Measurement method, program, and high-speed liquid chromatograph tandem mass spectrometer

Abstract

A method for measuring a creatinine correction value of a nucleoside according to the present disclosure includes a step (S10) for temporally separating a component, a step (S12) for acquiring a chromatogram of an ion derived from each component, a step (S14) for acquiring a nucleoside measurement value on the basis of a chromatogram of an ion of the nucleoside, a step (S16) for acquiring a creatinine measurement value on the basis of a chromatogram of a product ion other than the product ion having the highest ionic strength of creatinine, and a step (S18) for calculating a creatinine correction value of the nucleoside.

Description

Measurement method, program, and high-performance liquid chromatograph tandem mass spectrometer

[0001] The present disclosure relates to a measurement method, a program, and a high-performance liquid chromatograph tandem mass spectrometer, and more particularly to a technique for reducing the workload on a user involved in measuring the creatinine-corrected value of a nucleoside in a sample.

[0002] Measuring nucleosides in urine is considered important for research into various diseases, including infectious diseases. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is commonly used to analyze nucleosides in urine.

[0003] Urine tests performed to quantify urinary components, including the measurement of urinary nucleosides, are often evaluated using a single spot urine test because they are severely restricted by factors such as urine collection time, test location, testing equipment, and the physical burden on the subject. However, urinary components vary significantly within a single individual and vary significantly depending on gender, age, and dietary content, requiring careful evaluation. Therefore, when evaluating the amount of a specific component in urine, the ratio of the amount of the specific component to the amount of a reference component in urine is generally used. The reference component is a component whose daily excretion in urine is approximately constant, such as creatinine. The ratio of the measured value of the specific component to the measured value of creatinine is called the creatinine correction value.

[0004] Compared to nucleosides, creatinine is present in large amounts in urine. Therefore, it may be difficult to obtain measurements of both nucleosides and creatinine with the same accuracy through a single analysis of a urine sample diluted at a predetermined dilution rate. Generally, when analyzing creatinine using urine diluted to measure nucleosides, ions derived from creatinine exceeding the measurable range of the LC-MS / MS instrument are generated, resulting in reduced measurement accuracy. On the other hand, urine diluted to measure creatinine may not contain nucleosides in amounts greater than the lower limit measurable by the LC-MS / MS instrument. Therefore, to measure the creatinine correction value for nucleosides in a single urine sample, the user must separately analyze the sample diluted to measure creatinine and the sample diluted to measure nucleosides using the LC-MS / MS method, which may result in a long measurement time for the user.

[0005] Regarding a method for measuring the creatinine-corrected values ​​of urinary components using an LC-MS / MS device, Kaori Hosoda, Hiromi Shibasaki, Akitomo Yokokawa, and Kazuo Ishii, "Study on Simultaneous Analysis of Urinary Vanillylmandelic Acid, Homovanillic Acid, Metanephrine, Normetanephrine, and Creatinine by LC-MS / MS," Journal of the Kyorin Medical Society, Vol. 46, No. 3 (2015), pp. 207-212 (Non-Patent Document 1), discloses a method for measuring the creatinine-corrected values ​​of urinary components using an LC-MS / MS device in a single analysis, in which a sample is diluted 100-fold and a sample is diluted 50,000-fold.

[0006] Kaori Hosoda, Hiromi Shibasaki, Akitomo Yokokawa, Kazuo Ishii "Simultaneous Analysis of Urinary Vanillylmandelic Acid, Homovanillic Acid, Metanephrine, Normetanephrine, and Creatinine by LC-MS / MS" Journal of the Kyorin Medical Society, Vol. 46, No. 3 (2015), pp. 207-212

[0007] Non-Patent Document 1 discloses a method for measuring creatinine-corrected values ​​of urine components in a single analysis using LC-MS / MS. However, this method requires the user to dilute one sample at two different dilution rates, which can be cumbersome for the user.

[0008] The present disclosure has been devised in view of the above circumstances, and its purpose is to provide a technology that reduces the burden on users involved in the task of measuring the creatinine correction value of a nucleoside.

[0009] A measurement method according to a first aspect of the present disclosure is a method for measuring a creatinine-corrected value of a nucleoside in a sample using a high-performance liquid chromatography tandem mass spectrometer, which includes the steps of: (i) temporally separating creatinine and nucleosides contained in the sample by liquid chromatography; (ii) obtaining a chromatogram of product ions using ions derived from creatinine and nucleosides as precursor ions; (iii) obtaining a measurement value of the nucleoside based on the chromatogram of the product ions derived from the nucleoside; (iv) obtaining a measurement value of creatinine based on a chromatogram of product ions other than the product ion with the highest ion intensity among the chromatograms of the product ions derived from creatinine; and (v) calculating a creatinine-corrected value of the nucleoside based on the measurement values ​​of the nucleoside and the measurement values ​​of creatinine.

[0010] A program according to a second aspect of the present disclosure is a program executed by a processor installed in a computer. Execution of the program causes the computer to accept data obtained by measuring a sample containing creatinine and a nucleoside using a high-performance liquid chromatography tandem mass spectrometer, and to obtain a measurement value of the nucleoside based on a chromatogram of product ions derived from the nucleoside. Execution of the program causes the computer to obtain a measurement value of creatinine based on a chromatogram of product ions other than the product ion with the highest ion intensity among the chromatograms of product ions derived from creatinine, and to calculate a creatinine correction value for the nucleoside based on the measurement values ​​of the nucleoside and the creatinine.

[0011] A high-performance liquid chromatograph tandem mass spectrometer according to a third aspect of the present disclosure is a high-performance liquid chromatograph tandem mass spectrometer that measures creatinine-corrected values ​​of nucleosides in a sample. The high-performance liquid chromatograph tandem mass spectrometer includes a liquid chromatograph, a mass analyzer that analyzes components separated by the liquid chromatograph, and a data processor that receives measurement data from the mass analyzer. The liquid chromatograph temporally separates creatinine and nucleosides in the sample. The mass analyzer generates a chromatogram of product ions using ions derived from creatinine and nucleosides as precursor ions. The data processing unit obtains a measurement value of the nucleoside based on a chromatogram of the product ions derived from the nucleoside, obtains a measurement value of creatinine based on a chromatogram of product ions derived from creatinine other than the product ion with the strongest ion intensity, and calculates a creatinine correction value of the nucleoside based on the measurement values ​​of the nucleoside and the measurement values ​​of creatinine.

[0012] According to the data processing method of the present disclosure, it is possible to reduce the burden on the user involved in the task of measuring the creatinine correction value of a nucleoside.

[0013] 1 is a schematic diagram of an LC-MS / MS device according to an embodiment; 2 is a functional block diagram of an LC-MS / MS device according to an embodiment; 3 is a flowchart showing the steps of an LC-MS / MS method; 4 is a chromatogram of nucleosides produced by analyzing urine using LC-MS / MS; 5 is a chromatogram of creatinine produced by analyzing urine using LC-MS / MS; 6 is a mass spectrum produced by analyzing creatinine using tandem mass spectrometry; 7 is a diagram showing an example of a chromatogram of product ions produced using ions derived from creatinine as precursor ions; 8 is a diagram showing creatinine concentration and peak area values ​​according to Verification Example 1; 9 is a diagram showing the relationship between creatinine concentration and peak area according to Verification Example 1; 10 is a diagram showing the relationship between the measurement values ​​of creatinine contained in urine diluted 10 times, obtained by the enzymatic method and the LC-MS / MS method according to Verification Example 2; 11 is a diagram showing the relationship between the measurement values ​​of creatinine contained in urine diluted 100 times, obtained by the enzymatic method and the LC-MS / MS method according to Verification Example 2. 1 is a diagram showing the relationship between the measurement values ​​obtained by the enzymatic method and the LC-MS / MS method for creatinine contained in urine diluted 1000 times in Verification Example 2. 2 is a diagram showing the official names and abbreviations of nucleosides for which creatinine correction values ​​are measured. 3 is a diagram for explaining the process of measuring creatinine correction values ​​of nucleosides.

[0014] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail with reference to the accompanying drawings, in which the same or corresponding parts in the drawings are designated by the same reference numerals and the description thereof will not be repeated.

[0015] [Overall Configuration of LC-MS / MS Apparatus] Figure 1 is a diagram schematically illustrating the overall configuration of an LC-MS / MS apparatus 100 according to this embodiment. As shown in Figure 1, the LC-MS / MS apparatus 100 includes a liquid chromatograph 1, a mass spectrometer 2, a controller 3, a display 4, and an input unit 5. The controller 3, display 4, and input unit 5 may be incorporated into the mass spectrometer 2. Alternatively, the controller 3 may be a general-purpose computer located away from the liquid chromatograph 1 and the mass spectrometer 2. The LC-MS / MS apparatus 100 can separate components contained in a sample and detect ions derived from each component.

[0016] The sample is a liquid containing creatinine, such as urine collected from a living body. The urine collected from a living body may be used as the sample in its undiluted form, or may be used as the sample after being diluted at a given dilution rate. The given dilution rate may be selected from the range of 2 to 10,000 times, or may be 10 to 1,000 times.

[0017] The liquid chromatograph 1 includes a mobile phase container 10, a pump 11, an injector 12, and a column 13. The liquid chromatograph 1 can separate multiple types of components contained in a sample into individual types of components by utilizing differences in interactions between the components and the stationary phase and the mobile phase.

[0018] The mobile phase container 10 stores a mobile phase, which is a liquid that carries a sample injected into the liquid chromatograph 1. The mobile phase is, for example, an organic solvent or water, or a mixture thereof. Examples of organic solvents include 2-propanol, methanol, acetonitrile, chloroform, hexane, dichloromethane, and tetrahydrofuran. The mobile phase may contain an acidic solution (e.g., trifluoroacetic acid, formic acid, and ammonium formate) as an additive. The liquid chromatograph 1 may include a single mobile phase container or multiple mobile phase containers.

[0019] The pump 11 draws in the mobile phase stored in the mobile phase container 10 and delivers it at a predetermined flow rate. The flow rate of the mobile phase delivered by the pump 11 may be constant or may vary during one measurement. The liquid chromatograph 1 may include a single pump or multiple pumps.

[0020] The injector 12 injects a predetermined amount of a sample, which has been prepared in advance in a mobile phase flow path, into the liquid chromatograph 1. The sample is introduced from the injector 12 into the mobile phase delivered from the pump 11, and the mobile phase containing the sample is introduced into the column 13.

[0021] The column 13 is filled with a stationary phase, and the mobile phase passes through it. As the sample passes through the column 13, various components in the sample interact with the mobile phase and the stationary phase, causing them to be separated in the time direction. The separated components are eluted from the outlet of the column 13 and introduced into the mass spectrometer 2.

[0022] The mass analysis unit 2 includes an ionization chamber 20, a first intermediate chamber 21, a second intermediate chamber 22, and an analysis chamber 23. The mass analysis unit 2 performs mass analysis of the sample eluted from the liquid chromatograph 1. The analysis in the mass analysis unit 2 includes detecting peaks in the mass spectrum and measuring the mass-to-charge ratio of specific or non-specific substances contained in the sample.

[0023] The ionization chamber 20 has a probe 201 and a capillary 202. The interior of the ionization chamber 20 is at atmospheric pressure. The ionization chamber 20 is connected to the next-stage first intermediate chamber 21 via a thin-diameter capillary 202. The probe 201 sprays the sample introduced into the mass analysis unit 2 while imparting a biased charge to the sample. The charged microdroplets are split and refined by the action of electrostatic force, and the sample components in the droplets are ionized as the solvent evaporates. The generated ions pass through the capillary 202 and are introduced into the first intermediate chamber 21.

[0024] The first intermediate chamber 21 has an ion guide 211 and a skimmer 212. The inside of the first intermediate chamber 21 is at high vacuum. The first intermediate chamber 21 and the second intermediate chamber 22 in the next stage are connected through a small hole drilled in the top of the skimmer 212. The ion guide 211 focuses ions introduced from the ionization chamber 20 in the previous stage and transports them to the subsequent stage via the skimmer 212.

[0025] The second intermediate chamber 22 has an ion guide 221. The inside of the second intermediate chamber 22 is at high vacuum. The ion guide 221 focuses ions introduced from the first intermediate chamber 21 in the preceding stage and transports the ions to the subsequent stage.

[0026] The analysis chamber 23 includes quadrupole mass filters 231 and 233, a collision cell 232, and an ion detector 234. The quadrupole mass filter 231 is disposed before the collision cell 232, and the quadrupole mass filter 233 is disposed after the collision cell 232. The inside of the analysis chamber 23 is at atmospheric pressure. The analysis chamber 23 separates ions by mass and detects each of the separated ions to obtain a mass spectrum. Information regarding the molecular weight, molecular formula, and chemical structure of the compound can be obtained from the obtained mass spectrum.

[0027] The quadrupole mass filter 231 includes a main rod electrode 2312 and a pre-rod electrode 2311 located in front of it. The main rod electrode 2312 separates ions according to their mass-to-charge ratio. The pre-rod electrode 2311 corrects disturbances in the electric field at the entrance end and assists the function of the main rod electrode 2312.

[0028] The collision cell 232 includes a multipole ion guide 2321 therein. The collision cell 232 is connected to a collision-induced dissociation (CID) gas supply mechanism (not shown), which introduces CID gas into the collision cell 232. The CID gas promotes ion dissociation. The multipole ion guide 2321 focuses the dissociated ions and transports them to a subsequent stage. Examples of CID gas include argon, nitrogen, helium, and xenon.

[0029] The quadrupole mass filter 233 includes a main rod electrode 2332 and a pre-rod electrode 2331 disposed in front of it. The main rod electrode 2332 separates ions according to their mass-to-charge ratio. The pre-rod electrode 2331 corrects disturbances in the electric field at the entrance end and assists the function of the main rod electrode 2332.

[0030] The user can set the mass-to-charge ratio value of ions to be passed through the quadrupole mass filters 231 and 233 during mass analysis, as well as the allowable range for this value. The smaller the allowable range, the more limited the ions that can pass through. This improves the accuracy of ion detection, but reduces the detected intensity of ions.

[0031] The ion detector 234 is, for example, a pulse count detector, and generates a detection signal having a number of pulse signals corresponding to the number of incident ions. The detection signal is output to the control unit 3.

[0032] In the above-described embodiment, the LC-MS / MS device 100 is equipped with electrospray ionization as the ionization method, but the ionization method is not limited to electrospray ionization, and atmospheric pressure chemical ionization or atmospheric pressure photoionization may also be used.

[0033] The control unit 3 is communicatively connected to the liquid chromatograph 1 and the mass spectrometer 2, and is configured, for example, by a computer. The control unit 3 controls the operations of the liquid chromatograph 1 and the mass spectrometer 2, and receives measurement data acquired by the ion detector 234 of the mass spectrometer 2. The control unit 3 also acquires measurement values ​​of nucleosides and creatinine based on the measurement data, and calculates creatinine correction values ​​for the nucleosides.

[0034] The display unit 4 is configured by, for example, a liquid crystal display. In response to commands from the control unit 3, the display unit 4 displays the measurement data acquired by the ion detector 234, the measurement values ​​of nucleosides and creatinine acquired from the measurement data, and the creatinine correction values ​​of nucleosides.

[0035] The input unit 5 is configured with, for example, a keyboard, a mouse, etc. The input unit 5 receives instructions from the user for the liquid chromatograph 1 and the mass spectrometer 2 and outputs them to the control unit 3. A touch panel in which the display unit 4 and the input unit 5 are integrated may also be used.

[0036] 2 is a functional block diagram of the overall configuration of the LC-MS / MS system 100 according to this embodiment. As shown in Fig. 2, the control unit 3 has, as its main components, a CPU (Central Processing Unit) 30, a memory 31, a communication I / F (Interface) 32, a display I / F 33, and an input I / F 34. The components are interconnected by a data bus.

[0037] The CPU 30 is configured to execute predetermined arithmetic processing described in a program.

[0038] The memory 31 can non-temporarily store programs executed by the CPU 30, mass spectrum data created by the mass analysis unit 2, and component measurement values. The component measurement values ​​are calculated, for example, from the area value of a peak in the mass spectrum and the height of the peak in the mass spectrum. The programs stored in the memory 31 include a program for calculating the creatinine correction value of a nucleoside. The memory 31 includes volatile memory (e.g., RAM (Random Access Memory)) and non-volatile memory (e.g., ROM (Read Only Memory), a hard disk drive, and a solid state drive). The programs may also be stored in an external storage device accessible by the CPU 30.

[0039] The communication I / F 32 relays communication with external devices including the liquid chromatograph 1 and the mass spectrometer 2. The communication I / F 32 is implemented by, for example, a network adapter. The communication method may be wireless communication such as Bluetooth (registered trademark) or a wireless LAN, or wired communication using a USB (Universal Serial Bus) or the like.

[0040] The display I / F 33 relays data transmission between the CPU 30 and the display unit 4. In accordance with commands from the CPU 30, the display I / F 33 outputs to the display unit 4 signals for displaying the measurement data obtained by the mass spectrometric unit 2, the measurement values ​​of nucleosides and creatinine obtained from the measurement data, and the creatinine correction values ​​of the nucleosides.

[0041] The input I / F 34 relays data transmission between the CPU 30 and the input unit 5. The input I / F 34 receives commands for the liquid chromatograph 1 and the mass spectrometer unit 2.

[0042] [Quantitative Analysis of Components by LC-MS / MS Analysis] The procedure for detecting creatinine and nucleosides contained in a sample using the LC-MS / MS system 100 will be described with reference to Figures 1 and 3. Figure 3 is a flowchart showing the steps of the LC-MS / MS method. Of the processes shown in Figure 3, T10 is performed manually by an analyst using laboratory equipment commonly used in scientific experiments and mass spectrometry. Of the processes shown in Figure 3, T12 and subsequent steps are performed by the LC-MS / MS system 100.

[0043] 3, the user prepares a sample as shown in step T10. If the sample to be measured is urine collected from a living body, the sample may be diluted.

[0044] In step T12, the sample prepared in step T10 is introduced into liquid chromatograph 1 and separated into creatinine and nucleosides. Specifically, the sample introduced into liquid chromatograph 1 is introduced into column 13 together with the mobile phase delivered by pump 11. In column 13, each component interacts with the stationary phase and the mobile phase, with the degree of interaction varying depending on the type of component. The speed at which components move through column 13 is determined by this interaction, and therefore the speed of movement varies depending on the type of component. Therefore, due to the differences in the mobility of each component, liquid chromatograph 1 can separate the components by type.

[0045] In step T12, the eluate eluted from the liquid chromatograph 1 is introduced into the mass spectrometer 2. The processing in the mass spectrometer 2 is shown as step T14 in Fig. 3, and is further subdivided into steps T16 to T24.

[0046] In step T16, the components introduced into the mass analysis section 2 are ionized. The generated ions are introduced into the quadrupole mass filter 231.

[0047] In step T18, only selected ions from the ions introduced into the quadrupole mass filter 231 are introduced into the downstream collision cell 232. This selection is performed by the voltage applied to the main rod electrode 2312. That is, a voltage in which a predetermined radio frequency voltage and a DC voltage are superimposed is applied to the main rod electrode 2312, and the quadrupole mass filter 231 performs this selection by allowing only ions having a specific mass-to-charge ratio corresponding to the voltage applied to the main rod electrode 2312 to pass through among the various ions sent into the quadrupole mass filter 231. The ions that have passed through the quadrupole mass filter 231 are called precursor ions.

[0048] In step T20, precursor ions that have passed through the quadrupole mass filter 231 are introduced into the collision cell 232. A predetermined voltage is applied to electrodes disposed in the collision cell 232, and the precursor ions introduced into the collision cell 232 are accelerated within the collision cell 232 at an acceleration corresponding to the voltage applied to the electrodes. In addition, CID gas is supplied into the collision cell 232 at a predetermined pressure. As a result, the accelerated precursor ions collide with the CID gas with a predetermined collision energy, dissociating and fragmenting them. Ions generated from the precursor ions through fragmentation are called product ions.

[0049] In step T22, when the generated product ions are introduced into the quadrupole mass filter 233, only product ions having a specific mass-to-charge ratio according to the voltage applied to the main rod electrode 2332 of the quadrupole mass filter 233 pass through the quadrupole mass filter 233.

[0050] In step T24, the ion detector 234 detects the product ions that have passed through the quadrupole mass filter 233. The ion detector 234 transmits the detected data to the control unit 3. The control unit 3 generates a chromatogram for each product ion detected by the ion detector 234. Thereafter, the processing in FIG. 3 ends.

[0051] 3, a chromatogram can be generated for each product ion generated using ions derived from creatinine and nucleosides in the sample as precursor ions. The area of ​​a peak in the chromatogram is related to the amount of the substance that is the source of that peak in the sample. In this embodiment, the measured value of the corresponding substance is obtained from the area of ​​the peak in the chromatogram.

[0052] [Comparative Example] RNA (ribonucleic acid) is an important molecule for protein synthesis, and it has been found that some of this RNA is modified by enzymes. When RNA is broken down within cells, it becomes its component nucleosides. Nucleosides are a general term for chemical compounds containing a pentose sugar and a nitrogenous base, including chemically modified nucleosides. Some of the nucleosides produced by RNA breakdown are excreted outside the cell. The nucleosides are then excreted from the body in urine. Measuring nucleosides in urine is considered extremely important in researching various diseases, including infectious diseases. LC-MS / MS instruments are commonly used to analyze nucleosides in urine.

[0053] Urine tests performed to quantify urinary components, including the measurement of urinary nucleosides, are often evaluated using a single spot urine test due to significant constraints such as urine collection time, test location, testing equipment, and physical strain on the subject. However, urinary components vary significantly within the same individual and vary significantly depending on gender, age, and dietary content, requiring careful evaluation. Therefore, when assessing the amount of a specific component in urine, the ratio of the amount of the specific component to the amount of a reference component in urine is generally used. The reference component is a component whose daily urine excretion is approximately constant, such as creatinine. The ratio of the measured value of the specific component to the measured value of creatinine is called the creatinine correction value. Since the amount of creatinine excreted by an adult per day is approximately 1 g, the amount of the specific component per 1 g of creatinine corresponds to the 24-hour excretion amount of the specific component.

[0054] When measuring the creatinine-corrected value of nucleosides in urine, possible methods include (1) correcting (dividing) the nucleoside measurement value obtained by LC-MS / MS by the creatinine measurement value obtained by enzymatic analysis, and (2) correcting using values ​​(concentration, area value, etc.) obtained by measuring nucleosides and creatinine by LC-MS / MS. Method (1) requires measuring urine samples using two analytical methods, the enzymatic method and the LC-MS / MS, which may require a long user time. Method (2) may require two analyses using an LC-MS / MS instrument to measure the creatinine-corrected value of nucleosides in a single urine sample. This is because the amount of creatinine in urine is much greater than the amount of nucleosides.

[0055] The analysis of urinary nucleosides and creatinine using an LC-MS / MS system will be described with reference to Figures 4 and 5. Figures 4 and 5 show chromatograms generated by analyzing urine diluted at a predetermined dilution rate suitable for nucleoside analysis using an LC-MS / MS system.

[0056] 4 shows a chromatogram of cytidine, a type of nucleoside, produced by analyzing a urine sample diluted at a predetermined dilution rate using an LC-MS / MS system. The area surrounded by line L1 corresponds to the amount of cytidine, and it can be seen that the measured value of cytidine can be obtained from this chromatogram.

[0057] Figure 5 shows a chromatogram of creatinine obtained by LC-MS / MS analysis of a sample prepared by diluting urine at the same predetermined dilution rate as in Figure 4. In Figure 5, the ion intensity of ions derived from creatinine, indicated by line L2, exceeds the upper limit of measurable values ​​in the retention time range of 4.6 to 5.0. Therefore, the area enclosed by line L2 does not reflect the amount of creatinine, and a measurement value of creatinine cannot be obtained from this chromatogram with the same degree of accuracy as the measurement value of cytidine.

[0058] Furthermore, contrary to the cases described in FIGS. 4 and 5, in a urine sample prepared at a dilution rate suitable for creatinine analysis, the ionic strength of the nucleoside may be below the detection limit.

[0059] Non-Patent Document 1 discloses a method for obtaining measured values ​​of creatinine and other components in a single analysis using LC-MS / MS. However, in this method, a single sample is diluted at two different dilution rates, 100-fold and 50,000-fold, and then mixed and analyzed using an LC-MS / MS device. This requires the user to dilute a single specimen at two different dilution rates, one for measuring creatinine and one for measuring other components, which can be cumbersome for the user.

[0060] [Method for Measuring Nucleoside Creatinine Correction Values] In this embodiment, a urine sample diluted at a dilution ratio suitable for nucleoside measurement is analyzed by LC-MS / MS, and a creatinine measurement value is obtained using product ions other than the most intense product ion as an indicator. By selecting, from among the product ions derived from creatinine, product ions that do not exceed the upper limit of ion intensity detectable by the LC-MS / MS device, creatinine can be measured using urine diluted to suit nucleoside measurement. According to the measurement method of this embodiment, the nucleoside creatinine correction value can be measured in a single analysis without setting different dilution ratios for each component to be measured.

[0061] In this embodiment, product ions other than the most intense product ion derived from creatinine, which serve as indicators for creating a chromatogram, will be described. Fig. 6 shows a mass spectrum of product ions generated using ions derived from creatinine as precursor ions.

[0062] As shown in FIG. 6, creatinine has the molecular formula C ４ H ７ N ３ It is a substance expressed as 0. Therefore, its molecular weight is 113. The molecule contains hydrogen ions (H ＋ ) is detected by mass spectrometry, the mass-to-charge ratio of the precursor ion detected when creatinine is analyzed by a mass spectrometer is 114.1. In Figure 6, peak 510 indicates the precursor ion of creatinine.

[0063] 6 shows that in addition to peak 510, peaks 520, 530, and 540 were detected, which correspond to the peaks of product ions generated from the precursor ion of creatinine.

[0064] Peak 520 is a compound with the molecular formula CH ３N corresponds to a product ion having a mass-to-charge ratio of 44.05. The ion intensity of the product ion corresponding to peak 520 is the strongest among the produced product ions. In other words, the detection sensitivity of this product ion is better than that of the other product ions.

[0065] Peak 530 is a compound with the molecular formula C ３ H ８ N ３ This corresponds to a product ion with a mass-to-charge ratio of 86.01. The ion intensity of the product ion corresponding to peak 530 is the second strongest among the product ions produced.

[0066] Peak 540 is a compound with the molecular formula C ２ H ６ N ３ This corresponds to a product ion with a mass-to-charge ratio of 72.00. The ion intensity of the product ion corresponding to peak 540 is the third strongest among the product ions produced.

[0067] A chromatogram of creatinine is created based on at least one of these product ions. Generally, the product ion with the highest ionic intensity has the best detection sensitivity among the product ions. Therefore, by using a chromatogram created using the product ion with the highest ionic intensity as an index, the measurement sensitivity of the target component can be improved. Therefore, when creatinine is the target of analysis, a product ion with a mass-to-charge ratio of 44.05 is generally used as an index for creating a chromatogram.

[0068] In the measurement method according to this embodiment, a measurement value of creatinine is obtained using, as an indicator, a product ion other than the product ion with the strongest ion intensity and a mass-to-charge ratio of 44.05 among the product ions of creatinine shown in Figure 6. By using a product ion other than the product ion with the strongest ion intensity as an indicator, the detection intensity can be reduced, preventing the detection intensity from exceeding the detection limit of the LC-MS / MS apparatus 100. The product ions other than the product ion with the strongest ion intensity and a mass-to-charge ratio of 44.05 are product ions with a mass-to-charge ratio of 86.01 or a product ion with a mass-to-charge ratio of 72.00.

[0069] In the following description, the product ion with the second strongest ion intensity and a mass-to-charge ratio of 86.01 will be described as an example of a product ion other than the most intense product ion derived from creatinine.

[0070] The maximum value of the chromatogram to be created varies depending on the product ion used as an index when creating the chromatogram. Fig. 7 shows a chromatogram created using the product ion of creatinine as an index.

[0071] Figure 7 shows chromatograms of two types of product ions of creatinine generated when a urine sample diluted at a specified dilution rate is analyzed by LC-MS / MS. L3, shown by the solid line, represents the chromatogram of the product ion with the strongest ion intensity. L4, shown by the dashed line, represents the chromatogram of the product ion with the second strongest ion intensity.

[0072] As shown by line L3, in the chromatogram of the product ion with the strongest ion intensity, the ion intensity exceeds the detectable range in the retention time range of 3.5 to 4.0, so if creatinine is measured using the area value of line L3, the measurement accuracy will be reduced.

[0073] As shown by line L4, in the chromatogram of the product ion of creatinine, which has the second strongest ion intensity, the ion intensity does not exceed the detectable range. Therefore, by measuring creatinine using the area value of line L4, the accuracy of creatinine measurement can be improved.

[0074] As described above, when analyzing the same sample and detecting product ions, the intensity of a product ion with a strong ion intensity may exceed the upper limit of detection, while the intensity of a product ion with a weak ion intensity may be below the upper limit of detection. Therefore, even when urine diluted for nucleoside measurement is analyzed using the LC-MS / MS system 100, the ion intensity of the product ion derived from creatinine will be below the upper limit of detection, and a measurement value of creatinine can be obtained.

[0075] Therefore, by using the product ion with the second strongest ionic strength, rather than the product ion with the strongest ionic strength, as the product ion that serves as an indicator when creating a chromatogram, it becomes possible to obtain measurement values ​​even for samples for which measurement values ​​cannot be obtained using the product ion with the strongest ionic strength as an indicator.

[0076] Verification Example 1 The quantitativeness of the creatinine measurement value obtained by the measurement method described in this embodiment is verified using a creatinine standard.

[0077] In the measurements of Verification Examples 1 to 3 below, the analysis by the LC-MS / MS method was performed, for example, under the following conditions: (Apparatus used) LC: Nexera X2 system (manufactured by Shimadzu Corporation) MS: LCMS-8060NX (manufactured by Shimadzu Corporation) (LC conditions) Column: Shim-pack GIS C18 (2.1 mm I.D. x 150 mm L, 2 μm, manufactured by Shimadzu Corporation) Mobile phase A: acetic acid / ammonium acetate / ultrapure water Mobile phase B: acetonitrile / ultrapure water Flow mode: high-pressure gradient mode Flow rate: 0.4 mL / min Column temperature: 50°C Injection volume: 2 μL (MS conditions) Ionization mode: Ion Focus ESI Nebulizer gas flow rate: 3.0 L / min Heating gas flow rate: 10.0 L / min Drying gas flow rate: 10.0 L / min; Interface temperature: 300°C; Desolvation section (DL) temperature: 250°C; Heat block temperature: 400°C.

[0078] Standard samples with creatinine concentrations of 0.1 mg / L, 1 mg / L, 10 mg / L, and 100 mg / L are prepared using a creatinine standard. Each standard sample is subjected to LC-MS / MS to generate a chromatogram of the creatinine product ion, which has the second-highest ionic intensity. Area values ​​are obtained from each generated chromatogram. The prepared standard samples are mixed with an equal amount of a predetermined internal standard (IS). To eliminate analytical errors from the measured values, the area value of creatinine relative to the area value of the predetermined IS in the chromatogram is used as the measured value of creatinine for that sample.

[0079] Fig. 8 shows the ratio of the area value of creatinine obtained by analyzing a creatinine standard sample by LC-MS / MS using the chromatogram of the creatinine product ion, which has the second strongest ion intensity, as an index to the area value of a predetermined IS, which is the measured value of creatinine. As shown in Fig. 9, the measured value of creatinine increases by approximately 10 times when the creatinine concentration increases by 10 times.

[0080] 9 is a diagram showing the relationship between the creatinine concentration in the standard samples and the measured values. In FIG. 9, the horizontal axis represents the creatinine concentration in the standard samples, and the vertical axis represents the measured values ​​of the corresponding samples. Also shown in FIG. 9 is a linear equation expressing the relationship between the creatinine concentration and the measured values ​​of the four standard samples with different creatinine concentrations shown in FIG. 8. R is the correlation coefficient of the linear equation, and the closer the square of the correlation coefficient is to 1, the stronger the positive correlation.

[0081] In Figure 9, the square of the correlation coefficient R is 0.99999. Therefore, it can be said that there is a positive correlation between the creatinine concentration in the standard sample and its measured value. From the above, the measured value obtained using the chromatogram of the creatinine product ion, which has the second strongest ionic strength, as an indicator correlates with the creatinine concentration in the sample, at least in the creatinine concentration range of 0.1 to 100 mg / L. Therefore, the measured value of creatinine obtained by the measurement method described in this embodiment is a value related to the amount of creatinine in the sample.

[0082] Verification Example 2 Creatinine quantification is generally performed by an enzymatic method. Therefore, the results of measuring the same sample by the measurement method described in this embodiment and the results of measuring by the enzymatic method were compared.

[0083] 10 to 12 show the relationship between creatinine measurement values ​​obtained using the chromatogram of the creatinine product ion, which has the second-highest ion intensity, as an index when subjected to the LC-MS / MS system 100, and creatinine measurement values ​​obtained by the enzymatic method. In FIGS. 10 to 12, the horizontal axis represents the results obtained by the LC-MS / MS method, and the vertical axis represents the results obtained by the enzymatic method. One sample was subjected to measurement by the LC-MS / MS method and measurement by the enzymatic method, and the respective results are plotted as values ​​on the vertical and horizontal axes. Each figure also shows the measurement results of 30 urine samples. In FIG. 10, the sample was obtained from a living body and diluted 10 times; in FIG. 11, the sample was obtained from a living body and diluted 100 times; and in FIG. 12, the sample was obtained from a living body and diluted 1000 times.

[0084] 10, when one sample is subjected to measurement by the LC-MS / MS method and measurement by the enzymatic method and the relationship between the values ​​obtained by each method is expressed as a linear equation, the square of the correlation coefficient is 0.8648. From this, it can be said that there is a positive correlation between the measurement value by the LC-MS / MS method obtained by diluting a urine specimen 10 times and the measurement value by the enzymatic method.

[0085] 11, when one sample is subjected to measurement by the LC-MS / MS method and measurement by the enzymatic method and the relationship between the values ​​of the results is expressed as a linear equation, the square of the correlation coefficient is 0.8975. From this, it can be said that there is a positive correlation between the measurement value by the LC-MS / MS method obtained by diluting a urine specimen 100 times and the measurement value by the enzymatic method.

[0086] 12, when one sample is subjected to measurement by the LC-MS / MS method and measurement by the enzymatic method, and the relationship between the values ​​obtained by each method is expressed as a linear equation, the square of the correlation coefficient is 0.846. This indicates that there is a positive correlation between the measurement value obtained by the LC-MS / MS method using a urine sample diluted 1000-fold and the measurement value obtained by the enzymatic method.

[0087] From the above, the measurement value obtained using the chromatogram of the creatinine product ion, which has the second strongest ion intensity, as an index correlates with the measurement value of creatinine obtained by the enzymatic method, at least in a sample obtained by diluting urine 10 to 1000 times. Therefore, the creatinine-corrected value obtained by the measurement method described in this embodiment is equivalent to the creatinine-corrected value measured using the enzymatic method.

[0088] Verification Example 3: The measurement values ​​of urinary nucleosides and creatinine were measured using the measurement method described in this embodiment. Figure 13 shows the nucleosides for which creatinine-corrected values ​​were measured using the measurement method after analyzing a human urine sample using LC-MS / MS. Figure 13 also shows the full names and abbreviations of the nucleosides.

[0089] As shown in FIG. 13, the nucleosides for which the creatinine correction values ​​were measured by the measurement method described in this embodiment were pseudouridine (Y), dihydrouridine (D), 5-methylaminomethyl-2-thiouridine (mnm ５ s ２ U), 3-methyluridine (m ３ U), 5-methyluridine (m ５ U), 1-methylpseudouridine (m １ Y), 3-methylpseudouridine (m ３ Y), uridine (U), cytidine (C), guanosine (G), 3-methylcytidine (m ３ C), 5-methylcytidine (m ５ C), inosine (I), 2'-O-methylcytidine (Cm), 7-methylguanosine (m ７ G), 1-methylguanosine (m １ G), N2-methylguanosine (m ２ G), 1-methyladenosine (m １ A), N6-methyladenosine (m ６ A), 1-methylinosine (m １ I), 2'-O-methyluridine (Um), 2'-O-methylguanosine (Gm), N4-acetylcytidine (ac ４ C), 2'-O-methylinosine (Im), N2,N2-dimethylguanosine (m ２ , ２ G), adenosine (A), 5-methoxycarbonylmethyluridine (mcm ５ U), N6-threonylcarba moyladenosine (t ６ A), 2'-O-methyladenosine (Am), 2-methylthio-N6-threonylcarba moyladenosine (ms２ t ６ A), 2-thiocytidine(s ２ C), 5-formylcytidine (f ５ C), N4,2'-O-dimethylcytidine (m ４ Cm), N4,N4,2'-O-trimethylcytidine (m ４ , ４ Cm), 5-hydroxymethylcytidine (hm ５ C), uridine 5-oxyacetic acid methyl ester (mcmo ５ U), 5-methoxycarbonylmethyl-2-thiouridine (mcm ５ s ２ U), 5-carbamoylmethyl-2-thiouridine (ncm ５ s ２ U), 3-(3-amino-3-carboxypropyl)uridine (acp ３ U), 5-aminomethyluridine (nm ５ U), 5-Taurinomethyluridine (tm ５ U).

[0090] [Processing Flow] Figure 14 is a flowchart of an example of processing performed in the LC-MS / MS device 100 to measure creatinine-corrected values ​​of nucleosides based on creatinine measurement values ​​obtained by subjecting a urine sample to LC-MS / MS and using as an index a chromatogram of the creatinine product ion, which has the second strongest ion intensity. In one implementation example, of the processing in Figure 14, S10 is performed by the liquid chromatograph 1, and S12 and S14 are performed by the mass spectrometric unit 2. Of the processing in Figure 14, the processing from S16 onwards is called from the main routine and executed when the CPU 30 of the control unit 3 executes a given program.

[0091] In step S10, the liquid chromatograph 1 separates the creatinine and nucleosides contained in the introduced sample in terms of time. The separated creatinine and nucleosides are introduced into the mass spectrometer 2.

[0092] In step S12, the mass spectrometric unit 2 generates a chromatogram of product ions using ions derived from nucleosides and creatinine as precursor ions.

[0093] In step S14, the control unit 3 acquires a measurement value of the nucleoside based on the chromatogram of the nucleoside product ions generated in step S12 by the mass spectrometric unit 2. The measurement value of the nucleoside is, for example, the area of ​​the region surrounded by the product ion peak in the chromatogram.

[0094] In step S16, the control unit 3 acquires a measurement value of creatinine based on the chromatogram of the creatinine product ion having the second strongest ion intensity among the creatinine product ions generated in step S12 by the mass spectrometric unit 2. The measurement value of creatinine is, for example, the area of ​​the region surrounded by the product ion peak in the chromatogram.

[0095] In step S18, the control unit 3 calculates the creatinine correction value for the nucleoside based on the nucleoside measurement value obtained in step S14 and the creatinine measurement value obtained in step S16. Thereafter, the CPU 30 ends the creatinine correction value measurement subroutine and returns the process to the main routine.

[0096] In general, the amount of a urinary component per gram of creatinine is referred to as the 24-hour excretion amount of that urinary component. When converting the creatinine-corrected value of a nucleoside measured in the process shown in Figure 14 to the amount of nucleoside per gram of creatinine, it is necessary to prepare standard samples of creatinine and nucleoside with known contents, create calibration curves showing the relationship between the amount of each substance and the measured value obtained from the area of ​​the chromatogram, and convert the measured value into weight.

[0097] The measurement method according to the present disclosure allows for the measurement of creatinine-corrected values ​​of nucleosides in a sample through a single LC-MS / MS analysis of a urine sample diluted at a single dilution rate. Since there is no need to measure creatinine and nucleosides using separate measurement techniques, the time required for the user's measurement can be reduced. Furthermore, since there is no need to dilute a single sample at two different dilution rates, the user's workload is reduced. This reduction in the user's workload can prevent human error, such as incorrect dilution rates.

[0098] In the measurement method according to the present embodiment, the chromatogram of the ion with the second strongest ion intensity among the product ions detected using ions derived from creatinine as precursor ions is used as the index, but the index is not limited to the second strongest ion. Any product ion other than the product ion with the strongest ion intensity may be used, such as the third strongest product ion or the fourth strongest product ion.

[0099] By using an ion with a lower ionic strength as an indicator, the accuracy of creatinine measurement can be improved in samples with a higher creatinine content. Therefore, the accuracy of creatinine measurement can be improved in urine samples with a low dilution rate. Therefore, for example, when measuring the creatinine correction value of a nucleoside that is present in a small amount in urine, concentrated urine must be used for the analysis, and therefore, by using a product ion with a low ionic strength as an indicator, the accuracy of creatinine measurement can sometimes be improved.

[0100] In general, product ions with low ionic strength have lower detection sensitivity than product ions with high ionic strength. Therefore, the accuracy of creatinine measurement using product ions with low ionic strength as an indicator may be lower than the accuracy of creatinine measurement using product ions with high ionic strength as an indicator. Therefore, the accuracy of creatinine measurement can be improved by measuring creatinine using the most intense product ion, which does not exceed the ion intensity detectable by the LC-MS / MS instrument, among the product ions detected when a specific sample is analyzed using the LC-MS / MS instrument.

[0101] In the measurement method according to the present embodiment, the product ions of creatinine are classified into those having the molecular formula CH ３ N ion with a mass-to-charge ratio of 43.2, molecular formula C ３ H ８ N ３ and an ion with a mass-to-charge ratio of 86.01 and a molecular formula of C ２ H ６ N ３ The ion has a mass-to-charge ratio of 72.00. However, this order may change depending on the measurement conditions. ３ If the ion intensity of the ion having a mass-to-charge ratio of 43.2 at N is not the strongest among the product ions, the creatinine-corrected value of the nucleoside may be measured using the chromatogram of that ion as an index.

[0102] Nucleosides for which creatinine correction values ​​can be measured by the measurement method of the present disclosure are not limited to those shown in Figure 13. As long as the nucleoside can be measured by LC-MS / MS, the creatinine correction value can be measured by the measurement method of the present disclosure, regardless of the chemical modification of the nucleoside.

[0103] Furthermore, the components to be measured for creatinine correction values ​​are not limited to nucleosides, and the measurement method of the present disclosure can be applied to any component that can be measured by LC-MS / MS.

[0104] In this embodiment, a method for measuring the amount of a nucleoside corrected for the amount of creatinine using creatinine as a reference component has been described, but the reference component is not limited to creatinine. The reference component may be a substance whose amount excreted in urine per day is constant, a substance whose amount excreted in urine per day can be considered to be constant, or a substance whose amount excreted in urine per day is correlated with the amount of creatinine excreted.

[0105] [Modification] The detection intensity of product ions can be adjusted by adjusting the allowable range of the mass-to-charge ratio of ions that are allowed to pass through the quadrupole mass filters 231 and 233 during mass analysis. Specifically, by narrowing the allowable range, fewer product ions pass through the quadrupole mass filters 231 and 233, and therefore the ion intensity becomes weaker.

[0106] The tolerance range corresponds to the resolution of mass spectrometry, and a smaller tolerance range means higher resolution. The resolution of a quadrupole mass filter is expressed in terms of the molecular weights that can be distinguished. For example, a resolution of 7 Da means that ions derived from molecules with molecular weights that differ by 7 Da can be distinguished as different ions. Therefore, reducing the resolution value of the quadrupole filter reduces the detected intensity of ions.

[0107] If a user sets the resolution value of the quadrupole mass filter when analyzing creatinine to be smaller than the resolution value of the quadrupole mass filter when analyzing nucleosides and analyzes a sample with the LC-MS / MS system 100, the detection intensity of creatinine product ions can be reduced. Therefore, in the method described in the embodiment, the resolution value of the quadrupole mass filter when analyzing creatinine is set to be smaller than the resolution value of the quadrupole mass filter when analyzing nucleosides. Specifically, for example, the resolution value of the quadrupole mass filter when analyzing nucleosides is 0.7 Da, and the resolution value of the quadrupole mass filter when analyzing creatinine in that case is 0.4 Da.

[0108] The user can separately set the resolution value of the quadrupole mass filter 231 and the resolution value of the quadrupole mass filter 233. In a modified example, the resolution value of either the quadrupole mass filter 231 or the resolution value of the quadrupole mass filter 233 is smaller when analyzing creatinine than when analyzing nucleosides, and the other value is smaller when analyzing creatinine than when analyzing nucleosides, or is the same when analyzing creatinine and when analyzing nucleosides.

[0109] Aspects It will be understood by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0110] (Item 1) A measurement method in one aspect is a method for measuring a creatinine-corrected value of a nucleoside in a sample using a high-performance liquid chromatography tandem mass spectrometer, and may include the steps of: temporally separating the creatinine and the nucleoside contained in the sample by a liquid chromatograph; obtaining a chromatogram of product ions using each of ions derived from the creatinine and the nucleoside as precursor ions; obtaining a measurement value of the nucleoside based on the chromatogram of the product ions derived from the nucleoside; obtaining a measurement value of the creatinine based on a chromatogram of a product ion other than the product ion with the strongest ion intensity among the chromatograms of the product ions derived from the creatinine; and calculating a creatinine-corrected value of the nucleoside based on the measurement value of the nucleoside and the measurement value of the creatinine.

[0111] According to the measurement method described in paragraph 1, the creatinine-corrected values ​​of nucleosides in a sample can be measured by a single LC-MS / MS analysis without diluting one sample at two different dilution rates, thereby reducing the workload on the user and shortening the operation time.

[0112] (Item 2) In the measurement method described in Item 1, the product ion other than the product ion with the strongest ionic strength may be either the product ion with the second strongest ionic strength or the product ion with the third strongest ionic strength.

[0113] According to the measurement method described in paragraph 2, the measured value of creatinine is obtained based on the chromatogram of the product ion with the second strongest ion intensity or the product ion with the third strongest ion intensity among the product ions generated using ions derived from creatinine as precursor ions, thereby preventing the ion intensity from exceeding the detectable intensity of the device.

[0114] (Item 3) In the measurement method according to item 1 or 2, the mass-to-charge ratio of the product ions other than the product ion with the strongest ion intensity may be in the range of 70 or more and 90 or less.

[0115] According to the measurement method described in paragraph 3, a measurement value of creatinine is obtained based on a chromatogram of product ions, which are produced using ions derived from creatinine as precursor ions, and which have a mass-to-charge ratio in the range of 70 to 90.

[0116] (Item 4) In the measurement method according to any one of items 1 to 3, the mass-to-charge ratio of the product ions other than the product ion with the strongest ion intensity may be 86.01±0.5.

[0117] According to the measurement method described in paragraph 4, a measurement value of creatinine is obtained based on a chromatogram of a product ion having a mass-to-charge ratio of 86.01±0.5, among product ions produced using ions derived from creatinine as precursor ions.

[0118] (Item 5) In the measurement method according to any one of Items 1 to 4, the mass-to-charge ratio of the product ions other than the product ion with the strongest ion intensity may be 72.00±0.5.

[0119] According to the measurement method described in paragraph 5, a measurement value of creatinine is obtained based on a chromatogram of a product ion having a mass-to-charge ratio of 72.00±0.5, among product ions produced using ions derived from creatinine as precursor ions.

[0120] (Item 6) In the measurement method according to any one of Items 1 to 5, the molecule from which the product ions other than the product ion with the strongest ionic strength are derived has a molecular formula of C ３ H ８ N ３ It may be expressed as:

[0121] According to the measurement method described in item 6, among the product ions produced using ions derived from creatinine as precursor ions, there are ions having the molecular formula C ３ H ８ N ３ A measurement of creatinine is obtained based on a chromatogram of product ions derived from a molecule represented by:

[0122] (7) In the measurement method according to any one of items 1 to 6, the molecule from which the product ions other than the product ion with the strongest ionic strength are derived has a molecular formula of C ２ H ６ N ３ It may be expressed as:

[0123] According to the measurement method described in item 7, among the product ions produced using ions derived from creatinine as precursor ions, those having the molecular formula C ２ H ６ N ３ A measurement of creatinine is obtained based on a chromatogram of product ions derived from a molecule represented by:

[0124] (Item 8) In the measurement method described in any one of Items 1 to 7, the sample may be urine collected from a living body, and the method may further include a step of diluting the sample 2 to 10,000 times.

[0125] According to the measurement method described in item 8, it is possible to obtain creatinine-corrected values ​​of nucleosides in a sample obtained by diluting urine collected from a living body 2 to 10,000 times.

[0126] (Item 9) In the measurement method according to item 8, in the step of diluting the sample 2 to 10,000 times, the sample may be diluted 10 to 1,000 times.

[0127] According to the measurement method described in item 9, it is possible to obtain creatinine-corrected values ​​of nucleosides in a sample obtained by diluting urine collected from a living body 10 to 1000 times.

[0128] (Item 10) In the measurement method described in any one of Items 1 to 9, the high-performance liquid chromatograph tandem mass spectrometer includes two or more sets of quadrupole filters functioning as a mass spectrometer, and in the step of acquiring a chromatogram of the product ions, the resolution values ​​of the two or more sets of quadrupole filters when acquiring a chromatogram of the product ions derived from the creatinine may be equal to or less than the resolution values ​​of the two or more sets of quadrupole filters when acquiring a chromatogram of the product ions derived from the nucleoside, and the resolution value of at least one set of quadrupole filters out of the two or more sets of quadrupole filters when acquiring a chromatogram of the product ions derived from the creatinine may be smaller than the resolution value of the at least one set of quadrupole filters when acquiring a chromatogram of the product ions derived from the nucleoside.

[0129] According to the measurement method described in paragraph 10, the resolution value of the LC-MS / MS instrument when analyzing creatinine is smaller than the resolution value of the LC-MS / MS instrument when analyzing nucleosides. The smaller resolution value results in a lower ion intensity of the detected product ions. Therefore, the ion intensity of the product ions derived from creatinine can be prevented from exceeding the detectable ion intensity.

[0130] (Item 11) In the measurement method described in Item 10, the resolution value of at least one set of quadrupole filters among the two or more sets of quadrupole filters when acquiring a chromatogram of product ions derived from creatinine may be 0.5 Da or less.

[0131] According to the measurement method described in Item 11, by setting the resolution of the LC-MS / MS device when analyzing creatinine to 0.5 Da or less, the ion intensity of product ions derived from creatinine can be prevented from exceeding the detectable ion intensity.

[0132] (Item 12) In the measurement method according to any one of items 1 to 11, the nucleoside may include a chemically modified nucleoside.

[0133] According to the measurement method described in item 12, the creatinine-corrected value of a chemically modified nucleoside can be measured.

[0134] (Item 13) In the measurement method according to any one of items 1 to 12, the nucleoside is Y, D, mnm ５ s ２ U, m ３ U, m ５ U, m １ Y, m ３ Y, U, C, G, m ３ C, m ５ C, I, Cm, m ７ G, m １ G, m ２ G, m １ A, m ６ A, m １ I, Um, Gm, ac ４ C, Im, m ２ , ２ G, A, mcm ５ U, t ６ A, Am, ms ２ t ６ A, s ２ C, f ５ C, m ４ Cm, m ４ , ４ Cm, hm ５ C, mcmo ５ U, mcm ５ s２ U, ncm ５ s ２ U, acp ３ U, nm ５ U, tm ５ U may be selected from the group consisting of:

[0135] According to the measurement method described in paragraph 13, Y, D, mnm ５ s ２ U, m ３ U, m ５ U, m １ Y, m ３ Y, U, C, G, m ３ C, m ５ C, I, Cm, m ７ G, m １ G, m ２ G, m １ A, m ６ A, m １ I, Um, Gm, ac ４ C, Im, m ２ , ２ G, A, mcm ５ U, t ６ A, Am, ms ２ t ６ A, s ２ C, f ５ C, m ４ Cm, m ４ , ４ Cm, hm ５ C, mcmo ５ U, mcm ５ s ２ U, ncm ５ s ２ U, acp ３ U, nm ５ U, tm ５ The creatinine-corrected value of a nucleoside selected from the group consisting of U can be determined.

[0136] (Item 14) In one aspect, the program may be executed by a processor mounted on a computer, causing the computer to perform the following operations: accept data obtained by measuring a sample containing creatinine and a nucleoside using a high-performance liquid chromatography tandem mass spectrometer; obtain a measurement value of the nucleoside based on a chromatogram of product ions derived from the nucleoside; obtain a measurement value of the creatinine based on a chromatogram of product ions other than the product ion with the strongest ion intensity among the chromatograms of the product ions derived from the creatinine; and calculate a creatinine correction value for the nucleoside based on the measurement value of the nucleoside and the measurement value of the creatinine.

[0137] According to the program described in item 14, the creatinine-corrected value of the nucleoside in the sample can be determined based on the chromatogram obtained by LC-MS / MS analysis.

[0138] (Item 15) A high-performance liquid chromatograph tandem mass spectrometer in one aspect is a high-performance liquid chromatograph tandem mass spectrometer for measuring creatinine-corrected values ​​of nucleosides in a sample, the high-performance liquid chromatograph tandem mass spectrometer comprising a liquid chromatograph, a mass spectrometer that analyzes components separated by the liquid chromatograph, and a data processor that receives measurement data from the mass spectrometer, wherein the liquid chromatograph separates creatinine and the nucleosides in the sample in terms of time, and the mass spectrometer analyzes each of the ions derived from the creatinine and the nucleosides. A chromatogram of product ions may be generated as precursor ions, and the data processing unit may obtain a measurement value of the nucleoside based on a chromatogram of the product ions derived from the nucleoside, obtain a measurement value of the creatinine based on a chromatogram of a product ion other than the product ion with the strongest ionic intensity among the chromatograms of the product ions derived from the creatinine, and calculate a creatinine correction value for the nucleoside based on the measurement value of the nucleoside and the measurement value of the creatinine.

[0139] According to the high-performance liquid chromatography tandem mass spectrometer described in item 15, the creatinine-corrected values ​​of nucleosides in a sample can be measured in a single LC-MS / MS analysis without diluting one sample at two different dilution rates, thereby reducing the workload on the user and shortening the operation time.

[0140] The embodiments disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present disclosure is defined by the claims, not by the description of the above-described embodiments, and is intended to include all modifications within the meaning and scope of the claims. Furthermore, it is intended that each technique in the embodiments can be implemented alone or, if necessary, in combination with other techniques in the embodiments to the extent possible.

[0141] 1 Liquid chromatograph, 2 Mass spectrometry section, 3 Control section, 4 Display section, 5 Input section, 10 Mobile phase container, 11 Pump, 12 Injector, 13 Column, 20 Ionization chamber, 21 First intermediate chamber, 22 Second intermediate chamber, 23 Analysis chamber, 30 CPU, 31 Memory, 32 Communication I / F, 33 Display I / F, 34 Input I / F, 100 LC-MS / MS device, 201 Probe, 202 Capillary, 211, 221, 2321 Ion guide, 212 Skimmer, 231, 233 Quadrupole mass filter, 232 Collision cell, 234 Ion detector, 2311, 2331 Pre-rod electrode, 2312, 2332 Main rod electrode.

Claims

1. A method for measuring the creatinine correction value of a nucleoside in a sample using a high-performance liquid chromatography tandem mass spectrometer, comprising: a step of temporally separating the creatinine and the nucleoside contained in the sample using a liquid chromatograph; a step of obtaining a chromatogram of product ions using each of ions derived from the creatinine and the nucleoside as precursor ions; a step of obtaining a measurement value of the nucleoside based on the chromatogram of the product ions derived from the nucleoside; a step of obtaining a measurement value of the creatinine based on a chromatogram of a product ion other than the product ion with the strongest ion intensity among the chromatograms of the product ions derived from the creatinine; and a step of calculating the creatinine correction value of the nucleoside based on the measurement values ​​of the nucleoside and the creatinine.

2. The measurement method according to claim 1, wherein the product ion other than the product ion with the strongest ionic strength is either the product ion with the second strongest ionic strength or the product ion with the third strongest ionic strength.

3. A measurement method according to claim 1 or claim 2, wherein the mass-to-charge ratios of the product ions other than the product ion with the strongest ion intensity are in the range of 70 or more and 90 or less.

4. A measurement method according to claim 1 or claim 2, wherein the mass-to-charge ratio of the product ions other than the product ion with the strongest ion intensity is 86.01±0.

5.

5. A measurement method according to claim 1 or claim 2, wherein the mass-to-charge ratio of product ions other than the product ion with the strongest ion intensity is 72.00±0.

5.

6. The molecule from which the product ions other than the product ion with the strongest ionic strength are derived has the molecular formula C ３ H ８ N ３ The measurement method according to claim 1 or 2, wherein the measurement value is represented by the following formula:

7. The molecule from which the product ions other than the product ion with the strongest ionic strength are derived has the molecular formula C ２ H ６ N ３ The measurement method according to claim 1 or 2, wherein the measurement value is represented by the following formula:

8. The measurement method according to claim 1 or 2, wherein the sample is urine collected from a living body, and further comprising a step of diluting the sample 2 to 10,000 times.

9. The measurement method according to claim 8, wherein in the step of diluting the sample 2 to 10,000 times, the sample is diluted 10 to 1,000 times.

10. The measurement method of claim 1 or 2, wherein the high-performance liquid chromatograph tandem mass spectrometer includes two or more sets of quadrupole filters functioning as a mass spectrometer, and in the step of acquiring a chromatogram of the product ions, the resolution value of the two or more sets of quadrupole filters when acquiring a chromatogram of the product ions derived from the creatinine is equal to or less than the resolution value of the two or more sets of quadrupole filters when acquiring a chromatogram of the product ions derived from the nucleoside, and the resolution value of at least one set of quadrupole filters out of the two or more sets of quadrupole filters when acquiring a chromatogram of the product ions derived from the creatinine is smaller than the resolution value of the at least one set of quadrupole filters when acquiring a chromatogram of the product ions derived from the nucleoside.

11. The measurement method described in claim 10, wherein the resolution value of at least one set of quadrupole filters among the two or more sets of quadrupole filters when acquiring a chromatogram of product ions derived from creatinine is 0.5 Da or less.

12. The measurement method according to claim 1 or 2, wherein the nucleoside includes a chemically modified nucleoside.

13. The nucleoside is Y, D, mnm ５ s ２ U, m ３ U, m ５ U, m １ Y, m ３ Y, U, C, G, m ３ C, m ５ C, I, Cm, m ７ G, m １ G, m ２ G, m １ A, m ６ A, m １ I, Um, Gm, ac ４ C, Im, m ２ , ２ G, A, mcm ５ U, t ６ A, Am, ms ２ t ６ A, s ２ C, f ５ C, m ４ Cm, m ４ , ４ Cm, hm ５ C, mcmo ５ U, mcm ５ s ２ U, ncm ５ s ２ U, acp ３ U, nm ５ U, tm ５ The method according to claim 1 or 2, wherein the compound is selected from the group consisting of U.

14. A program that, when executed by a processor mounted on a computer, causes the computer to perform the following operations: accept data obtained by measuring a sample containing creatinine and a nucleoside using a high-performance liquid chromatography tandem mass spectrometer; obtain a measurement value of the nucleoside based on a chromatogram of product ions derived from the nucleoside; obtain a measurement value of the creatinine based on a chromatogram of product ions other than the product ion with the strongest ion intensity, among the chromatograms of product ions derived from the creatinine; and calculate a creatinine correction value for the nucleoside based on the measurement values ​​of the nucleoside and the creatinine.

15. A high-performance liquid chromatography tandem mass spectrometer for measuring creatinine-corrected values ​​of nucleosides in a sample, comprising: a liquid chromatograph; a mass spectrometer for analyzing components separated by the liquid chromatograph; and a data processor for receiving measurement data from the mass spectrometer, wherein the liquid chromatograph separates creatinine and the nucleosides in the sample in terms of time; the mass spectrometer generates a chromatogram of product ions using ions derived from the creatinine and the nucleoside as precursor ions, respectively; and the data processor obtains a measurement value of the nucleoside based on the chromatogram of the product ions derived from the nucleoside; and obtains a measurement value of the creatinine based on a chromatogram of product ions derived from the creatinine, other than the product ion with the strongest ion intensity, from the chromatogram of the product ions derived from the creatinine. A high-performance liquid chromatography tandem mass spectrometer that calculates a creatinine-corrected value of the nucleoside based on the measured value of the nucleoside and the measured value of creatinine.

Images