JP2012251937A - Preprocessor and automatic analyzer using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a preprocessor capable of improving analysis accuracy and reproducibility in an automatic analyzer by improving adjustment accuracy of a sample pH, and an automatic analyzer using the same.SOLUTION: In a pH adjustment processing having a step for measuring the pH of a sample housed by a solid-phase cartridge 6 for separating a specific component from the sample by a pH measuring instrument 15 to measure the pH of the sample, a step for adding a pH preparation solution for increasing the pH of the sample, and a step for adding a pH preparation solution for lowering the pH of the sample, a pH adjustment solution of a kind and quantity based on a measurement result of the pH measuring instrument 15 is dispensed to the sample.

Description

  The present invention relates to a pretreatment device for an apparatus for analyzing components in a sample such as blood, serum, plasma, urine and tissue, and an automatic analyzer using the same.

  One of the methods for analyzing target components contained in samples such as blood, serum, plasma, urine and tissue is immunoassay (immunoassay). In immunoassay analysis, the level of a substance contained in a sample is measured using a reaction between an antigen and an antibody. However, immunoassays require the production of antibodies against the target component, which increases the cost of testing, cannot handle cross-reactions with similar compounds such as metabolites, and support target components such as drugs that cannot produce antibodies. There was room for improvement in terms of being unable to do so. Therefore, in recent years, attempts have been made to physicochemically analyze target components using an automatic analyzer using a mass spectrometer. Also, before applying the sample to the analyzer, the pretreatment device performs pretreatment that purifies the target component to be analyzed from the sample containing a large amount of impurities to some extent, thereby improving the analysis accuracy of the automatic analyzer. ing.

  For example, Patent Document 1 (Japanese Patent Laid-Open No. 2010-60474) discloses a technique related to such a pretreatment device and an automatic analysis device for the purpose of enabling a large number of samples to be processed simultaneously in parallel. Separating agent that selectively separates specific components by letting liquid flow through, holding part that holds the separating agent inside, holding part that can hold a plurality of receiving parts and has an endless track, and continuous and random in the receiving part A preloading device comprising a pressure loading unit that can load pressure in an accessible manner, and an extraction solution receiving mechanism that selectively receives an extraction solution from a separating agent accommodated in the accommodating unit, and can be connected to the pretreatment device, An automatic analyzer including a mass spectrometer capable of pre-processing a sample and performing analytical measurement in a fully automatic manner is disclosed.

  Patent Document 2 (Japanese Patent Application Laid-Open No. 2003-121317) discloses that endocrine disrupting chemical substances contained in a water sample containing suspended solids (SS) can be simultaneously captured and extracted for quick and low-cost pretreatment. In order to provide a method for capturing and extracting endocrine disrupting chemicals in a water sample containing SS and a solid phase extraction column therefor, a water sample containing suspended material is contained in a suspended material capturing material. After passing water through a solid-phase extraction column and capturing the suspended matter adsorbed and dissolved components of the endocrine disrupting chemical contained in this water sample at the same time, washing the inside of the column to remove impurities, A technique for simultaneously recovering captured endocrine disrupting chemicals by solvent extraction is disclosed.

  Further, in Patent Document 3 (Japanese Patent Laid-Open No. 2006-53001), alkali metals and alkaline earth metals in a sample are efficiently excluded, and only the metal to be measured is easily eluted to obtain a sample for analysis. For the purpose of using a solid phase extraction part filled with a chelate resin having a polyaminopolycarboxylic acid (PAPC) type chelate functional group bonded to a base resin having a hydroxyl group via an imino group, In an analysis method using a chelate resin for extracting and analyzing a metal element, the measurement target sample is adjusted to be in the range of pH 3 to 6, and the adjusted measurement target sample is passed through the solid phase extraction unit. In addition, a technique is disclosed in which an aqueous acid solution is passed through a solid-phase extraction section after sample passage to elute the captured metal element, and the eluted metal element is analyzed by an element analyzer.

JP 2010-60474 A JP 2003-121317 A JP 2006-53001 A

  In the pretreatment device, for purification of the target component from the sample, for example, the sample (mobile phase) containing the analysis target component is passed through the solid phase cartridge (stationary phase), and the component is dissolved between the mobile phase and the stationary phase. A method is often used in which the target component is supplemented by the difference in sex, and after separating (removing) impurities, the target component is separated by passing through an eluate and eluting the target component. In this method, since the range of selection of the stationary phase is not so wide, for example, by changing the pH (hydrogen ion index) of the sample depending on the type (property) of the target component, it corresponds to diversification of separation. . At this time, since the accuracy of pH adjustment affects the recovery rate of the target component, and the recovery rate directly affects the analysis accuracy of the automatic analyzer, it is required to adjust the pH of the sample with high accuracy.

  However, when the conventional technology described in Patent Document 3 is applied to the automatic analyzer described in Patent Document 1 or Patent Document 2, a certain amount of pH adjustment solution is added to the sample, so that the original pH of the sample PH adjustment is affected by the amount of contaminants that vary between samples and samples, and there is a problem that the recovery rate of target components and, consequently, the accuracy of analysis varies, and there is room for improvement in this regard. It had been.

  The present invention has been made in view of the above, and by improving the accuracy of adjusting the pH of a sample, a pretreatment device capable of improving the analysis accuracy and reproducibility in an automatic analyzer and an automatic analysis using the same An object is to provide an apparatus.

  To achieve the above object, the present invention provides a solid phase cartridge for separating a specific component from a sample, a cartridge holding unit for holding the solid phase cartridge, and the sample contained in a sample container as the solid phase cartridge. A sample probe to be dispensed, a pressure loading unit for applying pressure to the solid phase cartridge held by the cartridge holding unit, a pH measuring device for measuring the pH of the sample, and a pH for preparing the sample A reagent probe for adding a pH adjustment solution to the sample, a step of measuring the pH of the sample, a step of adding a pH adjustment solution for increasing the pH of the sample, and a pH adjustment for lowering the pH of the sample And a controller for performing a pH adjustment process including a step of adding a solution, wherein the controller adjusts the pH adjustment process. Oite, on the basis of the measurement results of the pH meter, and controls the type and amount of the pH adjusting solution is dispensed into the sample.

  According to the present invention, it is possible to improve the analysis accuracy and reproducibility of the automatic analyzer by improving the pH adjustment accuracy of the sample.

It is a figure which shows the whole structure of the automatic analyzer which concerns on 1st Embodiment. It is a figure which shows the structure of a pressure load part roughly. It is a figure which shows notionally the control relationship between each part and control part of an automatic analyzer. It is a processing flow of the pre-process in the pre-processing apparatus of 1st Embodiment. It is a figure which shows the change of the recovery rate calculated | required from the detected amount of the object chemical | medical agent in a mass spectrometer at the time of changing pH in the sample of known concentration in pre-processing. It is a figure which shows the whole structure of the automatic analyzer which concerns on 2nd Embodiment.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

<First Embodiment>
FIG. 1 is a diagram showing an overall configuration of an automatic analyzer according to a first embodiment of the present invention, FIG. 2 is a diagram schematically showing a detailed configuration of a pressure load unit, and FIG. 3 is each part of the automatic analyzer. It is a figure which shows notionally the control relationship between and a control part.

  In FIG. 1, an automatic analyzer according to the present embodiment includes a pretreatment device 1 that performs preprocessing on a sample that is an analysis target, and a mass analysis unit that performs analysis processing for analyzing a target component in the pretreated sample. 2 schematically.

  The pretreatment device 1 accommodates a plurality of sample containers 4 that contain samples to be analyzed, a sample container transport unit 3 that holds and transports the plurality of sample containers 4, and various reagents used for sample pretreatment. A plurality of reagent containers 9, a reagent container transport unit 8 that holds and transports the plurality of reagent containers 9, a solid phase cartridge 6 that is a stationary phase used in a pretreatment (described later) in the pretreatment apparatus 1, and a solid phase A plurality of cartridge holding units 20 each holding the cartridge 6 are provided, the cartridge conveying unit 5 that conveys the solid phase cartridge 6 on an endless track, and the sample stored in the sample container 4 are held by the cartridge holding unit 20 The sample probe 7 to be dispensed into the solid phase cartridge 6 and the reagent contained in the reagent container 9 are stored in the cartridge holding unit 20. And a reagent probe 10 to be dispensed into a receiver 18 (described later) of the receiver transport unit 16, a stirring device 21 for stirring the contents (sample, reagent, or mixture thereof) of the solid phase cartridge 6, and a solid phase cartridge A pH measuring device 15 that measures the pH (hydrogen ion index) of the contained material 6, and a pressure loading unit 11 that applies air pressure to the solid phase cartridge 6 and causes the contained sample to pass through the solid phase cartridge 6. , A waste liquid tank 14 that stores the discharged liquid discharged from the solid phase cartridge 6 due to the pressure load by the pressure load section 11, and a receiver 18 that receives the extracted liquid extracted from the solid phase cartridge 6 by the pressure load by the pressure load section 11. A plurality of receiver holding parts 17 for holding the receivers 18 respectively, a receiver transporting part 16 for transporting the receivers 18 on an endless track, and an unused solid phase car A cartridge / receiver holder 19 that holds the ridge 6 and the receiver 18 and transfers the cartridge to the cartridge holder 20 and the receiver holder 17, and a controller 22 that controls the overall operation of the pretreatment device 1. Yes.

  The sample container 4 placed on the sample container transport unit 3 accommodates blood, serum, plasma, urine, tissue, and the like as samples. Dispensing from the sample container 4 to the solid phase cartridge 6 is performed by the sample probe 7 at a dispensing position provided on the transport track. In addition, the reagent container 9 placed on the reagent transport unit 8 includes a pH adjusting solution (trifluoroacetic acid, aqueous ammonia, etc.), an internal standard reagent (internal standard substance reagent), and elution as reagents used for pretreatment. Contains liquid, distilled water, ultrapure water, and the like. Dispensing from the reagent container 9 to the solid phase cartridge 6 is performed by the reagent probe 10 at a dispensing position provided on the start of conveyance.

  The stirrer 21 stirs the contents of the solid phase cartridge 6, and vibrates the contents, the solid phase cartridge 6 and the like with sound waves (ultrasonic waves) and performs stirring. In addition, it is good also as a structure which immerses a stirring rod in a stored thing and stirs.

  The solid-phase cartridge 6 separates the target component between the mobile phase (sample or the like) and the stationary phase (solid-phase cartridge 6) based on the difference in solubility. By loading the air pressure with the unit 11, the stored sample or reagent is passed through. Liquid passing through the solid-phase cartridge 6 by the pressure load unit 11 is performed at the extraction position and the discharge position provided on the transport track. The discharge position is a position where the solid phase cartridge 6 is washed and unnecessary liquid is discharged. The discharged liquid that has passed through the solid phase cartridge 6 is sent to the waste liquid tank 14. The extraction position is the target in the solid phase cartridge 6. This is the position where the components are eluted by the eluent, and the eluate that has passed through the solid phase cartridge 6 is sent to the receiver 18.

  The pressure load part 11 has a joint part 12 for applying a pressure load to the solid phase cartridge 6 at the extraction position and a joint part 13 for applying a pressure load to the solid phase cartridge 6 at the discharge position. The driving means integrally moves up and down to switch between a posture in which pressure can be applied to the solid phase cartridge 6 and the cartridge holding unit 20 and a posture in which the solid phase cartridge 6 and the cartridge holding unit 20 can be transported. As shown in FIG. 3, the pressure load unit 11 includes a syringe pump 30 that sucks and discharges air, an intake port 31 of air sucked into the syringe pump 30, an air filter 32 provided in the intake port 31, and a syringe Compressed air discharged from the pump 30 is circulated and sent to the joints 12 and 13, and a pressure gauge 33 and a flow meter 34 for measuring the pressure and flow rate, respectively, and an air flow path from the intake port 31 to the syringe pump 30 A suction state in which the air flow path from the syringe pump 30 in the direction of the joints 12 and 13 is blocked, and the air flow path from the intake port 31 to the syringe pump 30 is blocked, and the syringe pump And a three-way solenoid valve 35 for switching between a discharge state in which a flow path of air from 30 to the joints 12 and 13 is circulated. Then, the air is sucked by switching to a sucking state in a transportable posture, the air is discharged by switching to a discharging state in a posture capable of pressure loading, and pressure is applied to the solid phase cartridge 6 and the cartridge holding unit 20.

  The waste liquid tank 14 has a function of measuring the pH of the discharged liquid in the waste liquid tank 14 in addition to the function of storing the discharged liquid from the solid phase cartridge 6 and adds a neutralizing liquid to the discharged liquid in the waste liquid tank 14. It has a function. The discharged liquid in the waste liquid tank 14 is prepared in a substantially neutral state by appropriately adding a neutralizing liquid according to the measured pH.

  The receiver 18 that contains the extraction liquid from the solid phase cartridge 6 and is held by the receiver holder 17 is transported by the receiver transport unit 16 to an analysis position where the analysis by the mass analyzer 2 is performed. The extract of the receiver 16 conveyed to the analysis position is sent to the mass analyzer 2 and subjected to mass analysis.

  The mass spectrometric unit 2 introduces an extract and introduces the sample to the ionization unit, a separation unit that separates the sample ionized by the ionization unit by mass, and a detection unit that detects a sample (ion) separated by mass Etc. The analysis result in the mass analysis unit 2 (the detection result in the detection unit) is sent to the control unit 22.

  The control unit 22 controls the operation of the pre-processing apparatus 1 and at the same time controls the operation of the mass analysis unit 2, and thus controls the operation of the entire automatic analysis apparatus. The control unit 22 inputs various settings for preprocessing and analysis processing, and inputs an operation instruction of the automatic analyzer as necessary, and various setting / instruction screens and analysis. A display unit 23 for displaying the results and a storage unit (not shown) for storing various setting contents, analysis results, a program for executing an analysis protocol for the analysis target component, and the like, and execute pre-processing and analysis processing To do.

  As shown in FIG. 3, when an instruction to execute preprocessing or analysis processing is given, the control unit 22 configures each component of the automatic analyzer (sample container transport unit 3 based on the setting contents and programs stored in the storage unit). , Reagent container transport unit 8, cartridge transport unit 5, sample probe 7, stirring device 21, pressure load unit 11, receiver transport unit 16, cartridge / receptor holding unit 19, waste liquid tank 14) Operations (including pre-processing and analysis processing) are performed. In addition, the measurement results of the pH meter 15, the pressure gauge 33, and the flow meter 34 are fed back to the control unit 22 to control the operation of each component of the automatic analyzer.

  The principle and procedure of preprocessing in the preprocessing apparatus 1 of the present embodiment will be described with reference to the processing flow of FIG.

(1) Principle of pretreatment Here, a solid phase cartridge 6 that is a stationary phase is used with a solid-phase extraction material of a reverse phase system, and a sample using a hydrophilic eluent as a mobile phase is passed through the solid-phase cartridge 6. A case will be described in which the target component is captured, and after the contaminants are washed, only the target component is eluted and concentrated and purified. In addition, a pretreatment when gemcitabine (a kind of anticancer agent) in a serum sample is used as a target component will be described as an example.

  In the above concentration / purification, the capture efficiency of the target component to the solid-phase extraction material varies depending on the hydrophobicity of the target component (target drug). When the distribution coefficient (logP) that is an index of the degree of hydrophobicity is small, that is, when a target drug having a high degree of hydrophilicity is subjected to solid phase extraction, the target drug passes through without being captured by the solid phase extraction material. Therefore, when purifying a measurement target drug having a high degree of hydrophilicity, the target drug is held by the fixed extractant by adjusting the pH of the sample according to the target drug.

  Gemcitabine exemplified as a target drug is a therapeutic agent for non-small cell lung cancer, pancreatic cancer, biliary tract cancer, bladder cancer, metastatic breast cancer, and soft tissue sarcoma. The chemical properties are a drug having high hydrophilicity and acidity with a molecular weight of 262.1, pKa (acid dissociation constant) of 3.60, and log P (partition coefficient) of 0.14. Gemcitabine is a highly hydrophilic drug with a log P of 0.14, so even if a patient serum sample to which gemcitabine has been administered is subjected to solid phase extraction using a reversed phase solid phase extraction material, affinity to the solid phase Sexuality is weak and cannot be maintained. Gemcitabine is a highly acidic drug with a pKa of 3.60, so it is fixed using a patient acid sample and a mobile phase (equilibration solution and washing solution) that has been lowered in pH by adding strong acid to suppress ion dissociation. By strengthening the affinity for the phase, the target drug is retained. The affinity to the stationary phase here refers not only to the affinity to the reverse phase system, but also to the affinity of the solid phase extraction material to the base material. In the case of drugs with high acid dissociation constants, based on the same principle, ion dissociation is suppressed using a mobile phase (equilibrated solution and washing solution) whose patient serum sample and pH have been increased by adding strong alkali to the stationary phase. The target drug is retained by increasing the affinity for.

(2) Preprocessing Procedure and Control FIG. 4 is a processing flow showing the preprocessing of the present embodiment.

  The pretreatment according to the present embodiment has eight steps including a cartridge transport step, a conditioning step, an equilibration step, a sample addition step, a pH adjustment step, a washing step, an elution step, and a dilution step.

(2-1) Cartridge Transport Process First, the control unit 22 of the pretreatment device 1 transfers the solid phase cartridge 6 of the cartridge / receptor holding unit 19 to the cartridge holding unit 20 as a pretreatment preparation operation, and receives the receiver. 18 is transferred to the receiver holder 16.

(2-2) Conditioning process (steps S10 and S20)
The cartridge holding unit 20 holding the solid phase cartridge 6 is transferred to the drive range of the reagent probe 10 by the cartridge transfer unit 5, and 200 μL of methanol stored in the reagent container transfer unit 8 is added by dispensing (step S10). . The reagent container 9 of the reagent container transport unit 8 includes gemcitabine-13C, 15N2 hydrochloride (for example, Tronto Research Chemicals, # G305010), pH adjusted to 2000 ng / mL by diluting with distilled water as an internal standard substance of gemcitabine. Trifluoroacetic acid (for example, SIGMA, # 09653) prepared to 2% by diluting with distilled water as a preparation solution and ammonia water (for example, Wako Pure Chemical, # 010) prepared to 1% by diluting with distilled water. -20825), diluted with methanol (eg Wako Pure Chemicals, # 138-14521) used in the conditioning process and elution process of solid phase cartridges, and distilled water used in the equilibration process and washing process to 0.1% The prepared trifluoroacetic acid and distilled water for diluting the eluate (for example, Wako Pure Chemicals, # 046-16971) are stored. Next, the cartridge is transported to the discharge position of the pressure load section 11 by the cartridge transport section 5, pressure is applied, and the discharged liquid is discharged to the waste liquid tank 14 (step S20).

(2-3) Equilibration step (Steps S30 and S40)
The cartridge holding unit 20 holding the solid phase cartridge 6 is transferred to the dispensing position of the reagent probe 10 by the cartridge transfer unit 5, and 200 μL of 0.1% trifluoroacetic acid stored in the reagent container 9 is added ( Step S30). Subsequently, the cartridge is transported to the discharge position of the pressure load section 11 by the cartridge transport section 5, pressure is applied, and the discharged liquid is discharged to the waste liquid tank 14 (step S40).

(2-4) Sample addition process (steps S50 and S60)
The cartridge holding unit 20 holding the solid phase cartridge 6 is transferred to the dispensing position of the sample probe 7 by the cartridge transfer unit 5, and a 100 μL sample stored in the sample container 4 (in this example, a patient to which gemcitabine has been administered) Are added by dispensing (step S50). In addition to serum, it is also possible to use a sample that has been pretreated by a method using an analysis protocol optimized for each specimen type, such as plasma, whole blood, urine, saliva, tears, and cell tissue. Next, the cartridge holding unit 20 holding the solid phase cartridge 6 by the cartridge transfer unit 5 is transferred to the dispensing position of the reagent probe 10, and 5 μL of an internal standard substance (in this example, stored in the reagent container 9) Gemcitabine-13C, 15N2 hydrochloride prepared to 2000 ng / mL by diluting with distilled water is added by dispensing (step S60).

(2-5) pH adjustment step (steps S70 to S100)
This is a step of performing pH adjustment treatment. The cartridge holding unit 20 holding the solid phase cartridge 6 is transferred to the dispensing position of the reagent probe 10 by the cartridge transfer unit 5, and 5 μL of 2% trifluoroacetic acid, which is a pH adjustment solution stored in the reagent container 9, is dispensed. Add by pouring (step S70). In this example, trifluoroacetic acid and aqueous ammonia were used as the pH adjusting solution, but hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, ammonium bicarbonate, ammonium acetate, and the like can be used as the pH adjusting solution. Next, the sample accommodated in the solid phase cartridge 6 is stirred by the stirring device 21, and the pH is measured by the pH measuring device 15 (step 80). Next, it is determined whether or not the value measured by the pH measuring device 15 is within a predetermined range (in this example, the target drug is gemcitabine and has a pH of 5.0 (± 5% of likelihood)). (Step S90). If the determination result in step S90 is YES, the solid phase cartridge 6 is transported to the discharge position of the pressure load section by the cartridge transport section 5, pressure is applied, and the discharged liquid is discharged to the waste liquid tank 14 (step). S100). If the determination result in step S90 is NO, the processes in steps S70 and S80 are repeated until the determination result in step S90 is YES. If the determination result in step S90 is YES, the solid phase cartridge 6 is transported to the discharge position of the pressure load section by the cartridge transport section 5, pressure is applied, and the discharged liquid is discharged to the waste liquid tank 14 (step S100). In step S70, when the measured pH value exceeds the preset pH value on the acidic side (that is, when pH <4.75), 1 μL of 1% ammonia water is used as the pH adjustment solution. Add. When the measured pH value exceeds the preset pH value on the basic side (that is, when pH> 5.25), 1 μL of 2% trifluoroacetic acid is added.

(2-6) Cleaning step (Steps S110 and S120)
The cartridge holding unit 20 holding the solid phase cartridge 6 is conveyed to the dispensing position of the reagent probe 10 by the cartridge conveying unit 5, and 200 μL of 0.1% trifluoroacetic acid stored in the reagent container 9 is dispensed. Add (step S110). Next, the cartridge holding unit 20 holding the solid-phase cartridge 6 by the cartridge transfer unit 5 is transferred to the discharge position of the pressure load unit 11, pressure is applied, and the produced liquid is discharged to the waste liquid tank 14 (step S120). ). In addition, it can confirm that the waste liquid (discharge liquid) in the waste liquid tank 14 discharged | emitted in all the processes was neutralized suitably by adding the neutralization solution, and was neutralized by the pH measurement function.

(2-7) Elution process (steps S130 and S140)
The cartridge holding unit 20 holding the solid phase cartridge 6 is transferred to the dispensing position of the reagent probe 10 by the cartridge transfer unit 5, and 100 μL of methanol stored in the reagent container 9 is added as an eluent (step S130). Next, the cartridge holding unit 20 holding the solid phase cartridge 6 by the cartridge transfer unit 5 is transferred to the extraction position of the pressure load unit 11, and at the same time, the receiver 18 held by the receiver holding unit 17 is transferred to the receiver. The liquid is transferred to the lower part of the solid phase cartridge 6 by the part 16 and pressure is applied, and the eluate is received by the receiver 18 held by the receiver holder 17 (step S140). Note that the receiver transport unit 16 does not have to have the same shape as the cartridge transport unit 5, and the rotation axis may not be at the same position. In the present embodiment, the rotational axes are set at different positions, and the receiver transport unit 16 and the cartridge transport unit 5 intersect at the receiving position of the receiver transport unit 16 (that is, the extraction position of the cartridge transport unit 5) as viewed from above. With this configuration, a space is formed above the cartridge transport unit 5, and the cartridge transport unit 5 does not interfere when the reagent probe 10 accesses the receiver.

(2-8) Dilution process (step S150)
Since drugs with high hydrophilicity such as gemcitabine have low solubility in methanol, dilution is performed by adding distilled water to the eluate in the receiver 18. The receiver holding part 17 holding the receiver 18 is transported to the dispensing position of the reagent probe 10 by the receiver transporting part 16, and 900 μL of distilled water stored in the reagent container 9 is added (step S150). The reagent probe 10 is a probe that can rotate and move up and down. The pretreated sample is introduced into the mass analyzer 2 and analyzed.

  The operational effects of the present embodiment configured as described above will be described with reference to the drawings.

  When administering a drug to a patient in a clinical setting, it is important to make an individual administration plan according to the patient's constitution and symptoms in order to ensure the effectiveness and safety. At this time, since the blood concentration of the drug component varies depending on individual differences in drug pharmacokinetics, there may be a difference in the therapeutic effect of the same volume of drug. Therefore, blood drug monitoring (TDM; Therapeutic Drug Monitoring) is performed by measuring the blood concentration of each patient, and drug pharmacokinetic observation is performed to optimize the volume and usage so that the blood concentration falls within the therapeutic range. Is going.

  One of the methods for analyzing target components contained in samples such as blood, serum, plasma, urine and tissue is immunoassay (immunoassay). In immunoassay analysis, the level of a substance contained in a sample is measured using a reaction between an antigen and an antibody. However, immunoassays require the production of antibodies against the target component, which increases the cost of testing, cannot handle cross-reactions with similar compounds such as metabolites, and support target components such as drugs that cannot produce antibodies. There was room for improvement in terms of being unable to do so. Therefore, in recent years, attempts have been made to physicochemically analyze target components using an automatic analyzer using a mass spectrometer. Also, before applying the sample to the analyzer, the pretreatment device performs pretreatment that purifies the target component to be analyzed from the sample containing a large amount of impurities to some extent, thereby improving the analysis accuracy of the automatic analyzer. ing.

  In the pretreatment device, for purification of the target component from the sample, for example, the sample (mobile phase) containing the analysis target component is passed through the solid phase cartridge (stationary phase), and the component is dissolved between the mobile phase and the stationary phase. A method is often used in which the target component is supplemented by the difference in sex, and after separating (removing) impurities, the target component is separated by passing through an eluate and eluting the target component. In this method, since the range of selection of the stationary phase is not so wide, for example, by changing the pH (hydrogen ion index) of the sample depending on the type (property) of the target component, it corresponds to diversification of separation. . At this time, since the accuracy of pH adjustment affects the recovery rate of the target component, and the recovery rate directly affects the analysis accuracy of the automatic analyzer, it is required to adjust the pH of the sample with high accuracy.

  However, when the conventional technology described in Patent Document 3 is applied to the automatic analyzer described in Patent Document 1 or Patent Document 2, a certain amount of pH adjustment solution is added to the sample, so that the original pH of the sample PH adjustment is affected by the amount of contaminants that differ from sample to sample. FIG. 5 is a graph showing a change in the recovery rate obtained from the amount of gemcitabine (target drug) detected by the mass spectrometer when the pH in the sample having a known concentration was changed in the pretreatment, and the horizontal axis The pH of the sample in the pretreatment target drug supplementation step is shown, and the vertical axis represents the recovery rate (%). As can be seen from FIG. 5, when the pH adjustment solution (trifluoroacetic acid or the like) is changed and the pH in the sample is changed, the recovery rate is approximately 80% or more in the region where the pH is lower than 5.0. On the other hand, as the pH becomes higher than 5.0, the recovery rate decreases rapidly. That is, there is a problem that the recovery rate of the target component and, consequently, the analysis accuracy changes due to the fluctuation of the pH of the sample, and there remains room for improvement in this respect.

  On the other hand, in the present embodiment, the pH of the sample is measured by a pH measuring device, and a step of adding a pH adjusting solution for increasing the pH of the sample based on the measurement result and the pH of the sample are lowered. In the pH adjustment process including the step of adding a pH adjustment solution for the purpose, the type and amount of the pH adjustment solution dispensed to the sample are controlled, so that the adjustment accuracy of the pH of the sample can be improved. It is possible to improve the analysis accuracy and reproducibility in the automatic analyzer.

  In this embodiment, gemcitabine has been described as an example of a drug having high hydrophilicity and high acidity, but it is also effective as a technique for improving the capture rate of a measurement target drug having a high protein binding rate to a solid phase cartridge. It is. For example, an example of concentrating and purifying tamoxifen, which is a kind of anticancer agent, is shown. Tamoxifen has a high binding rate to proteins such as albumin, and its molecular size is larger than the pore size of the solid-phase extraction material (solid-phase cartridge 6), making it difficult to capture. For this reason, the affinity of the drug to be measured to the solid phase cartridge 6 can be increased by adding an acid to the sample to dissociate the bond between the drug to be tested and the protein. In this case, for example, trifluoroacetic acid prepared to 0.1% by dilution with distilled water is used as the acid added to the sample, and distilled water is used in the equilibration step and the washing step.

<Second Embodiment>
A second embodiment of the present invention will be described with reference to the drawings.

  In the present embodiment, the sample container transport unit 3 is configured to perform pH measurement, pH adjustment solution, and the like to the contents accommodated in the sample container 4.

  FIG. 6 is a diagram showing an overall configuration of an automatic analyzer according to the second embodiment of the present invention. In the figure, the same members as those in the first embodiment are denoted by the same reference numerals, and description thereof is omitted.

  In FIG. 6, the automatic analyzer of the present embodiment includes a pretreatment device 1 that performs preprocessing on a sample that is an analysis target, and a mass analysis unit that performs analysis processing for analyzing a target component in the preprocessed sample. 2 schematically.

  The pretreatment device 1 accommodates a plurality of sample containers 4 that contain samples to be analyzed, a sample container transport unit 3 that holds and transports the plurality of sample containers 4, and various reagents used for sample pretreatment. A plurality of reagent containers 9, a reagent container transport unit 8 that holds and transports the plurality of reagent containers 9, a solid phase cartridge 6 that is a stationary phase used in pretreatment in the pretreatment device 1, and a solid phase cartridge 6 A plurality of cartridge holding parts 20 each holding the cartridge, a cartridge carrying part 5 carrying the solid phase cartridge 6 on an endless track, and a sample container holding the reagent contained in the reagent container 9 in the sample container carrying part 3 4. Reagent probe 10A to be dispensed to solid phase cartridge 6 held in cartridge holding unit 20, and receiver 18 (described later) of receiver transport unit 16, and sample A stirrer 121 that stirs the contents (sample, reagent, or mixture thereof) of the container 4, a pH measuring device 15 A that measures the pH (hydrogen ion index) of the contents of the solid phase cartridge 6, and the sample container 4. A sample probe 7 for dispensing the stored sample into the solid phase cartridge 6 held in the cartridge holding unit 20, and a stirring device 21 for stirring the contents (sample, reagent, or mixture thereof) of the solid phase cartridge 6; The solid phase cartridge 6 is loaded with air pressure, and the loaded sample 11 is passed through the solid phase cartridge 6, and the discharged liquid is discharged from the solid phase cartridge 6 by the pressure load by the pressure load unit 11. And a receptacle for receiving the extract extracted from the solid phase cartridge 6 by the pressure load by the pressure load unit 11 8 and a plurality of receiver holding parts 17 for holding the receivers 18 respectively. The receiver transporting part 16 for transporting the receivers 18 on an endless track, the unused solid phase cartridge 6 and the receivers 18 A cartridge / receiver holder 19 that holds and transfers the cartridge to the cartridge holder 20 and the receiver holder 17 and a controller 22 that controls the overall operation of the pretreatment device 1 are provided.

  Next, the preprocessing procedure and control in the present embodiment will be described. The pretreatment according to the present embodiment has eight steps including a cartridge transport step, a conditioning step, an equilibration step, a pH adjustment step, a sample addition step, a washing step, an elution step, and a dilution step. In addition, about the process similar to 1st Embodiment, only a process name is shown and description is abbreviate | omitted.

  First, the control unit 22 of the pretreatment apparatus 1 performs a cartridge transport process, a conditioning process, and an equilibration process.

  Subsequently, a pH adjustment step is performed. This step is a step of performing pH adjustment treatment. First, the sample container 4 is transported to the dispensing position of the reagent probe 10A by the sample container transport unit 3, and 5 μL of an internal standard substance stored in the reagent container 9 (in this example, diluted with distilled water to 2000 ng / Gemcitabine-13C, 15N2 hydrochloride prepared in mL) is added by dispensing. Next, the sample container 4 is transported to the dispensing position of the reagent probe 10A by the sample container transporting section 3, and 5 μL of 2% trifluoroacetic acid, which is a pH adjusting solution stored in the reagent container 9, is added by dispensing ( Step S70). In this example, trifluoroacetic acid and aqueous ammonia were used as the pH adjusting solution, but hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, ammonium bicarbonate, ammonium acetate, and the like can be used as the pH adjusting solution. Next, the sample accommodated in the sample container 4 is stirred by the stirring device 121, and the pH is measured by the pH measuring device 15A. Next, it is determined whether or not the measured value by the pH measuring device 15A is within a predetermined range (in this example, the target drug is gemcitabine and has a pH of 5.0 (± 5% of likelihood)). . When the measured value is within the range, the process proceeds to the next step. When the measured value is out of the range, the pH adjustment solution addition and the pH measurement process are repeated until the measured value is within the range. When the measured value is within the range, the process proceeds to the next step. In addition, in the addition of the pH adjustment solution, when the measured value of the pH exceeds the preset pH value on the acidic side (that is, when pH <4.75), 1 μL of 1% as the pH adjustment solution Add ammonia water. When the measured pH value exceeds the preset pH value on the basic side (that is, when pH> 5.25), 1 μL of 2% trifluoroacetic acid is added.

  Next, a sample addition process from the sample container 4 to the solid phase cartridge 6 is performed. First, the cartridge holding unit 20 holding the solid phase cartridge 6 is conveyed to the dispensing position of the sample probe 7 by the cartridge conveying unit 5, and the sample stored in the sample container 4 is added by dispensing. Subsequently, the solid phase cartridge 6 is transported to the discharge position of the pressure load section by the cartridge transport section 5, pressure is applied, and the discharged liquid is discharged to the waste liquid tank 14.

  Thereafter, a washing process, an elution process, and a dilution process are performed, and the pretreatment is completed. The pretreated sample is introduced into the mass analyzer 2 and analyzed.

  Other configurations are the same as those of the first embodiment.

  Also in the present embodiment configured as described above, the same effects as those of the first embodiment can be obtained.

DESCRIPTION OF SYMBOLS 1 Pretreatment apparatus 2 Mass spectrometry part 3 Sample container conveyance part 4 Sample container 5 Cartridge conveyance part 6 Solid phase cartridge 7 Sample probe 8 Reagent container conveyance part 9 Reagent container 10, 10A Reagent probe 11 Pressure load part 12, 13 Junction part 14 Waste tanks 15 and 15A pH measuring device 16 Receptor transport unit 17 Receptor holding unit 18 Receptor 19 Cartridge / receptor holding unit 20 Cartridge holding unit 21 Stirrer 22 Control unit 23 Display unit 24 Operation unit 30 Syringe pump 31 Intake port 32 Air filter 33 Pressure gauge 34 Flow meter 35 Three-way solenoid valve

Claims (8)

A solid phase cartridge for separating specific components from a sample;
A cartridge holder for holding the solid phase cartridge;
A sample probe for dispensing the sample contained in a sample container into the solid phase cartridge;
A pressure loading unit for applying pressure to the solid phase cartridge held by the cartridge holding unit;
A pH meter for measuring the pH of the sample;
A reagent probe for adding a pH adjusting solution for adjusting the pH of the sample to the sample;
A pH adjustment process including a step of measuring the pH of the sample, a step of adding a pH adjusting solution for increasing the pH of the sample, and a step of adding a pH adjusting solution for decreasing the pH of the sample is performed. A control unit,
The control unit controls the type and amount of the pH adjusting solution to be dispensed to the sample based on the measurement result of the pH measuring device in the pH adjusting process. Processing equipment. In the pre-processing apparatus of the automatic analyzer of Claim 1,
The pretreatment apparatus for an automatic analyzer, wherein the pH adjustment process is performed on the sample dispensed in the solid phase cartridge. In the pre-processing apparatus of the automatic analyzer of Claim 1,
The pretreatment apparatus for an automatic analyzer, wherein the pH adjustment process is performed on the sample stored in the sample container. In the pre-processing apparatus of the automatic analyzer of Claim 1,
The pH adjusting solution for lowering the pH of the sample is any one of trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, or phosphoric acid,
The pretreatment apparatus for an automatic analyzer, wherein the pH adjusting solution for raising the pH of the sample is any one of ammonium bicarbonate and ammonium acetate. In the pre-processing apparatus of the automatic analyzer of Claim 1,
A pretreatment for an automatic analyzer comprising a neutralization mechanism for adding a neutralizing solution for neutralizing the waste liquid of the sample discharged from the solid phase cartridge by the pressure load from the pressure load section apparatus. In the pre-processing apparatus of the automatic analyzer of Claim 1,
Comprising target component information setting means for setting target component information to be separated from the sample by the solid phase cartridge;
The said control part selects the kind of said pH adjustment solution based on the information of the said objective component, The pre-processing apparatus of the automatic analyzer characterized by the above-mentioned. In the pre-processing apparatus of the automatic analyzer of Claim 6,
The control unit performs each step of the pH adjustment processing so that the measurement result of the control pH measuring device falls within a range predetermined in the information of the target component. Processing equipment.   An automatic analyzer comprising: the pretreatment device according to claim 1; and a mass analysis unit that performs mass analysis of a sample pretreated by the pretreatment device.

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