WO2021214916A1 - 物質分析方法及び物質分析システム - Google Patents

物質分析方法及び物質分析システム Download PDF

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Publication number
WO2021214916A1
WO2021214916A1 PCT/JP2020/017365 JP2020017365W WO2021214916A1 WO 2021214916 A1 WO2021214916 A1 WO 2021214916A1 JP 2020017365 W JP2020017365 W JP 2020017365W WO 2021214916 A1 WO2021214916 A1 WO 2021214916A1
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Prior art keywords
substance
flow path
unit
detection
column
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PCT/JP2020/017365
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English (en)
French (fr)
Japanese (ja)
Inventor
勇輝 長屋
峻 熊野
益之 杉山
平林　由紀子
信二 吉岡
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株式会社日立ハイテク
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Priority to PCT/JP2020/017365 priority Critical patent/WO2021214916A1/ja
Priority to JP2022516555A priority patent/JPWO2021214916A1/ja
Publication of WO2021214916A1 publication Critical patent/WO2021214916A1/ja

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers

Definitions

  • the present invention relates to a substance analysis method and a technique of a substance analysis system.
  • the liquid chromatograph mass spectrometry system is widely used for chemical analysis containing various substances such as biological samples.
  • a biological sample as a sample contains a substance to be measured (substance to be measured) and a substance such as a protein that interferes with measurement. Therefore, in the liquid chromatograph mass spectrometry system, the substance to be measured is measured after removing factors such as proteins that interfere with measurement by using a pretreatment column.
  • Patent Document 1 and Patent Document 2 are disclosed as techniques related to such a liquid chromatograph mass spectrometry system.
  • Patent Document 1 states, "The present invention provides a method for efficiently extracting a target protein contained in a biological sample such as serum or plasma, and makes it possible to perform highly accurate analysis. A method for removing a protein from a sample and extracting the target protein, which provides a method using a high concentration of salt and / or urea and a water-soluble organic solvent. ”A method for extracting a target protein from a biological sample and A method for analyzing the target protein is disclosed (see summary).
  • Patent Document 2 states, "Using the column switching method, pretreatment is performed using a low-pressure pump under optimum pressure that does not cause clogging, etc., and the treatment status is monitored using an ultraviolet detector. Only the sample that has been purified and purified is analyzed at high speed using a high-pressure pump to extend the life of the system and improve the throughput. ”A liquid chromatograph device and an analysis method are disclosed (see summary).
  • the sample is flowed through the pretreatment column, the low-molecular-weight measurement target substance is adsorbed on the pretreatment column, and the protein, which is a measurement interfering substance, is flowed through the ultraviolet detection device.
  • the flow path switching valve is switched so that the outflow route of the sample flows from the pretreatment column to the mass spectrometer.
  • the substance to be measured flowing out of the pretreatment column is separated in the column for analysis so as to be suitable for mass spectrometry, and the separated substance to be measured is introduced into the mass spectrometer.
  • the present invention has been made in view of such a background, and an object of the present invention is to perform efficient substance detection.
  • the present invention comprises a first substance detection unit that detects the amount of the first substance in the sample, and a substance other than the first substance in the sample, and the first substance.
  • Liquid is supplied by the second substance detection unit that detects the second substance that interferes with the measurement in the detection unit, the first liquid feed pump, the second liquid feed pump, and the first liquid feed pump.
  • a sample injection unit for injecting the sample into the liquid, a first column capable of selectively adsorbing and eluting the first substance, and permeating the second substance, and the first liquid feeding pump.
  • the second liquid feed pump, the first column, the first substance detection unit, and the flow path selection unit for switching the flow path connecting the second substance detection unit, and at least the first.
  • the first state having a flow path to which the first substance detection unit is connected, the first liquid feed pump, the sample injection unit, and the second substance detection unit are connected in series. It is possible to switch between such a flow path and a second state having a flow path such that the second liquid feeding pump, the first column, and the first substance detection unit are connected in series.
  • the control unit is the first detection result acquisition step for acquiring the first detection result which is the detection result by the second substance detection unit, and the detection result by the first substance detection unit. Timing for switching from the first state to the second state based on the second detection result acquisition step for acquiring the detection result 2, the first detection result, and the second detection result. It is characterized in that the switching timing determination step of determining the switching timing is executed. Other solutions will be described as appropriate in the embodiments.
  • efficient substance detection can be performed.
  • FIG. 1 is a diagram showing a configuration of a liquid chromatograph mass spectrometry system Z according to the first embodiment.
  • the liquid chromatograph mass spectrometer Z includes a liquid feed pump 8A, a liquid feed pump 8B, an autosampler 7, a UV detector 3, and a mass spectrometer 4. Further, the liquid chromatograph mass spectrometry system Z includes a pretreatment column 5, an analysis column 6, a flow path switching valve 2, and a control device 1.
  • Each of the liquid feed pump 8A, the liquid feed pump 8B, the autosampler 7, the UV detector 3, the mass spectrometer 4, and the flow path switching valve 2 communicates with the control device 1.
  • the control device 1 performs control, data acquisition, and the like with each of these devices.
  • the pretreatment column 5 and the analysis column 6 are collectively referred to as columns as appropriate.
  • the first buffer solution container C1 is filled with the first buffer solution. Further, the second buffer solution container C2 is filled with the second buffer solution.
  • the liquid feed pump 8A is connected to the first buffer solution container C1 and the second buffer solution container C2, and feeds the first buffer solution and the second buffer solution to the flow path F1 by applying a gradient.
  • the sent first buffer solution and second buffer solution are sent to the autosampler 7 through the flow path F1.
  • the autosampler 7 flows a certain amount of sample solution sucked up from one of a plurality of sample containers (not shown) set inside the autosampler 7 into the flow path F2.
  • the sample is mixed with the mixed solution of the first buffer solution and the second buffer solution.
  • the solution in which the sample is mixed is called the sample solution.
  • the mixed solution with each buffer solution and the sample solution are collectively collectively referred to as a solution.
  • the third buffer solution container C3 is filled with the third buffer solution.
  • the fourth buffer solution container C4 is filled with the fourth buffer solution.
  • the liquid feed pump 8B is connected to the third buffer solution container C3 and the fourth buffer solution container C4, and feeds the third buffer solution and the fourth buffer solution to the flow path F6 by applying a gradient.
  • the flow paths F3, F4, F5, F7 and F8 will be described later.
  • the flow paths F1 to F8 are collectively referred to as a flow path F as appropriate. again,
  • the liquid feed pump 8A feeds two types of buffer solutions, but one type or three or more types of buffer solutions may be fed.
  • the liquid feed pump 8B feeds two kinds of buffer solutions, but one kind or three or more kinds of buffer solutions may be fed.
  • the flow path switching valve 2 switches the flow path F.
  • switching of the flow path switching valve 2 will be described with reference to FIGS. 2A and 2B.
  • FIG. 2A is a diagram showing a first state of the flow path switching valve 2
  • FIG. 2B is a diagram showing a second state. Refer to FIG. 1 as appropriate.
  • the flow of the solution is indicated by a dashed arrow.
  • the sample solution introduced from the autosampler 7 is introduced into the pretreatment column 5 via the flow paths F2 and F3.
  • the pretreatment column 5 a large substance having a molecular weight of tens of thousands or more such as a protein is quickly discharged, but a substance having a molecular weight of several hundreds such as a drug or a hormone, that is, a substance to be measured (first substance).
  • the solution (containing the protein) discharged from the pretreatment column 5 is sent to the UV detector 3 via the flow paths F4 and F5. Thereby, the UV detector 3 detects the amount of the protein (second substance) which is an interfering substance in the sample.
  • the mixed solution of the third buffer solution and the fourth buffer solution sent from the liquid feed pump 8B is sent to the analysis column 6 via the flow paths F6 and F7. Be liquid. Further, the mixed solution of the third buffer solution and the fourth buffer solution is sent to the mass spectrometer 4 via the flow path F8. However, in the first state, since the solution flowing to the mass spectrometer 4 does not contain a sample, nothing is detected by the mass spectrometer 4.
  • the control device 1 determines that the protein is substantially discharged
  • the control device 1 switches the flow path switching valve 2 from the first state to the second state shown in FIG. 2B at a predetermined timing.
  • the timing of switching from the first state to the second state will be described later.
  • the flow path F2 and the flow path F5 are directly connected.
  • the sample solution sent from the autosampler 7 is sent to the UV detector 3 as it is.
  • the flow path F6 and the flow path F4 are connected, and further, the flow path F3 and the flow path F7 are connected. Therefore, the solution sent from the liquid feeding pump 8B is sent to the pretreatment column 5 via the flow paths F6 and F4. Then, the delivered solution slowly discharges the substance to be measured adsorbed on the pretreatment column 5.
  • the discharged solution containing the substance to be measured is introduced into the analytical column 6 via the flow paths F3 and F7.
  • the substance to be measured contained in the solution is separated so as to be suitable for analysis by the mass spectrometer 4. After that, the substance to be measured separated by the analytical column 6 is introduced into the mass spectrometer 4 via the flow path F8 and analyzed by the mass spectrometer 4.
  • the mass spectrometer 4 can detect a sharp peak by preventing diffusion of the substance to be measured in the flow path F and in the pretreatment column 5. As a result, a high S / N ratio can be obtained.
  • FIG. 3 is a functional block diagram showing the configuration of the control device 1 according to the first embodiment.
  • the control device 1 includes a memory 110 composed of a volatile memory and the like, an input device 151 such as a keyboard and a mouse, and a display device 152 such as a display. Further, the control device 1 has a communication device 153 such as a NIC (Network Interface Card), a CPU (Central Processing Unit) 154, and a storage device 160 such as an HD (Hard Disk).
  • NIC Network Interface Card
  • CPU Central Processing Unit
  • HD Hard Disk
  • the processing unit 111 includes a parameter setting unit 112, a UV detection control unit 113, a switching timing setting unit 114, a loop determination unit 115, a warning processing unit 116, a valve control unit 117, a mass spectrometry control unit 118, and a switching time determination unit 119. Has been done.
  • the parameter setting unit 112 sets each parameter required in the process of determining the switching timing of the flow path switching valve 2, which will be described later.
  • the UV detection control unit 113 controls the UV detector 3 to acquire UV detection results such as the amount of protein detected.
  • the switching timing setting unit 114 determines and sets the timing for switching the flow path switching valve 2 from the first state to the second state.
  • the loop determination unit 115 determines whether or not the processing loop exceeds a predetermined threshold value in the process of determining the switching timing of the flow path switching valve 2, which will be described later.
  • the warning processing unit 116 outputs a warning when the timing (switching time) for switching the flow path switching valve 2 from the first state to the second state cannot be determined.
  • the mass spectrometry control unit 118 controls the mass spectrometer to acquire the detected amount of the substance to be measured.
  • the switching time determination unit 119 determines whether or not the current switching time is appropriate.
  • FIG. 4A is a graph showing the amount of protein detected by the UV detector 3.
  • the horizontal axis represents time and the vertical axis represents the amount of protein.
  • the UV detector 3 draws a curve (graph L1) having a peak at the peak arrival time t1 as shown in FIG. 4A.
  • the amino acids that make up proteins tyrosine, tryptophan, and phenylalanine have aromatic groups such as benzene rings and absorb ultraviolet rays with a wavelength of around 280 nm.
  • the UV detector 3 quantifies the amount of protein by measuring the absorbance of ultraviolet rays at 280 nm.
  • the UV detector 3 may measure the absorbance at 260 nm and 280 nm and calculate the protein concentration corrected for the influence of the light absorption of the nucleic acid.
  • the time of the timing (switching timing) for switching the flow path switching valve 2 from the first state to the second state is set as the switching time t2.
  • the control device 1 determines whether or not a measurement interfering substance such as a protein is discharged based on the detection result of the UV detector 3. That is, when the switching time t2 shown in FIG. 4A is set to the timing for switching from the first state to the second state, the amount of protein detected by the UV detector 3 is almost nonexistent, so that the protein discharge is almost completed. It is thought that there is. Therefore, by switching the flow path switching valve 2 from the first state to the second state at the switching time t2, it is possible to prevent the inflow of protein into the analysis column 6 and the mass spectrometer 4.
  • the pretreatment column 5 adsorbs the substance to be measured, but it is not completely adsorbed, but the discharge time is delayed. That is, over time, the substance to be measured flows out from the pretreatment column 5.
  • the switching time t2 in FIG. 4A is set as the switching timing, the amount of the substance to be measured flowing out from the pretreatment column 5 also increases. That is, many substances to be measured flow out to the UV detector 3.
  • the amount of the substance to be measured flowing to the mass analyzer 4 is reduced, and the sensitivity is lowered.
  • the peak area A0 in FIG. 4A is the area of the portion surrounded by the curve L1 and the horizontal axis.
  • this switching time t2 is a timing at which the detected amount of protein has not completely decreased, in other words, the protein has not been completely discharged from the pretreatment column 5. Therefore, when the flow path switching valve 2 is switched from the first state to the second state at this switching time t2, proteins flowing into the flow paths F3, F7, and F8 are present. The proteins flowing into the flow paths F3 and F7 shown in FIG.
  • the peak area A1 in FIG. 4B is the area of the portion surrounded by the curve L1 and the horizontal axis (the area of the shaded portion) after the switching time t2.
  • the timing of switching the flow path switching valve 2 needs to be balanced between the amount of protein detected by the UV detector 3 (the amount of protein discharged) and the substance to be measured flowing out from the pretreatment column 5.
  • FIG. 5 is a mass spectrometric result plotting the relationship between the switching time t2 of the flow path switching valve 2 in FIGS. 4A and 4B and the detection peak height of the substance to be measured by the mass spectrometer 4.
  • the peak area of the substance to be measured is constant. That is, in FIG. 5, it is shown that the higher the detection peak height, the sharper the detection peak is, because the detection region is not diffused.
  • the earlier the time for switching the flow path switching valve 2 the higher the detection peak height. That is, if the time for switching the flow path switching valve 2 is earlier, the detection region is not diffused and a higher S / N ratio can be obtained.
  • the peak detected by the mass spectrometer 4 due to the influence of the interfering substance. May be low.
  • FIGS. 6A and 6B are flowcharts showing a procedure for determining the switching timing of the flow path switching valve 2 according to the first embodiment.
  • the processes of FIGS. 6A and 6B are processes performed by the control device 1.
  • the parameter setting unit 112 sets each parameter used in this flowchart (S101 in FIG. 6A).
  • the parameters set here are the threshold values (Nth, MPth, Rat) and the like used in steps S111, S131, and 133.
  • the setting parameters of the threshold the peak height, the peak area, the peak arrival time, the peak width, the temporary differential coefficient of the peak, the measurement time, and the sample in the UV detector 3 and the mass spectrometer 4 are used.
  • the amount of injection from the autosampler 7 may be set.
  • the UV detection control unit 113 sets the flow path switching valve 2 in the first state (S102), and measures with the UV detector 3 (UV measurement). Do (S103). This UV measurement is performed with the flow path switching valve 2 in the first state until the protein has completely flowed out.
  • the switching timing setting unit 114 records the peak area A0 (see FIG. 4A) and the peak arrival time t1 in the measurement result of step S103 (S104).
  • the switching timing setting unit 114 determines the switching time t2 based on the peak area A0 recorded in step S104 and the peak arrival time t1 (S105). At this time, the switching timing setting unit 114 sets a sufficiently large switching time t2 so that proteins exceeding a predetermined amount do not flow into the flow paths F3, F7, and F8. For example, the time at which the peak value ⁇ 0.2 in the graph L1 of the UV detector 3 shown in FIG. 4 is set as the switching time t2.
  • the loop determination unit 115 determines whether or not the number of repetitions (N) is equal to or less than the repetition threshold value (Nth) (S111).
  • the warning processing unit 116 When the number of repetitions (N) is larger than the repetition threshold (Nth) (S111 ⁇ No), the warning processing unit 116 outputs a warning (S112). In step S112, for example, the warning processing unit 116 displays a warning message on the display device 152 recommending the change of the liquid feeding condition or the replacement of the pretreatment column 5, assuming that the switching time t2 satisfying the condition does not exist.
  • the valve control unit 117 switches the flow path switching valve 2 to the first state (S121), and the UV detection control unit 113 detects UV.
  • the measurement (UV measurement) by the device 3 is started (S122).
  • the valve control unit 117 determines whether or not the time t from the start of injection of the sample by the autosampler 7 is equal to or greater than the switching time t2 (S123). When the time t from the start of injection of the sample is less than the switching time t2 (S123 ⁇ No), the processing unit 111 returns the processing to step S123. When the time t from the start of sample injection is equal to or longer than the switching time t2 (S123 ⁇ Yes), the valve control unit 117 switches the flow path switching valve 2 to the second state (S124), and the mass spectrometry control unit 118 determines. The measurement (MS measurement) by the mass spectrometer 4 is started (S125).
  • the switching time determination unit 119 determines whether or not the detection peak value (MP) by the mass spectrometer 4 is equal to or higher than the mass spectrometry threshold value (MPth). Judgment (S131 in FIG. 6B). When the peak value (MP) detected by the mass spectrometer 4 is less than the mass spectrometry threshold (MPth) (S131 ⁇ No), the switching timing setting unit 114 reduces the switching time t2 by a predetermined amount (S132), and the processing unit 111. Returns the process to step S111. As described above, a high detection peak value corresponds to a high S / N ratio.
  • the switching time determination unit 119 has a peak area ratio (RA) equal to or greater than the peak area ratio threshold (RAth). It is determined whether or not it is (S133).
  • the peak area ratio (RA) is the ratio of the peak area A1 of the UV detection result (see FIG. 4B) and the peak area A0 of the UV detection result (see FIG. 4A) after the switching time t2.
  • the peak area ratio (RA) is a value obtained by dividing the peak area A1 of the UV detection result after the switching time t2 by the peak area A0 of the UV detection result.
  • the peak area ratio (RA) corresponds to the protein removal rate.
  • the peak area ratio threshold value (RAth) is a value set by the parameter setting unit 112 in step S101.
  • the switching timing setting unit 114 When the peak area ratio (RA) is less than the peak area ratio threshold value (RAth) (S133 ⁇ No), the switching timing setting unit 114 increases the switching time t2 by a predetermined amount (S134), and the processing unit 111 goes to step S111. Return the process.
  • the switching timing setting unit 114 determines the switching time t2 with the current value (S135).
  • the switching timing setting unit 114 may increase the mass spectrometry threshold value (MPth) by a predetermined amount.
  • switching time t2 switching timing in which the protein removal rate (peak area ratio (RA)) and the peak value of the mass spectrometer 4 are balanced.
  • RA peak area ratio
  • FIG. 7 is a diagram showing the configuration of the liquid chromatograph mass spectrometry system Za according to the second embodiment.
  • the liquid chromatograph mass spectrometric system Za shown in FIG. 7 includes a first filter 9A and a second filter 9B. As shown in FIG. 7, when the flow path switching valve 2 is in the first state, the first filter 9A is provided in the front stage of the pretreatment column 5, and the second filter 9B is provided in the front stage of the analysis column 6. Be done.
  • the first filter 9A protects the pretreatment column 5
  • the second filter 9B protects the analysis column 6.
  • Other configurations and operations are the same as those in the first embodiment.
  • FIG. 8 is a diagram showing the configuration of the liquid chromatograph mass spectrometry system Zb according to the third embodiment.
  • a pretreatment column switching system 50 is provided in a portion of the pretreatment column 5.
  • an analysis column switching system 60 is provided in a portion of the analysis column 6.
  • a plurality of pretreatment columns 5A to 5D are connected to the first column switching valve 2A and the second column switching valve 2B.
  • the first column switching valve 2A and the second column switching valve 2B switch the pretreatment columns 5A to 5D so that any one of the pretreatment columns 5A to 5D is selected as the pretreatment column 5.
  • a plurality of analysis columns 6A to 6D are connected to the third column switching valve 2C and the fourth column switching valve 2D.
  • the third column switching valve 2C and the fourth column switching valve 2D switch the analytical columns 6A to 6D so that any one of the analytical columns 6A to 6D is selected as the analytical column 6.
  • FIG. 9 is a functional block diagram showing the configuration of the control device 1b according to the third embodiment.
  • the processing unit 111b of the control device 1b is different from the control device 1 shown in FIG. 3 in that it has a valve switching processing unit 120 and a deterioration determination unit 121.
  • the valve switching processing unit 120 controls the flow path switching of the first column switching valve 2A to the fourth column switching valve 2D.
  • the deterioration determination unit 121 determines the deterioration state of the pretreatment columns 5A to 5D and the analysis columns 6A to 6D.
  • FIG. 10 is a flowchart showing a procedure for determining the switching timing of the flow path switching valve 2 according to the third embodiment.
  • the same process as in FIG. 6A is assigned the same step number and the description thereof will be omitted. Further, since the processing after step S131 is the same as that in FIG. 6B, the illustration and description will be omitted.
  • the valve switching processing unit 120 includes all of the pretreatment columns 5A to 5D and the analysis columns 6A to 6D (all of the columns). Determines whether or not has been switched (S141). When all of the preprocessing columns 5A to 5D and the analysis columns 6A to 6D (all of the columns) have been switched (S141 ⁇ Yes), the warning processing unit 116 outputs a warning (S112).
  • the valve switching processing unit 120 is the first column switching valve 2A to the fourth column switching valve. A switching process for switching each of the 2Ds is performed (S142). At this time, the valve switching processing unit 120 switches the first column switching valve 2A to the fourth column switching valve 2D so as to use the unused pretreatment columns 5A to 5D and the analysis columns 6A to 6D. After that, the deterioration determination unit 121 performs deterioration determination processing of the pretreatment columns 5A to 5D and the analysis columns 6A to 6D (S143). Details of the deterioration determination process will be described later. Then, the processing unit 111 returns the processing to step S102.
  • the first column switching valve 2A to the fourth column switching valve 2D show an example of a four-way valve, but a six-way valve or a ten-way valve is used, and the pretreatment column 5 and the analysis column 6 are used. 4 or more types of each may be installed in parallel.
  • a plurality of pretreatment columns 5A to 5D and a plurality of analytical columns 6A to 6D if the number of times the sample is injected is the same, the frequency of use of each column is reduced and the liquid chromatograph The durability of the mass spectrometry system Zb is improved. Further, by using two or more pretreatment columns 5, the pretreatment can proceed in parallel. As a result, it is possible to improve the analysis throughput.
  • the pretreatment columns 5A to 5D and the analysis columns 6A to 6D are distributed.
  • the processing columns 5A to 5D and the analysis columns 6A to 6D can be specified.
  • the pretreatment columns 5A to 5D and the analysis columns 6A to 6D, which have no problem can be selected and measured.
  • the pretreatment columns 5A to 5D may be of the same type, and the analysis columns 6A to 6D may be of the same type.
  • the measurement is performed by the UV detector 3 or the mass spectrometer 4 while switching each of the pretreatment columns 5A to 5D.
  • the deterioration determination unit 121 confirms whether the pretreatment columns 5A to 5D and the analysis columns 6A to 6D have deteriorated. Specifically, first, the pretreatment column 5A and the analysis column 6A are selected, the protein (interfering substance) removal rate (A1 / A0 (see FIGS. 4A and 4B), and the signal of the substance to be measured).
  • the intensity and the peak arrival time of the protein (interfering substance) and the substance to be measured are observed.
  • the removal rate of the protein (interfering substance) is A1 / A0 (see FIGS. 4A and 4B.
  • the signal intensity of the substance is the detection peak (MP) in the mass analyzer 4, and the peak arrival time of the substance to be measured is the time from the injection of the sample to the detection of the detection peak in the mass analyzer 4. ..
  • the measurement is performed in a state of being fixed to the analysis column 6A while sequentially switching between the pretreatment column 5B, the pretreatment column 5C, and the pretreatment column 5D.
  • a t-test was performed on the protein removal rate obtained for each pretreatment column 5, the signal intensity of the substance to be measured, and the peak arrival time of the protein and the substance to be measured, and pretreatment with a significant difference. If there is a column 5, the deterioration determination unit 121 considers that the pretreatment column 5 has deteriorated. Then, when it is determined that the product has deteriorated, the warning processing unit 116 may output a warning message prompting the replacement.
  • first column switching valve 2A and the second column switching valve 2B were fixed to the pretreatment column 5 in which no deterioration was observed, and the analysis column 6A, the analysis column 6B, the analysis column 6C, and the analysis column 6D were formed.
  • the signal intensity of the substance to be measured and the peak arrival time of the substance to be measured are measured while sequentially switching.
  • the deterioration determination unit 121 performs a t-test on the obtained signal intensity of the small molecule and the peak arrival time of the small molecule for each of the analysis columns 6A to 6D.
  • the deterioration determination unit 121 considers that the analysis column 6 has deteriorated, and the warning processing unit 116 outputs a warning message prompting the replacement. May be good.
  • deterioration determination processing By performing such deterioration determination processing, it is possible to detect the deteriorated pretreatment column 5 and analysis column 6 and avoid such columns. As a result, highly accurate UV measurement and mass spectrometric measurement can be performed over a long period of time.
  • a reference threshold value may be set for the determination of deterioration, and if it is outside the range of the threshold value, the deterioration determination may be performed. Further, the deterioration determination process and the deterioration determination unit 121 can be omitted. The deterioration determination process may be performed independently of the process of FIG. 10, for example, on Saturdays and Sundays during a period when the plant is closed.
  • FIG. 11 is a flowchart showing a procedure for determining the switching timing of the flow path switching valve 2 according to the fourth embodiment.
  • the process of FIG. 11 is a process performed by the control device 1. Since the configurations of the liquid chromatograph mass spectrometry system Z and the control device 1 are the same as those in the first embodiment, the illustration and description thereof are omitted here.
  • the switching time t2 is determined while monitoring the protein intensity in real time.
  • the parameter setting unit 112 sets each parameter used in this flowchart (S201).
  • the parameters to be set are the threshold values (UVth, MPth) and the like used in steps S213 and S231. Other parameters are the same as those set in step S101 of FIG. 6A.
  • the loop determination unit 115 determines whether or not the number of repetitions (N) is equal to or less than the repetition threshold value (Nth) (S202).
  • the repeat threshold value (Nth) is a value set by the parameter setting unit 112 in step S201.
  • the warning processing unit 116 outputs a warning (S203).
  • Step S203 is the same process as step S112 of FIG. 6A.
  • the valve control unit 117 switches the flow path switching valve 2 to the first state (S211), and the UV detection control unit 113 detects UV.
  • the measurement (UV measurement) by the device 3 is started (S212).
  • the UV detection control unit 113 measures the peak value of the absorption chromatograph of the UV detector 3 and monitors the peak intensity.
  • the current time t exceeds the peak arrival time t1 of the UV detection result (t ⁇ t1), and the UV detection value (UV) is a predetermined UV. It is determined whether or not it is equal to or less than the detection threshold (UVth) (S213).
  • the switching timing setting unit 114 Increases the switching time t2 by a predetermined amount (S214). Then, the processing unit 111 returns the processing to step S213 and continues the measurement by the UV detector 3.
  • the bulb control unit 117 moves through the flow path.
  • the switching valve 2 is switched to the second state (S221).
  • the valve control unit 117 stores the switching time t2 at this time in the storage device 160 (S222).
  • the mass spectrometry control unit 118 starts the measurement (MS measurement) by the mass spectrometer 4 (S223).
  • the measurement by the UV detector 3 is also performed in parallel with the measurement by the mass spectrometer 4.
  • the switching timing setting unit 114 ends the measurement by the UV detector 3 and the measurement by the mass spectrometer 4 (S224), and as a result of the measurement by the mass spectrometer 4, the detection peak (MP) is the detection peak threshold. It is determined whether or not it is (MPth) or more (S231).
  • the detection peak (RAth) is a value set by the parameter setting unit 112 in step S201.
  • the switching timing setting unit 114 increases the value of the UV detection threshold value (UVth) used in step S213 (S232). After that, the processing unit 111 returns the processing to step S202.
  • the switching timing setting unit 114 determines the switching time t2 with the current value (S233).
  • the process shown in FIGS. 11A and 11B is a step of fixing the flow path switching valve 2 in FIG. 6A in the first state and performing measurement by the UV detector 3.
  • the processing of S102 and S103 is omitted. That is, it is possible to omit the process of performing only the measurement of the UV detector 3 without performing the measurement by the mass spectrometer 4 once, as in the process shown in FIGS. 6A and 6B.
  • the fourth embodiment can perform analysis with higher throughput than the first embodiment.
  • the protein removal rate (A1 / A0) is obtained by fitting the peak to the result of the chromatogram and dividing the area of the fitted peak from the integrated value of the signal values up to the switching time of the flow path switching valve 2. May be calculated.
  • FIG. 12 is a diagram showing a measurement result of the UV detector 3 according to the fourth embodiment and a measurement result of the mass spectrometer 4.
  • the horizontal axis represents time.
  • the graph L11 shows a time-lapse graph of the amount of protein (for example, protein in urine) detected by the UV detector 3
  • the graph L12 shows a substance to be measured (for example, in urine) detected by the mass spectrometer 4.
  • the time passage graph of the included metabolite (molecule A) is shown.
  • the intensity of the vertical axis is standardized. Further, in order to achieve high-speed analysis, each measurement of the UV detector 3 and the mass spectrometer 4 was performed under the condition that the peaks of both substances (protein and the substance to be measured) could be detected within 2 minutes.
  • the line L21 is the UV detection threshold (UVth). As shown in FIG. 12, the time after the peak of the graph L11 and the intersection of the graph L11 and the line L21 is the switching time t2.
  • the pretreatment column 5 used a filler having a particle size of 25 um, an inner diameter of 2.1 mm, and a length of 10 mm.
  • the pretreatment column 5 used a filler having a particle size of 25 um, an inner diameter of 2.1 mm, and a length of 10 mm.
  • UVth UV detection threshold
  • UVP UVP ⁇ 0.2
  • the method shown in the fifth embodiment relates to cleaning of the pretreatment column 5 and the analysis column 6.
  • FIG. 13 is a diagram showing the configuration of the liquid chromatograph mass spectrometry system Zd according to the fifth embodiment.
  • the same components as those in FIG. 1 are designated by the same reference numerals, and the description thereof will be omitted.
  • waste liquid valves V1 and V2 which are valves for switching waste liquid, are provided in the flow path F4 and the flow path F8.
  • a waste liquid flow path F9 is connected to each of the waste liquid valves V1 and V2.
  • a cleaning / equilibrium apparatus W1 for cleaning and equilibrating the pretreatment column 5 and a cleaning / equilibrium apparatus W2 for cleaning and equilibrating the analytical column 6 are provided.
  • the waste liquid valves V1 and V2 and the cleaning / balancing devices W1 and W2, respectively, are controlled by the control device 1d.
  • FIG. 14 is a functional block diagram showing the configuration of the control device 1d according to the fifth embodiment.
  • the processing unit 111d in the control device 1d is different from the control device 1 in FIG. 3 in that it has a cleaning / equilibration processing unit 122.
  • the cleaning / equilibration processing unit 122 controls the cleaning / balancing devices W1 and W2 (see FIG. 13) to perform cleaning and equilibration of the pretreatment column 5 and the analysis column 6.
  • the cleaning / equilibration processing unit 122 switches the waste liquid valves V1 and V2 to the side of the waste liquid flow path F9 at the time of cleaning the pretreatment column 5 and the analysis column 6, and wastes the cleaning liquid and the solution. Flow to the flow path F9. This makes it possible to prevent contamination of the UV detector 3 and the mass spectrometer 4.
  • FIG. 15 is a flowchart showing a procedure for determining the switching timing of the flow path switching valve 2 according to the fifth embodiment.
  • the process of FIG. 15 is a process performed by the control device 1.
  • the same steps as those in FIG. 6A are designated by the same step numbers, and the description thereof will be omitted. Further, since the processing after step S131 is the same as that in FIG. 6B, the illustration and description will be omitted.
  • the valve control unit 117 switches the flow path switching valve 2 to the first state (S161).
  • the cleaning / equilibration processing unit 122 performs cleaning and equilibration of the pretreatment column 5 and the analysis column 6 by the cleaning / equilibration devices W1 and W2 (see FIG. 13), respectively (S162). .. After that, the switching timing setting unit 114 performs the process of step S131 (FIG. 6B).
  • FIG. 16 is a flowchart in which the process of the fifth embodiment is applied to the process of FIG.
  • the same process as in FIG. 11 is assigned the same step number and the description thereof will be omitted.
  • the cleaning / equilibration processing unit 122 switches the flow path switching valve 2 to the first state (S261). After that, the cleaning / equilibration processing unit 122 performs cleaning and equilibration of the pretreatment column 5 and the analysis column 6 by the cleaning / equilibration devices W1 and W2 (see FIG. 13), respectively (S262). .. After that, the switching timing setting unit 114 performs the process of step S231.
  • the time required for cleaning the pretreatment column 5 and the analysis column 6 depends on the signal intensity of the UV detection result observed in step S103 in FIG. 15, step S212 in FIG. 16, and the peak area. May be changed.
  • the cleaning / balancing processing unit 122 may determine whether or not the time required for the cleaning step is appropriate by monitoring the background level of the detection signal of the UV detector 3. In this case, if the background level is equal to or less than the reference value, the cleaning / equilibration processing unit 122 determines that the pretreatment column 5 is not contaminated. If the background level is equal to or higher than the reference value, the cleaning / equilibration processing unit 122 continues the cleaning process or increases the cleaning time. Further, the cleaning / equilibration processing unit 122 may perform control such that the voltage of the ion source of the mass spectrometer 4 is not applied or heated during cleaning.
  • the robustness of the UV detector 3 and the mass spectrometer 4 is improved, and the analysis accuracy is stabilized. It has the effect of.
  • the flow rate of the flow path F is appropriately adjusted in order to solve the problem of column withstand voltage, which is a problem in aiming at measurement by a high-speed liquid chromatograph mass spectrometry system Z using online pretreatment. It is to do.
  • the configuration of the liquid chromatograph mass spectrometry system Z is the same as that shown in FIG. 1 except that the control device 1 becomes the control device 1e, and thus the illustration and description thereof are omitted here.
  • FIG. 17 is a functional block diagram showing the configuration of the control device 1e according to the sixth embodiment.
  • the processing unit 111e of the control device 1e is different from the control device 1 of FIG. 3 in that it has a flow rate control unit 123.
  • the flow rate control unit 123 controls the liquid feed pump 8B to increase the flow rate of the solution flowing through the flow paths F6 to F8.
  • FIG. 18 is a flowchart showing a procedure for determining the switching timing of the flow path switching valve 2 according to the sixth embodiment.
  • the same processing as in FIG. 6A is designated by the same reference numerals and the description thereof will be omitted. Further, since the processing after step S131 is the same as that in FIG. 6B, the illustration and description will be omitted.
  • the flow path F In order to solve the problem of the withstand voltage of the column, which is a problem in aiming at the measurement by the high-speed liquid chromatograph mass spectrometry system Z using the online pretreatment, the flow path F The flow rate is adjusted appropriately.
  • a packing material having a particle size of 1 to 5 ⁇ m is used for the analytical column 6. Since the high withstand voltage analytical column 6 generally has a withstand voltage of 50 MPa or more, it is possible to send a liquid at a high flow rate of 0.5 mL / min or more. On the other hand, since a large-sized substance such as a protein flows into the pretreatment column 5, a filler having a particle size of 10 um or more, which is larger than that of the analysis column 6, is generally used in order to prevent clogging. As a result, many of the pretreatment columns 5 have a lower withstand voltage than the analysis column 6, and the withstand voltage is about 10 to 30 MPa.
  • the pressure is most applied to the pretreatment column 5 in the second state in which the analysis column 6 and the pretreatment column 5 are connected in series. While the flow path switching valve 2 is in the second state, it is necessary to reduce the flow rate in order to use the pretreatment column 5 at a pressure equal to or lower than the withstand voltage. As a result, the measurement time increases.
  • step S124 the measurement by the mass spectrometer 4 is performed with the flow path switching valve 2 in the second state (S125). Then, the flow rate control unit 123 determines whether or not the substance to be measured has been completely eluted from the pretreatment column 5 (S171). When the substance to be measured has not been completely eluted from the pretreatment column 5 (S171 ⁇ No), the flow rate control unit 123 returns the treatment to step S171. When the substance to be measured has been completely eluted from the pretreatment column 5 (S171 ⁇ Yes), the valve control unit 117 switches the flow path switching valve 2 to the first state (S172). Then, the flow rate control unit 123 increases the flow rate within the range of the withstand voltage of the analysis column 6 (S173). After that, the process of step S131 is performed.
  • the valve control unit 117 takes too early to switch the flow path switching valve 2 from the second state to the first state. Is determined. Insufficient detection result of mass spectrometer means that the detection peak is not detected in the graph in the detection result of mass spectrometer 4, or the detection peak is detected but a part of the graph is missing. If you are.
  • the valve control unit 117 sets the flow path switching valve 2 from the second state to the first state in step S172. Controls to delay the timing of switching to the state.
  • the flow velocity of the solution flowing through the flow path F can be increased, so that the overall measurement time can be shortened.
  • GUI (Graphical User Interface) screen 200) 19A to 19C are diagrams showing an example of the GUI screen 200 in this embodiment.
  • the GUI screen 200 has a status screen area 210 for displaying the status of the liquid chromatograph mass spectrometric systems Z, Za, Zb, and Zd, and an input for instructing the execution of processing.
  • It has a screen area 220 and an optimization result (Result) screen area 230 for displaying the optimization result.
  • the optimization means the optimization of the switching time t2.
  • the process is any one of the processes shown in FIGS. 6A, 6B, 10, 11, 15, 16, and 18.
  • the state of the liquid chromatograph mass spectrometry system Z such as the temperature of the pretreatment column 5 and the analysis column 6, the internal pressure of the flow path F, the state of the flow path switching valve 2, and the ion source (not shown).
  • the state of the mass spectrometer 4 such as the temperature and the degree of vacuum in the vacuum chamber (not shown) may be displayed.
  • the input screen area 220 receives an instruction input for processing execution. When the input is made, the process is started.
  • the valve switching time t2 obtained as the execution result of each of the processes shown in FIGS. 6A, 6B, 10, 11, 15, 16 and 18, the number of times the process is executed, and the protein.
  • the removal rate of is displayed.
  • each process includes the number of loops in the process (N in FIGS. 6A, 10, 11, 15, and 18), a warning message issued when the switching time t2 of the flow path switching valve 2 satisfying the condition cannot be determined, and the like.
  • the information obtained by may be displayed.
  • the GUI screen 200 shown in FIG. 19A is a screen before the processing is started, and the optimization result screen area 230 includes “execution count: 0 times”, “switching time: 0s”, and “protein removal rate: 0”. It is displayed that it is “%".
  • the "switching time” is the switching time t2, which is the time of the switching timing for switching from the first state to the second state.
  • "preparing" indicating that the status is being prepared is displayed in the status screen area 210.
  • the protein removal rate corresponds to the peak area ratio (RA) as described above in FIG. 6A.
  • the GUI screen 200 shown in FIG. 19B is a screen displayed during processing, and nothing is displayed in the optimization result screen area 230. Further, in the status screen area 210, "execution” indicating that the process is being executed is displayed.
  • the GUI screen 200 shown in FIG. 19C is a screen displayed after the processing is completed, and "preparing” is displayed in the status screen area 210. Further, in the optimization result screen area, "execution count: 1 time”, “switching time: 100s”, and protein removal rate: 80% are displayed as the result of the processing.
  • the present invention is not limited to the above-described embodiment, and includes various modifications.
  • the above-described embodiment has been described in detail in order to explain the present invention in an easy-to-understand manner, and is not necessarily limited to those having all the described configurations.
  • an apparatus using an electrophoresis method may be used instead of the UV detector 3.
  • the peak width, the arrival time to the peak, and the like may be used.
  • each of the above-described configurations, functions, processing units 111, units 112 to 123, storage device 160, and the like may be realized by hardware, for example, by designing a part or all of them by an integrated circuit or the like.
  • the above-mentioned configurations, functions, and the like are realized by software by interpreting and executing a program in which a processor such as a CPU 154 realizes each function. You may. Information such as programs, tables, and files that realize each function is stored in HD as shown in FIGS. 3, 9, 14, and 17, and is recorded in memory, SSD (Solid State Drive), and the like.
  • control lines and information lines are shown as necessary for explanation, and not all the control lines and information lines are necessarily shown in the product. In practice, almost all configurations can be considered interconnected.

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