JP7205322B2 - Simultaneous analysis method for blood anticoagulants - Google Patents

Description

The present invention relates to a method for simultaneously analyzing multiple types of blood anticoagulants in a biological sample using chromatographic mass spectrometry.

In order to prevent the development of vascular diseases such as cerebral infarction and myocardial infarction, conventionally, vitamin K antagonists such as warfarin have been widely used as blood anticoagulants. Although warfarin is useful for preventing the onset of thromboembolism, it affects many coagulation factors and extensively suppresses the action of hemostasis and blood coagulation, which may cause complications such as cerebral hemorrhage. Therefore, when using this drug, it is necessary to repeatedly perform a prothrombin time-international normalized ratio (PT-INR) test to monitor blood levels.

On the other hand, in recent years, new blood anticoagulants such as thrombin inhibitors such as Dabigatran and factor Xa inhibitors such as apixaban, which act on one specific coagulation factor, have been developed and are now available for use. are provided. These new blood anticoagulants are called Direct Oral Anti-Coagulants (DOACs) to distinguish them from conventional vitamin K antagonists.

Direct oral anticoagulants are less likely to cause complications such as bleeding, and their actions and effects are less affected by food. For these reasons, the use of direct oral anticoagulants has been rapidly expanding in recent years. Initially, unlike vitamin K antagonists, it was said that blood level monitoring was not necessary when using direct oral anticoagulants. However, as the use of direct oral anticoagulants has expanded, serious side effects have also been confirmed. Monitoring of blood concentration using a device (hereinafter sometimes referred to as "LC-MS/MS") has been carried out (see Non-Patent Document 1, etc.).

WO2018/116443

Ito, Maruyama, ``How to evaluate the effect of oral anticoagulants?'', The Society for Bioanalytical Science, Biospecimen Analysis, Vol.40, No.2, 2017, pp.115-120

Depending on the condition of the patient to whom a blood anticoagulant is prescribed, multiple types of blood anticoagulants may be used in combination. When a vitamin K antagonist is used in combination with a direct oral anticoagulant, the PT-INR test, which is used to monitor the blood level of the vitamin K antagonist, shows that the interaction of these drugs may increase the blood level of the vitamin K antagonist. may show abnormal values. Therefore, recently, LC-MS/MS has come to be used not only for direct blood level monitoring of oral anticoagulants but also for blood level monitoring of vitamin K antagonists.

However, in the conventional analysis method of blood anticoagulants using LC-MS/MS, various blood anticoagulants cannot be comprehensively analyzed in one analysis, and are analyzed in multiple times. I needed it. The reason for this is that vitamin K antagonists and direct oral anticoagulants, in particular, have a large difference in blood concentration, which is the object of monitoring, and the measurement range in quantification by LC-MS/MS is completely different. Therefore, when comprehensively analyzing many types of blood anticoagulants, there are problems such as time-consuming analysis, long analysis time, low throughput, and high cost due to the need for large amounts of consumables such as mobile phases. there were.

In addition, in recent years, there have been cases of poisoning due to overdose of prescribed blood anticoagulants, and there is an increasing need to analyze various types of blood anticoagulants in a short time with high accuracy for emergency medical care. However, it was difficult to meet these demands with conventional analytical methods.

The present invention has been made in view of the above problems, and its object is to provide a method for treating multiple types of blood containing vitamin K antagonists and direct oral anticoagulants (thrombin inhibitors, factor Xa inhibitors, etc.). To provide a simultaneous analysis method for blood anticoagulants capable of simultaneously analyzing anticoagulants.

The first aspect of the present invention, which has been made to solve the above problems, is a method for simultaneous analysis of blood anticoagulants, comprising a vitamin K antagonist and a thrombin inhibitor and/or a factor Xa inhibitor in a biological sample. A method of analyzing multiple types of blood anticoagulants, including drugs, comprising:
A pretreatment step of performing a treatment to remove proteins from a biological sample;
The sample solution from which proteins have been removed in the pretreatment step is introduced into a liquid chromatograph-tandem mass spectrometer, and the liquid is separated from the plurality of types of blood anticoagulants in the sample solution by the liquid chromatograph. An analysis execution step of sequentially performing mass spectrometry on the eluate from the chromatograph with a mass spectrometer;
a qualitative processing step of performing signal processing for qualitatively identifying the plurality of types of blood anticoagulants based on the analysis results obtained in the analysis execution step;
In the analysis execution step, MRM measurement is performed for MRM transitions associated with each blood anticoagulant at a timing when each of the plurality of types of blood anticoagulants is estimated to elute from the liquid chromatograph, Further, the analysis parameters that affect the detection sensitivity in the mass spectrometer are determined so that the detection sensitivity is lower than the best state when performing MRM measurement for vitamin K antagonists. .

A second aspect of the present invention, which has been made to solve the above-mentioned problems, is a method for simultaneous analysis of blood anticoagulants, comprising a vitamin K antagonist and a thrombin inhibitor and/or factor Xa in a biological sample. A method of analyzing multiple types of blood anticoagulants, including inhibitors, comprising:
A pretreatment step of performing a treatment to remove proteins from a biological sample;
The sample solution from which proteins have been removed in the pretreatment step is introduced into a liquid chromatograph-tandem mass spectrometer, and the liquid is separated from the plurality of types of blood anticoagulants in the sample solution by the liquid chromatograph. An analysis execution step of sequentially performing mass spectrometry on the eluate from the chromatograph with a mass spectrometer;
a qualitative processing step of performing signal processing for qualitatively identifying the plurality of types of blood anticoagulants based on the analysis results obtained in the analysis execution step;
In the analysis execution step, MRM measurement is performed for MRM transitions associated with each blood anticoagulant at a timing when each of the plurality of types of blood anticoagulants is estimated to elute from the liquid chromatograph, And, the analysis parameters that affect the detection sensitivity in the mass spectrometer are the detection sensitivity when performing MRM measurement for vitamin K antagonists and the MRM measurement for thrombin inhibitors and factor Xa inhibitors. It is determined to be lower than the detection sensitivity.

The liquid chromatograph-tandem mass spectrometer here is typically a liquid chromatograph-triple quadrupole mass spectrometer, or a liquid chromatograph-quadrupole/time-of-flight (Q-TOF type) mass spectrometer. Normally, in such a liquid chromatograph-tandem mass spectrometer, each component in the eluate after being separated by the liquid chromatograph has the highest possible detection sensitivity, that is, the best condition. Analysis parameters are defined. On the other hand, when performing the simultaneous analysis methods according to the first and second aspects of the present invention, when performing MRM measurement for thrombin inhibitors and factor Xa inhibitors, for example, the detection sensitivity is in the best state. However, when performing MRM measurements for vitamin K antagonists, the analytical parameters are intentionally set so that the detection sensitivity is lower than best.

Vitamin K antagonists have significantly higher blood concentrations than thrombin inhibitors and factor Xa inhibitors. The detector may be saturated, or the ionic strength corresponding to thrombin inhibitors and factor Xa inhibitors may be too low to be detected.

On the other hand, according to the simultaneous analysis method for blood anticoagulants according to the first and second aspects of the present invention, the thrombin inhibitor and/or the thrombin inhibitor and/or the Xa phase are avoided while avoiding the saturation of the signal intensity corresponding to the vitamin K antagonist. Factor inhibitors can be detected with high sensitivity. This allows multiple types of blood anticoagulants, including vitamin K antagonists and direct oral anticoagulants, to be analyzed simultaneously, ie, by a single liquid chromatographic mass spectrometry analysis. As a result, it is possible to reduce the labor of analysis when analyzing multiple types of blood anticoagulants. In addition, since the analysis time can be shortened, the throughput can be improved, and it is possible to respond to urgent analysis. Furthermore, the use of various consumable materials such as the mobile phase used for analysis can be suppressed, and analysis costs can be reduced.

1 is a schematic configuration diagram of an embodiment of a liquid chromatograph-tandem mass spectrometer for carrying out the simultaneous analysis method for blood anticoagulants according to the present invention; FIG. A flow chart showing the procedure of work and processing in the simultaneous analysis method for blood anticoagulants, which is an embodiment of the present invention. A diagram showing an example of retention times and MRM transitions corresponding to nine types of blood anticoagulants to be analyzed in the simultaneous analysis method for blood anticoagulants according to the present embodiment. Schematic diagram showing the relationship between the chromatogram corresponding to each component eluted from the liquid chromatograph and the measurement time range for the component. Schematic diagram showing the relationship between the chromatogram corresponding to each component eluted from the liquid chromatograph and the measurement time range for the component. Experimental results showing the relationship between ionic strength (peak area value) and collision energy for vitamin K antagonists. Experimental results showing the relationship between ionic strength (peak area value) and DC voltage applied to the ion guide for vitamin K antagonists. Experimental results showing the relationship between ionic strength (peak area value) and DC voltage applied to the desolvation tube for vitamin K antagonists. Experimental results showing the relationship between ionic strength (peak area value) and DC voltage applied to the ESI probe for vitamin K antagonists.

Hereinafter, a simultaneous analysis method for blood anticoagulants, which is one embodiment of the present invention, will be described in detail with reference to the accompanying drawings.

[Device configuration]
FIG. 1 is a schematic configuration diagram of an example of a liquid chromatograph-tandem mass spectrometer (LC-MS/MS) for carrying out the simultaneous analysis method for blood anticoagulants according to this embodiment.

This LC-MS/MS comprises a liquid chromatograph unit 1, a mass spectrometer unit 2, a voltage generation unit 3, a data processing unit 4, a control unit 5, an input unit 6, and a display unit 7. .
The liquid chromatograph section 1 includes a mobile phase storage container 10, a liquid transfer pump 11, an injector 12, a column 13, and a column oven .

The mass spectrometer 2 has a chamber 20 in which an ionization chamber 21, a first intermediate vacuum chamber 22, a second intermediate vacuum chamber 23, and a high vacuum chamber 24 are formed. The inside of the ionization chamber 21 is in a substantially atmospheric pressure atmosphere, and the high vacuum chamber 24 is maintained in a high vacuum atmosphere by a rotary pump and a turbomolecular pump (not shown). Note that the number of intermediate vacuum chambers is not limited to "2" and can be reduced or increased.

An electrospray ionization (ESI) probe 25 is placed in the ionization chamber 21, which has a substantially atmospheric pressure atmosphere, and the ionization chamber 21 and the first intermediate vacuum chamber 22 are communicated through a desolvation pipe 26 heated to a high temperature. . An ion guide 27 called a Q-Array is arranged in the first intermediate vacuum chamber 22 , and the first intermediate vacuum chamber 22 and the second intermediate vacuum chamber 23 are small holes provided at the top of the skimmer 28 . communicated through A multipole ion guide 29 composed of a plurality of rod electrodes arranged so as to surround the central axis C is arranged in the second intermediate vacuum chamber 23 . In the high vacuum chamber 24, along the central axis C, there are a front quadrupole mass filter 30, a collision cell 31 having an ion guide 32 for converging and transporting ions, and a rear quadrupole mass filter. 33 and an ion detector 34 that outputs an ion intensity signal corresponding to the amount of incident ions as a detection signal.

Predetermined voltages are applied from the voltage generator 3 to the ESI probe 25, desolvation tube 26, ion guides 27, 29, 32, quadrupole mass filters 30, 33, collision cell 31, and the like. However, in order to avoid complicating the drawing, FIG. 1 omits part of the description of signal lines for applying voltage from the voltage generator 3 to the constituent elements of the mass spectrometer 2 .

A signal detected by the ion detector 34 is converted into digital data by an analog-digital converter (ADC) 35 and input to the data processing unit 4 . The data processing unit 4 includes a chromatogram creating unit 40, a qualitative analysis unit 41, and a quantitative analysis unit 42 as functional blocks. Further, the control unit 5 controls the liquid chromatograph unit 1, the mass spectrometer unit 2, the voltage generation unit 3, etc., and the control sequence storage unit 50, the parameter automatic adjustment unit 51, the analysis control unit 52, etc. are function blocks. include. The control unit 5 also provides a user interface through the input unit 6 and the display unit 7, as well as overall control of the entire system.

The data processing unit 4 and the control unit 5 use a personal computer or a more advanced work station as hardware resources, and execute dedicated control/processing software installed in advance on the computer. It can be configured to realize the function of In this case, the input unit 6 is a keyboard or pointing device (such as a mouse) attached to the computer, and the display unit 7 is a display monitor of the computer.

[General operation of the device]
A typical analytical operation in the LC-MS/MS shown in FIG. 1 is schematically described.
In the liquid chromatograph section 1, the liquid-sending pump 11 sucks the mobile phase from the mobile-phase storage container 10 and sends it to the column 13 at a constant flow rate. The injector 12 injects a sample prepared in advance into the mobile phase at a predetermined timing according to instructions from the control unit 5 . The injected sample is introduced into the column 13 along with the flow of the mobile phase. While passing through the column 13, various components contained in the sample are separated in the time direction and eluted from the outlet of the column 13. Column oven 14 keeps column 13 at a constant temperature during analysis.

In the mass spectrometry section 2, when a sample liquid (eluate) containing sample components is sent from the liquid chromatograph section 1 and a predetermined high voltage is applied to the ESI probe 25, the charged sample liquid is discharged from the tip of the ESI probe 25. When the droplets are atomized, the charged droplets collide with the atmosphere and are atomized, and the sample component molecules contained in the droplets are ionized. The generated ions originating from the sample components are sucked into the desolvation pipe 26 together with charged droplets in which the solvent is not sufficiently vaporized, and sent to the first intermediate vacuum chamber 22 . Evaporation of the solvent in the droplets in the desolvation tube 26 is further promoted, thereby promoting the generation of ions derived from the sample components. The generated ions are converged near the small hole of the skimmer 28 by the action of the electric field formed by the ion guide 27 and enter the second intermediate vacuum chamber 23 through the small hole. Further, the ions are converged and transported by the action of the electric field formed by the multipole ion guide 29 and enter the high vacuum chamber 24 .

In the high-vacuum chamber 24, the ions originating from the sample component are incident on the front quadrupole mass filter 30, and the mass-to-charge ratio m/z according to the voltage applied to the electrodes constituting the front quadrupole mass filter 30 is Only those ions that have ions pass through the pre-quadrupole mass filter 30 . The ions (precursor ions) that have passed through the front-stage quadrupole mass filter 30 enter the collision cell 31 and collide with the collision gas (usually an inert gas such as argon or nitrogen) introduced into the collision cell 31. Collision-induced dissociation (CID) occurs. Various product ions generated by this CID are transported while being converged by the ion guide 32 and enter the rear-stage quadrupole mass filter 33 . Only product ions having a mass-to-charge ratio corresponding to the voltage applied to the electrodes constituting the rear quadrupole mass filter 33 pass through the rear quadrupole mass filter 33 and enter the ion detector 34 . The ion detector 34 outputs an ion intensity signal having a magnitude corresponding to the amount of incident ions.

Specific product ions derived from specific components in the sample are detected by selectively allowing ions having a predetermined mass-to-charge ratio to pass through the front quadrupole mass filter 30 and the rear quadrupole mass filter 33, respectively. be able to. Thus, the method of selecting and detecting ions having a specific mass-to-charge ratio in each of the quadrupole mass filters 30 and 33 is the MRM measurement. MRM measurements are used to detect

[Analysis method of blood anticoagulant]
Next, a simultaneous analysis method for blood anticoagulants using the above LC-MS/MS will be described with reference to FIG. FIG. 2 is a flow chart showing the procedure of work and processing in the simultaneous analysis method for blood anticoagulants.

<Sample to be analyzed and blood anticoagulant>
A sample to be measured by the simultaneous analysis method of the present embodiment is plasma extracted from blood collected from a subject. However, serum may be used instead of plasma. The following nine types of blood anticoagulants are the components to be analyzed.
○Vitamin K antagonists: Warfarin, Acenocoumarol, and Fluindione.
○Thrombin inhibitors: Two types of dabigatran and argatroban.
○Factor Xa inhibitors: Four types: Edoxaban, Apixaban, Rivaroxaban, and Betrixaban.

<Measurement conditions>
Here, since the components to be analyzed are predetermined, the MRM transitions (combination of precursor ion m/z and product ion m/z) corresponding to each component are known. Further, the retention time corresponding to each component, that is, the timing of elution from the column 13 of the liquid chromatograph section 1 and introduction into the mass spectrometry section 2 can be roughly determined according to the LC separation conditions. In the analysis method of the present embodiment, the LC separation conditions in the liquid chromatograph section 1 are determined as follows based on the experimental results.

○ Kinds of mobile phase Mobile phase A: Water + 5 mM ammonium formate + formic acid (0.1%)
Mobile phase B: water:methanol = 1:9 + 5mM ammonium formate + formic acid (0.1%)
○ Gradient conditions (concentration % of mobile phase B): 0 min to 0.5 min: 2% → 0.5 min to 2.5 min: 2% to 50% → 2.5 min to 3 min: 50% to 98% → 3 min to 5 min : 98% → 5.01 min to 7 min: 2%
○ Autosampler (injector) rinsing liquid: water: methanol = 1: 1 + formic acid (0.1%)
○Type of column: ODS column By using an acidic rinsing liquid for the autosampler, it is possible to wash away the sample adsorbed to the needle part made of stainless steel and reduce carryover. Also, for the same reason, it is desirable that the column be free of stainless steel.

The retention times and MRM transitions (combination of precursor ion m/z and product ion m/z) under the above LC separation conditions for the nine blood anticoagulants are shown in FIG. . (K) indicated at the beginning of FIG. 3 indicates a vitamin K antagonist, (T) indicates a thrombin inhibitor, and the others indicate factor Xa inhibitors. Two MRM transitions are shown for each component, the quantification ion with the highest ionic strength on the top and the confirming ion with the second highest ionic strength on the bottom.

Since the retention time for each component is known as described above, ideally, the extraction ion chromatogram (commonly referred to as "mass chromatogram") corresponding to each component should center on the retention time of that component. A peak with an appropriate peak width appears. However, in practice, the elution time of the components varies due to various factors. Therefore, a measurement time range of a predetermined time width is defined for each component around the standard retention time shown in FIG. Perform measurements repeatedly. The time width for determining the measurement time range may be appropriately determined in advance in consideration of the expected peak spread (the time from the peak start point to the end point) and the expected maximum deviation of the elution time. . In the analysis method of the present embodiment, as an example, the measurement time range corresponding to each component was determined with a time width of 30 seconds before and after the standard retention time.

As can be seen from Figure 3, although the nine blood anticoagulants are temporally separated by liquid chromatography, some components have fairly close standard retention times. Therefore, when the measurement time range for each component is defined as described above, the measurement time range of one component and the measurement time range of another component overlap. FIG. 4 is a diagram schematically showing this state, showing the relationship between the chromatogram corresponding to each component and the measurement time range for that component.

In this example, since the retention time of component A and the retention time of component B are close, the measurement time range corresponding to component A and the measurement time range corresponding to component B overlap. In this overlapping time range, it is necessary to perform both MRM measurements on component A and MRM measurements on component B, which means multiple MRM measurements on different components, i.e. with different MRM transitions. This can be realized by repeating a routine that is executed one by one in order for a predetermined period of time. Of course, if the number of MRM measurements performed in one routine, that is, the number of components whose measurement time ranges overlap, the time per routine should be increased, or the execution time of one MRM measurement should be increased. It needs to be shortened, but in the analysis method of this embodiment, the number of components whose measurement time ranges overlap is not so large, so it does not pose a substantial problem.

As described above, in the analysis method of the present embodiment, the measurement time range (measurement start time and end time) and the MRM measurement performed within the measurement time range for each of the nine types of blood anticoagulants are performed in advance. A transition is determined and stored in the control sequence storage unit 50 as a control sequence.

On the other hand, the measurement conditions other than the MRM transition when performing MRM measurement in the mass spectrometry unit 2, specifically, each voltage parameter that affects the ion detection sensitivity, are determined by the LC-MS when a standard sample of a prescribed concentration is used. / is determined based on the results actually measured by MS. That is, the parameter automatic adjustment unit 51 executes the auto-tuning operation described in, for example, Patent Document 1. For example, while changing stepwise the DC bias voltage applied to the ion guide 27, the measurement of the standard sample is performed. is repeated, and the voltage parameter is determined by searching for the voltage condition that maximizes the ion intensity obtained. As a result, even if the state of the device changes, the voltage parameters are appropriately set so that the ion detection sensitivity for the standard sample is always maximized or close to it, enabling the detection and quantification of trace components.

However, when the voltage parameters are determined as described above, there are the following problems. Among the nine types of blood anticoagulants mentioned above, there is a large difference of more than one order of magnitude in blood concentration between the components belonging to vitamin K antagonists and the components belonging to other thrombin inhibitors and factor Xa inhibitors. . Therefore, when the voltage parameters are determined so that the components belonging to thrombin inhibitors and factor Xa inhibitors, which have relatively low blood concentrations, can be detected with high sensitivity, the blood concentrations are significantly higher than these. The ion intensity corresponding to components belonging to vitamin K antagonists may saturate the ion detector 34 . Conversely, if the voltage parameters are defined to ensure that saturation of the ionic strength corresponding to the components belonging to vitamin K antagonists is avoided, the components belonging to thrombin inhibitors and factor Xa inhibitors will not be detected or their quantification accuracy will be reduced. There is a risk of

Therefore, in the analysis method of the present embodiment, the collision energy, which greatly affects the ion detection sensitivity, is intentionally adjusted from the optimum value (that is, the value that gives the best sensitivity) in the MRM measurement corresponding to the component belonging to the vitamin K antagonist. By shifting, the ion detection sensitivity for components belonging to vitamin K antagonists is lowered. The collision energy is the energy possessed by the precursor ions that have passed through the front-stage quadrupole mass filter 30 and enters the collision cell 31, and this energy greatly affects the dissociation efficiency of the ions. Normally, the collision energy is applied to the DC bias voltage applied to the front-stage quadrupole mass filter 30 (strictly speaking, the post-rod electrodes constituting the mass filter 30) and the entrance electrode provided at the entrance end of the collision cell 31. Varying one or both of the DC bias voltages will change the collision energy, since it depends on the voltage difference from the applied DC bias voltage.

Here, the reason for using collision energy to adjust the ion detection sensitivity will be explained based on experimental results.

6, 7, 8, and 9 show the collision energy, the DC bias voltage applied to the ion guide 27, the DC voltage applied to the desolvation tube 26, and the ions applied to the ESI probe 25, respectively. This is a summary of the results of an experimental investigation of changes in the ionic strength of acenocoumarol and warfarin when the high voltage for production was adjusted. The ion intensity is indicated by the area value of the peak observed on the extracted ion chromatogram in the MRM transition of each component, and the numerical value in the Ave. column in the figure is the average of the peak area values from three measurements. value. In addition, the numerical values in the CV column in the figure indicate the variation (reproducibility) of the peak area values due to the three measurements.

“Original” in FIG. 6 indicates a state in which the collision energies are optimized for acenocoumarol and warfarin, respectively. At this time, the collision energy for acenocoumarol is −17 eV, and the collision energy for warfarin is −19 eV. . "+10" , " +5 ", "-10", and " -5 " in FIG. It is in a state of increasing eV. Here, the ions to be analyzed are positive ions, and the collision energy is a negative value with opposite polarity. Therefore, for example, "+10" means that the collision energy is lowered by 10 eV. For example, when the collision energy is decreased by +10 eV from the "original" state, the ionic strength of acenocoumarol is reduced to 1/5 or less, and the ionic strength of warfarin is also reduced to 1/3 or less. It is presumed that this is because the dissociation efficiency of ions decreased due to the decrease in collision energy.

As shown in FIG. 7, when the DC bias voltage applied to the ion guide 27 is changed from "0 V" to "-100 V", the ionic strength of acenocoumarol is reduced to about 1/3. It can be seen that the ionic strength of warfarin drops to about 1/3 or less. As shown in FIG. 8, when the DC voltage applied to the desolvation tube 26 is changed from "0 V" to "-100 V", the ionic strength of both acenocoumarol and warfarin is reduced to It can be seen that the value decreases to the same extent as shown in . On the other hand, as shown in FIG. 9, with respect to the DC high voltage applied to the ESI probe 25, even if the voltage was increased from the state of "1 kV" to "4 kV", the ionic strength of acenocoumarol and warfarin remained unchanged. The decrease in is limited to about 20 to 30%, which is a large difference from FIGS.

As described above, the difference in blood concentration between the vitamin K antagonist, the thrombin inhibitor, and the factor Xa inhibitor is as large as about one order of magnitude. For tuning, it is desirable that the change in sensitivity to changes in the voltage parameter be as large as possible. In this regard, it can be said that collision energy is the most desirable adjustment target based on the above experimental results. Further, it can be said that the DC bias voltage applied to the ion guide 27 and the DC voltage applied to the desolvation tube 26 are also desirable targets for adjustment. On the other hand, it can be concluded that a DC high voltage applied to the ESI probe 25 is not very effective for the above purpose.

As described above, when the collision energy is changed from the optimum value by a predetermined value (specifically, by +10 eV), the detection sensitivity for components belonging to vitamin K antagonists is greatly reduced. and a factor Xa inhibitor, the effect of the difference in blood concentration can be alleviated, and the difference in ionic strength can be reduced. Therefore, after searching for the optimal value of the collision energy using the standard sample of the vitamin K antagonist, the parameter automatic adjustment unit 51 reduces the optimal value by a predetermined value to obtain the collision energy corresponding to the component belonging to the vitamin K antagonist. Determined as On the other hand, for the components belonging to the thrombin inhibitor and the factor Xa inhibitor, the parameter automatic adjustment unit 51 searches for the optimum value of the collision energy using these standard samples, and then determines the optimum value for the thrombin inhibitor and the factor Xa inhibitor. It is determined as the collision energy corresponding to the component belonging to the factor Xa inhibitor.

The voltage parameter thus determined is added as a measurement condition of the control sequence stored in the control sequence storage unit 50. FIG. With such a control sequence stored in advance in the control sequence storage unit 50, measurement of unknown samples is performed.
Note that after searching for the optimum value of the collision energy, the parameter automatic adjustment unit 51 does not obtain the value of the collision energy for which the detection sensitivity is lowered by decreasing or increasing the predetermined value from the optimum value. A collision energy at which the ion intensity is reduced by a predetermined ratio from the ion intensity when is the optimum value may be searched for by measuring a standard sample.
The work and processing in simultaneous analysis will be described below with reference to FIG.

<Sample pretreatment>
When measuring an unknown sample, first, a blood sample (plasma) is subjected to deproteinization as a pretreatment to prepare a sample solution for measurement (step S1). Specifically, an extract solution having a composition of 40% acetonitrile, 40% methanol, and 20% water is added to the sample, stirred, and then allowed to stand for a predetermined period of time. This is centrifuged with a centrifuge to precipitate the precipitated protein, and the supernatant is collected and used as a sample. Of course, the method of protein removal is not limited to this, and various commonly used methods can be used.

<Measurement of sample>
Subsequently, the sample prepared as described above is set on the LC-MS/MS and measurement is started (step S2). At this time, the analysis control section 52 controls each section according to the control sequence stored in advance in the control sequence storage section 50 as described above. Thereby, the prepared sample is injected into the mobile phase in the injector 12 and introduced into the column 13 . While the sample passes through the column 13, various blood anticoagulants in the sample are separated in the time direction.

After the measurement is started, the analysis control unit 52 repeatedly determines whether or not the measurement time range corresponding to each blood anticoagulant has been reached (step S3). Then, when the measurement time range corresponding to a certain blood anticoagulant is reached (Yes in step S3), under the collision energy (and other voltage parameters) associated with that blood anticoagulant, , MRM measurement targeting the specified MRM transition is performed (step S4). If the blood anticoagulant is six components belonging to thrombin inhibitors or factor Xa inhibitors, the collision energy is set to an optimum value that maximizes the detection sensitivity. are three components belonging to vitamin K antagonists, the collision energies are set to values deviating from the optimum values that reduce the sensitivity of detection.

Then, the MRM measurement corresponding to the blood anticoagulant is repeatedly performed until the measurement time range ends, and when the measurement time range ends (Yes in step S5), the MRM measurement ends (step S6). If the predetermined analysis time has not ended at that point (No in step S7), the process returns to step S3, and MRM measurement is performed in the measurement time range corresponding to another blood anticoagulant.

However, although the flow chart shown in FIG. 2 does not consider the overlap of the measurement time ranges corresponding to a plurality of blood anticoagulants as described above, actually, as shown in FIG. The measurement time ranges corresponding to the coagulants overlap. Therefore, even while the determination in step S3 is Yes and the processing in steps S4 and S5 is being performed, the determination processing in step S3 is performed in parallel, and the measurement time range corresponding to another blood anticoagulant is determined. Upon arrival, time-shared MRM measurements for that blood anticoagulant are performed. That is, during the period when the measurement time ranges corresponding to the multiple blood anticoagulants overlap, the MRM measurements for detecting the multiple blood anticoagulants are performed substantially simultaneously.

As in FIG. 4, FIG. 5 shows a case where the measurement time range corresponding to component A and the measurement time range corresponding to component C overlap because the retention time of component A and the retention time of component C are close. It is the figure which showed the state typically. Here, component A is a component belonging to thrombin inhibitors or factor Xa inhibitors and component C is a component belonging to vitamin K antagonists. In this case, if the collision energy of MRM measurement for component C is set to an optimum value, there is a high possibility that the peak will be saturated on the extracted ion chromatogram as shown in FIG. 5(a). On the other hand, in the analysis method of the present embodiment, the collision energy in MRM measurement for component C deviates from the optimum value and the detection sensitivity is low. A well-shaped peak can be obtained without peak saturation.

In this way, by repeating steps S3 to S7, MRM measurements corresponding to all nine types of blood anticoagulants are performed, and when a predetermined analysis time comes (Yes in step S7), the analysis ends (step S8). In the data processing unit 4, the chromatogram creating unit 40 creates an extracted ion chromatogram corresponding to each blood anticoagulant using data obtained by MRM measurement for each blood anticoagulant (step S9). As shown in FIGS. 4 and 5(b), if a certain blood anticoagulant is contained in the sample, the extracted ion chromatogram corresponding to the blood anticoagulant will show A peak appears around the corresponding retention time. Therefore, the qualitative analysis unit 41 performs peak detection in each extracted ion chromatogram, and derives a qualitative result indicating that the blood anticoagulant is contained when there is a significant peak (step S10).

Furthermore, the quantitative analysis unit 42 calculates the area value of the peak on the extracted ion chromatogram for the blood anticoagulant confirmed to be present in the sample, and refers to a pre-created calibration curve to calculate the peak area value from the peak area value. Calculate the concentration value. That is, quantitative analysis is performed to obtain the concentration value for each blood anticoagulant (step S11). Then, the qualitative result and quantitative result are displayed on the display unit 7 .

As shown in FIG. 2, instead of creating an extraction chromatogram and performing qualitative and quantitative analysis after the analysis is completed, extraction chromatogram Gram preparation and qualitative/quantitative analysis may be performed. Thereby, the qualitative and quantitative results of the blood anticoagulant can be provided to the user more quickly.

The measurement conditions, particularly the LC separation conditions, in the above embodiments are merely examples, and are not limited to those described above. Normally, if the LC separation conditions are different, the retention time corresponding to each component will also change, so naturally the control sequence will also change accordingly. Further, in the above embodiment, nine types of blood anticoagulants are detected, but detection of some blood anticoagulants may be omitted depending on the purpose or situation. That is, simultaneous analysis of a plurality of types of blood anticoagulants, which is less than nine types, may be performed. In addition to these nine types of blood anticoagulants, other types of blood anticoagulants may also be detected.

Further, in the above embodiment, the detection sensitivity is adjusted by adjusting the collision energy. You may adjust a detection sensitivity by adjusting. Furthermore, detection sensitivity may be adjusted by adjusting multiple components, for example, both the collision energy and the DC bias voltage applied to the ion guide 27 . Thereby, the adjustment range of the detection sensitivity can be expanded.

In the above embodiment, LC-MS/MS using a triple quadrupole mass spectrometer was used for measurement, but LC/MS/MS using a Q-TOF mass spectrometer may be used. Further, as described above, when the detection sensitivity is adjusted by adjusting the DC bias voltage applied to the ion guide 27 or the DC voltage applied to the desolvation tube 26 instead of the collision energy, a collision cell is provided. LC-MS using a single quadrupole mass spectrometer, an ion trap time-of-flight mass spectrometer, or the like may be used, as it does not have to be a mass spectrometer.

Furthermore, since the above-described embodiment is merely an example of the present invention, any modifications, alterations, or additions made as appropriate within the spirit of the present invention are, of course, included in the scope of the claims of the present application.

[Various aspects]
The method for simultaneous analysis of blood anticoagulants according to the first aspect of the present invention includes a plurality of types of blood anticoagulants containing a vitamin K antagonist and a thrombin inhibitor and/or a factor Xa inhibitor in a biological sample. A method of analyzing an agent, comprising:
A pretreatment step of performing a treatment to remove proteins from a biological sample;
The sample solution from which the protein has been removed in the pretreatment step is introduced into a liquid chromatograph-tandem mass spectrometer, and the liquid is separated from the plurality of types of blood anticoagulants in the sample solution by the liquid chromatograph. An analysis execution step of sequentially performing mass spectrometry on the eluate from the chromatograph with a mass spectrometer;
a qualitative processing step of performing signal processing for qualitatively identifying the plurality of types of blood anticoagulants based on the analysis results obtained in the analysis execution step;
In the analysis execution step, MRM measurement is performed for MRM transitions associated with each blood anticoagulant at a timing when each of the plurality of types of blood anticoagulants is estimated to elute from the liquid chromatograph, In addition, the analysis parameters that affect the detection sensitivity of the mass spectrometer are determined so that the detection sensitivity is lower than the best when performing MRM measurement for vitamin K antagonists.

In addition, the method for simultaneous analysis of blood anticoagulants according to the second aspect of the present invention is characterized by the following: A method of analyzing a coagulant, comprising:
A pretreatment step of performing a treatment to remove proteins from a biological sample;
The sample solution from which proteins have been removed in the pretreatment step is introduced into a liquid chromatograph-tandem mass spectrometer, and the liquid is separated from the plurality of types of blood anticoagulants in the sample solution by the liquid chromatograph. An analysis execution step of sequentially performing mass spectrometry on the eluate from the chromatograph with a mass spectrometer;
a qualitative processing step of performing signal processing for qualitatively identifying the plurality of types of blood anticoagulants based on the analysis results obtained in the analysis execution step;
In the analysis execution step, MRM measurement is performed for MRM transitions associated with each blood anticoagulant at a timing when each of the plurality of types of blood anticoagulants is estimated to elute from the liquid chromatograph, And, the analysis parameters that affect the detection sensitivity in the mass spectrometer are the detection sensitivity when performing MRM measurement for vitamin K antagonists and the MRM measurement for thrombin inhibitors and factor Xa inhibitors. It is determined so as to be lower than the detection sensitivity.

According to the first and second aspects of the present invention, relatively low blood levels of thrombin inhibitors while avoiding signal intensity saturation corresponding to relatively high blood levels of vitamin K antagonists and/or can be detected with high sensitivity for factor Xa inhibitors. This allows multiple types of blood anticoagulants, including vitamin K antagonists and direct oral anticoagulants, to be analyzed simultaneously, ie, by a single liquid chromatographic mass spectrometry analysis. As a result, it is possible to reduce the labor of analysis when analyzing multiple types of blood anticoagulants. Also, analysis time can be shortened. Furthermore, the use of various consumables such as the mobile phase used for analysis can be suppressed, and analysis costs can be reduced.

According to a third aspect of the present invention, in the simultaneous analysis method for blood anticoagulants according to the first or second aspect, the biological sample may be plasma or serum.

According to the third aspect of the present invention, a sample can be collected from a subject relatively easily, and analysis can be performed quickly.

According to a fourth aspect of the present invention, in the simultaneous analysis method for blood anticoagulants according to any one of the first to third aspects, the analysis parameter is collision energy for dissociating ions. can do.

The "collision energy" actually consists of a DC voltage applied to a collision cell (or an ion guide electrode arranged in the collision cell) for dissociating ions, and a preceding stage such as a quadrupole mass filter. It is the voltage difference from the bias DC voltage applied to the rod electrode (main rod electrode or post-rod electrode), and this voltage difference forms an electric field that gives acceleration energy to the ions about to enter the collision cell. .

As described above, collision energy contributes more to changes in ion intensity than other analytical parameters. Therefore, according to the fourth aspect of the present invention, the effect of a large difference in blood concentration between a vitamin K antagonist and a direct oral anticoagulant can be effectively reduced, and a component belonging to the vitamin K antagonist and a direct It is possible to quantify with high accuracy any of the components belonging to the target oral anticoagulants.

In a fifth aspect of the present invention, in the simultaneous analysis method for blood anticoagulants according to any one of the first to fourth aspects, the plurality of types of blood anticoagulants are at least vitamin K antagonists. Warfarin, acenocoumarol, and fluindione; thrombin inhibitors dabigatran and argatroban; and factor Xa inhibitors edoxaban, apixaban, rivaroxaban, and betrixaban. can be done.

According to the fifth aspect of the present invention, analysis covering a wide range of drugs used as blood anticoagulants is possible.

In a sixth aspect of the present invention, in the simultaneous analysis method for blood anticoagulants according to any one of the first to fifth aspects, following the qualitative treatment by the qualitative treatment step, based on the analysis results , a quantitative processing step of performing quantitative processing on the blood anticoagulant whose presence has been confirmed and calculating the concentration.

According to the sixth aspect of the present invention, it is possible to confirm the presence of multiple types of blood anticoagulants and at the same time obtain information on the concentrations of the existing components.

DESCRIPTION OF SYMBOLS 1... Liquid chromatograph part 10... Mobile-phase storage container 11... Liquid-sending pump 12... Injector 13... Column 14... Column oven 2... Mass spectrometry part 20... Chamber 21... Ionization chamber 22... First intermediate vacuum chamber 23... Second Intermediate vacuum chamber 24 High vacuum chamber 25 ESI probe 26 Desolvation tube 27 Ion guide 28 Skimmer 29 Multipole ion guide 30 Front stage quadrupole mass filter 31 Collision cell 32 Ion guide 33 Rear stage Quadrupole mass filter 34 Detector 3 Voltage generation unit 4 Data processing unit 40 Chromatogram creation unit 41 Qualitative analysis unit 42 Quantitative analysis unit 5 Control unit 50 Control sequence storage unit 51 Automatic parameter adjustment Part 52...Analysis control part 6...Input part 7...Display part

Claims (6)

A method of analyzing a vitamin K antagonist and multiple blood anticoagulants, including thrombin inhibitors and/or factor Xa inhibitors, in a biological sample, comprising:
A pretreatment step of performing a treatment to remove proteins from a biological sample;
The sample solution from which proteins have been removed in the pretreatment step is introduced into a liquid chromatograph-tandem mass spectrometer, and the liquid is separated from the plurality of types of blood anticoagulants in the sample solution by the liquid chromatograph. An analysis execution step of sequentially performing mass spectrometry on the eluate from the chromatograph with a mass spectrometer;
a qualitative processing step of performing signal processing for qualitatively identifying the plurality of types of blood anticoagulants based on the analysis results obtained in the analysis execution step;
In the analysis execution step, MRM measurement is performed for MRM transitions associated with each blood anticoagulant at a timing when each of the plurality of types of blood anticoagulants is estimated to elute from the liquid chromatograph, And, the analysis parameter that affects the detection sensitivity in the mass spectrometer is determined so that the detection sensitivity is lower than the best state when performing MRM measurement for a vitamin K antagonist. Simultaneous analysis method for coagulants. A method of analyzing a vitamin K antagonist and multiple blood anticoagulants, including thrombin inhibitors and/or factor Xa inhibitors, in a biological sample, comprising:
A pretreatment step of performing a treatment to remove proteins from a biological sample;
The sample solution from which proteins have been removed in the pretreatment step is introduced into a liquid chromatograph-tandem mass spectrometer, and the liquid is separated from the plurality of types of blood anticoagulants in the sample solution by the liquid chromatograph. An analysis execution step of sequentially performing mass spectrometry on the eluate from the chromatograph with a mass spectrometer;
a qualitative processing step of performing signal processing for qualitatively identifying the plurality of types of blood anticoagulants based on the analysis results obtained in the analysis execution step;
In the analysis execution step, MRM measurement is performed for MRM transitions associated with each blood anticoagulant at a timing when each of the plurality of types of blood anticoagulants is estimated to elute from the liquid chromatograph, And, the analysis parameters that affect the detection sensitivity in the mass spectrometer are the detection sensitivity when performing MRM measurement for vitamin K antagonists and the MRM measurement for thrombin inhibitors and factor Xa inhibitors. A simultaneous analysis method for a blood anticoagulant, wherein the sensitivity is determined to be lower than the detection sensitivity. 3. The simultaneous analysis method for blood anticoagulants according to claim 1, wherein said biological sample is plasma or serum. The simultaneous analysis method for blood anticoagulants according to any one of claims 1 to 3, wherein said analysis parameter is collision energy for dissociating ions. The blood anticoagulants include at least the vitamin K antagonists warfarin, acenocoumarol, and fluindione; the thrombin inhibitors dabigatran and argatroban; and the factor Xa inhibitors edoxaban, apixaban, and liver. The simultaneous analysis method for blood anticoagulants according to any one of claims 1 to 4, wherein the nine types are loxaban and betrixaban. 6. The method according to any one of claims 1 to 5, further comprising: following the qualitative processing by the qualitative processing step, a quantitative processing is performed on the blood anticoagulant whose presence has been confirmed based on the analysis result to calculate the concentration. The simultaneous analysis method for a blood anticoagulant according to any one of items 1 and 2.

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