Amino acid and / or acyl carnitine analysis method

Abstract

The present invention provides a method for analyzing an amino acid and / or an acyl carnitine. In a method for analyzing an amino acid and / or acylcarnitine, a first MRM transition and a second MRM transition of a target substance, a first reference value for the measured intensity of the first MRM transition, and a second reference value for the ratio of the measured intensity of the first MRM transition to the measured intensity of the second MRM transition are set, and a sample to be analyzed is subjected to MRM measurement using the first MRM transition and the second MRM transition. A first intensity of the first MRM transition and a second intensity of the second MRM transition are measured (steps 11, 12), a first determination is made as to whether or not the target substance is included by comparing the value of the first intensity with a first reference value (step 14), and a second determination is made as to whether or not the target substance is included in the sample to be analyzed as determined in the first determination. A second determination is made as to whether or not the target substance is included by comparing the ratio of the value of the first intensity to the value of the second intensity with a second reference value (step 18).

Description

Technical Field

[0001] The present invention relates to a method for analyzing amino acids and / or acylcarnitines contained in a sample of an analyte. Background Art

[0002] In order to detect and treat congenital metabolic disorders in newborns, etc., newborn screening has been widely implemented. In recent years, with the rapid development of mass spectrometry technology, newborn mass spectrometry screening using a tandem mass spectrometry device has been popularized, contributing to the early detection of congenital metabolic disorders in newborns (for example, Non-Patent Document 1).

[0003] In such newborn mass spectrometry screening, multiple specific types of amino acids and acylcarnitines are respectively associated with different diseases, and a critical value is set for each disease. By comparing the amounts and / or concentrations of these specific types of amino acids and acylcarnitines contained in a sample sourced from blood collected from a newborn with the critical values corresponding to each substance, screening for each disease is performed.

[0004] As disclosed in Non-Patent Document 1, in screening using a tandem mass spectrometry device (tandem mass screening), multiple reaction monitoring (MRM) measurements are performed using MRM transitions (transitions) previously set for the above-mentioned multiple specific types of amino acids and acylcarnitines respectively to quantify each substance, and each quantitative value is compared with the above-mentioned critical value.

[0005] Prior Art Documents

[0006] Non-Patent Documents

[0007] Non-Patent Document 1: Yosuke Shigematsu, "The Reality of Newborn Mass Screening by Mass Spectrometry", The Mass Spectrometry Society of Japan, Journal of the Mass Spectrometry Society of Japan, Vol. 64, No. 4, 2016, pp. 127-131

[0008] Non-Patent Document 2: Takao Ishikawa, Michiko Tezuka, Miwa Yoshinaga, Shoji Nomachi, Shinji Hosomi, Koichi Yano, "Effect of Wax Coating on the Quantification of Leucine and Isoleucine in Tandem Mass Spectrometry", Journal of the Japanese Society for Mass Screening, Vol. 30 (No. 3), 2020, pp. 31(257)-43(269)

[0009] Non-patent document 3: Saito Ningko, Wada Yoichi, Maekawa Masamitsu, Wu Shigeo, "Essentials 3 cases of false positive of "ススクリーニングでアシルCoA dehydrase deficiency syndrome", Japanese Journal of Neonatal Screening, Vol.30(2), 2020, pp.102(196) Summary of the invention

[0010] Problem that the invention aims to solve

[0011] 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 placed in an autosampler was sequentially introduced into a mass spectrometer together with a mobile phase without passing through a column of a liquid chromatograph (LC) using a flow injection analysis (FIA) method. In the flow injection analysis method, each sample is introduced into a mass spectrometer without performing component separation based on a column, so various substances contained in the sample are simultaneously provided for MRM measurement.

[0012] For example, as described in Non-Patent Document 1, C5 acylcarnitine (acylcarnitine having an acyl group with a carbon number of 5) includes four isomers, namely, isovalerylcarnitine, pivaloylcarnitine, 2-methylbutyrylcarnitine, and n-valerylcarnitine. Among them, isovalerylcarnitine and 2-methylbutyrylcarnitine are metabolites associated with diseases, while pivaloylcarnitine is a metabolite derived from a drug (antibacterial agent) administered to newborns. Therefore, even if the quantitative value in the MRM assay of C5 acylcarnitine exceeds the critical value and is determined to be positive in the screening, this may be a false positive caused by pivaloylcarnitine derived from the drug.

[0013] The above is an example of a false positive originating from a substance (endogenous substance) contained in the sample itself, but a substance (exogenous substance) mixed in when collecting or preparing the sample may sometimes also lead to a false positive. For example, the following is recorded in non-patent document 2: In the screening of maple syrup urine disease, a false positive is generated because diethylene glycol monoethyl ether contained in the agent used for waxing is mixed in when preparing the sample and is detected together with leucine and isoleucine. In addition, the following is recorded in non-patent document 3: In the screening of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, a false positive is generated because a component contained in an essential oil used for aromatherapy for postpartum mothers is mixed in when collecting the sample and is detected together with octanoylcarnitine (C8) or decanoylcarnitine (C10). These exogenous substances are not isomers of the amino acids or acylcarnitines that are the targets of analysis. However, the tandem quadrupole mass spectrometers that are mostly used in newborn mass spectrometry screening have difficulty identifying ions with the same integer mass (mass-to-charge ratio). Therefore, false positives may also occur due to substances with the same integer mass as the amino acids or acylcarnitines that are the targets of analysis.

[0014] Therefore, in the past, when it is positive in screening, additional inspections based on other analytical methods such as chromatography mass spectrometry using a column are implemented, and detailed analysis is performed to determine the presence or absence of the disease. In general, blood is collected from newborns to prepare samples in newborn screening, so the implementation of additional inspections as described above causes a large burden on the body for newborns. In addition, if it is positive in screening, it causes a large burden psychologically for the parents of newborns. And, as the false positives generated increase, the number of samples that need additional inspections increases, so the workload of the analyst also increases, thereby increasing the burden. Therefore, a technology that reduces the false positives in screening compared to the past is sought.

[0015] Here, the problems in the prior art are described by taking specific amino acids and acylcarnitines as examples, but the same problems as described above also exist when performing screening by measuring other types of amino acids or acylcarnitines.

[0016] The problem to be solved by the present invention is to determine with higher accuracy whether a sample to be analyzed contains specific types of amino acids or acylcarnitines contained in the sample to be analyzed in an analysis method for performing MRM measurement on these substances.

[0017] Solutions for solving problems

[0018] The first aspect of the method for analyzing amino acids and / or acylcarnitines according to the present invention, which has been accomplished to solve the above-mentioned problems, is as follows:

[0019] For each of the one or more target substances that are amino acids or acylcarnitines, a first MRM transition and a second MRM transition that are a group of a mass-to-charge ratio of a precursor ion and a mass-to-charge ratio of a product ion, a first reference value related to a measured intensity of the first MRM transition, and a second reference value related to a ratio of the measured intensity of the first MRM transition to the measured intensity of the second MRM transition are set,

[0020] By performing MRM measurement on the analysis target sample using the first MRM transition and the second MRM transition set for each target substance, a first intensity as a measurement intensity of the first MRM transition and a second intensity as a measurement intensity of the second MRM transition are measured for each target substance,

[0021] performing a first determination for each of the target substances, wherein the first determination is performed by comparing the first intensity value with the first reference value to determine whether or not there is a possibility that the target substance is contained in the analysis object sample,

[0022] A second judgment is performed on the target substance that is determined in the first judgment to be likely to be contained in the analysis object sample. In the second judgment, whether the analysis object sample contains the target substance is determined by comparing the ratio of the first intensity value to the second intensity value with the second reference value.

[0023] In addition, a second embodiment of the method for analyzing amino acids and / or acylcarnitines according to the present invention, which has been accomplished to solve the above-mentioned problems, is as follows:

[0024] For each of the one or more target substances that are amino acids or acylcarnitines, a first MRM transition that is a set of a mass-to-charge ratio of a precursor ion and a mass-to-charge ratio of a product ion, a reference value related to a measurement intensity of the first MRM transition, and a second MRM transition in which an impurity that can be detected together with the target substance in an MRM measurement using the first MRM transition is not detected are set,

[0025] By performing MRM measurement on the analysis target sample using the first MRM transition and the second MRM transition set for each target substance, a first intensity as a measurement intensity of the first MRM transition and a second intensity as a measurement intensity of the second MRM transition are measured for each target substance,

[0026] performing a first determination for each of the target substances, wherein the first determination is performed by comparing the first intensity value with the reference value to determine whether or not there is a possibility that the target substance is contained in the analysis object sample,

[0027] A second determination is performed on the target substance determined in the first determination to be possibly contained in the analysis target sample. In the second determination, whether the analysis target substance is contained in the analysis target sample is determined based on the value of the second intensity.

[0028] Effects of the Invention

[0029] In the first and second aspects of the present invention, a first MRM transition and a second MRM transition, as well as a reference value (the first reference value in the first aspect) related to the measurement intensity of the first MRM transition are set for each target substance among one or more target substances that are amino acids or acylcarnitines, and the first MRM transition and the second MRM transition are used to perform MRM measurement on the sample to be analyzed for each target substance. For the first MRM transition, it is sufficient to set an MRM transition that can measure the target substance with high sensitivity in the same manner as the MRM transition used in the previous mass spectrometry screening. In addition, for the reference value (same as above) related to the measurement intensity of the first MRM transition, it is sufficient to set a value equivalent to the critical value used in the previous mass spectrometry screening.

[0030] In the first and second aspects of the present invention, first, for each target substance, the measured intensity obtained by the first MRM transition is compared with the reference value, thereby determining the possibility that the target substance is contained in the sample to be analyzed (first determination). In conventional mass spectrometry screening, the possibility that the target substance is contained in the sample to be analyzed is determined only by this determination, and therefore, it is not possible to distinguish between the case where the sample to be analyzed contains the target substance and the case where only impurities such as isomers detected together with the target substance by the first MRM transition exist but the target substance itself is not contained.

[0031] In the first mode of the present invention, a second reference value related to the ratio of the measured intensity of the first MRM transition to the measured intensity of the second MRM transition is set, and for the target substance that is determined (first determination) to be likely to be contained in the sample to be analyzed, the ratio of the first intensity value to the second intensity value is further compared with the second reference value, thereby determining whether the target substance is contained in the sample to be analyzed (second determination). Even in the case where there is an impurity that is detected together with the target substance through the first MRM transition, generally speaking, the ratio of the measured intensity obtained by the first MRM transition to the measured intensity obtained by the second MRM transition is different between the target substance and the impurity. In the first mode of the present invention, when it is determined in the first determination that there is a possibility of containing the target substance, by comparing the above ratio with the second reference value, it is determined whether the sample to be analyzed contains the target substance or the impurity (second determination), so it is possible to determine whether the sample to be analyzed contains the target substance with higher accuracy than before.

[0032] In the second aspect of the present invention, a second MRM transition is set in which an impurity that can be detected together with the target substance in the MRM measurement performed using the first MRM transition is not detected, and for each target substance among the target substances that are determined to be likely to be contained in the sample to be analyzed in the first determination, a determination (second determination) is made as to whether the target substance is contained in the sample to be analyzed based on the measurement intensity (second intensity value) obtained using the second MRM transition. In the second aspect of the present invention, based on the fact that the target substance is detected in the MRM measurement performed using the second MRM transition in which the impurity is not detected, it is determined (second determination) that the target substance is contained in the sample to be analyzed, and therefore it is possible to determine whether the target substance is contained in the sample to be analyzed with higher accuracy than before. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a diagram showing the main structure of a liquid chromatograph-mass spectrometer used in one embodiment of the method for analyzing amino acids and / or acylcarnitines according to the present invention.

[0034] Figure 2 This is an example of the compound database in this embodiment.

[0035] Figure 3 This is a diagram for explaining the first analysis mode of this embodiment.

[0036] Figure 4 This is another diagram for explaining the first analysis mode of this embodiment.

[0037] Figure 5 This is another diagram for explaining the first analysis mode of this embodiment.

[0038] Figure 6 This is a diagram for explaining the second analysis mode of this embodiment.

[0039] Figure 7 This is another diagram for explaining the second analysis mode of this embodiment.

[0040] Figure 8 This is another diagram for explaining the second analysis mode of this embodiment.

[0041] Fig. 9 This is a flowchart illustrating the process of the method for analyzing amino acids and / or acylcarnitines according to the present embodiment.

[0042] Fig.10 This is a flowchart illustrating the process of the first analysis mode in the analysis method of amino acids and / or acylcarnitines according to the present embodiment.

[0043] Fig.11 This is a flowchart illustrating the process of the second analysis mode in the analysis method of amino acids and / or acylcarnitines according to the present embodiment. DETAILED DESCRIPTION

[0044] One embodiment of the method for analyzing amino acids and / or acylcarnitines according to the present invention will be described with reference to the accompanying drawings. The method for analyzing amino acids and / or acylcarnitines according to this embodiment is used, for example, for implementing mass spectrometry screening of newborns.

[0045] Figure 1 1 is a diagram showing the main structure of a liquid chromatograph-mass spectrometer used in the present embodiment. The liquid chromatograph-mass spectrometer of the present embodiment includes a liquid chromatograph 1, a mass spectrometer 2, and a control / processing unit 4 for controlling the operations thereof.

[0046] The liquid chromatograph 1 includes a mobile phase container 11 storing a mobile phase, a pump 12 for sucking and delivering the mobile phase (solvent) at a constant flow rate, a syringe 13 for injecting a liquid sample into the mobile phase, and an automatic sampler 14 connected to the syringe 13. The liquid chromatograph has a column for separating components in the sample, but in this embodiment, the column is not used, and the liquid sample is introduced into the mass spectrometer 2 together with the mobile phase by the flow injection analysis (FIA) method.

[0047] The mass spectrometer 2 includes an ionization chamber 20 at a pressure substantially equal to atmospheric pressure and a vacuum chamber connected to the ionization chamber 20. The vacuum chamber is evacuated by a vacuum pump (not shown). Inside the vacuum chamber, there are a first intermediate vacuum chamber 21, a second intermediate vacuum chamber 22, and an analysis chamber 23, which are sequentially arranged from the ionization chamber 20 side, and a multi-stage differential exhaust system structure in which the vacuum degree gradually increases.

[0048] An electrospray ionization (ESI) probe 201 for applying electric charge to a sample solution and spraying the sample solution is provided in the ionization chamber 20. The ionization chamber 20 is connected to a first intermediate vacuum chamber 21 at a subsequent stage via a heating capillary (desolvation tube) 202 having a small diameter.

[0049] An ion lens 211 composed of a plurality of annular electrodes is disposed in the first intermediate vacuum chamber 21. The ion lens 211 converges the flight path of ions along the ion optical axis C (the central axis in the flight direction of ions). The first intermediate vacuum chamber 21 and the second intermediate vacuum chamber 22 are separated by a separator 212 having a small hole at the top.

[0050] An ion guide 221 composed of a plurality of rod electrodes is disposed in the second intermediate vacuum chamber 22. The ion guide 221 also converges the flight path of ions along the ion optical axis C, similarly to the ion lens 211. The second intermediate vacuum chamber 22 is separated from the analysis chamber by a partition wall having a small hole.

[0051] The analysis chamber 23 is provided with a front-stage quadrupole mass filter 231, a collision chamber 232, a rear-stage quadrupole mass filter 234, and an ion detector 235. An ion guide 233 is provided inside the collision chamber 232. In addition, a collision-induced dissociation (CID) gas is introduced into the collision chamber 232 from a gas source (not shown).

[0052] The mass spectrometer 2 can perform selected ion monitoring (SIM) measurement, MS / MS scanning (product ion scanning) measurement, multiple reaction monitoring (MRM) measurement, etc. In SIM measurement, the front-stage quadrupole mass filter 231 does not select ions (does not function as a mass filter), and the mass-to-charge ratio of ions passing through the rear-stage quadrupole mass filter 234 is fixed to detect ions.

[0053] In MS / MS scanning measurement and MRM measurement, both the front-stage quadrupole mass filter 231 and the rear-stage quadrupole mass filter 234 function as mass filters. In the front-stage quadrupole mass filter 231, only ions having a mass-to-charge ratio set as precursor ions are allowed to pass. In addition, CID gas is supplied to the interior of the collision chamber 232, and the precursor ions are introduced into the interior of the collision chamber 232 with acceleration, so that the precursor ions collide with the CID gas to promote the fragmentation of the precursor ions. In the MS / MS scanning measurement, the mass-to-charge ratio of the ions passing through the rear-stage quadrupole mass filter 234 is scanned, and in the MRM measurement, the mass-to-charge ratio of the ions passing through the rear-stage quadrupole mass filter 234 is fixed, so that only product ions having a specific mass-to-charge ratio are allowed to pass.

[0054] The control / processing unit 4 has a storage unit 41. The storage unit 41 stores a compound database (compound DB) 411 (preliminary preparation step, step 0. See Fig. 9 ).exist Figure 2 An example of information stored in the compound DB 411 is shown in FIG. The measurement conditions recorded in the compound DB 411 include, for example, the name of the compound, the magnitude of the collision energy when measuring the compound, the mass-to-charge ratio of the quantitative MRM transition used in the first determination of the first analysis mode and the second analysis mode described later, the reference value of the measurement intensity of the ion obtained by the quantitative MRM transition, the mass-to-charge ratio of the confirmation MRM transition (first analysis confirmation MRM transition) used in the first analysis mode, the reference value related to the ratio of the measurement intensity of the ion obtained by the first analysis confirmation MRM transition to the measurement intensity of the ion obtained by the quantitative MRM transition, the mass-to-charge ratio of the confirmation MRM transition (second analysis confirmation MRM transition) used in the second analysis mode, and the threshold value of the measurement intensity of the ion obtained by the second analysis confirmation MRM transition. The measurement intensity of the ion obtained by the quantitative MRM transition, the ratio of the above-mentioned measurement intensity, and the measurement intensity of the ion obtained by the second analysis confirmation MRM transition are respectively the index values ​​in the neonatal mass spectrometry screening. In addition, the storage unit 41 also stores information obtained by associating each compound with the disease associated with the compound.

[0055] Examples of amino acids included in compound DB 411 include phenylalanine, leucine, isoleucine, methionine, citrulline, and tyrosine. In addition, examples of acylcarnitines included in compound DB 411 include free carnitine (C0), acetylcarnitine (C2), propionylcarnitine (C3), butyrylcarnitine to palmitoylcarnitine (C4 to C18), isovalerylcarnitine, 2-methylbutyrylcarnitine, pivaloylcarnitine (C5), methylcrotonylcarnitine (C5:1), glutarylcarnitine (C5-DC), 3-hydroxyisovalerylcarnitine, 3-hydroxy-2-methylbutyryl-carnitine (C 5-OH), octanoyl carnitine (C8), decanoyl carnitine (C10), dodecanoyl carnitine (C12), tetradecenoyl carnitine (C14:1), tetradecanoyl carnitine (C14), palmitoyl carnitine (C16), stearoyl carnitine (C18), octadecenoyl carnitine (C18:1), hydroxyhexadecenoyl carnitine (C16-OH), hydroxyoctadecenoyl carnitine (C18:1-OH).

[0056] The above-mentioned MRM transitions are all a set of the mass-to-charge ratio of the precursor ion generated from the compound (amino acid or acylcarnitine) and the mass-to-charge ratio of the product ion generated by the fragmentation of the precursor ion. That is, in the MRM measurement, the front-stage quadrupole mass filter 231 is used to select ions having the mass-to-charge ratio of the precursor ion set by the MRM transition, and the ions are fragmented in the collision chamber 232 to generate product ions, and then, the rear-stage quadrupole mass filter 234 is used to select ions having the mass-to-charge ratio of the product ion set by the MRM transition, and detected in the ion detector 235. For quantitative MRM transitions, the MRM transition that can detect the compound (amino acid or acylcarnitine) with the highest sensitivity is usually selected.

[0057] For the first analytical confirmation MRM transition, for example, an MRM transition having the second highest measurement sensitivity compared to the quantitative MRM transition is selected. For the first analytical confirmation MRM transition, a reference value of a score related to the ratio of the measurement intensity of the first analytical confirmation MRM transition to the measurement intensity of the quantitative MRM transition is associated. In the first analytical mode, the measurement intensity of the ion obtained by the quantitative MRM transition becomes the first index value, and the score becomes the second index value.

[0058] For the second analytical confirmation MRM transition, an MRM transition is selected in which there is no generation from a compound (contaminant compound) that may be detected simultaneously when the compound is measured using a quantitative MRM transition. For the second analytical confirmation MRM transition, information on a threshold value of the measurement intensity of the second analytical confirmation MRM transition is associated. The threshold value is set to a value that can be distinguished from noise, for example. In addition, in the absence of noise considerations, the threshold value can also be set to 0. In the second analytical mode, the measurement intensity of the ion obtained using the quantitative MRM transition becomes the first index value, and the measurement intensity of the second analytical confirmation MRM transition becomes the second index value in newborn mass spectrometry screening.

[0059] The control / processing unit 4 includes an analysis condition setting unit 42, a measurement control unit 43, a first analysis unit 44, a second analysis unit 45, and an analysis result output unit 46 as functional blocks. The first analysis unit 44 includes a first determination unit 441 and a second determination unit 442. In addition, the second analysis unit 45 also includes a first determination unit 451 and a second determination unit 452. The entity of the control / processing unit 4 is a personal computer, and each functional block is realized by executing an amino acid and acylcarnitine analysis program pre-installed in the computer by a processor. In addition, the control / processing unit 4 is connected to an input unit 5 and a display unit 6.

[0060] Here, specific examples are described for each of the confirming MRM transition used in the first analysis mode (first analysis confirming MRM transition) and the confirming MRM transition used in the second analysis mode (second analysis confirming MRM transition).

[0061] The first analysis mode can be used to analyze Figure 3 Isovalerylcarnitine having the molecular structure shown in FIG. 1 is preferably used. Isovalerylcarnitine is a substance that serves as an indicator of isovaleric acidemia, but among its isomers, there are metabolites derived from antibiotics also administered to newborns, such as pivaloylcarnitine. The hydrogen addition ion ([M+H] + ) has a mass-to-charge ratio of 246.15 (integer mass is 246), and the quantitative MRM transition of isovalerylcarnitine can be measured with the highest sensitivity as 246.15>84.95 (for detecting the MRM transition of the product ion with a mass-to-charge ratio of 84.95 generated from the precursor ion with a mass-to-charge ratio of 246.15. Integer mass is 246>85). However, since the product ion of the same MRM transition is also generated from pivaloylcarnitine, in the screening using only the quantitative MRM transition, even if only pivaloylcarnitine is contained in the liquid sample, it will become positive (false positive) in the screening.

[0062] In the first analysis mode, in addition to the quantitative MRM transition, the first analysis confirmation MRM transition (246.15>187.05. The integer mass is 246>187) is also used for MRM measurement, and the ratio of the measurement intensity of the first analysis confirmation MRM transition (the height or area of ​​the peak in the mass chromatogram. The same applies hereinafter) to the measurement intensity of the quantitative MRM transition is calculated. Figure 4 As shown in FIG. 1 , the ratio of the measured intensity for isovaleryl carnitine is 17.5%, whereas the ratio of the measured intensity for pivaloyl carnitine is 25.5%, which is a difference sufficient to distinguish the two. Therefore, in the first analysis mode, by comparing the ratio of the measured intensity calculated from the actual measurement of the liquid sample with the value of the ratio of the measured intensity for isovaleryl carnitine, it is determined whether the substance contained in the liquid sample is isovaleryl carnitine or pivaloyl carnitine.

[0063] The above-mentioned determination can also be performed by comparing the ratio of the above-mentioned determination intensity obtained by the determination of the actual sample itself with the ratio of the above-mentioned determination intensity obtained by the determination of the standard sample of the target substance, but in this case, the value is different according to the compound, and in addition, the value is also different according to the difference of the confirmation MRM transformation used. Therefore, compared to the value of the difference between the reference value of the ratio of the determination intensity and the ratio of the determination intensity calculated according to the determination of the actual sample to determine whether it is a target substance, it is preferred to use an index common to all target substances and common to all confirmation MRM transformations. Therefore, for example, it is preferred to use the following formula (1) to calculate the score value that is set to 100 as completely consistent according to the ratio of the determination intensity. By calculating the score value in this way, it is possible to determine all compounds with the same benchmark.

[0064] Score = 100 - (|(actual measured strength ratio of the sample) - (reference value of the measured strength ratio)| / (reference value of the measured strength ratio) × 100)...(1)

[0065] exist Figure 5 An example of determination based on the score value is shown in FIG. This is an example of distinguishing isovaleryl carnitine as the target substance and pivaloyl carnitine as an impurity. The ratio of the measurement intensity of the first analytical confirmation MRM transition to the measurement intensity of the quantitative MRM transition calculated based on the standard sample of isovaleryl carnitine is 20.44% (in addition, since the ratio is the same as Figure 4 The examples illustrated in the example are different MRM transitions used as examples of the first analytical confirmatory MRM transitions, so the ratio of the measured intensities is different from Figure 4In the case where the ratios of the measurement intensity of the first analytical confirmation MRM transition to the measurement intensity of the quantitative MRM transition calculated for two test specimens (test specimens 1 and 2) as actual samples are 20.48% (test specimen 1) and 28.21% (test specimen 2), respectively, the score value of test specimen 1 calculated based on these values ​​and the above formula (1) is 99.78, and the score value of test specimen 2 is 61.99. Here, for example, if the reference value of the score (the critical value of the index value) is set to 75, it can be determined that test specimen 1 contains isovaleryl carnitine and test specimen 2 does not contain isovaleryl carnitine. The reference value can be appropriately set according to the purpose of screening, the required accuracy, etc. For example, in the case of screening in a manner that does not miss positive detections, a lower reference value can be set. Specifically, for example, the reference value of the score can be set to an appropriate value of greater than 70 and less than 80.

[0066] The second parsing mode can be used to analyze Figure 6 Glutarylcarnitine (C5-DC) having the molecular structure shown in FIG. 1 is preferably used. Glutarylcarnitine is a marker substance for glutaric acidemia type I, and its mass-to-charge ratio is 275.14 (integer mass is 275). Among glutarylcarnitine, there is an acylcarnitine, namely hydroxyhexanoylcarnitine (C6-OH, mass-to-charge ratio is 275.17, integer mass is 275), which has the same integer mass although the composition formula is different. In general, a tandem quadrupole type mass spectrometer is mostly used in neonatal mass spectrometry screening, and such a mass spectrometer is difficult to identify substances with the same integer mass.

[0067] Therefore, in the second analysis mode, an MRM transition in which a product ion is generated from glutarylcarnitine as a target substance and a product ion is not generated from hydroxyhexanoylcarnitine is used as a confirmatory MRM transition (second analysis confirmatory MRM transition).

[0068] exist Figure 7 The product ion spectra of glutarylcarnitine (C5-DC) and hydroxycaproylcarnitine (C6-OH) are shown in FIG. 1 (positive ion mode. The mass-to-charge ratio of the precursor ion is 276.1). Comparison of these product ion spectra shows that a product ion with a mass-to-charge ratio of 87.0 was detected with high intensity from glutarylcarnitine, whereas a product ion with a mass-to-charge ratio of 87.0 was not detected from hydroxycaproylcarnitine. Figure 8, mass spectra obtained by performing MRM measurements on standard samples of glutarylcarnitine and hydroxyhexanoylcarnitine respectively using the quantitative MRM transition (276.1>85.0) and the above-mentioned MRM transition (276.1>87.1) that measure glutarylcarnitine with the highest intensity. It can be seen that, for the quantitative MRM transition (276.1>85.0), product ions are detected from both substances, whereas, for the above-mentioned MRM transition (276.1>87.1), only glutarylcarnitine is detected. Therefore, by using 276.1>87.1 as the second analytical confirmation MRM transition, it is possible to identify whether glutarylcarnitine or hydroxyhexanoylcarnitine is contained in the specimen. In this example, glutarylcarnitine can also be measured by MRM measurement using only the second analytical confirmation MRM transition, but the measurement sensitivity of such MRM transition is not necessarily high. Therefore, in the second analysis mode, first, determination is made using the quantitative MRM having high measurement sensitivity, and then determination is made using the above-mentioned second analysis confirmation MRM transition.

[0069] Below, refer to Figures 9 to 11 A process of analyzing amino acids and acylcarnitines contained in a liquid sample as an analysis target using the liquid chromatograph-mass spectrometer of the present embodiment will be described. Fig. 9 This is a flowchart showing the overall flow of the analysis method according to this embodiment.

[0070] First, a test body (blood) is collected from a newborn as a test subject to prepare a liquid sample (step 1). For example, the filter paper blood collected from the newborn can be extracted using water and ethanol to prepare the liquid sample (recovery of amino acids and acylcarnitines) as described in Non-Patent Document 1. The prepared liquid sample is placed in the automatic sampler 14.

[0071] After placing the liquid sample in the automatic sampler 14, when the user instructs to start the analysis, the analysis condition setting unit 42 displays a screen for setting the analysis conditions on the display unit 6. In this screen, a list of amino acids and acylcarnitines included in the compound DB 411 is displayed, and the user selects a substance (target substance) to be analyzed from this list (step 2). Alternatively, the user may select a pre-grouped compound (e.g., a target compound group for neonatal mass spectrometry screening) at the same time.

[0072] When the target substance is selected, the analysis condition setting unit 42 then allows the user to select one of the first analysis mode and the second analysis mode (step 3). Here, first, the case where the first analysis mode is selected will be described.

[0073] When the first parsing mode is selected ("Yes" in step 4), the first parsing mode is executed in the following process (step 5). Fig.10 This is a flowchart showing the flow in the first analysis mode of this embodiment.

[0074] The analysis condition setting unit 42 reads the quantitative MRM transition and the first analytical confirmation MRM transition of each target substance from the compound DB 411, and creates a method file that records a method of executing them in sequence for a predetermined time. Then, a batch file for measuring the liquid sample placed in the automatic sampler 14 according to the method is created and stored in the storage unit 41.

[0075] After the batch file is created, when the user instructs to start measurement, the measurement control unit 43 injects the liquid sample from the autosampler 14 into the syringe 13. The liquid sample injected into the syringe 13 is introduced into the ESI probe 201 together with the mobile phase by the FIA ​​method.

[0076] After the liquid sample introduced into the ESI probe 201 is ionized in the ionization chamber 20, it converges along the ion optical axis C in the ion lens 211 of the first intermediate vacuum chamber 21 and the ion guide 221 of the second intermediate vacuum chamber 22 and is transported to the front-stage quadrupole mass filter 231. In the front-stage quadrupole mass filter 231, ions of a mass-to-charge ratio set for the quantitative MRM transition of the initial target substance are selected as precursor ions. The precursor ions selected by the front-stage quadrupole mass filter 231 collide with CID gas molecules in the collision chamber 232 and break apart, and enter the rear-stage quadrupole mass filter 234. In the rear-stage quadrupole mass filter 234, ions of a mass-to-charge ratio set for the quantitative MRM transition of the initial target substance are selected as product ions. The ions selected by the rear-stage quadrupole mass filter 234 are then incident on the ion detector 235 and detected (step 11). The ion detector 235 detects ions for a predetermined time and outputs signal data corresponding to the intensity thereof to the control / processing unit 4. The control / processing unit 4 sequentially stores the signal data received from the ion detector 235 in the storage unit 41.

[0077] When the quantitative MRM transition measurement of the first target substance is completed, the first analytical confirmation MRM of the same substance is measured in the same manner as above (step 12). When the quantitative MRM transition and the first analytical confirmation MRM transition measurement are completed for all target substances, the measurement of the first liquid sample is completed.

[0078] When the measurement of the first liquid sample is completed, the quantitative MRM transition and the first analytical confirmation MRM transition are measured for each target substance for the second liquid sample in the same manner as described above. When the measurement of all liquid samples is completed, the measurement operation is terminated.

[0079] When the measurement is completed (or, in parallel with the measurement operation), in the control / processing unit 4, the first analysis unit 44 first prepares a mass spectrum for the measurement of quantitative MRM transition for each target substance for the initial liquid sample, and calculates the height (or area. The same applies below.) of its peak (step 13). When the peak height is calculated for all target substances, the first determination unit 441 reads the threshold value (Tta, Ttb, Ttc, ...) set for each target substance from the compound DB 411, and compares it with the peak height value of the mass spectrum of each target substance (step 14). Then, the target substance whose peak height value exceeds the threshold is extracted (step 15).

[0080] Next, the first analysis unit 44 prepares a mass spectrum of the measurement of the first analysis confirmation MRM transition for each target substance extracted by the first determination unit 441, finds the height of the peak, and calculates the ratio of the peak height value relative to the quantitative MRM transition (step 16). When the ratio of the peak height value is calculated for each target substance extracted by the first determination unit 441, the second determination unit 442 reads the reference value (Ra, Rb, Rc, ...) of the peak height ratio (measurement intensity ratio) set for each target substance from the compound DB 411, and calculates the score for each target substance by the above formula (1) (step 17). Then, the value of the score of each target substance is compared with the reference value (step 18), and the target substance whose score value exceeds the reference value is extracted (step 19).

[0081] When the above series of processing is completed for all liquid samples, the analysis result output unit 46 displays a list obtained by associating the liquid sample from which the target substance is extracted by the second determination unit 442, the target substance extracted from the liquid sample by the second determination unit 442, and the disease name corresponding to the target substance on the screen of the display unit 6 (output analysis results. Step 7).

[0082] Next, the case where the user selects the second analysis mode will be described. When the second analysis mode is selected ("No" in step 4), the second analysis mode is executed in the following process (step 6). Fig.11 This is a flowchart showing the flow in the second analysis mode of this embodiment.

[0083] The analysis condition setting unit 42 reads the quantitative MRM transition and the second analysis confirmation MRM transition of each target substance from the compound DB 411, and creates a method file that records the method of executing them in sequence for a predetermined time. Then, a batch file for measuring the liquid samples placed in the automatic sampler 14 according to the method is created and stored in the storage unit 41.

[0084] After the batch file is created, when the user instructs the start of the measurement, the measurement control unit 43 injects the liquid sample from the automatic sampler 14 into the syringe 13. The liquid sample injected into the syringe 13 is introduced into the ESI probe 201 together with the mobile phase by the FIA ​​method for mass spectrometry analysis. Regarding the process of mass spectrometry, as in the case where the first analysis mode is selected, first, the MRM measurement is performed using the quantitative MRM transition (step 21), and then the MRM measurement is performed using the confirmation MRM transition (step 22) (wherein, in the second analysis mode, the second analysis confirmation MRM transition is used instead of the first analysis confirmation MRM transition). These measurement processes are the same as the first analysis mode, so detailed descriptions are omitted.

[0085] When the measurement of all liquid samples is completed (or, in parallel with the measurement operation), in the control / processing unit 4, the second analysis unit 45 first prepares a mass spectrum at the time of the measurement of the quantitative MRM transition for each target substance for the initial liquid sample, and calculates the height of its peak (step 23). When the peak height is calculated for all target substances, the first determination unit 451 reads the threshold value (Tta, Ttb, Ttc, ...) set for each target substance from the compound DB 411, and compares it with the value of the peak height of the mass spectrum of the quantitative MRM transition of each target substance (step 24). Then, the target substance whose peak height value exceeds the threshold is extracted (step 25).

[0086] Next, the second analysis unit 45 prepares a mass spectrum at the time of the measurement of the second analysis confirmation MRM transition for each target substance extracted by the first determination unit 451, and calculates the height of its peak (step 26). When the peak height is calculated for each target substance extracted by the first determination unit 451, the second determination unit 452 reads the peak height threshold (Tqa, Tqb, Tqc, ...) set for each target substance from the compound DB 411, and compares it with the value of the peak height of the mass spectrum of the second analysis confirmation MRM transition of each target substance (step 27). Then, the target substance whose peak height value exceeds the threshold is extracted (step 28).

[0087] When the above series of processing is completed for all liquid samples, the analysis result output unit 46 displays the liquid sample from which the target substance is extracted by the second determination unit 452, the target substance extracted by the second determination unit 452 for the liquid sample, and a list obtained by associating the disease name corresponding to the target substance on the screen of the display unit 6 (output analysis results. Step 7).

[0088] In the present embodiment, as described above, in either the first analysis mode or the second analysis mode, the measurement intensity obtained by the quantitative MRM transition is first compared with the threshold value to determine the possibility of each target substance being contained in the liquid sample (first determination). In conventional mass spectrometry screening, the possibility of the target substance being contained in the analysis object sample is determined only by this determination, and therefore, it is not possible to distinguish between the case where the analysis object sample contains the target substance and the case where only impurities such as isomers detected together with the target substance by the first MRM transition exist but the target substance itself is not contained.

[0089] In the first analysis mode of the present embodiment, a threshold value related to the ratio of the peak height value (measurement intensity) of the mass spectrum of the quantitative MRM transition to the peak height value (measurement intensity) of the mass spectrum of the first analysis confirmation MRM transition is set, and for a target substance whose peak height value is determined (first determination) to exceed the threshold value (i.e., there is a possibility of being contained in the liquid sample), the ratio of the peak height value of the mass spectrum of the first analysis confirmation MRM transition to the peak height value of the mass spectrum of the quantitative MRM transition is further compared with other threshold values, thereby further determining whether the target substance is contained in the liquid sample (second determination). Even in the case where there is an impurity detected by the quantitative MRM transition together with the target substance, generally speaking, the value of the ratio of the measurement intensity obtained by the quantitative MRM transition to the measurement intensity obtained by the first analysis confirmation MRM transition is different between the target substance and the impurity. In the first analysis mode of the present embodiment, when it is determined in the first judgment that there is a possibility of containing the target substance, by comparing the above-mentioned ratio with the threshold value, it is further determined whether the liquid sample contains the target substance or the included substance (second judgment), thereby being able to determine whether the liquid sample contains the target substance with higher accuracy than before.

[0090] In addition, in the second analysis mode of the present embodiment, a second analysis confirmation MRM transition is set in which an impurity that can be detected together with the target substance in the MRM measurement using the quantitative MRM transition is not detected, and for each target substance that is determined to be likely to be contained in the liquid sample in the first determination, a determination is further made as to whether the target substance is contained in the liquid sample based on the peak height value (measurement intensity) of the mass spectrum of the second analysis confirmation MRM transition (second determination). In the second analysis mode of the present embodiment, based on the detection of the target substance in the MRM measurement using the second analysis confirmation MRM transition in which the impurity is not detected, it is re-determined (second determination) that the target substance is contained in the liquid sample, so that it is possible to determine whether the target substance is contained in the liquid sample with higher accuracy than before.

[0091] The above embodiment is an example and can be appropriately changed according to the main purpose of the present invention. In the above embodiment, it is assumed that a large number of test bodies (samples) are analyzed. In order to improve the measurement efficiency, the sample is introduced into the mass spectrometer 2 by the FIA ​​method without using a column, but in the case of a small number of test bodies, the components in the sample can be separated by using a column and then introduced into the mass spectrometer 2. By separating the substances contained in the sample to be analyzed by using a column before MRM measurement, it is possible to determine with higher accuracy whether the sample contains the target substance.

[0092] In the above embodiment, it is assumed that a liquid chromatograph-mass spectrometer is used using the automatic sample injector 14 provided in the liquid chromatograph 1, but a sample may be directly introduced into the ESI probe 201 of the mass spectrometer 2, and in this case, the liquid chromatograph 1 may not be used. In addition, a gas chromatograph may be used instead of a liquid chromatograph depending on the characteristics of the sample to be analyzed.

[0093] In addition, in the above embodiment, the user selects either the first analysis mode or the second analysis mode, but the information obtained by associating each compound with the analysis mode may be stored in the compound DB 411, and the analysis mode may be automatically determined according to the compound (target substance) selected by the user. In addition, in the above embodiment, either the first analysis mode or the second analysis mode is selected, but the two analysis modes may be executed simultaneously.

[0094] In the above embodiment, an example is shown in which the first analysis confirmation MRM transition and the second analysis confirmation MRM transition are recorded in the compound DB 411 one by one for each compound ( Figure 2 ), but one or both of them may be set to multiple. In this case, a reference value of a score related to the ratio of the measured intensity is set for each of the multiple first analytical confirmation MRM transitions. For multiple second analytical confirmation MRM transitions, thresholds may be set separately or a common threshold may be set. Furthermore, the first analytical confirmation MRM transition and the second analytical confirmation MRM transition may be the same or different from each other.

[0095] [Way]

[0096] It is obvious to those skilled in the art that the above-described exemplary embodiments are specific examples of the following aspects.

[0097] (Item 1)

[0098] An analysis method for amino acids and / or acylcarnitines according to one embodiment of the present invention, wherein:

[0099] For each of the one or more target substances that are amino acids or acylcarnitines, a first MRM transition and a second MRM transition that are a group of a mass-to-charge ratio of a precursor ion and a mass-to-charge ratio of a product ion, a first reference value related to a measured intensity of the first MRM transition, and a second reference value related to a ratio of the measured intensity of the first MRM transition to the measured intensity of the second MRM transition are set,

[0100] By performing MRM measurement on the analysis target sample using the first MRM transition and the second MRM transition set for each target substance, a first intensity as a measurement intensity of the first MRM transition and a second intensity as a measurement intensity of the second MRM transition are measured for each target substance,

[0101] performing a first determination for each of the target substances, wherein the first determination is performed by comparing the first intensity value with the first reference value to determine whether or not there is a possibility that the target substance is contained in the analysis object sample,

[0102] A second judgment is performed on the target substance that is determined in the first judgment to be likely to be contained in the analysis object sample. In the second judgment, whether the analysis object sample contains the target substance is determined by comparing the ratio of the first intensity value to the second intensity value with the second reference value.

[0103] (Item 3)

[0104] Another aspect of the present invention relates to a method for analyzing amino acids and / or acylcarnitines, wherein:

[0105] For each of the one or more target substances that are amino acids or acylcarnitines, a first MRM transition that is a set of a mass-to-charge ratio of a precursor ion and a mass-to-charge ratio of a product ion, a reference value related to a measurement intensity of the first MRM transition, and a second MRM transition in which an impurity that can be detected together with the target substance in an MRM measurement using the first MRM transition is not detected are set,

[0106] performing MRM measurement on the analysis target sample using the first MRM transition and the second MRM transition set for each of the target substances, thereby acquiring a first intensity value as the measurement intensity of the first MRM transition and a second intensity value as the measurement intensity of the second MRM transition for each of the target substances,

[0107] performing a first determination for each of the target substances, wherein the first determination is performed by comparing the first intensity value with the reference value to determine whether or not there is a possibility that the target substance is contained in the analysis object sample,

[0108] A second determination is performed on the target substance determined in the first determination to be possibly contained in the analysis target sample. In the second determination, whether the analysis target substance is contained in the analysis target sample is determined based on the value of the second intensity.

[0109] In the analysis methods of amino acids and / or acylcarnitines of the first and third items, a first MRM transition and a second MRM transition, and a reference value related to the measurement intensity of the first MRM transition (the first reference value in the first item) are set for each of the one or more target substances that are amino acids or acylcarnitines, and the MRM measurement of the analysis object sample is performed using the first MRM transition and the second MRM transition for each target substance. For the first MRM transition, it is sufficient to set an MRM transition that can measure the target substance with high sensitivity in the same manner as the MRM transition used in the conventional mass spectrometry screening. In addition, for the reference value related to the measurement intensity of the first MRM transition (same as above), it is sufficient to set a value equivalent to the critical value used in the conventional mass spectrometry screening.

[0110] In the analysis methods of amino acids and / or acylcarnitines of the first and third items, first, for each target substance, the measured intensity obtained by the first MRM transition is compared with a reference value, thereby determining the possibility that the target substance is contained in the sample to be analyzed (first determination). In conventional mass spectrometry screening, the possibility that the target substance is contained in the sample to be analyzed is determined only by this determination, and therefore, it is not possible to distinguish between the case where the target substance is contained in the sample to be analyzed and the case where only impurities such as isomers detected together with the target substance by the first MRM transition exist but the target substance itself is not contained.

[0111] In the analysis method of amino acids and / or acylcarnitines of item 1, a second reference value related to the ratio of the measurement intensity of the first MRM transition to the measurement intensity of the second MRM transition is set, and for the target substance determined (first determination) to be likely to be contained in the sample to be analyzed, the ratio of the first intensity value to the second intensity value is further compared with the second reference value, thereby determining whether the target substance is contained in the sample to be analyzed (second determination). Even when there is a foreign substance detected by the first MRM transition together with the target substance, generally, the ratio of the measurement intensity obtained by the first MRM transition to the measurement intensity obtained by the second MRM transition is different between the target substance and the foreign substance. In the analysis method of amino acids and / or acylcarnitines of item 1, when it is determined in the first determination that the target substance is likely to be contained, by comparing the above ratio with the second reference value, it is determined whether the sample to be analyzed contains the target substance or the foreign substance (second determination), so it is possible to determine whether the sample to be analyzed contains the target substance with higher accuracy than before.

[0112] In the analysis method of amino acids and / or acylcarnitines of item 3, a second MRM transition is set in which a foreign substance that can be detected together with the target substance in the MRM measurement using the first MRM transition is not detected, and for each target substance that is determined in the first determination to be likely to be contained in the sample to be analyzed, a determination (second determination) is made as to whether the target substance is contained in the sample to be analyzed, based on the measurement intensity (second intensity value) obtained using the second MRM transition. In the analysis method of amino acids and / or acylcarnitines of item 3, based on the detection of the target substance in the MRM measurement using the second MRM transition in which the foreign substance is not detected, it is determined (second determination) that the target substance is contained in the sample to be analyzed, and therefore it is possible to determine whether the target substance is contained in the sample to be analyzed with higher accuracy than before.

[0113] (Item 2)

[0114] The method for analyzing amino acids and / or acylcarnitines according to Item 2 is the method for analyzing amino acids and / or acylcarnitines according to Item 1, wherein

[0115] In the second determination, the score of each of the target substances is calculated using the following formula:

[0116] Score = 100 - (|(actual measured strength ratio of the sample) - (reference value of the measured strength ratio)| / (reference value of the measured strength ratio) × 100)...(1)

[0117] By comparing the calculated score value with the second reference value, it is determined whether the target substance is contained in the analysis object sample.

[0118] In the analysis method of amino acids and / or acylcarnitines according to item 2, the second determination can be performed on all target substances using a common criterion.

[0119] (Item 4)

[0120] The method for analyzing amino acids and / or acylcarnitines according to item 4 is the method for analyzing amino acids and / or acylcarnitines according to any one of items 1 to 3, wherein

[0121] The MRM measurement is performed after substances contained in the sample to be analyzed are separated from each other using a column of a chromatograph.

[0122] In the analysis method of amino acids and / or acylcarnitines according to Item 4, substances contained in the sample to be analyzed are separated from each other before MRM measurement, so whether or not the sample contains the target substance can be determined with higher accuracy.

[0123] (Item 5)

[0124] The method for analyzing amino acids and / or acylcarnitines according to item 5 is the method for analyzing amino acids and / or acylcarnitines according to any one of items 1 to 3, wherein

[0125] The target substance is an amino acid selected from phenylalanine, leucine, isoleucine, methionine, citrulline and tyrosine, or free carnitine (C0), acetyl carnitine (C2), propionyl carnitine (C3), butyryl carnitine to palmitoyl carnitine (C4 to C18), isovaleryl carnitine, 2-methylbutyryl carnitine, pivaloyl carnitine (C5), methyl crotonyl carnitine (C5:1), glutaryl carnitine (C5-DC), 3-hydroxyisovaleryl carnitine, 3-hydroxy -Acylcarnitine selected from the group consisting of 2-methylbutyryl-carnitine (C5-OH), octanoylcarnitine (C8), decanoylcarnitine (C10), dodecanoylcarnitine (C12), tetradecenoylcarnitine (C14:1), tetradecanoylcarnitine (C14), palmitoylcarnitine (C16), stearoylcarnitine (C18), octadecenoylcarnitine (C18:1), hydroxyhexadecenoylcarnitine (C16-OH) and hydroxyoctadecenoylcarnitine (C18:1-OH).

[0126] The target substance in the method for analyzing amino acids and / or acylcarnitines according to Item 5 is an amino acid or acylcarnitine associated with a neonatal disease, and thus can be preferably used for neonatal mass spectrometry screening.

[0127] Description of Reference Numerals

[0128] 1: Liquid chromatograph; 11: Mobile phase container; 12: Pump; 13: Syringe; 14: Autosampler; 2: Mass spectrometer; 20: Ionization chamber; 201: ESI probe; 21: First intermediate vacuum chamber; 211: Ion lens; 212: Separator; 22: Second intermediate vacuum chamber; 221: Ion guide; 23: Analysis chamber; 231: Pre-quadrupole mass filter; 232: Collision chamber; 233: Ion guide; 234 : Post-stage quadrupole mass filter; 235: ion detector; 4: control / processing unit; 41: storage unit; 411: compound DB; 42: analysis condition setting unit; 43: measurement control unit; 44: first analysis unit; 441: first determination unit; 442: second determination unit; 45: second analysis unit; 451: first determination unit; 452: second determination unit; 46: analysis result output unit; 5: input unit; 6: display unit; C: ion optical axis.

Claims

1. A method for analyzing amino acids and / or acylcarnitines, wherein: For each of the one or more target substances that are amino acids or acylcarnitines, a first MRM transition and a second MRM transition that are a group of a mass-to-charge ratio of a precursor ion and a mass-to-charge ratio of a product ion, a first reference value related to a measured intensity of the first MRM transition, and a second reference value related to a ratio of the measured intensity of the first MRM transition to the measured intensity of the second MRM transition are set, By performing MRM measurement on the analysis target sample using the first MRM transition and the second MRM transition set for each target substance, a first intensity as a measurement intensity of the first MRM transition and a second intensity as a measurement intensity of the second MRM transition are measured for each target substance, performing a first determination for each of the target substances, wherein the first determination is performed by comparing the first intensity value with the first reference value to determine whether or not there is a possibility that the target substance is contained in the analysis object sample, A second judgment is performed on the target substance that is determined in the first judgment to be likely to be contained in the analysis object sample. In the second judgment, whether the analysis object sample contains the target substance is determined by comparing the ratio of the first intensity value to the second intensity value with the second reference value.

2. The method for analyzing amino acids and / or acylcarnitines according to claim 1, wherein: In the second determination, the score of each of the target substances is calculated using the following formula: Score = 100 - (|(actual measured strength ratio of the sample) - (reference value of the measured strength ratio)| / (reference value of the measured strength ratio) × 100)...(1) By comparing the calculated score value with the second reference value, it is determined whether the target substance is contained in the analysis object sample.

3. A method for analyzing amino acids and / or acylcarnitines, wherein: For each of the one or more target substances that are amino acids or acylcarnitines, a first MRM transition that is a set of a mass-to-charge ratio of a precursor ion and a mass-to-charge ratio of a product ion, a reference value related to a measurement intensity of the first MRM transition, and a second MRM transition in which an impurity that can be detected together with the target substance in an MRM measurement using the first MRM transition is not detected are set, By performing MRM measurement on the analysis target sample using the first MRM transition and the second MRM transition set for each target substance, a first intensity as a measurement intensity of the first MRM transition and a second intensity as a measurement intensity of the second MRM transition are measured for each target substance, performing a first determination for each of the target substances, wherein the first determination is performed by comparing the first intensity value with the reference value to determine whether or not there is a possibility that the target substance is contained in the analysis object sample, A second determination is performed on the target substance determined in the first determination to be possibly contained in the analysis target sample. In the second determination, whether the analysis target substance is contained in the analysis target sample is determined based on the value of the second intensity.

4. The method for analyzing amino acids and / or acylcarnitines according to claim 1 or 3, wherein: The MRM measurement is performed after substances contained in the sample to be analyzed are separated from each other using a column of a chromatograph.

5. The method for analyzing amino acids and / or acylcarnitines according to claim 1 or 3, wherein: The target substance is an amino acid selected from phenylalanine, leucine, isoleucine, methionine, citrulline and tyrosine, or free carnitine (C0), acetyl carnitine (C2), propionyl carnitine (C3), butyryl carnitine to palmitoyl carnitine (C4 to C18), isovaleryl carnitine, 2-methylbutyryl carnitine, pivaloyl carnitine (C5), methyl crotonyl carnitine (C5:1), glutaryl carnitine (C5-DC), 3-hydroxyisovaleryl carnitine, 3-hydroxy -Acylcarnitine selected from the group consisting of 2-methylbutyryl-carnitine (C5-OH), octanoylcarnitine (C8), decanoylcarnitine (C10), dodecanoylcarnitine (C12), tetradecenoylcarnitine (C14:1), tetradecanoylcarnitine (C14), palmitoylcarnitine (C16), stearoylcarnitine (C18), octadecenoylcarnitine (C18:1), hydroxyhexadecenoylcarnitine (C16-OH) and hydroxyoctadecenoylcarnitine (C18:1-OH).

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