Leucine and isoleucine analysis method and leucine and isoleucine analysis device

Abstract

To provide an analysis method with which it is possible to identify leucine and isoleucine in MRM measurement such as newborn infant mass screening.SOLUTION: Provided is a method for analyzing leucine and isoleucine by using a tandem mass spectrometer. The method includes: a measurement step in which a first MRM measurement regarding a multiple reaction monitoring transition where m / z is at least one of 132>69, 132>57, 132>56, 132>43, 132>41, 132>39, 132>30, 132>27 and a second MRM measurement regarding an MRM transition where m / z is 132>86 are carried out on an analyte, and information on strength of leucine- and isoleucine-derived ions is acquired; and a processing step in which leucine and isoleucine in the analyte are identified or the existence ratio of these is determined on the basis of a confirmed ion ratio representing the ratio of the ion signal strength obtained by the first MRM measurement to the signal strength obtained by the second MRM measurement.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method and an apparatus for analyzing leucine and isoleucine. [Background technology]

[0002] Newborn screening tests are widely carried out to detect and treat newborns with congenital metabolic disorders at an early stage. Recently, with the rapid development of mass spectrometry technology, newborn mass screening tests using tandem mass spectrometers have become widespread, and are proving to be extremely effective in the early detection of newborns with congenital metabolic disorders (see Non-Patent Document 1).

[0003] One such newborn mass screening test is the analysis of leucine and isoleucine to diagnose maple syrup urine disease. In general, quantitative analysis of leucine and isoleucine is performed by combining precursor ions derived from leucine and isoleucine with characteristic product ions of m / z 132>86 using multiple reaction monitoring (MRM) measurement with a tandem mass spectrometer. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Yosuke Shigematsu, "The Reality of Newborn Mass Screening by Mass Spectrometry", Journal of the Mass Spectrometry Society of Japan, Vol. 64, No. 4, 2016, pp. 127-131 Summary of the Invention [Problem to be solved by the invention]

[0005] Conventionally, in newborn mass screening tests, since it is necessary to analyze a large number of specimens (analyte samples) quickly, measurements are performed using flow injection analysis (FIA), in which each sample set in an autosampler is sequentially introduced into a mass spectrometer together with the mobile phase without passing through a liquid chromatograph (LC) column. In flow injection analysis, each sample is introduced into a mass spectrometer without component separation using a column, so various substances contained in the sample are simultaneously subjected to MRM measurement.

[0006] However, since leucine and isoleucine are structural isomers and have the same mass number, they cannot be separated and quantified by MRM measurement using the FIA ​​method. Therefore, maple syrup urine disease is diagnosed by a primary mass screening test using MRM measurement using the FIA ​​method and a secondary mass screening test using mass spectrometry (on-column analysis) involving separation of components by a column. Specifically, in the primary test, the combined value of leucine and isoleucine is compared with a predetermined cutoff value, and if the combined value is below the cutoff value, it is determined to be negative (i.e., not having maple syrup urine disease), and if it is equal to or greater than the cutoff value, a secondary test is performed.

[0007] In the secondary test, leucine, isoleucine, and alloisoleucine, which is an optical isomer of isoleucine, are each separated and quantified, and the quantitative value of leucine is compared with a preset cutoff value. If the quantitative value of leucine is below the cutoff value, it is determined to be negative. If it is above the cutoff value, it is further determined whether alloisoleucine is detected, and if alloisoleucine is detected, it is determined that "further examination is required," and if alloisoleucine is not detected, it is determined that "retest is required."

[0008] Thus, a two-stage mass screening test is necessary to diagnose maple syrup urine disease. Generally, newborn screening tests involve taking blood samples from newborns to prepare samples, so the second test imposes a large physical burden on newborns. In addition, the second test imposes a large psychological burden on parents of newborns who are not determined to be negative in the first test. Furthermore, the first and second tests are often performed at different facilities, and it is time-consuming to send samples to the facility where the second test is performed. Therefore, it is desirable to separate and detect leucine and isoleucine from the first test stage and reduce the number of samples to be transferred to the second test.

[0009] An object of the present invention is to provide an analytical method capable of discriminating between leucine and isoleucine in MRM measurements such as newborn mass screening. [Means for solving the problem]

[0010] The method for analyzing leucine and isoleucine of the present invention, which has been made to solve the above problems, comprises: A method for analyzing leucine and isoleucine using a tandem mass spectrometer, comprising the steps of: a measuring step of performing a first multiple reaction monitoring (MRM) measurement for at least one of m / z 132>69, m / z 132>57, m / z 132>56, m / z 132>43, m / z 132>41, m / z 132>39, m / z 132>30, and m / z 132>27 on a sample, and a second MRM measurement for an MRM transition of m / z 132>86 to obtain intensity information of ions derived from leucine and isoleucine; a processing step of discriminating between leucine and isoleucine in the sample or determining the abundance ratio of leucine and isoleucine in the sample based on a confirmation ion ratio which is a ratio between an ion signal intensity obtained by the first MRM measurement and a signal intensity obtained by the second MRM measurement; has.

[0011] In order to solve the above problems, the present invention provides an analytical device for leucine and isoleucine, a measurement unit which is a tandem mass spectrometer that performs a first multiple reaction monitoring (MRM) measurement on at least one of m / z 132>69, m / z 132>57, m / z 132>56, m / z 132>43, m / z 132>41, m / z 132>39, m / z 132>30, and m / z 132>27 on a sample, and a second MRM measurement on an MRM transition of m / z 132>86; a processing unit that distinguishes between leucine and isoleucine in the sample or determines the abundance ratio of leucine and isoleucine in the sample based on a confirmation ion ratio that is a ratio between a signal intensity obtained by the first MRM measurement in the measurement unit and a signal intensity obtained by the second MRM measurement; Equipped with. Effect of the Invention

[0012] According to the leucine and isoleucine analysis method and leucine and isoleucine analysis device of the present invention, even if a sample contains leucine or isoleucine, if the amount of leucine and isoleucine is the same, the MRM transition of the quantification ion (m / z 132>86) which shows almost the same ion intensity, and the MRM transition of the confirmation ion (m / z 132>69, m / z 132>57, m / z 132>56, m / z 132>43, m / z 132>41, m / z 132>39, m / z 132>30, m / z 132>27) which shows different ion intensities for leucine and isoleucine are used to distinguish between leucine and isoleucine or to determine the ratio of leucine and isoleucine. Therefore, it is possible to distinguish between leucine and isoleucine without separating them using a liquid chromatograph column. [Brief description of the drawings]

[0013] [Figure 1] 1 is a configuration diagram of a main part of an embodiment of a liquid chromatograph mass spectrometer according to the present invention; [Diagram 2]Chromatogram waveforms of the MRM transitions m / z 132.10>86.10 and m / z 132.10>69.20 for a sample containing only isoleucine (Leu:Ile=0:100). [Diagram 3] Chromatogram waveforms of the MRM transitions m / z 132.10>86.10 and m / z 132.10>69.20 for a sample containing only leucine (Leu:Ile=100:0). [Figure 4] Graphs (a) to (h) show the collision energy dependence of the confirmatory ion ratio for leucine and isoleucine, where the quantification ion is m / z 132.10>86.10 and the confirmatory ions are m / z 132.10>69.20, m / z 132.10>57.10, m / z 132.10>56.10, m / z 132.10>43.10, m / z 132.10>41.10, m / z 132.10>39.10, m / z 132.10>30.20, and m / z 132.10>27.20. [Diagram 5] Figures (a)-(h) show the relationship between the percentage of leucine in a sample and the confirming ion ratio, where the quantification ion is m / z 132.10>86.10 and the confirming ions are m / z 132.10>69.20, m / z 132.10>57.10, m / z 132.10>56.10, m / z 132.10>43.10, m / z 132.10>41.10, m / z 132.10>39.10, m / z 132.10>30.20, and m / z 132.10>27.20. [Figure 6] FIG. 1 shows analytical conditions for MRM measurement. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Hereinafter, one embodiment of the method for analyzing leucine and isoleucine according to the present invention will be described with reference to the accompanying drawings. The method for analyzing leucine and isoleucine according to the present embodiment is used in a newborn mass screening test for diagnosing maple syrup urine disease, for example.

[0015] 1 is a diagram showing the configuration of the main components of a liquid chromatograph mass spectrometer used in this embodiment. The liquid chromatograph mass spectrometer of this embodiment includes a liquid chromatograph 1, a mass spectrometer 2, a data processing unit 3, an analysis control unit 4, a central control unit 5, an input unit 6, and a display unit 7.

[0016] The liquid chromatograph 1 includes a mobile phase container 11 in which a mobile phase is stored, a pump 12 that draws in the mobile phase (solvent) and delivers it at a constant flow rate, an injector 13 that injects a liquid sample into the mobile phase, and an autosampler (not shown) connected to the injector 13. Although a liquid chromatograph has a column for separating components in a sample, in this embodiment, a column is not used, and the liquid sample is introduced into the mass spectrometer 2 together with the mobile phase by a flow injection analysis (FIA) method. In the case of a general newborn screening test, as described in Non-Patent Document 1, the sample is extracted and prepared from blood on a filter paper. However, the sample is not limited to this, and may be prepared from urine or other body fluids.

[0017] The mass spectrometer 2 is a triple quadrupole mass spectrometer, which is a type of tandem mass spectrometer, and includes an ionization chamber 201 maintained at approximately atmospheric pressure, and a first intermediate vacuum chamber 202, a second intermediate vacuum chamber 203, and a high vacuum chamber 204, each of which is evacuated by a vacuum pump (not shown). The ionization chamber 201 is provided with an ESI sprayer 21 that performs ionization by an ElectroSpray Ionization (ESI) method, and the ionization chamber 201 and the first intermediate vacuum chamber 202 at the next stage are connected by a desolvation tube 22. An ion guide 23 that focuses and transports ions is disposed in the first intermediate vacuum chamber 202, and the first intermediate vacuum chamber 202 and the second intermediate vacuum chamber 203 at the next stage are connected through a small hole formed at the top of a skimmer 24. A multipole ion guide 25 that focuses and transports ions is also disposed in the second intermediate vacuum chamber 203.

[0018] In the high vacuum chamber 204, a front quadrupole mass filter 26, a collision cell 27, a rear quadrupole mass filter 28, and an ion detector 29 are arranged along the ion flow. A quadrupole ion guide is arranged inside the collision cell 27. The front quadrupole mass filter 26 and the rear quadrupole mass filter 28 each have the function of selectively passing ions having a predetermined mass-to-charge ratio. An inert collision-induced dissociation (CID) gas such as argon is introduced into the collision cell 27 from the outside, and the collision cell 27 has the function of dissociating the introduced ions by contacting them with the CID gas to generate product ions.

[0019] The data processing unit 3 receives detection data from the ion detector 29 and performs processing based on the data, and includes as its functional blocks a data collecting unit 31, a peak intensity calculating unit 32, and a component determining unit 33. The analysis control unit 4 controls the operations of the liquid chromatograph 1 and the measuring unit 2 in accordance with an analysis condition file stored in the analysis condition storage unit 41. The central control unit 5 mainly executes overall control and user interface via the input unit 6, display unit 7, etc.

[0020] Generally, the data processing unit 3, analysis control unit 4, and central control unit 5 are actually computers called personal computers or more powerful workstations, and the functions of each of the above-mentioned functional blocks can be realized by running dedicated software (computer programs) pre-installed on the computer.

[0021] [Outline of MRM measurement operation] 1, when analyzing leucine and isoleucine, MRM measurements for predetermined MRM transitions are repeatedly carried out by the mass spectrometer 2. The operation during this MRM measurement will be briefly described below.

[0022] In the liquid chromatograph 1, the liquid delivery pump 12 draws the mobile phase from the mobile phase container 11 and delivers it to the injector 13 at a substantially constant flow rate. The injector 13 injects a predetermined amount of sample (specimen) into the mobile phase at a predetermined timing. The sample rides along the flow of the mobile phase and reaches the ESI spray 21 of the measurement unit 2. The sample diffuses in the forward and backward directions in the flow path until it reaches the ESI spray 21. Therefore, the amount of sample introduced into the ESI spray 21 is very small at first, but increases rapidly, and when it exceeds a maximum point, it rapidly decreases to zero. In other words, the concentration distribution of the sample exhibits a peak shape that is close to a Gaussian distribution over time.

[0023] In the ESI spray 21, the sample is sprayed into the ionization chamber 201 as fine charged droplets. The charged droplets come into contact with residual gas molecules and split, and in the process of the solvent in the droplets evaporating, the compound molecules in the sample are ionized. The generated ions are sent to the first intermediate vacuum chamber 202 through the desolvation tube 22, and further to the high vacuum chamber 204 through the ion guide 23, the small hole of the skimmer 24, and the multipole ion guide 25. The ions derived from the sample are introduced into the front quadrupole mass filter 26, and only ions having a predetermined mass-to-charge ratio corresponding to the voltage applied to the electrodes constituting the front quadrupole mass filter 26 selectively pass through as precursor ions. The precursor ions entering the collision cell 27 come into contact with the CID gas and are dissociated, generating various product ions.

[0024] The various product ions generated are introduced into a post-quadrupole mass filter 28, and only product ions having a predetermined mass-to-charge ratio corresponding to the voltage applied to the electrodes constituting the post-quadrupole mass filter 28 selectively pass through and reach an ion detector 29. The ion detector 29 generates a detection signal according to the amount of incident ions, and the detection data digitized by an analog-to-digital converter (not shown) is input to the data processing unit 3.

[0025] The analysis controller 4 controls the mass spectrometer 2 so that voltages corresponding to the target MRM transition are applied to the electrodes of the front quadrupole mass filter 26 and the rear quadrupole mass filter 28. This provides detection data indicating the ion intensity of ions corresponding to a specific MRM transition among ions derived from compounds contained in the sample, in other words, product ions having a specific mass-to-charge ratio generated by dissociation of a precursor ion having a specific mass-to-charge ratio.

[0026] [Principle of the analytical method for leucine and isoleucine] Next, the principle of the characteristic analytical method for leucine and isoleucine of this embodiment will be described. In the mass spectrometer 2, precursor ions are dissociated by CID in the collision cell 27, and the manner of dissociation differs depending on the kinetic energy of the precursor ion. This kinetic energy is the collision energy (CE). The collision energy is determined by the DC potential difference between the inlet end of the collision cell 27 and its upstream (the upstream quadrupole mass filter 26 in FIG. 1, but it may be another ion optical element such as an ion lens), and therefore the collision energy is usually indicated by this potential difference. Therefore, the collision energy can be adjusted by a DC bias voltage applied to either or both of the upstream quadrupole mass filter 26 and the upstream ion optical element.

[0027] As described above, when the collision energy is changed, the dissociation mode of the precursor ion changes, and the generation pattern of multiple types of product ions changes. In order to increase the detection sensitivity, it is desirable to select a product ion (i.e., an MRM transition) with the highest ion intensity. Therefore, in the conventional analysis of leucine and isoleucine, m / z 132.10>86.10, which has the highest ion intensity, has been used as the MRM transition. However, as already mentioned, leucine and isoleucine cannot be distinguished from each other at m / z 132.10>86.10. Therefore, the present inventors have experimentally and thoroughly investigated the relationship between the collision energy and the ion intensity for various MRM transitions related to leucine and isoleucine.

[0028] The above experiment was carried out according to the following procedure. A 100μg / L aqueous solution of leucine and isoleucine was prepared from each standard sample using ultrapure water, and this was used as the sample. A Shimadzu LCMS-8060 mass spectrometer was used, and product ion scan measurements were first performed on the common precursor ion (m / z 132.10) of leucine and isoleucine to search for product ions specific to each compound. As a result, no product ions specific to each compound were observed, and nine types of representative product ions with relatively high peak intensities were found to exist: m / z 86.10, m / z 69.20, m / z 57.10, m / z 56.10, m / z 43.10, m / z 41.10, m / z 39.10, m / z 30.20, and m / z 27.20.

[0029] The peak intensities of these nine product ions were measured when the collision energy setting was changed in 5 V steps in the range of -100 V to -5 V. As a result, in the MRM transition (m / z 132.10>86.10) conventionally used for the quantification of leucine and isoleucine, almost no difference was observed in the peak intensity patterns of leucine and isoleucine with respect to changes in collision energy. In contrast, the remaining eight MRM transitions (m / z 132.10>69.20, m / z 132.10>57.10, m / z 132.10>56.10, m / z 132.10>43.10, m / z 132.10>41.10, m / z 132.10>39.10, m / z 132.10>30.20, m / z 132.10>27.20) showed differences in the peak intensity patterns between leucine and isoleucine.

[0030] For example, Figures 2 and 3 are chromatogram waveform diagrams of the MRM transitions of m / z 132.10>86.10 and m / z 132.10>69.20 for a sample containing only isoleucine (Leu:Ile=0:100) and a sample containing only leucine (Leu:Ile=100:0), respectively. The component concentrations in both cases are 100 μg / L. As can be seen from Figures 2 and 3, the peak intensity patterns of m / z 132.10>86.10 for the sample containing leucine and the sample containing isoleucine were similar, and the peak intensity patterns of m / z 132.10>69.20 were different. In both the sample containing leucine and the sample containing isoleucine, the peak intensity of m / z 132.10>69.20 was smaller than that of m / z 132.10>86.10. These experimental findings indicate that it is possible to distinguish leucine from isoleucine by utilizing the difference in the peak intensity patterns of eight types of MRM transitions (m / z 132.10>69.20, m / z 132.10>57.10, m / z 132.10>56.10, m / z 132.10>43.10, m / z 132.10>41.10, m / z 132.10>39.10, m / z 132.10>30.20, m / z 132.10>27.20) relative to the peak intensity pattern of the MRM transition (m / z 132.10>86.10).

[0031] Therefore, the peak signal intensity A1 in the MRM transition of m / z 132.10>86.10 measured with the collision energy set at -10 V, and the peak signal intensity A2 in the above eight types of MRM transitions when the collision energy set was changed in the range of -100 V to -5 V in 5 V steps were measured, and the ratio of these (A2 / A1) was multiplied by 100 to calculate the value. Figure 4 shows the results.

[0032] In addition, if the ions detected in the m / z 132.10>86.10 MRM transition are called quantitative ions, and the ions detected in the other eight MRM transitions (m / z 132.10>69.20, m / z 132.10>57.10, m / z 132.10>56.10, m / z 132.10>43.10, m / z 132.10>41.10, m / z 132.10>39.10, m / z 132.10>30.20, m / z 132.10>27.20) are called confirmatory ions, then the intensity ratio between them is the so-called confirmatory ion ratio. Hereinafter, the above value ((A2 / A1)×100) is called the confirmatory ion ratio.

[0033] Figure 4 shows the collision energy dependence of the confirmation ion ratio for leucine and isoleucine in eight types of MRM transitions. In these figures, the horizontal axis is the collision energy, and the vertical axis is the confirmation ion ratio (A2 / A1 × 100). In addition, in the same figure, the solid line shows the confirmation ion ratio of leucine, and the dashed line shows the confirmation ion ratio of isoleucine. As can be seen from these figures, in all eight types of MRM transitions, the collision energy dependence patterns of the confirmation ion ratio were different between leucine and isoleucine. In particular, in the m / z 132.10>39.10 range, a clear difference was observed in the confirmation ion ratio over almost the entire range of collision energy (Figure 4(f)). In addition, in the m / z 132.10>41.10 range, a clear difference was observed in the confirmation ion ratio depending on the collision energy value (Figure 4(e)). Other than these, m / z 132.10>69.20, m / z 132.10>57.10, m / z 132.10>56.10, m / z 132.10>43.10, m / z 132.10>30.20, and m / z 132.10>27.200, the confirmed ion ratios showed significant differences depending on the collision energy value (Figures 4(a)-(d), (g), and (h)). For example, at m / z 132.10>69.20, the confirmed ion ratios of leucine and isoleucine peaked (maximum) when the collision energy was -20 V, and the difference between the confirmed ion ratios of the two was the largest at this peak (Figure 4(a)). Thus, when the MRM transitions were m / z 132.10>69.20, m / z 132.10>57.10, m / z 132.10>56.10, m / z 132.10>43.10, m / z 132.10>30.20, and m / z 132.10>27.200, there existed optimal collision energy values ​​at which the difference in the confirmatory ion ratios between leucine and isoleucine was significantly large.

[0034] 5(a)-(h) are diagrams showing the relationship between the ratio of leucine to leucine and isoleucine in a sample and the confirmation ion ratio, obtained from the actual measurement results, when eight types of MRM transitions are used. FIG. 6 shows the conditions of the MRM measurement, and the above-mentioned optimal value is used as the collision energy when the MRM transitions are m / z 132.10>69.20, m / z 132.10>57.10, m / z 132.10>56.10, m / z 132.10>43.10, m / z 132.10>30.20, and m / z 132.10>27.200. For m / z 132.10>41.10 and m / z 132.10>39.10, the collision energy value where the difference between the confirmation ion ratios of leucine and isoleucine is large is used near the maximum. As shown in Fig. 5(a) to (h), the proportion of leucine in the sample and the confirmation ion ratio are related by a linear equation, that is, a straight line. Therefore, in the device of the above embodiment, the above relationship obtained in advance by experiment is stored in the component determining section 33 as a calibration curve, and the proportion of leucine or isoleucine is estimated from the confirmation ion ratio obtained by actual measurement of the sample using the calibration curve. Note that it is sufficient that any one of the relational expressions in the case where eight types of MRM transitions are used is stored as a calibration curve in the component determining section 33. However, it is also possible to store a relational expression in the case where two or more types of MRM transitions are used as a calibration curve in the component determining section 33, and use the average value, median value, etc. of the values ​​estimated using each of these multiple calibration curves as the proportion of leucine or isoleucine.

[0035] [Specific example of how to distinguish between leucine and isoleucine] First, in the liquid chromatograph mass spectrometer of the above embodiment, the measurement unit 2 repeatedly performs MRM measurement at the MRM transition of a quantification ion and MRM measurement at the MRM transition of a predetermined confirmation ion on the same unknown sample under the control of the analysis control unit 4. The MRM measurement is repeatedly performed until a predetermined time has elapsed from the time the sample is injected by the injector 13. As a result, data constituting a chromatogram waveform showing the time course of ion intensity data, which is the result of each MRM measurement, is stored in the data collection unit 31.

[0036] The peak intensity calculation unit 32 creates chromatogram waveforms corresponding to the nine types of MRM transitions from the data stored in the data collection unit 31, and performs peak detection on the waveforms to obtain peak top values ​​(peak intensity values). Note that the peak intensity values ​​are used here as signal intensities for the computational processing described below, but instead of the peak intensity values, the area value of the peak from the peak start point to the peak end point may be calculated and used as the signal intensity.

[0037] Next, the component determining unit 33 calculates a confirmation ion ratio (A2 / A1×100) which is a ratio between a signal intensity A1 in an MRM transition (m / z 132.10>86.10) corresponding to a quantitative ion and a signal intensity A2 in an MRM transition corresponding to a confirmation ion. Then, the proportion of leucine or isoleucine is estimated from this confirmation ion ratio and a calibration curve stored in the component determining unit 33.

[0038] [Aspects] It will be apparent to those skilled in the art that the above-described exemplary embodiments are illustrative of the following aspects.

[0039] (Item 1) The method for analyzing leucine and isoleucine according to the present invention comprises: A method for analyzing leucine and isoleucine using a tandem mass spectrometer, comprising the steps of: a measuring step of performing a first multiple reaction monitoring (MRM) measurement for at least one of m / z 132>69, m / z 132>57, m / z 132>56, m / z 132>43, m / z 132>41, m / z 132>39, m / z 132>30, and m / z 132>27 on a sample, and a second MRM measurement for an MRM transition of m / z 132>86 to obtain intensity information of ions derived from leucine and isoleucine; a processing step of discriminating between leucine and isoleucine in the sample or determining the abundance ratio of leucine and isoleucine in the sample based on a confirmation ion ratio which is a ratio between an ion signal intensity obtained by the first MRM measurement and a signal intensity obtained by the second MRM measurement; has.

[0040] (Item 9) The leucine and isoleucine analyzing device according to the present invention comprises: a measurement unit which is a tandem mass spectrometer that performs a first multiple reaction monitoring (MRM) measurement on at least one of m / z 132>69, m / z 132>57, m / z 132>56, m / z 132>43, m / z 132>41, m / z 132>39, m / z 132>30, and m / z 132>27 on a sample, and a second MRM measurement on an MRM transition of m / z 132>86; a processing unit that distinguishes between leucine and isoleucine in the sample or determines the abundance ratio of leucine and isoleucine in the sample based on a confirmation ion ratio that is a ratio between a signal intensity obtained by the first MRM measurement in the measurement unit and a signal intensity obtained by the second MRM measurement; Equipped with.

[0041] According to the method for analyzing leucine and isoleucine described in item 1 or the analyzer for leucine and isoleucine described in item 9, regardless of whether a sample contains leucine or isoleucine in the same amount, it is possible to distinguish between leucine and isoleucine in the sample or to determine the ratio of leucine and isoleucine present in the sample based on the confirmation ion ratio, which is the ratio between the MRM transition of a quantification ion (m / z 132>86) that shows approximately the same ion intensity when the amounts of leucine and isoleucine are the same, and the MRM transitions of confirmation ions (m / z 132>69, m / z 132>57, m / z 132>56, m / z 132>43, m / z 132>41, m / z 132>39, m / z 132>30, m / z 132>27) that show different ion intensities for leucine and isoleucine.

[0042] (2) A method for analyzing leucine and isoleucine according to (1), comprising: In the measuring step, the first MRM measurement and the second MRM measurement are performed while changing collision energy to obtain intensity information of ions derived from leucine and isoleucine; The processing step may distinguish between leucine and isoleucine based on the collision energy dependence of the confirmatory ion ratio.

[0043] According to the method for analyzing leucine and isoleucine described in item 2, even if there is no difference in the confirmation ion ratio between leucine and isoleucine at a certain collision energy, leucine and isoleucine can be distinguished from each other.

[0044] (Item 3) A method for analyzing leucine and isoleucine according to item 1, comprising: The processing step may estimate the abundance ratio of leucine or isoleucine contained in the target specimen by utilizing information indicating a relationship between a confirmation ion ratio and the abundance ratio of leucine or isoleucine in the specimen, which information has been obtained in advance.

[0045] According to the method for analyzing leucine and isoleucine described in item 3, it is possible not only to distinguish between leucine and isoleucine but also to estimate the ratio of the two.

[0046] (Item 4) A method for analyzing leucine and isoleucine according to any one of items 1 to 3, The transition of the first MRM measurement may be any one of m / z 132>69, m / z 132>57, m / z 132>56, m / z 132>43, m / z 132>30, m / z 132>27.

[0047] According to the method for analyzing leucine and isoleucine described in item 4, there exists a range of collision energies in which the difference between the confirmation ion ratio for leucine and the confirmation ion ratio for isoleucine becomes large. Therefore, by carrying out the first and second MRM measurements while changing the collision energy within this range and using the obtained confirmation ion ratio, leucine and isoleucine can be more reliably distinguished from each other.

[0048] (Item 5) A method for analyzing leucine and isoleucine according to any one of items 1 to 3, The transition of the first MRM measurement may be m / z 132>41 or m / z 132>39.

[0049] According to the analytical method for leucine and isoleucine described in paragraph 5, peaks for both leucine and isoleucine are detected with sufficient intensity, and there is a range of collision energies in which a clear difference is observed between the confirmation ion ratio for leucine and the confirmation ion ratio for isoleucine. Therefore, by carrying out the first and second MRM measurements while changing the collision energy within this range, and using the obtained confirmation ion ratio, it is possible to reliably distinguish between leucine and isoleucine even if the sample contains impurities that inhibit peak detection.

[0050] (Item 6) A method for analyzing leucine and isoleucine according to any one of items 1 to 5, In the measuring step, the first MRM measurement and the second MRM measurement are repeatedly performed, In the processing step, the peak top value or peak area value of the peak showing the change over time in ion intensity derived from leucine and isoleucine may be used as the measurement result.

[0051] According to the method for analyzing leucine and isoleucine described in item 6, rapid processing is possible and screening efficiency can be improved by using the peak height (peak top value) as the measurement result. On the other hand, since the peak area value is generally more quantitative than the peak height, more accurate discrimination and determination can be performed by using the peak area value as the measurement result.

[0052] (Item 7) A method for analyzing leucine and isoleucine according to any one of items 1 to 6, The sample may be introduced into the tandem mass spectrometer using flow injection analysis.

[0053] According to the method for analyzing leucine and isoleucine described in item 7, since component separation using a column is not required, the measurement time for one sample can be shortened, and the method is suitable for screening tests that require rapidity.

[0054] (Item 8) A method for analyzing leucine and isoleucine according to any one of items 1 to 6, Components contained in a specimen may be separated by liquid chromatography and introduced into the tandem mass spectrometer.

[0055] According to the method for analyzing leucine and isoleucine described in Section 8, component separation using a column can also be utilized, thereby making it possible to further improve the accuracy of identification and judgment compared to the case where only the measurement results of specific MRM transitions are used. [Explanation of symbols]

[0056] 1...Sample introduction section 11...Mobile phase container 12...Liquid delivery pump 13...Injector 2…Measuring part 201…Ionization chamber 202…First intermediate vacuum chamber 203…Second intermediate vacuum chamber 204…High vacuum chamber 21…ESI Spray 22...Desolvation tube 23…Ion Guide 24…Skimmer 25...Multipole ion guide 26...Pre-quadrupole mass filter 27…Collision cell 28...Post-quadrupole mass filter 29...Ion detector 3. Data processing section 31…Data collection section 32...Peak intensity calculation section 33...component determination section 4. Analysis control section 41…Analysis condition storage unit 5...Central control unit 6. Input section 7…Display section

Claims

1. A method for analyzing leucine and isoleucine using a tandem mass spectrometer, A measurement step to obtain intensity information of ions derived from leucine and isoleucine by performing a first MRM measurement on a sample for at least one multiple reaction monitoring (MRM) transition among m / z 132>69, m / z 132>57, m / z 132>56, m / z 132>43, m / z 132>41, m / z 132>39, m / z 132>30, and m / z 132>27, and a second MRM measurement on an MRM transition at m / z 132>86, A processing step of determining whether the sample contains leucine or isoleucine, or determining the relative abundance of leucine and isoleucine in the sample, based on the confirmation ion ratio, which is the ratio of the signal intensity obtained by the first MRM measurement to the signal intensity obtained by the second MRM measurement, A method for analyzing leucine and isoleucine having [specific properties].

2. In the measurement step, the first MRM measurement and the second MRM measurement are performed while changing the collision energy to obtain intensity information of ions derived from leucine and isoleucine. The method for analyzing leucine and isoleucine according to claim 1, wherein the processing step identifies whether the sample contains leucine or isoleucine based on the collision energy dependence of the confirmation ion ratio.

3. The method for analyzing leucine and isoleucine according to claim 1, wherein the processing step involves estimating the proportion of leucine or isoleucine contained in a target sample by utilizing information obtained in advance that shows the relationship between the confirmation ion ratio and the proportion of leucine or isoleucine present in the sample.

4. The method for analyzing leucine and isoleucine according to claim 1, wherein the transition of the first MRM measurement is one of the following: m / z 132>69, m / z 132>57, m / z 132>56, m / z 132>43, m / z 132>30, and m / z 132>27.

5. The method for analyzing leucine and isoleucine according to claim 1, wherein the transition of the first MRM measurement is m / z 132>41 or m / z 132>39.

6. In the measurement step described above, the first MRM measurement and the second MRM measurement are repeatedly performed. The method for analyzing leucine and isoleucine according to claim 1, wherein in the processing step, the intensity value of the peak top or the peak area value of the peak showing the temporal change in ionic intensity derived from leucine and isoleucine is used as the measurement result.

7. The analytical method according to claim 1, wherein a sample is introduced into the tandem mass spectrometer using a flow injection analysis method.

8. The analytical method according to claim 1, wherein components contained in a sample are separated by liquid chromatography and introduced into the tandem mass spectrometer.

9. The measuring unit is a tandem mass spectrometer that performs a first MRM measurement on a sample for at least one multiple reaction monitoring (MRM) transition among m / z 132>69, m / z 132>57, m / z 132>56, m / z 132>43, m / z 132>41, m / z 132>39, m / z 132>30, and m / z 132>27, and a second MRM measurement on an MRM transition at m / z 132>86. A processing unit that identifies whether the sample contains leucine or isoleucine, or determines the relative abundance of leucine and isoleucine in the sample, based on a confirmation ion ratio which is the ratio of the signal intensity obtained by the first MRM measurement in the measurement unit to the signal intensity obtained by the second MRM measurement, A leucine and isoleucine analyzer equipped with [specific features / equipment].

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