Method and device for measuring blood concentrations of fat-soluble vitamins

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

[Technical Field]

[0001] 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, etc. [Background technology]

[0002] Vitamins are important nutrients for human survival and growth. It is generally known that deficiencies of vitamins D and K can impair the mineralization of bone matrix, accelerate the progression of osteoporosis, and increase the risk of fractures.

[0003] A known method for assessing vitamin D sufficiency is to measure the blood concentrations of vitamin D metabolites such as 24,25-dihydroxyvitamin D3 (hereinafter sometimes referred to as "24,25-(OH)2VitD3" or "24,25-(OH)2D3", etc.), 25-hydroxyvitamin D2 (hereinafter sometimes referred to as "25-(OH)VitD2" or "25-(OH)D2", etc.), and 25-hydroxyvitamin D3 (hereinafter sometimes referred to as "25-(OH)VitD3" or "25-(OH)D3", etc.) using a liquid chromatograph-tandem mass spectrometer (hereinafter sometimes abbreviated as "LC-MS / MS").

[0004] In LC-MS / MS for such measurements, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are widely used as ionization methods. ESI is the most commonly used ionization method in LC-MS / MS due to its applicability to a wide range of compounds and ease of instrument handling. However, as described in Patent Document 1 and elsewhere, vitamin D metabolites, particularly 24,25-dihydroxyvitamin D3, tend to have low ESI response and low ionization efficiency. Furthermore, the concentration of 24,25-dihydroxyvitamin D3 in blood is typically quite low. Therefore, LC-MS / MS using ESI often lacks detection sensitivity. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2018-54459 [Patent Document 2] Japanese Patent Application Publication No. 2018-81023 Summary of the Invention [Problem to be solved by the invention]

[0006] In response to this, a method for measuring vitamin D metabolites after derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), a Cookson-type derivatization reagent, has been known, as disclosed in Patent Documents 1 and 2. This derivatization improves the ionization efficiency of vitamin D metabolites, thereby enabling increased detection sensitivity.

[0007] However, these measurement methods require the blood sample to be deproteinized using solid-phase extraction or liquid-liquid extraction, followed by the complicated derivatization process described above. This makes the measurement time-consuming, labor-intensive, and costly. Furthermore, the complex workflow makes automated analysis without manual intervention difficult. Therefore, it is difficult to improve measurement throughput and reduce the risk of infection and other risks to workers.

[0008] Furthermore, in screening tests aimed at assessing the risk of osteoporosis, it is necessary to measure vitamin K (vitamin K1) and vitamin K metabolites such as vitamin K2MK-4 and vitamin K2MK-7, as well as the vitamin D metabolites described above, quickly and with as high a sensitivity as possible. To achieve this, it is necessary to simultaneously measure vitamin D metabolites, vitamin K, and its metabolites in a blood sample in a single measurement with high sensitivity, but such a measurement method has not been known until now.

[0009] The present invention has been made to solve these problems, and its main object is to provide a measurement method and a measurement device that can simultaneously measure fat-soluble vitamins such as vitamin D metabolites and vitamin K with high detection sensitivity without performing a complicated derivatization process that is difficult to automate. [Means for solving the problem]

[0010] One embodiment of the method for measuring blood concentrations of fat-soluble vitamins according to the present invention, which has been made to solve the above problems, is a method for measuring the concentrations 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 first pretreatment step of deproteinizing the blood sample by a denaturing method; a second pretreatment step of removing impurities contained in the sample after the first pretreatment step by solid phase extraction; a measuring step of performing LC / MS analysis using a liquid chromatograph-tandem mass spectrometer having an ion source based on electrospray ionization, in which components in the sample after the second pretreatment step are separated in time and singly charged protonated ions derived from each of the target components are selectively detected; It has.

[0011] 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-mentioned problems, is a device for measuring the concentrations 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, and a protein removal treatment section for removing proteins from a blood sample by sequentially adding an organic solvent to the sample, stirring the sample, and filtering the sample; an online solid-phase extraction unit that supplies the sample treated by the protein removal treatment unit to a solid-phase extraction column, retains the target component in the sample on the solid-phase extraction column, and then supplies a mobile phase to elute the target component from the solid-phase extraction column; a liquid chromatograph-tandem mass spectrometer having an ion source employing an electrospray ionization method, the liquid chromatograph-tandem mass spectrometer comprising: a measurement execution unit that executes an LC / MS analysis to selectively detect singly protonated ions derived from each of the target components while temporally separating components in a sample solution containing the target components eluted from the solid-phase extraction column; a data analysis unit that creates a chromatogram for each of the target components using the data obtained by the measurement execution unit and quantifies the target components based on the area or height of a peak observed in the chromatogram; Equipped with. [Effects of the Invention]

[0012] The present inventors conducted various experiments to find a method for highly sensitively detecting 24,25-dihydroxyvitamin D3, which is particularly difficult to detect among vitamin D metabolites, and discovered that one of the major factors behind the low sensitivity is the ion suppression effect of impurities such as lipids remaining in blood samples after deproteinization. In response to this, the inventors experimentally verified various methods for removing impurities and found that solid-phase extraction using a solid-phase extraction column can effectively and stably (with high reproducibility) remove impurities that particularly contribute to ion suppression, leading to the present invention.

[0013] The above-described aspects of the method and device for measuring blood concentrations of fat-soluble vitamins according to the present invention can suppress the ion suppression effect caused by impurities remaining in a blood sample after deproteinization by denaturation, thereby increasing the ionization efficiency of target components. As a result, vitamin D metabolites and vitamin K and their metabolites in blood can be measured simultaneously with high sensitivity without the need for a complicated and difficult-to-automate derivatization process using a Cookson-type derivatization reagent or the like.

[0014] Furthermore, according to the above-described aspects of the method and device for measuring blood concentrations of fat-soluble vitamins of the present invention, a sample solution containing a target component extracted online using solid-phase extraction can be directly analyzed by LC / MS, making it easy to automate a series of steps from deproteinization to measurement, thereby improving measurement throughput and stability, and making the device suitable for use in screening tests, etc. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a flowchart showing an example of a processing procedure of a method for measuring blood concentrations of fat-soluble vitamins according to one embodiment of the present invention. [Figure 2] 1 is a schematic block diagram of an apparatus for measuring blood concentrations of fat-soluble vitamins according to one embodiment of the present invention. [Figure 3]FIG. 1 shows examples of extracted ion chromatograms of vitamin D metabolites, vitamin K, and vitamin K metabolites obtained using the measurement device of this embodiment. [Figure 4] FIG. 1 shows an example of actual measurements of extracted ion chromatograms for multiple MRM transitions of 24,25-dihydroxyvitamin D3 obtained using the measurement device of this embodiment. [Figure 5] FIG. 1 shows an example of actual measurements of extracted ion chromatograms for multiple MRM transitions of 25-hydroxyvitamin D3 obtained using the measurement device of this embodiment. [Figure 6] FIG. 1 shows an example of actual measurements of extracted ion chromatograms for multiple MRM transitions of 25-hydroxyvitamin D2 obtained using the measurement device of this embodiment. [Figure 7] FIG. 1 shows an example of actual measurements of extracted ion chromatograms for multiple MRM transitions of vitamin K1 obtained using the measurement device of this embodiment. [Figure 8] FIG. 1 shows an example of actual measurements of extracted ion chromatograms for multiple MRM transitions of vitamin K2MK-4 obtained using the measurement device of this embodiment. [Figure 9] FIG. 1 shows an example of actual measurements of extracted ion chromatograms for multiple MRM transitions of vitamin K2MK-7, obtained using the measurement device of this embodiment. [Figure 10] FIG. 1 is a diagram showing an example of a calibration curve for 24,25-dihydroxyvitamin D3 obtained using the measurement device of this embodiment. [Figure 11] FIG. 1 is a diagram showing an example of a calibration curve for vitamin K1 obtained using the measurement device of this embodiment. [Figure 12] FIG. 1 shows the reproducibility results (low concentration control) of measurements of vitamin D metabolites, vitamin K, and vitamin K metabolites obtained using the measurement device of this embodiment. [Figure 13]FIG. 1 shows the reproducibility results (high concentration control) of measurements of vitamin D metabolites, vitamin K, and vitamin K metabolites obtained using the measurement device of this embodiment. [Figure 14] FIG. 1 shows an example of a chromatogram obtained using the measuring device of this embodiment, showing the effect of piping cleaning on reducing carryover of vitamin K and vitamin K metabolites. [Figure 15] FIG. 10 shows the results of comparing the retention time, peak area, peak height, and S / N ratio of 24,25-dihydroxyvitamin D3 when the RF voltage applied to the RF ion guide (Q array) is changed in the measurement device of this embodiment. [Figure 16] FIG. 1 shows an example of an extracted ion chromatogram measured for 24,25-dihydroxyvitamin D3 when the RF voltage applied to the RF ion guide (Q array) is changed in the measurement device of this embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0016] [Examples of means] In the present invention, the blood sample may be, for example, whole blood, serum, plasma, etc. The blood sample may also be collected from animals other than humans.

[0017] In the present invention, vitamin D metabolites include at least 24,25-dihydroxyvitamin D3 and may also include 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, etc. Meanwhile, in the present invention, vitamin K and its metabolites include at least vitamin K1 and may also include vitamin K2MK-4, vitamin K2MK-7, etc.

[0018] In the present invention, the deproteinization treatment by the denaturing method is a method in which an organic solvent is added to a blood sample to insolubilize and precipitate proteins, and examples of the organic solvent that can be used include methanol, ethanol, acetone, and acetonitrile.

[0019] In the present invention, the solid-phase extraction method involves, for example, first supplying a sample to a solid-phase extraction column to allow the target component to adsorb to the column's packing. Components that do not adsorb to the packing are then discharged. A mobile phase with elution properties is then supplied to the solid-phase extraction column, allowing the target component adsorbed to elute, resulting in a sample solution that is then sent directly to a liquid chromatograph column. This series of operations can be performed primarily by switching the flow path using valves, and online solid-phase extraction involves automatically and sequentially performing these operations. After the target component has been adsorbed to the solid-phase extraction column, a wash solution may be passed through the column, and components that were not adsorbed to the column but remain in the piping containing the column may be discharged.

[0020] In the present invention, the tandem mass spectrometer of the liquid chromatograph-tandem mass spectrometer can be, for example, a triple quadrupole mass spectrometer, a quadrupole time-of-flight mass spectrometer, an ion trap time-of-flight mass spectrometer, or an ion trap mass spectrometer equipped with an electrospray ion source.

[0021] Tandem mass spectrometers also have the function of dissociating ions generated in the ion source, but the method of dissociating ions is not particularly limited. Typically, collision-induced dissociation can be used as the method of dissociating ions.

[0022] In the present invention, the analysis for selectively detecting singly protonated ions derived from each target component is typically a multiple reaction monitoring (MRM) measurement targeting singly protonated product ions generated by ion dissociation. While MRM measurements are generally performed using a triple quadrupole mass spectrometer, when a product ion spectrum spanning a predetermined m / z range is obtained using a quadrupole-time-of-flight mass spectrometer, the intensities of ions with specific mass-to-charge ratios (m / z) can be obtained from the spectral data, and thus this can be considered to be essentially an MRM measurement.

[0023] Furthermore, the blood concentration measurement device for fat-soluble vitamins according to the present invention can be specifically configured by combining, for example, Shimadzu Corporation's fully automatic LCMS preprocessing device "CLAM-2030," Shimadzu Corporation's high-performance liquid chromatograph "Nexera series," Shimadzu Corporation's liquid chromatograph mass spectrometer "LCMS-8060NX," and a personal computer equipped with predetermined software for controlling them and processing data. In this case, the high-performance liquid chromatograph unit configuration can be configured to support online solid-phase extraction. Of course, the blood concentration measurement device according to the present invention can also be configured using various other devices and units.

[0024] [Measuring method and measuring device according to an embodiment] Hereinafter, an embodiment of a blood concentration measurement method and a measurement device according to the present invention will be described with reference to the accompanying drawings.

[0025] <Outline of measurement method> 1 is a flowchart showing an example of the processing procedure of the blood concentration measurement method according to this embodiment. The measurement method of this embodiment will be outlined with reference to FIG. In this example, the sample is serum extracted from blood collected from a subject, and the target components to be measured in this example are six types: 24,25-dihydroxyvitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin K1, vitamin K2MK-4, and vitamin K2MK-7.

[0026] First, a deproteinization process is performed on the blood sample as a pretreatment (step S1). For example, a denaturation method using a polar organic solvent that is miscible with water can be used for the deproteinization process. Examples of polar organic solvents that can be used include methanol, ethanol, and acetonitrile. In the deproteinization process, a specific polar organic solvent is added to the blood sample to be treated, followed by stirring and filtration using a filter or the like. This removes proteins precipitated by denaturation, and the supernatant is collected to obtain a deproteinized blood sample. When adding an organic solvent to the blood sample, an internal standard substance for calibration can also be added.

[0027] Next, impurities such as lipids contained in the deproteinized 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 captured by the packing material of the solid-phase extraction column. Most of the impurities in the blood sample are not captured by the packing material and are therefore passed through the solid-phase extraction column and discharged. Some impurities are not adsorbed by the packing material, but may adhere to the packing material or to the inside of the piping. Therefore, remaining impurities may be washed away by flowing a rinse solution that does not affect the target components through the piping containing the solid-phase extraction column.

[0028] Next, a mobile phase with elution properties is passed through the solid-phase extraction column to elute the target components adsorbed on the column, and the sample liquid containing the target components is carried along with the mobile phase flow and sent to the LC column. The target components in the sample liquid are separated in the time direction as they pass through the column (step S3).

[0029] The eluate from the column containing the separated target components is introduced into the ion source of a tandem mass spectrometer. The ion source utilizes ESI. Contaminants other than the target components contained in the blood sample cause ion suppression during ionization by ESI. In contrast, in this measurement method, the contaminants are effectively removed in step S2, suppressing the ion suppression effect caused by the contaminants and increasing the ionization efficiency of the target components. This allows more ions derived from the target components to be subjected to mass analysis. In the tandem mass spectrometer, for each target component, an MRM measurement (MS / MS analysis) is performed (step S4) targeting one or more MRM transitions (combinations of m / z values ​​of precursor ions and product ions) predetermined for that target component during the period in which the target component is eluted from the column (i.e., introduced into the ion source).

[0030] The measurements in steps S3 and S4 provide ion intensity data for one or more MRM transitions for each target component within a predetermined time range centered on its retention time. As described above, this measurement method provides data with high sensitivity and a good signal-to-noise ratio because the amount of ions derived from the target component subjected to mass spectrometry is relatively large. Quantitative analysis based on this data is then performed to calculate the concentration of each target component in the blood sample (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. The area or height of this chromatographic peak is then calculated, and the concentration of each target component is calculated using a calibration curve created in advance.

[0031] As described above, this measurement method can obtain the concentrations of six target components contained in a blood sample as measurement results.

[0032] <Device configuration> Next, the configuration and operation of an apparatus for carrying out the measurement method outlined above will be described. 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 measuring device includes a liquid chromatograph (LC) unit 1, a tandem mass spectrometry (MS / MS) unit 2, an online solid-phase extraction (SPE) unit 3, a deproteinization processing unit 4, a voltage generating unit 5, a data processing unit 6, a control unit 7, an operation unit 8, and a display unit 9.

[0033] The LC unit 1 includes a mobile phase reservoir 10, a pump 11, an injector 12, and a column 13. Although not shown here to avoid cluttering the drawing, in the actual measurement example described below, gradient elution is performed using two different mobile phases. To perform gradient elution, another mobile phase reservoir, a pump, a mixer, and other components are added to the configuration shown in Figure 2.

[0034] For example, a PFPP (Pentafluorophenylpropyl) column can be used as column 13. A PFPP column is a column into which PFPP groups have been introduced, and in recent years has been frequently used for separating components derived from living organisms as a reversed-phase column that exhibits good separation performance not only for hydrophobic compounds but also for hydrophilic compounds.

[0035] 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 sections, 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. The first intermediate vacuum chamber 202 is evacuated by a rotary pump (not shown), and a low vacuum atmosphere (for example, 10 2 The second intermediate vacuum chamber 203 is evacuated by a rotary pump and a turbo molecular pump (both not shown) and is maintained in a medium vacuum atmosphere (for example, 10 -1 ~10 -2 The high vacuum chamber 204 is also evacuated by a rotary pump and a turbo molecular pump (both not shown) to maintain a high vacuum atmosphere (for example, 10 -3 ~10 -4In this way, a multi-stage differential pumping system is adopted in which the degree of vacuum increases in order from the ionization chamber 201, which is at approximately atmospheric pressure.

[0036] An ESI spray 21 is disposed within the ionization chamber 201, and the ionization chamber 201 and a first intermediate vacuum chamber 202 are connected by a small-diameter desolvation tube 22. A first RF ion guide 23 that focuses and transports ions by the action of an RF (Radio Frequency) electric field is disposed within the first intermediate vacuum chamber 202, and the first intermediate vacuum chamber 202 and the second intermediate vacuum chamber 203 are connected by a small-diameter ion passage hole formed at the top of a skimmer 24. The first RF ion guide (hereinafter sometimes referred to as a "Q array" due to the structural features described below) 23 is composed of multiple (four in this example) rod electrodes arranged at approximately equal angular intervals to surround the ion optical axis C, and each rod electrode is structured to be composed of multiple short cylindrical (or disk-shaped) partial electrodes separated in the extension direction of the ion optical axis C. Of the multiple separated partial electrodes, the partial electrode located on the skimmer 24 side has a smaller radius of the inscribed circle centered on the ion optical axis C, thereby enabling ions to be efficiently focused near the ion optical axis C.

[0037] Inside the second intermediate vacuum chamber 203, there is arranged a second RF ion guide 25 consisting of a plurality of (e.g., eight) rod electrodes that are arranged at approximately equal angular intervals surrounding the ion optical axis C and that are parallel to the ion optical axis C. The second intermediate vacuum chamber 203 and the high vacuum chamber 204 are connected by a small-diameter ion passage hole.

[0038] Inside the high-vacuum chamber 204, a pre-quadrupole mass filter 26, a collision cell 27, a post-quadrupole mass filter 28, and an ion detector 29 are arranged in this order along the ion optical axis C. Each of the pre-quadrupole mass filter 26 and the post-quadrupole mass filter 28 includes four rod electrodes arranged parallel to the ion optical axis C at approximately equal angular intervals, surrounding the ion optical axis C. Here, pre-rod electrodes are provided before the main rod electrodes, but this can be omitted. Furthermore, post-rod electrodes may be provided after the main rod electrodes, if necessary. Inside the collision cell 27, an ion guide consisting of multiple rod electrodes is arranged parallel to the ion optical axis C at approximately equal angular intervals, surrounding the ion optical axis C. Furthermore, an inert CID gas, such as argon, is introduced into the collision cell 27 from the outside.

[0039] The online SPE unit 3 is essentially provided in front of the LC unit 1, and includes a mobile phase reservoir 30, a liquid delivery pump 31, an injector 12, a solid-phase extraction column 33, and a flow path switching unit 34 including a plurality of valves. The injector 12 is included in the LC unit 1. The solid-phase extraction column 33 may be, for example, a column having an ODS (Octa Decyl Silyl) group (C 18 H 37 An ODS column packed with chemically bonded porous spherical silica gel whose surface is modified with Si) as the stationary phase can be used.

[0040] The protein removal processing unit 4 is provided upstream of the online SPE unit 3. The actual entity of the protein removal processing unit 4 can be, for example, a sample pretreatment device that can perform various operations and processes on a sample contained in a vial, such as sample dispensing, reagent dispensing, stirring, filtration, heating, and transport to a sample collection position using an injector 12. In such a sample pretreatment device, protein removal can be performed by using predetermined reagents prepared in advance and performing each operation and process according to preset conditions. For example, the protein removal processing unit 4 can be realized using the fully automated LCMS pretreatment device "CLAM-2030" manufactured by Shimadzu Corporation, as mentioned above.

[0041] Under the control of the control unit 7, the voltage generation unit 5 applies a predetermined voltage to each unit of the MS / MS unit 2. 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.

[0042] The data processing unit 6 receives and processes detection signals 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.

[0043] The control unit 7 controls the protein removal processing unit 4, online SPE unit 3, LC unit 1, voltage generation unit 5, MS / MS unit 2, and data processing unit 6, thereby causing the measurement device to perform a series of processes from pretreatment of the blood sample to measurement and data analysis. The control unit 7 also has the function of input / output control via the operation unit 8 and display unit 9, which serve as user interfaces.

[0044] In this measuring device, the data processing unit 6 and control unit 7 are implemented using a personal computer as a hardware resource, and their respective functions can be realized by running dedicated data processing and control software pre-installed on the computer. The data processing and control software may include, for example, software for general measurement without limiting the measurement purpose or the components to be measured (although this software itself may consist of multiple pieces of software), and dedicated software for measuring the vitamin D metabolites and other components to be measured here. In Figure 2, this latter software is shown as a vitamin D / K blood concentration measurement program 70.

[0045] <Device operation during measurement> The operation of the measuring device shown in FIG. 2 when carrying out the above-described measuring method will be described. First, the user places a blood sample, which is a specimen, in a predetermined position in the protein removal processing unit 4 and performs a predetermined operation using the operation unit 8. In response to this operation, the control unit 7 operates each unit to perform a series of measurements.

[0046] First, the protein removal unit 4 sequentially performs operations such as dispensing a blood sample, dispensing a reagent into the blood sample, stirring, and filtering to obtain a blood sample from which proteins have been removed. When processing multiple blood samples, this protein removal process and subsequent measurement operations, including online 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.

[0047] In the online SPE unit 3, the liquid delivery pump 31 draws mobile phase from the mobile phase reservoir 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 deproteinized 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 by 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 and pass through the column 33, and are discharged via the flow path switching unit 34. As a result, the target components are collected in the solid-phase extraction column 33.

[0048] However, even if the impurities are not adsorbed to the packing material, some of the impurities may remain attached to the packing material or inside the piping. Therefore, for example, a cleaning solution may be aspirated from a cleaning solution reservoir (not shown) by a liquid delivery pump 31 and passed through the solid-phase extraction column 33 to a drain. This allows the impurities adhering to the packing material and inside the piping to be washed away, thereby improving the purity of the target component. During the period when the target component is being collected by the solid-phase extraction column 33, the mobile phase aspirated by the liquid delivery pump 11 may be passed through the column 13 using a flow path not shown in FIG. 2.

[0049] Next, the flow path is switched by the flow path switching unit 34, and the mobile phase sucked from the mobile phase reservoir 10 by the liquid delivery pump 11 is supplied to the solid-phase extraction column 33. The six target components collected in the solid-phase extraction column 33 are eluted into the mobile phase and are carried by the flow of the mobile phase into the column 13. The six target components introduced into the column 13 approximately simultaneously are separated in the time direction while passing through the column 13 and are eluted from the outlet of the column 13. The LC analysis conditions at this time, such as the type of mobile phase and the flow rate of the mobile phase, are specified in the vitamin D / K blood concentration measurement program 70.

[0050] Under the control of the control unit 7, the MS / MS unit 2 operates to perform MRM measurements targeting one or more MRM transitions corresponding to each of the six target components within a predetermined time range centered on the retention time of each of the six target components.

[0051] When an eluate containing one target component is introduced from the LC section 1 into the MS / MS section 2, a polarity-biased charge is imparted to the eluate in the ESI spray 21, and the eluate is sprayed into the ionization chamber 21, which is at approximately atmospheric pressure. The sprayed charged droplets come into contact with gas molecules and are atomized, and in the process of evaporation of the solvent from the charged droplets, the molecules of the target component are released with an electric charge and become gaseous ions. The generated ions are sucked into the desolvation tube 22 together with the minute charged droplets, and sent to the first intermediate vacuum chamber 202. The desolvation tube 22 is heated, which promotes evaporation from the droplets even within the desolvation tube 22, thereby promoting ionization of the target component.

[0052] Ions derived from the target component that enter the first intermediate vacuum chamber 202 are trapped in an RF electric field formed by the RF voltage applied to the first RF ion guide 23 from the voltage generator 5, and are focused near the ion optical axis C. The first intermediate vacuum chamber 202, which is the next stage after the ionization chamber 201, has a low degree of vacuum and contains many residual gas molecules, so ions derived from the target component are likely to come into contact with the residual gas molecules. This contact attenuates the kinetic energy of the ions derived from the target component, so the ions derived from the target component are easily trapped in the RF electric field and are well focused.

[0053] As is well known, ESI is a soft ionization method, which makes it difficult for ions to dissociate (fragment) during ionization. However, it is likely to generate not only singly protonated ions but also multiply charged ions and adduct ions with alkali metals or the like. Particularly when the amount of target component molecules is small, the increase in the number of ions derived from the target component during ionization is disadvantageous in terms of improving the detection sensitivity for specific MRM transitions. In response to this, the inventors discovered that the intensity of the singly protonated ions to be observed can be improved by adjusting the amplitude of the RF voltage applied to the first RF ion guide 23. Therefore, in the measurement apparatus of this embodiment, when measuring 24,25-dihydroxyvitamin D3, which requires particularly high detection sensitivity, a predetermined RF voltage is applied to the first RF ion guide 23 to increase the detection sensitivity for singly protonated ions. This point will be explained in detail later.

[0054] Ions derived from the target component focused by the RF electric field of the first RF ion guide 23 pass through a 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 the 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. Within the high vacuum chamber 204, the voltage generator 5 applies voltages to the front quadrupole mass filter 26 and the rear quadrupole mass filter 28, respectively, so as to selectively pass specific m / z values ​​in the MRM transition associated with the target component. CID gas is continuously or intermittently introduced into the collision cell 27 so that the CID gas pressure reaches a predetermined value.

[0055] Of the various ions introduced into the high vacuum chamber 204, only ions having a specific m / z value derived from the target component pass through the front quadrupole mass filter 26 and enter the collision cell 27 as precursor ions with a predetermined energy. The precursor ions that enter the collision cell 27 come into contact with the CID gas, causing dissociation to generate various product ions. Of the various product ions generated, only product ions having a specific m / z value pass through the rear quadrupole mass filter 28 and enter the ion detector 29. Ion detector 29 The LC unit 1 generates a detection signal every moment according to the amount of incident ions and sends it to the data processing unit 6. The concentration of one target component in the eluate sent from the LC unit 1 to the MS / MS unit 2 changes over time in a roughly mountain-like manner (theoretically according to a Gaussian distribution), so the detection signal (ion intensity signal) sent to the data processing unit 6 also changes over time in a roughly mountain-like manner.

[0056] The MS / MS unit 2 performs MRM measurement for each target component, targeting one or more MRM transitions that vary for each target component within a time range corresponding to that target component. This results in one or more detection signals for each target component that indicate the temporal changes in ion intensity, as described above. The MS / MS analysis conditions, including the MRM transitions, used in the MS / MS unit 2 are specified in the vitamin D / K blood concentration measurement program 70, similar to the LC analysis conditions. Therefore, when this program 70 is installed in a computer and measurements are performed, the user does not need to manually set the individual LC analysis conditions or MS / MS analysis conditions. Of course, the user may manually set the individual LC analysis conditions or MS / MS analysis conditions before performing the measurements.

[0057] The detection signals acquired by the MS / MS unit 2 are converted into digital data and stored in the data storage unit of the data acquisition unit 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 acquisition unit 60, and calculates peak information such as the retention time, area value, and height value of the peak observed in the chromatogram. The quantitative calculation unit 61 then confirms the target component from the peak retention time, and calculates the concentration from the peak area value or height value using a calibration curve created in advance. Note that, during quantification, peak area values ​​and height values ​​obtained by simultaneously measuring an internal standard substance added to the blood sample during deproteinization treatment can be used.

[0058] In this manner, the blood concentrations of six target components for one blood sample are determined. The measurement device shown in Figure 2 can obtain measurement results for each blood sample by automatically performing pretreatment and measurement according to the above-described procedure on a number of blood samples prepared in advance. The control unit 7 can display the measurement results thus obtained on the screen of the display unit 9 in a predetermined format.

[0059] <Measurement example> Next, an example of actual measurement using the measurement device of this embodiment will be described, showing specific parameter values ​​and the like.

[0060] The protein removal treatment was carried out according to the following procedure. In a filter container (a filter container with a 0.45 μm polytetrafluoroethylene (PTFE) 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 the internal standard sample) as a polar organic solvent. After all of the above (1) to (3) are added, the filtration vessel is stirred at 2200 rpm for 1 minute, and filtration is carried out for 1 minute. During filtration, the filtrate is collected in a collection vessel combined with the filtration vessel.

[0061] The conditions for online SPE are as follows: Solid-phase extraction column type: Shimadzu Shim-pack XR ODS 30L x 2.0 Mobile phase: Water: Methanol 50:50 ·Mobile phase flow rate: 0.5mL / min

[0062] The LC analysis conditions were as follows: Column type: Shimadzu Shim-Pack Velox PFPP 2.7μm 3.0 x 100mm Column temperature: 40℃ Sample injection volume: 30 μL Mobile phase A: Water + 5 mM ammonium formate + 0.1% formic acid Mobile phase B: Methanol ·Mobile phase flow rate: 0.7mL / min ·Analysis time: 12min

[0063] The MS / MS analysis conditions other than the MRM transitions were 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 (first RF ion guide) RF voltage: 100 V (24,25-(OH)2D3), default value (target components other than 24,25-(OH)2D3) The default value of the Q-array RF voltage is usually determined by automatic tuning of the device using a standard sample or the like, and is not necessarily constant.

[0064] Representative MRM transitions and collision energies (CE) for each target component are as follows: Representative MRM transitions are those used for quantification, and one or more MRM transitions of confirmation ions for component confirmation, for example, using confirmation ion ratios, can also be separately determined. ·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

[0065] Figure 3 shows representative extracted ion chromatograms for each target component obtained by actual measurements. The vertical positions of each chromatogram are appropriately shifted. These results show that the target components are observed in a state where they are sufficiently separated from each other. It also shows that 25(OH)VitD3 and its isomer (epi-) are properly separated.

[0066] Figures 4 to 9 show actual measurement examples of extracted ion chromatogram peaks for 0.5 ng / mL 24,25-dihydroxyvitamin D3, 4.9 ng / mL 25-hydroxyvitamin D3, 4.5 ng / mL 25-hydroxyvitamin D2, 0.1 ng / mL vitamin K1, 0.1 ng / mL vitamin K2MK-4, and 0.5 ng / mL vitamin K2MK-7, respectively. The concentrations shown are the lower limit of quantitation for each target component. In each figure, the peaks indicated by downward-pointing triangles are the peaks used for quantitation. The starting and ending points of the peaks and the lines connecting them (i.e., the baselines defining the peak areas) are also shown. It can be seen that peaks with good shapes were obtained for all target components.

[0067] Figure 10 shows a calibration curve for 24,25-dihydroxyvitamin D3 prepared using a calibration sample (calibrator). The calibrator was prepared using reagents manufactured by Golden West Diagnostics. The measurement concentration range is 0.5 to 120 ng / mL, which corresponds to the expected measurement concentration range in actual testing. Figure 11 shows an example of a calibration curve for vitamin K1 prepared using the same calibrator. The measurement concentration range is 0.1 to 24 ng / mL, which also corresponds to the expected measurement concentration range in actual testing.

[0068] Figure 12 shows the results of precision and reproducibility (intra-day reproducibility) for a low-concentration control sample prepared using a reagent manufactured by Golden West Diagnostics. Figure 13 shows the results of precision and reproducibility (intra-day reproducibility) for a high-concentration control sample prepared using a reagent manufactured by Golden West Diagnostics. The low-concentration control sample had a concentration five times the lower limit of quantitation, and the high-concentration control sample had a concentration of 18 ng / mL for vitamin K1 only and 90 ng / mL for the other target components.

[0069] The linearity of the calibration curves shown in Figures 10 and 11 is 2> 0.99 is good. Similarly, for the other four target components, calibration curves within the required measurement concentration ranges were verified for each, and in all cases, the linearity was r 2

[0070] It was confirmed that it was good at > 0.99. Also, as shown in FIGS. 12 and 13, the accuracy was ensured at 85 to 115% for all target components, and the reproducibility was within 13% except for vitamin K1 and vitamin K2 MK-7.

[0071] <Sensitivity improvement by adjusting the RF voltage of the Q array> As described above, by removing impurities in the sample by online solid-phase extraction, the ionization efficiency of the target component is improved and the detection sensitivity is enhanced. However, for 24,25-dihydroxyvitamin D3, which particularly has a low ESI responsiveness and requires low-concentration detection, further sensitivity improvement is desirable. In the process of experiments, the present inventor found that a large change in ion intensity occurred when the RF voltage applied to the first RF ion guide 23 was changed, and focused on this.

[0072] ​Figure 15 shows the retention time, peak area, height, and S / N ratio of the peak on the extracted ion chromatogram measured for 24,25-dihydroxyvitamin D3 when the RF voltage applied to the first RF ion guide 23 is changed to 0, 30, 60, 90, and 120 V. Note that a bias DC voltage is applied to the first RF ion guide 23 in addition to the RF voltage. Figure 16 shows an example of the measured extracted ion chromatogram when the RF voltage is changed. From these results, it can be seen that the peak area, height, and S / N ratio are significantly dependent on the RF voltage applied to the first RF ion guide 23. The reason for this can be inferred as follows.

[0073] In ESI, in addition to singly protonated ions derived from the target component, multiply charged ions and adduct ions derived from alkali metals or the like are likely to be generated, and various ions derived from the target component are introduced into the first intermediate vacuum chamber 202. Only singly protonated ions are selected as precursor ions by the front-stage quadrupole mass filter 26. Therefore, as the number of multiply charged ions and adduct ions generated increases, the number of singly charged protonated ions decreases accordingly, and the number of product ions generated from these precursor ions also decreases. Therefore, to improve the detection sensitivity of the target component, it is important to minimize the number of multiply charged ions and adduct ions derived from the target component and increase the number of singly charged protonated ions, i.e., to concentrate ions as much as possible into singly charged protonated ions.

[0074] Because many residual gas molecules exist in the first intermediate vacuum chamber 202, ions introduced into the intermediate vacuum chamber 202 come into contact with the residual gas molecules and are cooled (collisionally cooled). Because the ion's kinetic energy is attenuated by this cooling, the ions are more likely to be captured by an RF electric field. This is the main reason why ion focusing using an RF electric field in a low-vacuum atmosphere is effective. Ions derived from the target component lose their kinetic energy upon contact with residual gas molecules. Furthermore, a phenomenon similar to collision-induced dissociation occurs upon contact with residual gas molecules, resulting in the desorption of partial structures of the ions. For example, in the case of some adduct ions, when a protonated ion is further bonded to an adduct such as an alkali metal, the adduct portion is likely to desorb upon contact with the residual gas, resulting in the formation of protonated ions. Similarly, in the case of multivalent ions, contact with residual gas can result in the separation of multivalent ions to generate singly charged ions. That is, it is believed that the more opportunities there are for contact with residual gas in the first intermediate vacuum chamber 202, the less the proportion of adduct ions and multiply charged ions will be, and the more singly charged protonated ions will be.

[0075] The gas that flows into the first intermediate vacuum chamber 202 through the desolvation tube 22 expands as a supersonic free jet after leaving the outlet of the desolvation tube 22, but the density of the residual gas tends to be high near the ion optical axis C. Ions captured by the RF electric field oscillate periodically due to the action of that electric field, and 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 higher the RF voltage applied to the Q array, the more opportunities there are for ions derived from the target component to come into contact with the residual gas. As a result, the number of multiply charged ions and adduct ions derived from the target component decreases, and the number of singly charged protonated ions tends to increase relatively. This is thought to be the reason why the intensity of singly charged protonated ions derived from the target component is significantly affected by the magnitude of the RF voltage applied to the first RF ion guide 23.

[0076] However, increasing the RF voltage applied to the first RF ion guide 23 narrows the ion acceptance region at the entrance end of the ion guide 23. Therefore, increasing the RF voltage more than necessary may actually reduce the overall ion transmission efficiency. Experimental results show that, among the six target components, 24,25-dihydroxyvitamin D3 has the most stringent detection sensitivity. Therefore, in the measurement device of the above-described embodiment, when measuring 24,25-dihydroxyvitamin D3, the RF voltage is set to 100 V regardless of the default value of the RF voltage, and when measuring other target components, the RF voltage is set to the default value. This particularly enhances the detection sensitivity of 24,25-dihydroxyvitamin D3, while keeping the detection sensitivity of other target components within the target range.

[0077] In the measurement 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 D3, but the RF voltages for the other target components may also be set to predetermined values. In this case, the RF voltages may be individually different values, or some of them may be the same value.

[0078] Furthermore, the analytical conditions, including various parameter values, types of components such as columns, and consumables such as mobile phases, employed in the measurement method and measurement device of the above-described embodiment are merely examples and can be changed as appropriate. Furthermore, the six target components measured in the measurement method and measurement device of the above-described embodiment are merely examples, and target components can be deleted or added as appropriate, except that they include at least 24,25-dihydroxyvitamin D3 and vitamin K1.

[0079] Furthermore, the above embodiment is merely an example of the present invention, and it goes without saying that any appropriate modifications, alterations, additions, etc. made within the spirit of the present invention will also fall within the scope of the claims of the present application.

[0080] [Various aspects] It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0081] (Item 1) One aspect of the method for measuring blood concentrations of fat-soluble vitamins according to the present invention is a method for measuring the concentrations 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 first pretreatment step of deproteinizing the blood sample by a denaturing method; a second pretreatment step of removing impurities contained in the sample after the first pretreatment step by solid phase extraction; a measuring step of performing LC / MS analysis using a liquid chromatograph-tandem mass spectrometer having an ion source based on electrospray ionization, in which components in the sample after the second pretreatment step are separated in time and singly charged protonated ions derived from each of the target components are selectively detected; It has.

[0082] (Item 8) One aspect of the fat-soluble vitamin blood concentration measuring device according to the present invention is a device for measuring the concentrations 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, and the device comprises: a protein removal treatment section for removing proteins from a blood sample by sequentially adding an organic solvent to the sample, stirring the sample, and filtering the sample; an online solid-phase extraction unit that supplies the sample treated by the protein removal treatment unit to a solid-phase extraction column, retains the target component in the sample on the solid-phase extraction column, and then supplies a mobile phase to elute the target component from the solid-phase extraction column; a liquid chromatograph-tandem mass spectrometer having an ion source employing an electrospray ionization method, the liquid chromatograph-tandem mass spectrometer comprising: a measurement execution unit that executes an LC / MS analysis to selectively detect singly protonated ions derived from each of the target components while temporally separating components in a sample solution containing the target components eluted from the solid-phase extraction column; a data analysis unit that creates a chromatogram for each of the target components using the data obtained by the measurement execution unit and quantifies the target components based on the area or height of a peak observed in the chromatogram; Equipped with.

[0083] The blood concentration measurement method described in paragraph 1 and the blood concentration measurement device described in paragraph 8 can suppress the ion suppression effect caused by impurities remaining in a blood sample after deproteinization by denaturation, thereby increasing the ionization efficiency of the target component. This allows for simultaneous measurement of vitamin D metabolites, vitamin K, and its metabolites in blood with high sensitivity without the need for a complicated and difficult-to-automate derivatization process using a Cookson-type derivatization reagent or the like. Furthermore, since a sample solution containing the target component extracted online using solid-phase extraction can be directly analyzed by LC / MS, it is easy to automate the entire process from deproteinization to measurement. This improves measurement throughput and measurement stability, making the device suitable for use in screening tests, etc.

[0084] (Item 2) In the method for measuring blood concentration described in item 1, the vitamin D metabolites may include 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

[0085] According to the method for measuring blood vitamin D concentration described in item 2, it is possible to easily measure the concentration of the main vitamin D metabolites, which is particularly important in screening tests for osteoporosis and the like.

[0086] (Item 3) In the blood concentration measurement method according to item 1 or 2, the vitamin K and its metabolites may include vitamin K2MK-4 and vitamin K2MK-7.

[0087] According to the method for measuring blood vitamin K concentration described in item 3, it is possible to simply measure the concentration of the main vitamin K metabolites, which is particularly important in screening tests for osteoporosis and the like.

[0088] (Item 4) In the blood concentration measurement method according to any one of Items 1 to 3, the liquid chromatograph-tandem mass spectrometer has an RF ion guide, located next to the ion source, that focuses ions and transports them to the subsequent stage; In the measuring step, during the period in which 24,25-dihydroxyvitamin D3 is measured, the amplitude of the RF voltage applied to the RF ion guide may be set to a predetermined value corresponding to the detection of singly protonated ions derived from 24,25-dihydroxyvitamin D3. 。

[0089] (Item 5) In the blood concentration measurement method described in Item 4, the predetermined value may be a value that exhibits the effect of concentrating various ions derived from 24,25-dihydroxyvitamin D3 introduced into the RF ion guide into singly protonated ions.

[0090] Although 24,25-dihydroxyvitamin D3 is an important vitamin D metabolite in screening tests for osteoporosis, its blood concentration is lower than that of other vitamin D metabolites, making it difficult to measure. In contrast, the blood concentration measurement methods described in paragraphs 4 and 5 improve ionization efficiency by removing impurities from the sample using online solid-phase extraction. Furthermore, by appropriately setting the RF voltage, dispersion of ion species can be suppressed, thereby increasing the intensity of the monovalent protonated ions to be observed. This further enhances the detection sensitivity of 24,25-dihydroxyvitamin D3 in blood.

[0091] In the blood concentration measurement method described in paragraph 4, in the measurement step, the amplitude of the RF voltage applied to the RF ion guide during the period in which a target component other than 24,25-dihydroxyvitamin D3 is measured may be set to a standard value set in the liquid chromatograph-tandem mass spectrometer.

[0092] The standard value referred to here is, for example, a value determined in advance by an apparatus manufacturer or the like without considering a single specific compound, which provides good results on average, or a value set by an automatic tuning function of the apparatus using a standard sample, etc. In other words, this standard value is not a value optimized to, for example, maximize the detection sensitivity for each target component.

[0093] This allows the RF voltage applied to the RF ion guide to be constant during the period in which target components other than 24,25-dihydroxyvitamin D3 are measured, eliminating the need for a waiting time for voltage switching, even when, for example, the measurement periods for multiple target components overlap and it is necessary to switch the target components to be measured in a time-division manner. This shortens the time interval between repeated measurements of a single target component, improving the accuracy of the peak waveforms in the extracted ion chromatogram and enhancing quantitative performance.

[0094] (Item 6) In the blood concentration measurement method described in any one of Items 1 to 5, the second pretreatment step may use an ODS column as the solid-phase extraction column, and water and methanol as the mobile phase for supplying the sample to the column.

[0095] According to the blood concentration measurement method described in item 6, the target component contained in the blood can be efficiently collected, while the remaining impurities contained in the blood, particularly impurities that cause an ion suppression effect, can be suppressed. As a result, the impurities can be effectively removed from the blood, and the target component can be detected with high sensitivity.

[0096] (Item 7) In the blood concentration measurement method according to any one of items 1 to 6, the liquid chromatograph of the liquid chromatograph-tandem mass spectrometer may use a PFPP column.

[0097] The blood concentration measurement method described in item 7 allows for good separation of multiple vitamin D metabolites, including 24,25-dihydroxyvitamin D3, as well as vitamin K1 and one or more vitamin K metabolites, thereby improving the quantitative determination of each of these target components. [Explanation of symbols]

[0098] 1...Liquid chromatograph section (LC section) 10...Mobile phase storage section 11...Liquid transfer pump 12...Injector 13...Column 2...Tandem mass spectrometry section (MS / MS section) 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...Pre-quadrupole mass filter 27...Collision cell 28...Post-quadrupole mass filter 29...Ion detector 3. Online solid phase extraction (SPE) section 30...Mobile phase storage section 31...Liquid transfer pump 33...Solid phase extraction column 34...Flow path switching section 4...Deproteinization processing section 5...Voltage generating section 6...Data processing unit 60...Data collection section 61...Quantitative calculation section 7...Control unit 70... Vitamin D / K blood concentration measurement program 8...Operation unit 9…Display section

Claims

1. A blood concentration measurement method for measuring the concentration of a target component in a blood sample, comprising: a first pretreatment step of deproteinizing the blood sample by a denaturing method; a second pretreatment step of removing impurities contained in the blood sample after the first pretreatment step by solid phase extraction; Using a liquid chromatograph-tandem mass spectrometer having an ion source employing an electrospray ionization method and an RF ion guide that focuses ions at a stage subsequent to the ion source while transporting them to the subsequent stage, LC / MS analysis is performed to selectively detect singly protonated ions derived from vitamin D metabolites containing at least 24,25-dihydroxyvitamin D 3 , and vitamin K and its metabolites containing at least vitamin K 1 in the blood sample as target components while temporally separating the components in the blood sample after the second pretreatment step, thereby obtaining 24,25-dihydroxyvitamin D ３ During the measurement period, the amplitude of the RF voltage applied to the RF ion guide is adjusted to the value of 24,25-dihydroxyvitamin D introduced into the RF ion guide. ３ a measuring step of setting the value of the ion concentration to a predetermined value corresponding to the detection of singly charged protonated ions derived from the sample; A method for measuring the blood concentration of 24,25-dihydroxyvitamin D3.

2. The vitamin D metabolites further include 25-hydroxyvitamin D ２ and 25-hydroxyvitamin D ３ The blood concentration measuring method according to claim 1, comprising:

3. The vitamin K and its metabolites further include vitamin K ２ MK-4 and Vitamin K ２ The method for measuring blood concentration according to claim 2, comprising MK-7.

4. 4. The blood concentration measurement method according to claim 3, wherein in the second pretreatment step, an ODS column is used as the solid-phase extraction column, and water and methanol are used as the mobile phase for supplying the blood sample to the column.

5. 5. The blood concentration measurement method according to claim 4, wherein the liquid chromatograph of the liquid chromatograph-tandem mass spectrometer uses a PFPP column.

6. A blood concentration measuring device for measuring the concentration of a target component in a blood sample, a deproteinization treatment section for removing proteins from the blood sample by sequentially adding an organic solvent to the blood sample, stirring the blood sample, and filtering the blood sample; an online solid-phase extraction unit that supplies the blood sample treated by the protein removal treatment unit to a solid-phase extraction column, retains vitamin D metabolites containing at least 24,25-dihydroxyvitamin D3, and vitamin K and its metabolites containing at least vitamin K1 in the blood sample as target components in the solid-phase extraction column, and then supplies a mobile phase to elute the target components from the solid-phase extraction column; A liquid chromatograph-tandem mass spectrometer having an ion source using an electrospray ionization method and an RF ion guide that focuses ions at a stage next to the ion source while transporting them to the subsequent stage, is used to perform LC / MS analysis in which components in a sample solution containing the target component eluted from the solid-phase extraction column are separated in time and singly protonated ions derived from each of the target components are selectively detected, and 24,25-dihydroxyvitamin D is detected. ３ During the measurement period, the amplitude of the RF voltage applied to the RF ion guide is adjusted to the value of 24,25-dihydroxyvitamin D introduced into the RF ion guide. ３ a measurement execution unit that sets the value of the ion concentration to a predetermined value corresponding to the detection of singly charged protonated ions derived from the sample; a data analysis unit that creates a chromatogram for each of the target components using the data obtained by the measurement execution unit and quantifies the target components based on the area or height of a peak observed in the chromatogram; A blood concentration measuring device for 24,25-dihydroxyvitamin D3 comprising:

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