Method for acylcarnitine analysis and acylcarnitine analysis device

Abstract

One embodiment of the acylcarnitine analysis method according to the present application is a method for analyzing C5 acylcarnitine using a tandem mass spectrometer, the acylcarnitine analysis method including: a measurement step of performing MRM measurement based on at least one of MRM parent-daughter ion pairs of m / z 246 > 187, m / z 246 > 57, m / z 246 > 41, and m / z 246 > 29 on a test object; and a processing step of identifying isovalerylcarnitine and pivaloylcarnitine, which is an isomer of isovalerylcarnitine, in the test object, or determining whether pivaloylcarnitine is contained in C5 acylcarnitine in the test object, using a measurement result obtained in the measurement step. Thus, isovalerylcarnitine and pivaloylcarnitine, which have been difficult to identify in the past, can be favorably identified.

Description

TECHNICAL FIELD

[0001] The present application relates to an analysis method of acylcarnitines and an analysis device using the method. BACKGROUND

[0002] In order to find congenital metabolic abnormality diseases and the like of newborns at an early stage and perform treatment, newborn screening is being widely implemented. Recently, with the rapid development of mass spectrometry technology, newborn mass screening using a tandem mass spectrometry device has also become widespread, and has played a very large role in the early detection of congenital metabolic abnormality diseases of newborns (see Non-Patent Literature 1, 2).

[0003] As one of such newborn mass screenings, there is acylcarnitine analysis for diagnosing organic acid / fatty acid metabolic abnormality diseases. Acylcarnitines are carnitines accumulated in the body to which acyl groups derived from organic acids or fatty acids are bonded, and acylcarnitines of various structures that differ in the chain length of acyl groups are the objects of analysis.

[0004] As disclosed in Non-Patent Literature 2, in the usual acylcarnitine analysis, quantitative analysis of acylcarnitine molecules whose precursor ions differ according to the chain length of acyl groups in combination with characteristic product ions of m / z 85 (accurately 84.95) is performed using multiple reaction monitoring (MRM) measurement in a tandem mass spectrometry device.

[0005] PRIOR ART DOCUMENTS

[0006] NON-PATENT LITERATURE

[0007] Non-Patent Literature 1: Yutaka Nomachi, et al., “Tandem Mass Spectrometry-based Newborn Mass Screening Test Study 2008 (4th Year) Results”, Sapporo City Health Research Annual Report, Vol. 36, 2009, pp. 42-48

[0008] Non-Patent Literature 2: Yoichi Shigematsu, “Current Status of Newborn Mass Screening by Mass Spectrometry”, Journal of the Mass Spectrometry Society of Japan, Vol. 64, No. 4, 2016, pp. 127-131 SUMMARY

[0009] PROBLEMS TO BE SOLVED BY THE INVENTION

[0010] However, in the conventional acylcarnitine analysis method, the following problems exist.

[0011] In mass screening of newborns, a large number of test samples need to be analyzed quickly, and therefore, instead of separation using a liquid chromatograph (LC), a combination of flow injection analysis (FIA) and MRM measurement is used. Therefore, isomers having the same MRM transition (combination of mass-to-charge ratio of precursor ion and mass-to-charge ratio of product ion) cannot be distinguished.

[0012] For example, as also pointed out in Non-Patent Literature 2, C5 acylcarnitine (acylcarnitine having an acyl group having a carbon number of 5) includes isomers such as isovalerylcarnitine in addition to isovalerylcarnitine. Isovalerylcarnitine is a metabolite related to inborn diseases, and, in contrast, pivaloylcarnitine is mainly a metabolite derived from a medicament derived from a pivoxil group included in a part of antibacterial agents. Therefore, even in a case where an abnormal increase in C5 acylcarnitine is detected by acylcarnitine analysis and becomes a screening positive, it is possible that it is not an inborn factor that is originally intended to be detected but a false positive caused by the influence of a medicament that is not intended to be detected. The above antibacterial agent is a medicament that is prescribed to newborns at a relatively high frequency, and therefore, false positives derived from the medicament occur relatively frequently.

[0013] Accordingly, in a case where a screening positive is made with respect to C5 acylcarnitine, additional examination using other analysis methods such as liquid chromatography mass spectrometry needs to be performed to determine whether it is an inborn factor. In general newborn screening, blood is taken from a newborn to prepare a test sample, and therefore, the performance of the above additional examination is a particularly large physical burden on the newborn. In addition, when a positive is made in screening, the parents of the newborn are burdened with a very large psychological burden. Therefore, it is important to reduce false positives as much as possible, and even if additional examination is needed, it is possible to inform the parents of the possibility of a temporary false positive derived from a medicament.

[0014] The above problem is not limited to C5 acylcarnitine, and the same applies to, for example, C4 acylcarnitine including butyrylcarnitine, which is known as a metabolite related to inborn diseases such as short-chain acyl-CoA dehydrogenase deficiency, glutaric acidemia type II, and isobutyrylcarnitine, which is an isomer thereof.

[0015] In addition, in the case of the isomers described above, the molecular weights are completely the same, but if the molecular weights are not the same but are quite close, due to the performance of the mass spectrometry device, sometimes the different acylcarnitines cannot be identified based on the mass.

[0016] For example, regarding gultarylcarnitine and hydroxyhexanoylcarnitine, which are metabolites related to congenital diseases such as glutaric aciduria type I, although the chemical formulas are different, the integer masses are the same, and it is not possible to identify them by a mass spectrometry device with low mass resolution. In addition, regarding malonylcarnitine, which is a metabolite related to congenital diseases such as malonic acidemia, and 3-hydroxybutyrylcarnitine, which is a metabolite related to congenital diseases such as 3-hydroxyacyl-CoA dehydrogenase deficiency, although the chemical formulas are different, the integer masses are the same, and it is not possible to identify them by a mass spectrometry device with low mass resolution.

[0017] Regarding the identification of a plurality of acylcarnitines with the same integer mass, although it is generally possible to perform by a liquid chromatograph mass spectrometry device, the efficiency is poor, and it is not suitable for screening.

[0018] One embodiment of the present application, which was made in order to solve the above-described problems, has an object to provide an acylcarnitine analysis method and an analysis device capable of identifying acylcarnitines caused by congenital factors and isomers mainly derived from a medicament included in C5 acylcarnitines in mass screening of newborns and the like.

[0019] In addition, another embodiment of the present application, which was made in order to solve the above-described problems, has an object to provide an acylcarnitine analysis method and an analysis device capable of identifying a plurality of acylcarnitines with completely the same or substantially the same mass with good efficiency and good precision in mass screening of newborns and the like.

[0020] Solution to the problem

[0021] One embodiment of the acylcarnitine analysis method according to the present application, which was made in order to solve the above-described problems, is a method for analyzing C5 acylcarnitines, which are acylcarnitines having an acyl group with a carbon number of 5, using a tandem mass spectrometry device, the acylcarnitine analysis method including:

[0022] the measurement step, performing a multiple reaction monitoring (MRM) measurement based on at least one of MRM parent-daughter ion pairs of m / z 246 > 187, m / z 246 > 57, m / z 246 > 41, and m / z 246 > 29 on the test object; and

[0023] the processing step, using the measurement result obtained in the measurement step, to identify isovalerylcarnitine and pivaloylcarnitine as an isomer thereof in the test object, or to determine whether pivaloylcarnitine is contained in C5 acylcarnitine in the test object.

[0024] Another aspect of the acylcarnitine analysis method according to the present application, which has been achieved in order to solve the above-described problems, is an analysis method for identifying a plurality of different acylcarnitines having substantially the same mass using a tandem mass spectrometer, the acylcarnitine analysis method comprising:

[0025] the measurement step, performing a first MRM measurement for an MRM parent-daughter ion pair having the largest signal intensity and a second MRM measurement for an MRM parent-daughter ion pair different from the MRM parent-daughter ion pair having the largest signal intensity on the test object to obtain intensity information of ions derived from an acylcarnitine as a target; and

[0026] the processing step, identifying the plurality of different acylcarnitines or determining whether one of the plurality of different acylcarnitines is contained in the acylcarnitine as the target in the test object, based on a confirmation ion ratio that is a ratio of a signal intensity obtained by the first MRM measurement to a signal intensity obtained by the second MRM measurement.

[0027] Still another aspect of the acylcarnitine analysis method according to the present application, which has been achieved in order to solve the above-described problems, is an analysis method for identifying a plurality of different acylcarnitines having substantially the same mass using a tandem mass spectrometer, the acylcarnitine analysis method comprising:

[0028] the measurement step, performing MRM measurements for specific MRM parent-daughter ion pairs different from each other on the test object to obtain intensity information of ions derived from an acylcarnitine as a target, wherein the specific MRM parent-daughter ion pairs different from each other are respectively determined for the plurality of different acylcarnitines; and

[0029] the processing step, identifying the plurality of different acylcarnitines or determining whether one of the plurality of different acylcarnitines is contained in the acylcarnitine as the target in the test object, based on signal intensities for the specific MRM parent-daughter ion pairs obtained by the measurement step.

[0030] Another method for analyzing acylcarnitines according to the present application, which is achieved in order to solve the above-described problems, is a method for analyzing acylcarnitines, which is a method for analyzing a plurality of different acylcarnitines having substantially the same mass using a tandem mass spectrometer, the method for analyzing acylcarnitines comprising:

[0031] a measurement step of performing MRM measurement while changing collision energy for a specific MRM parent-daughter ion pair with respect to a test object, and acquiring intensity information of ions derived from acylcarnitines targeted as a result; and

[0032] a processing step of identifying the plurality of different acylcarnitines, or determining whether or not one of the plurality of different acylcarnitines is contained in the acylcarnitines targeted in the test object, on the basis of collision energy dependency of ion intensity obtained in the measurement step.

[0033] In addition, one embodiment of an acylcarnitine analysis device according to the present application, which is achieved in order to solve the above-described problems, includes:

[0034] a measurement unit which is a tandem mass spectrometer that performs MRM measurement based on at least one of MRM parent-daughter ion pairs of m / z 246 > 187, m / z 246 > 57, m / z 246 > 41, and m / z 246 > 29 with respect to a test object; and

[0035] a processing unit that identifies isovalerylcarnitine and neopentanoylcarnitine, which is an isomer of isovalerylcarnitine, in the test object, or determines whether or not neopentanoylcarnitine is contained in C5 acylcarnitine, which is acylcarnitine having an acyl group with a carbon number of 5, in the test object, using a measurement result obtained by the measurement unit.

[0036] Another embodiment of an acylcarnitine analysis device according to the present application, which is achieved in order to solve the above-described problems, includes:

[0037] a measurement unit which is a tandem mass spectrometer that performs first MRM measurement with respect to an MRM parent-daughter ion pair in which signal intensity is the largest, and second MRM measurement with respect to an MRM parent-daughter ion pair different from the MRM parent-daughter ion pair in which signal intensity is the largest, with respect to a test object; and

[0038] a processing unit that identifies a plurality of different acylcarnitines having substantially the same mass, or determines whether or not one of the plurality of different acylcarnitines is contained in acylcarnitines targeted in the test object, on the basis of a confirmation ion ratio which is a ratio of signal intensity obtained by the first MRM measurement to signal intensity obtained by the second MRM measurement in the measurement unit.

[0039] Another aspect of the acylcarnitine analysis device according to the present application, which has been achieved in order to solve the above-described problems, is characterized by comprising:

[0040] a measurement unit which is a tandem mass spectrometer that performs MRM measurement on the test subject for mutually different specific MRM parent-daughter ion pairs respectively determined for the plurality of different acylcarnitines; and

[0041] a processing unit that identifies a plurality of different acylcarnitines that are substantially identical in mass, or determines whether a certain one of the plurality of different acylcarnitines is contained in the acylcarnitine targeted in the test subject, on the basis of the signal intensity for the specific MRM parent-daughter ion pair obtained by the measurement unit.

[0042] Another aspect of the acylcarnitine analysis device according to the present application, which has been achieved in order to solve the above-described problems, is characterized by comprising:

[0043] a measurement unit which is a tandem mass spectrometer that performs MRM measurement on the test subject for mutually different specific MRM parent-daughter ion pairs respectively determined for the plurality of different acylcarnitines; and

[0044] a processing unit that identifies a plurality of different acylcarnitines that are substantially identical in mass, or determines whether a certain one of the plurality of different acylcarnitines is contained in the acylcarnitine targeted in the test subject, on the basis of the signal intensity for the specific MRM parent-daughter ion pair obtained by the measurement unit.

[0045] Here, the plurality of different acylcarnitines that are substantially identical in mass can include acylcarnitines that are completely identical in mass, such as isomers, and acylcarnitines that are not completely identical in mass but are identical in mass to the extent that they cannot be distinguished by the mass spectrometer used, such as acylcarnitines that are identical in mass by an integer.

[0046] In addition, in the above-described aspect of the present application, the tandem mass spectrometer is typically a triple quadrupole mass spectrometer or a quadrupole-time-of-flight (Q-TOF) mass spectrometer.

[0047] Here, the MRM measurement based on the MRM parent-daughter ion pair of m / z A > B is a measurement in which a precursor ion having m / z A is selected by a pre-mass separator, and a product ion of m / z B derived from the precursor ion of m / z A is detected. Thus, in the case where the tandem mass spectrometry device that performs this MRM measurement is a triple quadrupole type mass spectrometry device, the precursor ion of m / z A is selectively passed through by a pre- quadrupole filter, and the product ion of m / z B derived from the precursor ion of m / z A is selectively passed through and detected by a post- quadrupole filter. In addition, in the case where the tandem mass spectrometry device that performs this MRM measurement is a Q-TOF type mass spectrometry device, the precursor ion having m / z A is selectively passed through by a pre- quadrupole filter (Q), and the product ions including the product ion of m / z B derived from the precursor ion of m / z A are detected by a post- time-of-flight type mass separator (TOF) and an ion detector.

[0048] In addition, the values of the mass-to-charge ratios in the MRM parent-daughter ion pairs in the above-described one embodiment of the present application, m / z 246, m / z 187, m / z 57, m / z 41, m / z 29, and the value of the mass-to-charge ratio in the MRM parent-daughter ion pair described later, m / z 85, are integer values, but if expressed to two decimal places, they are m / z 246.15, m / z 187.05, m / z 57.00, m / z 41.10, m / z 29.15, and m / z 84.95, respectively, and generally they are the most desired (i.e., intended) values.

[0049] Here, in fact, the value of the mass-to-charge ratio of the ion selected by the mass separator such as a quadrupole filter certainly depends on the performance of the device used. Thus, in the case where, for example, m / z 246 is described in the present specification and claims, although the value of the mass-to-charge ratio of the most desired ion is m / z 246.15, ions within the range of m / z 246.15 ± 0.1, m / z 246.15 ± 0.2, m / z 246.15 ± 0.3, or m / z 246.15 ± 0.4 can also be included in the desired ions. In other words, when the value of the mass-to-charge ratio is expressed with an integer value, it can be set so as to include the ion that should be expressed as m / z 246, but not include the ions that should be expressed as m / z 245 and m / z 247. The same applies to other values of the mass-to-charge ratio.

[0050] Effects of the Invention

[0051] In one embodiment of the acylcarnitine analysis method and the acylcarnitine analysis device according to the present application, instead of the product ion of m / z 85, which is conventionally used in the quantification of acylcarnitines and which can be detected with high sensitivity, any one or more of the four product ions of m / z 187, m / z 57, m / z 41, and m / z 29, which have lower detection sensitivity than m / z 85, are used for the identification or determination of pivaloylcarnitine. Of course, the four product ions can be used in combination with the product ion of m / z 85.

[0052] According to one embodiment of the acylcarnitine analysis method and the acylcarnitine analysis device according to the present application, the identification of isovalerylcarnitine and pivaloylcarnitine, which has been difficult in the past, or the determination of whether pivaloylcarnitine is possibly contained in the detected C5 acylcarnitine, can be performed without performing component separation by the column of the liquid chromatograph. Thus, the false positives in the screening of C5 acylcarnitine can be reduced. In addition, even in the case where the screening of C5 acylcarnitine is positive, the user can be informed that the main cause is likely to be pivaloylcarnitine derived from a medicament. As a result, the physical burden on a newborn can be reduced, or the psychological burden on the parents of a newborn who is positive in the screening can be reduced.

[0053] In addition, according to another embodiment of the acylcarnitine analysis method and the acylcarnitine analysis device according to the present application, the identification of acylcarnitines that are isomers and have the same molecular weight, which has been difficult in the past, or the identification of acylcarnitines that have the same integer mass and are difficult to distinguish due to the mass resolution of the mass spectrometer, can be easily performed without performing component separation by the column of the liquid chromatograph. Thus, the screening of inborn errors of metabolism and the like caused by genetic factors can be efficiently and accurately performed. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 Structure diagram of the main part of one embodiment of the acylcarnitine analysis device according to the present application.

[0055] Figure 2 Graph showing the relationship between the collision energy and the peak intensity for isovalerylcarnitine (i-C5) and pivaloylcarnitine (p-C5) under the MRM parent-daughter ion pair: m / z 246.15 > 187.05.

[0056] Figure 3 Graph showing the relationship between the collision energy and the peak intensity for i-C5 and p-C5 under the MRM parent-daughter ion pair: m / z 246.15 > 84.95.

[0057] Figure 4A graph showing the relationship between collision energy and peak intensity for i-C5 and p-C5 for the MRM parent-daughter ion pair: m / z 246.15 > 57.00.

[0058] Figure 5 A graph showing the relationship between collision energy and peak intensity for i-C5 and p-C5 for the MRM parent-daughter ion pair: m / z 246.15 > 41.10.

[0059] Figure 6 A graph showing the relationship between collision energy and peak intensity for i-C5 and p-C5 for the MRM parent-daughter ion pair: m / z 246.15 > 29.15.

[0060] Figure 7 A graph showing a measured example of a chromatogram waveform in a case where it is determined that p-C5 is not contained in the sample.

[0061] Figure 8 A graph showing a measured example of a chromatogram waveform in a case where it is determined that there is a possibility that p-C5 is contained in the sample.

[0062] Figure 9 A graph showing the collision energy dependency of the confirmation ion ratio for i-C5 and p-C5 when the MRM parent-daughter ion pair: m / z 246.15 > 84.95 is set as the quantitative ion and the MRM parent-daughter ion pair: m / z 246.15 > 187.05 is set as the confirmation ion.

[0063] Figure 10 A graph showing the chromatogram waveforms obtained by measuring a standard sample of i-C5 and p-C5, respectively, using the MRM parent-daughter ion pairs: m / z 246.15 > 84.95 and m / z 246.15 > 187.05.

[0064] Figure 11 A graph showing a comparison of the chromatogram waveforms for a standard sample of i-C5 and p-C5 using the MRM parent-daughter ion pairs: m / z 246.15 > 84.95 and m / z 246.15 > 187.05.

[0065] Figure 12 A graph showing the relationship between the proportion of i-C5 in the sample and the confirmation ion ratio using the MRM parent-daughter ion pair: m / z 246.15 > 187.05.

[0066] Figure 13A graph showing the collision energy dependency of the ratio of the confirmatory ions for butyrylcarnitine (BCA) and isobutyrylcarnitine when the MRM parent-daughter ion pair: m / z 232 > 85 is set as the quantitative ion and the MRM parent-daughter ion pair: m / z 232 > 173 is set as the confirmatory ion.

[0067] Figure 14 A graph showing the collision energy dependency of the ratio of the confirmatory ions for butyrylcarnitine (BCA) and isobutyrylcarnitine when the MRM parent-daughter ion pair: m / z 232 > 85 is set as the quantitative ion and the MRM parent-daughter ion pair: m / z 232 > 57 is set as the confirmatory ion.

[0068] Figure 15 A graph showing the collision energy dependency of the ratio of the confirmatory ions for butyrylcarnitine (BCA) and isobutyrylcarnitine when the MRM parent-daughter ion pair: m / z 232 > 85 is set as the quantitative ion and the MRM parent-daughter ion pair: m / z 232 > 41 is set as the confirmatory ion.

[0069] Figure 16 A graph showing the collision energy dependency of the ratio of the confirmatory ions for BCA and IBCA when the MRM parent-daughter ion pair: m / z 232 > 85 is set as the quantitative ion and the MRM parent-daughter ion pair: m / z 232 > 29 is set as the confirmatory ion.

[0070] Figure 17 A graph showing the measured chromatogram waveform of a sample containing glutaroylcarnitine (C5-DC) under the MRM parent-daughter ion pair of m / z 276 > 87, m / z 276 > 69.

[0071] Figure 18 A graph showing the measured chromatogram waveform of a sample containing hydroxyhexanoylcarnitine (C6-OH) under the MRM parent-daughter ion pair of m / z 276 > 87, m / z 276 > 69.

[0072] Figure 19 A graph showing the collision energy dependency of the ratio of the confirmatory ions for malonylcarnitine (C3-DC) and 3-hydroxybutyrylcarnitine (C4-OH) when the MRM parent-daughter ion pair: m / z 248.2 > 85.1 is set as the quantitative ion and the MRM parent-daughter ion pair: m / z 248.2 > 189.1 is set as the confirmatory ion.

[0073] Figure 20The graph shows the collision energy dependence of the confirming ion ratio of C3-DC and C4-OH when the MRM parent-daughter ion pair: m / z 248.2>85.1 is set as the quantification ion and the MRM parent-daughter ion pair: m / z 248.2>144.1 is set as the confirming ion.

[0074] Figure 21 The graph shows the collision energy dependence of the confirming ion ratio of C3-DC and C4-OH when the MRM parent-daughter ion pair: m / z 248.2>85.1 is set as the quantification ion and the MRM parent-daughter ion pair: m / z 248.2>103.1 is set as the confirming ion.

[0075] Figure 22 The graph shows the collision energy dependence of the confirming ion ratio of C3-DC and C4-OH when the MRM parent-daughter ion pair: m / z 248.2>85.1 is set as the quantification ion and the MRM parent-daughter ion pair: m / z 248.2>58.1 is set as the confirming ion.

[0076] Figure 23 The graph shows the collision energy dependence of the confirming ion ratio of C3-DC and C4-OH when the MRM parent-daughter ion pair: m / z 248.2>85.1 is set as the quantification ion and the MRM parent-daughter ion pair: m / z 248.2>57.1 is set as the confirming ion.

[0077] Figure 24 The graph shows the collision energy dependence of the confirming ion ratio of C3-DC and C4-OH when the MRM parent-daughter ion pair: m / z 248.2>85.1 is set as the quantification ion and the MRM parent-daughter ion pair: m / z 248.2>45.1 is set as the confirming ion.

[0078] Figure 25 The graph shows the collision energy dependence of the confirming ion ratio of C3-DC and C4-OH when the MRM parent-daughter ion pair: m / z 248.2>85.1 is set as the quantification ion and the MRM parent-daughter ion pair: m / z 248.2>43.1 is set as the confirming ion.

[0079] Figure 26 The graph shows the collision energy dependence of the confirming ion ratio of C3-DC and C4-OH when the MRM parent-daughter ion pair: m / z 248.2>85.1 is set as the quantification ion and the MRM parent-daughter ion pair: m / z 248.2>29.2 is set as the confirming ion. DETAILED DESCRIPTION

[0080] An embodiment of the acylcarnitine analysis method and the acylcarnitine analysis device according to the present application will be described below with reference to the accompanying drawings.

[0081] [Structure of the acylcarnitine analysis device of the present embodiment]

[0082] Figure 1 An outline configuration diagram of an embodiment of the acylcarnitine analysis device according to the present application.

[0083] The acylcarnitine analysis device is mainly used for acylcarnitine analysis for diagnosing congenital organic acid / fatty acid metabolic abnormality diseases in neonatal screening. The analysis device includes a sample introduction section 1, a measurement section 2, a data processing section 3, an analysis control section 4, a central control section 5, an input section 6, and a display section 7.

[0084] The sample introduction section 1 is a device for introducing a sample based on the FIA method, and includes a mobile phase container 11 in which a mobile phase (solvent) is stored, a liquid delivery pump 12 that pumps and delivers the mobile phase from the mobile phase container 11, and an injector 13 that injects a sample into the mobile phase. Generally, these structural elements can use units in a liquid chromatograph. In addition, although not shown, an autosampler is generally connected to the injector 13 to sequentially analyze a plurality of test bodies. Furthermore, in the case of general neonatal screening, as described in Non-Patent Literature 2, a sample is extracted / prepared from filter paper blood. However, the sample is not limited thereto, and can be prepared from urine or other body fluids, etc.

[0085] The measurement section 2 is a triple quadrupole mass spectrometer, which is one type of tandem mass spectrometer, and includes an ionization chamber 201 maintained at substantially atmospheric pressure, and a first intermediate vacuum chamber 202, a second intermediate vacuum chamber 203, and a high vacuum chamber 204, each of which is vacuum-exhausted by a vacuum pump (not shown). An ESI sprayer 21 that ionizes by the ESI method is provided in the ionization chamber 201, and a desolvation tube 22 is connected between the ionization chamber 201 and the lower-stage first intermediate vacuum chamber 202. An ion guide 23 that converges and transports ions is disposed in the first intermediate vacuum chamber 202, and the first intermediate vacuum chamber 202 and the lower-stage second intermediate vacuum chamber 203 are connected by a small hole formed in the top of a skimmer 24. A multipole ion guide 25 that converges and transports ions is also disposed in the second intermediate vacuum chamber 203.

[0086] Within the high vacuum chamber 204, a front-stage quadrupole mass filter 26, a collision cell 27, a rear-stage quadrupole mass filter 28, and an ion detector 29 are arranged along the flow of ions. A quadrupole ion guide is arranged within the collision cell 27. The front-stage quadrupole mass filter 26 and the rear-stage quadrupole mass filter 28 each selectively allow ions with a predetermined mass-to-charge ratio to pass through. The collision cell 27 also introduces an inert collision-induced dissociation (CID) gas, such as argon, into the interior of the collision cell 27 from the outside, dissociating the introduced ions by contact with the CID gas to generate product ions.

[0087] The data processing unit 3 receives detection data from the ion detector 29 and processes this data. It includes a data collection unit 31, a peak intensity calculation unit 32, and a component determination unit 33 as functional blocks. The analysis control unit 4 controls the operations of the sample introduction unit 1 and the measurement unit 2 based on the analysis condition file stored in the analysis condition storage unit 41. The central control unit 5 primarily performs overall control and user interface functions through the input unit 6, display unit 7, and other components.

[0088] Generally speaking, the entities of the data processing unit 3, the analysis control unit 4 and the central control unit 5 are personal computers or higher-performance computers called workstations. The functions of the above-mentioned functional blocks can be realized by running the dedicated software (computer program) pre-installed on the computer on the computer.

[0089] [Overview of MRM measurement operation]

[0090] exist Figure 1 In the acylcarnitine analysis device shown, when analyzing acylcarnitines, MRM measurement for predetermined MRM parent and daughter ion pairs is repeatedly performed using the measurement unit 2. The operation during this MRM measurement will be briefly described.

[0091] In the sample introduction part 1, the liquid delivery pump 12 draws the mobile phase from the mobile phase container 11 and delivers the mobile phase to the injector 13 at a substantially constant flow rate. The injector 13 injects a specified amount of sample (test object) into the mobile phase at a specified time. The sample reaches the ESI sprayer 21 of the measuring part 2 along with the flow of the mobile phase. In the flow path until it reaches the ESI sprayer 21, the sample diffuses in the front-to-back direction. Therefore, although the amount of the sample introduced into the ESI sprayer 21 is very small at first, it increases rapidly and decreases rapidly to zero when it exceeds the maximum point. That is, the concentration distribution of the sample is peaked with respect to the passage of time, close to a Gaussian distribution.

[0092] In the ESI sprayer 21, the sample is sprayed into the ionization chamber 201 in the form of fine charged droplets. The charged droplets are split by contact with residual gas molecules, and in the process of vaporization of the solvent in the droplets, the compound molecules in the sample are ionized. The generated ions are sent to the first intermediate vacuum chamber 202 through the desolvation tube 22, and further sent to the high vacuum chamber 204 through the ion guide 23, the small hole of the separator 24, and the multipole ion guide 25. The ions originating from the sample are introduced into the precursor ion selection unit 26, and only the ions having a predetermined mass-to-charge ratio corresponding to the voltage applied to the electrodes constituting the precursor ion selection unit 26 selectively pass through as precursor ions. The precursor ions injected into the collision cell 27 are dissociated by contact with CID gas, and various product ions are generated.

[0093] The generated various product ions are introduced into the product ion selection unit 28, and only the product ions having a predetermined mass-to-charge ratio corresponding to the voltage applied to the electrodes constituting the product ion selection unit 28 selectively pass through to reach the ion detector 29. The ion detector 29 generates a detection signal corresponding to the amount of the incident ions, and the detection data digitized by an analog-digital converter not shown is input to the data processing section 3.

[0094] The analysis control section 4 controls the measurement section 2 so that the voltages corresponding to the MRM parent-daughter ion pairs as targets are respectively applied to the electrodes of the precursor ion selection unit 26 and the product ion selection unit 28. Thereby, detection data indicating the ion intensity of the product ions having a specific mass-to-charge ratio generated by dissociation of the precursor ions having a specific mass-to-charge ratio, which correspond to a specific MRM parent-daughter ion pair, among the ions originating from the compound contained in the sample, is obtained.

[0095] [Principle of the analysis method of C5 acylcarnitine]

[0096] Next, the principle of the analysis method characteristic of the acylcarnitine analysis device of the present embodiment will be described.

[0097] In the measurement section 2, the precursor ions are dissociated by CID in the collision cell 27, but the manner of dissociation differs depending on the kinetic energy possessed by the precursor ions. This kinetic energy is the collision energy (CE). The collision energy is determined by the direct-current potential difference between the entrance end of the collision cell 27 and the stage (preceding stage of the Figure 1 the precursor ion selection unit 26, but sometimes other ion optical elements such as an ion lens), and thus the collision energy is usually represented by this potential difference. Therefore, the collision energy can be adjusted using the direct-current bias voltage applied to either or both of the precursor ion selection unit 26 and the ion optical element of the preceding stage thereof.

[0098] As described above, when the collision energy is changed, the manner of dissociation of the precursor ion changes, and the generation pattern of the product ions changes. In order to improve the detection sensitivity, it is desirable to select a product ion (i.e., an MRM parent-daughter ion pair) having an ion intensity as large as possible. Therefore, in the conventional C5 acylcarnitine analysis, the MRM parent-daughter ion pair of m / z 246.15 > 84.95 having the largest ion intensity is used as a quantitative ion for determining the presence or absence of the component or calculating the content. However, as has been described, under m / z 246.15 > 84.95, isovalerylcarnitine associated with inborn metabolic abnormality and neopentanoylcarnitine mainly derived from antibacterial agents cannot be distinguished. Therefore, the present inventors carefully investigated the relationship between the collision energy and the ion intensity for each of various MRM parent-daughter ion pairs associated with C5 acylcarnitine through experiments.

[0099] Figures 2-6 A graph showing the relationship between the collision energy and the peak intensity for isovalerylcarnitine (hereinafter sometimes abbreviated as "i-C5") and neopentanoylcarnitine (hereinafter sometimes abbreviated as "p-C5") under each of different MRM parent-daughter ion pairs obtained through experiments.

[0100] The above experiment was performed by the following procedure.

[0101] A 100 pg / L aqueous solution was prepared from a standard of i-C5 and p-C5 using ultrapure water, respectively, and used as a sample. As a mass spectrometry device, LCMS-8060 manufactured by Shimadzu Corporation was used, and first, product ion scan measurement was performed on the precursor ion (m / z 246.15) common to i-C5 and p-C5, and product ions unique to each compound were searched. As a result, it was known that no product ion unique to each compound was observed, and as representative product ions capable of obtaining a certain degree of high peak intensity, there were five of m / z 187.05, m / z 84.95, m / z 57.00, m / z 41.10, and m / z 29.15.

[0102] Next, the peak intensity when the set value of the collision energy was changed in the range of -100 V to -10 V in steps of 5 V was measured for each of the five product ions. Figures 2-6 The results are shown. Among them, in Figures 2-6 In order to easily compare the peak intensity pattern with respect to the change in the collision energy, the pattern was normalized so that the maximum value of the peak intensity in each of the peak intensity patterns of i-C5 and p-C5 was 1.

[0103] In the conventional C5 acylcarnitine quantification, Figure 3The peak intensity pattern in i-C5 and p-C5 could hardly be distinguished in the case of the MRM parent-daughter ion pair of m / z 246.15 > 84.95. In contrast, the peak intensity pattern in i-C5 and p-C5 could be distinguished in the case of the MRM parent-daughter ion pairs of m / z 246.15 > 57.00, m / z 246.15 > 41.10, and m / z 246.15 > 29.15. In particular, in the case of m / z 246.15 > 41.10, a clear difference in the peak intensity could be seen over almost the entire range of collision energy. In the cases of m / z 246.15 > 57.00 and m / z 246.15 > 29.15, a sufficient difference in the peak intensity could be seen depending on the collision energy. From this experimental insight, it was known that the identification of i-C5 and p-C5 could be performed by utilizing the difference in the peak intensity or the peak intensity pattern in i-C5 and p-C5 in these specific MRM parent-daughter ion pairs.

[0104] [Specific example of the identification method of i-C5 and p-C5]

[0105] As described above, only from the fact that Figures 2-6 the MRM parent-daughter ion pair that produced the greatest difference in i-C5 and p-C5 was m / z 246.15 > 41.10. Therefore, in the acylcarnitine analysis device of the above-described embodiment, the measuring section 2 repeatedly performed the MRM measurement under CE = -22 V, m / z 246.15 > 84.95, and the MRM measurement under CE = -45 V, m / z 246.15 > 41.10, on the same unknown sample under the control of the analysis control section 4. The MRM measurement was repeatedly performed from the time when the sample was injected by the injector 13 to the time when a predetermined time had elapsed. Thereby, data constituting a chromatogram waveform indicating the time elapse of the ion intensity data as a result of each MRM measurement was stored in the data collection section 31.

[0106] The peak intensity calculation section 32 produced a chromatogram waveform corresponding to each of the above-described two MRM parent-daughter ion pairs from the data stored in the data collection section 31, and performed peak detection on the waveform to find the value of the peak top (the peak intensity value). Furthermore, the peak intensity value was used as the signal intensity for the operation processing described later, but instead of the peak intensity value, the area value of the peak from the start point to the end point of the peak could be calculated and used as the signal intensity.

[0107] In the case where the C5 acylcarnitine contained in the unknown sample is i-C5 and in the case where the C5 acylcarnitine contained in the unknown sample is p-C5, there is little difference in the signal intensity Al under the MRM parent-daughter ion pair of m / z 246.15 > 84.95. In contrast, there is a sufficient difference in the signal intensity A2 under the MRM parent-daughter ion pair of m / z 246.15 > 41.10 between the case where it is i-C5 and the case where it is p-C5. Therefore, the component determination section 33 calculates the intensity ratio A2 / Al between the signal intensity Al under the MRM parent-daughter ion pair of m / z 246.15 > 84.95 and the signal intensity A2 under the MRM parent-daughter ion pair of m / z 246.15 > 41.10.

[0108] Further, if the ion detected under the MRM parent-daughter ion pair of m / z 246.15 > 84.95 is set as a quantitative ion and the ion detected under the MRM parent-daughter ion pair of m / z 246.15 > 41.10 is set as a confirmation ion, the above intensity ratio A2 / Al is a so-called confirmation ion ratio. Therefore, the intensity ratio A2 / Al will be referred to as a confirmation ion ratio hereinafter.

[0109] From the above Figure 5 It is understood that the peak intensity of i-C5 is greater than that of p-C5 in the case where CE = -45 V. Therefore, if the C5 acylcarnitine is i-C5 (does not contain p-C5), the value of the above confirmation ion ratio is large, and if the C5 acylcarnitine is p-C5 or contains p-C5, the value of the confirmation ion ratio is relatively small.

[0110] Therefore, the component determination section 33 determines whether the confirmation ion ratio calculated based on the measurement result is within the determination range considered to be i-C5 with respect to the confirmation ion ratio. Also, in the case where the value of the confirmation ion ratio is within the determination range, it is determined that the C5 acylcarnitine contained in the unknown sample is i-C5. On the other hand, in the case where the value of the confirmation ion ratio deviates from the determination range, it is determined that the C5 acylcarnitine contained in the sample contains p-C5. That is, even in the case where it is determined to be positive by the detection of the C5 acylcarnitine, it is determined that there is a possibility of false positive. The central control section 5 receives the determination result of the component determination section 33 and displays it on the screen of the display section 7.

[0111] As described above, even in the case where it is determined that the C5 acylcarnitine contained in the sample contains p-C5, it cannot be determined that it does not contain i-C5, and thus re-examination is required. However, the doctor who confirms the result can determine that the detected C5 acylcarnitine is highly likely to originate from antibiotics based on, for example, the medication history of the examinee (newborn) or the like. Thus, even in the case where it is positive in the screening, it is possible to make a suggestion to the parents of the newborn that the cause is highly likely to be antibiotics rather than a congenital factor.

[0112] According to experiments by the present inventors, a range of 1.4% to 2.6% can be selected as a determination range of the value of the confirmation ion ratio under the above-described kinds of apparatus and analysis conditions. This is obtained by setting the center value to 2% and the allowable amplitude to ±30%. In this case, if the value of the confirmation ion ratio based on the measured data is within the range of 1.4% to 2.6%, it can be determined that the C5 acylcarnitine contained in the unknown sample is i-C5, and if the value of the confirmation ion ratio deviates from the range of 1.4% to 2.6%, it can be determined that the C5 acylcarnitine contained in the unknown sample is likely to be p-C5. Of course, the determination range depends on the kind of apparatus, the analysis conditions, and the like. Thus, it is necessary to determine an appropriate determination range in advance through experiments or the like.

[0113] Figure 7 and Figure 8 is a chromatogram waveform chart showing the results of experiments on whether i-C5 and p-C5 are correctly determined, Figure 7 is a chromatogram waveform chart for a sample containing i-C5, Figure 8 is a chromatogram waveform chart for a sample containing p-C5. The intensity of the peak of m / z 246.15>41.00 is small compared to the intensity of the peak of m / z 246.15>84.95 and is difficult to see, and thus, in Figure 7 and Figure 8 , the vertical axis (intensity axis) of the chromatogram of m / z 246.15>41.00 is enlarged by 30 times.

[0114] In the example shown in Figure 7 , the value of the confirmation ion ratio is within the determination range of 1.4% to 2.6%, and thus it is determined to be i-C5. In addition, in the example shown in Figure 8 , the value of the confirmation ion ratio deviates from the determination range of 1.4% to 2.6%, and thus it is determined to be likely to be p-C5. In this way, it is confirmed experimentally that i-C5 and p-C5 can be accurately distinguished by using the ratio of the signal intensities under the above-described two kinds of MRM parent-daughter ion pairs, that is, the confirmation ion ratio.

[0115] [Other examples of the method of distinguishing i-C5 and p-C5]

[0116] As long as you see Figure 5 It is known that the peak intensity pattern with respect to the change in collision energy is clearly different between i-C5 and p-C5. In addition, the collision energy value at which the peak intensity shows the maximum is different itself. Therefore, this phenomenon can also be utilized to identify i-C5 and p-C5.

[0117] Specifically, under the control of the analysis control section 4, the measurement section 2 acquires ion intensity data while changing the collision energy in the MRM parent-daughter ion pair of m / z 246.15 > 41.10 at a prescribed step width for the unknown sample. In the data processing section 3, the peak intensity calculation section 32 creates a peak intensity pattern based on the acquired data as shown in FIG. 6. The component determination section 33 determines whether it is only i-C5 or it can be p-C5 by comparing this peak intensity pattern with the peak intensity patterns that are respectively precalculated for i-C5 and p-C5. Figure 5 Alternatively, in the data processing section 3, the peak intensity calculation section 32 calculates the collision energy value at which the ion intensity becomes a peak (maximum) from the peak intensity pattern. Then, the component determination section 33 determines whether it is only i-C5 or it can be p-C5 by investigating whether the acquired collision energy value coincides with or is close to either of the collision energy values at which the peak intensity is the maximum that are respectively precalculated for i-C5 and p-C5.

[0118] In addition, in the explanation of the device based on the above-described embodiment, the MRM parent-daughter ion pair of m / z 246.15 > 41.10 in which the peak intensity pattern greatly differs between i-C5 and p-C5 is utilized, but it is known that

[0119] and Figure 4 In other two MRM parent-daughter ion pairs (m / z 246.15 > 57.00, m / z 246.15 > 29.15), the peak intensity also sufficiently differs between i-C5 and p-C5 depending on the value of the collision energy. Therefore, even if these MRM parent-daughter ion pairs (m / z 246.15 > 57.00, m / z 246.15 > 29.15) are used instead of m / z 246.15 > 41.10, i-C5 and p-C5 can be identified. Figure 6 In

[0120] In the above-described embodiment, the peak intensity pattern is calculated from the ion intensity data acquired while changing the collision energy in the MRM parent-daughter ion pair of m / z 246.15 > 41.10 at a prescribed step width, but the present application is not limited to this. For example, the peak intensity pattern can be calculated from the ion intensity data acquired while changing the collision energy in the MRM parent-daughter ion pair of m / z 246.15 > 41.10 at a prescribed step width, and the collision energy value at which the peak intensity is the maximum can be calculated from the peak intensity pattern. Then, it is determined whether it is only i-C5 or it can be p-C5 by investigating whether the acquired collision energy value coincides with or is close to either of the collision energy values at which the peak intensity is the maximum that are respectively precalculated for i-C5 and p-C5. Figure 2In the peak intensity pattern shown, no clear difference was observed between i-C5 and p-C5 in the MRM parent-daughter ion pair of m / z 246.15 > 187.05. In contrast, a graph of the collision energy dependence of the confirmation ion ratio was plotted with the ion detected in the MRM parent-daughter ion pair of m / z 246.15 > 84.95 as the quantitative ion and the ion detected in the MRM parent-daughter ion pair of m / z 246.15 > 187.05 as the confirmation ion. Figure 9 .

[0121] In addition, Figure 10 A graph showing the chromatogram waveforms obtained by measuring standard samples of i-C5 and p-C5 using the MRM parent-daughter ion pairs of m / z 246.15 > 84.95 and m / z 246.15 > 187.05, respectively, is shown in FIG. 6. In the chromatogram of i-C5 shown in (a) of FIG. 6, the confirmation ion ratio ((bl / al) x 100) was 17.5%, and in contrast, in the chromatogram of p-C5 shown in (b) of FIG. 6, the confirmation ion ratio ((b2 / a2) x 100) was 25.5%, a clear difference. Figure 10 Figure 10

[0122] From these results, it was found that i-C5 and p-C5 also have a clear difference in the confirmation ion ratio with the MRM parent-daughter ion pair of m / z 246.15 > 187.05 as the confirmation ion. That is, in addition to the three MRM parent-daughter ion pairs described above, the MRM parent-daughter ion pair of m / z 246.15 > 187.05 can also be used for the identification of i-C5 and p-C5.

[0123] Figure 11 A graph showing a comparison between the chromatogram waveforms for standard samples of i-C5 and p-C5 in the case of using the MRM parent-daughter ion pair of m / z 246.15 > 187.05 and the MRM parent-daughter ion pair of m / z 246.15 > 41.10 is shown in FIG. 7. The collision energy was set to the value at which the sensitivity was highest in each MRM parent-daughter ion pair.

[0124] From this measurement result, it was found that the MRM parent-daughter ion pair of m / z 246.15 > 187.05 has a detection sensitivity that is about 9 times higher for i-C5 and about 18 times higher for p-C5 than the MRM parent-daughter ion pair of m / z 246.15 > 41.10. That is, by using the MRM parent-daughter ion pair of m / z 246.15 > 187.05, it is possible to identify i-C5 and p-C5 with higher sensitivity than with the other MRM parent-daughter ion pairs.

[0125] Figure 12 ​​A graph showing the relationship between the ratio of i-C5 in the sample and the confirmation ion ratio in the case where the MRM parent-daughter ion pair using m / z 246.15 > 187.05 is used, which is calculated from the measurement results. As shown in FIG. 9, the ratio of i-C5 in the sample and the confirmation ion ratio are in a relationship represented by a straight line, i.e., a first order equation. Therefore, in the device of the above-described embodiment, the above-described relationship obtained by the preliminary experiment is set as a standard curve and stored in the component determination section 33, and it is possible to estimate the ratio of i-C5 or p-C5 from the confirmation ion ratio obtained from the measurement of the sample using the standard curve. Such estimation of the ratio of i-C5 or p-C5 using the confirmation ion ratio can also be performed using an MRM parent-daughter ion pair other than m / z 246.15 > 187.05. Figure 12

[0126] However, if the detection sensitivity of i-C5 or p-C5 is low, it is difficult to ensure sufficient accuracy. In contrast, as described above, under the MRM parent-daughter ion pair of m / z 246.15 > 187.05, the detection sensitivity is higher than that of other MRM parent-daughter ion pairs, and thus it is possible to improve the estimation accuracy of the ratio of i-C5 or p-C5.

[0127] In addition, in the above description, i-C5 and p-C5 are identified based on the measurement results of only one MRM parent-daughter ion pair, but it is also possible to improve the reliability of the determination by combining the information of the ion intensities obtained under a plurality of MRM parent-daughter ion pairs.

[0128] The identification of isovaleryl carnitine and neopentanoyl carnitine as its isomer was described, but other acyl carnitines can also be identified by the same method. Several examples are described.

[0129] [Identification of BCA and IBCA]

[0130] C4 acyl carnitine (acyl carnitine having an acyl group with a carbon number of 4) includes isobutyryl carnitine (sometimes abbreviated as "IBCA" hereinafter) and the like as isomers in addition to butyryl carnitine (sometimes abbreviated as "BCA" hereinafter). Butyryl carnitine is known as a metabolite related to congenital diseases such as short-chain acyl-CoA dehydrogenase deficiency and glutaric acidemia type II, but it is difficult to identify because it does not have specificity of product ions generated by dissociation in addition to the same mass as isobutyryl carnitine as its isomer.

[0131] Figures 13-16 ​A graph showing results obtained by showing collision energy dependency of confirmation ion ratios when product ions of MRM parent-daughter ion pairs based on m / z 232 > 85 having the highest sensitivity are set as quantitative ions and product ions under the four main MRM parent-daughter ion pairs other than this (m / z 232 > 173, m / z 232 > 57, m / z 232 > 41, m / z 232 > 29) are set as confirmation ions for actual measurement of BCA and IBCA.

[0132] From this result, it is known that there is a sufficient difference in confirmation ion ratio at a specific collision energy between BCA and IBCA under any of the four MRM parent-daughter ion pairs of m / z 232 > 173, m / z 232 > 57, m / z 232 > 41, and m / z 232 > 29. Accordingly, as with the identification of i-C5 and p-C5, it is possible to identify BCA and IBCA by using the confirmation ion ratio. In addition, if the size of the peak intensity observed in the chromatogram, that is, the detection sensitivity, and the size of the difference in confirmation ion ratio for BCA and IBCA are considered, it can be said that m / z 232 > 173 is preferable as the confirmation ion among the four MRM parent-daughter ion pairs described above.

[0133] [Identification of C5-DC and C6-OH]

[0134] The above examples are all examples for identifying acylcarnitines having the same mass because the chemical formulas of the compounds are the same, but even with acylcarnitines having different chemical formulas, there are cases where it is not possible to distinguish them by integral mass because the mass difference is extremely small.

[0135] For example, glutaroylcarnitine (sometimes abbreviated as "C5-DC" hereinafter) is known as a metabolite associated with a congenital disease such as glutaric aciduria type I, and the exact molecular weight is 275.1368806. On the other hand, although the exact molecular weight of hydroxylhexanoylcarnitine (sometimes abbreviated as "C6-OH" hereinafter) is 275.173264 and it is possible to identify C5-DC by the exact mass after the decimal point, it is not possible to identify it by mass using a general triple quadrupole mass spectrometer used in the device of the above embodiment. In addition, for C5-DC and C6-OH, the MRM parent-daughter ion pair with the highest sensitivity is m / z 276 > 85, and it is also difficult to identify them by the ion peak intensity under this MRM parent-daughter ion pair.

[0136] Therefore, it was revealed that when product ion scan measurements were performed on C5-DC and C6-OH, respectively, with the precursor ion set to m / z 276 while varying the collision energy, specific product ions were observed at specific collision energies. Specifically, at a collision energy of approximately -30 V, product ions were specifically detected for C5-DC with an MRM parent-daughter ion pair of m / z 276 > 87, and for C6-OH with an MRM parent-daughter ion pair of m / z 276 > 69.

[0137] Figure 17 and Figure 18 These graphs show chromatogram waveforms obtained for samples containing C5-DC and C6-OH, respectively, using two MRM parent and daughter ion pairs: m / z 276 > 87 and m / z 276 > 69. Each figure also shows chromatogram waveforms obtained for C5-DC and C6-OH using the MRM parent and daughter ion pair of m / z 276 > 85, which has been used as a quantification ion.

[0138] like Figure 17 As shown in A and B in FIG, for C5-DC, a clear peak was observed under the MRM parent-daughter ion pair of m / z 276>87, whereas no peak was observed under the MRM parent-daughter ion pair of m / z 276>69. Figure 18 As shown in Figures C and D, for C6-OH, a clear peak is observed for the MRM parent and daughter ion pair of m / z 276 > 69, whereas no peak is observed for the MRM parent and daughter ion pair of m / z 276 > 87. Therefore, based on the peak intensities of these two MRM parent and daughter ion pairs, m / z 276 > 69 and m / z 276 > 87, it is possible to identify whether the acylcarnitine contained in the sample is C5-DC or C6-OH.

[0139] In this way, C5-DC and C6-OH having the same integral mass can be identified using the signal intensity of a specific confirming ion, rather than using a common quantification ion for both.

[0140] [Identification of C3-DC and C4-OH]

[0141] As another example, malonylcarnitine (hereinafter sometimes abbreviated as "C3-DC") is known as a metabolite associated with a congenital disease such as malonic acidemia, and the exact molecular weight is 247.1055822. On the other hand, 3-hydroxybutyrylcarnitine (hereinafter sometimes abbreviated as "C4-OH") is known as a metabolite associated with a congenital disease such as 3-hydroxyacyl-CoA dehydrogenase deficiency, and the exact molecular weight is 247.1419656. Although C3-DC and C4-OH can be identified based on the exact mass below the decimal point, as in the above example, identification based on the integer mass cannot be performed. In addition, the MRM parent-daughter ion pair with the best sensitivity is m / z 248 > 85, and identification based on the intensity of this peak is also difficult.

[0142] As in the above example, the presence or absence of specific product ions was confirmed in C3-DC and C4-OH, but there was no specific product ion found between C5-DC and C6-OH. Therefore, whether or not identification can be performed using the difference in the confirmation ion ratio at the specific collision energy described above was investigated.

[0143] Figures 19-26 To show the results obtained by confirming the collision energy dependence of the confirmation ions when, for C3-DC and C4-OH, the product ions under the MRM parent-daughter ion pair with the highest sensitivity, m / z 248.2 > 85.1, were set as the quantitative ions, and the product ions under the eight main MRM parent-daughter ion pairs other than this (m / z 248.2 > 189.1, m / z 248.2 > 144.1, m / z 248.2 > 103.1, m / z 248.2 > 58.1, m / z 248.2 > 57.1, m / z 248.2 > 45.1, m / z 248.2 > 43.1, m / z 248.2 > 29.2) were set as the confirmation ions, a graph showing the results is shown.

[0144] From this result, it was found that for any of the eight MRM parent-daughter ion pairs, m / z 248.2 > 189.1, m / z 248.2 > 144.1, m / z 248.2 > 103.1, m / z 248.2 > 58.1, m / z 248.2 > 57.1, m / z 248.2 > 45.1, m / z 248.2 > 43.1, m / z 248.2 > 29.2, there was a sufficient difference in the confirmation ion ratio at a specific collision energy for C3-DC and C4-OH. Accordingly, as with the identification of BCA and IBCA, C3-DC and C4-OH can be identified by using the confirmation ion ratio at a prescribed confirmation ion.

[0145] As described above, the acylcarnitine analysis device of this embodiment and its variations can easily distinguish between acylcarnitines with identical molecular weights, such as i-C5 and p-C5, BCA and IBCA, or between acylcarnitines with identical integer masses, such as C5-DC and C6-OH, or C3-DC and C4-OH. Information available for this distinction includes the ratio of the signal intensity of the quantification ion to the signal intensity of the confirmation ion at a specific collision energy, i.e., the confirmation ion ratio; the pattern of peak intensity changes corresponding to changes in collision energy for a specific confirmation ion; and the signal intensity of the confirmation ion observed specifically for each acylcarnitine.

[0146] Furthermore, the acylcarnitine analysis device of the above embodiment can be modified as follows. Specifically, in the device of the above embodiment, sample introduction is performed using a flow injection analysis method. In this flow injection analysis method, a sample liquid is injected into a mobile phase delivered by a liquid delivery pump 12 without providing a column between the injector 13 and the ESI nebulizer 21. The sample liquid is then introduced into the ESI nebulizer 21 without using a column for component separation. However, the components in the sample may be separated using a liquid chromatograph and then introduced into a triple quadrupole mass spectrometer.

[0147] That is, in Figure 1 In the illustrated apparatus, a column can be placed between the injector 13 and the ESI nebulizer 21, and a sample solution (eluent) containing the components separated by the column can be introduced into the ESI nebulizer 21. Generally, i-C5 and p-C5 can be separated by liquid chromatography, but due to their similar retention times, sufficient separation may not be possible in some cases. Even in such cases, accurate identification of acylcarnitines such as i-C5 and p-C5 can be achieved by utilizing the MRM measurement results for specific MRM parent-daughter ion pairs as described above.

[0148] This type of analysis using a liquid chromatography-mass spectrometry device is often used in re-inspections. In this case, by using the above-described method, the recognition accuracy in the re-inspection can be improved.

[0149] The above-described embodiment and various modifications are merely examples of the present invention, and any modifications, changes, or additions made as appropriate within the scope of the gist of the present invention are clearly encompassed by the claims of the present application.

[0150] In addition, in the description of the above-described embodiments, the number of digits after the decimal point of the value of the mass-to-charge ratio of the MRM parent-daughter ion pair is sometimes set to one or two digits, but as already described, the accuracy of the mass-to-charge ratio value depends on the mass accuracy and the mass resolution of the device. Thus, the MRM parent-daughter ion pair in which the ion contained in the range of ±0.1, ±0.2, ±0.3, or ±0.4 with respect to the mass-to-charge ratio value expressed in one or two digits after the decimal point, for example, is also substantially equivalent to the MRM parent-daughter ion pair described in the claims of the present application.

[0151] [Various Modes]

[0152] It is obvious to those skilled in the art that the above-described exemplary embodiments are specific examples of the following modes.

[0153] (1) One mode of the acylcarnitine analysis method according to the present application is a method of analyzing C5 acylcarnitine using a tandem mass spectrometer, comprising:

[0154] a measurement step of performing MRM measurement based on at least one of MRM parent-daughter ion pairs of m / z 246 > 187, m / z 246 > 57, m / z 246 > 41, and m / z 246 > 29 on a test subject; and

[0155] a processing step of identifying isovalerylcarnitine and pivaloylcarnitine, which is an isomer thereof, in the test subject, or determining whether pivaloylcarnitine is contained in C5 acylcarnitine in the test subject, using the measurement result obtained in the measurement step.

[0156] (16) One mode of the acylcarnitine analysis device according to the present application is a device capable of performing the acylcarnitine analysis method described in item 1, comprising:

[0157] a measurement unit that is a tandem mass spectrometer that performs MRM measurement based on at least one of MRM parent-daughter ion pairs of m / z 246 > 187, m / z 246 > 57, m / z 246 > 41, and m / z 246 > 29 on a test subject; and

[0158] a processing unit that identifies isovalerylcarnitine and pivaloylcarnitine, which is an isomer thereof, in the test subject, or determines whether pivaloylcarnitine is contained in C5 acylcarnitine in the test subject, using the measurement result obtained by the measurement unit, wherein the C5 acylcarnitine is an acylcarnitine having an acyl group with a carbon number of 5.

[0159] The acylcarnitine analysis method according to the item 1 or the acylcarnitine analysis device according to the item 16 can accurately perform the identification of isovalerylcarnitine and neopentanoylcarnitine, which has been difficult in the past, or the determination of whether it is possible that neopentanoylcarnitine is contained in the detected C5 acylcarnitine, without performing component separation using a liquid chromatograph. Thus, it is possible to reduce false positives in the screening for C5 acylcarnitine. In addition, even in the case where it is positive in the screening for C5 acylcarnitine, it is possible to inform the user that the reason thereof is likely to be neopentanoylcarnitine derived from a medicament. As a result, it is possible to reduce the physical burden on a newborn due to reexamination, or to reduce the psychological burden on the parents of a newborn who is positive in the screening.

[0160] (2) In the acylcarnitine analysis method according to the item 1, it is possible to be configured so that:

[0161] In the measurement step, the first MRM measurement for one of the MRM parent-daughter ion pairs of m / z 246>187, m / z 246>57, m / z 246>41, and m / z 246>29, and the second MRM measurement for the MRM parent-daughter ion pair of m / z 246>85 are performed on the test subject to acquire intensity information of ions derived from C5 acylcarnitine,

[0162] In the processing step, the possibility of containing neopentanoylcarnitine is evaluated on the basis of a confirmation ion ratio that is the ratio of the signal intensity obtained by the first MRM measurement to the signal intensity obtained by the second MRM measurement.

[0163] The acylcarnitine analysis method according to the item 2 can evaluate the possibility of containing neopentanoylcarnitine with reference to the MRM parent-daughter ion pair (m / z 246>85) that shows approximately the same ion intensity, regardless of whether the C5 acylcarnitine contained in the sample is isovalerylcarnitine or neopentanoylcarnitine. Thus, the accuracy of the evaluation (including the identification or determination) in the processing step is not easily affected by variations in analysis conditions or variations in measurement environments, and accurate evaluation can always be performed.

[0164] (3) In the acylcarnitine analysis method according to the item 2, it is possible to be configured so that the MRM parent-daughter ion pair of the first MRM measurement is m / z 246>187 or m / z 246>41.

[0165] m / z 246>41 is the MRM parent-daughter ion pair having the largest difference in peak intensity between isovalerylcarnitine and pivaloylcarnitine. On the other hand, m / z 246>187 is an MRM parent-daughter ion pair having a relatively high sensitivity for isovalerylcarnitine and pivaloylcarnitine as compared with m / z 246>41. Thus, according to the acylcarnitine analysis method of item 3, the accuracy of the evaluation in the processing step can be further improved.

[0166] (4) In the acylcarnitine analysis method of item 1, it can be configured that:

[0167] In the processing step, the information representing the relationship between the confirmation ion ratio and the existence ratio of isovalerylcarnitine or pivaloylcarnitine in the test object is used to estimate the existence ratio of isovalerylcarnitine or pivaloylcarnitine contained in the test object as a target.

[0168] According to the acylcarnitine analysis method of item 4, not only isovalerylcarnitine and pivaloylcarnitine can be identified, but also the existence ratio of each compound can be estimated.

[0169] (5) In addition, in the acylcarnitine analysis method of item 1, it can be configured that:

[0170] In the measurement step, MRM measurement is performed while varying the collision energy under the MRM parent-daughter ion pair of m / z 246>41 for the test object, and intensity information of the ion derived from C5 acylcarnitine is acquired,

[0171] In the processing step, the possibility of containing pivaloylcarnitine is evaluated on the basis of the collision energy dependence of the ion intensity obtained in the measurement step.

[0172] According to the acylcarnitine analysis method of item 5, even in a case where, for example, MRM measurement for the MRM parent-daughter ion pair of m / z 246>85 described above cannot be performed for some reason, it is possible to determine whether pivaloylcarnitine is contained in the detected C5 acylcarnitine.

[0173] (6) In addition, in the acylcarnitine analysis method of any one of items 1 to 5, it can be configured that the test object is introduced into the tandem mass spectrometry device by flow injection analysis.

[0174] According to the acylcarnitine analysis method of item 6, since it does not require the use of a column for component separation, it is possible to shorten the measurement time for one test object, and is suitable for screening requiring rapidity.

[0175] (7) In the acylcarnitine analysis method described in item 6, it can be configured so that:

[0176] In the measurement step, MRM measurement is repeatedly performed,

[0177] In the processing step, the peak top value or the peak area value of the peak representing the change in ion intensity over time derived from the C5 acylcarnitine is used as the measurement result.

[0178] By using the height of the peak (the value of the peak top) as the measurement result, rapid processing can be performed, and the efficiency of the screening can be improved. On the other hand, generally, the quantification of the peak area value is more excellent than that of the height of the peak, and thus by using the peak area value as the measurement result, more accurate recognition or determination can be performed.

[0179] (8) In the acylcarnitine analysis method described in any one of items 1 to 5, it can be configured so that the components contained in the test body are separated by a liquid chromatograph and introduced into the tandem mass spectrometer.

[0180] According to the acylcarnitine analysis method described in item 8, the components are separated by using a column, and thus the accuracy of the recognition or determination of the measurement result using only a specific MRM parent-daughter ion pair can be further improved.

[0181] The method employed in the acylcarnitine analysis method described in item 1 and the acylcarnitine analysis apparatus described in item 16 is not limited to the recognition of isovalerylcarnitine and pivaloylcarnitine, and can be widely used for the recognition of acylcarnitines other than these, which have been difficult to recognize in the past.

[0182] (9) Another aspect of the acylcarnitine analysis method according to the present application is an analysis method for recognizing a plurality of different acylcarnitines having substantially the same mass using a tandem mass spectrometer, the method including:

[0183] a measurement step of performing, on a test body, first MRM measurement for an MRM parent-daughter ion pair that maximizes the signal intensity and second MRM measurement for an MRM parent-daughter ion pair different from the MRM parent-daughter ion pair that maximizes the signal intensity, to acquire intensity information of ions derived from an acylcarnitine that is a target; and

[0184] a processing step of recognizing the plurality of different acylcarnitines or determining whether or not one of the plurality of different acylcarnitines is contained in the acylcarnitine that is the target in the test body, on the basis of a confirmation ion ratio that is the ratio of the signal intensity obtained by the first MRM measurement to the signal intensity obtained by the second MRM measurement.

[0185] (10) In the acylcarnitine analysis method described in item 9, the plurality of different acylcarnitines can include isovalerylcarnitine and pivaloylcarnitine as an isomer thereof.

[0186] (11) In addition, in the acylcarnitine analysis method described in item 9, the plurality of different acylcarnitines can include butyrylcarnitine and isobutyrylcarnitine as an isomer thereof.

[0187] (17) Another aspect of the acylcarnitine analysis device according to the present application is a device that can implement the acylcarnitine analysis method described in item 9, and includes:

[0188] a measurement unit that is a tandem mass spectrometer that performs, on a test subject, first MRM measurement for an MRM parent-daughter ion pair that maximizes signal intensity, and second MRM measurement for an MRM parent-daughter ion pair that is different from the MRM parent-daughter ion pair that maximizes signal intensity; and

[0189] a processing unit that identifies a plurality of different acylcarnitines that are substantially identical in mass, or determines whether a certain one of the plurality of different acylcarnitines is included in an acylcarnitine that is a target in the test subject, on the basis of a confirmation ion ratio that is a ratio of signal intensity obtained by the first MRM measurement in the measurement unit to signal intensity obtained by the second MRM measurement.

[0190] As has been described, the "plurality of different acylcarnitines that are substantially identical in mass" described here can include cases where acylcarnitines that are completely identical in mass, such as isomers, exist, and cases where acylcarnitines that are not completely identical in mass, such as acylcarnitines that are identical in mass by an integer, exist. Isovalerylcarnitine and pivaloylcarnitine are an example of the former, and glutaroylcarnitine and hydroxylhexanoylcarnitine are an example of the latter.

[0191] The acylcarnitine analysis method described in item 9 and the acylcarnitine analysis device described in item 17 enable accurate identification of a plurality of different acylcarnitines that are substantially identical in mass by using a confirmation ion ratio that sets a specific product ion as a confirmation ion. In addition, it is not necessary to perform component separation using a column of a liquid chromatograph, and thus identification can be performed quickly and efficiently, and is particularly suitable for screening of inborn errors of metabolism and the like caused by genetic factors.

[0192] (12) In addition, another aspect of the acylcarnitine analysis method according to the present application is an analysis method that uses a tandem mass spectrometer to identify a plurality of different acylcarnitines that are substantially identical in mass, and includes:

[0193] the measuring step, performing MRM measurement on the test object for mutually different specific MRM parent-daughter ion pairs respectively determined for the plurality of different acylcarnitines, to obtain intensity information of ions derived from the acylcarnitine targeted as the object; and

[0194] the processing step, based on the signal intensity for the specific MRM parent-daughter ion pair obtained by the measuring step, identifying the plurality of different acylcarnitines, or determining whether the acylcarnitine targeted as the object in the test object contains any of the plurality of different acylcarnitines.

[0195] Item 13. The acylcarnitine analysis method according to item 12, wherein the plurality of different acylcarnitines include glutaryl carnitine and hydroxylhexanoyl carnitine.

[0196] Item 18. Another aspect of the acylcarnitine analysis device according to the present application is a device capable of performing the acylcarnitine analysis method according to item 12, and includes:

[0197] the measuring section, which is a tandem mass spectrometer that performs MRM measurement on a test object for mutually different specific MRM parent-daughter ion pairs respectively determined for the plurality of different acylcarnitines; and

[0198] the processing section, which, based on the signal intensity for the specific MRM parent-daughter ion pair obtained by the measuring section, identifies the plurality of different acylcarnitines having substantially the same mass, or determines whether the acylcarnitine targeted as the object in the test object contains any of the plurality of different acylcarnitines.

[0199] According to the acylcarnitine analysis method according to item 12 and the acylcarnitine analysis device according to item 18, by using the intensity information of the product ions observed uniquely in each acylcarnitine, the plurality of different acylcarnitines having substantially the same mass can be accurately and easily identified. In addition, since it is not necessary to separate components using a column of a liquid chromatograph, the identification can be performed quickly and efficiently, and is particularly suitable for screening of inborn errors of metabolism caused by genetic factors and the like.

[0200] Item 14. In addition, another aspect of the acylcarnitine analysis method according to the present application is an analysis method for identifying the plurality of different acylcarnitines having substantially the same mass using a tandem mass spectrometer, and includes:

[0201] The measurement step performs MRM measurement while changing the collision energy for the inspection object in a specific MRM parent-daughter ion pair, and acquires intensity information of ions derived from acylcarnitines as targets.

[0202] The processing step identifies the plurality of different acylcarnitines based on the collision energy dependence of the ion intensity obtained in the measurement step, or determines whether the inspection object contains one of the plurality of different acylcarnitines.

[0203] Item 15. The acylcarnitine analysis method according to item 14, wherein the plurality of different acylcarnitines include malonylcarnitine and 3-hydroxybutyrylcarnitine.

[0204] Item 19. Another aspect of the acylcarnitine analysis device according to the present application is a device that can perform the acylcarnitine analysis method according to item 14, and includes:

[0205] The measurement unit is a tandem mass spectrometer that performs MRM measurement while changing the collision energy for the inspection object in a specific MRM parent-daughter ion pair.

[0206] The processing unit identifies a plurality of different acylcarnitines that are substantially identical in mass based on the collision energy dependence of the ion intensity obtained by the measurement unit, or determines whether the inspection object contains one of the plurality of different acylcarnitines.

[0207] The acylcarnitine analysis method according to item 14 and the acylcarnitine analysis device according to item 19 can accurately and easily identify a plurality of different acylcarnitines that are substantially identical in mass by using the collision energy dependence of the confirmation ion ratio in which a specific product ion is set as a confirmation ion, or the collision energy dependence of the signal intensity of the confirmation ion. In particular, even in a case where a product ion that is observed uniquely in each acylcarnitine is not present, or a case where the confirmation ion ratio at a specific collision energy does not show a sufficient difference, the acylcarnitine analysis method according to item 12 and the acylcarnitine analysis device according to item 18 can appropriately perform identification. In addition, it is not necessary to separate components using a column of a liquid chromatograph, and thus identification can be performed quickly and efficiently, and is particularly suitable for screening of inborn errors of metabolism and the like caused by genetic factors.

[0208] Explanation of Reference Signs

[0209] 1: sample introduction section; 11: mobile phase container; 12: liquid delivery pump; 13: nebulizer; 2: measurement section; 201: ionization chamber; 202: first intermediate vacuum chamber; 203: second intermediate vacuum chamber; 204: high vacuum chamber; 21: ESI nebulizer; 22: desolvation tube; 23: ion guide; 24: separator; 25: multipole ion guide; 26: pre- quadrupole mass filter; 27: collision cell; 28: post-quadrupole mass filter; 29: ion detector; 3: data processing section; 31: data collection section; 32: peak intensity calculation section; 33: component determination section; 4: analysis control section; 41: analysis condition storage section; 5: central control section; 6: input section; 7: display section.

Claims

1. A method for analyzing acylcarnitines, which is a method for analyzing C5 acylcarnitines using a tandem mass spectrometer, wherein: The C5 acylcarnitine is an acylcarnitine having an acyl group with a carbon number of 5, and the acylcarnitine analysis method includes: a measuring step of performing a multiple reaction monitoring measurement on the specimen based on at least one multiple reaction monitoring parent-daughter ion pair of m / z 246>187, m / z 246>57, m / z 246>41, and m / z 246>29; and a processing step of distinguishing isovalerylcarnitine and its isomer pivaloylcarnitine in the specimen, or determining whether the C5 acylcarnitine in the specimen contains pivaloylcarnitine, using the measurement result obtained in the measurement step; wherein, in the measuring step, a first multiple reaction monitoring measurement is performed on the specimen for one of the multiple reaction monitoring parent-daughter ion pairs m / z 246>187, m / z 246>57, m / z 246>41, and m / z 246>29, and a second multiple reaction monitoring measurement is performed on the multiple reaction monitoring parent-daughter ion pair m / z 246>85, to obtain intensity information of ions derived from C5 acylcarnitines; and In the processing step, the possibility of containing pivaloylcarnitine is evaluated based on a confirmation ion ratio, wherein the confirmation ion ratio is a ratio of the signal intensity obtained by the first multiple reaction monitoring assay to the signal intensity obtained by the second multiple reaction monitoring assay. or, wherein, in the measurement step, multiple reaction monitoring measurement is performed on the specimen while varying the collision energy under multiple reaction monitoring parent-daughter ion pairs with m / z 246>41, thereby acquiring intensity information of ions derived from C5 acylcarnitines; and In the treating step, the possibility of containing pivaloylcarnitine is evaluated based on the collision energy dependence of the ion intensity obtained in the measuring step.

2. The acylcarnitine analysis method according to claim 1, wherein The multiple reaction monitoring parent and daughter ion pairs of the first multiple reaction monitoring assay are m / z 246>187 or m / z 246>41.

3. The acylcarnitine analysis method according to claim 1, wherein In the processing step, the abundance ratio of isovalerylcarnitine or pivaloylcarnitine contained in the target specimen is estimated using information indicating the relationship between the confirmation ion ratio and the abundance ratio of isovalerylcarnitine or pivaloylcarnitine in the specimen, which has been determined in advance.

4. The acylcarnitine analysis method according to any one of claims 1 to 3, wherein The sample was introduced into the tandem mass spectrometer using flow injection analysis.

5. The acylcarnitine analysis method according to claim 4, wherein In the measuring step, multiple reaction monitoring measurement is repeatedly performed, In the treatment step, the peak top value or peak area value of the peak indicating the temporal change in ion intensity derived from C5 acylcarnitine is used as the measurement result.

6. The acylcarnitine analysis method according to any one of claims 1 to 3, wherein: Components contained in the specimen are separated by liquid chromatography and introduced into the tandem mass spectrometer.

7. A method for analyzing acylcarnitines, comprising using a tandem mass spectrometer to identify a plurality of different acylcarnitines having substantially the same mass, the method comprising: a measurement step of performing a second MRM measurement on a MRM parent-daughter ion pair that maximizes signal intensity and a first MRM measurement on a MRM parent-daughter ion pair different from the MRM parent-daughter ion pair that maximizes signal intensity on the specimen, thereby acquiring intensity information of ions derived from the target acylcarnitine; as well as a processing step of identifying the plurality of different acylcarnitines or determining whether the target acylcarnitine in the specimen contains any one of the plurality of different acylcarnitines based on a confirmation ion ratio, wherein the confirmation ion ratio is a ratio of the signal intensity obtained by the first multiple reaction monitoring measurement to the signal intensity obtained by the second multiple reaction monitoring measurement; in, (i) the plurality of different acylcarnitines comprises isovalerylcarnitine and pivaloylcarnitine as an isomer thereof, performing the second multiple reaction monitoring assay on the multiple reaction monitoring parent and daughter ion pairs of m / z 246>85, and performing the first MRM assay on one of the following: m / z 246.15>187.05, m / z 246.15>57.00, m / z 246.15>41.00, and m / z 246.15>29.15; or (ii) the plurality of different acylcarnitines comprises butyrylcarnitine and isomer thereof, performing the second multiple reaction monitoring assay on the multiple reaction monitoring parent and daughter ion pairs of m / z 232>85, and performing the first multiple reaction monitoring assay on one of the multiple reaction monitoring parent and daughter ion pairs among m / z 232>173, m / z 232>57, m / z 232>41, and m / z 232>29; or (iii) the plurality of different acylcarnitines comprises malonylcarnitine and 3-hydroxybutyrylcarnitine, performing the second multiple reaction monitoring assay on the multiple reaction monitoring parent and daughter ion pair of m / z 248.2>85.1, and The first MRM assay was performed on one of the MRM parent and daughter ion pairs m / z 248.2>189.1, m / z 248.2>144.1, m / z 248.2>103.1, m / z 248.2>58.1, m / z 248.2>57.1, m / z 248.2>45.1, m / z 248.2>43.1, and m / z 248.2>29.

2.

8. A method for analyzing acylcarnitines, comprising using a tandem mass spectrometer to identify a plurality of different acylcarnitines having substantially the same mass, the method comprising: a measuring step of performing a multi-reaction monitoring measurement on a specimen for mutually different specific multi-reaction monitoring parent-daughter ion pairs to obtain intensity information of ions derived from target acylcarnitines, wherein the mutually different specific multi-reaction monitoring parent-daughter ion pairs are determined for each of the plurality of different acylcarnitines; and a processing step of identifying the plurality of different acylcarnitines based on the signal intensity of the specific multiple reaction monitoring parent-daughter ion pair obtained in the measuring step, or determining whether the target acylcarnitine in the specimen contains any one of the plurality of different acylcarnitines; in, The plurality of different acylcarnitines comprises glutarylcarnitine and hydroxycaproylcarnitine, and The specific MRM parent-daughter ion pair for the glutarylcarnitine is m / z 276>87, and the specific MRM parent-daughter ion pair for the hydroxyhexanoylcarnitine is m / z 276>69.

9. An acylcarnitine analysis method, which uses a tandem mass spectrometer to identify a plurality of different acylcarnitines having substantially the same mass, the acylcarnitine analysis method comprising: a measurement step of performing multiple reaction monitoring measurement on the specimen while varying the collision energy under specific multiple reaction monitoring parent-daughter ion pairs to obtain intensity information of ions derived from target acylcarnitines; as well as a processing step of identifying the plurality of different acylcarnitines based on the collision energy dependence of the ion intensity obtained in the measuring step, or determining whether the target acylcarnitine in the specimen contains any one of the plurality of different acylcarnitines; in, The plurality of different acylcarnitines include isovalerylcarnitine and pivaloylcarnitine as an isomer thereof, and The specific MRM parent and daughter ion pair is one of m / z 246.15>57.00, m / z 246.15>41.00, and m / z 246.15>29.

15.

10. An acylcarnitine analysis device comprising: a measuring unit, which is a tandem mass spectrometer that performs multi-reaction monitoring measurement of a sample based on at least one multi-reaction monitoring parent-daughter ion pair of m / z 246>187, m / z 246>57, m / z 246>41, and m / z 246>29; and a processing unit that uses the measurement result obtained by the measuring unit to distinguish isovalerylcarnitine and its isomer pivaloylcarnitine in the specimen, or determines whether the C5 acylcarnitine in the specimen contains pivaloylcarnitine, wherein: The C5 acylcarnitine is an acylcarnitine having an acyl group with a carbon number of 5, The measuring unit is configured to perform a first multiple reaction monitoring measurement on one multiple reaction monitoring parent-daughter ion pair among m / z 246>187, m / z 246>57, m / z 246>41, and m / z 246>29, and a second multiple reaction monitoring measurement on the multiple reaction monitoring parent-daughter ion pair of m / z 246>85 on the specimen, thereby acquiring intensity information of ions derived from C5 acylcarnitines; and The processing unit is configured to evaluate the possibility of containing pivaloylcarnitine based on a confirming ion ratio, the confirming ion ratio being a ratio of a signal intensity obtained by the first multiple reaction monitoring assay to a signal intensity obtained by the second multiple reaction monitoring assay, or, wherein the measuring unit is configured to perform MRM measurement on the specimen while varying the collision energy in MRM parent-daughter ion pairs with m / z 246>41, thereby acquiring intensity information of ions derived from C5 acylcarnitines; and The processing unit is configured to evaluate the possibility of containing pivaloylcarnitine based on the collision energy dependency of the ion intensity obtained by the measuring unit.

11. An acylcarnitine analysis device comprising: a measurement unit, which is a tandem mass spectrometer that performs, on a specimen, a second MRM measurement on a MRM parent-daughter ion pair that maximizes signal intensity, and a first MRM measurement on a MRM parent-daughter ion pair that is different from the MRM parent-daughter ion pair that maximizes signal intensity; and a processing unit that identifies a plurality of different acylcarnitines having substantially the same mass, or determines whether the target acylcarnitine in the specimen contains any one of the plurality of different acylcarnitines based on a confirmation ion ratio, wherein the confirmation ion ratio is a ratio of a signal intensity obtained by the first multiple reaction monitoring measurement to a signal intensity obtained by the second multiple reaction monitoring measurement in the measurement unit; in, (i) the plurality of different acylcarnitines comprises isovalerylcarnitine and pivaloylcarnitine as an isomer thereof, The measurement unit is configured to perform the second multiple reaction monitoring measurement on a multiple reaction monitoring parent-daughter ion pair with m / z 246>85, and to perform the first multiple reaction monitoring measurement on one of the multiple reaction monitoring parent-daughter ion pairs with m / z 246.15>187.05, m / z 246.15>57.00, m / z 246.15>41.00, and m / z 246.15>29.15; or (ii) the plurality of different acylcarnitines comprises butyrylcarnitine and isomer thereof, The measuring unit is configured to perform the second MRM measurement on the MRM parent-daughter ion pair with m / z 232>85, and to perform the first MRM measurement on one of the MRM parent-daughter ion pairs with m / z 232>173, m / z 232>57, m / z 232>41, and m / z 232>29; or (iii) the plurality of different acylcarnitines comprises malonylcarnitine and 3-hydroxybutyrylcarnitine, The measurement unit is configured to perform the second multiple reaction monitoring measurement on the multiple reaction monitoring parent-daughter ion pair of m / z 248.2>85.1, and to perform the first multiple reaction monitoring measurement on one of the multiple reaction monitoring parent-daughter ion pairs of m / z 248.2>189.1, m / z 248.2>144.1, m / z 248.2>103.1, m / z 248.2>58.1, m / z 248.2>57.1, m / z 248.2>45.1, m / z 248.2>43.1, and m / z 248.2>29.

2.

12. An acylcarnitine analysis device comprising: a measuring unit, which is a tandem mass spectrometer that performs MRM measurement on a specimen for mutually different specific MRM parent-daughter ion pairs, wherein the mutually different specific MRM parent-daughter ion pairs are determined for a plurality of different acylcarnitines; and a processing unit that identifies a plurality of different acylcarnitines having substantially the same mass based on the signal intensity for a specific multiple reaction monitoring parent-daughter ion pair obtained by the measuring unit, or determines whether the target acylcarnitine in the specimen contains any one of the plurality of different acylcarnitines; in, The plurality of different acylcarnitines comprises glutarylcarnitine and hydroxycaproylcarnitine, and The specific MRM parent-daughter ion pair for the glutarylcarnitine is m / z 276>87, and the specific MRM parent-daughter ion pair for the hydroxyhexanoylcarnitine is m / z 276>69.

13. An acylcarnitine analysis device comprising: a measurement unit, which is a tandem mass spectrometer that performs multi-reaction monitoring measurement on a specimen while varying collision energy under specific multi-reaction monitoring parent-daughter ion pairs; and a processing unit that identifies a plurality of different acylcarnitines having substantially the same mass based on the collision energy dependency of the ion intensity obtained by the measuring unit, or determines whether the target acylcarnitine in the specimen contains any one of the plurality of different acylcarnitines; in, The plurality of different acylcarnitines include isovalerylcarnitine and pivaloylcarnitine as an isomer thereof, and The specific MRM parent and daughter ion pair is one of m / z 246.15>57.00, m / z 246.15>41.00, and m / z 246.15>29.15.

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