WO2023195210A1 - Method and apparatus for measuring concentration of lipophilic vitamin component in blood - Google Patents
Method and apparatus for measuring concentration of lipophilic vitamin component in blood Download PDFInfo
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Images
Classifications
-
- 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
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
Definitions
- the present invention relates to a method and device for measuring blood concentrations of fat-soluble vitamins, and more particularly to a method and device for measuring blood concentrations of vitamin D metabolites, vitamin K, and the like.
- Vitamins are important nutrients for human survival and growth. It is generally known that deficiencies in vitamin D and vitamin K, among vitamins, may impede calcification of bone matrix, accelerate the progression of osteoporosis, and increase the risk of fractures and the like.
- 24,25-dihydroxyvitamin D 3 (hereinafter "24,25-(OH) 2 VitD 3 " or "24,25-(OH) 2 D”), which is a vitamin D metabolite, is used.
- ESI electrospray ionization
- APCI atmospheric pressure chemical ionization
- the ESI method is the most commonly used ionization method in LC-MS/MS because it is applicable to a wide range of compounds and the equipment is easy to handle.
- vitamin D metabolites especially 24,25-dihydroxyvitamin D 3
- the concentration of 24,25-dihydroxyvitamin D 3 in the blood is usually quite low. Therefore, LC-MS/MS using the ESI method often lacks detection sensitivity.
- Patent Documents 1 and 2 4-phenyl-1,2,4-triazoline-3,5-dione (4 A method is conventionally known in which vitamin D metabolites are derivatized using -Phenyl-1,2,4-triazoline-3,5-dione (PTAD) and then measured. Since such derivatization improves the ionization efficiency of vitamin D metabolites, detection sensitivity can be improved.
- PTAD -Phenyl-1,2,4-triazoline-3,5-dione
- vitamin K vitamin K 1
- vitamin K 2 MK-4 vitamin K 2 MK-7
- vitamin K 2 MK-7 etc.
- the present invention was made to solve these problems, and its main purpose is to simultaneously produce fat-soluble vitamins such as vitamin D metabolites and vitamin K without performing complex and difficult-to-automate derivatization processes.
- An object of the present invention is to provide a measuring method and a measuring device that can perform measurements with high detection sensitivity.
- a method for measuring the concentration of a target component which is vitamin K and its metabolites, including K1 , comprising: a first pretreatment step of performing protein removal treatment on the blood sample by a denaturation method; a second pretreatment step of removing impurities contained in the sample after the first pretreatment step by solid phase extraction; Using a liquid chromatograph-tandem mass spectrometer equipped with an ion source using electrospray ionization, the components in the sample after the second pretreatment step are temporally separated, and the components derived from each of the target components are separated in time.
- a measurement step of performing LC/MS analysis to selectively detect protonated ions of valence; has.
- one embodiment of the blood concentration measuring device for fat-soluble vitamins includes vitamin D metabolites containing 24,25-dihydroxyvitamin D 3 in a blood sample, and a device for measuring the concentration of target components, which are vitamin K and its metabolites, including vitamin K1 , a protein removal processing unit that removes proteins from the blood sample by sequentially adding an organic solvent to the sample, stirring, and filtering the sample;
- the sample processed by the protein removal processing unit is supplied to the solid-phase extraction column, and the target component in the sample is retained in the solid-phase extraction column, and then a mobile phase is supplied to extract the target component in the solid-phase extraction column.
- the present inventor has repeatedly conducted various experiments on a method for detecting 24,25-dihydroxyvitamin D3 with high sensitivity, which is particularly difficult to detect among vitamin D metabolites, and has found that one of the major factors for the low sensitivity is the removal of 24,25-dihydroxyvitamin D. It was discovered that this was due to the ion suppression effect due to impurities such as lipids remaining in the blood sample after protein. In response, we experimentally verified various methods for removing impurities, and by using solid-phase extraction using a solid-phase extraction column, we found that we could effectively remove the impurities that particularly contribute to ion suppression. The present inventors have discovered that it can be removed stably (with high reproducibility) and have achieved the present invention.
- the sample liquid containing the target component subjected to online solid-phase extraction can be directly analyzed by LC/MS. It is easy to automate a series of operations from protein processing to measurement. Thereby, it is possible to improve the measurement throughput and the measurement stability, and it can be suitably used for screening tests and the like.
- FIG. 1 is a flowchart showing an example of a processing procedure of a method for measuring blood concentration of fat-soluble vitamins, which is an embodiment of the present invention.
- 1 is a schematic block diagram of a fat-soluble vitamin blood concentration measuring device according to an embodiment of the present invention. The figure which shows the example of the extracted ion chromatogram of vitamin D metabolite, vitamin K, and vitamin K metabolite obtained using the measuring device of this embodiment.
- FIG. 3 is a diagram showing an actual measurement example of an extracted ion chromatogram for a plurality of MRM transitions for 24,25-dihydroxyvitamin D 3 obtained using the measuring device of the present embodiment.
- FIG. 3 is a diagram showing an actual measurement example of an extracted ion chromatogram for a plurality of MRM transitions for 25-hydroxyvitamin D 3 obtained using the measuring device of the present embodiment.
- FIG. 2 is a diagram showing an actual measurement example of an extracted ion chromatogram for a plurality of MRM transitions for 25-hydroxyvitamin D 2 obtained using the measuring device of the present embodiment.
- FIG. 3 is a diagram showing an example of an extracted ion chromatogram for a plurality of MRM transitions for vitamin K 2 MK-4 obtained using the measuring device of the present embodiment.
- FIG. 2 is a diagram illustrating an actual measurement example of an extracted ion chromatogram for a plurality of MRM transitions for vitamin K 2 MK-7 obtained using the measuring device of the present embodiment.
- FIG. 3 is a diagram showing an example of a calibration curve for 24,25-dihydroxyvitamin D 3 obtained using the measuring device of the present embodiment. The figure which shows an example of the calibration curve of vitamin K1 obtained using the measuring device of this embodiment.
- FIG. 3 is a diagram showing reproducibility results (low concentration control) of measurements of vitamin D metabolites, vitamin K, and vitamin K metabolites obtained using the measuring device of the present embodiment.
- FIG. 3 is a diagram showing reproducibility results (low concentration control) of measurements of vitamin D metabolites, vitamin K, and vitamin K metabolites obtained using the measuring device of the present embodiment.
- FIG. 3 is a diagram showing reproducibility results (high concentration control) of measurements of vitamin D metabolites, vitamin K, and vitamin K metabolites obtained using the measuring device of the present embodiment.
- the figure which shows the example of the actual measurement of the chromatogram which shows the carry-over reduction effect by piping cleaning with respect to vitamin K and vitamin K metabolites obtained using the measuring device of this embodiment.
- Comparison of retention time, peak area, peak height, and S/N ratio of 24,25-dihydroxyvitamin D 3 when changing the RF voltage applied to the RF ion guide (Q array) in the measurement device of this embodiment Diagram showing the results.
- FIG. 3 is a diagram showing an example of an extracted ion chromatogram for 24,25-dihydroxyvitamin D 3 measured when changing the RF voltage applied to the RF ion guide (Q array) in the measuring device of the present embodiment.
- the blood sample can be, for example, whole blood, serum, plasma, etc.
- the blood sample may be collected from an animal other than humans.
- vitamin D metabolites include at least 24,25-dihydroxyvitamin D 3 and may also include 25-hydroxyvitamin D 2 , 25-hydroxyvitamin D 3 and the like.
- vitamin K and its metabolites include at least vitamin K 1 and may also include vitamin K 2 MK-4, vitamin K 2 MK-7, and the like.
- protein removal treatment using a denaturation method is a method in which proteins are insolubilized and precipitated by adding an organic solvent to a blood sample.
- organic solvent include methanol, ethanol, acetone, and acetonitrile. can.
- a sample is first supplied to a column for solid phase extraction, and the target component is adsorbed on the packing material of the column. At this time, components that are not adsorbed to the filler are discharged. Thereafter, a mobile phase having an elution effect is supplied to the column for solid phase extraction, and the target component adsorbed on the column is eluted and taken out as a sample solution and sent as it is to a column of a liquid chromatograph.
- a series of such operations can be performed mainly by switching the flow paths using valves, and online solid-phase extraction is an automatic sequential execution of these operations.
- a cleaning solution is passed through the solid phase extraction column to discharge components that were not adsorbed by the column but remain in the piping containing the column. You may also add operations to do so.
- the tandem mass spectrometer of the liquid chromatograph-tandem mass spectrometer includes an electrospray ion source, for example, a triple quadrupole mass spectrometer, a quadrupole-time-of-flight mass spectrometer, It can be an ion trap-time-of-flight mass spectrometer, an ion trap mass spectrometer, or the like.
- tandem mass spectrometer has a function of dissociating ions generated by the ion source, but the method of ion dissociation is not particularly limited. Typically, collision-induced dissociation can be used as a method for the ion dissociation.
- MRM monitoring
- m/z mass-to-charge ratio
- the blood concentration measuring device for fat-soluble vitamins includes, for example, a fully automatic LCMS pretreatment device “CLAM-2030” manufactured by Shimadzu Corporation, and a high-performance liquid chromatograph “Nexera” manufactured by Shimadzu Corporation.
- LCMS-8060NX a liquid chromatograph mass spectrometer manufactured by Shimadzu Corporation
- a personal computer equipped with predetermined software that controls them and processes data In that case, the unit configuration of the high performance liquid chromatograph may be configured to be compatible with online solid phase extraction.
- the blood concentration measuring device according to the present invention can be configured using various other devices and units.
- FIG. 1 is a flowchart showing an example of the processing procedure of the blood concentration measuring method according to the present embodiment.
- the measurement method of this embodiment will be schematically explained according to FIG.
- the sample is serum extracted from blood drawn from a subject.
- the target components to be measured are 24,25-dihydroxyvitamin D 3 , 25-hydroxyvitamin D 2 , 25-hydroxyvitamin D 3 , vitamin K 1 , vitamin K 2 MK-4, vitamin K 2
- MK-7 There are 6 types of MK-7.
- a protein removal process is performed on a blood sample as a preprocess (step S1).
- a method for protein removal for example, a denaturation method using a polar organic solvent that is miscible with water can be used.
- a polar organic solvent methanol, ethanol, acetonitrile, etc.
- protein removal treatment a predetermined polar organic solvent is added to a blood sample to be treated, stirred, and then filtered using a filter or the like. Thereby, proteins precipitated by denaturation are removed, and the supernatant is collected to obtain a protein-depleted blood sample.
- an internal standard substance for calibration can also be added.
- step S2 impurities such as lipids contained in the protein-removed blood sample are removed by online solid-phase extraction (step S2).
- the blood sample is supplied to a solid-phase extraction column that has been conditioned in advance using a predetermined mobile phase, and the target components in the blood sample are collected on the packing material of the solid-phase extraction column. .
- Many of the contaminants in the blood sample are not captured by the packing material and are therefore discharged through the solid phase extraction column.
- some impurities are not adsorbed by the filler, there is a possibility that they may adhere to the filler or adhere to the inside of the pipe. Therefore, the remaining impurities may be washed away by flowing a rinsing liquid that does not affect the target component into the pipe containing the solid phase extraction column.
- a mobile phase with an elution effect is passed through a solid phase extraction column, the target components adsorbed on the column are eluted all at once, and the sample solution containing the target components is carried on the flow of the mobile phase and is then run on an LC. feed into the column.
- the target components in the sample liquid are separated in the time direction while passing through the column (step S3).
- the eluate from the column containing the separated target components is introduced into the ion source of the tandem mass spectrometer.
- the ion source utilizes the ESI method. Contaminants other than target components contained in a blood sample produce an ion suppression effect during ionization by the ESI method.
- the ion suppression effect due to impurities can be suppressed and the ionization efficiency of the target component can be increased. Thereby, more ions derived from the target component can be subjected to mass spectrometry.
- one or more MRM transitions are predetermined for each target component during the period during which the target component is eluted from the column (that is, introduced into the ion source).
- MRM measurement (MS/MS analysis) targeting (combination of m/z values of precursor ions and product ions) is performed (step S4).
- ion intensity data regarding one or more MRM transitions in a predetermined time range centered around the retention time is obtained for each target component.
- the concentration of each target component in the blood sample is calculated (step S5). Specifically, an extracted ion chromatogram is created for each target component, and a chromatographic peak corresponding to the target component is identified in each chromatogram. Then, the area or height of this chromatographic peak is calculated, and the concentration of each target component is calculated using a calibration curve created in advance.
- the concentrations of six types of target components contained in a blood sample can be obtained as measurement results.
- FIG. 2 is a schematic block diagram of the fat-soluble vitamin blood concentration measuring device according to this embodiment.
- this measurement device includes a liquid chromatograph section (LC section) 1, a tandem mass spectrometry section (MS/MS section) 2, an online solid phase extraction (SPE) section 3, and a protein removal processing section 4. , a voltage generation section 5, a data processing section 6, a control section 7, an operation section 8, and a display section 9.
- the LC section 1 includes a mobile phase storage section 10, a liquid feeding pump 11, an injector 12, and a column 13. Although the description is omitted here to avoid complicating the drawing, in the actual measurement example described later, gradient elution using two different types of mobile phases was performed. To perform gradient elution, another mobile phase reservoir, liquid pump, mixer, etc. are added to the configuration of FIG. 2.
- a PFPP (Pentafluorophenylpropyl) column can be used as the column 13, for example.
- a PFPP column is a column into which a PFPP group is introduced, and has been frequently used in recent years to separate biological components as a reversed-phase column that exhibits good separation performance not only for hydrophobic compounds but also for hydrophilic compounds.
- the MS/MS section 2 is a triple quadrupole mass spectrometer having an ESI ion source. Specifically, as shown in FIG. 2, the interior of the chamber 20 is divided into four parts, which are, in order from the LC section 1 side, an ionization chamber 201, a first intermediate vacuum chamber 202, a second intermediate vacuum chamber 203, and a high vacuum chamber 204. It becomes.
- the first intermediate vacuum chamber 202 is evacuated by a rotary pump (not shown) and maintained at a low vacuum atmosphere (about 10 2 [Pa] as an example).
- the second intermediate vacuum chamber 203 is evacuated by a rotary pump and a turbomolecular pump (both not shown) and maintained at a medium vacuum atmosphere (for example, about 10 -1 to 10 -2 [Pa]).
- the high vacuum chamber 204 is also evacuated by a rotary pump and a turbomolecular pump (both not shown) and maintained at a high vacuum atmosphere (about 10 -3 to 10 -4 [Pa] as an example).
- a multi-stage differential pumping system configuration is adopted in which the degree of vacuum increases sequentially starting from the ionization chamber 201, which is in an atmosphere of approximately atmospheric pressure.
- An ESI spray 21 is arranged inside the ionization chamber 201, and the ionization chamber 201 and the first intermediate vacuum chamber 202 are communicated through a small diameter desolvation tube 22.
- a first RF ion guide 23 that transports ions while converging them by the action of an RF (Radio Frequency) electric field is arranged, and the first intermediate vacuum chamber 202 and the second intermediate vacuum chamber 203 through a small diameter ion passage hole formed at the top of the skimmer 24.
- the first RF ion guide (hereinafter sometimes referred to as a "Q array" due to the structural characteristics described below) 23 includes a plurality of first RF ion guides (in this example, four Each rod electrode is composed of a plurality of short cylindrical (or disc-shaped) partial electrodes separated in the direction in which the ion optical axis C extends. Among the plurality of separated partial electrodes, the partial electrode located on the skimmer 24 side has a small radius of an inscribed circle centered on the ion optical axis C, thereby efficiently transferring ions to the ion optical axis C. I'm trying to have it converge nearby.
- a second RF electrode is provided inside the second intermediate vacuum chamber 203, which is composed of a plurality of rod electrodes (for example, eight rods) surrounding the ion optical axis C, arranged at approximately equal angular intervals, and parallel to the ion optical axis C.
- An ion guide 25 is arranged inside the second intermediate vacuum chamber 203.
- the second intermediate vacuum chamber 203 and the high vacuum chamber 204 communicate with each other through a small diameter ion passage hole.
- a front quadrupole mass filter 26, a collision cell 27, a rear quadrupole mass filter 28, and an ion detector 29 are arranged in order along the ion optical axis C.
- Both the front-stage quadrupole mass filter 26 and the rear-stage quadrupole mass filter 28 include four rod electrodes that surround the ion optical axis C and are arranged at approximately equal angular intervals and parallel to the ion optical axis C. include.
- a pre-rod electrode is provided before each main rod electrode, but this can be omitted.
- a post rod electrode may be provided after the main rod electrode if necessary.
- an ion guide consisting of a plurality of rod electrodes is arranged surrounding the ion optical axis C at approximately equal angular intervals and parallel to the ion optical axis C. Furthermore, CID gas, which is an inert gas such as argon, is introduced into the collision cell 27 from the outside.
- the online SPE section 3 is substantially provided upstream of the LC section 1, and includes a mobile phase storage section 30, a liquid feeding pump 31, an injector 12, a solid phase extraction column 33, and a flow path switching section 34 including a plurality of valves.
- a mobile phase storage section 30 includes a mobile phase storage section 30, a liquid feeding pump 31, an injector 12, a solid phase extraction column 33, and a flow path switching section 34 including a plurality of valves.
- the injector 12 one included in the LC section 1 is used.
- the column 33 for solid phase extraction for example, an ODS column packed with chemically bonded porous spherical silica gel whose surface is modified with ODS (Octa Decyl Silyl) groups (C 18 H 37 Si) as a stationary phase can be used. I can do it.
- the protein removal processing section 4 is provided further upstream of the online SPE section 3.
- the protein removal processing unit 4 performs various operations on the sample contained in a vial, such as dispensing the sample, dispensing a reagent, stirring, filtering, heating, and transporting the sample to the sample collection position using the injector 12.
- It can be a sample pretreatment device that can perform operations and treatments.
- protein removal treatment can be performed by using predetermined reagents prepared in advance and performing various operations and treatments according to preset conditions.
- the protein removal processing section 4 can be implemented by using the above-mentioned fully automatic LCMS pretreatment device "CLAM-2030" manufactured by Shimadzu Corporation.
- the voltage generation section 5 applies a predetermined voltage to each part of the MS/MS section 2 under the control of the control section 7.
- This voltage is either an RF voltage, an AC voltage with a lower frequency than the RF voltage, a DC voltage, or a combination thereof.
- the data processing unit 6 receives and processes a detection signal from the ion detector 29, and includes a data collection unit 60 including an analog-to-digital conversion unit and a data storage unit, and a quantitative calculation unit 61 as functional blocks.
- the control unit 7 controls the protein removal processing unit 4, the online SPE unit 3, the LC unit 1, the voltage generation unit 5, the MS/MS unit 2, and the data processing unit 6, thereby performing preprocessing, measurement, and measurement of the blood sample. Furthermore, it causes the measuring device to execute a series of processes up to data analysis.
- the control unit 7 also has an input/output control function through an operation unit 8 and a display unit 9, which are user interfaces.
- the data processing/control software includes, for example, software for performing general measurements without limiting the purpose of measurement or the components to be measured (however, this software itself may consist of multiple pieces of software); It includes dedicated software for measuring vitamin D metabolites, etc., which are the measurement targets here. In FIG. 2, this latter software is shown as a vitamin D/K blood concentration measurement program 70.
- the protein removal processing unit 4 sequentially performs operations such as dispensing a blood sample, dispensing a reagent to the blood sample, stirring, and filtration to obtain a blood sample from which proteins have been removed.
- this protein removal process and subsequent measurement operations including on-line SPE can be performed in parallel.
- the container containing the processed blood sample is transported to a sample collection position by the injector 12 at a predetermined timing.
- the liquid pump 31 sucks the mobile phase from the mobile phase storage unit 30 and sends it to the solid phase extraction column 33 through the flow path switching unit 34.
- the injector 12 injects the protein-removed blood sample into the mobile phase.
- the injected sample is supplied to the solid-phase extraction column 33, and the target components contained in the sample are mainly adsorbed on the packing material of the solid-phase extraction column 33.
- the target components contained in the sample are not adsorbed by the packing material, so they pass through the column 33 and are discharged via the flow path switching section 34. Thereby, the target component is collected in the solid phase extraction column 33.
- the cleaning liquid may be sucked from a cleaning liquid reservoir (not shown) using the liquid feeding pump 31, and may be caused to flow through the solid phase extraction column 33 to the drainage path. This makes it possible to wash away impurities adhering to the filler and inside the piping, thereby increasing the purity of the target component. Note that during the period in which the target component is collected in the solid-phase extraction column 33, the mobile phase sucked by the liquid pump 11 is caused to flow into the column 13 using a channel not shown in FIG. It's good to do that.
- the flow path is switched by the flow path switching unit 34, and the mobile phase sucked from the mobile phase storage unit 10 by the liquid feeding pump 11 is supplied to the solid phase extraction column 33.
- the above-mentioned six target components collected in the solid phase extraction column 33 are eluted into the mobile phase and sent to the column 13 along with the flow of the mobile phase.
- the six target components introduced into the column 13 at approximately the same time are separated in the time direction while passing through the column 13 and eluted from the outlet of the column 13.
- LC analysis conditions such as the type of mobile phase and the flow rate of the mobile phase at this time are defined in the vitamin D/K blood concentration measurement program 70.
- the MS/MS unit 2 targets one or more MRM transitions corresponding to the target components in a predetermined time range centered on the retention time of each of the six types of target components. It operates to perform MRM measurements.
- the ESI spray 21 imparts an electric charge with biased polarity to the eluate, and the eluate is heated at approximately atmospheric pressure. It is sprayed into a certain ionization chamber 21.
- the sprayed charged droplets come into contact with gas molecules and become fine, and in the process of vaporizing the solvent from the charged droplets, molecules of the target component fly out with charge and become gas ions.
- the generated ions are sucked into the desolvation tube 22 together with fine charged droplets and sent to the first intermediate vacuum chamber 202.
- the desolvation tube 22 is heated, and vaporization from droplets is promoted in the desolvation tube 22 as well, so ionization of the target component is promoted.
- Ions derived from the target component that have entered the first intermediate vacuum chamber 202 are captured by the RF electric field formed by the RF voltage applied from the voltage generator 5 to the first RF ion guide 23, and are moved near the ion optical axis C. Converged.
- the degree of vacuum inside the first intermediate vacuum chamber 202, which is the next stage of the ionization chamber 201, is low and there are many residual gas molecules, so ions derived from the target component easily come into contact with the residual gas molecules. Since the kinetic energy of the ions derived from the target component is attenuated by the contact, the ions derived from the target component are easily captured by the RF electric field, and convergence is also performed well.
- the ESI method is a soft ionization method, and ion dissociation (cleavage) is less likely to occur during ionization.
- ion dissociation cleavage
- not only monovalent protonated ions but also multivalent ions and adduct ions added with alkali metals are likely to be generated.
- increasing the number of types of ions derived from the target component during ionization is disadvantageous in increasing detection sensitivity for a specific MRM transition.
- the inventors of the present invention have found that by adjusting the amplitude of the RF voltage applied to the first RF ion guide 23, the intensity of monovalent protonated ions to be observed can be improved.
- the detection sensitivity of monovalent protonated ions is predetermined to be high.
- the RF voltage is applied to the first RF ion guide 23. Note that this point will be explained in detail later.
- Ions derived from the target component focused by the RF electric field of the first RF ion guide 23 pass through the small hole at the top of the skimmer 24 and enter the second intermediate vacuum chamber 203.
- a predetermined RF voltage is applied to the second RF ion guide 25 from the voltage generator 5, and ions derived from the target component are captured and transported by the RF electric field thus formed, and sent to the high vacuum chamber 204.
- the front quadrupole mass filter 26 and the rear quadrupole mass filter 28 are each configured to selectively pass a specific m/z value in the MRM transition associated with the target component.
- a voltage is applied from the voltage generator 5.
- CID gas is continuously or intermittently introduced into the collision cell 27 so that the CID gas pressure becomes a predetermined value.
- the concentration of one target component in the eluate sent from the LC section 1 to the MS/MS section 2 changes roughly in a mountain shape (theoretically according to a Gaussian distribution) over time
- the data processing section 6 The detection signal (ion intensity signal) sent to the sensor also changes roughly in the shape of a mountain over time.
- the MS/MS unit 2 executes MRM measurement targeting one or more MRM transitions that are different for each target component within a time range corresponding to the target component. As a result, one or more detection signals indicating temporal changes in ion intensity as described above are obtained for each target component.
- the MS/MS analysis conditions including the MRM transition in the MS/MS unit 2 are defined by the vitamin D/K blood concentration measurement program 70, similar to the LC analysis conditions. Therefore, when the program 70 is introduced into a computer to perform measurements, the user does not need to manually set individual LC analysis conditions and MS/MS analysis conditions. Of course, the user may manually set the individual LC analysis conditions and MS/MS analysis conditions before performing the measurement.
- the detection signal acquired by the MS/MS section 2 is converted into digital data and stored in the data storage section of the data acquisition section 60.
- the quantitative calculation unit 61 creates one or more extracted ion chromatograms for each target component based on the data stored in the data collection unit 60, and calculates the retention time, area value, and peak value of the peak observed in the chromatogram. Calculate peak information such as height values. Then, after confirming the target component from the retention time of the peak, the quantitative calculation unit 61 calculates the concentration from the peak area value or height value using a calibration curve created in advance. In addition, in the case of quantitative determination, it is possible to utilize the peak area value and height value obtained by simultaneously measuring the internal standard substance added to the blood sample during protein removal treatment.
- measurement results for each blood sample can be obtained by automatically performing preprocessing and measurement on a large number of blood samples prepared in advance according to the above-described procedures.
- the control unit 7 can display the measurement results obtained in this manner on the screen of the display unit 9 in a predetermined format.
- Protein removal treatment was performed according to the following procedure.
- a filtration container a filtration container with a 0.45 ⁇ m polytetrafluoroethylene (PTFE) filtration filter
- IPA isopropyl alcohol
- sample a sample
- methanol methanol (including internal standard sample) as a polar organic solvent
- LC analysis conditions are as follows. ⁇ Column type: Shimadzu Shim-Pack Velox PFPP 2.7 ⁇ m 3.0 ⁇ 100mm ⁇ Column temperature: 40°C ⁇ Sample injection volume: 30 ⁇ L ⁇ Mobile phase A: Water + 5mM ammonium formate + 0.1% formic acid ⁇ Mobile phase B: Methanol ⁇ Mobile phase flow rate: 0.7mL/min ⁇ Analysis time: 12min
- MS/MS analysis conditions other than MRM transition are as follows.
- ⁇ Equipment Shimadzu LCMS-8060
- ⁇ Ionization method ESI method ⁇ Nebulization gas flow rate: 2L/min
- ⁇ Drying gas flow rate 17L/min
- ⁇ Heating gas flow rate 3L/min
- Desolvation tube temperature 250°C
- ⁇ Heat block temperature 300°C
- Interface temperature 300°C ⁇ Collision cell gas pressure: 190kPa
- ⁇ ESI spray applied voltage 3kV ⁇ Q array (1st RF ion guide)
- RF voltage 100V (24,25-(OH) 2 D 3 ), default value (target components other than 24,25-(OH) 2 D 3 )
- the default value of the Q array RF voltage is usually a value determined by automatic tuning of the device using a standard sample or the like, and is not necessarily constant.
- Typical MRM transitions and collision energies (CE) for each target component are as follows.
- a typical MRM transition is an MRM transition used for quantification, and in addition to this, one or more MRM transitions of confirmation ions for component confirmation using confirmation ion ratios can be separately determined, for example.
- FIG. 3 is a representative extracted ion chromatogram for each target component obtained through actual measurements. The vertical position of each chromatogram is shifted as appropriate. From this result, it can be seen that each target component is observed while being sufficiently separated from each other. It can also be seen that 25(OH)VitD 3 and its isomer (epi-) can be separated appropriately.
- Figures 4 to 9 show 24,25-dihydroxyvitamin D 3 at a concentration of 0.5 ng/mL, 25-hydroxyvitamin D 3 at a concentration of 4.9 ng/mL, and 25-hydroxyvitamin D at a concentration of 4.5 ng/mL, respectively.
- concentration is the lower limit of quantification for each target component.
- the peak indicated by a downward triangular mark is the peak used for quantification, and the starting point and ending point of the peak and the line connecting them (that is, the baseline defining the peak area) are also shown. It can be confirmed that peaks with good shapes were obtained for all target components.
- FIG. 10 is a calibration curve for 24,25-dihydroxyvitamin D 3 created using a calibration curve sample (calibrator).
- the calibrator was prepared using reagents manufactured by Golden West Diagnostics.
- the measured concentration range is 0.5 to 120 ng/mL, which corresponds to the measured concentration range expected in actual testing.
- FIG. 11 is an example of a vitamin K 1 calibration curve created using the same calibrator.
- the measured concentration range is 0.1 to 24 ng/mL, which also corresponds to the measured concentration range expected in actual tests.
- FIG. 12 is a diagram showing the results of accuracy and reproducibility (within-day reproducibility) for a low concentration control sample prepared using a reagent manufactured by Golden West Diagnostics.
- FIG. 13 is a diagram showing the accuracy and reproducibility (within-day reproducibility) results for a high concentration control sample also prepared using a reagent manufactured by Golden West Diagnostics.
- the low concentration control sample has a concentration 5 times the lower limit of quantitation
- the high concentration control sample has a concentration of 18 ng/mL for vitamin K1 only, and 90 ng/mL for other target components.
- the linearity of the calibration curves shown in FIGS. 10 and 11 is good, with r 2 >0.99.
- the calibration curves for the other four target components were similarly verified in the measurement concentration range required for each, and it was confirmed that the linearity was good with r 2 > 0.99 in all cases. .
- accuracy of 85 to 115% is ensured for all target components, and reproducibility is within 13% except for vitamin K 1 and vitamin K 2 MK-7.
- FIG. 14 is an extracted ion chromatogram showing the effect of reducing carryover when washing with a washing liquid is performed.
- the upper part of FIG. 14 is an extracted ion chromatogram for the QC sample with the highest concentration of vitamin K, and the lower part of FIG. 14 is an extracted ion chromatogram when washed with methanol. From these figures, it can be confirmed that carryover can be reduced to a substantially non-problematic level by cleaning using an appropriate cleaning liquid such as methanol. For this reason, it is important to reduce carryover through washing, especially in improving the precision and reproducibility of vitamin K compounds.
- FIG. 15 shows extracted ion chromatograms actually measured for 24,25-dihydroxyvitamin D3 when the RF voltage applied to the first RF ion guide 23 was changed to 0, 30, 60, 90, and 120V.
- FIG. 2 is a diagram showing the retention time of the peak, the area, height, and SN ratio of the peak. Note that a bias DC voltage is applied to the first RF ion guide 23 in addition to the RF voltage.
- FIG. 16 is an example of an extracted ion chromatogram measured when changing the RF voltage.
- the amount of multivalent ions and adduct ions derived from the target component should be reduced as much as possible, and the amount of monovalent protonated ions should be increased. It is important to consolidate as much as possible.
- the ions introduced into the intermediate vacuum chamber 202 come into contact with the residual gas molecules and are cooled (collision cooling). Since the kinetic energy of the ions is thus attenuated by cooling, the ions are more likely to be captured by the RF electric field. This is a major reason why ion focusing using an RF electric field is effective in a low vacuum atmosphere. Ions derived from the target component lose kinetic energy due to contact with residual gas molecules, but in addition, a phenomenon similar to collision-induced dissociation occurs due to contact with residual gas molecules, and the partial structure of the ion is detached.
- adduct such as an alkali metal
- the adduct portion is likely to be detached by contact with residual gas and become a protonated ion.
- multivalent ions can be separated and monovalent ions can be generated by contact with residual gas. That is, it is considered that the more opportunities for contact with residual gas in the first intermediate vacuum chamber 202, the lower the proportion of adduct ions and multivalent ions, and the more monovalent protonated ions increase.
- the gas that has flowed into the first intermediate vacuum chamber 202 through the desolvation tube 22 becomes a supersonic free jet and spreads after leaving the exit of the desolvation tube 22, but the density of the residual gas is low near the ion optical axis C. It can easily become expensive. Ions captured by the RF electric field oscillate periodically due to the action of the electric field, but the stronger the electric field, the more likely they are to be confined in a narrow region near the ion optical axis C. Therefore, the stronger the RF electric field, that is, the greater the RF voltage applied to the Q array, the greater the chance that ions derived from the target component will come into contact with the residual gas.
- 24,25-dihydroxyvitamin D 3 has the most severe detection sensitivity among the above six types of target components. Therefore, in the measuring device of the embodiment described above, when measuring 24,25-dihydroxyvitamin D 3 , the RF voltage is set to 100 V regardless of the default value of the RF voltage, and other target components are measured. In some cases, the RF voltage is set to a default value. As a result, while the detection sensitivity of 24,25-dihydroxyvitamin D 3 is particularly increased, the detection sensitivity of other target components can also be kept within the target range.
- the RF voltage applied to the first RF ion guide 23 is set to a predetermined value only when measuring 24,25-dihydroxyvitamin D 3 .
- the RF voltage may also be set to a predetermined value for each target component. In that case, these values may be individually different values, or some of them may be the same value.
- the various parameter values adopted in the measurement method and measurement device of the above embodiment, the analysis conditions including the types of constituent elements such as columns and consumable materials such as mobile phase, etc. are merely examples, and may be changed as appropriate. It is possible.
- the six types of target components to be measured in the measuring method and measuring device of the above embodiment are merely examples, and the target components can be adjusted as appropriate except for containing at least 24,25-dihydroxyvitamin D 3 and vitamin K 1 . can be deleted or added to.
- One embodiment of the method for measuring blood concentration of fat-soluble vitamins according to the present invention includes vitamin D metabolites including 24,25-dihydroxyvitamin D 3 and vitamin K 1 in a blood sample.
- a method for measuring the concentration of a target component, which is vitamin K and its metabolites comprising: a first pretreatment step of performing protein removal treatment on the blood sample by a denaturation method; a second pretreatment step of removing impurities contained in the sample after the first pretreatment step by solid phase extraction; Using a liquid chromatograph-tandem mass spectrometer equipped with an ion source using electrospray ionization, the components in the sample after the second pretreatment step are temporally separated, and the components derived from each of the target components are separated in time.
- a measurement step of performing LC/MS analysis to selectively detect protonated ions of valence has.
- One embodiment of the fat-soluble vitamin blood concentration measuring device measures vitamin D metabolites including 24,25-dihydroxyvitamin D 3 and vitamins including vitamin K 1 in a blood sample.
- a device for measuring the concentration of target components, which are K and its metabolites a protein removal processing unit that removes proteins from the blood sample by sequentially adding an organic solvent to the sample, stirring, and filtering the sample; The sample processed by the protein removal processing unit is supplied to the solid-phase extraction column, and the target component in the sample is retained in the solid-phase extraction column, and then a mobile phase is supplied to extract the target component in the solid-phase extraction column.
- the ion suppression effect caused by impurities remaining in the blood sample after protein removal treatment by the denaturation method can be suppressed.
- ionization efficiency of the target component can be increased.
- vitamin D metabolites, vitamin K, and their metabolites in blood can be simultaneously measured with high sensitivity without performing derivatization using Cookson-type derivatization reagents, which are complicated and difficult to automate. be able to.
- a sample solution containing target components subjected to online solid-phase extraction can be directly analyzed by LC/MS, it is easy to automate a series of operations from protein removal treatment to measurement. Thereby, it is possible to improve measurement throughput and measurement stability, and it can be suitably used for screening tests and the like.
- the vitamin D metabolite may include 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 .
- the vitamin K and its metabolites may include vitamin K 2 MK-4 and vitamin K 2 MK-7.
- the liquid chromatograph-tandem mass spectrometer focuses ions on the next stage of the ion source. It has an RF ion guide that transports the ions to the subsequent stage while In the measurement step, during the period of measuring 24,25-dihydroxyvitamin D 3 , the amplitude of the RF voltage applied to the RF ion guide is adjusted to the level of the monovalent protonated ion derived from 24,25-dihydroxyvitamin D 3 . It may be set to a predetermined value in response to detection.
- the predetermined value is determined by monovalent protonation of various ions derived from 24,25-dihydroxyvitamin D 3 introduced into the RF ion guide. It can be set to a value that indicates the action of concentrating into ions.
- 24,25-dihydroxyvitamin D3 is an important vitamin D metabolite in screening tests for osteoporosis, it has a low blood concentration compared to other vitamin D metabolites and is difficult to measure. It was a metabolite.
- the RF voltage By appropriately setting , it is possible to mainly suppress the dispersion of ion types and increase the intensity of monovalent protonated ions to be observed. Thereby, the detection sensitivity of 24,25-dihydroxyvitamin D 3 present in blood can be further increased.
- the amplitude of the RF voltage applied to the RF ion guide is changed during the period of measuring target components other than 24,25-dihydroxyvitamin D3 . , it may be set to a standard value set in the liquid chromatograph-tandem mass spectrometer.
- the standard value here is, for example, a value predetermined by an equipment manufacturer, etc., without assuming one specific type of compound, and which yields good results on average, or a value determined by an equipment using a standard sample, etc.
- the value set by the automatic tuning function is not a value optimized for each target component so that the detection sensitivity is the highest, for example.
- the RF voltage applied to the RF ion guide can be kept constant during the period when target components other than 24,25-dihydroxyvitamin D 3 are measured, so that, for example, the measurement periods for multiple target components overlap. Even if it is necessary to switch the target component of the measurement target on a time-sharing basis, there is no need to provide a waiting time for switching the voltage. Thereby, the time interval between repeated measurements for one target component can be shortened, the accuracy of the peak waveform of the extracted ion chromatogram can be improved, and quantitative performance can be improved.
- the target components contained in blood can be efficiently collected, while contaminants contained in blood, particularly contaminants that cause an ion suppression effect, can be collected.
- the residual amount can be suppressed.
- impurities can be effectively removed from the blood, and target components can be detected with high sensitivity.
- Mobile phase storage section 31 Liquid pump 33...Column for solid phase extraction 34...Flow path switching section 4
- Protein removal processing section 5 ...Voltage generation section 6
- Data processing section 60 ...Data collection section 61
- Quantitative calculation section 7 ...Control section 70
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Abstract
Provided is a method for measuring the concentration of a lipophilic vitamin component in blood, which is a method for measuring the concentrations of target components that are a vitamin D metabolite comprising 24,25-dihydroxyvitamin D3 and a vitamin K component comprising vitamin K1 and a metabolite thereof in a blood sample, the method comprising: a first pretreatment step (S1) for subjecting the blood sample to protein removal by a denaturation method; a second pretreatment step (S2) for removing contaminants contained in the sample obtained after the first pretreatment step by solid phase extraction; and a measurement step (S3,S4) for performing LC/MS analysis for selectively detecting a monovalent protonated ion derived from each of the target components while separating the components in the sample obtained after the second pretreatment step over time by employing LC-MS/MS having an ion source by ESI method.
Description
本発明は、脂溶性ビタミン類の血中濃度を測定する方法及び装置に関し、さらに詳しくは、ビタミンＤ代謝物やビタミンＫ等の血中濃度を測定する方法及び装置に関する。
The present invention relates to a method and device for measuring blood concentrations of fat-soluble vitamins, and more particularly to a method and device for measuring blood concentrations of vitamin D metabolites, vitamin K, and the like.
ビタミン類はヒトの生存や生育において重要な栄養素である。ビタミン類の中でビタミンＤやビタミンＫの不足は骨基質の石灰化の障害をもたらすおそれがあり、骨粗しょう症の進行を促進し、骨折等のリスクを高めることが一般に知られている。
Vitamins are important nutrients for human survival and growth. It is generally known that deficiencies in vitamin D and vitamin K, among vitamins, may impede calcification of bone matrix, accelerate the progression of osteoporosis, and increase the risk of fractures and the like.
ビタミンＤの充足度を評価する方法として、ビタミンＤ代謝物である２４，２５－ジヒドロキシビタミンＤ3（以下「24,25-(OH)2VitD3」又は「24,25-(OH)2D3」等と記す場合がある）、２５－ヒドロキシビタミンＤ2（以下「25-(OH)VitD2」又は「25-(OH)D2」等と記す場合がある）、２５－ヒドロキシビタミンＤ3（以下「25-(OH)VitD3」又は「25-(OH)D3」等と記す場合がある）などの血中濃度を、液体クロマトグラフ-タンデム型質量分析装置（以下「ＬＣ－ＭＳ／ＭＳ」と略す場合がある）を用いて測定する方法が知られている。
As a method for evaluating the degree of vitamin D sufficiency, 24,25-dihydroxyvitamin D 3 (hereinafter "24,25-(OH) 2 VitD 3 " or "24,25-(OH) 2 D"), which is a vitamin D metabolite, is used. 3 ), 25-hydroxyvitamin D 2 (hereinafter sometimes referred to as ``25-(OH)VitD 2 '' or ``25-(OH)D 2' '), 25-hydroxyvitamin D 3 (hereinafter sometimes referred to as ``25-(OH)VitD 3 '' or ``25-(OH)D 3' '), etc., was measured using a liquid chromatograph-tandem mass spectrometer (hereinafter referred to as ``LC- A method of measuring using MS/MS (sometimes abbreviated as "MS/MS") is known.
こうした測定のためのＬＣ－ＭＳ／ＭＳでは、イオン化法としてエレクトロスプレーイオン化（ElectroSpray Ionization：ＥＳＩ）法や大気圧化学イオン化（Atmospheric Pressure Chemical Ionization：ＡＰＣＩ）法が広く用いられている。ＥＳＩ法は、適用可能である化合物の範囲が広いこと、装置の取り扱いが容易であること、などから、ＬＣ－ＭＳ／ＭＳにおいて最も一般的に利用されているイオン化法である。しかしながら、特許文献１等にも記載されているように、ビタミンＤ代謝物、特に２４，２５－ジヒドロキシビタミンＤ3はＥＳＩ応答性が低く、イオン化効率が低い傾向にある。また、通常、血中の２４，２５－ジヒドロキシビタミンＤ3の濃度はかなり低い。そのため、ＥＳＩ法を利用したＬＣ－ＭＳ／ＭＳでは、検出感度が不足することが多い。
In LC-MS/MS for such measurements, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are widely used as ionization methods. The ESI method is the most commonly used ionization method in LC-MS/MS because it is applicable to a wide range of compounds and the equipment is easy to handle. However, as described in Patent Document 1 and the like, vitamin D metabolites, especially 24,25-dihydroxyvitamin D 3 , tend to have low ESI responsiveness and low ionization efficiency. Also, the concentration of 24,25-dihydroxyvitamin D 3 in the blood is usually quite low. Therefore, LC-MS/MS using the ESI method often lacks detection sensitivity.
これに対し、特許文献１、２等に開示されているように、クックソン（Cookson）型誘導体化試薬の一つである４－フェニル－１，２，４－トリアゾリン－３，５－ジオン（4-Phenyl-1,2,4-triazoline-3,5-dione：ＰＴＡＤ）を用いてビタミンＤ代謝物を誘導体化したあとに測定する方法が従来知られている。こうした誘導体化によってビタミンＤ代謝物のイオン化効率が改善されるため、検出感度を向上させることが可能である。
On the other hand, as disclosed in Patent Documents 1 and 2, 4-phenyl-1,2,4-triazoline-3,5-dione (4 A method is conventionally known in which vitamin D metabolites are derivatized using -Phenyl-1,2,4-triazoline-3,5-dione (PTAD) and then measured. Since such derivatization improves the ionization efficiency of vitamin D metabolites, detection sensitivity can be improved.
しかしながら、こうした測定方法では、血液サンプルに対して固相抽出法や液液抽出法による除タンパク処理を実施したあとに、さらに上述したような煩雑な誘導体化処理を行う必要がある。そのため、測定に時間、手間、及びコストが掛かる。また、作業工程が複雑であるため、人手を介さない自動分析が困難である。そのため、測定のスループットの向上が難しく、また感染症などの作業者のリスクを低減することも難しい。
However, in such a measurement method, it is necessary to perform a complicated derivatization process as described above after a blood sample is subjected to protein removal treatment using a solid-phase extraction method or a liquid-liquid extraction method. Therefore, measurement takes time, effort, and cost. Furthermore, since the work process is complex, automatic analysis without human intervention is difficult. Therefore, it is difficult to improve measurement throughput, and it is also difficult to reduce risks to workers, such as infectious diseases.
また、骨粗しょう症のリスク評価等を目的としたスクリーニング検査では、上述したようなビタミンＤ代謝物と共に、ビタミンＫ（ビタミンＫ1）や、ビタミンＫ2ＭＫ－４、ビタミンＫ2ＭＫ－７などのビタミンＫ代謝物を迅速に且つできるだけ高い感度で測定することが求められる。そのためには、血液サンプル中のビタミンＤ代謝物並びにビタミンＫ及びその代謝物を、１回の測定で同時に且つ高感度で測定する必要があるものの、そうした測定方法は従来知られていない。
In addition, in screening tests aimed at assessing the risk of osteoporosis, vitamin K (vitamin K 1 ), vitamin K 2 MK-4, vitamin K 2 MK-7, etc. are used in addition to the vitamin D metabolites mentioned above. There is a need to quickly and with the highest possible sensitivity measure the vitamin K metabolites of the human body. To this end, it is necessary to measure vitamin D metabolites and vitamin K and its metabolites in a blood sample simultaneously and with high sensitivity in one measurement, but such a measurement method has not been known so far.
本発明はこうした課題を解決するためになされたものであり、その主たる目的は、煩雑で自動化が難しい誘導体化処理を行うことなく、ビタミンＤ代謝物やビタミンＫなどの脂溶性ビタミン類を同時に且つ高い検出感度で以て測定することができる測定方法及び測定装置を提供することである。
The present invention was made to solve these problems, and its main purpose is to simultaneously produce fat-soluble vitamins such as vitamin D metabolites and vitamin K without performing complex and difficult-to-automate derivatization processes. An object of the present invention is to provide a measuring method and a measuring device that can perform measurements with high detection sensitivity.
上記課題を解決するためになされた本発明に係る脂溶性ビタミン類の血中濃度測定方法の一態様は、血液サンプル中の、２４，２５－ジヒドロキシビタミンＤ3を含むビタミンＤ代謝物、並びにビタミンＫ1を含むビタミンＫ及びその代謝物、である目的成分の濃度を測定する方法であって、
血液サンプルに対し変性法により除タンパク処理を行う第１前処理工程と、
前記第１前処理工程後のサンプルに含まれる夾雑物を固相抽出により除去する第２前処理工程と、
エレクトロスプレーイオン化法によるイオン源を有する液体クロマトグラフ－タンデム型質量分析装置を用いて、前記第２前処理工程後のサンプル中の成分を時間的に分離しつつ、前記目的成分それぞれに由来する１価のプロトン付加イオンを選択的に検出するＬＣ／ＭＳ分析を実行する測定工程と、
を有する。 One embodiment of the method for measuring the blood concentration of fat-soluble vitamins according to the present invention, which has been made to solve the above problems, detects vitamin D metabolites including 24,25-dihydroxyvitamin D 3 and vitamin D in a blood sample. A method for measuring the concentration of a target component, which is vitamin K and its metabolites, including K1 , comprising:
a first pretreatment step of performing protein removal treatment on the blood sample by a denaturation method;
a second pretreatment step of removing impurities contained in the sample after the first pretreatment step by solid phase extraction;
Using a liquid chromatograph-tandem mass spectrometer equipped with an ion source using electrospray ionization, the components in the sample after the second pretreatment step are temporally separated, and the components derived from each of the target components are separated in time. a measurement step of performing LC/MS analysis to selectively detect protonated ions of valence;
has.
血液サンプルに対し変性法により除タンパク処理を行う第１前処理工程と、
前記第１前処理工程後のサンプルに含まれる夾雑物を固相抽出により除去する第２前処理工程と、
エレクトロスプレーイオン化法によるイオン源を有する液体クロマトグラフ－タンデム型質量分析装置を用いて、前記第２前処理工程後のサンプル中の成分を時間的に分離しつつ、前記目的成分それぞれに由来する１価のプロトン付加イオンを選択的に検出するＬＣ／ＭＳ分析を実行する測定工程と、
を有する。 One embodiment of the method for measuring the blood concentration of fat-soluble vitamins according to the present invention, which has been made to solve the above problems, detects vitamin D metabolites including 24,25-dihydroxyvitamin D 3 and vitamin D in a blood sample. A method for measuring the concentration of a target component, which is vitamin K and its metabolites, including K1 , comprising:
a first pretreatment step of performing protein removal treatment on the blood sample by a denaturation method;
a second pretreatment step of removing impurities contained in the sample after the first pretreatment step by solid phase extraction;
Using a liquid chromatograph-tandem mass spectrometer equipped with an ion source using electrospray ionization, the components in the sample after the second pretreatment step are temporally separated, and the components derived from each of the target components are separated in time. a measurement step of performing LC/MS analysis to selectively detect protonated ions of valence;
has.
また、上記課題を解決するためになされた本発明に係る脂溶性ビタミン類の血中濃度測定装置の一態様は、血液サンプル中の、２４，２５－ジヒドロキシビタミンＤ3を含むビタミンＤ代謝物、並びにビタミンＫ1を含むビタミンＫ及びその代謝物、である目的成分の濃度を測定するための装置であって、
血液サンプルに対し有機溶媒の添加、撹拌、及び濾過を順に行うことにより該サンプル中のタンパクを除去する除タンパク処理部と、
前記除タンパク処理部による処理済みのサンプルを固相抽出用カラムに供給し該サンプル中の目的成分を該固相抽出用カラムに保持したあと、移動相を供給して目的成分を該固相抽出用カラムから溶出させるオンライン固相抽出部と、
エレクトロスプレーイオン化法によるイオン源を有する液体クロマトグラフ－タンデム型質量分析装置であって、前記固相抽出用カラムから溶出した目的成分が含まれる試料液中の成分を時間的に分離しつつ、前記目的成分それぞれに由来する１価のプロトン付加イオンを選択的に検出するＬＣ／ＭＳ分析を実行する測定実行部と、
前記測定実行部により得られたデータを利用して前記目的成分毎にクロマトグラムを作成し、該クロマトグラムにおいて観測されるピークの面積又は高さに基いて該目的成分を定量するデータ解析部と、
を備える。 In addition, one embodiment of the blood concentration measuring device for fat-soluble vitamins according to the present invention, which has been made to solve the above problems, includes vitamin D metabolites containing 24,25-dihydroxyvitamin D 3 in a blood sample, and a device for measuring the concentration of target components, which are vitamin K and its metabolites, including vitamin K1 ,
a protein removal processing unit that removes proteins from the blood sample by sequentially adding an organic solvent to the sample, stirring, and filtering the sample;
The sample processed by the protein removal processing unit is supplied to the solid-phase extraction column, and the target component in the sample is retained in the solid-phase extraction column, and then a mobile phase is supplied to extract the target component in the solid-phase extraction column. an online solid-phase extraction section for elution from a column for
A liquid chromatograph-tandem mass spectrometer having an ion source using an electrospray ionization method, which temporally separates components in a sample solution containing target components eluted from the solid phase extraction column, and a measurement execution unit that executes LC/MS analysis that selectively detects monovalent protonated ions originating from each target component;
a data analysis unit that creates a chromatogram for each target component using the data obtained by the measurement execution unit and quantifies the target component based on the area or height of a peak observed in the chromatogram; ,
Equipped with
血液サンプルに対し有機溶媒の添加、撹拌、及び濾過を順に行うことにより該サンプル中のタンパクを除去する除タンパク処理部と、
前記除タンパク処理部による処理済みのサンプルを固相抽出用カラムに供給し該サンプル中の目的成分を該固相抽出用カラムに保持したあと、移動相を供給して目的成分を該固相抽出用カラムから溶出させるオンライン固相抽出部と、
エレクトロスプレーイオン化法によるイオン源を有する液体クロマトグラフ－タンデム型質量分析装置であって、前記固相抽出用カラムから溶出した目的成分が含まれる試料液中の成分を時間的に分離しつつ、前記目的成分それぞれに由来する１価のプロトン付加イオンを選択的に検出するＬＣ／ＭＳ分析を実行する測定実行部と、
前記測定実行部により得られたデータを利用して前記目的成分毎にクロマトグラムを作成し、該クロマトグラムにおいて観測されるピークの面積又は高さに基いて該目的成分を定量するデータ解析部と、
を備える。 In addition, one embodiment of the blood concentration measuring device for fat-soluble vitamins according to the present invention, which has been made to solve the above problems, includes vitamin D metabolites containing 24,25-dihydroxyvitamin D 3 in a blood sample, and a device for measuring the concentration of target components, which are vitamin K and its metabolites, including vitamin K1 ,
a protein removal processing unit that removes proteins from the blood sample by sequentially adding an organic solvent to the sample, stirring, and filtering the sample;
The sample processed by the protein removal processing unit is supplied to the solid-phase extraction column, and the target component in the sample is retained in the solid-phase extraction column, and then a mobile phase is supplied to extract the target component in the solid-phase extraction column. an online solid-phase extraction section for elution from a column for
A liquid chromatograph-tandem mass spectrometer having an ion source using an electrospray ionization method, which temporally separates components in a sample solution containing target components eluted from the solid phase extraction column, and a measurement execution unit that executes LC/MS analysis that selectively detects monovalent protonated ions originating from each target component;
a data analysis unit that creates a chromatogram for each target component using the data obtained by the measurement execution unit and quantifies the target component based on the area or height of a peak observed in the chromatogram; ,
Equipped with
本発明者は、ビタミンＤ代謝物の中で特に検出が難しい２４，２５－ジヒドロキシビタミンＤ3を高感度で検出するための手法について様々な実験を繰り返し、低感度の大きな要因の一つが、除タンパク後に血液サンプルに残存する脂質などの夾雑物によるイオンサプレッション効果であるとの知見を得た。これに対し、夾雑物を除去するための様々な方法を実験的に検証し、固相抽出用カラムを用いた固相抽出を用いることで、イオンサプレッションに特に寄与している夾雑物を効果的に且つ安定的に（高い再現性を以て）除去し得ることを見出し、本発明を得るに至った。
The present inventor has repeatedly conducted various experiments on a method for detecting 24,25-dihydroxyvitamin D3 with high sensitivity, which is particularly difficult to detect among vitamin D metabolites, and has found that one of the major factors for the low sensitivity is the removal of 24,25-dihydroxyvitamin D. It was discovered that this was due to the ion suppression effect due to impurities such as lipids remaining in the blood sample after protein. In response, we experimentally verified various methods for removing impurities, and by using solid-phase extraction using a solid-phase extraction column, we found that we could effectively remove the impurities that particularly contribute to ion suppression. The present inventors have discovered that it can be removed stably (with high reproducibility) and have achieved the present invention.
本発明に係る脂溶性ビタミン類の血中濃度測定方法及び測定装置の上記態様によれば、変性法による除タンパク処理の後に血液サンプル中に残存する夾雑物に起因するイオンサプレッション効果を抑制することができ、目的成分のイオン化効率を高めることができる。それにより、煩雑で自動化が難しい、クックソン型誘導体化試薬等を用いた誘導体化処理を行うことなく、血中のビタミンＤ代謝物やビタミンＫ及びその代謝物を高い感度で以て且つ同時に測定することができる。
According to the above aspects of the method and apparatus for measuring blood concentration of fat-soluble vitamins according to the present invention, it is possible to suppress the ion suppression effect caused by impurities remaining in a blood sample after protein removal treatment by a denaturation method. It is possible to improve the ionization efficiency of the target component. As a result, vitamin D metabolites, vitamin K, and their metabolites in blood can be simultaneously measured with high sensitivity without performing derivatization using Cookson-type derivatization reagents, which are complicated and difficult to automate. be able to.
また、本発明に係る脂溶性ビタミン類の血中濃度測定方法及び測定装置の上記態様によれば、オンライン固相抽出した目的成分を含む試料液をそのままＬＣ／ＭＳ分析することができるので、除タンパク処理から測定までの一連の作業を自動化することが容易である。それによって、測定のスループット向上と測定の安定性を図ることができ、スクリーニング検査などに好適に用いることができる。
Furthermore, according to the above-mentioned aspects of the blood concentration measuring method and measuring device for fat-soluble vitamins according to the present invention, the sample liquid containing the target component subjected to online solid-phase extraction can be directly analyzed by LC/MS. It is easy to automate a series of operations from protein processing to measurement. Thereby, it is possible to improve the measurement throughput and the measurement stability, and it can be suitably used for screening tests and the like.
［手段の例示］
本発明において、血液サンプルとは例えば、全血、血清、血漿などとすることができる。なお、血液サンプルはヒト以外の動物から採取されたものでもよい。 [Example of means]
In the present invention, the blood sample can be, for example, whole blood, serum, plasma, etc. Note that the blood sample may be collected from an animal other than humans.
本発明において、血液サンプルとは例えば、全血、血清、血漿などとすることができる。なお、血液サンプルはヒト以外の動物から採取されたものでもよい。 [Example of means]
In the present invention, the blood sample can be, for example, whole blood, serum, plasma, etc. Note that the blood sample may be collected from an animal other than humans.
本発明において、ビタミンＤ代謝物とは、少なくとも２４，２５－ジヒドロキシビタミンＤ3を含み、それ以外に、２５－ヒドロキシビタミンＤ2、２５－ヒドロキシビタミンＤ3などを含むものとすることができる。一方、本発明において、ビタミンＫ及びその代謝物とは、少なくともビタミンＫ1を含み、それ以外に、ビタミンＫ2ＭＫ－４、ビタミンＫ2ＭＫ－７などを含むものとすることができる。
In the present invention, vitamin D metabolites include at least 24,25-dihydroxyvitamin D 3 and may also include 25-hydroxyvitamin D 2 , 25-hydroxyvitamin D 3 and the like. On the other hand, in the present invention, vitamin K and its metabolites include at least vitamin K 1 and may also include vitamin K 2 MK-4, vitamin K 2 MK-7, and the like.
本発明において、変性法による除タンパク処理は、有機溶媒を血液サンプルに添加することでタンパクを不溶化して沈澱させる方法であり、有機溶媒としては例えばメタノール、エタノール、アセトン、アセトニトリルなどを用いることができる。
In the present invention, protein removal treatment using a denaturation method is a method in which proteins are insolubilized and precipitated by adding an organic solvent to a blood sample. Examples of the organic solvent include methanol, ethanol, acetone, and acetonitrile. can.
本発明において、固相抽出法は例えば、まず固相抽出用カラムへサンプルを供給して該カラムの充填剤に目的成分を吸着させる。このとき、充填剤に吸着しない成分は排出される。その後、固相抽出用カラムに溶出作用を有する移動相を供給し、該カラムに吸着されていた目的成分を溶出させて試料液として取り出しそのまま液体クロマトグラフのカラムに送る。こうした一連の操作は、主としてバルブによる流路の切り替えによって実行することができ、それを自動的に順次実行するのがオンライン固相抽出である。なお、固相抽出用カラムに目的成分を吸着させたあと、該固相抽出用カラムに洗浄液を流通させ、該カラムに吸着されなかったものの該カラムを含む配管中に残存している成分を排出する操作を加えてもよい。
In the present invention, in the solid phase extraction method, for example, a sample is first supplied to a column for solid phase extraction, and the target component is adsorbed on the packing material of the column. At this time, components that are not adsorbed to the filler are discharged. Thereafter, a mobile phase having an elution effect is supplied to the column for solid phase extraction, and the target component adsorbed on the column is eluted and taken out as a sample solution and sent as it is to a column of a liquid chromatograph. A series of such operations can be performed mainly by switching the flow paths using valves, and online solid-phase extraction is an automatic sequential execution of these operations. After adsorbing the target component on the solid phase extraction column, a cleaning solution is passed through the solid phase extraction column to discharge components that were not adsorbed by the column but remain in the piping containing the column. You may also add operations to do so.
本発明において、液体クロマトグラフ－タンデム型質量分析装置のタンデム型質量分析装置は、エレクトロスプレーイオン源を搭載した、例えばトリプル四重極型質量分析装置、四重極－飛行時間型質量分析装置、イオントラップ－飛行時間型質量分析装置、イオントラップ型質量分析装置などとすることができる。
In the present invention, the tandem mass spectrometer of the liquid chromatograph-tandem mass spectrometer includes an electrospray ion source, for example, a triple quadrupole mass spectrometer, a quadrupole-time-of-flight mass spectrometer, It can be an ion trap-time-of-flight mass spectrometer, an ion trap mass spectrometer, or the like.
また、タンデム型質量分析装置は、イオン源で生成されたイオンを解離させる機能を有するが、そのイオン解離の手法は特に限定されない。典型的には、そのイオン解離の手法として衝突誘起解離を用いることができる。
Further, the tandem mass spectrometer has a function of dissociating ions generated by the ion source, but the method of ion dissociation is not particularly limited. Typically, collision-induced dissociation can be used as a method for the ion dissociation.
本発明において、目的成分それぞれに由来する１価のプロトン付加イオンを選択的に検出する分析とは、典型的には、イオン解離によって生成される１価のプロトン付加プロダクトイオンをターゲットとする多重反応モニタリング（ＭＲＭ）測定である。一般的にＭＲＭ測定はトリプル四重極型質量分析装置で実施されるが、四重極－飛行時間型質量分析装置のように所定のm/z値範囲に亘るプロダクトイオンスペクトルが得られる場合には、そのスペクトルデータから特定の質量電荷比（m/z）を有するイオンの強度を得ることができるから、これは実質的にＭＲＭ測定であるとみなすことができる。
In the present invention, analysis that selectively detects monovalent protonated ions derived from each target component typically involves multiple reactions targeting monovalent protonated product ions generated by ion dissociation. This is a monitoring (MRM) measurement. Generally, MRM measurements are performed with a triple quadrupole mass spectrometer, but when a product ion spectrum covering a predetermined m/z value range can be obtained, such as with a quadrupole-time-of-flight mass spectrometer, Since the intensity of ions with a specific mass-to-charge ratio (m/z) can be obtained from the spectral data, this can essentially be considered an MRM measurement.
また、本発明に係る脂溶性ビタミン類の血中濃度測定装置は、具体的には、例えば島津製作所製の全自動ＬＣＭＳ前処理装置「CLAM-2030」、島津製作所製の高速液体クロマトグラフ「Nexeraシリーズ」、島津製作所製の液体クロマトグラフ質量分析計「LCMS-8060NX」、及びそれらを制御すると共にデータ処理を行う所定のソフトウェアが搭載されたパーソナルコンピューター、などを組み合わせて構成することができる。その場合、高速液体クロマトグラフのユニット構成を、オンライン固相抽出に対応した構成とすればよい。勿論、本発明に係る血中濃度測定装置はそれ以外の様々な装置やユニットを用いて構成され得る。
Further, the blood concentration measuring device for fat-soluble vitamins according to the present invention includes, for example, a fully automatic LCMS pretreatment device “CLAM-2030” manufactured by Shimadzu Corporation, and a high-performance liquid chromatograph “Nexera” manufactured by Shimadzu Corporation. LCMS-8060NX, a liquid chromatograph mass spectrometer manufactured by Shimadzu Corporation, and a personal computer equipped with predetermined software that controls them and processes data. In that case, the unit configuration of the high performance liquid chromatograph may be configured to be compatible with online solid phase extraction. Of course, the blood concentration measuring device according to the present invention can be configured using various other devices and units.
［一実施形態の測定方法及び測定装置］
以下、本発明に係る血中濃度測定方法及び測定装置の一実施形態について、添付図面を参照して説明する。 [Measuring method and measuring device of one embodiment]
EMBODIMENT OF THE INVENTION Hereinafter, one embodiment of the blood concentration measuring method and measuring device according to the present invention will be described with reference to the accompanying drawings.
以下、本発明に係る血中濃度測定方法及び測定装置の一実施形態について、添付図面を参照して説明する。 [Measuring method and measuring device of one embodiment]
EMBODIMENT OF THE INVENTION Hereinafter, one embodiment of the blood concentration measuring method and measuring device according to the present invention will be described with reference to the accompanying drawings.
＜測定方法の概略＞
図１は、本実施形態による血中濃度測定方法の処理手順の一例を示すフローチャートである。図１に従って、本実施形態の測定方法を概略的に説明する。
この例では、サンプルは被検者から採取した血液から取り出した血清である。また、この例において測定対象である目的成分は、２４，２５－ジヒドロキシビタミンＤ3、２５－ヒドロキシビタミンＤ2、２５－ヒドロキシビタミンＤ3、ビタミンＫ1、ビタミンＫ2ＭＫ－４、ビタミンＫ2ＭＫ－７の６種類である。 <Outline of measurement method>
FIG. 1 is a flowchart showing an example of the processing procedure of the blood concentration measuring method according to the present embodiment. The measurement method of this embodiment will be schematically explained according to FIG.
In this example, the sample is serum extracted from blood drawn from a subject. In this example, the target components to be measured are 24,25-dihydroxyvitamin D 3 , 25-hydroxyvitamin D 2 , 25-hydroxyvitamin D 3 , vitamin K 1 , vitamin K 2 MK-4, vitamin K 2 There are 6 types of MK-7.
図１は、本実施形態による血中濃度測定方法の処理手順の一例を示すフローチャートである。図１に従って、本実施形態の測定方法を概略的に説明する。
この例では、サンプルは被検者から採取した血液から取り出した血清である。また、この例において測定対象である目的成分は、２４，２５－ジヒドロキシビタミンＤ3、２５－ヒドロキシビタミンＤ2、２５－ヒドロキシビタミンＤ3、ビタミンＫ1、ビタミンＫ2ＭＫ－４、ビタミンＫ2ＭＫ－７の６種類である。 <Outline of measurement method>
FIG. 1 is a flowchart showing an example of the processing procedure of the blood concentration measuring method according to the present embodiment. The measurement method of this embodiment will be schematically explained according to FIG.
In this example, the sample is serum extracted from blood drawn from a subject. In this example, the target components to be measured are 24,25-dihydroxyvitamin D 3 , 25-hydroxyvitamin D 2 , 25-hydroxyvitamin D 3 , vitamin K 1 , vitamin K 2 MK-4, vitamin K 2 There are 6 types of MK-7.
まず、血液サンプルに対し、前処理として除タンパク処理が実行される（ステップＳ１）。除タンパクの方法としては、例えば水と混和可能である極性有機溶媒を用いた変性法を用いることができる。極性有機溶媒としては、メタノール、エタノール、アセトニトリルなどを用いることができる。除タンパク処理においては、処理対象の血液サンプルに所定の極性有機溶媒を添加し、撹拌したあとに濾過フィルター等を用いた濾過を行う。これにより、変性によって析出したタンパク質を除去し、上清を回収することで除タンパク済みの血液サンプルを得る。なお、血液サンプルに有機溶媒を添加する際に、併せて検量のための内部標準物質を添加することができる。
First, a protein removal process is performed on a blood sample as a preprocess (step S1). As a method for protein removal, for example, a denaturation method using a polar organic solvent that is miscible with water can be used. As the polar organic solvent, methanol, ethanol, acetonitrile, etc. can be used. In protein removal treatment, a predetermined polar organic solvent is added to a blood sample to be treated, stirred, and then filtered using a filter or the like. Thereby, proteins precipitated by denaturation are removed, and the supernatant is collected to obtain a protein-depleted blood sample. Note that when adding the organic solvent to the blood sample, an internal standard substance for calibration can also be added.
次に、オンライン固相抽出法により、除タンパク済みの血液サンプルに含まれる脂質等の夾雑物が除去される（ステップＳ２）。具体的には、該血液サンプルは所定の移動相を用いて事前にコンディショニングされた固相抽出用カラムに供給され、血液サンプル中の目的成分が固相抽出用カラムの充填剤に捕集される。血液サンプル中の夾雑物の多くは充填剤に捕集されないため、固相抽出用カラムを通り抜けて排出される。一部の夾雑物は、充填剤に吸着されないものの充填剤に付着したり配管内部に付着したりする可能性がある。そこで、目的成分に影響を与えないリンス液を固相抽出用カラムを含む配管に流すことにより、残存している夾雑物を洗い流すようにしてもよい。
Next, impurities such as lipids contained in the protein-removed blood sample are removed by online solid-phase extraction (step S2). Specifically, the blood sample is supplied to a solid-phase extraction column that has been conditioned in advance using a predetermined mobile phase, and the target components in the blood sample are collected on the packing material of the solid-phase extraction column. . Many of the contaminants in the blood sample are not captured by the packing material and are therefore discharged through the solid phase extraction column. Although some impurities are not adsorbed by the filler, there is a possibility that they may adhere to the filler or adhere to the inside of the pipe. Therefore, the remaining impurities may be washed away by flowing a rinsing liquid that does not affect the target component into the pipe containing the solid phase extraction column.
引き続いて、溶出作用を有する移動相を固相抽出用カラムに流し、該カラムに吸着されている目的成分をまとめて溶出させ、その目的成分を含む試料液を移動相の流れに乗せてＬＣのカラムに送り込む。試料液中の目的成分はカラムを通過する間に、時間方向に分離される（ステップＳ３）。
Subsequently, a mobile phase with an elution effect is passed through a solid phase extraction column, the target components adsorbed on the column are eluted all at once, and the sample solution containing the target components is carried on the flow of the mobile phase and is then run on an LC. feed into the column. The target components in the sample liquid are separated in the time direction while passing through the column (step S3).
分離された目的成分を含むカラムからの溶出液は、タンデム型質量分析装置のイオン源に導入される。イオン源はＥＳＩ法を利用したものである。血液サンプルに含まれる目的成分以外の夾雑物は、ＥＳＩ法によるイオン化の際にイオンサプレッション効果を生じる。これに対し、本測定方法では、ステップＳ２において効果的に夾雑物が除去されるため、夾雑物によるイオンサプレッション効果を抑制し、目的成分のイオン化効率を高めることができる。それによって、目的成分由来のイオンをより多く質量分析に供することができる。タンデム型質量分析装置では、目的成分毎に、その目的成分がカラムから溶出する（つまりはイオン源に導入される）期間にその目的成分に対応して予め決められている一又は複数のＭＲＭトランジション（プリカーサーイオンとプロダクトイオンのm/z値の組合せ）をターゲットとするＭＲＭ測定（ＭＳ／ＭＳ分析）が実行される（ステップＳ４）。
The eluate from the column containing the separated target components is introduced into the ion source of the tandem mass spectrometer. The ion source utilizes the ESI method. Contaminants other than target components contained in a blood sample produce an ion suppression effect during ionization by the ESI method. In contrast, in this measurement method, since impurities are effectively removed in step S2, the ion suppression effect due to impurities can be suppressed and the ionization efficiency of the target component can be increased. Thereby, more ions derived from the target component can be subjected to mass spectrometry. In a tandem mass spectrometer, one or more MRM transitions are predetermined for each target component during the period during which the target component is eluted from the column (that is, introduced into the ion source). MRM measurement (MS/MS analysis) targeting (combination of m/z values of precursor ions and product ions) is performed (step S4).
ステップＳ３、Ｓ４での測定により、目的成分毎に、その保持時間を中心とする所定の時間範囲における一又は複数のＭＲＭトランジションについてのイオン強度データが得られる。上述したように、本測定方法では、質量分析に供される目的成分由来のイオンの量が相対的に多いため、高い感度で且つＳＮ比が良好なデータが得られる。このデータに基く定量解析が実施されることで、血液サンプル中の各目的成分の濃度が算出される（ステップＳ５）。具体的には、目的成分毎に抽出イオンクロマトグラムが作成され、各クロマトグラムにおいて目的成分に対応するクロマトピークが特定される。そして、このクロマトピークの面積又は高さが算出され、予め作成された検量線を利用して各目的成分の濃度が算出される。
Through the measurements in steps S3 and S4, ion intensity data regarding one or more MRM transitions in a predetermined time range centered around the retention time is obtained for each target component. As described above, in this measurement method, since the amount of ions derived from the target component subjected to mass spectrometry is relatively large, data with high sensitivity and a good signal-to-noise ratio can be obtained. By performing quantitative analysis based on this data, the concentration of each target component in the blood sample is calculated (step S5). Specifically, an extracted ion chromatogram is created for each target component, and a chromatographic peak corresponding to the target component is identified in each chromatogram. Then, the area or height of this chromatographic peak is calculated, and the concentration of each target component is calculated using a calibration curve created in advance.
以上のようにして、本測定方法では、血液サンプルに含まれる６種類の目的成分の濃度を測定結果として得ることができる。
As described above, in this measurement method, the concentrations of six types of target components contained in a blood sample can be obtained as measurement results.
＜装置の構成＞
次に、上記概説した測定方法を実施する装置の構成と動作について説明する。
図２は、本実施形態による脂溶性ビタミン類血中濃度測定装置の概略ブロック構成図である。図２に示すように、この測定装置は、液体クロマトグラフ部（ＬＣ部）１、タンデム型質量分析部（ＭＳ／ＭＳ部）２、オンライン固相抽出（ＳＰＥ）部３、除タンパク処理部４、電圧発生部５、データ処理部６、制御部７、操作部８、及び表示部９、を含む。 <Device configuration>
Next, the configuration and operation of an apparatus that implements the measurement method outlined above will be explained.
FIG. 2 is a schematic block diagram of the fat-soluble vitamin blood concentration measuring device according to this embodiment. As shown in FIG. 2, this measurement device includes a liquid chromatograph section (LC section) 1, a tandem mass spectrometry section (MS/MS section) 2, an online solid phase extraction (SPE)section 3, and a protein removal processing section 4. , a voltage generation section 5, a data processing section 6, a control section 7, an operation section 8, and a display section 9.
次に、上記概説した測定方法を実施する装置の構成と動作について説明する。
図２は、本実施形態による脂溶性ビタミン類血中濃度測定装置の概略ブロック構成図である。図２に示すように、この測定装置は、液体クロマトグラフ部（ＬＣ部）１、タンデム型質量分析部（ＭＳ／ＭＳ部）２、オンライン固相抽出（ＳＰＥ）部３、除タンパク処理部４、電圧発生部５、データ処理部６、制御部７、操作部８、及び表示部９、を含む。 <Device configuration>
Next, the configuration and operation of an apparatus that implements the measurement method outlined above will be explained.
FIG. 2 is a schematic block diagram of the fat-soluble vitamin blood concentration measuring device according to this embodiment. As shown in FIG. 2, this measurement device includes a liquid chromatograph section (LC section) 1, a tandem mass spectrometry section (MS/MS section) 2, an online solid phase extraction (SPE)
ＬＣ部１は、移動相貯留部１０、送液ポンプ１１、インジェクター１２、カラム１３、を含む。ここでは図面が煩雑になるのを避けるため記載を省略しているが、後述する実測例では、異なる２種類の移動相を用いたグラジエント溶離を行っている。グラジエント溶離を実施するためには、別の移動相貯留部、送液ポンプ、ミキサーなどが図２の構成に追加される。
The LC section 1 includes a mobile phase storage section 10, a liquid feeding pump 11, an injector 12, and a column 13. Although the description is omitted here to avoid complicating the drawing, in the actual measurement example described later, gradient elution using two different types of mobile phases was performed. To perform gradient elution, another mobile phase reservoir, liquid pump, mixer, etc. are added to the configuration of FIG. 2.
カラム１３としては例えば、ＰＦＰＰ（Pentafluorophenylpropyl）カラムを用いることができる。ＰＦＰＰカラムは、ＰＦＰＰ基を導入したカラムであり、疎水性化合物のみならず親水性化合物にも良好な分離性能を示す逆相カラムとして、近年、生体由来成分の分離にしばしば利用されている。
As the column 13, for example, a PFPP (Pentafluorophenylpropyl) column can be used. A PFPP column is a column into which a PFPP group is introduced, and has been frequently used in recent years to separate biological components as a reversed-phase column that exhibits good separation performance not only for hydrophobic compounds but also for hydrophilic compounds.
ＭＳ／ＭＳ部２は、ＥＳＩイオン源を有するトリプル四重極型質量分析装置である。詳しくは、図２に示すように、チャンバー２０の内部は四つに区画され、ＬＣ部１側から順に、イオン化室２０１、第１中間真空室２０２、第２中間真空室２０３、高真空室２０４となっている。第１中間真空室２０２はロータリーポンプ（図示せず）により真空排気され、低真空雰囲気（一例として１０2[Pa]程度）に維持される。第２中間真空室２０３はロータリーポンプ及びターボ分子ポンプ（ともに図示せず）により真空排気され、中真空雰囲気（一例として１０-1～１０-2[Pa]程度）に維持される。高真空室２０４もロータリーポンプ及びターボ分子ポンプ（ともに図示せず）により真空排気され、高真空雰囲気（一例として１０-3～１０-4[Pa]程度）に維持される。このように、略大気圧雰囲気であるイオン化室２０１から順に真空度が高まる多段差動排気系の構成が採られている。
The MS/MS section 2 is a triple quadrupole mass spectrometer having an ESI ion source. Specifically, as shown in FIG. 2, the interior of the chamber 20 is divided into four parts, which are, in order from the LC section 1 side, an ionization chamber 201, a first intermediate vacuum chamber 202, a second intermediate vacuum chamber 203, and a high vacuum chamber 204. It becomes. The first intermediate vacuum chamber 202 is evacuated by a rotary pump (not shown) and maintained at a low vacuum atmosphere (about 10 2 [Pa] as an example). The second intermediate vacuum chamber 203 is evacuated by a rotary pump and a turbomolecular pump (both not shown) and maintained at a medium vacuum atmosphere (for example, about 10 -1 to 10 -2 [Pa]). The high vacuum chamber 204 is also evacuated by a rotary pump and a turbomolecular pump (both not shown) and maintained at a high vacuum atmosphere (about 10 -3 to 10 -4 [Pa] as an example). In this way, a multi-stage differential pumping system configuration is adopted in which the degree of vacuum increases sequentially starting from the ionization chamber 201, which is in an atmosphere of approximately atmospheric pressure.
イオン化室２０１内にはＥＳＩスプレー２１が配置され、イオン化室２０１と第１中間真空室２０２とは細径の脱溶媒管２２により連通している。第１中間真空室２０２の内部には、ＲＦ（Radio Frequency）電場の作用によりイオンを収束させつつ輸送する第１ＲＦイオンガイド２３が配置されており、第１中間真空室２０２と第２中間真空室２０３とはスキマー２４の頂部に形成された小径のイオン通過孔により連通している。第１ＲＦイオンガイド（下記のような構造上の特徴から、以下「Ｑアレー」という場合がある）２３は、イオン光軸Ｃを取り囲むように略等角度間隔で配置された複数（この例では４本）のロッド電極からなり、各ロッド電極はイオン光軸Ｃの延伸方向に複数に分離された短い円柱状（又は円盤状）の部分電極から成る構造である。その分離された複数の部分電極のうち、スキマー２４側に位置する部分電極はイオン光軸Ｃを中心とする内接円の半径が小さくなっており、これによって、イオンを効率良くイオン光軸Ｃ付近に収束させるようにしている。
An ESI spray 21 is arranged inside the ionization chamber 201, and the ionization chamber 201 and the first intermediate vacuum chamber 202 are communicated through a small diameter desolvation tube 22. Inside the first intermediate vacuum chamber 202, a first RF ion guide 23 that transports ions while converging them by the action of an RF (Radio Frequency) electric field is arranged, and the first intermediate vacuum chamber 202 and the second intermediate vacuum chamber 203 through a small diameter ion passage hole formed at the top of the skimmer 24. The first RF ion guide (hereinafter sometimes referred to as a "Q array" due to the structural characteristics described below) 23 includes a plurality of first RF ion guides (in this example, four Each rod electrode is composed of a plurality of short cylindrical (or disc-shaped) partial electrodes separated in the direction in which the ion optical axis C extends. Among the plurality of separated partial electrodes, the partial electrode located on the skimmer 24 side has a small radius of an inscribed circle centered on the ion optical axis C, thereby efficiently transferring ions to the ion optical axis C. I'm trying to have it converge nearby.
第２中間真空室２０３の内部には、イオン光軸Ｃを取り囲んで略等角度間隔に、且つ該イオン光軸Ｃに平行に配置された複数本（例えば８本）のロッド電極からなる第２ＲＦイオンガイド２５が配置されている。第２中間真空室２０３と高真空室２０４とは小径のイオン通過孔により連通している。
Inside the second intermediate vacuum chamber 203, a second RF electrode is provided, which is composed of a plurality of rod electrodes (for example, eight rods) surrounding the ion optical axis C, arranged at approximately equal angular intervals, and parallel to the ion optical axis C. An ion guide 25 is arranged. The second intermediate vacuum chamber 203 and the high vacuum chamber 204 communicate with each other through a small diameter ion passage hole.
高真空室２０４の内部には、イオン光軸Ｃに沿って順に、前段四重極マスフィルター２６、コリジョンセル２７、後段四重極マスフィルター２８、及びイオン検出器２９、が配置されている。前段四重極マスフィルター２６及び後段四重極マスフィルター２８はいずれも、イオン光軸Ｃを取り囲んで略等角度間隔に、且つ該イオン光軸Ｃに平行に配置された４本のロッド電極を含む。ここでは、それぞれメインロッド電極の前段にプリロッド電極を設けているが、これは省くことができる。また、必要に応じてメインロッド電極の後段にポストロッド電極を設けてもよい。コリジョンセル２７の内部には、イオン光軸Ｃを取り囲んで略等角度間隔に、且つ該イオン光軸Ｃに平行に複数本のロッド電極からなるイオンガイドが配置されている。また、コリジョンセル２７の内部には、外部からアルゴンなどの不活性ガスであるＣＩＤガスが導入されるようになっている。
Inside the high vacuum chamber 204, a front quadrupole mass filter 26, a collision cell 27, a rear quadrupole mass filter 28, and an ion detector 29 are arranged in order along the ion optical axis C. Both the front-stage quadrupole mass filter 26 and the rear-stage quadrupole mass filter 28 include four rod electrodes that surround the ion optical axis C and are arranged at approximately equal angular intervals and parallel to the ion optical axis C. include. Here, a pre-rod electrode is provided before each main rod electrode, but this can be omitted. Further, a post rod electrode may be provided after the main rod electrode if necessary. Inside the collision cell 27, an ion guide consisting of a plurality of rod electrodes is arranged surrounding the ion optical axis C at approximately equal angular intervals and parallel to the ion optical axis C. Furthermore, CID gas, which is an inert gas such as argon, is introduced into the collision cell 27 from the outside.
オンラインＳＰＥ部３は実質的にＬＣ部１の前段に設けられ、移動相貯留部３０、送液ポンプ３１、インジェクター１２、固相抽出用カラム３３、複数のバルブを含む流路切替部３４、を含む。インジェクター１２はＬＣ部１に含まれるものが用いられる。固相抽出用カラム３３としては例えば、ＯＤＳ（Octa Decyl Silyl）基（Ｃ18Ｈ37Ｓｉ）で表面が修飾された化学結合型多孔性球状シリカゲルが固定相として充填されているＯＤＳカラムを用いることができる。
The online SPE section 3 is substantially provided upstream of the LC section 1, and includes a mobile phase storage section 30, a liquid feeding pump 31, an injector 12, a solid phase extraction column 33, and a flow path switching section 34 including a plurality of valves. include. As the injector 12, one included in the LC section 1 is used. As the column 33 for solid phase extraction, for example, an ODS column packed with chemically bonded porous spherical silica gel whose surface is modified with ODS (Octa Decyl Silyl) groups (C 18 H 37 Si) as a stationary phase can be used. I can do it.
除タンパク処理部４は、オンラインＳＰＥ部３のさらに前段に設けられている。除タンパク処理部４の実体は例えば、バイアルに収容されたサンプルに対し、サンプルの分注、試薬の分注、撹拌、濾過、加温、インジェクター１２によるサンプル採取位置への搬送、などの各種の操作や処理を実行可能である試料前処理装置とすることができる。こうした試料前処理装置において、予め用意された所定の試薬を用い、予め設定された条件に従って各操作や処理を実行することで、除タンパク処理を実施することができる。例えば、上述した島津製作所製の全自動ＬＣＭＳ前処理装置「CLAM-2030」などを用いることで除タンパク処理部４を具現化することができる。
The protein removal processing section 4 is provided further upstream of the online SPE section 3. The protein removal processing unit 4 performs various operations on the sample contained in a vial, such as dispensing the sample, dispensing a reagent, stirring, filtering, heating, and transporting the sample to the sample collection position using the injector 12. It can be a sample pretreatment device that can perform operations and treatments. In such a sample pretreatment device, protein removal treatment can be performed by using predetermined reagents prepared in advance and performing various operations and treatments according to preset conditions. For example, the protein removal processing section 4 can be implemented by using the above-mentioned fully automatic LCMS pretreatment device "CLAM-2030" manufactured by Shimadzu Corporation.
電圧発生部５は制御部７の制御の下で、ＭＳ／ＭＳ部２の各部に所定の電圧を印加する。この電圧は、ＲＦ電圧、ＲＦ電圧よりも周波数が低いＡＣ電圧、ＤＣ電圧のいずれか、又はその組合せである。
The voltage generation section 5 applies a predetermined voltage to each part of the MS/MS section 2 under the control of the control section 7. This voltage is either an RF voltage, an AC voltage with a lower frequency than the RF voltage, a DC voltage, or a combination thereof.
データ処理部６は、イオン検出器２９から検出信号を受けて処理するものであり、アナログデジタル変換部やデータ記憶部を含むデータ収集部６０と、定量演算部６１と、を機能ブロックとして含む。
The data processing unit 6 receives and processes a detection signal from the ion detector 29, and includes a data collection unit 60 including an analog-to-digital conversion unit and a data storage unit, and a quantitative calculation unit 61 as functional blocks.
制御部７は、除タンパク処理部４、オンラインＳＰＥ部３、ＬＣ部１、電圧発生部５、ＭＳ／ＭＳ部２、データ処理部６をそれぞれ制御することにより、血液サンプルに対する前処理から測定、さらにはデータ解析までの一連の処理を測定装置に実行させるものである。また、制御部７は、ユーザーインターフェイスである操作部８及び表示部９を通した入出力制御の機能も有する。
The control unit 7 controls the protein removal processing unit 4, the online SPE unit 3, the LC unit 1, the voltage generation unit 5, the MS/MS unit 2, and the data processing unit 6, thereby performing preprocessing, measurement, and measurement of the blood sample. Furthermore, it causes the measuring device to execute a series of processes up to data analysis. The control unit 7 also has an input/output control function through an operation unit 8 and a display unit 9, which are user interfaces.
本測定装置において、データ処理部６及び制御部７はパーソナルコンピューターをハードウェア資源とし、該コンピューターに予めインストールされた専用のデータ処理・制御用のソフトウェアを該コンピューター上で実行することにより、それぞれの機能を実現するものとすることができる。そのデータ処理・制御用のソフトウェアは、例えば測定目的や測定対象の成分を限定することなく一般的な測定を行うためのソフトウェア（但し、このソフトウェア自体が複数のソフトウェアであってもよい）と、ここで測定対象としているビタミンＤ代謝物等を測定するための専用のソフトウェアと、を含む。図２では、この後者のソフトウェアを、ビタミンＤ／Ｋ血中濃度測定用プログラム７０として示している。
In this measuring device, the data processing section 6 and the control section 7 use a personal computer as a hardware resource, and each of them is The function can be realized. The data processing/control software includes, for example, software for performing general measurements without limiting the purpose of measurement or the components to be measured (however, this software itself may consist of multiple pieces of software); It includes dedicated software for measuring vitamin D metabolites, etc., which are the measurement targets here. In FIG. 2, this latter software is shown as a vitamin D/K blood concentration measurement program 70.
＜測定実行における装置の動作＞
図２に示した測定装置において、上述した測定方法を実行する際の動作を説明する。
まずユーザーは、検体である血液サンプルを除タンパク処理部４の所定位置にセットし、操作部８で所定の操作を実行する。この操作を受けて制御部７は、一連の測定を実行するために各部を動作させる。 <Operation of the device during measurement execution>
The operation of the measuring device shown in FIG. 2 when performing the above-mentioned measuring method will be described.
First, the user sets a blood sample, which is a specimen, at a predetermined position in the proteinremoval processing section 4, and executes a predetermined operation using the operation section 8. In response to this operation, the control section 7 operates each section to perform a series of measurements.
図２に示した測定装置において、上述した測定方法を実行する際の動作を説明する。
まずユーザーは、検体である血液サンプルを除タンパク処理部４の所定位置にセットし、操作部８で所定の操作を実行する。この操作を受けて制御部７は、一連の測定を実行するために各部を動作させる。 <Operation of the device during measurement execution>
The operation of the measuring device shown in FIG. 2 when performing the above-mentioned measuring method will be described.
First, the user sets a blood sample, which is a specimen, at a predetermined position in the protein
まず、除タンパク処理部４は、血液サンプルの分注、該血液サンプルへの試薬の分注、撹拌、濾過等の操作を順に実行し、タンパクが除去された血液サンプルを得る。多数の血液サンプルを処理する場合、この除タンパク処理とそれ以降のオンラインＳＰＥを含む測定動作とは並行して実行され得る。処理済みの血液サンプルが収容された容器は、所定のタイミングでインジェクター１２によるサンプル採取位置へと搬送される。
First, the protein removal processing unit 4 sequentially performs operations such as dispensing a blood sample, dispensing a reagent to the blood sample, stirring, and filtration to obtain a blood sample from which proteins have been removed. When processing a large number of blood samples, this protein removal process and subsequent measurement operations including on-line SPE can be performed in parallel. The container containing the processed blood sample is transported to a sample collection position by the injector 12 at a predetermined timing.
オンラインＳＰＥ部３では、送液ポンプ３１が移動相貯留部３０から移動相を吸引し、流路切替部３４を通して固相抽出用カラム３３へと送る。所定のタイミングでインジェクター１２は、除タンパク処理済みの血液サンプルを移動相中に注入する。注入されたサンプルは固相抽出用カラム３３に供給され、該サンプルに含まれる主として目的成分は、固相抽出用カラム３３の充填剤に吸着される。他方、サンプルに含まれる夾雑物の多くは、充填剤に吸着されないために該カラム３３を通り抜け、流路切替部３４を経て排出される。これにより、目的成分が固相抽出用カラム３３に捕集される。
In the online SPE unit 3, the liquid pump 31 sucks the mobile phase from the mobile phase storage unit 30 and sends it to the solid phase extraction column 33 through the flow path switching unit 34. At a predetermined timing, the injector 12 injects the protein-removed blood sample into the mobile phase. The injected sample is supplied to the solid-phase extraction column 33, and the target components contained in the sample are mainly adsorbed on the packing material of the solid-phase extraction column 33. On the other hand, most of the impurities contained in the sample are not adsorbed by the packing material, so they pass through the column 33 and are discharged via the flow path switching section 34. Thereby, the target component is collected in the solid phase extraction column 33.
但し、夾雑物が充填剤に吸着はされなくても、夾雑物の一部は充填剤や配管内部に付着した状態で残存する場合がある。そこで、例えば送液ポンプ３１により図示しない洗浄液貯留部から洗浄液を吸引し、固相抽出用カラム３３を通して排液路へ流すようにしてもよい。これにより、充填剤や配管内部に付着していた夾雑物を洗い流し、目的成分の純粋性を高めることができる。なお、固相抽出用カラム３３に目的成分を捕集させる期間中には、図２では示されていない流路を用いて、送液ポンプ１１で吸引された移動相をカラム１３に流すようにするとよい。
However, even if the contaminants are not adsorbed by the filler, some of the contaminants may remain attached to the filler or inside the pipe. Therefore, for example, the cleaning liquid may be sucked from a cleaning liquid reservoir (not shown) using the liquid feeding pump 31, and may be caused to flow through the solid phase extraction column 33 to the drainage path. This makes it possible to wash away impurities adhering to the filler and inside the piping, thereby increasing the purity of the target component. Note that during the period in which the target component is collected in the solid-phase extraction column 33, the mobile phase sucked by the liquid pump 11 is caused to flow into the column 13 using a channel not shown in FIG. It's good to do that.
次いで、流路切替部３４により流路が切り替えられ、送液ポンプ１１により移動相貯留部１０から吸引された移動相が固相抽出用カラム３３に供給される。固相抽出用カラム３３に捕集されていた上記６種類の目的成分は移動相中に溶出し、移動相の流れに乗ってカラム１３に送り込まれる。略同時にカラム１３に導入された６種類の目的成分は、カラム１３を通過する間に時間方向に分離されて該カラム１３の出口から溶出する。このときの移動相の種類や移動相の流量等のＬＣ分析条件は、ビタミンＤ／Ｋ血中濃度測定用プログラム７０で規定されている。
Next, the flow path is switched by the flow path switching unit 34, and the mobile phase sucked from the mobile phase storage unit 10 by the liquid feeding pump 11 is supplied to the solid phase extraction column 33. The above-mentioned six target components collected in the solid phase extraction column 33 are eluted into the mobile phase and sent to the column 13 along with the flow of the mobile phase. The six target components introduced into the column 13 at approximately the same time are separated in the time direction while passing through the column 13 and eluted from the outlet of the column 13. LC analysis conditions such as the type of mobile phase and the flow rate of the mobile phase at this time are defined in the vitamin D/K blood concentration measurement program 70.
制御部７による制御の下で、ＭＳ／ＭＳ部２は、６種類の目的成分それぞれの保持時間を中心とする所定の時間範囲において、その目的成分に対応する一又は複数のＭＲＭトランジションをターゲットとするＭＲＭ測定を実行するように動作する。
Under the control of the control unit 7, the MS/MS unit 2 targets one or more MRM transitions corresponding to the target components in a predetermined time range centered on the retention time of each of the six types of target components. It operates to perform MRM measurements.
いま、一つの目的成分を含む溶出液がＬＣ部１からＭＳ／ＭＳ部２に導入されると、ＥＳＩスプレー２１において該溶出液に対して、極性が片寄った電荷が付与され、略大気圧であるイオン化室２１に噴霧される。噴霧された帯電液滴はガス分子に接触して微細化され、帯電液滴から溶媒が気化する過程で目的成分の分子は電荷を以て飛び出し気体イオンとなる。生成されたイオンは微細な帯電液滴と共に脱溶媒管２２中に吸い込まれ、第１中間真空室２０２に送られる。脱溶媒管２２は加熱されており、脱溶媒管２２中においても液滴からの気化が促進されるため、目的成分のイオン化が促進される。
Now, when an eluate containing one target component is introduced from the LC section 1 to the MS/MS section 2, the ESI spray 21 imparts an electric charge with biased polarity to the eluate, and the eluate is heated at approximately atmospheric pressure. It is sprayed into a certain ionization chamber 21. The sprayed charged droplets come into contact with gas molecules and become fine, and in the process of vaporizing the solvent from the charged droplets, molecules of the target component fly out with charge and become gas ions. The generated ions are sucked into the desolvation tube 22 together with fine charged droplets and sent to the first intermediate vacuum chamber 202. The desolvation tube 22 is heated, and vaporization from droplets is promoted in the desolvation tube 22 as well, so ionization of the target component is promoted.
第１中間真空室２０２内に入った目的成分由来のイオンは、電圧発生部５から第１ＲＦイオンガイド２３に印加されているＲＦ電圧によって形成されるＲＦ電場に捕捉され、イオン光軸Ｃ付近に収束される。イオン化室２０１の次段である第１中間真空室２０２内は真空度が低く、残留ガス分子が多いため、目的成分由来のイオンは残留ガス分子に接触し易い。その接触により目的成分由来イオンが持つ運動エネルギーが減衰するため、目的成分由来のイオンはＲＦ電場に捕捉され易く、また収束も良好に行われる。
Ions derived from the target component that have entered the first intermediate vacuum chamber 202 are captured by the RF electric field formed by the RF voltage applied from the voltage generator 5 to the first RF ion guide 23, and are moved near the ion optical axis C. Converged. The degree of vacuum inside the first intermediate vacuum chamber 202, which is the next stage of the ionization chamber 201, is low and there are many residual gas molecules, so ions derived from the target component easily come into contact with the residual gas molecules. Since the kinetic energy of the ions derived from the target component is attenuated by the contact, the ions derived from the target component are easily captured by the RF electric field, and convergence is also performed well.
よく知られているようにＥＳＩ法はソフトなイオン化法であり、イオン化に際してイオンの解離（開裂）が生じにくい。一方で、１価のプロトン付加イオンのみならず、多価イオンや、アルカリ金属などが付加したアダクトイオンが生成され易い。特に目的成分分子の量が少ない場合、イオン化において目的成分由来のイオンの種類が増えることは、特定のＭＲＭトランジションについての検出感度を高めるうえで不利である。これに対し、本発明者は、第１ＲＦイオンガイド２３に印加するＲＦ電圧の振幅を調整することで、観測したい１価のプロトン付加イオンの強度を向上できることを見出した。そこで、本実施形態の測定装置では、特に高感度の検出が要求される２４，２５－ジヒドロキシビタミンＤ3を測定する際に、１価のプロトン付加イオンの検出感度が高くなるように予め決められたＲＦ電圧を第１ＲＦイオンガイド２３に印加するようにしている。なお、この点については後で詳しく説明する。
As is well known, the ESI method is a soft ionization method, and ion dissociation (cleavage) is less likely to occur during ionization. On the other hand, not only monovalent protonated ions but also multivalent ions and adduct ions added with alkali metals are likely to be generated. Particularly when the amount of target component molecules is small, increasing the number of types of ions derived from the target component during ionization is disadvantageous in increasing detection sensitivity for a specific MRM transition. On the other hand, the inventors of the present invention have found that by adjusting the amplitude of the RF voltage applied to the first RF ion guide 23, the intensity of monovalent protonated ions to be observed can be improved. Therefore, in the measurement device of this embodiment, when measuring 24,25-dihydroxyvitamin D 3 which requires particularly high sensitivity detection, the detection sensitivity of monovalent protonated ions is predetermined to be high. The RF voltage is applied to the first RF ion guide 23. Note that this point will be explained in detail later.
第１ＲＦイオンガイド２３によるＲＦ電場によって収束された目的成分由来のイオンは、スキマー２４頂部の小孔を通過して第２中間真空室２０３に入射する。第２ＲＦイオンガイド２５には電圧発生部５から所定のＲＦ電圧が印加され、目的成分由来のイオンは、これにより形成されたＲＦ電場に捕捉されて輸送され、高真空室２０４へと送られる。高真空室２０４内において、前段四重極マスフィルター２６及び後段四重極マスフィルター２８にはそれぞれ、目的成分に対応付けられたＭＲＭトランジションにおける特定のm/z値を選択的に通過させるような電圧が電圧発生部５から印加される。コリジョンセル２７には、ＣＩＤガス圧が所定値になるようにＣＩＤガスが連続的に又は間欠的に導入される。
Ions derived from the target component focused by the RF electric field of the first RF ion guide 23 pass through the small hole at the top of the skimmer 24 and enter the second intermediate vacuum chamber 203. A predetermined RF voltage is applied to the second RF ion guide 25 from the voltage generator 5, and ions derived from the target component are captured and transported by the RF electric field thus formed, and sent to the high vacuum chamber 204. In the high vacuum chamber 204, the front quadrupole mass filter 26 and the rear quadrupole mass filter 28 are each configured to selectively pass a specific m/z value in the MRM transition associated with the target component. A voltage is applied from the voltage generator 5. CID gas is continuously or intermittently introduced into the collision cell 27 so that the CID gas pressure becomes a predetermined value.
高真空室２０４内に導入された各種イオンのうち、目的成分由来の特定のm/z値を有するイオンのみが前段四重極マスフィルター２６を通り抜け、プリカーサーイオンとして所定のエネルギーを有してコリジョンセル２７に入射する。コリジョンセル２７に入射したプリカーサーイオンはＣＩＤガスに接触し、解離を生じて各種のプロダクトイオンが生成される。生成された各種のプロダクトイオンのうち、特定のm/z値を有するプロダクトイオンのみが後段四重極マスフィルター２８を通り抜けてイオン検出器２９に入射する。イオン検出器２０は、入射したイオンの量に応じた検出信号を時々刻々と生成し、データ処理部６に送る。ＬＣ部１からＭＳ／ＭＳ部２に送られる溶出液中の一つの目的成分の濃度は、時間経過に伴って概ね山状に（理論的にはガウス分布に従って）変化するから、データ処理部６に送られる検出信号（イオン強度信号）も時間経過に伴って概ね山状に変化する。
Of the various ions introduced into the high vacuum chamber 204, only ions with a specific m/z value derived from the target component pass through the front quadrupole mass filter 26 and collide as precursor ions with a predetermined energy. The light enters the cell 27. The precursor ions that have entered the collision cell 27 come into contact with the CID gas and are dissociated to generate various product ions. Among the various product ions generated, only product ions having a specific m/z value pass through the second-stage quadrupole mass filter 28 and enter the ion detector 29. The ion detector 20 momentarily generates a detection signal corresponding to the amount of incident ions and sends it to the data processing section 6. Since the concentration of one target component in the eluate sent from the LC section 1 to the MS/MS section 2 changes roughly in a mountain shape (theoretically according to a Gaussian distribution) over time, the data processing section 6 The detection signal (ion intensity signal) sent to the sensor also changes roughly in the shape of a mountain over time.
ＭＳ／ＭＳ部２では、目的成分毎に、その目的成分に対応する時間範囲内で目的成分毎に異なる一又は複数のＭＲＭトランジションをターゲットとするＭＲＭ測定を実行する。それにより、目的成分毎に一又は複数の、上述したようなイオン強度の時間的変化を示す検出信号が得られる。このＭＳ／ＭＳ部２における、ＭＲＭトランジションを含むＭＳ／ＭＳ分析条件は、ＬＣ分析条件と同様に、ビタミンＤ／Ｋ血中濃度測定用プログラム７０で規定されている。従って、このプログラム７０をコンピューターに導入して測定を実施する場合には、ユーザーは個々のＬＣ分析条件やＭＳ／ＭＳ分析条件を手動で設定する必要はない。勿論、ユーザーが個々のＬＣ分析条件やＭＳ／ＭＳ分析条件を手動で設定したうえで測定を実行するようにしてもよい。
The MS/MS unit 2 executes MRM measurement targeting one or more MRM transitions that are different for each target component within a time range corresponding to the target component. As a result, one or more detection signals indicating temporal changes in ion intensity as described above are obtained for each target component. The MS/MS analysis conditions including the MRM transition in the MS/MS unit 2 are defined by the vitamin D/K blood concentration measurement program 70, similar to the LC analysis conditions. Therefore, when the program 70 is introduced into a computer to perform measurements, the user does not need to manually set individual LC analysis conditions and MS/MS analysis conditions. Of course, the user may manually set the individual LC analysis conditions and MS/MS analysis conditions before performing the measurement.
ＭＳ／ＭＳ部２により取得された検出信号はデジタルデータに変換され、データ収集部６０のデータ記憶部に保存される。定量演算部６１は、データ収集部６０に保存されたデータに基いて、目的成分毎に一又は複数の抽出イオンクロマトグラムを作成し、該クロマトグラムにおいて観測されるピークの保持時間、面積値、高さ値などのピーク情報を算出する。そして、定量演算部６１は、ピークの保持時間から目的成分を確認したうえで、予め作成しておいた検量線を利用してピーク面積値又は高さ値から濃度を算出する。なお、定量の際には、除タンパク処理の際に血液サンプルに添加した内部標準物質を同時に測定することで得られたピーク面積値や高さ値を利用することができる。
The detection signal acquired by the MS/MS section 2 is converted into digital data and stored in the data storage section of the data acquisition section 60. The quantitative calculation unit 61 creates one or more extracted ion chromatograms for each target component based on the data stored in the data collection unit 60, and calculates the retention time, area value, and peak value of the peak observed in the chromatogram. Calculate peak information such as height values. Then, after confirming the target component from the retention time of the peak, the quantitative calculation unit 61 calculates the concentration from the peak area value or height value using a calibration curve created in advance. In addition, in the case of quantitative determination, it is possible to utilize the peak area value and height value obtained by simultaneously measuring the internal standard substance added to the blood sample during protein removal treatment.
以上のようにして、一つの血液サンプルに対する６種類の目的成分の血中濃度が求まる。図２に示した測定装置では、予め用意された多数の血液サンプルに対し上述した手順で自動的に前処理及び測定をそれぞれ実施することで、各血液サンプルについての測定結果を得ることができる。制御部７は、こうして得られた測定結果を、所定形式で表示部９の画面上に表示することができる。
As described above, the blood concentrations of six types of target components for one blood sample are determined. In the measurement apparatus shown in FIG. 2, measurement results for each blood sample can be obtained by automatically performing preprocessing and measurement on a large number of blood samples prepared in advance according to the above-described procedures. The control unit 7 can display the measurement results obtained in this manner on the screen of the display unit 9 in a predetermined format.
＜実測例＞
次に、上述した本実施形態の測定装置を利用した実測例について、具体的なパラメーター値等を示しながら説明する。 <Actual measurement example>
Next, an actual measurement example using the measuring device of the present embodiment described above will be explained while showing specific parameter values and the like.
次に、上述した本実施形態の測定装置を利用した実測例について、具体的なパラメーター値等を示しながら説明する。 <Actual measurement example>
Next, an actual measurement example using the measuring device of the present embodiment described above will be explained while showing specific parameter values and the like.
除タンパク処理は次の手順に従って実施した。
濾過容器（０．４５μmのポリテトラフルオロエチレン（ＰＴＦＥ）濾過フィルター付きの濾過容器）内に、
（１）フィルターのコンディショニングのために７５％のイソプロピルアルコール（ＩＰＡ）を２０μL添加
（２）３０μLのサンプルを添加
（３）極性有機溶媒として９０μLのメタノール（内部標準試料を含む）を添加。
上記（１）～（３）の全ての添加の後に、２２００rpm、１minの条件で濾過容器の撹拌を行い、１min間、濾過を実行する。濾過の際には、濾過容器と組み合わせられた回収容器内に濾過液が回収される。 Protein removal treatment was performed according to the following procedure.
In a filtration container (a filtration container with a 0.45 μm polytetrafluoroethylene (PTFE) filtration filter),
(1) Add 20 μL of 75% isopropyl alcohol (IPA) to condition the filter. (2) Add 30 μL of sample. (3) Add 90 μL of methanol (including internal standard sample) as a polar organic solvent.
After all the additions in (1) to (3) above, the filtration container is stirred at 2200 rpm for 1 minute, and filtration is performed for 1 minute. During filtration, the filtrate is collected into a collection container combined with the filtration container.
濾過容器（０．４５μmのポリテトラフルオロエチレン（ＰＴＦＥ）濾過フィルター付きの濾過容器）内に、
（１）フィルターのコンディショニングのために７５％のイソプロピルアルコール（ＩＰＡ）を２０μL添加
（２）３０μLのサンプルを添加
（３）極性有機溶媒として９０μLのメタノール（内部標準試料を含む）を添加。
上記（１）～（３）の全ての添加の後に、２２００rpm、１minの条件で濾過容器の撹拌を行い、１min間、濾過を実行する。濾過の際には、濾過容器と組み合わせられた回収容器内に濾過液が回収される。 Protein removal treatment was performed according to the following procedure.
In a filtration container (a filtration container with a 0.45 μm polytetrafluoroethylene (PTFE) filtration filter),
(1) Add 20 μL of 75% isopropyl alcohol (IPA) to condition the filter. (2) Add 30 μL of sample. (3) Add 90 μL of methanol (including internal standard sample) as a polar organic solvent.
After all the additions in (1) to (3) above, the filtration container is stirred at 2200 rpm for 1 minute, and filtration is performed for 1 minute. During filtration, the filtrate is collected into a collection container combined with the filtration container.
オンラインＳＰＥの条件は次の通りである。
・固相抽出用カラムの種類 ：島津製作所製 Shim-pack XR ODS 30L × 2.0
・移動相 ：水：メタノール ５０：５０
・移動相流量 ：０．５mL/min The conditions for online SPE are as follows.
・Type of column for solid phase extraction: Shimadzu Shim-pack XR ODS 30L × 2.0
・Mobile phase: water: methanol 50:50
・Mobile phase flow rate: 0.5mL/min
・固相抽出用カラムの種類 ：島津製作所製 Shim-pack XR ODS 30L × 2.0
・移動相 ：水：メタノール ５０：５０
・移動相流量 ：０．５mL/min The conditions for online SPE are as follows.
・Type of column for solid phase extraction: Shimadzu Shim-pack XR ODS 30L × 2.0
・Mobile phase: water: methanol 50:50
・Mobile phase flow rate: 0.5mL/min
ＬＣ分析条件は次の通りである。
・カラムの種類 ：島津製作所製 Shim-Pack Velox PFPP 2.7μm 3.0 × 100mm
・カラム温度 ：４０℃
・サンプル注入量 ：３０μL
・移動相Ａ ：水＋５mM ギ酸アンモニウム＋０．１％ギ酸
・移動相Ｂ ：メタノール
・移動相流量 ：０．７mL/min
・分析時間 :１２min The LC analysis conditions are as follows.
・Column type: Shimadzu Shim-Pack Velox PFPP 2.7μm 3.0 × 100mm
・Column temperature: 40℃
・Sample injection volume: 30μL
・Mobile phase A: Water + 5mM ammonium formate + 0.1% formic acid ・Mobile phase B: Methanol ・Mobile phase flow rate: 0.7mL/min
・Analysis time: 12min
・カラムの種類 ：島津製作所製 Shim-Pack Velox PFPP 2.7μm 3.0 × 100mm
・カラム温度 ：４０℃
・サンプル注入量 ：３０μL
・移動相Ａ ：水＋５mM ギ酸アンモニウム＋０．１％ギ酸
・移動相Ｂ ：メタノール
・移動相流量 ：０．７mL/min
・分析時間 :１２min The LC analysis conditions are as follows.
・Column type: Shimadzu Shim-Pack Velox PFPP 2.7μm 3.0 × 100mm
・Column temperature: 40℃
・Sample injection volume: 30μL
・Mobile phase A: Water + 5mM ammonium formate + 0.1% formic acid ・Mobile phase B: Methanol ・Mobile phase flow rate: 0.7mL/min
・Analysis time: 12min
ＭＲＭトランジション以外のＭＳ／ＭＳ分析条件は次の通りである。
・装置 ：島津製作所製 LCMS-8060
・イオン化法 ：ＥＳＩ法
・ネブライズガス流量 ：２L/min
・ドライイングガス流量 ：１７L/min
・ヒーティングガス流量 ：３L/min
・脱溶媒管温度 ：２５０℃
・ヒートブロック温度 ：３００℃
・インターフェイス温度 ：３００℃
・コリジョンセルガス圧 ：１９０kPa
・ＥＳＩスプレー印加電圧：３kV
・Ｑアレー（第１ＲＦイオンガイド）ＲＦ電圧：１００Ｖ（24,25-(OH)2D3）、デフォルト値（24,25-(OH)2D3以外の目的成分）
なお、ＱアレーＲＦ電圧のデフォルト値とは、通常、標準試料等を用いた装置の自動チューニングによって決まる値であり、必ずしも一定ではない。 MS/MS analysis conditions other than MRM transition are as follows.
・Equipment: Shimadzu LCMS-8060
・Ionization method: ESI method ・Nebulization gas flow rate: 2L/min
・Drying gas flow rate: 17L/min
・Heating gas flow rate: 3L/min
・Desolvation tube temperature: 250℃
・Heat block temperature: 300℃
・Interface temperature: 300℃
・Collision cell gas pressure: 190kPa
・ESI spray applied voltage: 3kV
・Q array (1st RF ion guide) RF voltage: 100V (24,25-(OH) 2 D 3 ), default value (target components other than 24,25-(OH) 2 D 3 )
Note that the default value of the Q array RF voltage is usually a value determined by automatic tuning of the device using a standard sample or the like, and is not necessarily constant.
・装置 ：島津製作所製 LCMS-8060
・イオン化法 ：ＥＳＩ法
・ネブライズガス流量 ：２L/min
・ドライイングガス流量 ：１７L/min
・ヒーティングガス流量 ：３L/min
・脱溶媒管温度 ：２５０℃
・ヒートブロック温度 ：３００℃
・インターフェイス温度 ：３００℃
・コリジョンセルガス圧 ：１９０kPa
・ＥＳＩスプレー印加電圧：３kV
・Ｑアレー（第１ＲＦイオンガイド）ＲＦ電圧：１００Ｖ（24,25-(OH)2D3）、デフォルト値（24,25-(OH)2D3以外の目的成分）
なお、ＱアレーＲＦ電圧のデフォルト値とは、通常、標準試料等を用いた装置の自動チューニングによって決まる値であり、必ずしも一定ではない。 MS/MS analysis conditions other than MRM transition are as follows.
・Equipment: Shimadzu LCMS-8060
・Ionization method: ESI method ・Nebulization gas flow rate: 2L/min
・Drying gas flow rate: 17L/min
・Heating gas flow rate: 3L/min
・Desolvation tube temperature: 250℃
・Heat block temperature: 300℃
・Interface temperature: 300℃
・Collision cell gas pressure: 190kPa
・ESI spray applied voltage: 3kV
・Q array (1st RF ion guide) RF voltage: 100V (24,25-(OH) 2 D 3 ), default value (target components other than 24,25-(OH) 2 D 3 )
Note that the default value of the Q array RF voltage is usually a value determined by automatic tuning of the device using a standard sample or the like, and is not necessarily constant.
各目的成分についての代表的なＭＲＭトランジション及びコリジョンエネルギー（ＣＥ）は次の通りである。代表的なＭＲＭトランジションとは定量に利用されるＭＲＭトランジションであり、それ以外に、例えば確認イオン比を用いた成分確認のための確認イオンのＭＲＭトランジションを別途、一以上定めることができる。
・24,25(OH)2VitD3：４１７．５０００＞３８１．２０００、ＣＥ：－１０．０
・25(OH)VitD3：３８３．４０００＞２５７．２０００、ＣＥ：－１５．０
・25(OH)VitD2：３９５．３０００＞２６９．３０００、ＣＥ：－１７．０
・VitK1 ：４５１．４０００＞１８７．３０００、ＣＥ：－２７．０
・VitK2(MK-4)：４４５．３０００＞１８７．００００、ＣＥ：－２５．０
・VitK2(MK-7)：６４９．５０００＞１８７．１５００、ＣＥ：－３８．０ Typical MRM transitions and collision energies (CE) for each target component are as follows. A typical MRM transition is an MRM transition used for quantification, and in addition to this, one or more MRM transitions of confirmation ions for component confirmation using confirmation ion ratios can be separately determined, for example.
・24,25(OH) 2 VitD 3 :417.5000>381.2000, CE:-10.0
・25(OH)VitD 3 :383.4000>257.2000, CE:-15.0
・25(OH)VitD 2 :395.3000>269.3000, CE:-17.0
・VitK 1 :451.4000>187.3000, CE:-27.0
・VitK 2 (MK-4): 445.3000>187.0000, CE: -25.0
・VitK 2 (MK-7): 649.5000>187.1500, CE: -38.0
・24,25(OH)2VitD3：４１７．５０００＞３８１．２０００、ＣＥ：－１０．０
・25(OH)VitD3：３８３．４０００＞２５７．２０００、ＣＥ：－１５．０
・25(OH)VitD2：３９５．３０００＞２６９．３０００、ＣＥ：－１７．０
・VitK1 ：４５１．４０００＞１８７．３０００、ＣＥ：－２７．０
・VitK2(MK-4)：４４５．３０００＞１８７．００００、ＣＥ：－２５．０
・VitK2(MK-7)：６４９．５０００＞１８７．１５００、ＣＥ：－３８．０ Typical MRM transitions and collision energies (CE) for each target component are as follows. A typical MRM transition is an MRM transition used for quantification, and in addition to this, one or more MRM transitions of confirmation ions for component confirmation using confirmation ion ratios can be separately determined, for example.
・24,25(OH) 2 VitD 3 :417.5000>381.2000, CE:-10.0
・25(OH)VitD 3 :383.4000>257.2000, CE:-15.0
・25(OH)VitD 2 :395.3000>269.3000, CE:-17.0
・VitK 1 :451.4000>187.3000, CE:-27.0
・VitK 2 (MK-4): 445.3000>187.0000, CE: -25.0
・VitK 2 (MK-7): 649.5000>187.1500, CE: -38.0
図３は、実測により得られた、各目的成分についての代表的な抽出イオンクロマトグラムである。各クロマトグラムの縦方向の位置は適宜ずらしてある。
この結果から、各目的成分は互いに十分に分離された状態で観測されていることが分かる。また、25(OH)VitD3とその異性体（epi-）とが適切に分離できていることも分かる。 FIG. 3 is a representative extracted ion chromatogram for each target component obtained through actual measurements. The vertical position of each chromatogram is shifted as appropriate.
From this result, it can be seen that each target component is observed while being sufficiently separated from each other. It can also be seen that 25(OH)VitD 3 and its isomer (epi-) can be separated appropriately.
この結果から、各目的成分は互いに十分に分離された状態で観測されていることが分かる。また、25(OH)VitD3とその異性体（epi-）とが適切に分離できていることも分かる。 FIG. 3 is a representative extracted ion chromatogram for each target component obtained through actual measurements. The vertical position of each chromatogram is shifted as appropriate.
From this result, it can be seen that each target component is observed while being sufficiently separated from each other. It can also be seen that 25(OH)VitD 3 and its isomer (epi-) can be separated appropriately.
図４～図９はそれぞれ、濃度０．５ng/mLの２４，２５－ジヒドロキシビタミンＤ3、濃度４．９ng/mLの２５－ヒドロキシビタミンＤ3、濃度４．５ng/mLの２５－ヒドロキシビタミンＤ2、濃度０．１ng/mLのビタミンＫ1、濃度０．１ng/mLのビタミンＫ2ＭＫ－４、濃度０．５ng/mLのビタミンＫ2ＭＫ－７に対する抽出イオンクロマトグラムのピークの実測例である。濃度は各目的成分の定量下限である。各図において下向きの三角印で示したピークが定量に用いられるピークであり、該ピークの始点及び終点とそれを結ぶ線（つまりはピーク面積を規定するベースライン）も併せて記載してある。全ての目的成分について、良好な形状のピークが得られていることが確認できる。
Figures 4 to 9 show 24,25-dihydroxyvitamin D 3 at a concentration of 0.5 ng/mL, 25-hydroxyvitamin D 3 at a concentration of 4.9 ng/mL, and 25-hydroxyvitamin D at a concentration of 4.5 ng/mL, respectively. 2. Actual measurement example of extracted ion chromatogram peaks for vitamin K 1 at a concentration of 0.1 ng/mL, vitamin K 2 MK-4 at a concentration of 0.1 ng/mL, and vitamin K 2 MK-7 at a concentration of 0.5 ng/mL. It is. The concentration is the lower limit of quantification for each target component. In each figure, the peak indicated by a downward triangular mark is the peak used for quantification, and the starting point and ending point of the peak and the line connecting them (that is, the baseline defining the peak area) are also shown. It can be confirmed that peaks with good shapes were obtained for all target components.
図１０は、検量線用サンプル（キャリブレーター）を用いて作成された２４，２５－ジヒドロキシビタミンＤ3の検量線である。キャリブレーターは、Golden West Diagnostics社製の試薬を用いて調製されたものである。測定濃度範囲は０．５～１２０ng/mLであり、これは実際の検査で想定される測定濃度範囲に対応している。図１１は、同じくキャリブレーターを用いて作成されたビタミンＫ1の検量線の一例である。測定濃度範囲は０．１～２４ng/mLであり、これも実際の検査で想定される測定濃度範囲に対応している。
FIG. 10 is a calibration curve for 24,25-dihydroxyvitamin D 3 created using a calibration curve sample (calibrator). The calibrator was prepared using reagents manufactured by Golden West Diagnostics. The measured concentration range is 0.5 to 120 ng/mL, which corresponds to the measured concentration range expected in actual testing. FIG. 11 is an example of a vitamin K 1 calibration curve created using the same calibrator. The measured concentration range is 0.1 to 24 ng/mL, which also corresponds to the measured concentration range expected in actual tests.
また、図１２は、Golden West Diagnostics社製の試薬を用いて調製された低濃度コントロール試料についての精度及び再現性（日内再現性）の結果を示す図である。図１３は、同じくGolden West Diagnostics社製の試薬を用いて調製された高濃度コントロール試料についての精度及び再現性（日内再現性）の結果を示す図である。低濃度コントール試料は定量下限の５倍の濃度、高濃度コントロール試料はビタミンＫ1のみ１８ng/mL、それ以外の目的成分では９０ng/mLである。
Moreover, FIG. 12 is a diagram showing the results of accuracy and reproducibility (within-day reproducibility) for a low concentration control sample prepared using a reagent manufactured by Golden West Diagnostics. FIG. 13 is a diagram showing the accuracy and reproducibility (within-day reproducibility) results for a high concentration control sample also prepared using a reagent manufactured by Golden West Diagnostics. The low concentration control sample has a concentration 5 times the lower limit of quantitation, the high concentration control sample has a concentration of 18 ng/mL for vitamin K1 only, and 90 ng/mL for other target components.
図１０、図１１に示した検量線の直線性はいずれもｒ2＞０．９９で良好である。それ以外の４種類の目的成分についても同様に、それぞれに必要とされる測定濃度範囲における検量線を検証したが、いずれも直線性はｒ2＞０．９９で良好であることが確認できた。また、図１２、図１３に示すように、全ての目的成分について精度は８５～１１５％が確保されており、再現性はビタミンＫ1、ビタミンＫ2ＭＫ－７を除き１３％以内である。
The linearity of the calibration curves shown in FIGS. 10 and 11 is good, with r 2 >0.99. The calibration curves for the other four target components were similarly verified in the measurement concentration range required for each, and it was confirmed that the linearity was good with r 2 > 0.99 in all cases. . Furthermore, as shown in FIGS. 12 and 13, accuracy of 85 to 115% is ensured for all target components, and reproducibility is within 13% except for vitamin K 1 and vitamin K 2 MK-7.
ビタミンＤ代謝物に比べてビタミンＫ及びその代謝物の再現性が劣る大きな要因は、ＬＣ部１におけるキャリーオーバーであることが判明した。よく知られているように、これは、インジェクター１２に用いられているニードルなどを洗浄液で洗浄することにより解消され得る。図１４は、洗浄液による洗浄を行った場合のキャリーオーバー低減効果を示す抽出イオンクロマトグラムである。図１４の上は、ビタミンＫ類における最も濃度の高いＱＣサンプルに対する抽出イオンクロマトグラム、図１４の下は、メタノールで洗浄した場合の抽出イオンクロマトグラムである。これら図から、メタノール等の適切な洗浄液を用いた洗浄によって、キャリーオーバーは実質的に問題ない程度まで低減できることが確認できる。こうしたことから、特にビタミンＫ類における精度や再現性を高めるうえで、洗浄によるキャリーオーバーの低減は重要である。
It has been found that carryover in the LC section 1 is a major factor in the inferior reproducibility of vitamin K and its metabolites compared to vitamin D metabolites. As is well known, this can be resolved by cleaning the needle and the like used in the injector 12 with a cleaning liquid. FIG. 14 is an extracted ion chromatogram showing the effect of reducing carryover when washing with a washing liquid is performed. The upper part of FIG. 14 is an extracted ion chromatogram for the QC sample with the highest concentration of vitamin K, and the lower part of FIG. 14 is an extracted ion chromatogram when washed with methanol. From these figures, it can be confirmed that carryover can be reduced to a substantially non-problematic level by cleaning using an appropriate cleaning liquid such as methanol. For this reason, it is important to reduce carryover through washing, especially in improving the precision and reproducibility of vitamin K compounds.
＜ＱアレーのＲＦ電圧調整による感度改善＞
上述したように、オンライン固相抽出によりサンプル中の夾雑物を除去することで、目的成分についてのイオン化効率は改善され検出感度は向上する。但し、特にＥＳＩ応答性が低く、低濃度の検出が要求される２４，２５－ジヒドロキシビタミンＤ3については、さらなる感度改善が望ましい。本発明者は実験の過程で、第１ＲＦイオンガイド２３に印加するＲＦ電圧を変化させるとイオン強度に大きな変化があることを見つけ、これに着目した。 <Improvement of sensitivity by adjusting the RF voltage of Q array>
As described above, by removing impurities in the sample by online solid-phase extraction, the ionization efficiency for the target component is improved and the detection sensitivity is improved. However, further sensitivity improvement is desirable, especially for 24,25-dihydroxyvitamin D 3 , which has low ESI response and requires detection of low concentrations. In the course of experiments, the inventor of the present invention found that there was a large change in ion intensity when the RF voltage applied to the firstRF ion guide 23 was changed, and focused on this.
上述したように、オンライン固相抽出によりサンプル中の夾雑物を除去することで、目的成分についてのイオン化効率は改善され検出感度は向上する。但し、特にＥＳＩ応答性が低く、低濃度の検出が要求される２４，２５－ジヒドロキシビタミンＤ3については、さらなる感度改善が望ましい。本発明者は実験の過程で、第１ＲＦイオンガイド２３に印加するＲＦ電圧を変化させるとイオン強度に大きな変化があることを見つけ、これに着目した。 <Improvement of sensitivity by adjusting the RF voltage of Q array>
As described above, by removing impurities in the sample by online solid-phase extraction, the ionization efficiency for the target component is improved and the detection sensitivity is improved. However, further sensitivity improvement is desirable, especially for 24,25-dihydroxyvitamin D 3 , which has low ESI response and requires detection of low concentrations. In the course of experiments, the inventor of the present invention found that there was a large change in ion intensity when the RF voltage applied to the first
図１５は、第１ＲＦイオンガイド２３に印加するＲＦ電圧を０、３０、６０、９０、１２０Ｖに変化させた場合に、２４，２５－ジヒドロキシビタミンＤ3に対して実測される抽出イオンクロマトグラム上のピークの保持時間、該ピークの面積、高さ、及びＳＮ比を示す図である。なお、第１ＲＦイオンガイド２３には、ＲＦ電圧以外に、バイアスＤＣ電圧が印加されている。図１６は、ＲＦ電圧を変化させた場合の抽出イオンクロマトグラムの実測例である。これら結果から、ピーク面積、高さ、ＳＮ比は、第１ＲＦイオンガイド２３へ印加されるＲＦ電圧に大きく依存していることが分かる。その理由は次のように推測され得る。
FIG. 15 shows extracted ion chromatograms actually measured for 24,25-dihydroxyvitamin D3 when the RF voltage applied to the first RF ion guide 23 was changed to 0, 30, 60, 90, and 120V. FIG. 2 is a diagram showing the retention time of the peak, the area, height, and SN ratio of the peak. Note that a bias DC voltage is applied to the first RF ion guide 23 in addition to the RF voltage. FIG. 16 is an example of an extracted ion chromatogram measured when changing the RF voltage. These results show that the peak area, height, and SN ratio are largely dependent on the RF voltage applied to the first RF ion guide 23. The reason can be inferred as follows.
ＥＳＩ法では、目的成分に由来する１価のプロトン付加イオンのほか、多価イオンや、アルカリ金属等が付加したアダクトイオンが生成され易く、目的成分由来の様々なイオンが第１中間真空室２０２に導入される。前段四重極マスフィルター２６でプリカーサーイオンとして選択されるのは１価のプロトン付加イオンのみである。そのため、多価イオンやアダクトイオンの生成量が増えると、それだけ１価のプロトン付加イオンの量は相対的に減少し、そのプリカーサーイオンから生成されるプロダクトイオンの量も減少する。従って、目的成分の検出感度を高めるには、目的成分由来である多価イオンやアダクトイオンの量をできるだけ減らし、１価のプロトン付加イオンの量を増加させる、つまりイオンを１価のプロトン付加イオンにできるだけ集約させることが重要である。
In the ESI method, in addition to monovalent protonated ions derived from the target component, multivalent ions and adduct ions to which alkali metals and the like are added are likely to be generated, and various ions derived from the target component enter the first intermediate vacuum chamber 202. will be introduced in Only monovalent protonated ions are selected as precursor ions in the front quadrupole mass filter 26. Therefore, as the amount of multivalent ions and adduct ions generated increases, the amount of monovalent protonated ions relatively decreases, and the amount of product ions generated from the precursor ions also decreases. Therefore, in order to increase the detection sensitivity of the target component, the amount of multivalent ions and adduct ions derived from the target component should be reduced as much as possible, and the amount of monovalent protonated ions should be increased. It is important to consolidate as much as possible.
第１中間真空室２０２には残留ガス分子が多く存在するため、該中間真空室２０２内に導入されたイオンは残留ガス分子に接触しクーリング（衝突冷却）される。このようにクーリングによってイオンが有する運動エネルギーが減衰するために、該イオンはＲＦ電場に捕捉され易くなる。これが、低真空雰囲気の下でＲＦ電場を利用したイオン収束が有効である大きな理由である。残留ガス分子との接触によって目的成分由来のイオンは運動エネルギーを失うが、それ以外に、残留ガス分子との接触によって衝突誘起解離に類似した現象が生じ、イオンの部分構造が脱離する。例えば一部のアダクトイオンのようにプロトン付加イオンにさらにアルカリ金属等のアダクトが結合している場合、残留ガスとの接触によってアダクト部分が脱離しプロトン付加イオンになり易い。また、多価イオンについても同様に、残留ガスとの接触によって、多価イオンが分離して１価のイオンが生成され得る。即ち、第１中間真空室２０２において残留ガスとの接触の機会が多いほど、アダクトイオンや多価イオンの割合が減って、その分、１価のプロトン付加イオンが増加すると考えられる。
Since there are many residual gas molecules in the first intermediate vacuum chamber 202, the ions introduced into the intermediate vacuum chamber 202 come into contact with the residual gas molecules and are cooled (collision cooling). Since the kinetic energy of the ions is thus attenuated by cooling, the ions are more likely to be captured by the RF electric field. This is a major reason why ion focusing using an RF electric field is effective in a low vacuum atmosphere. Ions derived from the target component lose kinetic energy due to contact with residual gas molecules, but in addition, a phenomenon similar to collision-induced dissociation occurs due to contact with residual gas molecules, and the partial structure of the ion is detached. For example, when an adduct such as an alkali metal is further bonded to a protonated ion as in some adduct ions, the adduct portion is likely to be detached by contact with residual gas and become a protonated ion. In addition, in the same way, multivalent ions can be separated and monovalent ions can be generated by contact with residual gas. That is, it is considered that the more opportunities for contact with residual gas in the first intermediate vacuum chamber 202, the lower the proportion of adduct ions and multivalent ions, and the more monovalent protonated ions increase.
脱溶媒管２２を経て第１中間真空室２０２内に流れ込んだガスは、脱溶媒管２２の出口を出たあと超音速自由噴流となって拡がるものの、残留ガスの密度はイオン光軸Ｃ付近が高くなり易い。ＲＦ電場によって捕捉されたイオンはその電場の作用によって周期的に振動するが、その電場が強い方がイオン光軸Ｃ付近の狭い領域に閉じ込められ易い。そのため、ＲＦ電場が強い方が、つまりはＱアレーに印加されるＲＦ電圧が大きい方が、目的成分由来のイオンが残留ガスに接触する機会が増加する。それによって、目的成分由来の多価イオンやアダクトイオンが減少し、１価のプロトン付加イオンが相対的に多くなり易い。これが、目的成分由来の１価のプロトン付加イオンの強度が、第１ＲＦイオンガイド２３に印加されるＲＦ電圧の大きさの影響を大きく受ける原因であると考えられる。
The gas that has flowed into the first intermediate vacuum chamber 202 through the desolvation tube 22 becomes a supersonic free jet and spreads after leaving the exit of the desolvation tube 22, but the density of the residual gas is low near the ion optical axis C. It can easily become expensive. Ions captured by the RF electric field oscillate periodically due to the action of the electric field, but the stronger the electric field, the more likely they are to be confined in a narrow region near the ion optical axis C. Therefore, the stronger the RF electric field, that is, the greater the RF voltage applied to the Q array, the greater the chance that ions derived from the target component will come into contact with the residual gas. As a result, multivalent ions and adduct ions derived from the target component decrease, and monovalent protonated ions tend to increase relatively. This is considered to be the reason why the intensity of monovalent protonated ions derived from the target component is greatly influenced by the magnitude of the RF voltage applied to the first RF ion guide 23.
但し、第１ＲＦイオンガイド２３に印加するＲＦ電圧を大きくすると、該イオンガイド２３の入口端におけるイオンのアクセプタンス領域が狭くなる。そのため、ＲＦ電圧を必要以上に大きくすると、却って総合的なイオン透過効率が下がるおそれがある。実験的に確認した結果では、上記６種類の目的成分の中で検出感度が最も厳しいのが２４，２５－ジヒドロキシビタミンＤ3である。そのため、上述した実施形態の測定装置では、２４，２５－ジヒドロキシビタミンＤ3を測定する際に、ＲＦ電圧のデフォルト値に拘わらず該ＲＦ電圧を１００Ｖに設定し、それ以外の目的成分を測定する際には、ＲＦ電圧をデフォルト値に設定している。これにより、２４，２５－ジヒドロキシビタミンＤ3の検出感度を特に高める一方、それ以外の目的成分の検出感度も目標とする範囲に収めることができている。
However, if the RF voltage applied to the first RF ion guide 23 is increased, the ion acceptance region at the entrance end of the ion guide 23 becomes narrower. Therefore, if the RF voltage is increased more than necessary, there is a risk that the overall ion transmission efficiency will decrease. According to the experimentally confirmed results, 24,25-dihydroxyvitamin D 3 has the most severe detection sensitivity among the above six types of target components. Therefore, in the measuring device of the embodiment described above, when measuring 24,25-dihydroxyvitamin D 3 , the RF voltage is set to 100 V regardless of the default value of the RF voltage, and other target components are measured. In some cases, the RF voltage is set to a default value. As a result, while the detection sensitivity of 24,25-dihydroxyvitamin D 3 is particularly increased, the detection sensitivity of other target components can also be kept within the target range.
なお、上記実施形態の測定装置では、２４，２５－ジヒドロキシビタミンＤ3を測定する際にのみ、第１ＲＦイオンガイド２３に印加するＲＦ電圧を予め決められた値に設定していたが、他の目的成分についてもそれぞれＲＦ電圧を予め決められた値に設定するようにしてもよい。その場合、それら値は個別に異なる値でもよいし、一部が同じ値であってもよい。
Note that in the measuring device of the above embodiment, the RF voltage applied to the first RF ion guide 23 is set to a predetermined value only when measuring 24,25-dihydroxyvitamin D 3 . The RF voltage may also be set to a predetermined value for each target component. In that case, these values may be individually different values, or some of them may be the same value.
また、上記実施形態の測定方法及び測定装置において採用した各種のパラメーター値、カラム等の構成要素や移動相などの消費材の種類などを含む分析条件は一例であって、適宜に変更することが可能である。また、上記実施形態の測定方法及び測定装置において測定の対象とした６種類の目的成分は一例であり、少なくとも２４，２５－ジヒドロキシビタミンＤ3とビタミンＫ1を含むこと以外は、目的成分を適宜に削除又は追加することができる。
In addition, the various parameter values adopted in the measurement method and measurement device of the above embodiment, the analysis conditions including the types of constituent elements such as columns and consumable materials such as mobile phase, etc. are merely examples, and may be changed as appropriate. It is possible. In addition, the six types of target components to be measured in the measuring method and measuring device of the above embodiment are merely examples, and the target components can be adjusted as appropriate except for containing at least 24,25-dihydroxyvitamin D 3 and vitamin K 1 . can be deleted or added to.
また、上記実施形態はあくまでも本発明の一例にすぎず、本発明の趣旨の範囲で適宜変形、修正、追加等を行っても本願特許請求の範囲に包含されることは当然である。
Further, the above-described embodiment is merely an example of the present invention, and it goes without saying that any changes, modifications, additions, etc. made within the scope of the spirit of the present invention will still fall within the scope of the claims of the present application.
［種々の態様］
上述した例示的な実施形態は、以下の態様の具体例であることが当業者により理解される。 [Various aspects]
It will be appreciated by those skilled in the art that the exemplary embodiments described above are specific examples of the following aspects.
上述した例示的な実施形態は、以下の態様の具体例であることが当業者により理解される。 [Various aspects]
It will be appreciated by those skilled in the art that the exemplary embodiments described above are specific examples of the following aspects.
（第１項）本発明に係る脂溶性ビタミン類の血中濃度測定方法の一態様は、血液サンプル中の、２４，２５－ジヒドロキシビタミンＤ3を含むビタミンＤ代謝物、並びにビタミンＫ1を含むビタミンＫ及びその代謝物、である目的成分の濃度を測定する方法であって、
血液サンプルに対し変性法により除タンパク処理を行う第１前処理工程と、
前記第１前処理工程後のサンプルに含まれる夾雑物を固相抽出により除去する第２前処理工程と、
エレクトロスプレーイオン化法によるイオン源を有する液体クロマトグラフ－タンデム型質量分析装置を用いて、前記第２前処理工程後のサンプル中の成分を時間的に分離しつつ、前記目的成分それぞれに由来する１価のプロトン付加イオンを選択的に検出するＬＣ／ＭＳ分析を実行する測定工程と、
を有する。 (Section 1) One embodiment of the method for measuring blood concentration of fat-soluble vitamins according to the present invention includes vitamin D metabolites including 24,25-dihydroxyvitamin D 3 and vitamin K 1 in a blood sample. A method for measuring the concentration of a target component, which is vitamin K and its metabolites, comprising:
a first pretreatment step of performing protein removal treatment on the blood sample by a denaturation method;
a second pretreatment step of removing impurities contained in the sample after the first pretreatment step by solid phase extraction;
Using a liquid chromatograph-tandem mass spectrometer equipped with an ion source using electrospray ionization, the components in the sample after the second pretreatment step are temporally separated, and the components derived from each of the target components are separated in time. a measurement step of performing LC/MS analysis to selectively detect protonated ions of valence;
has.
血液サンプルに対し変性法により除タンパク処理を行う第１前処理工程と、
前記第１前処理工程後のサンプルに含まれる夾雑物を固相抽出により除去する第２前処理工程と、
エレクトロスプレーイオン化法によるイオン源を有する液体クロマトグラフ－タンデム型質量分析装置を用いて、前記第２前処理工程後のサンプル中の成分を時間的に分離しつつ、前記目的成分それぞれに由来する１価のプロトン付加イオンを選択的に検出するＬＣ／ＭＳ分析を実行する測定工程と、
を有する。 (Section 1) One embodiment of the method for measuring blood concentration of fat-soluble vitamins according to the present invention includes vitamin D metabolites including 24,25-dihydroxyvitamin D 3 and vitamin K 1 in a blood sample. A method for measuring the concentration of a target component, which is vitamin K and its metabolites, comprising:
a first pretreatment step of performing protein removal treatment on the blood sample by a denaturation method;
a second pretreatment step of removing impurities contained in the sample after the first pretreatment step by solid phase extraction;
Using a liquid chromatograph-tandem mass spectrometer equipped with an ion source using electrospray ionization, the components in the sample after the second pretreatment step are temporally separated, and the components derived from each of the target components are separated in time. a measurement step of performing LC/MS analysis to selectively detect protonated ions of valence;
has.
（第８項）本発明に係る脂溶性ビタミン類血中濃度測定装置の一態様は、血液サンプル中の、２４，２５－ジヒドロキシビタミンＤ3を含むビタミンＤ代謝物、並びにビタミンＫ1を含むビタミンＫ及びその代謝物、である目的成分の濃度を測定するための装置であって、
血液サンプルに対し有機溶媒の添加、撹拌、及び濾過を順に行うことにより該サンプル中のタンパクを除去する除タンパク処理部と、
前記除タンパク処理部による処理済みのサンプルを固相抽出用カラムに供給し該サンプル中の目的成分を該固相抽出用カラムに保持したあと、移動相を供給して目的成分を該固相抽出用カラムから溶出させるオンライン固相抽出部と、
エレクトロスプレーイオン化法によるイオン源を有する液体クロマトグラフ－タンデム型質量分析装置であって、前記固相抽出用カラムから溶出した目的成分が含まれる試料液中の成分を時間的に分離しつつ、前記目的成分それぞれに由来する１価のプロトン付加イオンを選択的に検出するＬＣ／ＭＳ分析を実行する測定実行部と、
前記測定実行部により得られたデータを利用して前記目的成分毎にクロマトグラムを作成し、該クロマトグラムにおいて観測されるピークの面積又は高さに基いて該目的成分を定量するデータ解析部と、
を備える。 (Section 8) One embodiment of the fat-soluble vitamin blood concentration measuring device according to the present invention measures vitamin D metabolites including 24,25-dihydroxyvitamin D 3 and vitamins including vitamin K 1 in a blood sample. A device for measuring the concentration of target components, which are K and its metabolites,
a protein removal processing unit that removes proteins from the blood sample by sequentially adding an organic solvent to the sample, stirring, and filtering the sample;
The sample processed by the protein removal processing unit is supplied to the solid-phase extraction column, and the target component in the sample is retained in the solid-phase extraction column, and then a mobile phase is supplied to extract the target component in the solid-phase extraction column. an online solid-phase extraction section for elution from a column for
A liquid chromatograph-tandem mass spectrometer having an ion source using an electrospray ionization method, which temporally separates components in a sample solution containing target components eluted from the solid phase extraction column, and a measurement execution unit that executes LC/MS analysis that selectively detects monovalent protonated ions originating from each target component;
a data analysis unit that creates a chromatogram for each target component using the data obtained by the measurement execution unit and quantifies the target component based on the area or height of a peak observed in the chromatogram; ,
Equipped with
血液サンプルに対し有機溶媒の添加、撹拌、及び濾過を順に行うことにより該サンプル中のタンパクを除去する除タンパク処理部と、
前記除タンパク処理部による処理済みのサンプルを固相抽出用カラムに供給し該サンプル中の目的成分を該固相抽出用カラムに保持したあと、移動相を供給して目的成分を該固相抽出用カラムから溶出させるオンライン固相抽出部と、
エレクトロスプレーイオン化法によるイオン源を有する液体クロマトグラフ－タンデム型質量分析装置であって、前記固相抽出用カラムから溶出した目的成分が含まれる試料液中の成分を時間的に分離しつつ、前記目的成分それぞれに由来する１価のプロトン付加イオンを選択的に検出するＬＣ／ＭＳ分析を実行する測定実行部と、
前記測定実行部により得られたデータを利用して前記目的成分毎にクロマトグラムを作成し、該クロマトグラムにおいて観測されるピークの面積又は高さに基いて該目的成分を定量するデータ解析部と、
を備える。 (Section 8) One embodiment of the fat-soluble vitamin blood concentration measuring device according to the present invention measures vitamin D metabolites including 24,25-dihydroxyvitamin D 3 and vitamins including vitamin K 1 in a blood sample. A device for measuring the concentration of target components, which are K and its metabolites,
a protein removal processing unit that removes proteins from the blood sample by sequentially adding an organic solvent to the sample, stirring, and filtering the sample;
The sample processed by the protein removal processing unit is supplied to the solid-phase extraction column, and the target component in the sample is retained in the solid-phase extraction column, and then a mobile phase is supplied to extract the target component in the solid-phase extraction column. an online solid-phase extraction section for elution from a column for
A liquid chromatograph-tandem mass spectrometer having an ion source using an electrospray ionization method, which temporally separates components in a sample solution containing target components eluted from the solid phase extraction column, and a measurement execution unit that executes LC/MS analysis that selectively detects monovalent protonated ions originating from each target component;
a data analysis unit that creates a chromatogram for each target component using the data obtained by the measurement execution unit and quantifies the target component based on the area or height of a peak observed in the chromatogram; ,
Equipped with
第１項に記載の血中濃度測定方法及び第８項に記載の血中濃度測定装置によれば、変性法による除タンパク処理の後に血液サンプル中に残存する夾雑物に起因するイオンサプレッション効果を抑制することができ、目的成分のイオン化効率を高めることができる。それにより、煩雑で自動化が難しい、クックソン型誘導体化試薬等を用いた誘導体化処理を行うことなく、血中のビタミンＤ代謝物やビタミンＫ及びその代謝物を高い感度で以て且つ同時に測定することができる。また、オンライン固相抽出した目的成分を含む試料液をそのままＬＣ／ＭＳ分析することができるので、除タンパク処理から測定までの一連の作業を自動化することが容易である。それにより、測定のスループット向上と測定の安定性を図ることができ、スクリーニング検査などに好適に用いることができる。
According to the blood concentration measuring method described in item 1 and the blood concentration measuring device described in item 8, the ion suppression effect caused by impurities remaining in the blood sample after protein removal treatment by the denaturation method can be suppressed. ionization efficiency of the target component can be increased. As a result, vitamin D metabolites, vitamin K, and their metabolites in blood can be simultaneously measured with high sensitivity without performing derivatization using Cookson-type derivatization reagents, which are complicated and difficult to automate. be able to. Furthermore, since a sample solution containing target components subjected to online solid-phase extraction can be directly analyzed by LC/MS, it is easy to automate a series of operations from protein removal treatment to measurement. Thereby, it is possible to improve measurement throughput and measurement stability, and it can be suitably used for screening tests and the like.
（第２項）第１項に記載の血中濃度測定方法において、前記ビタミンＤ代謝物は、２５－ヒドロキシビタミンＤ2及び２５－ヒドロキシビタミンＤ3を含むものとし得る。
(Section 2) In the method for measuring blood concentration according to Item 1, the vitamin D metabolite may include 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 .
第２項に記載の血中濃度測定方法によれば、骨粗しょう症のスクリーニング検査等において特に重要である、主たるビタミンＤ代謝物の濃度測定を簡便に行うことができる。
According to the method for measuring blood concentration described in Section 2, it is possible to easily measure the concentration of the main vitamin D metabolite, which is particularly important in screening tests for osteoporosis.
（第３項）第１項又は第２項に記載の血中濃度測定方法において、前記ビタミンＫ及びその代謝物は、ビタミンＫ2ＭＫ－４及びビタミンＫ2ＭＫ－７を含むものとし得る。
(Section 3) In the method for measuring blood concentration according to Item 1 or 2, the vitamin K and its metabolites may include vitamin K 2 MK-4 and vitamin K 2 MK-7.
第３項に記載の血中濃度測定方法によれば、骨粗しょう症のスクリーニング検査等において特に重要である、主たるビタミンＫ代謝物の濃度測定を簡便に行うことができる。
According to the blood concentration measurement method described in Section 3, it is possible to easily measure the concentration of the main vitamin K metabolite, which is particularly important in screening tests for osteoporosis.
（第４項）第１項～第３項のいずれか１項に記載の血中濃度測定方法において、前記液体クロマトグラフ－タンデム型質量分析装置は、前記イオン源の次段に、イオンを収束しつつ後段へ輸送するＲＦイオンガイドを有し、
前記測定工程において、２４，２５－ジヒドロキシビタミンＤ3を測定する期間には、前記ＲＦイオンガイドに印加するＲＦ電圧の振幅を、２４，２５－ジヒドロキシビタミンＤ3由来の１価のプロトン付加イオンの検出に対応して予め決められた所定の値に設定するものとし得る (Section 4) In the method for measuring blood concentration according to any one ofItems 1 to 3, the liquid chromatograph-tandem mass spectrometer focuses ions on the next stage of the ion source. It has an RF ion guide that transports the ions to the subsequent stage while
In the measurement step, during the period of measuring 24,25-dihydroxyvitamin D 3 , the amplitude of the RF voltage applied to the RF ion guide is adjusted to the level of the monovalent protonated ion derived from 24,25-dihydroxyvitamin D 3 . It may be set to a predetermined value in response to detection.
前記測定工程において、２４，２５－ジヒドロキシビタミンＤ3を測定する期間には、前記ＲＦイオンガイドに印加するＲＦ電圧の振幅を、２４，２５－ジヒドロキシビタミンＤ3由来の１価のプロトン付加イオンの検出に対応して予め決められた所定の値に設定するものとし得る (Section 4) In the method for measuring blood concentration according to any one of
In the measurement step, during the period of measuring 24,25-dihydroxyvitamin D 3 , the amplitude of the RF voltage applied to the RF ion guide is adjusted to the level of the monovalent protonated ion derived from 24,25-dihydroxyvitamin D 3 . It may be set to a predetermined value in response to detection.
（第５項）第４項に記載の血中濃度測定方法において、前記所定の値は、前記ＲＦイオンガイドに導入された２４，２５－ジヒドロキシビタミンＤ3由来の各種イオンを１価のプロトン付加イオンに集約する作用を示すような値であるものとすることができる。
(Section 5) In the method for measuring blood concentration according to Item 4, the predetermined value is determined by monovalent protonation of various ions derived from 24,25-dihydroxyvitamin D 3 introduced into the RF ion guide. It can be set to a value that indicates the action of concentrating into ions.
２４，２５－ジヒドロキシビタミンＤ3は骨粗しょう症のスクリーニング検査等において重要なビタミンＤ代謝物であるものの、他のビタミンＤ代謝物に比べても血中濃度が低く、従来、測定することが難しい代謝物であった。これに対し、第４項及び第５項に記載の血中濃度測定方法によれば、オンライン固相抽出によりサンプル中の夾雑物を除去することでイオン化効率を向上させるのに加えて、ＲＦ電圧を適切に設定することで主としてイオンの種類の分散を抑制し、観測対象である１価のプロトン付加イオンの強度を高めることができる。それにより、血中に存在する２４，２５－ジヒドロキシビタミンＤ3の検出感度をさらに一層高めることができる。
Although 24,25-dihydroxyvitamin D3 is an important vitamin D metabolite in screening tests for osteoporosis, it has a low blood concentration compared to other vitamin D metabolites and is difficult to measure. It was a metabolite. On the other hand, according to the blood concentration measurement method described in Sections 4 and 5, in addition to improving the ionization efficiency by removing impurities in the sample by online solid-phase extraction, the RF voltage By appropriately setting , it is possible to mainly suppress the dispersion of ion types and increase the intensity of monovalent protonated ions to be observed. Thereby, the detection sensitivity of 24,25-dihydroxyvitamin D 3 present in blood can be further increased.
なお、第４項に記載の血中濃度測定方法において、前記測定工程では、２４，２５－ジヒドロキシビタミンＤ3以外の目的成分を測定する期間に、前記ＲＦイオンガイドに印加するＲＦ電圧の振幅を、前記液体クロマトグラフ－タンデム型質量分析装置において設定されている標準的な値に設定するものとし得る。
In the blood concentration measuring method according to item 4, in the measuring step, the amplitude of the RF voltage applied to the RF ion guide is changed during the period of measuring target components other than 24,25-dihydroxyvitamin D3 . , it may be set to a standard value set in the liquid chromatograph-tandem mass spectrometer.
ここでいう標準的な値とは、例えば特定の一種類の化合物を想定せずに予め装置メーカー等が定めた、平均的に良好な結果が得られる値、又は、標準試料等を用いた装置の自動チューニングの機能によって設定された値などである。即ち、この標準的な値とは、目的成分それぞれについて、例えば検出感度が最も高くなるように最適化された値ではない。
The standard value here is, for example, a value predetermined by an equipment manufacturer, etc., without assuming one specific type of compound, and which yields good results on average, or a value determined by an equipment using a standard sample, etc. For example, the value set by the automatic tuning function. That is, this standard value is not a value optimized for each target component so that the detection sensitivity is the highest, for example.
これによれば、２４，２５－ジヒドロキシビタミンＤ3以外の目的成分を測定する期間中、ＲＦイオンガイドに印加するＲＦ電圧を一定にすることができるので、例えば複数の目的成分の測定期間が重なっていて時分割で測定対象の目的成分を切り替える必要がある場合でも電圧の切替えに伴う待ち時間を設ける必要がない。それにより、一つの目的成分に対する繰り返し測定の時間間隔を短くすることができ、抽出イオンクロマトグラムのピーク波形の精度を向上させ、定量性を高めることができる。
According to this, the RF voltage applied to the RF ion guide can be kept constant during the period when target components other than 24,25-dihydroxyvitamin D 3 are measured, so that, for example, the measurement periods for multiple target components overlap. Even if it is necessary to switch the target component of the measurement target on a time-sharing basis, there is no need to provide a waiting time for switching the voltage. Thereby, the time interval between repeated measurements for one target component can be shortened, the accuracy of the peak waveform of the extracted ion chromatogram can be improved, and quantitative performance can be improved.
（第６項）第１項～第５項のいずれか１項に記載の血中濃度測定方法において、前記第２前処理工程では、固相抽出用カラムとしてＯＤＳカラムを用い、該カラムにサンプルを供給する移動相として水及びメタノールを用いるものとし得る。
(Section 6) In the method for measuring blood concentration according to any one of Items 1 to 5, in the second pretreatment step, an ODS column is used as the column for solid phase extraction, and the sample is placed on the column. Water and methanol may be used as the mobile phase to provide the .
第６項に記載の血中濃度測定方法によれば、血液に含まれる上記目的成分を効率良く捕集することができる一方、血液に含まれる夾雑物、特にイオンサプレッション効果をもたらすような夾雑物の残留を抑えることができる。それによって、血液中から夾雑物を良好に除去することができ、目的成分を高い感度で検出することができる。
According to the blood concentration measuring method described in item 6, the target components contained in blood can be efficiently collected, while contaminants contained in blood, particularly contaminants that cause an ion suppression effect, can be collected. The residual amount can be suppressed. Thereby, impurities can be effectively removed from the blood, and target components can be detected with high sensitivity.
（第７項）第１項～第６項のいずれか１項に記載の血中濃度測定方法において、前記液体クロマトグラフ－タンデム型質量分析装置の液体クロマトグラフは、ＰＦＰＰカラムを用いたものとし得る。
(Section 7) In the method for measuring blood concentration according to any one of Items 1 to 6, the liquid chromatograph of the liquid chromatograph-tandem mass spectrometer uses a PFPP column. obtain.
第７項に記載の血中濃度測定方法によれば、２４，２５－ジヒドロキシビタミンＤ3を含む複数種類のビタミンＤ代謝物、並びにビタミンＫ1と１種類以上のビタミンＫ代謝物とを良好に分離することができる。それによって、それら各目的成分の定量性を高めることができる。
According to the blood concentration measurement method described in Section 7, multiple types of vitamin D metabolites including 24,25-dihydroxyvitamin D 3 as well as vitamin K 1 and one or more types of vitamin K metabolites can be well detected. Can be separated. Thereby, the quantitative nature of each of these target components can be improved.
１…液体クロマトグラフ部（ＬＣ部）
１０…移動相貯留部
１１…送液ポンプ
１２…インジェクター
１３…カラム
２…タンデム型質量分析部（ＭＳ／ＭＳ部）
２０…チャンバー
２０１…イオン化室
２０２…第１中間真空室
２０３…第２中間真空室
２０４…高真空室
２１…ＥＳＩスプレー
２２…脱溶媒管
２３…第１ＲＦイオンガイド（Ｑアレー）
２４…スキマー
２５…第２ＲＦイオンガイド
２６…前段四重極マスフィルター
２７…コリジョンセル
２８…後段四重極マスフィルター
２９…イオン検出器
３…オンライン固相抽出（ＳＰＥ）部
３０…移動相貯留部
３１…送液ポンプ
３３…固相抽出用カラム
３４…流路切替部
４…除タンパク処理部
５…電圧発生部
６…データ処理部
６０…データ収集部
６１…定量演算部
７…制御部
７０…ビタミンＤ／Ｋ血中濃度測定用プログラム
８…操作部
９…表示部 1...Liquid chromatograph section (LC section)
10...Mobilephase storage unit 11...Liquid pump 12...Injector 13...Column 2...Tandem type mass spectrometry unit (MS/MS unit)
20...Chamber 201...Ionization chamber 202...First intermediate vacuum chamber 203...Second intermediate vacuum chamber 204...High vacuum chamber 21...ESI spray 22...Desolvation tube 23...First RF ion guide (Q array)
24...Skimmer 25... Second RF ion guide 26... Front stage quadrupole mass filter 27... Collision cell 28... Back stage quadrupole mass filter 29... Ion detector 3... Online solid phase extraction (SPE) section 30... Mobile phase storage section 31...Liquid pump 33...Column for solid phase extraction 34...Flow path switching section 4...Protein removal processing section 5...Voltage generation section 6...Data processing section 60...Data collection section 61...Quantitative calculation section 7...Control section 70... Vitamin D/K blood concentration measurement program 8...Operation section 9...Display section
１０…移動相貯留部
１１…送液ポンプ
１２…インジェクター
１３…カラム
２…タンデム型質量分析部（ＭＳ／ＭＳ部）
２０…チャンバー
２０１…イオン化室
２０２…第１中間真空室
２０３…第２中間真空室
２０４…高真空室
２１…ＥＳＩスプレー
２２…脱溶媒管
２３…第１ＲＦイオンガイド（Ｑアレー）
２４…スキマー
２５…第２ＲＦイオンガイド
２６…前段四重極マスフィルター
２７…コリジョンセル
２８…後段四重極マスフィルター
２９…イオン検出器
３…オンライン固相抽出（ＳＰＥ）部
３０…移動相貯留部
３１…送液ポンプ
３３…固相抽出用カラム
３４…流路切替部
４…除タンパク処理部
５…電圧発生部
６…データ処理部
６０…データ収集部
６１…定量演算部
７…制御部
７０…ビタミンＤ／Ｋ血中濃度測定用プログラム
８…操作部
９…表示部 1...Liquid chromatograph section (LC section)
10...Mobile
20...
24...
Claims (8)
- 血液サンプル中の、２４，２５－ジヒドロキシビタミンＤ3を含むビタミンＤ代謝物、並びにビタミンＫ1を含むビタミンＫ及びその代謝物、である目的成分の濃度を測定する方法であって、
血液サンプルに対し変性法により除タンパク処理を行う第１前処理工程と、
前記第１前処理工程後のサンプルに含まれる夾雑物を固相抽出により除去する第２前処理工程と、
エレクトロスプレーイオン化法によるイオン源を有する液体クロマトグラフ－タンデム型質量分析装置を用いて、前記第２前処理工程後のサンプル中の成分を時間的に分離しつつ、前記目的成分それぞれに由来する１価のプロトン付加イオンを選択的に検出するＬＣ／ＭＳ分析を実行する測定工程と、
を有する脂溶性ビタミン類の血中濃度測定方法。 A method for measuring the concentration of target components, which are vitamin D metabolites, including 24,25-dihydroxyvitamin D3 , and vitamin K and its metabolites, including vitamin K1 , in a blood sample, the method comprising:
a first pretreatment step of performing protein removal treatment on the blood sample by a denaturation method;
a second pretreatment step of removing impurities contained in the sample after the first pretreatment step by solid phase extraction;
Using a liquid chromatograph-tandem mass spectrometer equipped with an ion source using electrospray ionization, the components in the sample after the second pretreatment step are temporally separated, and the components derived from each of the target components are separated in time. a measurement step of performing LC/MS analysis to selectively detect protonated ions of valence;
A method for measuring blood concentration of fat-soluble vitamins. - 前記ビタミンＤ代謝物は、２５－ヒドロキシビタミンＤ2及び２５－ヒドロキシビタミンＤ3を含む、請求項１に記載の脂溶性ビタミン類の血中濃度測定方法。 2. The method for measuring blood concentration of fat-soluble vitamins according to claim 1, wherein the vitamin D metabolites include 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 .
- 前記ビタミンＫ及びその代謝物は、ビタミンＫ2ＭＫ－４及びビタミンＫ2ＭＫ－７を含む、請求項２に記載の脂溶性ビタミン類の血中濃度測定方法。 The method for measuring blood concentration of fat-soluble vitamins according to claim 2, wherein the vitamin K and its metabolites include vitamin K 2 MK-4 and vitamin K 2 MK-7.
- 前記液体クロマトグラフ－タンデム型質量分析装置は、前記イオン源の次段に、イオンを収束しつつ後段へ輸送するＲＦイオンガイドを有し、
前記測定工程において、２４，２５－ジヒドロキシビタミンＤ3を測定する期間には、前記ＲＦイオンガイドに印加するＲＦ電圧の振幅を、２４，２５－ジヒドロキシビタミンＤ3由来の１価のプロトン付加イオンの検出に対応して予め決められた所定の値に設定する、請求項１～３のいずれか１項に記載の脂溶性ビタミン類の血中濃度測定方法。 The liquid chromatograph-tandem mass spectrometer has an RF ion guide next to the ion source that focuses ions and transports them to a subsequent stage,
In the measurement step, during the period of measuring 24,25-dihydroxyvitamin D 3 , the amplitude of the RF voltage applied to the RF ion guide is adjusted to the level of the monovalent protonated ion derived from 24,25-dihydroxyvitamin D 3 . The method for measuring blood concentration of fat-soluble vitamins according to any one of claims 1 to 3, wherein the blood concentration of fat-soluble vitamins is set to a predetermined value corresponding to the detection. - 前記所定の値は、前記ＲＦイオンガイドに導入された２４，２５－ジヒドロキシビタミンＤ3由来の各種イオンを１価のプロトン付加イオンに集約する作用を示すような値である、請求項４に記載の脂溶性ビタミン類の血中濃度測定方法。 5. The predetermined value is a value that exhibits an effect of concentrating various ions derived from 24,25-dihydroxyvitamin D 3 introduced into the RF ion guide into monovalent protonated ions. A method for measuring blood levels of fat-soluble vitamins.
- 前記第２前処理工程では、固相抽出用カラムとしてＯＤＳカラムを用い、該カラムにサンプルを供給する移動相として水及びメタノールを用いる、請求項３に記載の脂溶性ビタミン類の血中濃度測定方法。 The blood concentration measurement of fat-soluble vitamins according to claim 3, wherein in the second pretreatment step, an ODS column is used as a column for solid phase extraction, and water and methanol are used as a mobile phase for supplying the sample to the column. Method.
- 前記液体クロマトグラフ－タンデム型質量分析装置の液体クロマトグラフは、ＰＦＰＰカラムを用いたものである、請求項６に記載の脂溶性ビタミン類の血中濃度測定方法。 The method for measuring blood concentration of fat-soluble vitamins according to claim 6, wherein the liquid chromatograph of the liquid chromatograph-tandem mass spectrometer uses a PFPP column.
- 血液サンプル中の、２４，２５－ジヒドロキシビタミンＤ3を含むビタミンＤ代謝物、並びにビタミンＫ1を含むビタミンＫ及びその代謝物、である目的成分の濃度を測定するための装置であって、
血液サンプルに対し有機溶媒の添加、撹拌、及び濾過を順に行うことにより該サンプル中のタンパクを除去する除タンパク処理部と、
前記除タンパク処理部による処理済みのサンプルを固相抽出用カラムに供給し該サンプル中の目的成分を該固相抽出用カラムに保持したあと、移動相を供給して目的成分を該固相抽出用カラムから溶出させるオンライン固相抽出部と、
エレクトロスプレーイオン化法によるイオン源を有する液体クロマトグラフ－タンデム型質量分析装置であって、前記固相抽出用カラムから溶出した目的成分が含まれる試料液中の成分を時間的に分離しつつ、前記目的成分それぞれに由来する１価のプロトン付加イオンを選択的に検出するＬＣ／ＭＳ分析を実行する測定実行部と、
前記測定実行部により得られたデータを利用して前記目的成分毎にクロマトグラムを作成し、該クロマトグラムにおいて観測されるピークの面積又は高さに基いて該目的成分を定量するデータ解析部と、
を備える脂溶性ビタミン類の血中濃度測定装置。 An apparatus for measuring the concentration of target components, which are vitamin D metabolites including 24,25-dihydroxyvitamin D3 , and vitamin K and its metabolites including vitamin K1 , in a blood sample, comprising:
a protein removal processing unit that removes proteins from the blood sample by sequentially adding an organic solvent to the sample, stirring, and filtering the sample;
The sample processed by the protein removal processing unit is supplied to the solid-phase extraction column, and the target component in the sample is retained in the solid-phase extraction column, and then a mobile phase is supplied to extract the target component in the solid-phase extraction column. an online solid-phase extraction section for elution from a column for
A liquid chromatograph-tandem mass spectrometer having an ion source using an electrospray ionization method, which temporally separates components in a sample solution containing target components eluted from the solid phase extraction column, and a measurement execution unit that executes LC/MS analysis that selectively detects monovalent protonated ions originating from each target component;
a data analysis unit that creates a chromatogram for each target component using the data obtained by the measurement execution unit and quantifies the target component based on the area or height of a peak observed in the chromatogram; ,
A device for measuring blood concentration of fat-soluble vitamins.
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