Method for analyzing amino acid and / or acylcarnitine

Abstract

To determine whether amino acid or acylcarnitine is contained in an analysis object sample with high accuracy.SOLUTION: A method for analyzing amino acid and acylcarnitine includes: setting first and second MRM transitions for a target substance, a first reference value of a measured intensity of the first MRM transition and a second reference value of a measured intensity ratio between the measured intensities of the first MRM transition and the second MRM transition; performing MRM measurements of an analysis object sample using the first and second MRM transitions to measure a first intensity of the first MRM transition and a second intensity of the second MRM transition (steps 11 and 12); comparing a value of the first intensity with the first reference value to perform first determination of whether the target substance is contained (step 14); and comparing, for the target substance determined to be contained in the analysis object sample in the first determination, a ratio between a value of the first intensity and a value of the second intensity with the second reference value to perform second determination of whether or not the target substance is contained (step 18).SELECTED DRAWING: Figure 10

Description

[Technical field]

[0001] The present invention relates to a method for analyzing amino acids and / or acylcarnitines contained in a sample to be analyzed. [Background technology]

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

[0003] In these newborn mass screening tests, multiple specific types of amino acids and acylcarnitines are associated with different diseases, and cutoff values ​​are set for each disease. Screening for each disease is performed by comparing the amounts and concentrations of these specific types of amino acids and acylcarnitines contained in a blood sample taken from a newborn with the cutoff values ​​corresponding to each substance.

[0004] As disclosed in Non-Patent Document 1, in screening using a tandem mass spectrometer (tandem mass screening), multiple reaction monitoring (MRM) measurements are performed using MRM transitions preset for each of the specific types of amino acids and acylcarnitines, and each substance is quantified, and the quantitative values ​​are compared with the cutoff values. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Yosuke Shigematsu, "The Reality of Newborn Mass Screening by Mass Spectrometry", Journal of the Mass Spectrometry Society of Japan, Vol. 64, No. 4, 2016, pp. 127-131 [Non-Patent Document 2] Takao Ishikawa, Michiko Tezuka, Miwa Yoshinaga, Shosuke Nomachi, Nobuhito Hosoumi, and Koichi Yano, "Effect of waxing on leucine and isoleucine quantification in tandem mass screening," Journal of the Japanese Society for Mass Screening, Vol. 30(3), 2020, pp.31(257)-43(269) [Non-Patent Document 3] Saito, Y., Wada, Y., Maekawa, M., and Kure, S., "Three cases of false positives for medium-chain acyl-CoA dehydrogenase deficiency in neonatal mass screening due to essential oils," Japanese Journal of Neonatal Screening, Vol. 30(2), 2020, pp. 102(196) Summary of the Invention [Problem to be solved by the invention]

[0006] Conventionally, in newborn mass screening tests, since it is necessary to analyze a large number of specimens (analyte samples) quickly, measurements are performed using flow injection analysis (FIA), in which each sample set in an autosampler is sequentially introduced into a mass spectrometer together with the mobile phase without passing through a liquid chromatograph (LC) column. In flow injection analysis, each sample is introduced into a mass spectrometer without component separation using a column, so various substances contained in the sample are simultaneously subjected to MRM measurement.

[0007] For example, as described in Non-Patent Document 1, C5 acylcarnitine (acylcarnitine having an acyl group with 5 carbon atoms) includes four isomers, pivaloylcarnitine, 2-methylbutyrylcarnitine, and n-valerylcarnitine, in addition to isovalerylcarnitine. Of these, isovalerylcarnitine and 2-methylbutyrylcarnitine are metabolites related to diseases, while pivaloylcarnitine is a metabolite derived from a drug (antibacterial agent) administered to a neonate. Therefore, even if the quantitative value in the MRM measurement of C5 acylcarnitine exceeds the cutoff value and is determined to be positive in screening, it may be a false positive due to pivaloylcarnitine derived from the drug.

[0008] The above are examples of false positives caused by substances (endogenous substances) contained in the sample itself to be analyzed, but substances (exogenous substances) mixed in during sample collection or preparation may also cause false positives. For example, Non-Patent Document 2 describes that in a screening test for maple syrup urine disease, diethylene glycol monoethyl ether contained in a waxing agent is mixed in during sample preparation and detected together with leucine and isoleucine, resulting in a false positive. In addition, Non-Patent Document 3 describes that in a screening test for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, a component contained in an essential oil used in aromatherapy for postpartum mothers is mixed in during sample collection and is measured together with octanoylcarnitine (C8) and decanoylcarnitine (C10), resulting in a false positive. Although these exogenous substances are not isomers of the amino acids or acylcarnitines being analyzed, false positives can still occur due to substances that have the same nominal mass as the amino acids or acylcarnitines being analyzed, because the tandem quadrupole mass spectrometers used in many neonatal mass screening tests have difficulty distinguishing between ions with the same nominal mass (mass-to-charge ratio).

[0009] Therefore, in the past, when a screening test was positive, additional tests were performed using other analytical methods, such as column-based chromatography mass spectrometry, and detailed analysis was performed to confirm the presence or absence of the disease. Generally, newborn screening tests involve taking blood samples from newborns to prepare samples, so performing such additional tests places a heavy physical burden on the newborn. In addition, a positive screening test places a heavy psychological burden on the parents of the newborn. Furthermore, the more false positives there are, the more samples need to be tested, which increases the workload of the analyst and puts a greater burden on them. Therefore, there is a demand for technology that can reduce false positives in screening tests more than ever before.

[0010] Although the problems in the conventional technology have been explained here by taking specific amino acids and acylcarnitines as examples, similar problems exist when performing screening tests to measure other types of amino acids and acylcarnitines.

[0011] The problem to be solved by the present invention is to determine with higher accuracy whether or not a sample to be analyzed contains specific types of amino acids or acylcarnitines contained in the sample to be analyzed, in an analytical method for MRM measurement of these substances. [Means for solving the problem]

[0012] The first aspect of the method for analyzing amino acids and acylcarnitines according to the present invention, which has been made to solve the above problems, is for each of one or more target substances which are amino acids or acylcarnitines, a first MRM transition and a second MRM transition each being a pair of a mass-to-charge ratio of a precursor ion and a mass-to-charge ratio of a product ion, a first reference value relating to the measured intensity of the first MRM transition, and a second reference value relating to the ratio of the measured intensity of the first MRM transition to the measured intensity of the second MRM transition; performing MRM measurement of an analyte sample using the first MRM transition and the second MRM transition set for each of the target substances, thereby measuring a first intensity, which is a measurement intensity of the first MRM transition, and a second intensity, which is a measurement intensity of the second MRM transition, for each of the target substances; performing a first determination for each of the target substances in order to determine whether or not the target substance is likely to be contained in the analysis target sample by comparing the first intensity value with the first reference value; A second determination is made to determine whether or not the target substance is contained in the analysis target sample by comparing a ratio of the first intensity value to the second intensity value with the second reference value for the target substance determined in the first determination as possibly being contained in the analysis target sample. It is something.

[0013] In addition, a second aspect of the method for analyzing amino acids and acylcarnitines according to the present invention, which has been made to solve the above problems, is For each of one or more target substances which are amino acids or acylcarnitines, a first MRM transition which is a pair of a mass-to-charge ratio of a precursor ion and a mass-to-charge ratio of a product ion, a reference value for the measurement intensity of the first MRM transition, and a second MRM transition which does not detect impurities which may be detected together with the target substance in an MRM measurement using the first MRM transition, are set; performing MRM measurement of an analyte sample using the first MRM transition and the second MRM transition set for each of the target substances, thereby measuring a first intensity, which is a measurement intensity of the first MRM transition, and a second intensity, which is a measurement intensity of the second MRM transition, for each of the target substances; performing a first determination for each of the target substances in order to determine whether or not the target substance is likely to be contained in the analysis target sample by comparing the value of the first intensity with the reference value; A second determination is made on the basis of the second intensity value as to whether or not the target substance is contained in the analysis target sample, for the target substance determined in the first determination as possibly being contained in the analysis target sample. It is something. Effect of the Invention

[0014] In the first and second aspects of the present invention, a first MRM transition and a second MRM transition, and a reference value for the measurement intensity of the first MRM transition (first reference value in the first aspect) are set for each of one or more target substances that are amino acids or acylcarnitines, and an analysis sample is subjected to MRM measurement using the first MRM transition and the second MRM transition for each target substance. The first MRM transition may be set to one that can measure the target substance with high sensitivity, similar to the MRM transitions used in conventional mass screening. In addition, the reference value for the measurement intensity of the first MRM transition (same as above) may be set to one that corresponds to the cutoff value used in conventional mass screening.

[0015] In the first and second aspects of the present invention, first, for each target substance, the measurement intensity obtained by the first MRM transition is compared with a reference value to determine the possibility that the target substance is contained in the analysis target sample (first determination). Conventional mass screening determined the possibility that the target substance is contained in the analysis target sample only by this determination, and therefore could not distinguish between a case in which the target substance is contained in the analysis target sample and a case in which only impurities such as isomers detected together with the target substance in the first MRM transition are present and the target substance itself is not contained.

[0016] In the first aspect of the present invention, 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 is set, and for a target substance determined (first determination) to possibly be contained in the sample to be analyzed, the ratio of the first intensity value to the second intensity value is compared with the second reference value to determine (second determination) whether the target substance is contained in the sample to be analyzed. Even if there is an impurity substance detected together with the target substance in the first MRM transition, the target substance and the impurity usually have different values ​​for the ratio of the measured intensity by the first MRM transition to the measured intensity by the second MRM transition. In the first aspect of the present invention, when it is determined in the first determination that the target substance may be contained, the ratio is compared with the second reference value to determine (second determination) whether the target substance is contained in the sample to be analyzed. Therefore, it is possible to determine whether the target substance is contained in the sample to be analyzed with higher accuracy than before.

[0017] In a second aspect of the present invention, a second MRM transition is set in which impurity substances that may be detected together with the target substance are not detected in an MRM measurement using the first MRM transition, and for each target substance determined in the first determination to be possibly contained in the analysis sample, a determination is made (second determination) as to whether or not the target substance is contained in the analysis sample based on the measurement intensity (second intensity value) by the second MRM transition. In the second aspect of the present invention, based on the detection of a target substance in an MRM measurement using the second MRM transition in which impurity substances are not detected, it is determined (second determination) that the target substance is contained in the analysis sample, so that it is possible to determine with higher accuracy than before whether or not the target substance is contained in the analysis sample. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a diagram showing the configuration of the main parts of a liquid chromatograph mass spectrometer used in one embodiment of the method for analyzing amino acids and acylcarnitines according to the present invention. [Diagram 2] 3 shows an example of a compound database in this embodiment. [Diagram 3] 5A to 5C are diagrams for explaining a first analysis mode according to the present embodiment. [Figure 4] FIG. 4 is another diagram for explaining the first analysis mode of the embodiment. [Diagram 5] FIG. 11 is yet another diagram for explaining the first analysis mode of the embodiment. [Figure 6] 5A to 5C are diagrams for explaining a second analysis mode according to the present embodiment. [Figure 7] FIG. 11 is another diagram for explaining the second analysis mode of the embodiment. [Figure 8] FIG. 11 is still another diagram for explaining the second analysis mode of the embodiment. [Figure 9] 1 is a flowchart illustrating the steps of the method for analyzing amino acids and acylcarnitines according to the present embodiment. [Figure 10] 3 is a flowchart for explaining the procedure of a first analysis mode in the method for analyzing amino acids and acylcarnitines of the present embodiment. [Figure 11] 4 is a flowchart for explaining the procedure of a second analysis mode in the method for analyzing amino acids and acylcarnitines of the present embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] An embodiment of the method for analyzing amino acids and acylcarnitines according to the present invention will be described with reference to the drawings. The method for analyzing amino acids and acylcarnitines according to the present embodiment is used, for example, to perform a newborn mass screening test.

[0020] 1 is a diagram showing the configuration of the main components of a liquid chromatograph mass spectrometer used in this embodiment. The liquid chromatograph mass spectrometer of this embodiment includes a liquid chromatograph 1, a mass spectrometer 2, and a control / processing unit 4 that controls the operations of these components.

[0021] The liquid chromatograph 1 includes a mobile phase container 11 in which a mobile phase is stored, a pump 12 that draws in the mobile phase (solvent) and delivers it at a constant flow rate, an injector 13 that injects a liquid sample into the mobile phase, and an autosampler 14 connected to the injector 13. Although a liquid chromatograph has a column for separating components in a sample, this embodiment does not use a column, and instead introduces the liquid sample together with the mobile phase into the mass spectrometer 2 by flow injection analysis (FIA).

[0022] The mass spectrometer 2 comprises an ionization chamber 20 which is at approximately 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, from the ionization chamber 20 side, there are provided a first intermediate vacuum chamber 21, a second intermediate vacuum chamber 22, and an analysis chamber 23, in that order, and the structure is a multi-stage differential pumping system in which the degree of vacuum increases in this order.

[0023] An electrospray ionization (ESI) probe 201 that applies an electric charge to a sample solution and sprays it is installed in the ionization chamber 20. The ionization chamber 20 and a first intermediate vacuum chamber 21 in the rear stage are connected via a thin-diameter heated capillary (desolvation tube) 202.

[0024] An ion lens 211 composed of multiple ring electrodes is disposed in the first intermediate vacuum chamber 21. The ion lens 211 converges the flight path of the ions along the ion optical axis C (the central axis in the flight direction of the ions). The first intermediate vacuum chamber 21 and the second intermediate vacuum chamber 22 are separated by a skimmer 212 having a small hole at the top.

[0025] An ion guide 221 composed of a plurality of rod electrodes is disposed in the second intermediate vacuum chamber 22. Like the ion lens 211, the ion guide 221 also converges the flight path of the ions along the ion optical axis C. The second intermediate vacuum chamber 22 and the analysis chamber are separated by a partition wall having small holes formed therein.

[0026] The analysis chamber 23 is provided with a front-stage quadrupole mass filter 231, a collision cell 232, a rear-stage quadrupole mass filter 234, and an ion detector 235. An ion guide 233 is provided inside the collision cell 232. A collision-induced dissociation (CID) gas is introduced into the collision cell 232 from a gas source (not shown).

[0027] The mass spectrometer 2 can perform selected ion monitoring (SIM) measurements, MS / MS scan (product ion scan) measurements, multiple reaction monitoring (MRM) measurements, etc. In SIM measurements, 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.

[0028] In the MS / MS scan measurement and the MRM measurement, both the front quadrupole mass filter 231 and the rear quadrupole mass filter 234 function as mass filters. The front quadrupole mass filter 231 passes only ions having a mass-to-charge ratio set as precursor ions. In addition, CID gas is supplied to the inside of the collision cell 232, and the precursor ions are accelerated and introduced into the collision cell 232, where the precursor ions collide with the CID gas to promote fragmentation of the precursor ions. In the MS / MS scan measurement, the mass-to-charge ratio of the ions passing through the rear quadrupole mass filter 234 is scanned, and in the MRM measurement, the mass-to-charge ratio of the ions passing through the rear quadrupole mass filter 234 is fixed to pass only product ions having a specific mass-to-charge ratio.

[0029] The control / processing unit 4 has a storage unit 41. The storage unit 41 stores a compound database (compound DB) 411 that stores information including the measurement conditions and analysis methods of a plurality of known amino acids and acylcarnitines (preparatory step, step 0. See FIG. 9). FIG. 2 shows an example of information stored in the compound DB 411. The measurement conditions stored in the compound DB 411 include, for example, the compound name, the magnitude of collision energy when the compound is measured, the mass-to-charge ratio of the quantitative MRM transition ion used in the first judgment of the first analysis and the second analysis described later, the reference value of the measured intensity of the ion 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, the reference value regarding the ratio of the measured intensity of the ion by the first analysis confirmation MRM transition to the measured intensity of the ion by the quantitative MRM transition ion, the mass-to-charge ratio of the confirmation MRM transition (second analysis confirmation MRM transition) used in the second analysis, and the threshold value of the measured intensity of the ion by the second analysis confirmation MRM transition. The measurement intensities of the ions by the quantitative MRM measurement, the ratio of the measurement intensities, and the measurement intensities of the ions by the second analytical confirmation MRM transition are index values ​​in the newborn mass screening test. The memory unit 41 also stores information that associates each compound with a disease associated with the compound.

[0030] Examples of amino acids included in the compound DB411 are phenylalanine, leucine, isoleucine, methionine, citrulline, and tyrosine. Examples of acylcarnitines included in the compound DB411 are free carnitine (C0), acetylcarnitine (C2), propionylcarnitine (C3), butyrylcarnitine to palmitoylcarnitine (C4 to C18), isovalerylcarnitine, 2-methylbutyrylcarnitine, pivaloylcarnitine (C5), tiglylcarnitine (C5:1), glutarylcarnitine (C5-DC), 3-hydroxyisovalerylcarnitine, 3-hydroxy-2-methylbutyryl- Carnitine (C5-OH), octanoylcarnitine (C8), decanoylcarnitine (C10), dodecanoylcarnitine (C12), tetradecenoylcarnitine (C14:1), tetradecanoylcarnitine (C14), palmitoylcarnitine (C16), stearylcarnitine (C18), octadecenoylcarnitine (C18:1), hydroxyhexadecanoylcarnitine (C16-OH), hydroxyoctadecenoylcarnitine (C18:1-OH).

[0031] The above MRM transitions are each a combination of the mass-to-charge ratio of a precursor ion generated from the compound (amino acid or acylcarnitine) and the mass-to-charge ratio of a product ion generated by fragmentation of the precursor ion. That is, in the MRM measurement, ions having the mass-to-charge ratio of the precursor ion set in the MRM transition are selected by the front-stage quadrupole mass filter 231, and product ions are generated by fragmentation in the collision cell 232, and then ions having the mass-to-charge ratio of the product ion set in the MRM transition are selected by the rear-stage quadrupole mass filter 234 and detected by the ion detector 235. For the quantitative MRM transition, one that can detect the compound (amino acid or acylcarnitine) with the highest sensitivity is usually selected.

[0032] The first analytical confirmation MRM transition is selected to have the second highest measurement sensitivity after the quantitative MRM transition. The first analytical confirmation MRM transition is associated with a reference score value related to the ratio of the measured intensity of the first analytical confirmation MRM transition to the measured intensity of the quantitative MRM transition. In the first analysis mode, the measured intensity of the ion by the quantitative MRM transition is the first index value, and this score is the second index value.

[0033] The second analysis confirmation MRM transition is selected so that it is not generated from compounds (impurity compounds) that may be detected simultaneously when the compound is measured using the quantitative MRM transition. The second analysis confirmation MRM transition is associated with information on the threshold value of the measurement intensity of the second analysis confirmation MRM transition. This threshold is set, for example, to a value that can be distinguished from noise. If noise does not need to be taken into consideration, the threshold may be set to 0 (i.e., the second threshold does not need to be set). In the second analysis mode, the measurement intensity of the ion by the quantitative MRM transition becomes the first index value, and the measurement intensity of the second analysis confirmation MRM transition becomes the index value in the newborn mass screening test.

[0034] The control and processing unit 4 includes, as functional blocks, 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. The first analysis unit 44 includes a first judgment unit 441 and a second judgment unit 442. The second analysis unit 45 also includes a first judgment unit 451 and a second judgment unit 452. The actual entity of the control and processing unit 4 is a personal computer, and each functional block is realized by executing an amino acid and acylcarnitine analysis program preinstalled in the computer with a processor. Furthermore, an input unit 5 and a display unit 6 are connected to the control and processing unit 4.

[0035] Here, a specific example of each of the confirmation MRM transitions used in the first analysis mode (first analysis confirmation MRM transitions) and the confirmation MRM transitions used in the second analysis mode (second analysis confirmation MRM transitions) will be described.

[0036] The first analysis mode can be suitably used, for example, when analyzing isovalerylcarnitine, which has the molecular structure shown in FIG. 3. Isovalerylcarnitine is a substance that is an indicator of isovaleric acidemia, and its isomers include pivaloylcarnitine, which is a metabolite derived from an antibacterial drug that is also administered to newborns. The hydrogen adduct ion ([M+H] + ) has a mass-to-charge ratio of 246.15 (integer mass is 246), and the quantitative MRM transition that can measure isovalerylcarnitine with the highest sensitivity is 246.15>84.95 (an MRM transition that detects a product ion with a mass-to-charge ratio of 84.95 generated from a precursor ion with a mass-to-charge ratio of 246.15. Integer mass is 246>85). However, since a product ion with the same MRM transition is also generated from pivaloylcarnitine, screening using only quantitative MRM transitions will result in a positive (false positive) result in a screening test even if the liquid sample contains only pivaloylcarnitine.

[0037] In the first analysis mode, in addition to the quantitative MRM transition, the first analysis confirmation MRM transition (246.15>187.05; 246>187 in integer mass) is also subjected to MRM measurement, and the ratio of the measurement intensity of the first analysis confirmation MRM transition to the measurement intensity of the quantitative MRM transition (peak height or area of ​​the mass chromatogram; the same applies below) is calculated. As shown in FIG. 4, the ratio of the above measurement intensities is 17.5% for isovalerylcarnitine, whereas the ratio of the above measurement intensities is 25.5% for pivaloylcarnitine, which is a sufficient difference to distinguish between the two. Therefore, in the first analysis mode, the ratio of the measurement intensities calculated from the actual measurement of the liquid sample is compared with the value of the ratio of the measurement intensities of isovalerylcarnitine to determine whether the substance contained in the liquid sample is isovalerylcarnitine or pivaloylcarnitine.

[0038] The above judgment may be performed by comparing the value of the ratio of the measured intensities obtained by measuring an actual sample with the value of the ratio of the measured intensities obtained by measuring a standard sample of the target substance, but in that case, the value varies depending on the compound and also varies depending on the confirmation MRM transition used. Therefore, rather than judging whether or not a target substance is present based on the difference between the reference value of the ratio of the measured intensities and the value of the ratio of the measured intensities calculated from the measurement of an actual sample, it is preferable to use an index that is common to all target substances and common to all confirmation MRM transitions. Therefore, for example, it is preferable to calculate a score value from the ratio of the measured intensities using the following formula (1), with 100 being a perfect match. By calculating the score value in this way, judgment can be performed for all compounds based on the same criteria. Score = 100 - (| (actual sample measured intensity ratio value) - (measured intensity ratio reference value) | / (measured intensity ratio reference value) x 100) ... (1)

[0039] FIG. 5 shows an example of judgment based on score values. This is an example of distinguishing between the target substance isovalerylcarnitine and impurity substances such as pivaloylcarnitine. The ratio of the measurement intensity of the first analysis confirmation MRM transition to the measurement intensity of the quantitative MRM transition calculated from a standard sample of isovalerylcarnitine is 20.44% (note that since this is an example in which a different MRM transition from the example described in FIG. 4 is used as the first analysis confirmation MRM transition, the value of the measurement intensity ratio is different from that in FIG. 4). When the values ​​of the ratio of the measurement intensity of the first analysis confirmation MRM transition to the measurement intensity of the quantitative MRM transition calculated for two specimens (specimens 1 and 2) that are actual samples are 20.48% (specimen 1) and 28.21% (specimen 2), respectively, the score value of specimen 1 calculated from these values ​​and the above formula (1) is 99.78, and the score value of specimen 2 is 61.99. Here, for example, if the reference value of the score (cutoff value of the index value) is set to 75, it can be determined that sample 1 contains isovalerylcarnitine and sample 2 does not contain isovalerylcarnitine. This reference value may be set appropriately depending on the purpose of screening, the required accuracy, etc. For example, when a screening test is performed so as not to miss positive results, a lower reference value may be set. Specifically, for example, the reference value of the score may be set to an appropriate value between 70 and 80.

[0040] The second analysis mode can be suitably used, for example, when analyzing glutarylcarnitine (C5-DC) having the molecular structure shown in FIG. 6. Glutarylcarnitine is a marker substance for glutaric acidemia type I, and its mass-to-charge ratio is 275.14 (integer mass is 275). Glutarylcarnitine has a different composition formula, but exists as an acylcarnitine with the same integer mass, hydroxyhexanoylcarnitine (C6-OH. Mass-to-charge ratio is 275.17. Integer mass is 275). Generally, tandem quadrupole mass spectrometers are often used in neonatal mass screening tests, and it is difficult for such mass spectrometers to distinguish substances with the same integer mass.

[0041] Therefore, in the second analysis mode, an MRM transition that produces product ions from the target substance glutarylcarnitine, but does not produce product ions from hydroxyhexanoylcarnitine, is used as a confirmation MRM transition (second analysis confirmation MRM transition).

[0042] Figure 7 shows the product ion spectra (positive ion mode, mass-to-charge ratio of precursor ion is 276.1) of glutaryl carnitine (C5-DC) and hydroxyhexanoyl (C6-OH). Comparing these product ion spectra, it can be seen that a product ion with a mass-to-charge ratio of 87.0 from glutaryl carnitine is measured at high intensity, whereas a product ion with a mass-to-charge ratio of 87.0 from hydroxyhexanoyl is not detected. Figure 8 shows the quantitative MRM transition (276.1>85.0) in which glutaryl carnitine is measured at the highest intensity, and the mass chromatograms obtained by MRM measurement of standard samples of glutaryl carnitine and hydroxyhexanoyl using the above MRM transition (276.1>87.1). It can be seen that while the quantitative MRM transition (276.1>85.0) detects product ions from both substances, the above MRM transition (276.1>87.1) detects only glutaryl carnitine. Therefore, by using 276.1>87.1 as the second analysis confirmation MRM transition, it is possible to distinguish whether the sample contains glutaryl carnitine or hydroxyhexanoyl. In this example, it is also possible to measure glutaryl carnitine by MRM measurement using only the second analysis confirmation MRM transition, but such MRM transitions do not necessarily have high measurement sensitivity. Therefore, in the second analysis mode, first, a judgment is made using quantitative MRM, which has high measurement sensitivity, and then a judgment is made using the above second analysis MRM transition.

[0043] Hereinafter, a procedure for analyzing amino acids and acylcarnitines contained in a liquid sample to be analyzed using the liquid chromatograph mass spectrometer of this embodiment will be described with reference to Figures 9 to 11. Figure 9 is a flow chart showing the overall flow of the analysis method of this embodiment.

[0044] First, a specimen (blood) is collected from a newborn subject to prepare a liquid sample (step 1). Preparation of the liquid sample (recovery of amino acids and acylcarnitines) can be performed by subjecting a blood sample collected from a newborn to an extraction procedure using water and ethanol, as described in Non-Patent Document 1. The prepared liquid sample is set in the autosampler 14.

[0045] After the liquid sample is set in the autosampler 14, when the user issues an instruction to start the analysis, the analysis condition setting unit 42 displays a screen for setting the analysis conditions on the display unit 6. On this screen, a list of amino acids and acylcarnitines recorded in the compound DB 411 is displayed, and the user selects from the list those to be analyzed (target substances) (Step 2). Alternatively, the user may select all compounds grouped in advance (for example, a group of compounds to be targeted in a newborn mass screening test) at once.

[0046] Once the target substance has been selected, the analysis condition setting section 42 then prompts the user to select either the first analysis mode or the second analysis mode (step 3). First, the case where the first analysis mode is selected will be described.

[0047] When the first analysis mode is selected (YES in step 4), the first analysis mode is executed in the following procedure (step 5). Fig. 10 is a flowchart showing the flow in the first analysis mode of this embodiment.

[0048] The analysis condition setting unit 42 reads out the quantitative MRM transitions and the first analysis confirmation MRM transitions of each target substance from the compound DB 411, and creates a method file describing a method for executing them in sequence for a predetermined time each. Then, a batch file is created for measuring each of the liquid samples set in the autosampler 14 using that method, and is stored in the storage unit 41.

[0049] After the batch file is created, when the user issues an instruction to start measurement, the measurement control unit 43 injects the liquid sample from the autosampler 14 into the injector 13. The liquid sample injected into the injector 13 is introduced into the ESI probe 201 together with the mobile phase by the FIA ​​method.

[0050] The liquid sample introduced into the ESI probe 201 is ionized in the ionization chamber 20, and then is transported to the front-stage quadrupole mass filter 231 while being converged along the ion optical axis C by the ion lens 211 in the first intermediate vacuum chamber 21 and the ion guide 221 in the second intermediate vacuum chamber 22. The front-stage quadrupole mass filter 231 selects ions having a mass-to-charge ratio set in the quantitative MRM transition of the first target substance as precursor ions. The precursor ions selected by the front-stage quadrupole mass filter 231 collide with CID gas molecules in the collision cell 232 and are fragmented, and enter the rear-stage quadrupole mass filter 234. The rear-stage quadrupole mass filter 234 selects ions having a mass-to-charge ratio set in the quantitative MRM transition of the first target substance 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 according to the intensity of the ions to the control / processing unit 4. The control / processing unit 4 stores the signal data received from the ion detector 235 in the storage unit 41 in sequence.

[0051] After the measurement of the quantitative MRM transition of the first target substance is completed, the first analysis confirmation MRM transition of the same substance is then measured in the same manner as above (step 12). When the measurement of the quantitative MRM transitions and the first analysis confirmation MRM transitions of all target substances is completed, the measurement of the first liquid sample is terminated.

[0052] After the measurement of the first liquid sample is completed, the quantitative MRM transition and the first analysis MRM transition are measured for each target substance for the second liquid sample in the same manner as described above. When the measurements for all liquid samples are completed, the measurement operation ends.

[0053] When the measurement is completed (or in parallel with the measurement operation), in the control and processing unit 4, the first analysis unit 44 first creates a mass chromatogram for each target substance in the first liquid sample during the measurement of the quantitative MRM transition, and calculates the peak height (or area; the same applies below) (step 13). When the peak heights for all target substances are calculated, the first judgment unit 441 reads out the thresholds (Tta, Ttb, Ttc, ...) set for each target substance from the compound DB 411, and compares them with the peak height values ​​of the mass chromatograms for each target substance (step 14). Then, target substances whose peak height values ​​exceed the thresholds are extracted (step 15).

[0054] Next, the first analysis unit 44 creates a mass chromatogram for each of the target substances extracted by the first judgment unit 441 when the first analytical confirmation amount MRM transition is measured, obtains the peak height, and further calculates the ratio to the peak height value of the quantitative MRM transition (step 16). When the ratio of the peak height value is calculated for each of the target substances extracted by the first judgment unit 441, the second judgment unit 442 reads out 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 score value of each target substance is compared with the reference value (step 18), and target substances whose score value exceeds the reference value are extracted (step 19).

[0055] When the above series of processes has been completed for all liquid samples, the analysis result output unit 46 displays on the screen of the display unit 6 a list correlating the liquid sample from which the target substance was 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 (output of analysis results; step 7).

[0056] Next, a case where the user selects the second analysis mode will be described. When the second analysis mode is selected (NO in step 4), the first analysis mode is executed in the following procedure (step 6). Fig. 11 is a flowchart showing the flow in the second analysis mode of this embodiment.

[0057] The analytical condition setting unit 42 reads out the quantitative MRM transitions and the second analysis confirmation MRM transitions of each target substance from the compound DB 411, and creates a method file describing a method for executing them in sequence for a predetermined time each. Then, a batch file is created for measuring each of the liquid samples set in the autosampler 14 using that method, and is stored in the storage unit 41.

[0058] After the batch file is created, when the user issues an instruction to start measurement, the measurement control unit 43 injects the liquid sample from the autosampler 14 into the injector 13. The liquid sample injected into the injector 13 is introduced into the ESI probe 201 together with the mobile phase by the FIA ​​method and subjected to mass spectrometry. The mass spectrometry procedure is the same as when the first analysis mode is selected, first, an MRM measurement using a quantitative MRM transition (step 21), and then an MRM measurement using a confirmatory MRM transition (step 22) (however, in the second analysis mode, a second analysis confirmatory MRM transition is used instead of the first analysis confirmatory MRM transition). These measurement procedures are the same as those in the first analysis mode, so detailed explanations will be omitted.

[0059] When the measurement of all liquid samples is completed (or in parallel with the measurement operation), the second analysis unit 45 in the control and processing unit 4 first creates a mass chromatogram for each target substance at the time of measurement of the quantitative MRM transition for the first liquid sample, and calculates the peak height (step 23). When the peak heights for all target substances are calculated, the second judgment unit 452 reads out the thresholds (Tta, Ttb, Ttc, ...) set for each target substance from the compound DB 411, and compares them with the peak height values ​​of the mass chromatograms of the quantitative MRM transitions of each target substance (step 24). Then, target substances whose peak height values ​​exceed the thresholds are extracted (step 25).

[0060] Next, the second analysis unit 45 creates a mass chromatogram for each target substance extracted by the first judgment unit 451 at the time of measuring the second analysis confirmation amount MRM transition, and obtains the peak height (step 26). When the peak height is calculated for each target substance extracted by the first judgment unit 451, the second judgment unit 452 reads out the peak height thresholds (Tqa, Tqb, Tqc, ...) set for each target substance from the compound DB 411, and compares the peak height values ​​of the mass chromatograms of the second analysis confirmation MRM transitions of each target substance (step 27). Then, target substances whose peak height values ​​exceed the thresholds are extracted (step 28).

[0061] When the above series of processes has been completed for all liquid samples, the analysis result output unit 46 displays on the screen of the display unit 6 a list correlating the liquid sample from which the target substance has been extracted by the second determination unit 452, the target substance extracted from the liquid sample by the second determination unit 452, and the disease name corresponding to the target substance (output of analysis results; step 7).

[0062] In this embodiment, as described above, in both the first and second analysis modes, the measurement intensity obtained by the quantitative MRM transition is compared with a threshold value to determine the possibility that each target substance is contained in the liquid sample (first determination). In conventional mass screening, the possibility that the target substance is contained in the analysis target sample was determined only by this determination, and therefore it was not possible to distinguish between a case where the target substance is contained in the analysis target sample and a case where only impurities such as isomers detected together with the target substance in the first MRM transition are present and the target substance itself is not contained.

[0063] In the first analysis mode of this embodiment, a threshold is set for the ratio of the peak height value (measurement intensity) of the mass chromatogram of the quantitative MRM transition to the peak height value (measurement intensity) of the mass chromatogram of the first analysis confirmation MRM transition, and for a target substance that is determined (first determination) to have a peak height value that exceeds the threshold (i.e., that may be contained in the liquid sample), the ratio of the peak height value of the mass chromatogram of the quantitative MRM transition to the peak height value of the mass chromatogram of the first analysis confirmation MRM transition is compared with another threshold to further determine (second determination) whether or not the target substance is contained in the liquid sample. Even if there is an impurity substance that is detected together with the target substance by the quantitative MRM transition, the ratio of the measurement intensity by the quantitative MRM transition to the measurement intensity by the first analysis confirmation MRM transition is usually different between the target substance and the impurity substance. In the first analysis mode of this embodiment, if the first judgment determines that the target substance may be contained, a further judgment (second judgment) is made as to whether the liquid sample contains the target substance or an impurity substance by comparing the above ratio with a threshold value, so that it is possible to determine whether or not the liquid sample contains the target substance with greater accuracy than conventional methods.

[0064] In the second analysis mode of the present embodiment, a second analysis confirmation MRM transition is set in which no impurity substances that may be detected together with the target substance are detected in the MRM measurement using the quantitative MRM transition, and for each target substance determined in the first determination to be possibly contained in the liquid sample, a further determination (second determination) is made as to whether or not the target substance is contained in the liquid sample based on the peak density (measurement intensity) of the mass chromatogram of the second analysis confirmation MRM transition. In the second analysis mode of the present embodiment, based on the detection of a target substance in the MRM measurement using the second analysis confirmation MRM transition in which no impurity substances are detected, the target substance is re-determined (second determination) to be contained in the liquid sample, so that it is possible to determine whether or not the target substance is contained in the liquid sample with higher accuracy than in the past.

[0065] The above embodiment is merely an example, and can be modified as appropriate in accordance with the spirit of the present invention. In the above embodiment, assuming the case where a large amount of specimens (samples) are analyzed, the samples are introduced into the mass spectrometer 2 by the FIA ​​method without using a column in order to increase the measurement efficiency, but when the number of specimens is small, the components in the sample may be separated using a column before being introduced into the mass spectrometer 2. By separating substances contained in the sample to be analyzed using a column before MRM measurement, it is possible to determine with higher accuracy whether the sample contains a target substance.

[0066] In the above embodiment, a liquid chromatograph mass spectrometer was used assuming the use of an autosampler 14 provided in the liquid chromatograph 1, but a sample may be directly introduced into the ESI probe 201 of the mass spectrometer 2, in which case it is not necessary to use the liquid chromatograph 1. Also, depending on the characteristics of the sample to be analyzed, a gas chromatograph may be used instead of the liquid chromatograph.

[0067] In the above embodiment, the user is allowed to select either the first analysis mode or the second analysis mode, but information associating each compound with an 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 the above embodiment, the user is allowed to select either the first analysis mode or the second analysis mode, but both analysis modes may be executed.

[0068] In the above embodiment, an example was shown in which one each of the first analysis confirmation MRM transition and the second analysis confirmation MRM transition was recorded in the compound DB411 for each compound (FIG. 2), but one or both of these may be set in multiples. In that case, a reference value for the score related to the ratio of the measured intensities is set for each of the multiple first analysis confirmation MRM transitions. A threshold value may be set for each of the multiple second analysis confirmation MRM transitions, or a common threshold value may be set. Furthermore, the first analysis confirmation MRM transition and the second analysis confirmation MRM transition may be the same or different from each other.

[0069] [Aspects] It will be apparent to those skilled in the art that the above-described exemplary embodiments are illustrative of the following aspects.

[0070] (Section 1) A method for analyzing amino acids and acylcarnitines according to one embodiment of the present invention comprises the steps of: for each of one or more target substances which are amino acids or acylcarnitines, a first MRM transition and a second MRM transition each being a pair of a mass-to-charge ratio of a precursor ion and a mass-to-charge ratio of a product ion, a first reference value relating to the measured intensity of the first MRM transition, and a second reference value relating to the ratio of the measured intensity of the first MRM transition to the measured intensity of the second MRM transition; performing MRM measurement of an analyte sample using the first MRM transition and the second MRM transition set for each of the target substances, thereby measuring a first intensity, which is a measurement intensity of the first MRM transition, and a second intensity, which is a measurement intensity of the second MRM transition, for each of the target substances; performing a first determination for each of the target substances in order to determine whether or not the target substance is likely to be contained in the analysis target sample by comparing the first intensity value with the first reference value; A second determination is made to determine whether or not the target substance is contained in the analysis target sample by comparing a ratio of the first intensity value to the second intensity value with the second reference value for the target substance determined in the first determination as possibly being contained in the analysis target sample. It is something.

[0071] (Section 3) A method for analyzing amino acids and acylcarnitines according to another embodiment of the present invention comprises the steps of: For each of one or more target substances which are amino acids or acylcarnitines, a first MRM transition which is a pair of a mass-to-charge ratio of a precursor ion and a mass-to-charge ratio of a product ion, a reference value for the measurement intensity of the first MRM transition, and a second MRM transition which does not detect impurities which may be detected together with the target substance in an MRM measurement using the first MRM transition, are set; performing MRM measurement of an analyte sample using the first MRM transition and the second MRM transition set for each of the target substances, thereby obtaining, for each of the target substances, a first intensity value which is a measurement intensity of the first MRM transition and a second intensity value which is a measurement intensity of the second MRM transition; performing a first determination for each of the target substances in order to determine whether or not the target substance is likely to be contained in the analysis target sample by comparing the value of the first intensity with the reference value; A second determination is made on the basis of the second intensity value as to whether or not the target substance is contained in the analysis target sample, for the target substance determined in the first determination as possibly being contained in the analysis target sample. It is something.

[0072] In the method for analyzing amino acids and acylcarnitines according to paragraphs 1 and 3, a first MRM transition and a second MRM transition, and a reference value for the measurement intensity of the first MRM transition (first reference value in paragraph 1) are set for each of one or more target substances that are amino acids or acylcarnitines, and an analysis sample is subjected to MRM measurement using the first MRM transition and the second MRM transition for each target substance. The first MRM transition may be set to one that can measure the target substance with high sensitivity, similar to the MRM transitions used in conventional mass screening. In addition, the reference value for the measurement intensity of the first MRM transition (same as above) may be set to one that corresponds to the cutoff value used in conventional mass screening.

[0073] In the amino acid and acylcarnitine analysis methods of paragraphs 1 and 3, first, for each target substance, the measurement intensity obtained by the first MRM transition is compared with a reference value to determine the possibility that the target substance is contained in the analysis sample (first determination). Conventional mass screening determined the possibility that the target substance is contained in the analysis sample only by this determination, and therefore was unable to distinguish between a case in which the target substance is contained in the analysis sample and a case in which only contaminants such as isomers detected together with the target substance in the first MRM transition are present and the target substance itself is not contained.

[0074] In the method for analyzing amino acids and acylcarnitines of paragraph 1, a second reference value for the ratio of the measurement intensity of the first MRM transition to the measurement intensity of the second MRM transition is set, and for a target substance determined to possibly be contained in the sample to be analyzed (first determination), the ratio of the first intensity value to the second intensity value is compared with the second reference value to determine whether or not the target substance is contained in the sample to be analyzed (second determination). Even if there is an impurity substance detected together with the target substance in the first MRM transition, the target substance and the impurity usually have different values ​​for the ratio of the measurement intensity by the first MRM transition to the measurement intensity by the second MRM transition. In the method for analyzing amino acids and acylcarnitines of paragraph 1, when it is determined to possibly be contained in the sample to be analyzed by the first determination, the ratio is compared with the second reference value to determine whether the target substance is contained in the sample to be analyzed or not (second determination), so that it is possible to determine whether or not the target substance is contained in the sample to be analyzed with higher accuracy than before.

[0075] In the method for analyzing amino acids and acylcarnitines of paragraph 3, a second MRM transition is set in which no impurity substances that may be detected together with the target substance are detected in an MRM measurement using the first MRM transition, and for each target substance determined in the first determination to be possibly contained in the sample to be analyzed, a determination is made (second determination) as to whether or not the target substance is contained in the sample to be analyzed based on the measurement intensity (second intensity value) by the second MRM transition.In the method for analyzing amino acids and acylcarnitines of paragraph 3, a determination is made (second determination) that the target substance is contained in the sample to be analyzed based on the detection of the target substance in an MRM measurement using the second MRM transition in which no impurity substances are detected, so that it is possible to determine with higher accuracy than before whether or not the target substance is contained in the sample to be analyzed.

[0076] (Section 2) The method for analyzing amino acids and acylcarnitines according to the second aspect of the present invention is the method for analyzing amino acids and acylcarnitines according to the first aspect of the present invention, In the second determination, a score for each of the target substances is calculated using the following formula: Score = 100 - (| (actual sample measured intensity ratio value) - (measured intensity ratio reference value) | / (measured intensity ratio reference value) x 100) ... (1) The calculated score is compared with the second reference value to determine whether or not the target substance is contained in the analysis sample.

[0077] In the analytical method for amino acids and acylcarnitines in paragraph 2, a second judgment can be made for all target substances using common criteria.

[0078] (Section 4) The method for analyzing amino acids and acylcarnitines according to item 4 is a method for analyzing amino acids and acylcarnitines according to any one of items 1 to 3, comprising: The substances contained in the sample to be analyzed are separated from each other using a chromatographic column, and then the MRM measurement is carried out.

[0079] In the method for analyzing amino acids and acylcarnitines according to paragraph 4, the substances contained in the sample to be analyzed are separated from each other before MRM measurement, so that it is possible to determine with higher accuracy whether or not the sample contains the target substance.

[0080] (Section 5) The method for analyzing amino acids and acylcarnitines according to item 5 is a method for analyzing amino acids and acylcarnitines according to any one of items 1 to 3, comprising: The target substance is an amino acid selected from phenylalanine, leucine, isoleucine, methionine, citrulline, and tyrosine, or free carnitine (C0), acetylcarnitine (C2), propionylcarnitine (C3), butyrylcarnitine to palmitoylcarnitine (C4 to C18), isovalerylcarnitine, 2-methylbutyrylcarnitine, pivaloylcarnitine (C5), tiglylcarnitine (C5:1), glutarylcarnitine (C5-DC), 3-hydroxyisovalerylcarnitine, 3-hydroxy-2- The acylcarnitine is selected from methylbutyryl-carnitine (C5-OH), octanoylcarnitine (C8), decanoylcarnitine (C10), dodecanoylcarnitine (C12), tetradecenoylcarnitine (C14:1), tetradecanoylcarnitine (C14), palmitoylcarnitine (C16), stearylcarnitine (C18), octadecenoylcarnitine (C18:1), hydroxyhexadecanoylcarnitine (C16-OH), and hydroxyoctadecenoylcarnitine (C18:1-OH).

[0081] The target substance in the amino acid and acylcarnitine analysis method according to paragraph 5 is an amino acid or acylcarnitine associated with a neonatal disease, and therefore can be suitably used in neonatal mass screening tests. [Explanation of symbols]

[0082] 1. Liquid chromatograph 11...Mobile phase container 12…Pump 13...Injector 14…Autosampler 2...Mass spectrometer 20…Ionization chamber 201…ESI probe 21…First intermediate vacuum chamber 211…Ion lens 212…Skimmer 22…Second intermediate vacuum chamber 221…Ion Guide 23…Analysis room 231...Pre-quadrupole mass filter 232…Collision cell 233…Ion Guide 234...Post-quadrupole mass filter 235…Ion detector 4. Control and processing section 41...Storage section 411…Compound DB 42…Analysis condition setting section 43...Measurement control section 44...1st analysis section 441...1st judgment section 442…Second judgment section 45...Second analysis section 451...1st judgment section 452...Second judgment section 46…Analysis result output section 5. Input section 6...Display section C…Ion optical axis

Claims

1. For each of the one or more target substances, which are amino acids or acylcarnitines, a first MRM transition and a second MRM transition are set, which are pairs of the mass-charge ratio of the precursor ion and the mass-charge ratio of the product ion, respectively, a first reference value for the measurement intensity of the first MRM transition and a second reference value for the ratio of the measurement intensity of the first MRM transition to the measurement intensity of the second MRM transition are set, By performing MRM measurement on the analyte sample using the first MRM transition and the second MRM transition set for each of the target substances, a first intensity, which is the measurement intensity of the first MRM transition, and a second intensity, which is the measurement intensity of the second MRM transition, are measured for each of the target substances. For each of the aforementioned target substances, a first determination is made to determine whether or not the sample to be analyzed may contain the target substance by comparing the first intensity value with the first reference value. For the target substance that was determined to be potentially present in the analyte sample in the first determination, a second determination is made to determine whether or not the analyte sample contains the target substance by comparing the ratio of the first intensity value to the second intensity value with a second reference value. A method for analyzing amino acids and / or acylcarnitines.

2. In the second determination, the score for each of the target substances is calculated using the following formula: Score = 100 - (|(Ratio of actual sample intensity) - (Reference value of the ratio of measurement intensity)| / (Reference value of the ratio of measurement intensity) x 100) ... (1) The method for analyzing amino acids and / or acylcarnitines according to claim 1, wherein the value of the calculated score is compared with the second reference value to determine whether or not the target substance is contained in the sample to be analyzed.

3. For each of the one or more target substances, which are amino acids or acylcarnitines, a first MRM transition is set, which is a pair of the mass-charge ratio of the precursor ion and the mass-charge ratio of the product ion; a reference value for the measurement intensity of the first MRM transition is set; and a second MRM transition is set, in which no interfering substances that may be detected together with the target substance in the MRM measurement using the first MRM transition are detected. By performing MRM measurement on the analyte sample using the first MRM transition and the second MRM transition set for each of the target substances, a first intensity, which is the measurement intensity of the first MRM transition, and a second intensity, which is the measurement intensity of the second MRM transition, are measured for each of the target substances. For each of the aforementioned target substances, a first determination is made to determine whether or not the sample to be analyzed may contain the target substance by comparing the first intensity value with the reference value. For the target substance that was determined to be potentially present in the analyte sample in the first determination, a second determination is made to determine whether or not the target substance is present in the analyte sample based on the second intensity value. A method for analyzing amino acids and / or acylcarnitines.

4. A method for analyzing amino acids and / or acylcarnitines according to claim 1 or 3, wherein the substances contained in the analyte sample are separated from each other using a chromatographic column, and then the MRM measurement is performed.

5. The target substance is an amino acid selected from phenylalanine, leucine, isoleucine, methionine, citrulline, and tyrosine, or free carnitine (C0), acetylcarnitine (C2), propionylcarnitine (C3), butyrylcarnitine to palmitoylcarnitine (C4 to C18), isovalerylcarnitine, 2-methylbutyrylcarnitine, pivaloylcarnitine (C5), tigrylcarnitine (C5:1), glutarylcarnitine (C5-DC), 3-hydroxyisovalerylcarnitine, 3-hydroxy-2-methylbutyrylcarnitine (C5- A method for analyzing amino acids and / or acylcarnitines according to claim 1 or 3, wherein the acylcarnitine is selected from OH), octanoylcarnitine (C8), decanoylcarnitine (C10), dodecanoylcarnitine (C12), tetradecenoylcarnitine (C14:1), tetradecanoylcarnitine (C14), palmitoylcarnitine (C16), stearylcarnitine (C18), octadecenoylcarnitine (C18:1), hydroxyhexadecanoylcarnitine (C16-OH), and hydroxyoctadecenoylcarnitine (C18:1-OH).

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