Modified nucleoside analysis method

Abstract

A method for analyzing a modified nucleoside according to the present invention comprises: an elution step for introducing a sample containing a target component that is a modified nucleoside of which the hydrophobicity is increased by modification and a reference component that is a different component from the target component into a liquid chromatography column, and then separating the target component and the reference component from each other and eluting the target component and the reference component from the column by gradient elution such that the mixing ratio of a plurality of solvents constituting a mobile phase is changed over time; a step for detecting the target component and the reference component separately by mass spectrometry; and a step for calculating the ratio of a detected value for the target component to a detected value for the reference component. In the elution step, the mixing ratio of the solvents constituting the mobile phase to be introduced into the column is changed in such a manner that a third period in which the change ratio of the mixing ratio of the solvents constituting the mobile phase as measured at an outlet port of the column is larger than that in a first period in which the target component is eluted from the column and that in a second period in which the reference component is eluted from the column is provided between the first period and the second period.

Description

[Technical Field]

[0001] The present invention relates to a method for analyzing modified nucleosides contained in biological samples such as serum and urine. [Background technology]

[0002] Human mitochondria contain 22 types of transfer RNA (mt-tRNA), and these mt-tRNAs are known to contain many chemical modifications. In recent years, the relationship between chemical modifications in human mt-tRNA and disease has been pointed out. For example, it has been reported that the taurine modification introduced at the 34th uridine of human mt-tRNA is deficient in mt-tRNA derived from tissues collected from patients with mitochondrial disease.

[0003] Therefore, it has been considered to analyze the nucleosides of mt-tRNA, and use the results of analyzing the presence or absence of chemical modifications such as taurine modification and the amount of modified nucleosides in the diagnosis of mitochondrial disease. For example, Patent Document 1 describes a method for diagnosing mitochondrial disease in which biological samples such as urine and serum collected from a subject are analyzed using liquid chromatography (LC) or liquid chromatography mass spectrometry (LC / MS) to measure the amount of modified nucleosides contained in the sample. The method described in Patent Document 1 was devised based on the finding that the amount of modified nucleosides derived from mt-tRNA contained in urine, serum, etc. is significantly higher in patients with mitochondrial disease than in healthy individuals. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. WO2018 / 124235 [Non-patent literature]

[0005] [Non-Patent Document 1] Wei et al., J Clin Invest. 2011; 121(9):3598-3608 Summary of the Invention [Problem to be solved by the invention]

[0006] When measuring the amount of a target component in a biological sample using LC or LC / MS, it is common to detect an internal standard component (hereinafter referred to as a reference component) in the biological sample along with the target component, and express the amount of the target component as the ratio of the detected value of the reference component to the detected value of the target component. This allows the amount (concentration) of the target component in the sample to be accurately measured while minimizing the effects of changes in the concentration of the biological sample itself, deterioration of the biological sample, pretreatment, etc. on the analytical results of the target component. For example, if the biological sample is urine, creatinine is used as the reference component to minimize the effects of urine concentration, and if the biological sample is serum, a metabolic precursor of the target component is used as the reference component.

[0007] When analyzing multiple components contained in a biological sample using LC / MS, a commonly used method is to use a mixture of multiple solvents with different elution strengths as the mobile phase, and to separate and elute the multiple components from the column using gradient elution, in which the mixing ratio of the solvents is continuously changed over time.

[0008] The inventors have discovered that a modified nucleoside (6-threonylcarbamoyl adenosine (t)) having a specific chemical structure contained in urine or serum samples collected from subjects is 6 A), 2-thiomethyl, 6-threonylcarbamoyl adenosine (ms 2 t 6 The present inventors have found that the amount of modified nucleosides (t) having the specific chemical structure described above can be used to predict whether or not the subject is suffering from an infectious disease called COVID-19 and is likely to become severe, based on the results of analyzing the amount of modified nucleosides (t) by LC / MS. 6 A,ms 2 t 6A) is a modified nucleoside that was discovered by the inventors of the present invention, Tomizawa and Nagayoshi, et al., to be abundant in urine and serum collected from COVID-19 patients.

[0009] However, it was discovered that the modified nucleosides described above, due to their specific chemical structure (i.e., chemical modification), are more hydrophobic than nucleosides that do not have such a chemical structure (i.e., non-chemically modified nucleosides). Because the reference components typically used in LC / MS analysis of components contained in urine and serum are highly hydrophilic, separating the modified nucleosides and the reference components by LC takes time. As LC separation takes time, it also takes longer to determine the amounts of modified nucleosides and reference components, resulting in the problem of longer times required to diagnose COVID-19 or predict the severity of the disease.

[0010] Here, we have used the example of analyzing modified nucleosides, which are useful for diagnosing COVID-19 or predicting the severity of the disease, using LC / MS. However, similar problems can arise when measuring modified nucleosides, which become more hydrophobic due to the modification, together with reference components using LC / MS.

[0011] The problem to be solved by the present invention is to shorten the time required to correctly measure the amount of a specific modified nucleoside contained in a sample by LC / MS. [Means for solving the problem]

[0012] The method for analyzing modified nucleosides according to the present invention, which has been made to solve the above problems, comprises: an elution step of introducing a sample containing a target component, which is a modified nucleoside whose hydrophobicity is increased by modification, and a reference component, which is a component different from the target component, into a liquid chromatography column, and separating and eluting the target component and the reference component from each other by gradient elution in which the mixing ratio of multiple solvents constituting the mobile phase is changed over time; detecting the target component and the reference component by mass spectrometry; calculating a ratio between the detection value of the target component and the detection value of the reference component; The elution step The mixing ratio of the solvents constituting the mobile phase introduced into the column is changed so that there is a third period between a first period in which the target component is eluted from the column and a second period in which the reference component is eluted from the column, in which the rate of change of the mixing ratio of the solvents constituting the mobile phase at the outlet of the column is greater than the rate of change of the mixing ratio of the solvents constituting the mobile phase in each of the first period and the second period. [Effects of the Invention]

[0013] In the modified nucleoside analysis method of the present invention, in the step of separating and eluting a target component and a reference component from an LC column, a third period is provided between the first and second periods, in which the rate of change in the mixture ratio of the solvents constituting the mobile phase at the column outlet is greater than the rate of change in the first and second periods. This shortens the time required to separate and elute the target component and reference component from the column in the elution step, thereby shortening the time required to accurately measure the amount of the target component contained in a sample using LC / MS. [Brief explanation of the drawings]

[0014] [Figure 1] A diagram showing the structure of t6A and ms2t6A. [Figure 2] 1 shows the gradient schedule for the LC / MS / MS analysis in Example 1. [Figure 3] MRM chromatogram of the sample. [Figure 4] Gradient schedule for LC / MS / MS analysis in Example 2. [Figure 5] Of the MRM chromatograms of the sample, (a) shows the vicinity of the acp3U peak, (b) shows the vicinity of the t6A peak, and (c) shows the vicinity of the ms2t6A peak. [Figure 6]This graph shows the analysis results of Example 3, and shows the amount of t6A, the target component, contained in the serum of COVID-19 patients and healthy individuals, expressed as a ratio to the measured value of adenosine, the reference component. [Figure 7] This graph shows the analytical results of Example 3, and represents the amount of t6A, the target component, contained in the serum of COVID-19 patients and healthy individuals, as measured values ​​of t6A. [Figure 8] This graph shows the analysis results of Example 4, and shows the amount of ms2t6A, the target component, contained in the serum of COVID-19 patients and healthy individuals, expressed as a ratio to the measured value of adenosine, the reference component. [Figure 9] This graph shows the analysis results of Example 4, and represents the amount of ms2t6A, the target component, contained in the serum of COVID-19 patients and healthy individuals, as measured values ​​of ms2t6A. DETAILED DESCRIPTION OF THE INVENTION

[0015] The method for analyzing modified nucleosides of the present invention is a method for analyzing the amount of a target component in a sample by LC / MS, using a modified nucleoside, particularly one whose hydrophobicity is increased by modification, as a target component and a component different from the target component as a reference component. In the present invention, the amount of the target component can be accurately determined by calculating the ratio of the detected value of the target component to the detected value of the reference component.

[0016] In the present invention, it is preferable that a modified nucleoside having a predetermined chemical structure among modified nucleosides contained in the sample is used as the target component, and a component contained in the sample that does not have the predetermined chemical structure is used as the reference component. Modified nucleosides having a predetermined chemical structure have increased hydrophobicity compared to nucleosides that do not have the predetermined chemical structure (unmodified nucleosides). Therefore, the predetermined chemical structure is a structure that increases the hydrophobicity of the modified nucleoside. The reference component may be a modified nucleoside or an unmodified nucleoside, as long as it does not have the predetermined chemical structure. Alternatively, it may be another component.

[0017] The sample used in the modified nucleoside analysis method of the present invention is typically a biological sample such as serum, plasma, or urine collected from a mammal (especially a human), and may be one that has undergone a predetermined pretreatment. When the biological sample that may contain a modified nucleoside is urine, creatinine is generally used as the reference component, and when it is blood (whole blood, plasma, or serum), a metabolic precursor of the target component is generally used as the reference component. All of these reference components are known to be highly hydrophilic (low hydrophobic).

[0018] The predetermined chemical structure is preferably a structure that not only affects the hydrophobicity of the modified nucleoside but also affects the retention strength of the target component and reference component on an LC column. In this case, a target component having such a chemical structure has the property of being more easily retained (stronger retention) or less easily retained (weaker retention) on a column than a reference component that does not have the chemical structure.

[0019] In the modified nucleoside analysis method of the present invention, the target component and the reference component are separated from each other and eluted from the column by gradient elution. If the target component has a stronger retention on the column than the reference component, the first period in which the target component elutes from the column occurs after the second period in which the reference component elutes, and if the target component has a weaker retention than the reference component, the first period occurs before the second period.

[0020] During the period between the first and second periods, components that are neither the target component nor the reference component, i.e., components that are not necessary for measuring the amount of the target component (unnecessary components, impurity components), are eluted from the column. In the present invention, a third period is provided between the first and second periods, during which the rate of change in the mixture ratio of the solvent in the mobile phase at the column outlet is greater than the rate of change in the mixture ratio of the solvent in the mobile phase at the column outlet during both the first and second periods. This shortens the time it takes for the unnecessary components or impurity components to elute from the column. As a result, the overall time required to accurately measure the amount of the target component using LC / MS can be shortened.

[0021] The rate of change in the mixing ratio of the solvent in the mobile phase during each of the first to third periods may or may not be constant. If the rate of change in the mixing ratio of the solvent in the mobile phase during each period is not constant, the average rate of change (average value of the rate of change) can be defined as the rate of change during that period. Furthermore, the third period does not need to be strictly separated from the first and second periods, and part or all of the third period may overlap with the first and / or second periods.

[0022] In the method for analyzing modified nucleosides according to the present invention, the elution step is performed according to a gradient program in which the rate of change in the mobile phase solvent ratio at the column outlet during the first to third periods satisfies the above-described relationship. Such a gradient program can be determined by calculating the time required for the mobile phase to migrate from the mobile phase solvent mixing section to the column outlet based on, for example, the length of the flow path from the mobile phase solvent mixing section to the column, the length, type, and characteristics of the column, and other factors that affect the mobile phase flow rate, and adjusting the timing for changing the mobile phase solvent mixing section so that the target component and reference component are eluted from the column outlet at the desired timing. In this case, multiple rates of change in the mobile phase solvent ratio may be prepared, and the rates of change in the mobile phase solvent ratio at the column outlet when the mobile phase is introduced into the column at each of these rates may be determined, and the gradient program may be determined based on the relationship between the rate of change in the mobile phase solvent ratio at the column inlet and the rate of change in the mobile phase solvent ratio at the column outlet.

[0023] When the LC column is a column for reversed-phase chromatography, examples of chemical structures that can affect the retention on the column include adenosine derivatives in which threonine is bound to the 6-position of the adenosine via a carbonyl group. Modified nucleosides having such a chemical structure include 6-threonylcarbamoyl adenosine (tRNA), which is a modified adenosine derived from mitochondrial tRNA (mt-tRNA). 6 A), 2-thiomethyl, 6-threonylcarbamoyl adenosine (ms 2 t 6 A) is shown in Figure 1. 6 A and ms 2 t 6 The structure of A is shown.

[0024] t 6A is a modified base present at position 37 of tRNA, which decodes the ANN codon. It is a modified nucleoside conserved in almost all organisms and essential for the growth of many organisms. t6A is known to play important roles in various steps of protein synthesis, including tRNA aminoacylation, translocation reactions, accurate codon recognition, and maintenance of the reading frame.

[0025] ms 2 t 6 A is t 6 The chemical structure of A is that the 2-position of the adenine is thiomethylated. 2 t 6 A is a modified base at position 37 of tRNA whose anticodon is UUU, and is transduced by the methylthiotransferase Cdkal1. 6 The present inventors have reported that it is biosynthesized from A (Non-Patent Document 1).

[0026] Furthermore, by using the method of the present invention, it is possible to detect t 6 A and ms 2 t 6 Therefore, the amount of t A in serum and urine can be measured accurately and in a short time. 6 A and ms 2 t 6 By comparing the amount of A with an index value representing the possibility that the subject has COVID-19 and / or an index value representing the possibility that the subject will have COVID-19 and become severe, it is possible to diagnose whether the subject has COVID-19 and, if so, to predict the severity of the disease early and with high reliability. Therefore, the modified nucleoside analysis method of the present invention can be said to be a useful method for early diagnosis and prediction of the severity of COVID-19.

[0027] The present invention will be described in detail below based on examples, but these examples do not limit the present invention in any way.

[0028] [Example 1] First, in order to examine whether it is possible to measure a modified nucleoside having a specific chemical structure contained in a biological sample by MRM (multiple reaction monitoring) measurement using a liquid chromatography mass spectrometer (LC / MS / MS), a sample was prepared by dissolving standards of the target component and the reference component in water, and MRM measurement was performed on this sample. The standards used for the measurement are as follows. <Target component> · 6-Threonylcarbamoyladenosine (t 6 A) (manufactured by Cosmo Bio Co., Ltd.) · 2-Thiomethyl, 6-threonylcarbamoyladenosine (ms 2 t 6 A) (manufactured by Santa Cruz Biotechnology) <Reference component> · 3-Amino 3-carboxypropyluridine (acp 3 U) (manufactured by Carbosynth) · Adenosine (manufactured by Sigma-Aldrich Japan LLC)

[0029] acp 3 U is one of the modified nucleosides. Also, adenosine is one of the nucleosides.

[0030] Also, the names of the apparatuses and the analysis conditions used for the measurement are as follows. In this specification, "%" represents volume % (%(v / v)). <Apparatus> Liquid chromatograph: Ultra-high speed liquid chromatograph Nexera X3 (manufactured by Shimadzu Corporation) Mass spectrometer: Ultra-high speed triple quadrupole mass spectrometer LCMS-8060 (manufactured by Shimadzu Corporation)

[0031] <LC analysis conditions>[[ID=4,1]] Column: Mastro2 C18 (inner diameter 2.1 mm × length 150 mm, particle size 3 μm, manufactured by Shimadzu GL Sciences Inc.) Mobile phase A: 0.1% formic acid - water Mobile phase B: 0.1% formic acid - acetonitrile Gradient: Mobile phase B concentration 10% (0 - 1.0 min) → 20% (1.2 min) → 35% (3.8 min) → 90% (4.0 - 4.5 min) → 10% (4.7 - 5.7 min) Flow rate: 0.3 mL / min Column temperature: 40 °C Injection volume: 2 μL

[0032] Ionization mode: ESI Nebulizer gas flow rate: 3. L / min Drying gas flow rate: 10 L / min Heating gas flow rate: 10 L / min Interface temperature: 300 °C DL temperature: 250 °C Heat block temperature: [400 °C

[0033] Figure 2 is a graph showing the time change of the mixing ratio of the solvents constituting the mobile phase in gradient elution (hereinafter also referred to as "the mixing ratio of the mobile phase"). Table 1 shows the retention times of the target component and the reference component under the above LC analysis conditions, and the MRM transitions of the quantitative ions and the confirmation ions. In Table 1, "polarity" represents the polarity of the measured ion.

Table 1

[0034] Figure 2 shows the time change of the mixing ratio of the mobile phase when the mobile phase is introduced into the column. As shown in Figure 2, in this example, the components retained in the column by the mobile phase introduced into the column during the period from time 0 min to 3.80 min are eluted, and the column is washed by the mobile phase introduced into the column during the period from 3.80 min to 4.7 min.

[0035] Note: There seems to be a formatting issue in the original text where the "400 °C" in line ID=24 is split. It should be a single continuous value in the translation as well, but the format of the original text makes it a bit unclear. I've translated it as best as possible while maintaining the integrity of the information.When a mobile phase is introduced into a column, components are eluted from the column as the mobile phase moves from the inlet to the outlet, and the column is washed. Therefore, the process of eluting components from the column and the process of washing the column occur later in time than the timing at which the mobile phase is introduced into the column. However, for convenience in the following explanation, the processes in which the mobile phase involved in the elution process and the washing process are introduced into the column are referred to as the elution process and the washing process, respectively.

[0036] In this example, the mixing ratio of the mobile phase changed suddenly during the period from 1.0 min to 1.2 min of the elution step (the period marked with reference numeral 100 in FIG. 2), and the reference component (acp 3 During the second period, the target component (t 6 A, and ms 2 t 6 A) is eluted from the column. That is, period 100 corresponds to a third period in which the rate of change of the mobile phase mixing ratio is greater than the rate of change of the mobile phase mixing ratio in each of the first and second periods. Furthermore, the mobile phase mixing ratio changes rapidly in the early and late periods of the cleaning step (period 101 from 3.8 min to 4.0 min, and period 102 from 4.5 min to 4.7 min), and the rate of change of the mobile phase mixing ratio is greater than the rate of change of the mobile phase mixing ratio in each of the first and second periods. Therefore, these periods 101 and 102 correspond to a fourth period of the present invention.

[0037] Figure 3 shows the acp 3 U, adenosine, t 6 A, and ms 2 t 6 3 shows the MRM chromatogram obtained for a sample containing A. As shown in FIG. 3, as a result of analysis under the analytical conditions of this example, it was found that the two reference components and the two target components contained in the sample could be separated and eluted from the column, and each component could be analyzed individually without interfering with each other.

[0038] As can be seen from FIG. 2, in this embodiment, the time taken for the target component and the reference component contained in the sample to elute from the column was 4.7 minutes in total for the elution step and the washing step. Also, as can be seen from FIG. 3, the reference component eluted during the period from 1.25 min to 1.55 min, and the target component eluted during the period from 3.20 min to 3.75 min. Actually, the time taken for all of the reference component and the target component to elute from the column was 2.50 minutes. Compared with the fact that the conventional method took 30 minutes or more in total for the elution step and the washing step, it was found that the method of this embodiment with the third period and the fourth period provided can significantly shorten the measurement time of the target component.

[0039] [Example 2] Under the same conditions as in Example 1 except that the conditions for gradient elution in LC analysis were changed, MRM (multiple reaction monitoring) measurement using a liquid chromatograph mass spectrometer (LC / MS / MS) was performed on a biological sample collected from a subject. In this embodiment, t 6 A and ms 2 t 6 A was used as the target component, and acp 3 U was used as the reference component.

[0040] The biological sample used for the analysis was serum collected from a patient diagnosed with COVID-19.

[0041] The name of the apparatus used for the analysis and the analysis conditions are as follows. <Apparatus> Liquid chromatograph: Ultra-high speed liquid chromatograph Nexera X2 (manufactured by Shimadzu Corporation) Mass spectrometer: Ultra-high speed triple quadrupole mass spectrometer LCMS-8045 (manufactured by Shimadzu Corporation)

[0042] <LC analysis conditions> Column: Mastro2 C18 (inner diameter 2.1 mm × length 150 mm, particle size 3 μm, manufactured by Shimadzu GL Sciences Inc.) Mobile phase A: 0.1% formic acid - water Mobile Phase B: 0.1% formic acid - acetonitrile Gradient: Concentration of Mobile Phase B 5% (0 - 2.0 min) → 25% (2.01 min) → 30% (4.3 - 4.5 min) → 90% (4.5 - 5.0 min) → 5% (5.01 - 6.0 min) Flow rate: 0.3 mL / min Column temperature: 40 °C Injection volume: 2 μL

[0043] Ionization mode: ESI Nebulizer gas flow rate: 3.0 L / min Drying gas flow rate: 10 L / min Heating gas flow rate: 10 L / min Interface temperature: 300 °C DL temperature: 250 °C Heat block temperature: 400 °C

[0044] Figure 4 is a graph showing the time change of the mixing ratio of the mobile phase in gradient elution. Also, Table 2 shows the retention times of the target component and the reference component, and the MRM transitions of the quantitative ions and confirmation ions under the above LC analysis conditions. In Table 2, "polarity" represents the polarity of the measured ions.

Table 2

[0045] Similar to Figure 2, Figure 4 shows the time change of the mixing ratio of the mobile phase when the mobile phase is introduced into the column. As shown in the graph of Figure 4, in this example, the period from time 0 min to 4.30 min corresponds to the elution process, and the period from time 4.30 min to 5.00 min corresponds to the cleaning process. During the period from time 2.0 min to 2.01 min during the elution process (the period marked 200 in Figure 4), the mixing ratio of the mobile phase changes rapidly, and in the periods before and after this period 200, the reference component (acp 3 U) elutes from the column in the second period, and the target component (t6 A, and ms 2 t 6 A) is eluted from the column during the second period 200. This period 200 corresponds to the third period of the present invention. Furthermore, during the washing process, the mixing ratio of the mobile phase changes suddenly during period 201 from 4.30 minutes to 4.50 minutes and during period 202 from 5.0 minutes to 5.01 minutes, and these periods 201 and 202 correspond to the fourth period of the present invention.

[0046] Figure 5 shows the acp 3 U, t 6 A, and ms 2 t 6 5 shows the MRM chromatogram obtained for A. As can be seen from Figure 5, the analysis results using the analytical conditions of this example showed that one reference component and two target components contained in the sample could be separated and eluted from the column, and each component could be analyzed individually without interfering with each other. Furthermore, in this example, the time required to elute one reference component and two target components contained in the sample from the column, including the elution and washing steps, was 5.0 minutes, of which the time required for all three components to actually elute from the column was approximately 2.6 minutes (1.60 min - 4.20 min). Compared to the conventional method, which required more than 30 minutes for the elution and washing steps, the method of this example, which includes the third and fourth periods, was found to significantly shorten the measurement time for the target components.

[0047] [Example 3] Urine samples prepared from patients diagnosed with COVID-19 and healthy individuals were subjected to MRM (multiple reaction monitoring) measurement using a liquid chromatograph mass spectrometer (LC / MS / MS) under the same conditions as in Example 2. 6 A and ms 2 t 6 The ratio of the measured value of A to the measured value of adenosine is calculated, and this value is used as the t 6 A and ms 2 t 6 The amount was A.

[0048] Figures 6 and 8 show the 6 A and ms 2 t 6 The t value was calculated from the ratio of the measured value of A to the measured value of adenosine. 6 Amount of A and ms 2 t 6 On the other hand, Fig. 7 and Fig. 9 show the amount of t 6 A and ms 2 t 6 t calculated from the A measurement (i.e., without using adenosine measurement) 6 Amount of A and ms 2 t 6 It shows the amount of A.

[0049] From Figures 6 to 9, t 6 A and ms 2 t 6 For each of the target components in A, when the measured values ​​were expressed as a ratio to the measured values ​​of the reference component adenosine, a significant difference was observed in the amount of the target component contained in the serum between COVID-19 patients and healthy subjects. On the other hand, when the measured values ​​of the reference component were not used, no significant difference was observed between COVID-19 patients and healthy subjects. Furthermore, when the ratio of the measured values ​​of the target component to the measured values ​​of the reference component was used, the amount of the target component contained in the urine of healthy individuals was almost zero, and the variability was smaller than when the measured values ​​of the reference component were not used. The above results suggest that the amount of a target component contained in a sample can be correctly evaluated by expressing the measured value of the target component as a ratio to the measured value of a reference component, and therefore the modified nucleoside analysis method of the present invention is useful for determining the possibility of infection with COVID-19 and / or predicting the severity of COVID-19 patients.

[0050] [Various aspects] It will be apparent to those skilled in the art that the above-described exemplary embodiments are examples of the following aspects.

[0051] (Item 1) The method for analyzing modified nucleosides of the present invention comprises: an elution step of introducing a sample containing a target component, which is a modified nucleoside whose hydrophobicity is increased by modification, and a reference component, which is a component different from the target component, into a liquid chromatography column, and separating and eluting the target component and the reference component from each other by gradient elution in which the mixing ratio of multiple solvents constituting the mobile phase is changed over time; detecting the target component and the reference component by mass spectrometry; calculating a ratio of the detected value of the target component to the detected value of the reference component; The elution step The mixing ratio of the solvents constituting the mobile phase introduced into the column is changed so that there is a third period between a first period in which the target component is eluted from the column and a second period in which the reference component is eluted from the column, in which the rate of change of the mixing ratio of the solvents constituting the mobile phase at the outlet of the column is greater than the rate of change of the mixing ratio of the solvents in the mobile phase in each of the first period and the second period.

[0052] According to the modified nucleoside analysis method of paragraph 1, in the step of separating and eluting a target component and a reference component from an LC column, the mixing ratio of the solvents constituting the mobile phase introduced into the column is changed so that there is a third period between the first and second periods in which the rate of change in the mixing ratio of the solvents constituting the mobile phase at the outlet of the column is greater than the rate of change in the first and second periods. This shortens the time required to separate and elute the target component and reference component from the column in the elution step. Therefore, the time required to accurately measure the amount of the target component contained in a sample using the detected values ​​of the target component and the reference component obtained using LC / MS can be shortened. Although components in the sample are also eluted from the column during the third period, these components are not necessary for measuring the amount of the target component contained in the sample. Therefore, increasing the rate of change in the mixing ratio of the solvents in the mobile phase during the third period does not adversely affect the accurate measurement of the amount of the target component in the sample.

[0053] (Item 2) In the modified nucleoside analysis method of item 1, the target component may be a modified nucleoside that is a derivative of adenosine and has a chemical structure in which threonine is bound to the adenosine via a carbonyl group, and the reference component may be a component that does not have the chemical structure.

[0054] According to the method for analyzing modified nucleosides of item 2, the time interval between the elution time of the target component and the elution time of the reference component can be adjusted relatively easily by utilizing the properties of the chemical structure.

[0055] (Item 3) In the method for analyzing modified nucleosides according to item 1, the target component is 6-threonylcarbamoyladenosine and / or 2-thiomethyl,6-threonylcarbamoyladenosine, The method may further include a step of comparing the determined amount of the target component with at least one of an index value representing the possibility that the subject from whom the sample was collected has COVID-19 and an index value representing the possibility that the subject will contract COVID-19 and become severely ill.

[0056] According to the modified nucleoside analysis method of paragraph 3, based on the comparison results between the amount of the target component and the index value, it is possible to quickly determine whether the subject from whom the sample was collected is infected with COVID-19, or whether a subject infected with COVID-19 is likely to become seriously ill.

[0057] (Item 4) In the method for analyzing a modified nucleoside according to any one of Items 1 to 3, the sample is urine, The reference component may be at least one selected from the group consisting of creatinine, urea nitrogen, uric acid, adenosine, and 3-amino-3-carboxypropyluridine.

[0058] According to the modified nucleoside analysis method of item 4, the influence of urine concentration on the analysis results of the target component can be reduced.

[0059] (Item 5) In the method for analyzing a modified nucleoside according to any one of Items 1 to 3, the sample is plasma or serum, The reference moiety may be at least one of adenosine and 3-amino-3-carboxypropyluridine.

[0060] According to the modified nucleoside analysis method of paragraph 5, the influence of the time taken for the plasma or serum collected from the subject to be analyzed, or the pretreatment performed on the plasma or serum, on the analysis results of the target component can be reduced.

[0061] (Item 6) In any one of the modified nucleoside analysis methods 1 to 5, The mobile phase may be a mixture of formic acid, acetonitrile, and water.

[0062] (Item 7) In any one of the modified nucleoside analysis methods 1 to 6, The liquid chromatography may be reverse phase chromatography.

[0063] According to the method for analyzing modified nucleosides of the sixth or seventh aspect, the target component and the reference component can be reliably separated and eluted from the column.

[0064] (Item 8) In any one of the modified nucleoside analysis methods of items 1 to 7, a washing step of washing the column with the mobile phase after the elution step, The washing step may be configured to change the mixing ratio of the solvents constituting the mobile phase introduced into the column in an early or late stage thereof so as to have a fourth period in which the rate of change of the mixing ratio of the solvents constituting the mobile phase is greater than the rate of change of the mixing ratio of the solvents constituting the mobile phase in each of the first period and the second period.

[0065] According to the modified nucleoside analysis method of item 8, the time required for transition from the elution step to the washing step, or from the washing step to the next elution step, can be shortened.

Claims

1. an elution step of introducing a sample containing a target component, which is a modified nucleoside whose hydrophobicity is increased by modification, and a reference component, which is a component different from the target component, into a liquid chromatography column, and separating and eluting the target component and the reference component from each other by gradient elution in which the mixing ratio of multiple solvents constituting the mobile phase is changed over time; detecting the target component and the reference component by mass spectrometry; calculating a ratio between the detection value of the target component and the detection value of the reference component; The elution step the mixing ratio of the solvent constituting the mobile phase introduced into the column is changed so that there is a third period between a first period in which the target component is eluted from the column and a second period in which the reference component is eluted from the column, in which the rate of change of the mixing ratio of the solvent constituting the mobile phase at an outlet of the column is greater than the rates of change of the mixing ratio of the solvent constituting the mobile phase in each of the first period and the second period; the target component is 6-threonylcarbamoyladenosine and / or 2-thiomethyl,6-threonylcarbamoyladenosine, which is a derivative of adenosine and is a modified nucleoside having a chemical structure in which threonine is bound to the adenosine via a carbonyl group, the reference component is a component that does not have the chemical structure; The modified nucleoside analysis method further comprises a step of comparing the determined amount of the target component with at least one of an index value representing the possibility that the subject from whom the sample was collected is infected with COVID-19 and an index value representing the possibility that the subject will be infected with COVID-19 and develop severe symptoms.

2. the sample is urine, 2. The method for analyzing a modified nucleoside according to claim 1, wherein the reference component is at least one selected from the group consisting of creatinine, urea nitrogen, uric acid, adenosine, and 3-amino-3-carboxypropyluridine.

3. the sample is plasma or serum, 2. The method of claim 1, wherein the reference component is at least one of adenosine and 3-amino-3-carboxypropyluridine.

4. 2. The method for analyzing modified nucleosides according to claim 1, wherein the mobile phase is a mixture of formic acid, acetonitrile, and water.

5. 2. The method for analyzing modified nucleosides according to claim 1, wherein the liquid chromatography is reversed-phase chromatography.

6. a washing step of washing the column with the mobile phase after the elution step, The modified nucleoside analysis method of claim 1, wherein the washing step changes the mixing ratio of the solvents constituting the mobile phase introduced into the column so that the washing step has a fourth period in which the rate of change of the mixing ratio of the solvents constituting the mobile phase is greater than the rate of change of the mixing ratio of the solvents constituting the mobile phase in each of the first period and the second period.

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