Calculation method, program, quantification method, and high performance liquid chromatography tandem mass spectrometry device

Abstract

This calculation method for calculating a relative sensitivity coefficient of a second lipid to a first lipid in a high performance liquid chromatography tandem mass spectrometry (LC-MS / MS) device comprises: a step (S10) for acquiring a quantitative value of the first lipid contained in a first sample; a step (S12) for acquiring a quantitative value of the second lipid contained in a second sample; a step (S14) for measuring the first sample by the LC-MS / MS, and obtaining a detection intensity of the first lipid; a step (S16) for measuring the second sample by the LC-MS / MS, and obtaining a detection intensity of the second lipid; and a step (S18) for calculating the relative sensitivity coefficient of the second lipid to the first lipid from the quantitative value of the first lipid, the quantitative value of the second lipid, the detection intensity of the first lipid, and the detection intensity of the second lipid.

Description

Calculation method, program, quantification method, and high-performance liquid chromatograph tandem mass spectrometer

[0001] The present disclosure relates to a calculation method, a program, a quantification method, and a high-performance liquid chromatograph tandem mass spectrometer, and more particularly to a technique for improving the accuracy of lipid quantification.

[0002] Lipids are substances that are soluble in nonpolar solvents but insoluble in water, and include substances with a variety of structures. For example, triacylglycerol, in which one glycerol molecule is ester-bonded with three fatty acid molecules, can be found in a wide variety of types depending on the type and combination of fatty acids bound to it and the position of the fatty acid bond to the glycerol.

[0003] Some lipids produced by plants and algae have physiological activities that are beneficial to humans, and some can be used as biofuels. Therefore, when examining plant growth conditions for the purpose of improving the production efficiency of target lipids, it is desirable to quantitatively analyze the different types of lipids in samples extracted from the plants, each containing multiple types of lipids.

[0004] When analyzing lipids by type in a sample containing multiple types of lipids, measurement by high-performance liquid chromatography tandem mass spectrometry (LC-MS / MS) is widely used. This is because, in lipid measurement by LC-MS / MS, the lipids contained in the sample are separated based on differences in chemical properties using high-performance liquid chromatography in the upstream stage, and the separated lipids can be detected with high sensitivity using a mass spectrometer in the downstream stage. In addition, in a tandem mass spectrometer, the lipid to be measured is decomposed and ions derived from the decomposition products are detected, allowing the structure of the lipid to be measured to be estimated, making it useful for analyzing samples containing unknown lipids.

[0005] Regarding the analysis of lipid components, examples of lipid measurement methods using LC-MS / MS are disclosed in Jouhet J, Lupette J, Clerc O, Magneschi L, Bedhomme M, Collin S, Roy S, Marechal E, and Rebeille F, "LC-MS / MS versus TLC plus GC methods: Consistency of glycerolipid and fatty acid profiles in microalgae and higher plant cells and the effect of a nitrogen starvation," PLoS ONE, 13(10):e0206397, 2018 (Non-Patent Document 1), and Koivusalo M, Haimi P, Heikinheimo L, Kostiainen R, and Somerharju P, "Quantitative determination of phospholipid compositions by ESI-MS: effects of acyl chain length, unsaturation, and lipid concentration on instrument response," J Lipid Res 42(4):663-72, 2001 (Non-Patent Document 2).

[0006] Regarding the estimation of lipid types, Shiva S, Vu HS, Roth MR, Zhou Z, Marepally SR, Nune DS, Lushington GH, Visvanathan M, Welti R "Lipidomic analysis of plant membrane lipids by direct infusion tandem mass spectrometry," Methods Mol Biol. 1009:79-91, 2013. (Non-Patent Document 3) discloses an example of a method for estimating lipid structures using a tandem mass spectrometer.

[0007] Jouhet J, Lupette J, Clerc O, Magneschi L, Bedhomme M, Collin S, Roy S, Marechal E, Rebeille F "LC-MS / MS versus TLC plus GC methods:Consistency of glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation." PLoS ONE, 13(10):e0206397, 2018.Koivusalo M, Haimi P, Heikinheimo L, Kostiainen R, Somerharju P "Quantitative determination of phospholipid compositions by ESI-MS: effects of acyl chain length, unsaturation, and lipid concentration on instrument response." J Lipid Res 42(4):663-72, 2001.Shiva S, Vu HS, Roth MR, Zhou Z, Marepally SR, Nune DS, Lushington GH, Visvanathan M, Welti R "Lipidomic analysis of plant membrane lipids by direct infusion tandem mass spectrometry." Methods Mol Biol. 1009:79-91, 2013.

[0008] One method for quantifying lipids in a sample using LC-MS / MS is a quantification method based on comparison with a standard substance. Specifically, a standard substance and the lipid to be measured are simultaneously measured, and the lipid is quantified by comparing the detection intensity with that of the standard substance. In this method, since a different standard substance corresponds to each type of lipid, measurement costs may be high when multiple lipids are to be measured.

[0009] On the other hand, there is also a method of analyzing lipids by measuring a sample containing multiple types of lipids by LC-MS / MS and comparing the detection intensities derived from each lipid. However, since the ionization efficiency differs for each type of lipid, lipids with high detection intensities are not necessarily contained in greater amounts in the sample than lipids with lower detection intensities. In other words, quantification of lipids by comparing the detection intensities may not be able to achieve sufficient quantitative accuracy.

[0010] Therefore, when analyzing multiple types of lipids by LC-MS / MS, there is a need for a quantitative determination method that can quantify multiple types of lipids with sufficient accuracy using standard substances of some of the lipids to be measured.

[0011] The present disclosure has been devised in light of the above-described circumstances, and its purpose is to provide a technology for improving the quantitative accuracy of lipids when measuring samples containing multiple types of lipids by LC-MS / MS, by using standard substances for some of the lipids to be measured.

[0012] A calculation method according to a first aspect of the present disclosure is a method for calculating a relative sensitivity coefficient of a second lipid of a type different from the first lipid to a first lipid in an LC-MS / MS device, and includes the steps of: acquiring a quantitative value of the first lipid contained in a first sample containing the first lipid; acquiring a quantitative value of a second lipid contained in a second sample containing the second lipid; measuring the first sample by LC-MS / MS and determining the detection intensity of the first lipid from the obtained chromatogram; measuring the second sample by LC-MS / MS and determining the detection intensity of the second lipid from the obtained chromatogram; and calculating the relative sensitivity coefficient of the second lipid to the first lipid from the quantitative value of the first lipid, the quantitative value of the second lipid, the detection intensity of the first lipid, and the detection intensity of the second lipid.

[0013] A program according to a second aspect of the present disclosure is a program executed by a processor installed in a computer, causing the computer to execute the above calculation method.

[0014] A calculation method according to a third aspect of the present disclosure is a method for calculating a lipid sensitivity coefficient in an LC-MS / MS device, and includes the steps of obtaining a quantitative value of a lipid contained in a lipid-containing sample, measuring the sample by LC-MS / MS and determining a detection intensity derived from the lipid contained in the sample from the obtained chromatogram, and determining the lipid sensitivity coefficient from the quantitative value and the detection intensity.

[0015] A quantification method according to a fourth aspect of the present disclosure is a method for quantifying a second lipid in a third sample containing a first lipid and a second lipid by a relative sensitivity coefficient calculated in an LC-MS / MS device using a first sample containing a first lipid and a second sample containing a second lipid of a different type from the first lipid, the method comprising the steps of: acquiring a quantitative value of the first lipid contained in the first sample; acquiring a quantitative value of the second lipid contained in the second sample; measuring the first sample by LC-MS / MS and determining the detection intensity of the first lipid from the obtained chromatogram; measuring the second sample by LC-MS / MS and determining the detection intensity of the second lipid from the obtained chromatogram; and quantifying the first lipid contained in the first sample. the step of measuring the third sample by LC-MS / MS to determine the detection intensities of the first lipid and the second lipid contained in the third sample; the step of measuring the quantitative value of the first lipid contained in the third sample by LC-MS / MS using a standard substance of the first lipid; and the step of quantifying the second lipid contained in the third sample using the relative sensitivity coefficient, the detection intensities of the first lipid and the second lipid contained in the third sample, and the quantitative value of the first lipid contained in the third sample.

[0016] An LC-MS / MS device according to a fifth aspect of the present disclosure is an LC-MS / MS device that quantifies a second lipid in a third sample containing a first lipid and a second lipid, and includes a liquid chromatograph, a mass spectrometer that analyzes components separated by the liquid chromatograph, and a data processor that receives measurement data from the mass spectrometer, wherein the mass spectrometer measures the third sample to determine the detection intensities of the first lipid and the second lipid contained in the third sample, and measures the quantitative value of the first lipid contained in the third sample using a standard substance for the first lipid, and the data processor calculates the quantitative value of the second lipid contained in the third sample using the detection intensities and quantitative values ​​of the first lipid and the second lipid contained in the third sample, and the relative sensitivity coefficient calculated by the calculation method described in the first aspect.

[0017] According to the present disclosure, in the measurement of multiple types of lipids by LC-MS / MS, the accuracy of lipid quantification can be improved by using standard substances for some of the lipids to be measured.

[0018] FIG. 1 is a schematic configuration diagram of an LC-MS / MS device according to an embodiment. FIG. 2 is a functional block diagram of an LC-MS / MS device according to an embodiment. FIG. 3 is a flowchart showing steps of lipid analysis by LC-MS / MS. FIG. 4 is a diagram for explaining a method for calculating a relative sensitivity coefficient. FIG. 5 is a flowchart showing a procedure for preparing a sample for calculating a relative sensitivity coefficient. FIG. 6 is a flowchart showing a procedure for calculating a relative sensitivity coefficient from a prepared sample. FIG. 7 is a diagram for explaining a method for quantifying lipids using a relative sensitivity coefficient. FIG. 8 is a flowchart showing processing related to the method for quantifying lipids using a relative sensitivity coefficient.

[0019] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail with reference to the accompanying drawings, in which the same or corresponding parts are designated by the same reference numerals and will not be described repeatedly.

[0020] [Overall Configuration of LC-MS / MS Apparatus] FIG. 1 is a diagram schematically illustrating the overall configuration of an LC-MS / MS apparatus 100 according to this embodiment. As shown in FIG. 1, the LC-MS / MS apparatus 100 includes a liquid chromatograph 1, a mass spectrometer 2, a controller 3, a display 4, and an input unit 5. The controller 3, display 4, and input unit 5 may be incorporated into the mass spectrometer 2. Alternatively, the controller 3 may be a general-purpose computer located away from the liquid chromatograph 1 and the mass spectrometer 2. The LC-MS / MS apparatus 100 can separate lipids contained in a sample into individual types of lipids and obtain detection intensities corresponding to each lipid.

[0021] Lipids are substances that are poorly soluble in water but easily soluble in organic solvents, specifically, for example, simple lipids, complex lipids, cholesterol, steroids, carotenoids, and substances structurally similar to these. Simple lipids include those in which alcohol and fatty acid are bonded, such as triacylglycerol. Complex lipids are those in which alcohol and fatty acid are bonded with at least one of phosphate, sulfur, nitrogen base, and sugar, such as glycerophospholipids, sphingophospholipids, glyceroglycolipids, and sphingoglycolipids.

[0022] The liquid chromatograph 1 includes a mobile phase container 10, a pump 11, an injector 12, and a column 13. The liquid chromatograph 1 can separate multiple types of lipids contained in a sample into individual types of lipids by utilizing differences in interactions between the stationary phase and the mobile phase.

[0023] The mobile phase container 10 stores a mobile phase, which is a liquid that carries a sample injected into the liquid chromatograph 1. The mobile phase is, for example, an organic solvent or water, or a mixture thereof. Examples of organic solvents include 2-propanol, methanol, acetonitrile, chloroform, hexane, dichloromethane, and tetrahydrofuran. The mobile phase may contain an acidic solution (e.g., trifluoroacetic acid, formic acid, and ammonium formate) as an additive. The liquid chromatograph 1 may include a single mobile phase container or multiple mobile phase containers.

[0024] The pump 11 draws in the mobile phase stored in the mobile phase container 10 and delivers it at a predetermined flow rate. The flow rate of the mobile phase delivered by the pump 11 may be constant or may vary during one measurement. The liquid chromatograph 1 may include a single pump or multiple pumps.

[0025] The injector 12 injects a predetermined amount of a sample, which has been prepared in advance in a mobile phase flow path, into the liquid chromatograph 1. The sample is introduced from the injector 12 into the mobile phase delivered from the pump 11, and the mobile phase containing the sample is introduced into the column 13.

[0026] The column 13 is filled with a stationary phase, and the mobile phase passes through the column 13. When passing through the column 13, various lipids in the sample interact with the mobile phase and the stationary phase, causing separation in the time direction. The separated lipids are eluted from the outlet of the column 13 and introduced into the mass spectrometry unit 2. The type of stationary phase is not limited as long as it is used for separating lipids.

[0027] The mass analysis unit 2 includes an ionization chamber 20, a first intermediate chamber 21, a second intermediate chamber 22, and an analysis chamber 23. The mass analysis unit 2 performs mass analysis of the sample eluted from the liquid chromatograph 1. The analysis in the mass analysis unit 2 includes detecting peaks in the mass spectrum and measuring the mass-to-charge ratio of specific or non-specific substances contained in the sample.

[0028] The ionization chamber 20 has a probe 201 and a capillary 202. The interior of the ionization chamber 20 is at atmospheric pressure. The ionization chamber 20 is connected to the next-stage first intermediate chamber 21 via a thin-diameter capillary 202. The probe 201 sprays the sample introduced into the mass analysis unit 2 while imparting a biased charge to the sample. The charged microdroplets are split and refined by the action of electrostatic force, and lipids in the sample in the droplets are ionized as the solvent evaporates. The generated ions pass through the capillary 202 and are introduced into the first intermediate chamber 21.

[0029] The first intermediate chamber 21 has an ion guide 211 and a skimmer 212. The inside of the first intermediate chamber 21 is at high vacuum. The first intermediate chamber 21 and the second intermediate chamber 22 in the next stage are connected through a small hole drilled in the top of the skimmer 212. The ion guide 211 focuses ions introduced from the ionization chamber 20 in the previous stage and transports them to the subsequent stage via the skimmer 212.

[0030] The second intermediate chamber 22 has an ion guide 221. The inside of the second intermediate chamber 22 is at high vacuum. The ion guide 221 focuses ions introduced from the first intermediate chamber 21 in the preceding stage and transports the ions to the subsequent stage.

[0031] The analysis chamber 23 includes quadrupole mass filters 231 and 233, a collision cell 232, and an ion detector 234. The quadrupole mass filter 231 is disposed before the collision cell 232, and the quadrupole mass filter 233 is disposed after the collision cell 232. The inside of the analysis chamber 23 is at atmospheric pressure. The analysis chamber 23 separates ions by mass and detects each of the separated ions to obtain a mass spectrum. Information regarding the molecular weight, molecular formula, and chemical structure of the compound can be obtained from the obtained mass spectrum.

[0032] The quadrupole mass filter 231 includes a main rod electrode 2312 and a pre-rod electrode 2311 located in front of it. The main rod electrode 2312 separates ions according to their mass-to-charge ratio. The pre-rod electrode 2311 corrects disturbances in the electric field at the entrance end and assists the function of the main rod electrode 2312.

[0033] The collision cell 232 includes a multipole ion guide 2321 therein. The collision cell 232 is connected to a collision-induced dissociation (CID) gas supply mechanism (not shown), which introduces CID gas into the collision cell 232. The CID gas promotes ion dissociation. The multipole ion guide 2321 focuses the dissociated ions and transports them to a subsequent stage. Examples of CID gas include argon, nitrogen, helium, and xenon.

[0034] The quadrupole mass filter 233 includes a main rod electrode 2332 and a pre-rod electrode 2331 disposed in front of it. The main rod electrode 2332 separates ions according to their mass-to-charge ratio. The pre-rod electrode 2331 corrects disturbances in the electric field at the entrance end and assists the function of the main rod electrode 2332.

[0035] The ion detector 234 is, for example, a pulse count detector, and generates a detection signal having a number of pulse signals corresponding to the number of incident ions. The detection signal is output to the control unit 3.

[0036] In the above-described embodiment, the LC-MS / MS device 100 is equipped with electrospray ionization as the ionization method, but the ionization method is not limited to electrospray ionization, and atmospheric pressure chemical ionization or atmospheric pressure photoionization may also be used.

[0037] The control unit 3 is communicatively connected to the liquid chromatograph 1 and the mass spectrometer 2, and is configured, for example, by a computer. The control unit 3 controls the operations of the liquid chromatograph 1 and the mass spectrometer 2, and receives measurement data acquired by the ion detector 234 of the mass spectrometer 2. The control unit 3 also calculates the quantitative values ​​of lipids based on the relative sensitivity coefficients and the measurement data.

[0038] The display unit 4 is configured by, for example, a liquid crystal display. In response to commands from the control unit 3, the display unit 4 displays the measurement data acquired by the ion detector 234 and the quantitative values ​​of lipids calculated based on the measurement data.

[0039] The input unit 5 is configured with, for example, a keyboard, a mouse, etc. The input unit 5 receives instructions from the user for the liquid chromatograph 1 and the mass spectrometer 2 and outputs the instructions to the control unit 3. A touch panel in which the display unit 4 and the input unit 5 are integrated may also be used.

[0040] 2 is a functional block diagram of the overall configuration of the LC-MS / MS system 100 according to this embodiment. As shown in FIG. 2, the control unit 3 has, as its main components, a CPU (Central Processing Unit) 30, a ROM (Read Only Memory) 31, a RAM (Random Access Memory) 32, a HDD (Hard Disk Drive) 33, a communication I / F (Interface) 34, a display I / F 35, and an input I / F 36. The components are interconnected by a data bus.

[0041] The communication I / F 34 relays communication with external devices including the liquid chromatograph 1 and the mass spectrometer 2. The communication I / F 34 is implemented by, for example, a network adapter. The communication method may be wireless communication such as Bluetooth (registered trademark) or wireless LAN, or wired communication using a USB (Universal Serial Bus) or the like.

[0042] The display I / F 35 relays data transmission between the CPU 30 and the display unit 4. In response to commands from the CPU 30, the display I / F 35 outputs to the display unit 4 a signal for displaying the measurement data obtained by the mass spectrometry unit 2 and the quantitative values ​​of lipids calculated based on the measurement data.

[0043] The input I / F 36 relays data transmission between the CPU 30 and the input unit 5. The input I / F 36 receives various information provided by operating the input unit 5, such as relative sensitivity coefficients, and commands for the liquid chromatograph 1 and the mass spectrometer 2.

[0044] The ROM 31 can non-temporarily store programs executed by the CPU 30. The RAM 32 can temporarily store data generated by the execution of the programs in the CPU 30, data input via the communication I / F 34, and instructions for the liquid chromatograph 1 and the mass spectrometer 2 input by the user via the input unit 5, and can function as a primary storage device. The HDD 33 is a non-volatile storage device and can store relative sensitivity coefficients. The control unit 3 may have a semiconductor storage device such as a flash memory instead of or in addition to the HDD 33.

[0045] [Lipid Selectivity in LC-MS / MS Analysis] Extracts from living organisms generally contain multiple types of lipids. Among these lipids, some have similar chemical properties and roughly the same molecular weight. Therefore, it is often difficult to detect a single type of lipid in a sample containing multiple types of lipids. However, by using the LC-MS / MS device 100 described above, it is possible to detect a single type of lipid from a sample containing multiple types of lipids. The procedure for detecting a single type of lipid from a sample containing multiple types of lipids using the LC-MS / MS device 100 is described using Figures 1 and 3 . Figure 3 is a flowchart showing the steps of lipid analysis by LC-MS / MS. Of the processes shown in Figure 3 , T10 is performed manually by an analyst using laboratory equipment commonly used in general scientific experiments and mass spectrometry. Of the processes shown in Figure 3 , T12 and subsequent steps are performed by the LC-MS / MS device 100.

[0046] The lipid to be detected is assumed to be a complex lipid having one polar head and two fatty acids, with glycerol as the linking moiety. Figure 3 includes illustrations of lipids and ions to more specifically explain the procedure. In lipids 61, 62, 63, and 64, the line segment represents the glycerol backbone, the ellipse represents the fatty acid, and the diamond represents the polar head. Lipids 61, 62, 63, and 64 are lipids with a structure in which two fatty acid molecules, represented by two ellipses, are bound to the glycerol backbone represented by the line segment. The length of the ellipse represents the length of the fatty acid carbon chain. In other words, different ellipse lengths represent different types of fatty acids. The shaded diamonds represent different types of polar heads than the unshaded diamonds. In other words, lipids 61 and 63 have the same polar head, but different fatty acids bound to them, making them different types of lipids. Furthermore, lipids 61 and 64 have the same type of fatty acid bound to them and the same binding position, but different polar heads, making them different types of lipids. Therefore, each of the lipids 61, 62, 63, and 64 represents a different type of lipid.

[0047] In FIG. 3, as shown in step T10, the user prepares a sample containing multiple types of lipids.

[0048] In step T12, the sample prepared in step T10 is separated by type of lipid using a liquid chromatograph. Specifically, the sample introduced into the liquid chromatograph 1 is introduced into the column 13 together with the mobile phase delivered by the pump 11. In the column 13, the lipids interact with the stationary phase and the mobile phase, but the degree of interaction varies depending on the type of lipid. The speed at which the lipids move within the column 13 is determined by this interaction, so the speed of movement varies depending on the type of lipid. Therefore, due to the difference in mobility of each lipid, the liquid chromatograph 1 can separate the lipids by type. On the other hand, lipids 61, 62, 63, and 64 with the same or approximately the same mobility are eluted simultaneously.

[0049] In step T12, the eluate containing the lipids 61, 62, 63, and 64 is introduced into the mass spectrometric section 2. The processing in the mass spectrometric section 2 is shown as step T14 in Fig. 3, and is further subdivided into steps T16 to T24.

[0050] In step T16, the lipids 61, 62, 63, and 64 introduced into the mass spectrometric section 2 are ionized to generate ions 611, 621, 631, and 641, respectively. The generated ions are introduced into the quadrupole mass filter 231.

[0051] In step T18, of the ions 611, 621, 631, and 641 introduced into the quadrupole mass filter 231, only ion 621 is selected and introduced into the downstream collision cell 232. This selection is performed by the voltage applied to the main rod electrode 2312. That is, a voltage in which a predetermined radio frequency voltage and a DC voltage are superimposed is applied to the main rod electrode 2312, and the quadrupole mass filter 231 performs this selection by allowing only ions having a specific mass-to-charge ratio corresponding to the voltage applied to the main rod electrode 2312 to pass through among the various ions sent into the quadrupole mass filter 231. The ions that have passed through the quadrupole mass filter 231 are called precursor ions.

[0052] In step T20, precursor ions 621 that have passed through the quadrupole mass filter 231 are introduced into the collision cell 232. A predetermined voltage is applied to electrodes disposed in the collision cell 232, and the ions 621 introduced into the collision cell 232 are accelerated within the collision cell 232 at an acceleration rate that corresponds to the voltage applied to the electrodes. Furthermore, CID gas is supplied into the collision cell 232 at a predetermined pressure. As a result, the accelerated ions 621 collide with the CID gas with a predetermined collision energy and dissociate, generating ions 6211, 6212, 6213, 6214, and 6215. The ions generated from the precursor ions are called product ions.

[0053] In step T22, when ions 6211, 6212, 6213, 6214, and 6215, which are product ions generated by dissociation of ion 621, are introduced into the quadrupole mass filter 233, only ion 6213, which is a product ion having a specific mass-to-charge ratio according to the voltage applied to the main rod electrode 2332 of the quadrupole mass filter 233, passes through the quadrupole mass filter 233.

[0054] In step T24, the ion detector 234 detects the ion 6213, which is a product ion that has passed through the quadrupole mass filter 233, and the process in FIG. 3 ends.

[0055] In the MS / MS analysis described above, ions to be detected are selected in two stages: the quadrupole mass filter 231 and the quadrupole mass filter 233. This highly selective analysis makes it possible to distinguish and detect ions derived from specific lipids from other ions. For example, in FIG. 3, if there is another ion having the same mass-to-charge ratio as ion 621, it may be difficult to select and detect only one of them in step T18. However, by detecting a product ion characteristic of one of these ions, it is possible to distinguish and detect two types of lipids with the same mass.

[0056] Comparative Example of a Lipid Quantification Method Using LC-MS / MS In a comparative example of a lipid quantification method using LC-MS / MS, the lipid to be measured and a standard substance corresponding to the lipid, whose quantitative value is known, are measured by LC-MS / MS, and the lipid to be measured is quantified by comparing the detection intensities of each. A standard substance is, for example, a substance in which some of the atoms constituting the lipid to be measured are replaced with a predetermined isotope. This substance has the same ionization efficiency as the lipid to be measured, but has a molecular weight different from that of the lipid to be measured, so that it can be distinguished and detected in a mass spectrometer. Therefore, the ratio of the detection intensities obtained from the product ions of each substance in the LC-MS / MS measurement corresponds to the quantitative ratio of each substance, and therefore the lipid to be measured can be quantified.

[0057] In the above-described method, it is necessary to prepare a standard substance for each type of lipid to be measured, which may impose a burden on the user to prepare the standard substances and increase the cost of measurement.

[0058] Another method is to measure multiple types of lipids by LC-MS / MS and compare their detection intensities to estimate their quantitative relationship. However, since the ionization efficiency differs for each type of lipid, lipids with higher detection intensities than other lipids are not necessarily contained in the largest amount in the sample. Therefore, simply comparing the detection intensities obtained by LC-MS / MS may not provide sufficient quantitative accuracy.

[0059] [Lipid quantification method according to an embodiment] Therefore, in the lipid quantification method according to this embodiment, a relative sensitivity coefficient is calculated, which quantifies the difference in ionization efficiency of each lipid in LC-MS / MS, and each lipid is quantified from the detection intensity obtained by LC-MS / MS measurement using this. By using the relative sensitivity coefficient, the difference in ionization efficiency for each type of lipid is corrected, thereby improving the accuracy of quantification based on the detection intensity. Furthermore, since it is possible to quantitate other lipids based on the quantitative value of a predetermined lipid as a reference, it is possible to quantitate lipids other than the predetermined lipid without using a corresponding standard substance.

[0060] [Method for calculating relative sensitivity coefficient] A method for calculating the relative sensitivity coefficient will be described. The relative sensitivity coefficient is the sensitivity coefficient of another lipid relative to the sensitivity coefficient of a reference lipid. The sensitivity coefficient is the quantitative value of that lipid relative to the detection intensity obtained by LC-MS / MS.

[0061] <1. Preparation of Samples for Calculating the Relative Sensitivity Coefficient> When calculating the relative sensitivity coefficient, a sample containing a first lipid as a reference and a sample containing a different type of lipid from the first lipid are prepared. In this case, it is preferable that each sample contains a single type of lipid. This is because when a sample contains a single type of lipid, the quantification accuracy of the lipid contained in the sample is improved compared to when a sample contains multiple types of lipid. The lipid may be produced by chemical synthesis or may be extracted from a living organism. Examples of living organisms include animals, plants, and bacteria.

[0062] 2. Quantification of lipids contained in a sample> Quantitative values ​​of the first lipid and a type of lipid different from the first lipid contained in the sample are obtained. Quantitative values ​​include, for example, concentration, amount of substance, and weight. Furthermore, when the lipid contains fatty acids, the amount of lipid is measured by quantifying the fatty acids that constitute the lipid. The device used to quantify lipids is not limited, and may be, for example, at least one of gas chromatography, liquid chromatography, liquid chromatography mass spectrometer, gas chromatography mass spectrometry, high-performance liquid chromatography tandem mass spectrometer, gas chromatography tandem mass spectrometer, balance, and electronic balance.

[0063] 3. Identifying the detection intensity of lipids contained in the sample Next, the sample is measured by LC-MS / MS, and the detection intensity of each lipid is determined from a chromatogram derived from the first lipid and a type of lipid different from the first lipid contained in the sample. The detection intensity is, for example, at least one of the peak area and the peak intensity.

[0064] 4. Calculation of Sensitivity Coefficient The sensitivity coefficient of the first lipid is calculated as the ratio of the detection intensity to the quantitative value of the first lipid contained in the sample. Similarly, sensitivity coefficients for lipids other than the first lipid are calculated.

[0065] 5. Calculation of relative sensitivity coefficient using sensitivity coefficient> The sensitivity coefficient for the first lipid can be calculated by correcting the calculated sensitivity coefficient so that the sensitivity coefficient for the first lipid becomes 1.

[0066] The method for calculating the relative sensitivity coefficient will be specifically described with reference to Fig. 4. Fig. 4 is a diagram for explaining the method for calculating the relative sensitivity coefficient.

[0067] In Fig. 4, the samples assumed are sample A containing lipid A, which is a reference lipid, sample B containing lipid B, sample C containing lipid C, and sample D containing lipid D. In the example of Fig. 4, the quantitative value of lipid A contained in sample A is 4 mol / L, the quantitative value of lipid B contained in sample B is 3 mol / L, the quantitative value of lipid C contained in sample C is 10 mol / L, and the quantitative value of lipid D contained in sample D is 2 mol / L.

[0068] 4 also shows the peak areas identified from the chromatograms obtained by analyzing each sample by LC-MS / MS. The peak area derived from lipid A contained in sample A is 8, the peak area derived from lipid B contained in sample B is 3, the peak area derived from lipid C contained in sample C is 5, and the peak area derived from lipid D contained in sample D is 8.

[0069] 4 also shows the sensitivity coefficients obtained by dividing the quantitative values ​​of lipids contained in each sample by the peak areas obtained. The sensitivity coefficients for lipid A, lipid B, lipid C, and lipid A are calculated to be 0.5, 1, 2, and 0.25, respectively.

[0070] 4 shows the sensitivity coefficient for the first lipid, calculated by correcting the calculated sensitivity coefficient so that the sensitivity coefficient for the first lipid is 1. That is, the sensitivity coefficients for lipids A, B, C, and D were calculated to be 0.5, 1, 2, and 0.25, respectively, and by dividing each sensitivity coefficient by 0.5, which is the sensitivity coefficient for lipid A, the relative sensitivity coefficients of lipids B, C, and D to lipid A were calculated to be 2, 4, and 0.5, respectively.

[0071] [Procedure for Calculating Relative Sensitivity Coefficients] As a specific example of calculating relative sensitivity coefficients, the procedure for calculating the relative sensitivity coefficients of lipids in Arabidopsis thaliana will be described with reference to FIGS. 5 and 6. FIG. 5 is a flowchart of the procedure for preparing a sample from Arabidopsis thaliana to be used for calculating relative sensitivity coefficients. FIG. 6 is a flowchart of the procedure for calculating relative sensitivity coefficients using the prepared sample. As in FIG. 3, FIG. 5 includes illustrations of lipids and ions. In FIGS. 5 and 6, it is assumed that lipids consist of fatty acids and glycerol, and that the relative sensitivity coefficient of glycerophospholipids is to be calculated.

[0072] Lipids 65, 66, 67, and 68 in Figure 5 are complex lipids with one polar head group and two fatty acids, with glycerol as the linking moiety, as in Figure 3. Also, in Figure 5, unshaded diamonds indicate polar head groups of phosphate, while shaded diamonds indicate polar head groups other than phosphate. That is, lipids 66 and 68, which have a polar head group of phosphate and two fatty acids, are classified as glycerophospholipids. The process shown in Figure 5 is performed manually by an analyst using laboratory equipment commonly used in scientific experiments and mass spectrometry, or by partially automated laboratory equipment.

[0073] In FIG. 5, as shown in step U10, an extract containing lipids 65, 66, 67, and 68 is obtained from Arabidopsis thaliana by extraction with an organic solvent.

[0074] In step U12, lipids are separated by thin layer chromatography based on the difference in the polar head group of the lipid, and lipids 66 and 68, which are glycerophospholipids, are separated from lipids 65 and 67.

[0075] As shown in step U14, the fraction corresponding to the glycerophospholipids containing the lipids 66 and 68 physically separated on the solid phase of the thin layer chromatography is collected and dissolved in a solvent.

[0076] As shown in step U16, the solution obtained in step U14 is separated into fractions 71, 72, 73, 74, 75, and 76 by a fractionation system consisting of a liquid chromatograph, according to the fatty acid composition of the lipids 66 and 68. Specifically, the eluate from the fractionation system is divided into fixed volumes, thereby dividing the solution obtained in step U14 into fractions 71, 72, 73, 74, 75, and 76.

[0077] As shown in step U18, fractions 71, 72, 73, 74, 75, and 76 obtained in step U16 are analyzed by LC-MS / MS to confirm whether each fraction contains lipids. As a result, it is revealed that fraction 72 contains lipid 66 and fraction 75 contains lipid 68.

[0078] As shown in step U20, fractions 72 and 75 are concentrated in step U18 and the resulting concentrates are redissolved to obtain sample 81 containing lipid 66 and sample 82 containing lipid 68. Samples 81 and 82 are then used in the process of step V10 in FIG.

[0079] In the following process, sample 81 and sample 82 are each subjected to the same process in parallel. The following description will be given taking the process of sample 81 containing lipids 66 as an example. Note that, of the processes shown in FIG. 6, steps other than step V38 are performed manually by an analyst using experimental equipment used in general scientific experiments and mass spectrometry, or by partially automated experimental equipment. Of the processes shown in FIG. 6, step V38 is performed by control unit 3. Note that the process corresponding to step V38 will be described in detail in steps S10 to S18 in FIG. 8, which will be described later.

[0080] In FIG. 6, as shown in step V10, the sample 81 containing the separated lipids 66 is concentrated by removing the solvent.

[0081] In step V12, 1 mL of methanol is added to the container to dissolve the lipids 66. In step V14, 800 μL of the solution obtained in step V12 is taken.

[0082] In step V16, 50 μL of 1 mM pentadecanoic acid (C15:0) solution is added to the lysate collected in step V14 as a standard for measuring fatty acids. Note that the fatty acids used as the standard are not limited to the fatty acids described above, and any fatty acids other than those constituting lipids 66 can be used.

[0083] In step V18, 900 μL of 3M hydrochloric acid in methanol is added to the solution obtained in step V16.

[0084] In step V20, the solution obtained in step V18 is heated at 85°C for 1 hour. The fatty acids that make up the lipid 66 are liberated by heating with hydrochloric acid. Each of the liberated fatty acids then becomes a fatty acid methyl ester. In FIG. 5, the lipid 66 contains two types of fatty acids with different lengths, so two types of fatty acid methyl esters derived from each fatty acid are produced. At the same time, the pentadecanoic acid added in step V16 is also methyl-esterified. Note that methyl-esterifying the fatty acids suppresses tailing of the detection peak in gas chromatography (GC) analysis, which will be described later, improves the volatility of the analyte, and improves the sensitivity of the GC analysis.

[0085] In step V22, the lipids 66 obtained in step V20 and fatty acid methyl esters derived from pentadecanoic acid are extracted with hexane.

[0086] In step V24, the solvent of the extract extracted in step V22 is removed, and then the extract is dissolved in 100 μL of hexane.

[0087] In step V26, the solution obtained in step V24 is analyzed by GC. Fatty acid methyl esters derived from lipid 66 and pentadecanoic acid can be separated by GC. Each separated fatty acid methyl ester is detected by a flame ionization detector (FID). As a result, a chromatogram derived from the fatty acid methyl esters derived from lipid 66 and pentadecanoic acid can be obtained.

[0088] In step V28, the molar concentration of the fatty acids of the lipids 66 contained in the sample 81 is calculated. Specifically, the mass concentration of the fatty acid methyl esters derived from the lipids 66 obtained in step V26 is quantified using an external calibration curve, and then the molar concentration of each fatty acid is calculated by correcting the mass concentration of the fatty acid methyl esters derived from pentadecanoic acid added as an internal standard in step V16.

[0089] In step V30, 10 μL of the solution obtained in step V12 is taken and dried.

[0090] In step V32, the dried product obtained in step V30 is dissolved in 200 μL of 40% 2-propanol.

[0091] In step V34, insoluble precipitates are removed from the lysate obtained in step V32.

[0092] In step V36, the solution from which the precipitate has been removed in step V34 is subjected to LC-MS / MS to obtain the area of ​​the ion peak derived from the lipid 66 contained in the sample 81.

[0093] In step V38, the control unit 3 acquires the molar concentration of lipid 66 contained in sample 81 calculated from the molar concentration of fatty acid obtained in step V28 and the area of ​​the ion peak derived from lipid contained in sample 81 obtained in step V36, and calculates the molar concentration relative to the peak area of ​​lipid 66. The molar concentration relative to the peak area of ​​lipid 68 obtained by a similar process is divided by the molar concentration relative to the peak area of ​​lipid 66 to calculate the relative sensitivity coefficient of lipid 68 relative to lipid 66. Thereafter, the LC-MS / MS apparatus 100 ends the process of FIG.

[0094] [Method for quantifying lipids using relative response coefficients] Next, a method for quantifying lipids contained in a sample using relative response coefficients will be described. This quantification method can quantify lipids contained in a sample for which relative response coefficients have been calculated. The lipids contained in the sample may be chemically synthesized or may be synthesized by a living organism. Examples of living organisms include animals, plants, and fungi. The sample may also contain lipids other than those for which relative response coefficients have been calculated.

[0095] A predetermined amount of a standard substance corresponding to at least one lipid among the lipids contained in the sample and for which a relative sensitivity coefficient has been calculated is mixed with the sample. The lipid selected here corresponds to the "reference lipid." This allows the reference lipid contained in the sample to be quantified by comparing the detection intensity of the reference lipid contained in the sample with the detection intensity derived from the standard substance for a predetermined amount of the reference lipid mixed with the lipid. Note that the reference lipid is not limited to one type, and multiple types may be used. The detection intensity is, for example, peak area and peak intensity.

[0096] The sample is subjected to LC-MS / MS measurement to obtain chromatograms derived from each lipid contained in the sample and the standard substance. The detection intensities of each lipid and the standard substance are determined from the obtained chromatograms.

[0097] The detection intensity of the above-mentioned standard substance is compared with the detection intensity of the reference lipid to obtain the quantitative value of the reference lipid.

[0098] The relative amount ratio of each lipid contained in the sample is calculated by multiplying the detection intensity of each lipid by the corresponding relative sensitivity coefficient.

[0099] The quantitative value of each lipid contained in the sample is calculated by multiplying the quantitative value of the reference lipid quantified using a standard substance by the ratio of the relative amounts of each lipid contained in the sample described above.

[0100] The method for quantifying lipids using the above-mentioned relative sensitivity coefficient will be specifically described with reference to Fig. 7, taking the relative sensitivity coefficient calculated in Fig. 4 as an example. Fig. 7 is a diagram for explaining the method for quantifying lipids using the relative sensitivity coefficient.

[0101] 7, it is assumed that there is a sample X containing at least lipids A, B, C, and D. Lipid A', a standard substance for lipid A, is mixed with sample X to a concentration of 1 mol / L, and the sample X mixed with lipid A' is subjected to LC-MS / MS measurement.

[0102] 7 shows the peak areas identified from the chromatogram obtained by LC-MS / MS measurement. The peak areas of lipids A, B, C, D, and A' are 4, 6, 10, 4, and 2, respectively. Furthermore, by comparing the peak areas of lipid A and lipid A', it can be calculated that the concentration of lipid A in sample X is 2 mol / L.

[0103] Figure 7 shows the relative ratio of the amount of each lipid contained in sample X. By multiplying the obtained peak area by the relative sensitivity coefficient calculated in Figure 4, it is calculated that lipids B, C, and D are contained in sample X in amounts 3 times, 10 times, and 0.5 times the amount of lipid A.

[0104] 7 shows the quantitative values ​​of lipids contained in sample X. From the concentration of lipid A in sample X obtained by comparison with lipid A' and the relative amounts of lipids B, C, and D to lipid A, the concentrations of lipids B, C, and D in sample X are calculated to be 6 mol / L, 20 mol / L, and 1 mol / L.

[0105] In the above-mentioned quantification method, by calculating a relative sensitivity coefficient prepared in advance, it is possible to quantify each lipid in a sample (lipids B, C, and D in the above example) other than the reference lipid (lipid A in the above example) without using a corresponding standard substance. Furthermore, the above-mentioned quantification method corrects for differences in ionization efficiency of each lipid, compared to quantification methods that simply compare peak areas, thereby improving the accuracy of quantification.

[0106] In addition, by calculating the sensitivity coefficient of each lipid in advance, it is possible to calculate the amount of the lipid in the sample in subsequent measurements from the detection intensity. For example, for lipid B in Figures 4 and 7, the peak area of ​​lipid B in Figure 7 is 6, and the sensitivity coefficient of lipid B in Figure 4 is 1, so it can be calculated that sample X in Figure 7 contains 6 mol / L of lipid B. However, the absolute value of the detection intensity is easily affected by the state of the mass spectrometry unit 2 (for example, the degree of contamination of the probe 201), and the value is likely to fluctuate depending on the timing of the measurement. Therefore, although it is possible to quantify lipids using the sensitivity coefficient, if time has passed since the sensitivity coefficient was created and the state of the mass spectrometry unit 2 is different from when the sensitivity coefficient was created, the accuracy of the quantification may be insufficient.

[0107] On the other hand, the relative sensitivity coefficient is unlikely to fluctuate even if the timing of measurement is different. Therefore, as in the above example, after calculating the relative values ​​between lipids using the relative sensitivity coefficient, at least one of the lipids is quantified using a standard substance to quantify multiple types of lipids, thereby improving the accuracy of quantification compared to the measurement method according to the comparative example and the measurement method using the sensitivity coefficient.

[0108] The relative sensitivity coefficient is a value that fluctuates little when the measurement conditions are kept constant. Therefore, the LC-MS / MS device used can be used in another LC-MS / MS device by using the same measurement conditions (for example, the solvent, column, and ionization method).

[0109] [Processing Flow] Figure 8 is a flowchart of an example of a process performed to measure the amount of lipid contained in a sample using a relative response coefficient. In one implementation example, the process of Figure 8 is called from the main routine and executed when the CPU 30 of the control unit 3 executes a given program. Note that the first sample and the second sample are samples used to calculate the relative response coefficient, and the third sample is a sample to be subjected to lipid quantification using the relative response coefficient. In addition, the first lipid corresponds to the reference lipid described above.

[0110] In step S10, the CPU 30 receives a quantitative value of the first lipid in the first sample containing the first lipid, obtained by a predetermined quantification method. The predetermined method is, for example, a method in which the first sample is separated by gas chromatography, and then fatty acids derived from the first lipid are measured using a flame ionization detector to quantify the first lipid.

[0111] In step S12, the CPU 30 receives a quantitative value of the second lipid in the second sample containing the second lipid, obtained by a predetermined quantification method. The predetermined method is, for example, a method in which the second sample is separated by gas chromatography, and then fatty acids derived from the second lipid are measured by a flame ionization detector to quantify the second lipid.

[0112] In step S14, the CPU 30 receives from the ion detector 234 a chromatogram derived from the first lipid in the first sample introduced into the liquid chromatograph 1 by the injector 12, and determines the detection intensity of the first lipid in the first sample.

[0113] In step S16, the CPU 30 receives from the ion detector 234 a chromatogram derived from the second lipid in the second sample introduced into the liquid chromatograph 1 by the injector 12, and determines the detection intensity of the second lipid in the second sample.

[0114] In step S18, the CPU 30 calculates a relative sensitivity coefficient of the second lipid to the first lipid using the amount of the first lipid contained in the first sample received in step S10, the amount of the second lipid contained in the second sample received in step S12, the detection intensity derived from the first lipid contained in the first sample calculated in step S14, and the detection intensity derived from the second lipid contained in the second sample calculated in step S16. The calculated relative sensitivity coefficient of the second lipid to the first lipid is stored in the HDD 33.

[0115] In step S20, the CPU 30 calculates the quantitative value of the first lipid contained in the third sample from the detection intensity derived from the first lipid in the third sample mixed with the standard substance of the first lipid and the standard substance.

[0116] In step S22, the CPU 30 obtains the respective detection intensities from the chromatograms derived from the first lipid and the second lipid of the third sample introduced into the liquid chromatograph 1 by the injector 12.

[0117] In step S24, CPU 30 calculates the quantitative value of the second lipid contained in the third sample from the relative sensitivity coefficient of the second lipid to the first lipid calculated in step S18, the quantitative value of the first lipid contained in the third sample calculated in step S20, and the detection intensities of the first lipid and the second lipid in the third sample determined in step S22. CPU 30 then ends the lipid quantification subroutine and returns the process to the main routine.

[0118] When the amount of lipid contained in a sample is quantified using the relative sensitivity coefficient once calculated, the process starts from step S20.

[0119] In addition, in the above example, the amount of the first lipid contained in the third sample is measured using a standard substance, but the amount of the second lipid may be measured using a standard substance corresponding to the second lipid, and the amount of the first lipid may be determined using a relative sensitivity coefficient.

[0120] The method for quantifying lipids using the relative sensitivity coefficient according to the present disclosure corrects for differences in ionization efficiency depending on the type of lipid, thereby improving the accuracy of quantification using the detection sensitivity obtained by LC-MS / MS.

[0121] Furthermore, the method for quantifying lipids using relative response coefficients according to the present disclosure eliminates the need to prepare standard substances for all lipids to be measured, thereby reducing the cost of measurement.

[0122] For example, when measuring lipids produced by a specific organism, the relative response coefficient can be calculated using the organism, allowing the exact relative response coefficient for the lipids contained in the organism to be created. Furthermore, even if it is difficult to obtain a standard substance for the lipid to be measured, the lipid can be quantified without using the standard substance by using the measurement method using the relative response coefficient according to the present disclosure.

[0123] Aspects It will be understood by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0124] (Item 1) In one aspect, a calculation method is a method for calculating a relative sensitivity coefficient of a second lipid, which is a different type of lipid, to a first lipid in an LC-MS / MS device, and may include the steps of: acquiring a quantitative value of the first lipid contained in a first sample containing the first lipid; acquiring a quantitative value of the second lipid contained in a second sample containing the second lipid; measuring the first sample by the LC-MS / MS and determining a detection intensity of the first lipid from the obtained chromatogram; measuring the second sample by the LC-MS / MS and determining a detection intensity of the second lipid from the obtained chromatogram; and calculating a relative sensitivity coefficient of the second lipid to the first lipid from the quantitative value of the first lipid, the quantitative value of the second lipid, the detection intensity of the first lipid, and the detection intensity of the second lipid.

[0125] According to the calculation method described in paragraph 1, a technique is provided for improving the quantitative accuracy of lipids in the measurement of multiple types of lipids by LC-MS / MS without using lipid standards other than the reference lipid.

[0126] (Item 2) In the calculation method according to item 1, each of the first lipid and the second lipid may be a glycerolipid.

[0127] According to the calculation method described in Section 2, a technique is provided for improving the quantitative accuracy of glycerolipids in the measurement of multiple types of glycerolipids by LC-MS / MS without using a glycerolipid standard substance other than the reference glycerolipid.

[0128] (Item 3) In the calculation method described in Item 2, the quantitative value may be a quantitative value of the glycerolipid obtained by separating the fatty acids constituting the glycerolipid by gas chromatography and quantifying the fatty acids by a flame ionization detector.

[0129] According to the calculation method described in Section 3, in the measurement of multiple types of glycerolipids by LC-MS / MS, the fatty acids that constitute the glycerolipids are separated by gas chromatography, and the fatty acids are quantified by a flame ionization detector. This method uses the quantitative values ​​of the glycerolipids obtained, thereby providing a technology for improving the quantitative accuracy of glycerolipids without using any standard substance for glycerolipids other than the reference glycerolipid.

[0130] (4) In the calculation method described in any one of paragraphs 1 to 3, the quantitative values ​​of each of the first lipid and the second lipid may include at least one of concentration, amount of substance, and weight.

[0131] According to the calculation method described in Section 4, a technique is provided for improving the accuracy of lipid quantification in the measurement of multiple types of lipids by LC-MS / MS by using at least one of concentration, substance amount, and weight as a quantitative value, without using lipid standard substances other than the reference lipid.

[0132] (Item 5) In the calculation method according to any one of items 1 to 4, the detection intensity may be calculated based on at least one of a peak intensity and a peak area calculated from the chromatogram.

[0133] According to the calculation method described in Section 5, a technique is provided for improving the quantitative accuracy of lipids in the measurement of multiple types of lipids by LC-MS / MS, using a detection intensity calculated based on at least one of the peak intensity and peak area calculated from a chromatogram obtained by LC-MS / MS measurement, without using a lipid standard substance other than the reference lipid.

[0134] (Item 6) In the calculation method described in any one of Items 1 to 5, each of the first sample and the second sample may be extracted from at least one of plants, algae, and animals.

[0135] According to the calculation method described in Section 6, a technique is provided for improving the quantitative accuracy of lipids in the measurement of multiple types of lipids by LC-MS / MS, using samples extracted from at least one of plants, algae, and animals, without using lipid standards other than the reference lipids.

[0136] (7) In one aspect, the program may be executed by a processor installed in a computer, and may cause the computer to execute the calculation method described in any one of the 1st to 6th paragraphs.

[0137] According to the program described in paragraph 7, a technique is provided for improving the quantitative accuracy of lipids in the measurement of multiple types of lipids by LC-MS / MS without using lipid standards other than the reference lipid.

[0138] (Item 8) In one aspect, the calculation method is a method for calculating a sensitivity coefficient of a lipid in an LC-MS / MS device, and may include the steps of acquiring a quantitative value of the lipid contained in a sample containing the lipid, measuring the lipid contained in the sample by the LC-MS / MS and determining a detection intensity derived from the lipid contained in the sample from the obtained chromatogram, and determining the sensitivity coefficient of the lipid from the quantitative value and the detection intensity.

[0139] According to the calculation method described in Section 8, a technique is provided for quantifying lipids in lipid measurement by LC-MS / MS without using lipid standards other than the reference lipid.

[0140] (Item 9) In one aspect, a quantification method is a method for quantifying a second lipid in a third sample containing a first lipid and the second lipid by a relative sensitivity coefficient calculated in an LC-MS / MS device using a first sample containing the first lipid and a second sample containing a second lipid of a type different from the first lipid, the method comprising the steps of: acquiring a quantitative value of the first lipid contained in the first sample; acquiring a quantitative value of the second lipid contained in the second sample; measuring the first sample by the LC-MS / MS and determining a detection intensity of the first lipid from the obtained chromatogram; measuring the second sample by the LC-MS / MS and determining a detection intensity of the second lipid from the obtained chromatogram; and determining the quantitative value of the first lipid contained in the first sample, a first lipid contained in the second sample, and a second lipid contained in the second sample. The method may include the steps of: calculating a relative sensitivity coefficient of the second lipid with respect to the first lipid from the quantitative value of the second lipid, the detection intensity of the first lipid contained in the first sample, and the detection intensity of the second lipid contained in the second sample; measuring the third sample by the LC-MS / MS and determining the detection intensities of each of the first lipid and the second lipid contained in the third sample; measuring the quantitative value of the first lipid contained in the third sample by the LC-MS / MS using a standard substance of the first lipid; and quantifying the second lipid contained in the third sample using the relative sensitivity coefficient, the detection intensities of each of the first lipid and the second lipid contained in the third sample, and the quantitative value of the first lipid contained in the third sample.

[0141] According to the quantification method described in paragraph 9, in the measurement of multiple types of lipids by LC-MS / MS, a technique is provided for improving the accuracy of lipid quantification without using lipid standards other than the reference lipid.

[0142] (Item 10) In the method for quantifying the amount of a substance according to item 9, the third sample may be extracted from at least one of a plant, an alga, and an animal.

[0143] According to the quantification method described in paragraph 10, a technique is provided for improving the accuracy of lipid quantification in LC-MS / MS measurement of lipids extracted from at least one of plants, algae, and animals, without using lipid standards other than the reference lipid.

[0144] (Item 11) An LC-MS / MS device in one aspect is an LC-MS / MS device that quantifies the second lipid in a third sample containing the first lipid and the second lipid, and includes a liquid chromatograph, a mass spectrometer that analyzes components separated by the liquid chromatograph, and a data processor that receives measurement data from the mass spectrometer, wherein the mass spectrometer determines detection intensities of the first lipid and the second lipid contained in the third sample by measuring the third sample, and measures a quantitative value of the first lipid contained in the third sample using a standard substance of the first lipid, and the data processor calculates the quantitative value of the second lipid contained in the third sample using the detection intensities of the first lipid and the second lipid contained in the third sample, the quantitative values, and a relative sensitivity coefficient calculated by the calculation method described in item 1.

[0145] According to the LC-MS / MS device described in paragraph 11, a technique is provided for improving the quantitative accuracy of lipids in the measurement of lipids by LC-MS / MS without using lipid standards other than the reference lipids.

[0146] The embodiments disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present disclosure is defined by the claims, not by the description of the above-described embodiments, and is intended to include all modifications within the meaning and scope of the claims. Furthermore, it is intended that each technique in the embodiments can be implemented alone or, if necessary, in combination with other techniques in the embodiments to the extent possible.

[0147] 1 Liquid chromatograph, 2 Mass spectrometry section, 3 Control section, 4 Display section, 5 Input section, 10 Mobile phase container, 11 Pump, 12 Injector, 13 Column, 20 Ionization chamber, 21 First intermediate chamber, 22 Second intermediate chamber, 23 Analysis chamber, 30 CPU, 31 ROM, 32 RAM, 33 HDD, 34 Communication I / F, 35 Display I / F, 36 Input I / F, 100 LC-MS / MS device, 201 Probe, 202 Capillary, 211, 221, 2321 Ion guide, 212 Skimmer, 231, 233 Quadrupole mass filter, 232 Collision cell, 234 Ion detector, 2311, 2331 Pre-rod electrode, 2312, 2332 Main rod electrode.

Claims

1. A method for calculating a relative sensitivity coefficient of a second lipid of a type different from the first lipid to a first lipid in a high performance liquid chromatography tandem mass spectrometry (LC-MS / MS) device, comprising the steps of: acquiring a quantitative value of the first lipid contained in a first sample containing the first lipid; acquiring a quantitative value of the second lipid contained in a second sample containing the second lipid; measuring the first sample by the LC-MS / MS and determining the detection intensity of the first lipid from the obtained chromatogram; measuring the second sample by the LC-MS / MS and determining the detection intensity of the second lipid from the obtained chromatogram; and calculating the relative sensitivity coefficient of the second lipid to the first lipid from the quantitative value of the first lipid, the quantitative value of the second lipid, the detection intensity of the first lipid, and the detection intensity of the second lipid.

2. The calculation method according to claim 1, wherein each of the first lipid and the second lipid is a glycerolipid.

3. The method of claim 2, wherein the quantitative value is a quantitative value of the glycerolipid obtained by separating fatty acids constituting the glycerolipid by gas chromatography and quantifying the fatty acids by a flame ionization detector.

4. A calculation method according to claim 1 or claim 2, wherein the quantitative values ​​of each of the first lipid and the second lipid include at least one of concentration, amount of substance, and weight.

5. The calculation method according to claim 1 or 2, wherein the detection intensity is calculated based on at least one of a peak intensity and a peak area calculated from the chromatogram.

6. The calculation method according to claim 1 or 2, wherein each of the first sample and the second sample is extracted from at least one of a plant, an algae, and an animal.

7. A program executed by a processor installed in a computer, causing the computer to execute the calculation method according to claim 1 or 2.

8. A method for calculating a sensitivity coefficient of a lipid in a high performance liquid chromatography tandem mass spectrometry (LC-MS / MS) device, comprising the steps of: acquiring a quantitative value of the lipid contained in a sample containing the lipid; measuring the sample by the LC-MS / MS and determining a detection intensity attributable to the lipid contained in the sample from the obtained chromatogram; and determining the sensitivity coefficient of the lipid from the quantitative value and the detection intensity.

9. A method for quantifying a first lipid and a third sample containing the second lipid by a relative sensitivity coefficient calculated using a first sample containing a first lipid and a second sample containing a second lipid different from the first lipid in a high performance liquid chromatography tandem mass spectrometry (LC-MS / MS) device, comprising the steps of: acquiring a quantitative value of the first lipid contained in the first sample; acquiring a quantitative value of the second lipid contained in the second sample; measuring the first sample by the LC-MS / MS and determining a detection intensity of the first lipid from the obtained chromatogram; measuring the second sample by the LC-MS / MS and determining a detection intensity of the second lipid from the obtained chromatogram; and calculating a relative sensitivity coefficient of the second lipid to the first lipid from the quantitative value of the first lipid contained in the first sample, the quantitative value of the second lipid contained in the second sample, the detection intensity of the first lipid contained in the first sample, and the detection intensity of the second lipid contained in the second sample; The quantitative method includes the steps of: measuring the third sample by the LC-MS / MS and determining the detection intensities of the first lipid and the second lipid contained in the third sample; measuring a quantitative value of the first lipid contained in the third sample by the LC-MS / MS using a standard substance of the first lipid; and quantifying the second lipid contained in the third sample using the relative response coefficient, the detection intensities of the first lipid and the second lipid contained in the third sample, and the quantitative value of the first lipid contained in the third sample.

10. The quantitative method according to claim 9, wherein the third sample is extracted from at least one of a plant, an algae, and an animal.

11. An LC-MS / MS apparatus for quantifying the second lipid in a third sample containing the first lipid and the second lipid, comprising: a liquid chromatograph; a mass spectrometer for analyzing components separated by the liquid chromatograph; and a data processor for receiving measurement data from the mass spectrometer, wherein the mass spectrometer determines detection intensities of the first lipid and the second lipid contained in the third sample by measuring the third sample; measures a quantitative value of the first lipid contained in the third sample using a standard substance for the first lipid; and the data processor calculates the quantitative value of the second lipid contained in the third sample using the detection intensities of the first lipid and the second lipid contained in the third sample, the quantitative value, and a relative sensitivity coefficient calculated by the calculation method described in claim 1.

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