polyamines with the measured amount of an internal standard. When the measured amount of polyamines is obtained by mass spectrometry, for example, it is obtained as a peak area area. Such measured amounts such as peak area areas are often not values ​​that directly indicate the concentration of polyamines in a measurement sample or a biological sample, and it is preferable to calculate the standardized concentration by correction. The standardized concentration can be used, for example, as an estimated value of the concentration of polyamines in a measurement sample or a biological sample.

[0034] The internal standard is contained in a predetermined amount in the measurement sample, and the concentration is known. By performing correction using the correlation between the measured amount of such an internal standard and the measured amount of polyamines, the measured amount of polyamines can be corrected to an estimated value of the concentration in the measurement sample. For example, the correction method may be a method of calculating the concentration y of polyamines in the measurement sample by a predetermined calculation formula y=f(x) using the ratio x of the measured amount of polyamines to the measured amount of the internal standard.

[0035] The predetermined calculation formula may be appropriately set, for example, from information showing the correspondence between the measured amount corresponding to each concentration of polyamines and the measured amount corresponding to a predetermined amount of internal standard. Such information may be, for example, a calibration curve, and in this case, the predetermined calculation formula may be y=ax+b (wherein a indicates the slope of the calibration curve and b indicates the y-intercept of the calibration curve). A method for generating the calibration curve will be described later in the section "Calibration curve generation process".

[0036] In the standardization step, the ratio of the measured amount of polyamines to the measured amount of the internal standard may simply be calculated as the standardized concentration. In this case, the standardized concentration may be used as it is as a preliminary value related to the concentration of polyamines in the measurement sample or biological sample. For example, when biological samples are collected from a large number of subjects to obtain the measured amounts of polyamines, such a value may be used for preliminary grouping of subjects.

[0037] [Calibration curve generation process] The calibration curve generating step is a step of generating a calibration curve showing the correspondence relationship between the measured amount of polyamines and the measured amount of the internal standard for each concentration of polyamines. The calibration curve generating step is performed using a calibration curve sample.

[0038] The calibration curve sample is used to obtain a calibration curve, and contains polyamines of known concentrations and a predetermined amount of an internal standard substance as well as a chelating agent. The polyamines contained in the calibration curve sample are the same type as the polyamines to be measured in the biological sample. The chelating agent contained in the calibration curve sample is also preferably the same type as the chelating agent contained in the measurement sample.

[0039] The calibration curve sample is preferably composed of a plurality of samples each containing at least two types of polyamines at different concentrations. The calibration curve sample may also have a sample containing polyamines at a concentration of 0 (zero). In other words, the calibration curve sample may be composed of a sample that does not substantially contain polyamines and contains a predetermined amount of an internal standard and a chelating agent.

[0040] As an example of a method for generating a calibration curve in the calibration curve generating step, the measured amount of polyamines in the calibration curve sample and the measured amount of the internal standard are obtained by the same method for obtaining the measured amount as in the measurement step, such as mass spectrometry. Then, the ratio of the measured amount of polyamines to the measured amount of the internal standard is calculated for each calibration curve sample. The ratio value is plotted on a graph with the y-axis and the concentration value of polyamines on the x-axis, and the regression line calculated from each plot may be used as the calibration curve. The regression line may be calculated using a regression model such as the least squares method, and the regression model may be appropriately weighted. The method for generating a calibration curve is not limited to the above-mentioned method, and may be generated by various other known methods.

[0041] [Collection kit] A collection kit according to one embodiment of the present invention is a biological sample collection kit for carrying out the above-mentioned method for quantifying polyamines. The collection kit includes a container capable of containing a biological sample and a chelating agent.

[0042] The container capable of accommodating the biological sample is not particularly limited, and is preferably a container made of a synthetic resin such as polyethylene, polypropylene, polystyrene, or polyethylene terephthalate, or may be a container made of a material other than synthetic resin such as glass. The type of container may be appropriately selected depending on the type of biological sample.

[0043] The chelating agent may be the same type of chelating agent as that used in the measurement step of the method for quantifying polyamines according to one embodiment of the present invention. The collection kit may include the chelating agent as a solution or as a powder. When the chelating agent is a powder, the collection kit may further include a solvent such as a buffer for dissolving the chelating agent.

[0044] The chelating agent may be contained in a container capable of containing the biological sample, or may be contained in a separate container. When the chelating agent is contained in a separate container, the collection kit may further include a pipette or the like for adding the chelating agent to the biological sample.

[0045] The collection kit may further include a biological sample collection tool such as a urine collection cup or an oral swab, an instruction manual describing a method for collecting the biological sample, a seal for sealing the container after collection of the biological sample, and a packaging material for the container after collection of the biological sample, etc. The collection kit may further include a method for obtaining the measured amount of polyamines and various reagents used in the calibration curve generation step, etc.

[0046] [Application example] It is known that the concentration of polyamines in a biological sample is useful as a disease marker for tumors and the like. According to the method for quantifying polyamines according to one embodiment of the present invention, the concentration of polyamines in a biological sample can be quantified more accurately than in the past. Therefore, the results obtained by the method for quantifying polyamines according to one embodiment of the present invention may be used, for example, to estimate whether or not a subject is suffering from a target disease.

[0047] The diseases that can be predicted using polyamines as markers include, for example, solid cancer and blood cancer.The solid cancer includes, for example, breast cancer, stomach cancer, colon cancer, colon cancer, small intestine cancer, bladder cancer, esophageal cancer, kidney cancer, liver cancer, prostate cancer, lung cancer, pancreatic cancer, ovarian cancer, head and neck cancer, brain tumor, cervical cancer, thyroid cancer, laryngeal cancer and skin cancer.The blood cancer includes, for example, leukemia, malignant lymphoma and myeloma.

[0048] 〔summary〕 A method for quantifying polyamines according to a first aspect of the present invention includes a measurement step of obtaining measured amounts of polyamines and measured amounts of the internal standard in a measurement sample containing a biological sample collected from a living body, a predetermined amount of an internal standard, and a chelating agent, and a standardization step of correcting the measured amounts of the polyamines with the measured amount of the internal standard to calculate a standardized concentration, wherein the internal standard is 1,6-diaminohexane.

[0049] The method for quantifying polyamines according to aspect 2 of the present invention, in accordance with aspect 1, further includes a calibration curve generating step of generating a calibration curve showing a correspondence relationship between the measured amount of the polyamines and the measured amount of the internal standard for each concentration of the polyamines, and in the calibration curve generating step, a calibration curve sample for obtaining the calibration curve may contain the chelating agent.

[0050] A method for quantifying polyamines according to aspect 3 of the present invention, in accordance with aspect 1 or 2, may be such that the polyamines include at least one selected from the group consisting of spermine, N1-acetylspermine, spermidine, N1-acetylspermidine, N8-acetylspermidine, cadaverine, N1N12-diacetylspermine, N1N8-diacetylspermidine, putrescine, N1-acetylputrescine, and diaminopropane.

[0051] A fourth aspect of the present invention relates to a method for quantifying polyamines in any one of the first to third aspects, in which the biological sample is saliva.

[0052] A fifth aspect of the present invention relates to a method for quantifying polyamines in any one of the first to fourth aspects, wherein the chelating agent is ethylenediaminetetraacetic acid or a metal salt thereof.

[0053] A biological sample collection kit according to aspect 6 of the present invention is a biological sample collection kit for carrying out the quantification method of any one of aspects 1 to 5, comprising a container capable of holding the biological sample and the chelating agent. EXAMPLES

[0054] An embodiment of the present invention will now be described.

[0055] Example 1. Storage stability of saliva samples containing chelating agents Saliva samples were used as biological samples, and ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) was added as a chelating agent at 80 mol / m 3 The storage stability of the stability samples was examined by adding the following: The storage temperatures were -30°C, 4°C, room temperature (approximately 23°C), 37°C, or 60°C. The storage periods were 1 day, 2 days, 3 days, 6 days, 8 days, and 10 days.

[0056] The polyamines measured were spermine (SPM), acetylspermine (Ac-SPM), spermidine (SPD), N1-acetylspermidine (N1-Ac-SPD), cadaverine (CAD), N1-N12-diacetylspermine (DAc-SPM), N1-N8-diacetylspermidine (DAc-SPD) and N1-acetylputrescine (Ac-PUT).

[0057] 1,6-Diaminohexane (DAH) was used as an internal standard.

[0058] After the storage period, the stability samples were each diluted with 100 μmol / m 3DAH was added. Deproteinization and derivatization with DBD-F were performed according to the method described in Non-Patent Document 1, and the amounts of polyamines and DAH were obtained as peak area areas by UPLC-ESI-MS / MS.

[0059] The ratio of the peak area of ​​polyamines to the peak area of ​​DAH (area rate, peak area of ​​polyamines / peak area of ​​DAH) was calculated. The obtained ratio was applied to the calibration curves previously prepared for each polyamine to obtain the estimated concentration of each polyamine in the stability sample (unit: μmol / m 3 The results are shown in Figures 1 and 2.

[0060] As shown in Figures 1 and 2, no decrease in polyamine concentration was observed in the stability samples stored at -30°C for any storage period from 1 to 10 days. Next, the concentrations of each polyamine under storage conditions of 4°C, room temperature, and 37°C were compared with those under storage conditions of -30°C. As a result, no clear decrease in polyamine concentration was observed with the passage of storage time.

[0061] Next, the stability of each polyamine when stored at 60° C. was compared with that when stored at -30° C. As a result, a decrease in concentration was observed in the stability samples for SPM, Ac-SPM, and SPD on the 8th and 10th days of storage, but no decrease in concentration was observed for the other polyamines.

[0062] From the above, it was shown that polyamines in saliva samples containing EDTA-2Na are stably maintained for at least 10 days not only at -30°C, but also at 4°C, room temperature, and 37°C. In addition, when saliva samples are transported to testing institutions, it is expected that they will become hot in mailboxes or transport vehicles in summer. Therefore, storage stability at 60°C was also examined, and it was shown that all polyamines were stably maintained for 6 days. This result shows a sufficient practical storage stability, considering the period usually required for transportation, and the period during which the temperature may be high, such as 60°C.

[0063] Example 2. Effect of chelating agent on calibration curve The effect of adding a chelating agent to the calibration curve samples on the calibration curve was examined.

[0064] EDTA-2Na was used as the chelating agent.

[0065] As polyamines, in addition to SPM, Ac-SPM, SPD, N1-Ac-SPD, CAD, DAc-SPM, DAc-SPD and PUT used in Example 1, N8-acetylspermidine (N8-Ac-SPD), N1-acetylputrescine (Ac-PUT) and diaminopropane (DAP) were used.

[0066] DAH was used as an internal standard substance.

[0067] For each polyamine, the calibration curve samples were prepared at the concentrations (unit: μmol / m 3 ) polyamines and 100 μmol / m 3 DAH and 0 mol / m 3 (not added), 1mol / m 3 , 10 mol / m 3 , 40mol / m 3 or 60 mol / m 3Each mixed sample was subjected to a deproteinization treatment and derivatization with DBD-F by the method described in Non-Patent Document 1. Then, the measured amounts of polyamines and DAH were obtained as peak area areas by UPLC-ESI-MS / MS.

[0068] [Table 1]

[0069] The ratio of the peak area of ​​polyamines to the peak area of ​​DAH (area rate, peak area of ​​polyamines / peak area of ​​DAH) was calculated. The x-axis represents the concentration of polyamines (unit: μmol / m 3 The ratios obtained for each polyamine were plotted on the y-axis with Area Rate as the axon, and a (1 / x)-weighted calibration curve was generated. The coefficient of determination (R 2 The coefficients of determination obtained for each polyamine are shown in Table 2 below.

[0070] [Table 2]

[0071] As shown in Table 2, when EDTA-2Na was not added, the coefficient of determination for eight types other than CAD, PUT, and Ac-PUT was low, significantly below 0.9. On the other hand, when EDTA-2Na was added, the coefficient of determination for all polyamines was high, at 0.95 or higher. The lower the coefficient of determination, the lower the reliability of the calibration curve.

[0072] These results show that the addition of EDTA-2Na to the standard curve samples significantly improved the coefficient of determination for polyamines, which had low coefficients of determination without the addition of EDTA-2Na. In other words, it was shown that the addition of EDTA-2Na to the samples can reduce the variability in the standardized concentrations of polyamines.

[0073] In this Example 2, the results obtained for SPM among the polyamines were used to further carry out the following study.

[0074] Example 3. Effect of chelating agents on the measured amounts of polyamines and internal standards In order to confirm the effect of EDTA-2Na, the peak area of ​​SPM before correction by the peak area of ​​DAH was extracted from the results of SPM in Example 2, and a calibration curve was generated again. Here, the x-axis represents the SPM concentration (unit: μmol / m 3 ) and the y-axis is the peak area. Figure 3 shows the generated calibration curves for each concentration of EDTA-2Na.

[0075] From the results in Figure 3, the calibration curves generated from the peak area of ​​SPM showed high coefficients of determination of 0.95 or more, regardless of whether EDTA-2Na was added or not. This suggests that EDTA-2Na may be affecting the variability in the measured amount of DAH, the internal standard, rather than polyamines such as SPM.

[0076] Therefore, only the peak area of ​​DAH was extracted from the results of SPM in Example 2, and the average value and the coefficient of variation of the peak area of ​​DAH at each concentration of polyamines were calculated for each concentration of EDTA-2Na. The results are shown in Table 3 below.

[0077] [Table 3]

[0078] As shown in Table 3, without the addition of EDTA-2Na, the coefficient of variation in the DAH peak area was as large as 63%, whereas the addition of EDTA-2Na resulted in a relatively small coefficient of variation in the DAH peak area within the range of 4.2% to 16.4%.

[0079] The above results indicate that the chelating agent acts to reduce the variability in the measured amount of DAH, the internal standard, and stabilize the measured amount.

[0080] [Additional Notes] The present invention is not limited to the above-described embodiments or examples, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in the different embodiments and examples are also included in the technical scope of the present invention. [Industrial Applicability]

[0081] An embodiment of the present invention can be used, for example, in disease testing.

Claims

1. a measuring step of acquiring the measured amount of polyamines and the measured amount of the internal standard in a measurement sample containing a biological sample collected from a living body, a predetermined amount of an internal standard, and a chelating agent; A standardization step of correcting the measured amount of the polyamines with the measured amount of the internal standard to calculate a standardized concentration, The method for quantifying polyamines, wherein the internal standard is 1,6-diaminohexane.

2. The method further includes a calibration curve generating step of generating a calibration curve showing a correspondence relationship between the measured amount of the polyamines and the measured amount of the internal standard for each concentration of the polyamines; The quantitative method according to claim 1 , wherein in the calibration curve generating step, the chelating agent is contained in a calibration curve sample for obtaining the calibration curve.

3. The method according to claim 1, wherein the polyamines include at least one selected from the group consisting of spermine, N1-acetylspermine, spermidine, N1-acetylspermidine, N8-acetylspermidine, cadaverine, N1N12-diacetylspermine, N1N8-diacetylspermidine, putrescine, N1-acetylputrescine, and diaminopropane.

4. The quantitative method according to claim 1 , wherein the biological sample is saliva.

5. The method according to claim 1 , wherein the chelating agent is ethylenediaminetetraacetic acid or a metal salt thereof.

6. A biological sample collection kit for carrying out the quantitative determination method according to any one of claims 1 to 5, comprising: A container capable of containing the biological sample; A collection kit comprising the chelating agent.

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