CN110967439A - Liquid chromatography method and liquid chromatography apparatus - Google Patents
Liquid chromatography method and liquid chromatography apparatus Download PDFInfo
- Publication number
- CN110967439A CN110967439A CN201910915871.9A CN201910915871A CN110967439A CN 110967439 A CN110967439 A CN 110967439A CN 201910915871 A CN201910915871 A CN 201910915871A CN 110967439 A CN110967439 A CN 110967439A
- Authority
- CN
- China
- Prior art keywords
- liquid chromatography
- components
- component
- measurement
- elution time
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000004811 liquid chromatography Methods 0.000 title claims abstract description 84
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000005259 measurement Methods 0.000 claims abstract description 72
- 238000010828 elution Methods 0.000 claims abstract description 46
- 238000000926 separation method Methods 0.000 claims description 27
- 238000004458 analytical method Methods 0.000 claims description 25
- 239000003480 eluent Substances 0.000 claims description 19
- 229940079593 drug Drugs 0.000 claims description 17
- 239000003814 drug Substances 0.000 claims description 17
- 210000004369 blood Anatomy 0.000 claims description 12
- 239000008280 blood Substances 0.000 claims description 12
- 230000005856 abnormality Effects 0.000 claims description 7
- 230000002159 abnormal effect Effects 0.000 claims description 5
- 238000011002 quantification Methods 0.000 claims description 3
- 239000012071 phase Substances 0.000 description 29
- 239000000523 sample Substances 0.000 description 26
- 239000007790 solid phase Substances 0.000 description 20
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- 238000012545 processing Methods 0.000 description 11
- 238000010586 diagram Methods 0.000 description 7
- LOUPRKONTZGTKE-LHHVKLHASA-N quinidine Chemical compound C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@H]2[C@@H](O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-LHHVKLHASA-N 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000005517 L01XE01 - Imatinib Substances 0.000 description 3
- FFGPTBGBLSHEPO-UHFFFAOYSA-N carbamazepine Chemical compound C1=CC2=CC=CC=C2N(C(=O)N)C2=CC=CC=C21 FFGPTBGBLSHEPO-UHFFFAOYSA-N 0.000 description 3
- 229960000623 carbamazepine Drugs 0.000 description 3
- LOUPRKONTZGTKE-UHFFFAOYSA-N cinchonine Natural products C1C(C(C2)C=C)CCN2C1C(O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-UHFFFAOYSA-N 0.000 description 3
- 238000011067 equilibration Methods 0.000 description 3
- KTUFNOKKBVMGRW-UHFFFAOYSA-N imatinib Chemical compound C1CN(C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)C=C1 KTUFNOKKBVMGRW-UHFFFAOYSA-N 0.000 description 3
- 229960002411 imatinib Drugs 0.000 description 3
- PYZRQGJRPPTADH-UHFFFAOYSA-N lamotrigine Chemical compound NC1=NC(N)=NN=C1C1=CC=CC(Cl)=C1Cl PYZRQGJRPPTADH-UHFFFAOYSA-N 0.000 description 3
- 229960001848 lamotrigine Drugs 0.000 description 3
- 229960001404 quinidine Drugs 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- BCEHBSKCWLPMDN-MGPLVRAMSA-N voriconazole Chemical compound C1([C@H](C)[C@](O)(CN2N=CN=C2)C=2C(=CC(F)=CC=2)F)=NC=NC=C1F BCEHBSKCWLPMDN-MGPLVRAMSA-N 0.000 description 3
- 229960004740 voriconazole Drugs 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000007781 pre-processing Methods 0.000 description 2
- ZBAFFZBKCMWUHM-UHFFFAOYSA-N propiram Chemical compound C=1C=CC=NC=1N(C(=O)CC)C(C)CN1CCCCC1 ZBAFFZBKCMWUHM-UHFFFAOYSA-N 0.000 description 2
- 229950003779 propiram Drugs 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000013307 optical fiber Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- 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
- G01N30/86—Signal analysis
- G01N30/8658—Optimising operation parameters
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/49—Blood
- G01N33/491—Blood by separating the blood components
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8804—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 automated systems
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8822—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving blood
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8895—Independent juxtaposition of embodiments; Reviews
Abstract
The invention provides a liquid chromatography method and a liquid chromatography apparatus, which can accurately, simply and quickly measure a plurality of components contained in a sample by using liquid chromatography. In a liquid chromatography method for quantifying a plurality of components contained in a measurement sample by liquid chromatography, the liquid chromatography method includes: a pretreatment step of separating each component from a plurality of components; and a quantitative determination step of adjusting the measurement conditions of the liquid chromatography of each component so that the elution time is less than T0 and the difference between the elution times of the components is within. + -. 10%, respectively, for each of the separated components, and performing quantitative determination by the liquid chromatography, when the elution time for determining each component by the primary liquid chromatography in a state where the plurality of components are mixed is T0.
Description
Technical Field
The present invention relates to a liquid chromatography method and a liquid chromatography apparatus.
Background
In the field of Drug therapy, the implementation of Therapeutic Drug Monitoring (TDM) for measuring the blood concentration of an administered Drug is considered to be important for the prevention of the discovery of Therapeutic effects and side effects.
In TDM, since the drug to be administered to each patient is known in advance, it is required to perform quantitative analysis efficiently and quickly by using the drug as a measurement target.
Conventionally, the blood concentration of a drug is quantified by an immunochemical method, an immunoassay method, or the like using an antibody against the target drug.
In these methods, an antibody and a dedicated reagent are required, professional knowledge is required, and since an antibody is used, a substance different from the target drug may cross-react with the antibody, and thus the quantitative accuracy may be insufficient.
Therefore, there is a method of quantifying the concentration of a drug in blood by liquid chromatography. In this case, a technique of removing unnecessary components in measurement by performing pretreatment and extracting necessary components is known (patent document 1).
Patent document 1: japanese patent No. 6264465
In addition, in the liquid chromatography analysis, it is necessary to appropriately set the pretreatment conditions, the separation column, or the flow-equivalent measurement conditions. Further, since the elution time varies depending on the type of the drug to be measured and the measurement conditions, the time required for one measurement for each drug varies, and there is a problem that the operation efficiency is lowered due to the occurrence of a waiting time for the operation.
On the other hand, in the TDM, for example, since a drug to be administered to each patient is known in advance, it is required to perform quantitative analysis efficiently and quickly by using the drug as a measurement target. Since the sample is whole blood or serum and contains a complex matrix in the sample, it is considered more efficient if the target component can be obtained as a single peak in the analysis.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide a liquid chromatography method and a liquid chromatography apparatus capable of accurately, simply, and in a short time, measuring a plurality of components contained in a sample by liquid chromatography.
In order to achieve the above object, a liquid chromatography method according to the present invention is a liquid chromatography method for quantifying a plurality of components contained in a measurement sample by liquid chromatography, the liquid chromatography method including: a pretreatment step of separating each component from the plurality of components; and a quantitative determination step of adjusting the measurement conditions of the liquid chromatography for each of the separated components so that the elution time is less than T0 and the difference between the elution times of the components is within. + -. 10%, when the elution time for determining the components by a single liquid chromatography in a state where the components are mixed is T0.
According to this liquid chromatography analysis method, the measurement conditions are adjusted so that the elution time T1 of each component falls within a range of ± 10%, whereby the measurement time can be shortened as compared with the case where each component is quantified by one-time liquid chromatography in a state where a plurality of components are mixed, and since the measurement times of the components are substantially similar, the waiting time for analysis of a specific component does not become long, the measurement can be performed quickly, and the target or schedule for completion of the measurement can be easily prepared. In addition, since each component is a single peak, the time window for identification can be fixed, reproducibility of the peak area or elution time can be improved, quantitative accuracy can be improved, and the degree of separation of the peaks can be stabilized.
In addition, since the elution times T1 of the respective components are similar, the measurement conditions are not greatly different, and the quantitative accuracy is improved.
Further, when the difference between the elution times T1 of the respective components is within. + -. 10%, the analysis time is substantially constant, so that the influence on the analysis of the sample is constant, and the cause can be easily analyzed when there is a measurement failure. When the difference between the elution times T1 of the respective components is within. + -. 10%, it is easy to select a compound to be added as an internal standard.
In addition, when a plurality of components are mixed and measured by a single liquid chromatography, the measurement must be performed at a single wavelength, and the detection sensitivity may not be optimal for each component. Therefore, if the conditions (optimum wavelength, optimum mobile phase composition, etc.) optimum for each component are adjusted by separating each component in advance, the detection sensitivity is improved.
In the measurement conditions, the separation column may be the same as the mobile phase.
According to this liquid chromatography method, it is not necessary to change the separation column or the mobile phase for each component, and it is easy to measure each component in 1 liquid chromatography apparatus. In addition, the measurement of each component can be automated.
The mobile phase may contain two or more kinds of eluents, and the mixing ratio of each eluent may be changed for each component.
According to this liquid chromatography method, it is not necessary to physically replace the mobile phase for each bottle containing the eluent, and it is only necessary to control the mixer (mixer) for each component and change the mixing ratio of the eluent, and it is easy to measure each component in 1 liquid chromatography apparatus. In addition, the measurement of each component can be automated.
The method may further include the following abnormality determination step: and determining that the analysis is abnormal when the elution time of each component exceeds a predetermined threshold.
The extent of the elution time T1 is known by preliminary experiments, and when T1 exceeds the threshold value, it is considered that some problems have occurred in the liquid chromatography apparatus, and it is possible to notify the user of an abnormality.
The plurality of components may be drugs in the blood.
The liquid chromatography analysis apparatus of the present invention quantifies a plurality of components contained in a measurement sample by liquid chromatography, characterized in that the liquid chromatography device comprises a measurement condition storage means and a quantification means, when the elution time for quantifying each component by primary liquid chromatography in a state where the plurality of components are mixed in a state where each component is separated from the plurality of components by pretreatment is T0, a measurement condition storage unit that stores the measurement conditions of the liquid chromatography for each of the separated components, the measurement conditions are such that the quantification by the liquid chromatography is possible in such a manner that the elution times T1 are less than T0 and the difference in the elution times T1 of the respective components is within. + -. 10%, respectively, the quantifying unit quantifies the respective components by the liquid chromatography with reference to the measurement conditions.
In the measurement conditions of the liquid chromatography apparatus of the present invention, the separation column may be the same as the mobile phase.
In the liquid chromatography apparatus of the present invention, the mobile phase may contain two or more kinds of eluents, and the mixing ratio of each of the eluents may be changed for each of the components.
The liquid chromatography apparatus of the present invention may further include abnormality determination means for determining that the analysis is abnormal when the elution time T1 of each component exceeds a predetermined threshold value Tt.
In the liquid chromatography apparatus of the present invention, the plurality of components may be blood drugs.
According to the present invention, a plurality of components contained in a sample can be measured accurately, simply, and in a short time by liquid chromatography.
Drawings
Fig. 1 is a diagram showing the configuration of a liquid chromatography apparatus according to an embodiment of the present invention.
Fig. 2 is a diagram showing a chromatogram when 6 components contained in blood as a measurement sample are mixed and each component is separated by primary liquid chromatography and quantified.
Fig. 3 is a diagram showing a chromatogram of the isolated carbamazepine.
Fig. 4 is a diagram showing a chromatogram of isolated lamotrigine.
Fig. 5 is a diagram showing a chromatogram of the isolated quinidine.
Fig. 6 is a graph showing a chromatogram of isolated voriconazole.
Fig. 7 is a graph showing a chromatogram of the isolated imatinib.
Fig. 8 is a graph showing a chromatogram of the isolated propylpiramide.
Fig. 9 is a diagram showing an example of the elution time T1 of each component, the pretreatment conditions, and the measurement conditions stored in the storage unit.
Description of the reference symbols
3. 4: a mobile phase; 7: a separation column; 6: an autosampler; 9: a detector; 10: a data processing device; 100: liquid chromatography device
Detailed Description
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
Fig. 1 is a diagram showing the configuration of a liquid chromatography apparatus 100 according to an embodiment of the present invention.
The liquid chromatography apparatus 100 includes: a data processing device (control unit) 10 that controls the whole; two mobile phases 3, 4; pumps 1, 2, which deliver mobile phases 3, 4, respectively; a mixer 5, which mixes the respective mobile phases 3, 4 in a ratio of 100: 0 to 0: mixing in the range of 100 (%); an auto-sampler 6 which injects a sample; a separation column 7 for separating components; a column oven 8 for keeping the separation column 7 at a constant temperature; a detector 9 that detects the separated component; and a waste liquid bottle 11.
The data processing apparatus 10 is constituted by a computer having: a control unit (CPU) that executes analysis and analyzes the analysis result; a storage unit (hard disk or the like) 10a for storing analysis results or analysis results; and a display unit (monitor) for displaying the analysis result or the analysis result.
The data processing device 10 corresponds to "a quantifying unit" and "an abnormality determining unit" in the claims. The storage unit 10a corresponds to "measurement condition storage means" in the claims.
The autosampler 6 has a rack 6a on which a plurality of samples can be set, and a collection container 21 can be set in each holding unit for each sample of the rack 6 a. Above the collection container 21, a solid-phase filter 20 is disposed, and the solid-phase filter 20 is used to separate each component from a plurality of components contained in the measurement sample.
Further, the following pretreatment is performed in the specific solid-phase filter 20: the target components are adsorbed and the impurity components are removed, and the target components adsorbed on the solid phase filter 20 are eluted with a predetermined elution solution, and then recovered in the recovery container 21 and separated. The components separated by the pretreatment are directly introduced from the rack 6a into the autosampler 6 as they are and used for measurement.
The solid phase filter 20 and eluent are selected according to the component to be separated. In addition, the solid-phase filter 20 is detached from the collection container 21 and used for analysis.
As a specific example of the pretreatment, in the case of using acetonitrile as the eluent, acetonitrile is first added to the solid phase filter 20 (the collection container 21 is attached), and the solid phase filter 20 on each collection container 21 is centrifuged using a desktop centrifuge or the like, and the added solution is passed through the solid phase filter 20 (activation step).
Subsequently, water containing as little impurity as possible, such as distilled water or purified water, is similarly added to the solid phase filter 20, and the solid phase filter 20 in each collection container 21 is centrifuged to remove the remaining acetonitrile from the solid phase filter 20. The measurement sample is put into the solid-phase filter 20 in this state. By this operation, the specific component in the measurement sample is adsorbed to the solid-phase filter 20 (adsorption step).
After the solid phase filter 20 is centrifuged again, the liquid (serum) passing through the centrifuge is discarded, and distilled water, purified water, or the like is added to the solid phase filter 20 to perform centrifugation again and washing (washing step).
Next, in order to elute and separate the components adsorbed on the solid phase filter, an acetonitrile aqueous solution is added as an eluent to the solid phase filter 20, and the target component is eluted by performing a centrifugal operation on the solid phase filter 20, thereby recovering a solution containing the target component (elution step).
The collection container 21 may be directly set on the rack 6a of the auto-sampler 6, or the measurement sample collected in the collection container 21 may be transferred to a container (so-called sample vial) holding another sample solution and used.
The detector 9 is a three-dimensional detector as follows: the optical fiber has a plurality of elements for detecting signal intensity, and can simultaneously obtain signal intensity with respect to time at a plurality of wavelengths.
As described above, a plurality of components contained in the measurement sample are pretreated in advance and separated into individual components. The pretreated solutions each containing each separated component are injected individually from an injector (not shown) of the auto-sampler 6, and pass through the separation column 7 together with the mixed solutions of the mobile phases 3 and 4 fed from the pumps 1 and 2, whereby each component (single component) is developed in the separation column 7. In this regard, the main purpose of the separation column 7 is not to separate a plurality of components in a sample under normal HPLC analysis into respective peaks.
The respective components are detected by a detector 9. The signal of the detector 9 is sent to a data processing device 10 for data processing.
Here, in the present embodiment, the mobile phases 3 and 4 are two types of eluents, the mixing ratio of the mobile phases 3 and 4 by the mixer 5 is determined according to the components to be separated, and if the separated components to be measured are specified, the measurement conditions including the mixing ratio and the equilibration conditions before measurement (conditions for equilibration and stabilization of the column (adjustment conditions)) are automatically called, and the equilibration and measurement are performed in this order.
The separation column 7 may use a device that is generally used as a separation section for separating components of a sample present in a mobile phase. As the separation column 7, there are a packed-type separation column, a monolithic separation column, and the like, but a monolithic separation column is preferable. As the column packing of the separation column 7, various types such as an adsorption type, a distribution type, and an ion exchange type can be used. In order to keep the separation column 7 at a constant temperature and to separate a sample with good reproducibility, the separation column 7 is preferably provided in a column oven 8.
Next, an example of a liquid chromatography method using the liquid chromatography apparatus 100 will be described. In this example, the measurement sample is serum, the plurality of components are blood drugs, and the blood concentration of the drugs is quantified. Specifically, the multiple components are 6 of carbamazepine, lamotrigine, propiram, quinidine, voriconazole, and imatinib.
Fig. 2 is a chromatogram in which the six components contained in blood as a measurement sample are mixed and each component is separated by primary liquid chromatography and quantified. The elution time T0 for the 6 components of the chromatogram to end elution was about 10 minutes.
Here, in the present invention, the measurement conditions of the liquid chromatography of each component are adjusted for each separated component, and the components are quantified so that the elution times are less than T0 and the difference between the elution times of the components is within ± 10%.
Chromatograms of the isolated carbamazepine, lamotrigine, quinidine, voriconazole, imatinib, and propiram are shown in fig. 3-8, respectively.
Fig. 9 shows an example of the elution time T1 of each component, the pretreatment conditions, and the measurement conditions.
In addition, the "mixing ratio of mobile phases (eluents)" in fig. 9 is the mixing ratio (corresponding to the volume ratio) of the mobile phases 3 and 4, the mobile phase 3 is acetonitrile, and the mobile phase 4 is 10mM ammonium acetate buffer. Thus, for example, 40 of fig. 9: 60 shows the following: a mixed solution of 40% mobile phase 3 and 60% mobile phase 4 was set as a mobile phase.
In addition, the same separation column was used for each component.
As described above, by separating each component in advance by pretreating a plurality of components contained in a measurement sample, and adjusting the measurement conditions for each separated component so that the elution time is less than T0 and the difference between the elution times T1 of the components is within ± 10%, the measurement time can be shortened as compared with the case where each component is quantified by single liquid chromatography in a state where a plurality of components are mixed together, and the measurement times of the components are substantially similar, and therefore, the waiting time for analysis of a specific component does not become long, the measurement can be performed quickly, and the target or schedule for completion of the measurement can be easily prepared. Further, since each component has a single peak as shown in fig. 3 to 8, the time window for identification can be fixed, reproducibility of the peak area and elution time can be improved, quantitative accuracy can be improved, and the degree of separation of the peaks can be stabilized.
In addition, since the elution times T1 of the respective components are similar, the measurement conditions are not greatly different, and the quantitative accuracy is improved.
Further, when the difference between the elution times T1 of the respective components is within ± 10%, the analysis time is substantially constant, so that the influence on the analysis of the sample is also constant, and the cause can be easily analyzed when there is a measurement failure. When the difference between the elution times T1 of the respective components is within. + -. 10%, it is easy to select a compound to be added as an internal standard.
In addition, when a plurality of components are mixed and measured by a single liquid chromatography, the measurement must be performed at a single wavelength, and the detection sensitivity may not be optimal for each component. Therefore, if the conditions (optimum wavelength, optimum mobile phase composition, etc.) optimum for each component are adjusted by separating each component in advance, the detection sensitivity is improved.
Preferably, the difference in elution times T1 for each component is within. + -. 5%, more preferably the difference in elution times T1 for each component is within. + -. 3%.
In the liquid chromatography apparatus 100 according to the present embodiment, the threshold Tt of the elution time, the pretreatment condition, and the measurement condition, which will be described later, are stored in the storage unit 10a of the data processing apparatus 10 for each component in fig. 9. However, the threshold value Tt may be a predetermined value between the lower limit and the upper limit of fig. 9.
Thus, for example, when a user specifies a predetermined component on the liquid chromatography apparatus 100, the data processing apparatus 10 reads the pretreatment conditions from the storage unit 10a and displays the pretreatment conditions on the screen, so that the user can prepare the solid phase filter 20 and the eluent with the screen as a guide.
When a user designates a predetermined component, the data processing device 10 reads out the measurement condition from the storage unit 10a and sets the measurement condition, thereby enabling automatic measurement. For example, in the example of fig. 9, the data processing device 10 automatically sets the flow rates of the mobile phases 3 and 4 to be transferred to the separation column 7, the mixing ratio of the mobile phases, and the UV wavelength of the detector 9, and then performs measurement.
In the present embodiment, as shown in fig. 9, the separation column and the mobile phase are made the same under the measurement conditions. This eliminates the need to change the separation column or the mobile phase for each component, and facilitates measurement of each component in 1 liquid chromatography apparatus. In addition, the measurement of each component can be automated.
In the present embodiment, as shown in fig. 9, the mobile phase contains two or more kinds of eluents, and the mixing ratio of each eluent is changed for each component. Thus, it is not necessary to physically replace the mobile phase for each bottle, and it is only necessary to control the mixer 5 for each component and change the mixing ratio of the eluents, and it is easy to measure each component in 1 liquid chromatography apparatus. In addition, the measurement of each component can be automated.
In addition, the data processing device 10 may determine that the analysis is abnormal when the elution time T1 of each component exceeds the predetermined threshold value Tt. The extent of the elution time T1 can be known by preliminary experiments, and when T1 exceeds Tt, it is considered that some problems have occurred in the liquid chromatography apparatus 100, and an abnormality can be notified to the user.
In addition, regarding the magnitude relationship between T1 and Tt, for example, when the peak height of the chromatogram at the time Tt is equal to or higher than a predetermined value, it is sufficient to determine that T1 exceeds Tt, considering that elution is not completed.
In addition, the pretreatment conditions of fig. 9 may be added as barcode information to the holding container for each separated component, and if the barcode is read at the time of pretreatment, the pretreatment conditions may be displayed. Further, the reading may be performed again after the end of the preprocessing, so that whether the preprocessing is correct or not can be confirmed.
Further, the positional information of each component on the rack 6a of the auto-sampler 6 may be added as barcode information to the holding container of each component, and if the barcode is read at the time of measurement, it is possible to determine whether or not each sample is arranged at the correct position on the rack 6 a.
This prevents erroneous sample collection and forgetting to set a sample.
The present invention is not limited to the above-described embodiments, and naturally, it relates to various modifications and equivalents included in the spirit and scope of the present invention.
Claims (10)
1. A liquid chromatography method for quantifying a plurality of components contained in a measurement sample by liquid chromatography,
the liquid chromatography method comprises:
a pretreatment step of separating each component from the plurality of components; and
and a quantitative determination step of adjusting the measurement conditions of the liquid chromatography for each of the separated components so that the elution time is less than T0 and the difference between the elution times of the components is within. + -. 10%, when the elution time for determining the components by a single liquid chromatography in a state where the components are mixed is T0.
2. The liquid chromatography analysis method according to claim 1,
in the measurement conditions, the separation column was made the same as the mobile phase.
3. The liquid chromatography analysis method according to claim 1 or 2,
the mobile phase contains two or more kinds of eluents, and the mixing ratio of the eluents is changed for each of the components.
4. The liquid chromatography method according to any one of claims 1 to 3, wherein,
the liquid chromatography analysis method further comprises the following abnormality determination step: and determining that the analysis is abnormal when the elution time of each component exceeds a predetermined threshold.
5. The liquid chromatography method according to any one of claims 1 to 4, wherein,
the plurality of components are drugs in the blood.
6. A liquid chromatography apparatus for quantifying a plurality of components contained in a measurement sample by liquid chromatography,
the liquid chromatography device comprises a measurement condition storage unit and a quantitative unit,
in a state where each component is separated from the plurality of components by the pretreatment,
a measurement condition storage means for storing, for each of the separated components, measurement conditions of the liquid chromatography for each component which enable quantification by the liquid chromatography such that an elution time T1 is less than T0 and a difference in the elution times T1 of the components is within. + -. 10%, where T0 is an elution time when each component is quantified by a single liquid chromatography in a state where the plurality of components are mixed together,
the quantifying unit quantifies the respective components by the liquid chromatography with reference to the measurement conditions.
7. The liquid chromatography apparatus according to claim 6,
in the measurement conditions, the separation column was made the same as the mobile phase.
8. The liquid chromatography apparatus according to claim 7,
the mobile phase contains two or more kinds of eluents, and the mixing ratio of each eluent is changed for each component.
9. The liquid chromatography apparatus according to any one of claims 6 to 8, wherein,
the liquid chromatography apparatus further includes an abnormality determination unit that determines that the analysis is abnormal when the elution time T1 of each component exceeds a predetermined threshold value Tt.
10. The liquid chromatography apparatus according to any one of claims 6 to 9, wherein,
the plurality of components are drugs in the blood.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018-183188 | 2018-09-28 | ||
| JP2018183188A JP7240704B2 (en) | 2018-09-28 | 2018-09-28 | LIQUID CHROMATOGRAPH ANALYSIS METHOD AND LIQUID CHROMATOGRAPH ANALYZER |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110967439A true CN110967439A (en) | 2020-04-07 |
Family
ID=69781646
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910915871.9A Pending CN110967439A (en) | 2018-09-28 | 2019-09-26 | Liquid chromatography method and liquid chromatography apparatus |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JP7240704B2 (en) |
| CN (1) | CN110967439A (en) |
| DE (1) | DE102019214127A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112362795A (en) * | 2020-08-19 | 2021-02-12 | 中谱安信(杭州)检测科技有限公司 | Method for measuring low molecular mass resistance maltodextrin in food by high performance liquid chromatography |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116699020A (en) * | 2023-05-29 | 2023-09-05 | 山东英盛生物技术有限公司 | Method and kit for detecting antiarrhythmic drugs in blood plasma by HPLC-MS |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01221663A (en) * | 1988-02-29 | 1989-09-05 | Shimadzu Corp | Dispensing method for preparative liquid chromatograph |
| US5277871A (en) * | 1989-10-20 | 1994-01-11 | Hitachi, Ltd. | Liquid chromatographic analyzer, sample feeder and prelabeling reaction treating method |
| CN101191790A (en) * | 2006-12-01 | 2008-06-04 | 株式会社日立高新技术 | Liquid chromatographic analysis apparatus |
| US20170284982A1 (en) * | 2014-09-02 | 2017-10-05 | Shimadzu Corporation | Preprocessing device and analysis system provided with same |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0618504A (en) * | 1992-06-30 | 1994-01-25 | Kyoto Daiichi Kagaku:Kk | Method for stabilizing measured value of high speed liquid chromatography |
| JP5262482B2 (en) * | 2008-09-12 | 2013-08-14 | 株式会社島津製作所 | Gas chromatograph |
| WO2017216934A1 (en) * | 2016-06-16 | 2017-12-21 | 株式会社日立ハイテクノロジーズ | Chromatographic mass analysis device and control method |
-
2018
- 2018-09-28 JP JP2018183188A patent/JP7240704B2/en active Active
-
2019
- 2019-09-17 DE DE102019214127.7A patent/DE102019214127A1/en active Pending
- 2019-09-26 CN CN201910915871.9A patent/CN110967439A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01221663A (en) * | 1988-02-29 | 1989-09-05 | Shimadzu Corp | Dispensing method for preparative liquid chromatograph |
| US5277871A (en) * | 1989-10-20 | 1994-01-11 | Hitachi, Ltd. | Liquid chromatographic analyzer, sample feeder and prelabeling reaction treating method |
| CN101191790A (en) * | 2006-12-01 | 2008-06-04 | 株式会社日立高新技术 | Liquid chromatographic analysis apparatus |
| US20170284982A1 (en) * | 2014-09-02 | 2017-10-05 | Shimadzu Corporation | Preprocessing device and analysis system provided with same |
Non-Patent Citations (1)
| Title |
|---|
| 王立 等: "色谱分析样品处理", 化学工业出版社, pages: 1 - 6 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112362795A (en) * | 2020-08-19 | 2021-02-12 | 中谱安信(杭州)检测科技有限公司 | Method for measuring low molecular mass resistance maltodextrin in food by high performance liquid chromatography |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2020051960A (en) | 2020-04-02 |
| DE102019214127A1 (en) | 2020-04-02 |
| JP7240704B2 (en) | 2023-03-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gallay et al. | LC–MS/MS method for the simultaneous analysis of seven antimalarials and two active metabolites in dried blood spots for applications in field trials: Analytical and clinical validation | |
| CN110967439A (en) | Liquid chromatography method and liquid chromatography apparatus | |
| Mantikou et al. | Newborn screening for hemoglobinopathies using capillary electrophoresis technology: Testing the Capillarys® Neonat Fast Hb device | |
| JP6221458B2 (en) | Method for determining the injection state of a specimen | |
| US20210063363A1 (en) | Techniques for checking state of analyzers | |
| WO2020012571A1 (en) | Analysis system, auto sampler, and template sheet for auto sampler | |
| US10359405B2 (en) | Analyzing-device controller | |
| JP7232841B2 (en) | automatic analyzer | |
| JP6856434B2 (en) | Liquid chromatographic measurement method, liquid chromatography measurement device, and liquid chromatography measurement program | |
| EP4266059A1 (en) | Method for controlling sample pretreatment device | |
| JP2008241517A (en) | Sample analyzer | |
| WO2014168865A1 (en) | Automated analysis systems | |
| JP4438597B2 (en) | Liquid chromatographic dissolution test equipment | |
| US20220082533A1 (en) | Liquid chromatograph and dissolution test system | |
| JPH049756A (en) | Liquid chromatograph analyser | |
| JP7070724B2 (en) | Liquid chromatograph and dissolution test system | |
| US20230384269A1 (en) | Column device | |
| JPS63212861A (en) | Method and apparatus for simultaneous analysis of vanillylmandelic acid, homovanillic acid and creatinine | |
| JP2023117671A (en) | Method for measuring calibrator and liquid chromatograph apparatus | |
| James et al. | Fast turnaround bioanalysis in discovery and early clinical development | |
| CN107305204B (en) | Liquid chromatography measuring method, liquid chromatography measuring apparatus, and liquid chromatography measuring program storage medium | |
| CN117110508A (en) | Method and apparatus for measuring stabilized glycosylated hemoglobin, and non-transitory computer-readable storage medium | |
| JP2023172913A (en) | Stable glycohemoglobin measurement method, stable glycohemoglobin measurement apparatus, and stable glycohemoglobin measurement program | |
| JP2023015939A (en) | Device for hemolyzing and diluting puncture blood, and glycosylated hemoglobin analysis method using the same | |
| CN117007696A (en) | Method and kit for monitoring citalopram blood concentration |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |