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WO2025047369A1 - Procédé de calcul, programme, procédé de quantification et dispositif de spectrométrie de masse en tandem à chromatographie liquide haute performance - Google Patents

Procédé de calcul, programme, procédé de quantification et dispositif de spectrométrie de masse en tandem à chromatographie liquide haute performance Download PDF

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Publication number
WO2025047369A1
WO2025047369A1 PCT/JP2024/028378 JP2024028378W WO2025047369A1 WO 2025047369 A1 WO2025047369 A1 WO 2025047369A1 JP 2024028378 W JP2024028378 W JP 2024028378W WO 2025047369 A1 WO2025047369 A1 WO 2025047369A1
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Prior art keywords
lipid
sample
contained
quantitative value
lipids
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Japanese (ja)
Inventor
雄太 井原
美恵 下嶋
修一 中家
真希 山田
啓之 太田
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Shimadzu Corp
Phytolipid Technologies Co Ltd
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Shimadzu Corp
Phytolipid Technologies Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers

Definitions

  • the present disclosure relates to a calculation method, a program, a quantification method, and a high-performance liquid chromatography tandem mass spectrometer, and more specifically to a technology for improving the accuracy of lipid quantification.
  • Lipids are substances that are soluble in non-polar solvents and insoluble in water, and include substances with a variety of structures.
  • triacylglycerol in which one molecule of glycerol is linked by an ester bond to three molecules of fatty acid, can be of many different types depending on the types and combinations of fatty acids linked together, and the position of the fatty acids linked to the glycerol.
  • Some lipids produced by plants and algae have physiological activities that are useful to humans, and some can be used as biofuels. Therefore, when examining plant growth conditions with the aim of improving the production efficiency of a target lipid, for example, it is desirable to distinguish and quantitatively analyze the types of lipids in a sample containing multiple types of lipids extracted from the plant.
  • LC-MS/MS high performance liquid chromatography tandem mass spectrometry
  • the tandem mass spectrometer breaks down the lipids to be measured and detects the ions derived from the breakdown products, making it possible to estimate the structure of the lipids to be measured, which is also useful for analyzing samples containing unknown lipids.
  • Non- Patent Document 1 discloses an example of a method for measuring lipids using LC-MS/MS.
  • Non-Patent Document 3 discloses an example of a method for estimating lipid structure using a tandem mass spectrometer.
  • One method for quantifying lipids in a sample using LC-MS/MS is a quantitative method that involves comparison with a standard substance. Specifically, a standard substance and the lipid to be measured are measured simultaneously, and the lipid is quantified by comparing the detection intensity with that of the standard substance. In this method, a different standard substance corresponds to each type of lipid, so when there are multiple lipids to be measured, the measurement costs can be high.
  • the present disclosure has been devised in light of this current situation, and its purpose is to provide a technology that improves the quantitative accuracy of lipids when measuring samples containing multiple types of lipids using LC-MS/MS, by using standard substances for some of the lipids being measured.
  • the calculation method is a method for calculating a relative sensitivity coefficient of a second lipid of a different type to a first lipid in an LC-MS/MS device, and includes the steps of: acquiring a quantitative value of a 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 a 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.
  • the program according to the second aspect of the present disclosure is a program executed by a processor installed in a computer, and causes the computer to execute the above calculation method.
  • the calculation method 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 lipids contained in a lipid-containing sample, measuring the sample by LC-MS/MS and determining the detection intensity derived from the lipids contained in the sample from the obtained chromatogram, and determining the lipid sensitivity coefficient from the quantitative value and the detection intensity.
  • the method includes a step of calculating a relative sensitivity coefficient of the second lipid to the first lipid from the amount value, 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; a step of measuring the third sample by LC-MS/MS and determining the detection intensity of each of the first lipid and the second lipid contained in the third sample; a 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 a step of quantifying the second lipid contained in the third sample using the relative sensitivity coefficient, the detection intensity 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.
  • the LC-MS/MS device 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, the mass spectrometer determining the detection intensities of the first lipid and the second lipid contained in the third sample by measuring the third sample, and measuring the quantitative value of the first lipid contained in the third sample using a standard substance for the first lipid, and the data processor calculating 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.
  • the quantitative accuracy of lipids can be improved by using standard substances for some of the lipids being measured.
  • FIG. 1 is a schematic diagram of an LC-MS/MS device according to an embodiment.
  • FIG. 1 is a functional block diagram of an LC-MS/MS device according to an embodiment.
  • 1 is a flow chart showing the steps of lipid analysis by LC-MS/MS.
  • FIG. 13 is a diagram for explaining a method of calculating a relative sensitivity coefficient.
  • 1 is a flowchart showing a procedure for preparing a sample for calculating a relative response coefficient.
  • 1 is a flowchart showing a procedure for calculating a relative sensitivity coefficient from a prepared sample.
  • FIG. 1 is a diagram for explaining a method for quantifying lipids using a relative response coefficient.
  • 1 is a flowchart showing a process of a lipid quantification method using a relative response coefficient.
  • Fig. 1 is a diagram showing a schematic overall configuration of an LC-MS/MS device 100 according to this embodiment.
  • the LC-MS/MS device 100 includes a liquid chromatograph 1, a mass spectrometer 2, a controller 3, a display 4, and an input unit 5.
  • the controller 3, the display 4, and the input unit 5 may be incorporated in the mass spectrometer 2.
  • the controller 3 may be a general-purpose computer provided at a position separate from the liquid chromatograph 1 and the mass spectrometer 2.
  • the LC-MS/MS device 100 can separate lipids contained in a sample into individual types of lipids and obtain detection intensities corresponding to each lipid.
  • Lipids are substances that are poorly soluble in water and easily soluble in organic solvents, and specific examples include simple lipids, complex lipids, cholesterol, steroids, carotenoids, and substances with structures similar to these.
  • Simple lipids include those in which alcohol and fatty acids are combined, such as triacylglycerol.
  • Complex lipids include those in which at least one of phosphate, sulfur, nitrogen base, and sugar is combined with alcohol and fatty acid, such as glycerophospholipids, sphingophospholipids, glyceroglycolipids, and sphingoglycolipids.
  • 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 each type of lipid by utilizing differences in interactions between the stationary phase and the mobile phase.
  • the mobile phase container 10 stores the mobile phase, which is a liquid that carries the sample injected into the liquid chromatograph 1.
  • the mobile phase is, for example, an organic solvent or water, or a mixture of these.
  • the organic solvent is, for example, 2-propanol, methanol, acetonitrile, chloroform, hexane, dichloromethane, and tetrahydrofuran.
  • the mobile phase may contain an acidic solution (for example, trifluoroacetic acid, formic acid, and ammonium formate) as an additive.
  • the liquid chromatograph 1 may contain a single mobile phase container or multiple mobile phase containers.
  • Pump 11 draws in the mobile phase stored in mobile phase container 10 and delivers it at a predetermined flow rate.
  • the flow rate of the mobile phase delivered by pump 11 may be constant during one measurement or may vary.
  • Liquid chromatograph 1 may include a single pump or multiple pumps.
  • the injector 12 injects a predetermined amount of sample, which has been prepared in advance in the mobile phase flow path, into the liquid chromatograph 1.
  • the sample is introduced from the injector 12 into the mobile phase delivered by the pump 11, and the mobile phase containing the sample is introduced into the column 13.
  • Column 13 is filled with a stationary phase, through which the mobile phase passes. As the sample passes through column 13, various lipids in the sample interact with the mobile phase and stationary phase, causing them to be separated in the time direction. The separated lipids are eluted from the outlet of column 13 and introduced into mass spectrometry section 2.
  • stationary phase There are no limitations on the type of stationary phase, so long as it is one that can be used to separate lipids.
  • the mass spectrometry section 2 includes an ionization chamber 20, a first intermediate chamber 21, a second intermediate chamber 22, and an analysis chamber 23.
  • the mass spectrometry section 2 performs mass analysis of the sample eluted from the liquid chromatograph 1.
  • the analysis in the mass spectrometry section 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.
  • the ionization chamber 20 has a probe 201 and a capillary 202.
  • the inside of the ionization chamber 20 is at atmospheric pressure.
  • the ionization chamber 20 is connected to the next stage first intermediate chamber 21 through 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 tiny droplets are split and made fine by the action of electrostatic force, and the lipids of the sample in the droplets are ionized in the process of evaporating the solvent.
  • the generated ions pass through the capillary 202 and are introduced into the first intermediate chamber 21.
  • the first intermediate chamber 21 has an ion guide 211 and a skimmer 212.
  • the inside of the first intermediate chamber 21 is a 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 the ions introduced from the ionization chamber 20 in the previous stage and transports them to the subsequent stage via the skimmer 212.
  • the second intermediate chamber 22 has an ion guide 221.
  • the inside of the second intermediate chamber 22 is a high vacuum.
  • the ion guide 221 focuses the ions introduced from the first intermediate chamber 21 in the preceding stage and transports them to the following stage.
  • 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. From the obtained mass spectrum, information regarding the molecular weight, molecular formula, and chemical structure of the compound can be obtained.
  • the quadrupole mass filter 231 comprises a main rod electrode 2312 and a pre-rod electrode 2311 placed 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 inlet end and assists the function of the main rod electrode 2312.
  • the collision cell 232 includes a multipole ion guide 2321 inside.
  • 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.
  • CID gas promotes dissociation of ions.
  • the multipole ion guide 2321 focuses the dissociated ions and transports them to the rear stage.
  • the CID gas is, for example, argon, nitrogen, helium, and xenon.
  • the quadrupole mass filter 233 comprises a main rod electrode 2332 and a pre-rod electrode 2331 placed 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 inlet end and assists the function of the main rod electrode 2332.
  • the ion detector 234 is, for example, a pulse count detector, and generates a detection signal whose number of pulse signals corresponds to the number of incident ions. This detection signal is output to the control unit 3.
  • 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.
  • the control unit 3 is connected to and communicates with the liquid chromatograph 1 and the mass spectrometry unit 2, and is configured, for example, by a computer.
  • the control unit 3 controls the operation of the liquid chromatograph 1 and the mass spectrometry unit 2, and receives the measurement data acquired by the ion detector 234 of the mass spectrometry unit 2.
  • the control unit 3 also calculates the quantitative value of lipids based on the relative sensitivity coefficient and the measurement data.
  • the display unit 4 is configured, for example, with 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 value of lipids calculated based on the measurement data.
  • the input unit 5 is composed of, for example, a keyboard, a mouse, etc.
  • the input unit 5 receives instructions from the user for the liquid chromatograph 1 and the mass spectrometry unit 2, and outputs them 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.
  • FIG. 2 is a functional block diagram of the overall configuration of the LC-MS/MS device 100 according to this embodiment.
  • 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.
  • a CPU Central Processing Unit
  • ROM Read Only Memory
  • RAM Random Access Memory
  • HDD Hard Disk Drive
  • Communication I/F Interface
  • the communication I/F 34 relays communication with external devices including the liquid chromatograph 1 and the mass spectrometry unit 2.
  • the communication I/F 34 is realized, for example, by 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.
  • the input I/F 36 relays data transmission between the CPU 30 and the input unit 5.
  • the input I/F 36 accepts various information provided by operating the input unit 5, such as the relative sensitivity coefficient, and commands for the liquid chromatograph 1 and the mass spectrometry unit 2.
  • the ROM 31 can non-temporarily store the 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 commands input by the user via the input unit 5 for the liquid chromatograph 1 and the mass analysis unit 2, 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.
  • FIG. 3 is a flow chart showing the steps of lipid analysis by LC-MS/MS.
  • T10 is performed manually by an analyst using experimental equipment used in general scientific experiments and mass spectrometry.
  • T12 and subsequent steps are performed by the LC-MS/MS device 100.
  • the lipids to be detected are assumed to be complex lipids with one polar head and two fatty acids, with glycerol as the linking part.
  • Fig. 3 illustrations of lipids and ions are added to explain the procedure in more detail.
  • the line segment represents the glycerol backbone
  • the ellipse represents the fatty acid
  • the diamond represents the polar head.
  • Lipids 61, 62, 63, and 64 are lipids having a structure in which two molecules of fatty acid, 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 carbon chain of the fatty acid.
  • lipids 61 and 63 have the same polar head, but the fatty acids bound to them are different, so they are different types of lipids.
  • lipid 61 and lipid 64 have the same type of fatty acid bound to them and the same binding position, but have different polar head groups, making them different types of lipids. Therefore, lipids 61, 62, 63, and 64 each represent a different type of lipid.
  • step T10 the user prepares a sample containing multiple types of lipids.
  • step T12 the sample prepared in step T10 is separated into different types of lipids by liquid chromatography.
  • the sample introduced into liquid chromatograph 1 is introduced into column 13 together with the mobile phase delivered by pump 11.
  • column 13 lipids interact with the stationary phase and the mobile phase, but the degree of interaction differs depending on the type of lipid.
  • the speed at which lipids move through column 13 is determined by this interaction, so the speed at which lipids move differs depending on the type of lipid. Therefore, due to the difference in mobility of each lipid, lipids can be separated into different types in liquid chromatograph 1. Meanwhile, lipids 61, 62, 63, and 64, which have the same or nearly the same mobility, are eluted simultaneously.
  • step T12 the eluate containing lipids 61, 62, 63, and 64 is introduced into the mass spectrometry unit 2.
  • the processing in the mass spectrometry unit 2 is shown as step T14 in FIG. 3, and is further broken down into steps T16 to T24.
  • step T16 the lipids 61, 62, 63, and 64 introduced into the mass spectrometry unit 2 are ionized to generate ions 611, 621, 631, and 641, respectively.
  • the generated ions are introduced into the quadrupole mass filter 231.
  • 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 subsequent 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 passing only ions having a specific mass-to-charge ratio according to the voltage applied to the main rod electrode 2312 among the various ions sent to the quadrupole mass filter 231.
  • the ions that have passed through the quadrupole mass filter 231 are called precursor ions.
  • ion 621 which is a precursor ion that has passed through quadrupole mass filter 231
  • a predetermined voltage is applied to the electrodes arranged in collision cell 232, and ion 621 introduced into collision cell 232 is accelerated in collision cell 232 at an acceleration corresponding to the voltage applied to the electrodes.
  • CID gas is supplied into collision cell 232 at a predetermined pressure.
  • accelerated ion 621 collides with CID gas with a predetermined collision energy and dissociates, generating ions 6211, 6212, 6213, 6214, and 6215. Ions generated from precursor ions are called product ions.
  • step T22 when the product ions 6211, 6212, 6213, 6214, and 6215 generated by dissociation of ion 621 are introduced into the quadrupole mass filter 233, only ion 6213, 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.
  • step T24 the ion detector 234 detects ion 6213, which is a product ion that has passed through the quadrupole mass filter 233, and the process in FIG. 3 ends.
  • the ions to be detected are selected in two stages, at 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 those ions, it is possible to distinguish and detect two types of lipids with the same mass.
  • 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 the respective substances.
  • the 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.
  • a relative sensitivity coefficient is calculated by quantifying the difference in ionization efficiency of each lipid in LC-MS/MS, and each lipid is quantified from the detection intensity obtained by the measurement of LC-MS/MS using the relative sensitivity coefficient.
  • the relative sensitivity coefficient the difference in ionization efficiency for each type of lipid is corrected, so that the accuracy of quantification based on the detection intensity can be improved.
  • it is possible to quantify other lipids based on the quantitative value of a predetermined lipid as a standard it is possible to quantify lipids other than the predetermined lipid without using a standard substance corresponding to them.
  • the relative response coefficient is the response coefficient of another lipid relative to the response coefficient of a reference lipid.
  • the response coefficient is the quantitative value of the lipid relative to the detection intensity obtained by LC-MS/MS.
  • each sample contains a single type of lipid. This is because when a single type of lipid is contained in a sample, the quantitative accuracy of the lipid contained in the sample is improved compared to when a plurality of types of lipid are contained.
  • the lipid may be a lipid produced by chemical synthesis or may be a lipid extracted from a living body.
  • the living body may be, for example, an animal, a plant, or a bacterial body.
  • the quantitative value of the first lipid and the lipid different from the first lipid contained in the sample is obtained.
  • the quantitative value is, for example, concentration, amount of substance, and weight.
  • the amount of lipid is measured by quantifying the fatty acid constituting the lipid.
  • the device used for quantifying the lipid is not limited, and 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 is used.
  • the sample is measured by LC-MS/MS, and the detection intensity of each lipid is obtained 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 a peak area and a peak intensity.
  • sensitivity coefficient of the first lipid is calculated as a ratio of the detection intensity to the quantitative value of the first lipid contained in the sample. In the same manner, sensitivity coefficients are calculated for lipids of a type different from the first lipid.
  • Figure 4 is a diagram for explaining the method for calculating the relative sensitivity coefficient.
  • 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.
  • 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
  • the quantitative value of lipid D contained in sample D is 2 mol/L.
  • the example in Figure 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
  • the peak area derived from lipid D contained in sample D is 8.
  • Figure 4 also shows the sensitivity coefficients obtained by dividing the quantitative value of lipids contained in each sample by the peak area obtained.
  • the sensitivity coefficient of lipid A is calculated to be 0.5
  • the sensitivity coefficient of lipid B is 1
  • the sensitivity coefficient of lipid C is 2
  • the sensitivity coefficient of lipid A is 0.25.
  • FIG. 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 the sensitivity coefficient for lipid A, which is 0.5, the relative sensitivity coefficients for lipids B, C, and D to lipid A are calculated to be 2, 4, and 0.5, respectively.
  • Fig. 5 is a flow chart of the procedure for preparing a sample used for calculating the relative response coefficient from Arabidopsis thaliana.
  • Fig. 6 is a flow chart of the procedure for calculating the relative response coefficient using the prepared sample.
  • Fig. 5, like Fig. 3, is accompanied by pictures representing lipids and ions.
  • Figs. 5 and 6 it is assumed that the lipids are composed of fatty acids and glycerol, and that the relative response coefficient of glycerophospholipid is to be calculated.
  • Lipids 65, 66, 67, and 68 in Figure 5 are complex lipids with one polar head and two fatty acids, with glycerol as the linking moiety, as in Figure 3.
  • unshaded diamonds indicate a phosphate polar head
  • shaded diamonds indicate a polar head other than phosphate.
  • lipids 66 and 68 which have a phosphate polar head and two fatty acids, are classified as glycerophospholipids.
  • the process shown in Figure 5 is performed manually by an analyst using laboratory equipment used in general scientific experiments and mass spectrometry, or by partially automated laboratory equipment.
  • an extract containing lipids 65, 66, 67, and 68 is obtained from Arabidopsis thaliana by extraction with an organic solvent.
  • step U12 the 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.
  • step U14 the fraction corresponding to glycerophospholipids containing lipids 66 and 68 physically separated on the solid phase of the thin layer chromatography is collected and dissolved in a solvent.
  • step U16 the solution obtained in step U14 is separated into the fatty acid compositions that make up lipids 66 and lipids 68 by a separation system consisting of a liquid chromatograph. Specifically, the solution obtained in step U14 is divided into fractions 71, 72, 73, 74, 75, and 76 by dividing the eluate from the separation system into fixed volumes.
  • 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.
  • step U20 fractions 72 and 75 are concentrated in step U18, and the concentrates obtained 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. 6.
  • sample 81 and sample 82 are each subjected to the same process in parallel.
  • the following describes the process of sample 81 containing lipids 66 as an example.
  • 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.
  • 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.
  • step V10 the sample 81 containing the separated lipids 66 is concentrated by removing the solvent.
  • step V12 1 mL of methanol is added to the vessel to dissolve the lipids 66.
  • step V14 800 ⁇ L of the lysate obtained in step V12 is taken.
  • step V16 50 ⁇ L of 1 mM pentadecanoic acid (C15:0) solution is added to the solution separated in step V14 as a standard for measuring fatty acids.
  • the fatty acids used as the standard are not limited to the fatty acids mentioned above, and any fatty acid other than those constituting lipid 66 can be used.
  • step V18 900 ⁇ L of 3 M hydrochloric acid in methanol is added to the solution obtained in step V16.
  • step V20 the solution obtained in step V18 is heated at 85°C for 1 hour.
  • the fatty acids that make up lipid 66 are liberated by heating together with hydrochloric acid. Each of the liberated fatty acids becomes a fatty acid methyl ester.
  • lipid 66 has two types of fatty acids with different lengths, so two types of fatty acid methyl esters derived from each fatty acid are produced.
  • the pentadecanoic acid added in step V16 is also methyl esterified. Note that methyl esterification of fatty acids suppresses tailing of detection peaks in gas chromatography (GC) analysis described below, improves the volatility of the analyte, and improves the sensitivity of GC analysis.
  • GC gas chromatography
  • step V22 the lipids 66 obtained in step V20 and fatty acid methyl esters derived from pentadecanoic acid are extracted with hexane.
  • step V24 after removing the solvent from the extract extracted in step V22, the extract is dissolved in 100 ⁇ L of hexane.
  • 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).
  • FID flame ionization detector
  • 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 the pentadecanoic acid added as an internal standard in step V16.
  • step V30 10 ⁇ L of the solution obtained in step V12 is removed and dried.
  • step V32 the dried material obtained in step V30 is dissolved in 200 ⁇ L of 40% 2-propanol.
  • step V34 insoluble precipitates are removed from the solution obtained in step V32.
  • step V36 the solution from which the precipitate was removed in step V34 is subjected to LC-MS/MS to obtain the area of the ion peak derived from lipid 66 contained in sample 81.
  • step V38 the control unit 3 obtains 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.
  • the LC-MS/MS device 100 ends the process of FIG. 6.
  • lipids contained in a sample using the relative sensitivity coefficient may be chemically synthesized or may be synthesized by a living organism.
  • the living organism may be, for example, an animal, a plant, or a fungus.
  • the sample may also contain lipids other than the lipids for which the relative sensitivity coefficient has been calculated.
  • a predetermined amount of a standard substance corresponding to at least one of the lipids contained in the sample and for which the 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.
  • the reference lipid is not limited to one type, and may be multiple types.
  • the detection intensity is, for example, peak area and peak intensity.
  • the sample is subjected to LC-MS/MS measurement, and chromatograms derived from each lipid contained in the sample and the above-mentioned standard substance are obtained.
  • the detection intensity of each lipid and the above-mentioned standard substance is obtained from the obtained chromatograms.
  • 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.
  • the quantitative value of each lipid contained in the sample is calculated by multiplying the quantitative value of the reference lipid quantified using the standard substance by the relative amount ratio of each lipid contained in the sample described above.
  • Figure 7 is a diagram for explaining the method for quantifying lipids using relative sensitivity coefficients.
  • sample X that contains at least lipids A, B, C, and D.
  • Lipid A' a standard substance for lipid A, is mixed into sample X at 1 mol/L, and the sample X mixed with lipid A' is subjected to LC-MS/MS measurement.
  • Figure 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 lipids A and A', it can be calculated that the concentration of lipid A in sample X is 2 mol/L.
  • 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.
  • Figure 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.
  • the above-mentioned quantitative method by calculating the 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 the corresponding standard substance. Furthermore, the above-mentioned quantitative method can improve the accuracy of quantification compared to a quantitative method that simply compares peak areas, because the difference in ionization efficiency of each lipid is corrected.
  • the sensitivity coefficient of each lipid in advance, it is possible to calculate the amount of that lipid in the sample in subsequent measurements from the detection intensity.
  • 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 lipid B is contained in sample X in Figure 7 at 6 mol/L.
  • the absolute value of the detection intensity is easily affected by the state of the mass spectrometry unit 2 (for example, the degree of dirt on the probe 201), and the value is likely to vary depending on the timing of the measurement.
  • the relative sensitivity coefficient is unlikely to fluctuate even if the timing of the measurement is different. Therefore, as in the above example, by calculating the relative values between lipids using the relative sensitivity coefficient, and then quantifying at least one of the lipids using a standard substance to quantify multiple types of lipids, the accuracy of quantification can be improved compared to the measurement method in the comparative example and the measurement method using the sensitivity coefficient.
  • the relative sensitivity coefficient is a value that fluctuates little when the measurement conditions are kept constant, so it can be used in other LC-MS/MS devices by keeping the measurement conditions (for example, the solvent, column, and ionization method used) the same.
  • FIG. 8 is a diagram showing a flowchart of an example of a process performed to measure the amount of lipid contained in a sample using a relative response coefficient.
  • the process of Fig. 8 is called from the main routine and executed when the CPU 30 of the control unit 3 executes a given program.
  • the first sample and the second sample are samples used for calculating the relative response coefficient
  • the third sample is a sample that is the target of lipid quantification using the relative response coefficient.
  • the first lipid corresponds to the lipid that is the reference described above.
  • step S10 the CPU 30 receives the quantitative value of the first lipid in the first sample containing the first lipid obtained by a predetermined quantitative 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 by a flame ionization detector to quantify the first lipid.
  • step S12 the CPU 30 receives the quantitative value of the second lipid in the second sample containing the second lipid obtained by a predetermined quantitative 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.
  • 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.
  • 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 calculates the detection intensity of the second lipid in the second sample.
  • 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 determined in step S14, and the detection intensity derived from the second lipid contained in the second sample determined in step S16.
  • the calculated relative sensitivity coefficient of the second lipid to the first lipid is stored in the HDD 33.
  • step S20 the CPU 30 calculates a 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 first lipid standard substance and the standard substance.
  • step S22 the CPU 30 determines the detection intensities of the first and second lipids from the chromatograms derived from the third sample introduced into the liquid chromatograph 1 by the injector 12.
  • step S24 the 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.
  • the CPU 30 then ends the lipid quantification subroutine and returns processing to the main routine.
  • step S20 If the amount of lipids contained in a sample is quantified using the relative sensitivity coefficient once calculated, processing begins with step S20.
  • 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 calculated using a relative sensitivity coefficient.
  • the lipid quantification method using the relative sensitivity coefficients disclosed herein corrects for differences in ionization efficiency due to the type of lipid, thereby improving the accuracy of quantification using the detection sensitivity obtained by LC-MS/MS.
  • the lipid quantification method using the relative sensitivity coefficients disclosed herein eliminates the need to prepare standard substances for all lipids to be measured, thereby reducing the cost of measurement.
  • the organism when measuring lipids produced by a specific organism, the organism can be used to calculate the relative sensitivity coefficient, allowing the exact relative sensitivity coefficient to be created for the lipids contained in the organism. 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 sensitivity coefficient according to the present disclosure.
  • the calculation method is a method for calculating a relative sensitivity coefficient of a second lipid, which is different from the first 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 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.
  • the calculation method described in paragraph 1 provides a technology for improving the quantitative accuracy of lipids when measuring multiple types of lipids by LC-MS/MS without using lipid standard substances other than the reference lipid.
  • each of the first lipid and the second lipid may be a glycerolipid.
  • the calculation method described in paragraph 2 provides a technology for improving the quantitative accuracy of glycerolipids in the measurement of multiple types of glycerolipids by LC-MS/MS without using a standard substance for glycerolipids other than the reference glycerolipid.
  • 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.
  • the calculation method described in paragraph 3 provides a technology for improving the quantitative accuracy of glycerolipids in the measurement of multiple types of glycerolipids by LC-MS/MS, by using the quantitative values of glycerolipids obtained by separating the fatty acids that constitute the glycerolipids by gas chromatography and quantifying the fatty acids using a flame ionization detector, without using a standard substance for glycerolipids other than the reference glycerolipid.
  • the quantitative values of the first lipid and the second lipid may include at least one of concentration, amount of substance, and weight.
  • the calculation method described in paragraph 4 provides a technology for improving the quantitative accuracy of lipids in the measurement of multiple types of lipids by LC-MS/MS by using at least one of the concentration, amount of substance, and weight as a quantitative value, without using lipid standard substances other than the reference lipid.
  • the detection intensity may be calculated based on at least one of the peak intensity and the peak area calculated from the chromatogram.
  • the calculation method described in paragraph 5 provides a technology for improving the quantitative accuracy of lipids in the measurement of multiple types of lipids by LC-MS/MS, using detection intensity calculated based on at least one of the peak intensity and peak area calculated from the chromatogram obtained by LC-MS/MS measurement, without using lipid standard substances other than the reference lipid.
  • each of the first sample and the second sample may be extracted from at least one of plants, algae, and animals.
  • the calculation method described in paragraph 6 provides a technique 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 standard substances other than the reference lipid.
  • the program is a program executed by a processor installed in a computer, and may cause the computer to execute any one of the calculation methods described in any one of the items 1 to 6.
  • the program described in paragraph 7 provides a technology for improving the quantitative accuracy of lipids when measuring multiple types of lipids by LC-MS/MS without using lipid standard substances other than the reference lipid.
  • the calculation method is a method for calculating a lipid sensitivity coefficient 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.
  • the calculation method described in paragraph 8 provides a technique for quantifying lipids in lipid measurements by LC-MS/MS without using lipid standard substances other than the reference lipid.
  • the method may include a step of calculating a relative sensitivity coefficient of the second lipid 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; a step 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; a step of 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 a 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.
  • the quantitative method described in paragraph 9 provides a technology for improving the quantitative accuracy of lipids in the measurement of multiple types of lipids by LC-MS/MS without using lipid standard substances other than the reference lipid.
  • the third sample may be extracted from at least one of a plant, an algae, and an animal.
  • the quantitative method described in paragraph 10 provides a technology for improving the quantitative accuracy of lipids in LC-MS/MS measurements of lipids extracted from at least one of plants, algae, and animals, without using lipid standard substances other than the reference lipid.
  • the LC-MS/MS device 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.
  • the mass spectrometer determines the detection intensities of the first lipid and the second lipid contained in the third sample by measuring 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.
  • the data processor may calculate 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 the relative sensitivity coefficient calculated by the calculation method described in Item 1.
  • the LC-MS/MS device described in paragraph 11 provides a technology for improving the quantitative accuracy of lipids in lipid measurement by LC-MS/MS without using lipid standard substances other than the reference lipid.

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Abstract

L'invention concerne un procédé permettant de calculer un coefficient de sensibilité relative d'un second lipide à un premier lipide dans un dispositif de spectrométrie de masse en tandem à chromatographie liquide (LC-MS/MS) haute performance comprenant les étapes suivantes : l'étape (S10) consistant à acquérir une valeur quantitative du premier lipide contenu dans un premier échantillon ; l'étape (S12) consistant à acquérir une valeur quantitative du second lipide contenu dans un second échantillon ; l'étape (S14) consistant à mesurer le premier échantillon par LC-MS/MS, et à obtenir une intensité de détection du premier lipide ; l'étape (S16) consistant à mesurer le second échantillon par LC-MS/MS, et à obtenir une intensité de détection du second lipide ; et une étape (S18) consistant à calculer le coefficient de sensibilité relative du second lipide au premier lipide à partir de la valeur quantitative du premier lipide, de la valeur quantitative du second lipide, de l'intensité de détection du premier lipide et de l'intensité de détection du second lipide.
PCT/JP2024/028378 2023-08-29 2024-08-08 Procédé de calcul, programme, procédé de quantification et dispositif de spectrométrie de masse en tandem à chromatographie liquide haute performance Pending WO2025047369A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090134323A1 (en) * 2002-06-26 2009-05-28 Gross Richard W Multidimensional mass spectrometry of serum and cellular lipids directly from biologic extracts
JP2017187469A (ja) * 2016-03-30 2017-10-12 花王株式会社 皮膚の健康の評価方法
JP2021119345A (ja) * 2014-10-30 2021-08-12 ウオーターズ・テクノロジーズ・コーポレイシヨン 標識化グリコシルアミンの迅速調製およびそれを生成するグリコシル化生体分子の分析方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090134323A1 (en) * 2002-06-26 2009-05-28 Gross Richard W Multidimensional mass spectrometry of serum and cellular lipids directly from biologic extracts
JP2021119345A (ja) * 2014-10-30 2021-08-12 ウオーターズ・テクノロジーズ・コーポレイシヨン 標識化グリコシルアミンの迅速調製およびそれを生成するグリコシル化生体分子の分析方法
JP2017187469A (ja) * 2016-03-30 2017-10-12 花王株式会社 皮膚の健康の評価方法

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