Method for analyzing leucine and isoleucine, and device for analyzing leucine and isoleucine

Abstract

The invention provides a method for analyzing leucine and isoleucine, and a device for analyzing leucine and isoleucine. And leucine and isoleucine can be identified in MRM (Magnetic Resonance Mirror) determination such as newborn mass spectrum screening. The analysis method includes a measurement step of performing a first MRM measurement of at least one multi-reaction monitoring transition 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, m / z 132 > 27, and a second MRM measurement of an MRM transition of m / z 132 > 86 on a specimen to acquire intensity information of ions derived from leucine and isoleucine; and a processing step for identifying whether the specimen contains leucine or isoleucine or determining the presence ratio of leucine and isoleucine in the specimen on the basis of a confirmation ion ratio, which is the ratio of the signal intensity of the first MRM measurement to the signal intensity of the second MRM measurement.

Description

Technical Field

[0001] The present invention relates to a method and device for analyzing leucine and isoleucine. Background Art

[0002] Neonatal screening is widely implemented to detect and treat inborn errors of metabolism in newborns at an early stage. In recent years, with the rapid development of mass spectrometry technology, neonatal mass spectrometry screening using tandem mass spectrometry devices has also become popular and is very effective for the early detection of inborn errors of metabolism in newborns (see Non-Patent Document 1).

[0003] As one of such newborn mass spectrometry screening, there is the analysis of leucine and isoleucine for diagnosing maple syrup urine disease. In general, the 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 by multiple reaction monitoring (MRM) measurement using a tandem mass spectrometer to perform quantitative analysis.

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-patent literature 1: Shigematsu Yosuke, "Mass Analysis Method for the New Generation of Mass Spectrometry", Journal of the Mass Spectrometry Society of Japan, Vol.64, No.4, 2016, pp.127-131 Summary of the invention

[0007] Problem that the invention aims to solve

[0008] In the past, in mass spectrometry screening of newborns, a large number of test specimens (analyzed samples) needed to be analyzed quickly, so each sample set in an automatic sampler was sequentially introduced into a mass spectrometer together with a mobile phase without passing through a chromatographic column of a liquid chromatograph (LC) using a flow injection analysis (FIA) method for measurement. In the flow injection analysis method, each sample is introduced into a mass spectrometer without performing component separation based on a chromatographic column, so various substances contained in the sample are used simultaneously in the MRM measurement.

[0009] However, leucine and isoleucine are structural isomers and have the same mass number, so they cannot be separated and quantified in the MRM measurement based on the FIA ​​method. Therefore, maple syrup urine disease is diagnosed by primary mass spectrometry screening using MRM measurement based on the FIA ​​method and secondary mass spectrometry screening using mass spectrometry analysis (on-column analysis) with component separation based on a chromatographic column. Specifically, in the primary test, the total value of the quantitative value of leucine and the quantitative value of isoleucine is compared with a predetermined critical value. If the total value is lower than the critical value, it is determined to be negative (that is, not maple syrup urine disease), and if the total value is higher than the critical value, a secondary test is performed.

[0010] In the secondary inspection, leucine, isoleucine, and allo-isoleucine, which is an optical isomer of isoleucine, are separated and quantified, and the quantitative value of leucine is compared with a preset critical value. Moreover, if the quantitative value of leucine is lower than the critical value, it is determined to be negative. If the quantitative value of leucine is higher than the critical value, it is further determined whether allo-isoleucine is detected. If allo-isoleucine is detected, it is determined to be "needing detailed inspection", and if allo-isoleucine is not detected, it is determined to be "needing re-inspection".

[0011] As such, in the diagnosis of maple syrup urine disease, two stages of mass spectrometry screening are required. Generally speaking, in neonatal screening, since blood samples are collected from newborns to prepare samples, the implementation of the secondary examination causes a large physical burden on the newborn. In addition, for the parents of newborns who are not judged as negative in the primary examination, the implementation of the secondary examination causes a large psychological burden. In addition, the facilities for performing the primary examination are mostly different from the facilities for performing the secondary examination, and the labor and time required to transfer the inspection body to the facilities for implementing the secondary inspection are required. Therefore, it is expected that leucine and isoleucine will be separated and detected from the stage of the primary examination to reduce the number of inspection bodies transferred to the secondary examination.

[0012] The problem to be solved by the present invention is to provide an analytical method capable of identifying leucine and isoleucine in MRM determination such as neonatal mass spectrometry screening.

[0013] Solutions for solving problems

[0014] The method for analyzing leucine and isoleucine of the present invention, which has been completed in order to solve the above-mentioned problems, is a method for analyzing leucine and isoleucine using a tandem mass spectrometer, and the method for analyzing leucine and isoleucine comprises the following steps:

[0015] a measuring step of performing, on the specimen, a first multiple reaction monitoring (MRM) measurement of 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, and a second MRM measurement of an MRM transition of m / z 132>86, to obtain intensity information of ions derived from the leucine and the isoleucine; and

[0016] The processing step is to identify whether the specimen contains leucine or isoleucine, or to determine the abundance ratio of leucine to isoleucine in the specimen based on the ratio of the signal intensity obtained by the first MRM measurement to the signal intensity obtained by the second MRM measurement, i.e., the confirming ion ratio.

[0017] The leucine and isoleucine analysis device of the present invention, which has been made to solve the above-mentioned problems, comprises:

[0018] A measuring unit as a tandem mass spectrometer, which performs a first MRM measurement on at least one of the multiple reaction monitoring (MRM) transitions 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 test object, and a second MRM measurement on m / z 132>86; and

[0019] A processing unit that identifies whether the specimen contains leucine or isoleucine, or determines the existence ratio of leucine and isoleucine in the specimen based on 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, i.e., the confirming ion ratio.

[0020] Effects of the Invention

[0021] According to the leucine and isoleucine analysis method and leucine and isoleucine analysis device of the present invention, MRM transitions of quantitative ions (m / z132>86) showing substantially the same ion intensity regardless of whether leucine or isoleucine is contained in a specimen as long as the amount of the quantification ions is the same, and MRM transitions of confirmation ions (m / z132>69, m / z132>57, m / z132>56, m / z132>43, m / z132>41, m / z132>39, m / z132>30, m / z132>27) showing different ion intensities for leucine and isoleucine can be used to identify leucine and isoleucine or to determine the abundance ratio of leucine and isoleucine. Therefore, leucine and isoleucine can be identified without separating them using a chromatographic column of a liquid chromatograph. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a structural diagram of the main parts of one embodiment of the liquid chromatography-mass spectrometry analysis device involved in the present invention.

[0023] Figure 2 The chromatograms are of the MRM transitions of m / z 132.10>86.10 and m / z 132.10>69.20 in a sample containing only isoleucine (Leu:Ile=0:100).

[0024] Figure 3 The chromatograms are of the MRM transitions of m / z 132.10>86.10 and m / z 132.10>69.20 in a sample containing only leucine (Leu:Ile=100:0).

[0025] Figure 4 Graphs (a) to (h) show the collision energy dependence of the confirming ion ratio of leucine and isoleucine when the MRM transition: m / z 132.10>86.10 is set as the quantitative ion and the 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, and m / z 132.10>27.20 are set as the confirming ions.

[0026] Figure 5Graphs (a) to (h) show the relationship between the proportion of leucine in a sample and the confirming ion ratio when the MRM transition: m / z 132.10>86.10 is set as the quantitative ion, and the 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, and m / z 132.10>27.20 are set as the confirming ions.

[0027] Figure 6 It is a diagram showing the analysis conditions of MRM measurement. DETAILED DESCRIPTION

[0028] An embodiment of the method for analyzing leucine and isoleucine according to the present invention will be described below with reference to the accompanying drawings. The method for analyzing leucine and isoleucine according to this embodiment is used, for example, in mass spectrometry screening of newborns for diagnosing maple syrup urine disease.

[0029] Figure 1 1 is a diagram showing the main parts of the liquid chromatography-mass spectrometry analysis device used in this embodiment. The liquid chromatography-mass spectrometry analysis device 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.

[0030] The liquid chromatograph 1 includes a mobile phase container 11 storing a mobile phase, a pump 12 for sucking the mobile phase (solvent) and delivering the mobile phase (solvent) at a constant flow rate, an injector 13 for injecting a liquid sample into the mobile phase, and an automatic sampler (not shown) connected to the injector 13. The liquid chromatograph has a chromatographic column for separating components in a sample, but in this embodiment, a chromatographic column is not used, and the liquid sample is introduced into the mass spectrometer 2 together with the mobile phase by flow injection analysis (FIA). In addition, in the case of general newborn screening, as described in non-patent document 1, the sample is extracted and prepared from filter paper blood. However, this is not limited to this, and the sample can also be prepared from urine, other body fluids, etc.

[0031] The mass spectrometer 2 is a triple quadrupole mass spectrometer as a type of tandem mass spectrometer, and includes an ionization chamber 201 maintained at substantially 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). An ESI sprayer 21 for ionization by an electrospray ionization (ESI) method is provided in the ionization chamber 201, and the ionization chamber 201 is connected to the first intermediate vacuum chamber 202 of the next stage by a desolvation tube 22. An ion guide 23 for converging and transporting ions is arranged in the first intermediate vacuum chamber 202, and the first intermediate vacuum chamber 202 and the second intermediate vacuum chamber 203 of the next stage are connected through a small hole formed at the top of a separator 24. A multipole ion guide 25 for converging and transporting ions is also arranged in the second intermediate vacuum chamber 203.

[0032] In the high vacuum chamber 204, a front-stage quadrupole mass filter 26, a collision cell 27, a rear-stage quadrupole mass filter 28, and an ion detector 29 are arranged along the flow of ions. A quadrupole-type ion guide 271 is arranged inside the collision cell 27. The front-stage quadrupole mass filter 26 and the rear-stage quadrupole mass filter 28 each have the function of selectively allowing ions with a specified mass-to-charge ratio to pass through. An inactive collision-induced dissociation (CID) gas such as argon is introduced from the outside into the collision cell 27, so that the introduced ions are dissociated by contact with the CID gas to generate product ions.

[0033] The data processing unit 3 receives the detection data from the ion detector 29 and performs processing based on the data. The data processing unit 3 includes a data collection unit 31, a peak intensity calculation unit 32, and a component determination unit 33 as functional blocks. The analysis control unit 4 controls the operation of the liquid chromatograph 1 and the measurement unit 2 according to the analysis condition file stored in the analysis condition storage unit 41. The central control unit 5 mainly performs comprehensive control and control via the user interface such as the input unit 6 and the display unit 7.

[0034] Generally speaking, the entities of the data processing unit 3, the analysis control unit 4 and the central control unit 5 are personal computers or higher-performance computers called workstations, and the functions of the above-mentioned functional blocks are realized by running dedicated software (computer programs) pre-installed on the computer on the computer.

[0035] [Overview of MRM measurement operation]

[0036] exist Figure 1In the illustrated liquid chromatography-mass spectrometry analysis apparatus, when analyzing leucine and isoleucine, MRM measurement regarding a predetermined MRM transition is repeatedly performed by the mass spectrometer 2. The operation during the MRM measurement will be schematically described.

[0037] In the liquid chromatograph 1, the liquid delivery pump 12 draws the mobile phase from the mobile phase container 11 and delivers the mobile phase to the injector 13 at a substantially constant flow rate. The injector 13 injects a specified amount of sample (test body) into the mobile phase at a specified time. The sample reaches the ESI sprayer 21 of the measuring unit 2 by virtue of the flow of the mobile phase. In the flow path until reaching the ESI sprayer 21, the sample diffuses in the front-to-back direction. Therefore, the amount of the sample introduced into the ESI sprayer 21 is initially very small, but increases sharply, and when it exceeds the maximum point, it decreases sharply and becomes zero. That is, the concentration distribution of the sample is peak-shaped with respect to the passage of time, close to a Gaussian distribution.

[0038] In the ESI sprayer 21, the sample is sprayed into the ionization chamber 201 as fine charged droplets. The charged droplets come into contact with the residual gas molecules and split, and in the process of the solvent in the droplets being vaporized, the compound molecules in the sample are ionized. The generated ions are transported to the first intermediate vacuum chamber 202 through the desolvation tube 22, and are transported to the high vacuum chamber 204 through the ion guide 23, the small holes of the separator 24, and the multipole ion guide 25. The ions from the sample are introduced into the front quadrupole mass filter 26, and only ions with a specified mass-to-charge ratio corresponding to the voltage applied to the electrodes constituting the front quadrupole mass filter 26 are selectively passed as precursor ions. The precursor ions incident on the collision cell 27 come into contact with the CID gas and are dissociated to generate various product ions.

[0039] The various product ions generated are introduced into the post-stage 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-stage quadrupole mass filter 28 selectively pass through and reach the ion detector 29. The ion detector 29 generates a detection signal corresponding to the amount of incident ions, and inputs the detection data obtained by digitizing by an analog-to-digital converter (not shown) into the data processing unit 3.

[0040] The analysis control unit 4 controls the mass spectrometer 2 in such a manner that voltages corresponding to the targeted MRM transition are applied to the electrodes of the front-stage quadrupole mass filter 26 and the rear-stage quadrupole mass filter 28. Thus, detection data indicating the ion intensity of product ions having a specific mass-to-charge ratio, which are generated by dissociation of ions corresponding to the specific MRM transition, that is, precursor ions having a specific mass-to-charge ratio, from among ions of the compound contained in the sample, are obtained.

[0041] [Principle of the analysis method of leucine and isoleucine]

[0042] Next, the principle of the characteristic analysis method of leucine and isoleucine according to the present embodiment will be described.

[0043] In the mass spectrometer 2, the precursor ions are dissociated by CID in the collision cell 27, but the dissociation method varies depending on the kinetic energy of the precursor ions. This kinetic energy is the collision energy (CE). The collision energy is determined by the inlet end of the collision cell 27 and its front stage (in Figure 1 The collision energy is usually expressed by the potential difference of the DC voltage (in this case, the front quadrupole mass filter 26, but sometimes also other ion lenses and plasma optical elements). Therefore, the collision energy can be adjusted by applying a DC bias voltage to either or both of the front quadrupole mass filter 26 and the front ion optical element.

[0044] As described above, when the collision energy is changed, the dissociation mode of the precursor ion changes, and the generation pattern of multiple product ions changes. In order to improve the detection sensitivity, it is desirable to select product ions (i.e., MRM transitions) with the largest ion intensity as possible. Therefore, in the previous analysis of leucine and isoleucine, m / z 132.10>86.10 with the largest ion intensity was used as MRM transition. However, as already described, in m / z 132.10>86.10, leucine and isoleucine cannot be distinguished. Therefore, the present inventors and others conducted experiments to investigate in detail the relationship between collision energy and ion intensity for various MRM transitions associated with leucine and isoleucine.

[0045] The above experiment was carried out according to the following process.

[0046] Ultrapure water was used to prepare 100 μg / L aqueous solution from the standard of leucine and isoleucine, and it was set as sample. LCMS-8060 manufactured by Shimadzu Corporation was used as liquid chromatography-mass spectrometry analysis device. First, the common precursor ion (m / z 132.10) of leucine and isoleucine was subjected to product ion scanning measurement to search for product ions peculiar to each compound. As a result, it was known that the peculiar product ions derived from each compound were not observed. As the mass-to-charge ratio of the representative product ions that could obtain peak intensity high to a certain extent, there were nine kinds of 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.

[0047] The peak intensities of the nine product ions were measured when the set value of the collision energy was changed in steps of 5 V within the range of -100 V to -5 V. As a result, in the MRM transition (m / z 132.10>86.10) used for the quantification of leucine and isoleucine in the past, there was almost no difference between the peak intensity pattern of leucine corresponding to the change in collision energy and the peak intensity pattern of isoleucine. In contrast, in 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), the peak intensity pattern of leucine is different from that of isoleucine.

[0048] For example, Figure 2 and Figure 3 The chromatographic waveforms are respectively 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). The concentration of the components is 100μg / L. Figure 2 and Figure 3 It can be seen that the peak intensity patterns of m / z 132.10>86.10 are similar between the sample containing leucine and the sample containing isoleucine, and the peak intensity patterns of m / z 132.10>69.20 are different. In addition, in both the sample containing leucine and the sample containing isoleucine, the intensity of the peak of m / z 132.10>69.20 is smaller than the intensity of the peak of m / z 132.10>86.10. Based on such experimental insights, it can be known that leucine and isoleucine can be distinguished by utilizing the difference between the peak intensity patterns of 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) and the peak intensity pattern of the MRM transition (m / z 132.10>86.10).

[0049] Therefore, the signal intensity A1 of the MRM transition peak of m / z 132.10>86.10 measured when the collision energy setting value was set to -10 V, and the signal intensity A2 of the above eight MRM transition peaks when the collision energy setting value was changed in the range of -100 V to -5 V in steps of 5 V were measured respectively, and the value obtained by multiplying their ratio (A2 / A1) by 100 was calculated. Figure 4 The results are shown.

[0050] In addition, if the ion detected in the MRM transition of m / z 132.10>86.10 is set as the quantitative ion, 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 set as the confirming ion, then the intensity ratio between them is the so-called confirming ion ratio. Hereinafter, the above value ((A2 / A1)×100) is referred to as the confirming ion ratio.

[0051] Figure 4 Graphs showing the collision energy dependence of the confirmation ion ratio for eight MRM transitions for leucine and isoleucine. In these graphs, the horizontal axis is the collision energy and the vertical axis is the confirmation ion ratio (A2 / A1×100). In addition, in the figure, the solid line represents the confirmation ion ratio of leucine and the dotted line represents the confirmation ion ratio of isoleucine. As can be seen from these graphs, in all eight MRM transitions, there is a difference between the pattern of the collision energy dependence of the confirmation ion ratio of leucine and the pattern of the collision energy dependence of the confirmation ion ratio of isoleucine. In particular, in m / z 132.10>39.10, there is a clear difference in the confirmation ion ratio over almost the entire range of the collision energy ( Figure 4 (f)). In addition, in m / z 132.10>41.10, it was confirmed that the ion ratio had a clear difference depending on the collision energy value ( Figure 4 (e)). In the other cases of 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>27.20, it was confirmed that the ion ratios had significant differences depending on the collision energy values ​​( Figure 4For example, in m / z 132.10>69.20, when the collision energy is -20V, the confirmation ion ratio of leucine and isoleucine becomes a peak (maximum), and at this peak, the difference in the confirmation ion ratio of the two is the largest ( Figure 4 (a)). As such, 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>27.20, there is an optimal collision energy value at which the difference between the confirmation ion ratio of leucine and the confirmation ion ratio of isoleucine becomes significantly large.

[0052] Figure 5 (a) to (h) are graphs showing the relationship between the ratio of leucine in leucine and isoleucine in a sample and the confirming ion ratio when eight types of MRM transitions are used, which were obtained based on actual measurement results. Figure 6 The conditions for MRM measurement are shown. 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>27.20, the above-mentioned optimal value is used as the collision energy. When m / z 132.10>41.10 and m / z 132.10>39.10, a collision energy value with a large difference between leucine and isoleucine near the maximum confirmation ion ratio is used. Figure 5 As shown in (a) to (h), the relationship between the proportion of leucine in the sample and the confirmation ion ratio is expressed by a linear equation, that is, a straight line. Therefore, in the device of the above-mentioned embodiment, the above-mentioned relationship obtained in advance through experiments is stored in the component determination unit 33 as a calibration curve, and the calibration curve is applied to the confirmation ion ratio obtained from the actual measurement of the sample to estimate the proportion of leucine or isoleucine. In addition, any one of the relationship equations in the case of using eight MRM transitions can be stored in the component determination unit 33 as a calibration curve. However, the relationship equation in the case of using more than two MRM transitions can also be stored in the component determination unit 33 as a calibration curve, and the average value, central value, etc. of the values ​​estimated using each of these multiple calibration curves can be set as the proportion of leucine or isoleucine.

[0053] [Specific example of the method for identifying leucine and isoleucine]

[0054] First, in the liquid chromatography-mass spectrometry analysis apparatus of the above-mentioned embodiment, the measurement unit 2 repeatedly performs MRM measurement of MRM transitions for quantitative ions and MRM measurement of MRM transitions for predetermined confirmation ions on the same unknown sample under the control of the analysis control unit 4. The MRM measurement is repeatedly performed from the time point when the sample is injected by the injector 13 until the time point when the predetermined time has passed. Thus, data constituting a chromatographic waveform representing the time passage of each MRM measurement result, i.e., ion intensity data, is stored in the data collection unit 31.

[0055] The peak intensity calculation unit 32 creates a chromatographic waveform corresponding to each of the nine MRM transitions described above based on the data stored in the data collection unit 31, and performs peak detection on the waveform to obtain the value of the peak top (peak intensity value). In addition, here, the peak intensity value is used as the signal intensity for the calculation processing described later, but the area value of the peak from the peak start point to the end point can also be calculated instead of the peak intensity value and used as the signal intensity.

[0056] Next, the component determination unit 33 calculates the ratio of the signal intensity A1 of the MRM transition corresponding to the quantitative ion (m / z 132.10>86.10) to the signal intensity A2 of the MRM transition corresponding to the confirmation ion, that is, the confirmation ion ratio (A2 / A1×100). Then, the calibration curve stored in the component determination unit 33 is applied to the confirmation ion ratio to estimate the ratio of leucine or isoleucine.

[0057] [Way]

[0058] It is obvious to those skilled in the art that the above-mentioned exemplary embodiments are specific examples of the following aspects.

[0059] (Item 1) The method for analyzing leucine and isoleucine according to the present invention is a method for analyzing leucine and isoleucine using a tandem mass spectrometer, and the method for analyzing leucine and isoleucine comprises the following steps:

[0060] a measuring step of performing, on the specimen, a first MRM measurement of at least one multiple reaction monitoring transition (MRM transition) 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, and a second MRM measurement of an MRM transition of m / z 132>86, to obtain intensity information of ions derived from the leucine and the isoleucine; and

[0061] The processing step is to identify whether the specimen contains leucine or isoleucine, or to determine the abundance ratio of leucine to isoleucine in the specimen based on the ratio of the signal intensity obtained by the first MRM measurement to the signal intensity obtained by the second MRM measurement, i.e., the confirming ion ratio.

[0062] (Item 9) The leucine and isoleucine analysis device according to the present invention comprises:

[0063] A measuring unit of a tandem mass spectrometer, wherein the measuring unit performs, on a test object, a first MRM measurement of at least one multiple reaction monitoring transition (MRM transition) 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, and a second MRM measurement of an MRM transition of m / z 132>86; and

[0064] A processing unit that identifies whether the specimen contains leucine or isoleucine, or determines the existence ratio of leucine and isoleucine in the specimen based on 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, i.e., the confirming ion ratio.

[0065] According to the analysis method for leucine and isoleucine described in item 1 or the analysis apparatus for leucine and isoleucine described in item 9, whether the specimen contains leucine or isoleucine or the existence ratio of leucine and isoleucine in the specimen can be identified based on the ratio of the signal intensity in the MRM transition of the quantitative ion (m / z 132>86) which shows substantially the same ion intensity regardless of whether leucine or isoleucine is contained in the specimen as long as the amount of the leucine and isoleucine is the same, i.e., the confirmation ion ratio, and the signal intensity in 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.

[0066] (Item 2) In the method for analyzing leucine and isoleucine according to Item 1, the method may be:

[0067] 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 the ions derived from the leucine and isoleucine,

[0068] In the processing step, whether the specimen contains leucine or isoleucine is identified based on the collision energy dependency of the confirming ion ratio.

[0069] According to the analysis method of leucine and isoleucine described in the second item, even when there is no difference between the confirming ion ratio of leucine and the confirming ion ratio of isoleucine at a certain collision energy, leucine and isoleucine can be distinguished.

[0070] (Item 3) In the method for analyzing leucine and isoleucine according to Item 1, the method may be:

[0071] In the processing step, the abundance ratio of leucine or isoleucine contained in the target specimen is estimated using information indicating the relationship between the confirming ion ratio and the abundance ratio of leucine or isoleucine in the specimen obtained in advance.

[0072] According to the analytical method for leucine and isoleucine described in Item 3, not only leucine and isoleucine can be identified, but also the existence ratio of the two can be estimated.

[0073] (Item 4) In the method for analyzing leucine and isoleucine according to any one of Items 1 to 3, the method may be:

[0074] The transition determined by the first MRM is any one of 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.

[0075] According to the analysis method of leucine and isoleucine described in Item 4, since there is a range of collision energy in which the difference between the confirmation ion ratio of leucine and the confirmation ion ratio of isoleucine becomes large, the collision energy is changed within this range and the first MRM measurement and the second MRM measurement are performed. By using the obtained confirmation ion ratio, leucine and isoleucine can be identified more reliably.

[0076] (Item 5) In the method for analyzing leucine and isoleucine according to any one of Items 1 to 3, the method may be:

[0077] The transition measured by the first MRM is m / z 132>41 or m / z 132>39.

[0078] According to the analysis method of leucine and isoleucine described in Item 5, since there is a range of collision energy in which peaks of both leucine and isoleucine are detected with sufficient intensity and there is a clear difference between the confirmation ion ratio of leucine and the confirmation ion ratio of isoleucine, the collision energy is changed within this range and the first MRM measurement and the second MRM measurement are performed. By using the obtained confirmation ion ratio, leucine and isoleucine can be reliably identified even when the specimen contains impurities that hinder peak detection.

[0079] (Item 6) In the method for analyzing leucine and isoleucine according to any one of Items 1 to 5, the method may be:

[0080] In the measuring step, the first MRM measurement and the second MRM measurement are repeatedly performed,

[0081] In the processing step, the intensity value or the peak area value of the peak top of the peak indicating the temporal change in the intensity of the ions derived from leucine and isoleucine is used as the measurement result.

[0082] According to the analysis method of leucine and isoleucine described in Item 6, by using the peak height (peak top intensity value) as the measurement result, rapid processing can be performed, thereby improving the efficiency of screening. On the other hand, in general, the peak area value is more excellent in quantitative properties than the peak height, so by using the peak area value as the measurement result, more accurate identification and determination can be implemented.

[0083] (Item 7) In the method for analyzing leucine and isoleucine according to any one of Items 1 to 6, the method may be:

[0084] The specimen is introduced into the tandem mass spectrometer by flow injection analysis.

[0085] According to the analysis method of leucine and isoleucine described in Item 7, since separation of components using a chromatographic column is not required, the measurement time for one specimen can be shortened, and thus it is suitable for screening requiring rapidity.

[0086] (Item 8) In the method for analyzing leucine and isoleucine according to any one of Items 1 to 6, the method may be:

[0087] Components contained in the specimen are separated by liquid chromatography and introduced into the tandem mass spectrometer.

[0088] According to the analysis method of leucine and isoleucine described in Item 8, component separation using a chromatographic column can also be utilized, and therefore the accuracy can be further improved compared to identification and determination using only measurement results of specific MRM transitions.

[0089] Description of Reference Numerals

[0090] 1: Liquid chromatograph; 11: Mobile phase container; 12: Liquid delivery pump; 13: Injector; 2: Mass spectrometer; 201: Ionization chamber; 202: First intermediate vacuum chamber; 203: Second intermediate vacuum chamber; 204: High vacuum chamber; 21: ESI sprayer; 22: Desolventizing tube; 23: Ion guide; 24: Separator; 25: Multipolar ion guide; 26: Front quadrupole mass filter; 27: Collision cell; 28: Post quadrupole mass filter; 29: Ion detector; 3: Data processing unit; 31: Data collection unit; 32: Peak intensity calculation unit; 33: Component determination unit; 4: Analysis control unit; 41: Analysis condition storage unit; 5: Central control unit; 6: Input unit; 7: Display unit.

Claims

1. A method for analyzing leucine and isoleucine, which is a method for analyzing leucine and isoleucine using a tandem mass spectrometer, and the method for analyzing leucine and isoleucine comprises the following steps: a measuring step of performing, on the specimen, a first MRM measurement of at least one multiple reaction monitoring transition (MRM transition) 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, and a second MRM measurement of an MRM transition of m / z 132>86, to obtain intensity information of ions derived from the leucine and the isoleucine; as well as The processing step is to identify whether the specimen contains leucine or isoleucine, or to determine the abundance ratio of leucine to isoleucine in the specimen based on the ratio of the signal intensity obtained by the first MRM measurement to the signal intensity obtained by the second MRM measurement, i.e., the confirming ion ratio.

2. The method for analyzing leucine and isoleucine according to claim 1, wherein: 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 the ions derived from the leucine and isoleucine, In the processing step, whether the specimen contains leucine or isoleucine is identified based on the collision energy dependency of the confirming ion ratio.

3. The method for analyzing leucine and isoleucine according to claim 1, wherein: In the processing step, the abundance ratio of leucine or isoleucine contained in the target specimen is estimated using information indicating the relationship between the confirming ion ratio and the abundance ratio of leucine or isoleucine in the specimen obtained in advance.

4. The method for analyzing leucine and isoleucine according to claim 1, wherein: The transition determined by the first MRM is any one of 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 measured by the first MRM is m / z 132>41 or m / z 132>39.

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

7. The method for analyzing leucine and isoleucine according to any one of claims 1 to 6, wherein: The specimen is introduced into the tandem mass spectrometer by flow injection analysis.

8. The method for analyzing leucine and isoleucine according to any one of claims 1 to 6, wherein: Components contained in the specimen are separated by liquid chromatography and introduced into the tandem mass spectrometer.

9. An analytical device for leucine and isoleucine, comprising: A measuring unit of a tandem mass spectrometer, wherein the measuring unit performs, on a specimen, a first MRM measurement of at least one multiple reaction monitoring transition (MRM transition) 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, and a second MRM measurement of an MRM transition of m / z 132>86; and A processing unit that identifies whether the specimen contains leucine or isoleucine, or determines the existence ratio of leucine and isoleucine in the specimen based on 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, i.e., the confirming ion ratio.

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