WO2018092818A1 - 多次元クロマトグラフィー分析方法及び分析システム - Google Patents
多次元クロマトグラフィー分析方法及び分析システム Download PDFInfo
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/38—Flow patterns
- G01N30/46—Flow patterns using more than one column
- G01N30/461—Flow patterns using more than one column with serial coupling of separation columns
- G01N30/462—Flow patterns using more than one column with serial coupling of separation columns with different eluents or with eluents in different states
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/38—Flow patterns
- G01N30/46—Flow patterns using more than one column
- G01N30/461—Flow patterns using more than one column with serial coupling of separation columns
- G01N30/463—Flow patterns using more than one column with serial coupling of separation columns for multidimensional chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/38—Flow patterns
- G01N30/46—Flow patterns using more than one column
- G01N30/461—Flow patterns using more than one column with serial coupling of separation columns
- G01N30/465—Flow patterns using more than one column with serial coupling of separation columns with specially adapted interfaces between the columns
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/38—Flow patterns
- G01N30/46—Flow patterns using more than one column
- G01N30/468—Flow patterns using more than one column involving switching between different column configurations
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/96—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 using ion-exchange
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—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
- G01N2030/8877—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 optical isomers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/96—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 using ion-exchange
- G01N2030/965—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 using ion-exchange suppressor columns
Definitions
- the present invention relates to an analysis method for optical isomers of a plurality of components in a sample by chromatography, and an analysis system for executing this analysis method.
- Chromatography uses a mobile phase introduced into the analysis system and a stationary phase held in a column or the like, introduces a sample containing a plurality of components together with the mobile phase into the analysis system, and each component in the mobile phase. This is a method for separating and detecting a plurality of components by utilizing the difference in physical and chemical interaction between the solid phase and the stationary phase.
- chromatographic peak fractions containing the target component separated by the first-dimensional chromatography are collected and introduced into the second-dimensional chromatography to accurately separate the target components.
- online column switching method a method of switching columns with a series of devices
- offline column switching method a method of once fractionating the fraction outside the analysis system and introducing it into another device system
- Patent Document 1 a two-dimensional liquid that can analyze multiple components simultaneously, simultaneously, and automatically by combining two columns and a multi-loop that holds the separated fractions individually.
- Chromatographic methods and systems have been developed (Patent Document 1).
- the components separated in the time from the start of the rise of the peak of each component separated by the first-dimensional chromatography from the baseline to the end of the return to the baseline are sequentially transferred to the holding unit.
- a liquid chromatography system has been developed in which components that have been separated and subjected to separation are subjected to a second-dimensional chromatographic analysis (Patent Document 2).
- Patent Documents 1 and 2 were intended to separate optical isomers that are difficult to separate by one-dimensional chromatography by second-dimensional chromatography.
- Patent Document 2 discloses that the components fractionated at the time from the start of rising of the peak of each component from the baseline to the end of the return to the baseline are sequentially collected into the holding unit. However, this was based on the premise that each component to be analyzed had no peak overlap with components other than optical isomers.
- Japanese Patent No. 4291628 Japanese Patent No. 4980740 Japanese Patent Laying-Open No. 2015-48332
- optical isomers of a plurality of components It was possible to separate and quantify optical isomers of a plurality of components by conventional two-dimensional chromatography. However, when there is a large amount of complex matrix components, such as amino acids contained in a sample in a living body, and one optical isomer is significantly different from the other optical isomer The present inventors have found a problem that overlapping chromatographic peaks caused by slight fluctuations in retention time due to differences in conditions such as temperature and temperature may cause fluctuations in measured values of the relatively smaller optical isomer. It was.
- the present inventors sought the cause to solve such a problem.
- a slight overlap of peaks separated in the first dimension chromatography resulted in a separation result in the second dimension chiral chromatography. It was found to have a big effect. Therefore, as a result of further examination of the chromatography system by the present inventors, in the chromatogram obtained by one-dimensional chromatography, the start of rising from the baseline and the end of return to the baseline are strictly observed.
- the fraction containing the multiple components is A system has been devised that is subjected to chromatography and repeats chromatographic analysis until complete separation is achieved for a single component.
- the fraction corresponding to the peak at which the target separation has been achieved is subjected to chromatographic analysis (chiral chromatography analysis) intended to sequentially separate optical isomers, so that a minute amount in a complex matrix such as a biological sample can be obtained.
- chromatographic analysis chiral chromatography analysis
- the present invention relates to the following inventions: [1] A method for analyzing optical isomers of a plurality of components in a sample by chromatography, (I) separating and detecting a sample containing a plurality of components by chromatography using a column and a mobile phase to obtain a chromatogram; (Ii) determining the start of rising from the baseline and the end of return to the baseline for one or more chromatographic peaks; (Iii) The step of sequentially collecting the corresponding fractions separately from the start of the rise from the baseline to the end of the return to the baseline, (Iv) If the peak from the start of the rise to the baseline to the end of the return to the baseline is a single peak, proceed to the next step, and from the start of the rise from the baseline to the end of the return to the baseline When the fraction corresponding to the above includes a plurality of peaks, the step of repeating steps (i) to (iv); (V) Separation and detection of each optical isomer by chiral column
- any one of items 1 to 5 wherein the column is selected from the group consisting of a reverse phase column, a normal phase column, a cation exchange column, an anion exchange column, a chiral column, and a mixed mode column thereof. Analysis method described. [7] The analysis method further includes the following: (Vi) The analysis method according to any one of items 1 to 6, comprising a step of quantifying each optical isomer of each component from a chromatogram of each optical isomer. [8] The analysis method according to any one of items 1 to 7, wherein the sample is a biological sample. [9] The analysis method according to any one of items 1 to 8, wherein the components to be analyzed are amino acids constituting the protein and optical isomers thereof.
- One or more chromatography units including a column, a mobile phase introduction unit, and a detection unit, a chiral chromatography unit including a chiral column, a mobile phase introduction unit, and a detection unit, a channel, and a channel switching unit
- a system for analyzing a plurality of components comprising one or a plurality of liquid storage units and a control unit, (I) The controller obtains a chromatogram by driving the chromatography unit to separate and detect a plurality of components in the sample; (Ii) the control unit determines the start of rising from the baseline and the end of return to the baseline for a chromatographic peak corresponding to one or more components in the chromatogram; (Iii) The control unit drives the flow path switching unit to sequentially introduce corresponding fractions from the start of rising from the base line to the end of return to the base line into one or a plurality of liquid storage units.
- the control unit drives the flow path switching unit to change the chromatography unit from one liquid storage unit to a chromatography unit different from the chromatography unit used so far. Introducing and repeating steps (i) to (iv), (V) The controller drives the flow path switching unit to introduce a fraction corresponding to a single peak from the start of rising from the baseline to the end of return to the baseline into the chiral chromatography unit.
- the control unit drives the flow path switching unit, and the fraction corresponding to the peak from the start of the rise from the base line to the end of the return to the base line is determined as a multi-loop.
- the system is introduced into a different chromatography unit in step (iii) instead of being introduced into step 10.
- the liquid storage section is a loop.
- a chromatography unit different from the chromatography unit used so far has the same column as the column of the chromatography unit used so far, but the mobile phase of the chromatography unit used so far.
- Item 16 The system according to any one of Items 10 to 15, wherein the system has a different mobile phase.
- the chromatography is high-performance liquid chromatography.
- the column according to any one of items 10 to 17, wherein the column is selected from the group consisting of a reverse feed column, a normal phase column cation exchange column, an anion exchange column, a chiral column, and a mixed mode column thereof. System.
- FIG. 1 shows an example of a block diagram of an analysis system for performing the chromatography analysis method of the present invention.
- FIG. 2 shows an example of a configuration diagram of an analysis system for performing the chromatography analysis method of the present invention.
- FIG. 3 is an example of a configuration diagram illustrating details of the mobile phase introduction unit 10.
- FIG. 4 is an example of a configuration diagram showing the flow path switching unit 4 and the liquid storage unit 50 / multi-loop unit 55 in more detail.
- FIG. 5 shows a chromatogram obtained by the chromatographic analysis method carried out according to the present invention. Three-dimensional chromatography was performed.
- FIG. 5A shows the first-dimensional chromatogram. In one dimension, glutamine and serine could not achieve complete separation for a single component.
- FIG. 5 shows a chromatogram obtained by the chromatographic analysis method carried out according to the present invention. Three-dimensional chromatography was performed.
- FIG. 5A shows the first-dimensional chromatogram. In one dimension, glutamine and serine could not achieve complete
- FIG. 5B shows a second-dimensional chromatogram. Complete separation of peaks was achieved in the second dimension.
- FIG. 5C shows a chromatogram obtained by subjecting a preparative fraction that has achieved complete separation to chiral chromatography.
- FIG. 6 shows a chromatogram obtained by a conventional two-dimensional chromatographic analysis method.
- FIG. 6A shows a one-dimensional chromatogram. Glutamine and serine did not achieve complete separation for a single component in the first dimension, but fractionated fractions at the separation time estimated based on the Gaussian distribution.
- FIG. 6B shows a chromatogram obtained by subjecting the fractionated fraction to chiral chromatography.
- FIG. 7 shows a chromatogram obtained by the chromatographic analysis method carried out according to the present invention.
- FIG. 7A shows a first-dimensional chromatogram. In the first dimension, glutamine and serine could not achieve complete separation for a single component.
- FIG. 7B shows a second-dimensional chromatogram. Complete separation of peaks was achieved in the second dimension.
- FIG. 7C shows a chromatogram obtained by subjecting a preparative fraction that has achieved complete separation to chiral chromatography.
- FIG. 8 shows a chromatogram obtained by a conventional two-dimensional chromatographic analysis method.
- A Glutamine and serine did not achieve complete peak separation in the first dimension, but fractionated fractions were separated at the separation time estimated based on the Gaussian distribution.
- B A chromatogram obtained by subjecting the collected fraction to chiral chromatography is shown.
- FIG. 9 shows a chromatogram obtained by conventional two-dimensional chromatographic analysis using human urine as a sample. Chromatograms of asparagine (A), serine (B), alanine (C), and proline (D) are shown.
- FIG. 10 shows a chromatogram obtained by three-dimensional chromatographic analysis using human urine as a sample for the purpose of separating asparagine, serine, alanine, and proline.
- FIG. 10A is a first-dimensional chromatogram
- FIG. 10B is a second-dimensional chromatogram
- FIG. 10C represents a third-dimensional chromatogram.
- FIG. 11 shows a chromatogram obtained by two-dimensional chromatographic analysis using mouse urine as a sample.
- FIG. 10A is a first-dimensional chromatogram
- FIG. 10B is a second-dimensional chromatogram
- FIG. 10C represents a third-dimensional chromatogram.
- FIG. 11 shows a chromatogram obtained by two-dimensional chromatographic analysis using mouse urine as
- FIG. 11A is a first-dimensional chromatogram
- FIG. 11B is a second-dimensional chromatogram
- FIG. 12 shows a chromatogram obtained by three-dimensional chromatographic analysis using mouse urine as a sample.
- FIG. 12A is a first-dimensional chromatogram
- FIG. 12B is a second-dimensional chromatogram
- FIG. 12C represents a third-dimensional chromatogram.
- FIG. 13 shows a chromatogram obtained by three-dimensional chromatographic analysis using rat urine as a sample.
- FIG. 13A is a first-dimensional chromatogram
- FIG. 13B is a second-dimensional chromatogram
- FIG. 13C represents a third-dimensional chromatogram.
- FIG. 14 shows a chromatogram obtained by three-dimensional chromatographic analysis using human urine as a sample.
- FIG. 14A is a first-dimensional chromatogram
- FIG. 14B is a second-dimensional chromatogram
- FIG. 14C represents a third-dimensional chromatogram.
- the present invention relates to a method for analyzing optical isomers of a plurality of components in a sample by chromatography. More specifically, the present invention includes the following steps: (I) separating and detecting a sample containing a plurality of components by chromatography using a column and a mobile phase to obtain a chromatogram; (Ii) determining the start of rising from the baseline and the end of returning to the baseline, excluding matrix components, for the chromatographic peak of one or more components for analysis purposes; (Iii) A step of sequentially collecting fractions corresponding to the period from the start of rising from the baseline to the end of return to the baseline, excluding matrix components, (Iv) If the peak from the start of the rise to the baseline to the end of the return to the baseline consists of a single peak, proceed to the optical isomer separation process and start from the start of the rise to the baseline.
- chromatography may be performed using a combination of a column and / or mobile phase different from the combination of the column and / or mobile phase used so far. preferable.
- the invention provides one or more chromatographic units comprising a column, a mobile phase inlet, and a detector, a chiral chromatography unit comprising a chiral column, a mobile phase tank, a pump, and a detector,
- the present invention relates to a system for analyzing a plurality of components, comprising a channel, a channel switching unit, one or a plurality of liquid storage units, and a control unit.
- the system of the present invention comprises the following steps: (I) The controller obtains a chromatogram by driving the chromatography unit to separate and detect a plurality of components in the sample; (Ii) the control unit determines the start of rising from the baseline and the end of return to the baseline for one or more chromatograms in the chromatogram; (Iii) The control unit drives the flow path switching unit to sequentially introduce corresponding fractions from the start of rising from the base line to the end of return to the base line into one or a plurality of liquid storage units.
- the control unit drives the flow path switching unit so that the fraction corresponding to the peak from the start of the rise from the baseline to the end of the return to the baseline is obtained.
- the chromatography may be any chromatography, for example, liquid chromatography or gas chromatography.
- Liquid chromatography uses a liquid introduced into an analysis system as a mobile phase and a column or the like as a stationary phase, and introduces a sample containing a plurality of components into the analysis system. This is a technique for separating and detecting a plurality of components by utilizing the difference in physical and chemical interaction between the two.
- the chromatographic analysis method of the present invention relates to performing multidimensional chromatography.
- N refers to an integer of 2 or more, and 3-dimensional, 4-dimensional, 5-dimensional, or higher-dimensional chromatography can be realized).
- a system 1 for performing N-dimensional chromatographic analysis will be described with reference to the drawings.
- the system 1 of the present invention includes a control unit 2, a chromatography unit 3, a flow channel switching unit 4, and a flow channel 5, and the control unit 2 includes these chromatography unit 3, the flow channel switching unit 4, and the like. To control.
- the chromatography unit 3 and the flow path switching unit 4 are connected via a flow path 5.
- the sample introduced into the first chromatography unit 3- (1) is repeatedly introduced n times in the repeated chromatography unit 3- (n) (n represents an integer of 0 or more), and the last chiral chromatography unit 3- (f) is introduced and analyzed.
- the repetition number n is represented by (N ⁇ 2). Iterations are performed after each chromatographic analysis until complete separation of a single component containing only optical isomers is achieved. If complete separation of a single component containing only the optical isomers is achieved, it is subjected to the final chiral chromatography unit 3- (f) without further repetition.
- the chromatography unit used is preferably a chromatography unit that differs in terms of the column 30 and / or mobile phase used previously.
- a chromatography unit different from the chromatography unit used so far refers to using chromatography of a combination of a column and a mobile phase different from the combination of a column and a mobile phase used so far. Therefore, it is also possible to use a chromatography unit in which the columns are the same while only the mobile phase is different. In that case, the method is carried out using the previously used column and only the mobile phase being different.
- the chromatography unit 3 usually includes a mobile phase introduction unit 10, a sample introduction unit 20, a column 30, and a detector 40.
- a chromatogram can be generated from the measured value of the detector of the liquid chromatography analysis unit and recorded in the recorder 45.
- a column temperature controller 35 may be attached to the column 30.
- a mobile phase tank 11 and a pump 12 are connected to the mobile phase introduction unit 10 to introduce a mobile phase.
- the mobile phase to be introduced may be a combination of two or more solvents.
- the mobile phase mixing unit 13 can be used by changing the concentration gradient over time.
- the phases can also be fed and mixed. Since the gas contained in the mobile phase pumped up by the pump 12 affects the interaction with the stationary phase and causes noise, a deaeration device 14 may be further installed.
- the mobile phase introduced from the mobile phase introduction unit 10 is sent to the column 30 through the sample introduction unit 20.
- a sample can be injected from the sample introduction unit 20.
- the sample may be pretreated or the sample may be used as it is.
- a sample temporarily stored in the liquid storage unit 50 is introduced from the liquid storage unit 50.
- Each component contained in the sample is separated by a combination of a stationary phase such as a column and a mobile phase to be introduced, and the components eluted in order from the component with the smallest retention are detected by the detector.
- the sample component signal detected by the detector is sent to a recorder to be recorded, and a chromatogram can be obtained regarding the amount of the detected sample component relative to the retention time.
- the chromatographic unit 3 differs in terms of the previously used column 30 and mobile phase until complete separation is achieved for components containing only the desired optical isomer except for the matrix component. Separation and analysis using is repeated. Accordingly, in addition to the first chromatography unit 3- (1), the chromatography unit 3- (n) is provided repeatedly for the number of repetitions. Finally, it is connected to the chiral chromatography unit 3- (f). Components separated after passing through the chromatography column can be temporarily stored in the liquid storage unit 50- (n) by switching the flow path by the flow path switching unit 4- (n). As an example, the liquid reservoir 50- (n) is a multi-loop unit 55 including a plurality of loops 56.
- the flow path switching unit 4 can be realized, for example, by controlling the six-way switching valve in FIG.
- the switch position S1 the eluate from the column is connected to the drain tank, and when the detector 45 detects a fraction to be sorted, the column is switched to the switch position S2.
- the switch position S2 the eluate from the column is connected to the liquid storage unit 50 / multiloop unit 55, and the storage destination is determined by the switching means 57a and 57b of the liquid storage unit 50 / multiloop unit 55.
- the multi-loop unit 55 includes a plurality of loops 56 and switching means 57a and 57b for connecting one selected loop among the plurality of loops 56 to the flow paths 5a and 5b.
- each of the components of the sample separated by the column 30 can be individually held in the loop 56. That is, if the switch position of the flow path switching unit 4 is switched from S1 to S2 in accordance with the detection of the sample component in the detector, and further switched according to the loop 56 connecting the switching means 57a and 57b, each loop is switched. Every 56, each of the sample components can be retained.
- the switch position in the flow path switching unit 4 is switched again to S1, and at the same time, the mobile phase introduction unit 10 of the next column unit is driven to The loop 56 holding the fraction can be introduced into the next chromatography unit.
- the liquid chromatograph apparatus in the liquid chromatograph apparatus according to the present invention, only one sample optical isomer is removed by using one or a plurality of chromatographic analysis units in one sample injection into the first liquid. Each component to be contained is completely separated, and finally the D-form and L-form can be separated and quantified by optical resolution using a chiral column chromatography analysis unit for all the desired components. Therefore, when the liquid chromatograph apparatus according to the present invention is used, optical separation and detection can be performed on the components of a plurality of samples online after a single sample injection, so that conventional column switching chiral HPLC can be used offline. In comparison with the method that is repeated a plurality of times, the amount of sample to be discarded is small, the amount of sample may be small, and the analysis of the optical isomers in the components of the sample can be performed rapidly.
- the control unit 2 included in the system of the present invention can control the chromatography unit 3 and the flow path switching unit 4 respectively. More specifically, the control unit 2 controls the mobile phase introduction unit 10, the sample introduction unit 20, the column 30, the column temperature adjustment unit 35, the detector 40, and the recorder 45 included in the chromatography unit 3. The chromatography unit can be driven. Then, the control unit 2 can switch the flow path switching unit 4 based on the chromatogram recorded in the recording unit 35. More specifically, the control unit 2 determines the time when the chromatogram corresponding to one or more components in the chromatogram starts from the start of the rise from the baseline except for the matrix component, and ends when the return to the baseline ends except for the matrix component. To do.
- the control unit 2 controls the flow path switching unit 4 to sequentially introduce corresponding fractions from the start of rising from the base line to the end of return to the base line into one or a plurality of liquid storage units. it can.
- the control unit 2 determines whether or not the corresponding fraction from the start of the rise from the baseline to the end of the return to the baseline corresponds to a chromatographic peak derived from a single component containing only optical isomers. To do. This determination is performed by determining whether the peak is a single peak or a plurality of peaks on the chromatogram.
- the control unit 2 determines the last chiral The chromatogram is obtained by separating and detecting optical isomers of the fractions that have been completely separated into single components by driving the chiral chromatography unit. . Fractions corresponding from the start of baseline rise to the end of baseline return do not correspond to chromatographic peaks derived from a single component containing only optical isomers (i.e., multiple non-optical isomers).
- the control unit drives the flow path switching unit, the chromatograph different from the chromatographic unit used so far can be obtained by driving the flow path switching unit.
- a chromatography unit different from the chromatography unit used so far refers to using chromatography of a combination of a column and a mobile phase different from the combination of a column and a mobile phase used so far.
- chromatography units that have the same column but differ only in the mobile phase. In that case, it is also possible to configure the system so that only the mobile phase is different using the previously used column.
- the chromatography column used in the present invention is not particularly limited, and for example, a reverse phase column, a normal phase column, a cation exchange column, an anion exchange column, a chiral column, or a mixed mode column thereof can be used. Those skilled in the art can appropriately select the packing material and mobile phase used in these columns.
- the liquid chromatograph apparatus by this invention can be comprised using the apparatus of the following nano space series by Shiseido Co., Ltd.
- the pump 12 has 3301 as the column 3, ML-1000 (0.53 mm id ⁇ ⁇ 1000 mm), ML-KSAAAX-00001250-003 (1.5 mm id ⁇ ⁇ 250 mm), final chiral column 3- (F) is KSAACSP-001S15250-070 (1.5 mm id ⁇ 250 mm), the sample injection unit 20 is an autosampler 3033, the mobile phase mixing unit 13 is MPV, and the degassing device 14 3010 and 3011 can be used for the column selection unit 4.
- the multi-loop unit 55 can be manufactured by connecting, for example, ten loops 56 having an inner diameter of 0.8 mm and a length of 80 cm (volume of 400 ⁇ L) to the MLV.
- 3013 can be used for the fluorescence detector as the detector 40, and 3750 can be used for the recorder 45.
- a system having a three-dimensional chromatography analysis unit was created.
- parameters such as mobile phase, column, column temperature, mobile phase flow rate, etc.
- optical resolution detection of 20 types of amino acid optical isomers and / or derivatives thereof can be performed with high accuracy. Is possible.
- suitable conditions for optical resolution were examined for the optical isomers of glutamine (Gln) and serine (Ser).
- suitable conditions for optical resolution were examined for optical isomers of amino acids in plasma samples and urine samples.
- a sample containing each component can be reacted with an optical resolution reagent in advance or prior to analysis by chiral chromatography.
- an optical resolution reagent for optical resolution, a reagent containing a compound such as 2,5-dioxopyrrolidin-1-yl (2- (6-methoxy-4-oxoquinolin-1 (4H) -yl) ethyl carbonate is used.
- the optical resolution reagent the reaction method with the reagent, and the separation conditions in the chiral chromatography column, the method described in Patent Document 3 (Japanese Patent Application Laid-Open No. 2015-48332) can be used. It is not intended to use these reagents.
- the start of rising from the baseline refers to the first time of rising from the baseline due to the presence of a peak group including one or more peaks of the target component excluding the matrix component.
- the end of the return to the baseline refers to a point in time when a peak that has risen due to the presence of a peak group including one or more peaks returns to the baseline for the first time excluding the matrix component. Therefore, from the start of the rise from the baseline to the end of the return to the baseline, this means that one or more peaks are included, but there is no overlap with the peaks of other target components. .
- the fraction corresponding to the time from the start of the rise to the baseline until the end of the return to the baseline corresponds to one or more peaks included from the start of the rise from the baseline to the end of the return to the baseline
- the fraction before the start of rising from the baseline and the fraction after the end of returning to the baseline are included. It may be.
- “Complete separation” is a peak in the Japanese Pharmacopoeia, meaning “separation degree of 1.5 or more”. In the present invention, it is preferably 1.2 or more, more preferably 1.5 or more. In the present invention, the definition of complete separation is used to determine the start of rising from the baseline and the end of return to the baseline. Thus, it is not necessary for a single peak to be separated. When complete separation is achieved between a peak group including one or more peaks having an overlap and another peak, a preparative sample corresponding to the peak group is provided to the next chromatographic analysis unit. .
- the present invention uses chiral column chromatography for each fraction of the fraction corresponding to a single peak from the start of the rise from the baseline to the end of the return to the baseline.
- the method includes a step of separating and detecting isomers to obtain a chromatogram, and the amount or concentration of the optical isomer of a specific component is measured based on the chromatogram obtained in the step. Therefore, as long as it does not affect the quantification of the amount or concentration of the optical isomer of the specific component, it may be acceptable that the “single peak” in the last step (v) includes an undesired peak.
- a chromatogram is obtained in a form that includes a matrix component specific to the type of biological sample in addition to the chromatopeak of the target component. Therefore, when analyzing a biological sample, there is a case where it is not possible to determine the end of the return from the baseline to the end of the baseline due to the background due to the matrix component. In such a case, with a standard product that includes multiple target components, the start time determined when the start of rising from the baseline and the end time of returning to the baseline are determined in advance, and the biological sample is chromatographed. The fraction can be acquired at the time and at the end of return.
- the standard product refers to a sample prepared by adding a target component.
- a sample to which an equal amount of D-form and L-form of each amino acid except glycine is added can be used as a standard product.
- the step of determining the start of rising from the baseline and the end of return to the baseline for the plurality of chromatogram peaks in the analysis method of the present invention is the standard method. This is based on the product chromatogram. Then, in the step (iii), fractions are sequentially and separately collected at a time corresponding to the time from the start of rising from the baseline determined by this step to the end of return to the baseline.
- the analytical method of the present invention may include the following steps: (I) separating and detecting a sample containing a plurality of components by chromatography using a column and a mobile phase to obtain a chromatogram; (Ii) determining the start of rising from the baseline and the end of return to the baseline for one or more chromatographic peaks; (Iii) The step of sequentially collecting the corresponding fractions separately from the start of the rise from the baseline to the end of the return to the baseline, (Iv) Use a combination of column and mobile phase that is different from the combination of the column and mobile phase used so far for the fractions that correspond from the start of the rise from the baseline to the end of the return to the baseline.
- the analysis system of the present invention includes a plurality of chromatography units including a column, a mobile phase tank, a pump, and a detection unit, and a chiral column, a mobile phase tank, a pump, and a detection.
- a chiral chromatography unit comprising a section, a flow path, a flow path switching unit, one or a plurality of liquid storage sections, and a control section;
- the controller obtains a chromatogram by driving the chromatography unit to separate and detect a plurality of components in the sample;
- the control unit determines the start of rising from the baseline and the end of return to the baseline for one or more chromatograms in the chromatogram;
- the control unit drives the flow path switching unit to sequentially introduce corresponding fractions from the start of rising from the base line to the end of return to the base line into one or a plurality of liquid storage units.
- control unit drives the flow path switching unit, the control unit introduces from one liquid storage unit to a chromatography unit different from the chromatography unit used so far;
- the control unit drives the different chromatography unit to further separate and detect the fraction introduced into one liquid storage unit to obtain a chromatogram;
- the control unit determines the start of rising from the baseline and the end of return to the baseline for one or more chromatograms in the chromatogram;
- the control unit drives the flow path switching unit to sequentially introduce the corresponding fractions from the start of the rise from the baseline to the end of the return to the baseline into one or more liquid storage units.
- the control unit drives the flow path switching unit so that the fraction corresponding to the peak from the start of the rise from the baseline to the end of the return to the baseline is obtained.
- the control unit drives the flow path switching unit to introduce a fraction containing a single component into the chiral chromatography unit;
- the controller executes the chiral chromatographic unit to separate and detect optical isomers and acquire chromatograms for the fraction containing a single component.
- the first-dimensional chromatography can be performed, for example, under the following conditions: Column: ML-1000 (0.53 mm id x 1000 mm) Flow rate: 25 ⁇ L / min Temperature: 45 ° C Mobile phase: 5% MeCN, 0.05% TFA aqueous solution
- the second-dimensional chromatography can be performed, for example, under the following conditions: Column: ML-KSAAAX-00001250-003 (1.5 mm id x 250 mm) Flow rate: 150 ⁇ L / min Temperature: 10 ° C. Mobile phase: 0.15% formic acid methanol / acetonitrile (85/15)
- the third-dimensional chiral chromatography can be performed, for example, under the following conditions: Column: KSAACSP-001S15250-070 (1.5 mm id x 250 mm) Flow rate: 150 ⁇ L / min Temperature: 25 ° C. Mobile phase: glutamine For analysis: 0.5% formic acid methanol / acetonitrile (50/50) For serine analysis: 0.6% formic acid methanol / acetonitrile (40/60)
- the analysis method of the present invention may determine the start of rise and the end of return to the baseline in a chromatogram obtained in advance by analyzing the target component standard product.
- a chromatogram is generated in real time, and from the chromatogram, the start time of rising and the end time of returning to the baseline may be determined.
- the sample containing a plurality of components may be any sample such as a chemical sample or a biological sample.
- the sample is preferably a biological sample, for example, body fluid such as blood, plasma, serum, ascites, amniotic fluid, lymph, saliva, semen, urine, excrement such as feces, sweat, nasal discharge, hair, nails, Skin tissues, body tissues such as visceral tissues, and the like can be included.
- media obtained from cultured cells, cell disruptions, and the like can also be used as biological samples.
- the sample of the present invention can be appropriately pretreated depending on the type of sample and the component to be separated for the purpose of removing insoluble fractions and improving separation ability. Such pretreatment can be arbitrarily selected by those skilled in the art. As an example, when blood is used as a sample, the following steps are performed: Plasma / serum separation step; A pretreatment may be performed including a slow protein step; and a fluorescent derivatization step.
- the component to be analyzed may be any component having an optical isomer, for example, an in vivo metabolite such as amino acid, lactic acid, malic acid and the like.
- it is an amino acid, particularly a proteinogenic amino acid.
- amino acids other than protein-constituting amino acids include citrulline, ornithine, kynurenine, hydroxyproline, DOPA, and the like.
- 20 kinds of protein-constituting amino acids are known, and optical isomers (L-amino acids, D-amino acids) exist except glycine.
- L-amino acids are mainly present in living organisms, while it has been known in recent years that a small amount of D-amino acids have functions in living organisms. -It is desired to accurately measure the amount of amino acids. It has been clarified that D-amino acids have various known or unknown actions and functions as disease markers in living bodies (WO2013 / 140785). These D-amino acids are often present in very small amounts in the living body as compared with L-amino acids. The present invention enables accurate quantitative analysis and is very useful in terms of physiological function analysis and diagnostic use.
- the pretreated sample was subjected to chromatography under the following conditions.
- the first dimension chromatography was performed, for example, under the following conditions: Column: ML-1000 (0.53 mm id x 1000 mm) Flow rate: 25 ⁇ L / min Temperature: 45 ° C Mobile phase: 5% MeCN, 0.05% TFA aqueous solution The obtained chromatogram is shown in FIG. 5A.
- Second dimension chromatography was performed under the following conditions: Column: ML-KSAAAX-00001250-003 (1.5 mm id x 250 mm) Flow rate: 150 ⁇ L / min Temperature: 10 ° C. Mobile phase: 0.15% formic acid methanol / acetonitrile (85/15). The obtained chromatogram is shown in FIG. 5B.
- the third-dimensional chiral chromatography was performed, for example, under the following conditions: Column: KSAACSP-001S15250-070 (1.5 mm id x 250 mm) Flow rate: 150 ⁇ L / min Temperature: 25 ° C. Mobile phase: glutamine For analysis: 0.5% formic acid methanol / acetonitrile (50/50) For serine analysis: 0.6% formic acid methanol / acetonitrile (40/60) The resulting chromatogram is shown in FIG. 5C.
- Comparative Example 1 As a comparative example, two-dimensional chromatography combining reverse phase chromatography and chiral chromatography was performed. The chromatographic conditions used are as follows:
- the first dimension chromatography was performed, for example, under the following conditions: Column: ML-1000 (0.53 mm id x 1000 mm) Flow rate: 25 ⁇ L / min Temperature: 45 ° C Mobile phase: 5% MeCN, 0.05% TFA aqueous solution The obtained chromatogram is shown in FIG. 6A.
- the second-dimensional chiral chromatography was performed, for example, under the following conditions: Column: KSAACSP-001S15250-070 (1.5 mm id x 250 mm) Flow rate: 150 ⁇ L / min Temperature: 25 ° C. Mobile phase: glutamine For analysis: 0.5% formic acid methanol / acetonitrile (50/50) For serine analysis: 0.6% formic acid methanol / acetonitrile (40/60) The obtained chromatogram is shown in FIG. 6B.
- Example 2 separation analysis of D-serine and L-serine, and D-glutamine and L-glutamine in human plasma was performed. As pretreatment, plasma samples were subjected to gradual protein and fluorescence derivatization treatment.
- the first dimension chromatography was performed, for example, under the following conditions: Column: ML-1000 (0.53 mm id x 1000 mm) Flow rate: 25 ⁇ L / min Temperature: 45 ° C Mobile phase: 5% MeCN, 0.05% TFA aqueous solution The obtained chromatogram is shown in FIG. 7A.
- Samples corresponding to the peak group including the D, L-serine peak and the D, L-glutamine peak based on the conditions of Example 1 from the start of rising from the baseline to the end of return to the baseline were sorted.
- Second dimension chromatography was performed under the following conditions: Column: ML-KSAAAX-00001250-003 (1.5 mm id x 250 mm) Flow rate: 150 ⁇ L / min Temperature: 10 ° C. Mobile phase: 0.15% formic acid methanol / acetonitrile (85/15). The obtained chromatogram is shown in FIG. 7B.
- the fractionated sample was subjected to third-dimensional chiral chromatography.
- Third dimension chiral chromatography was performed under the following conditions: Column: KSAACSP-001S15250-070 (1.5 mm id x 250 mm) Flow rate: 150 ⁇ L / min Temperature: 25 ° C.
- Mobile phase glutamine
- the obtained chromatogram is shown in FIG. 7C.
- Comparative Example 2 As a comparative example, two-dimensional chromatography combining reverse phase chromatography and chiral chromatography was performed. The chromatographic conditions used are as follows:
- the first dimension chromatography was performed, for example, under the following conditions: Column: ML-1000 (0.53 mm id x 1000 mm) Flow rate: 25 ⁇ L / min Temperature: 45 ° C Mobile phase: 5% MeCN, 0.05% TFA aqueous solution The obtained chromatogram is shown in FIG. 8A.
- the second-dimensional chiral chromatography was performed, for example, under the following conditions: Column: KSAACSP-001S15250-070 (1.5 mm id x 250 mm) Flow rate: 150 ⁇ L / min Temperature: 25 ° C. Mobile phase: glutamine For analysis: 0.5% formic acid methanol / acetonitrile (50/50) For serine analysis: 0.6% formic acid methanol / acetonitrile (40/60) The obtained chromatogram is shown in FIG. 8B. Single peaks of glutamine and serine optical isomers were not obtained.
- Comparative Example 3 As a comparative example, human urine was used as a sample for two-dimensional chromatography combining reverse phase chromatography and chiral chromatography. As pretreatment, the urine sample was subjected to gradual protein and fluorescence derivatization treatment.
- a 20-fold volume of methanol was added to the urine sample, and 10 ⁇ L of the supernatant obtained from the methanol homogenate was dried under reduced pressure, and 20 ⁇ L of 200 mM sodium borate buffer (pH 8.0) and 5 ⁇ L of fluorescence induction reagent (40 mM of 4 -Fluoro-7-nitro 2,1,3-benzooxadiazole (NBD-F) in anhydrous MeCN) was added, and the mixture was heated at 60 ° C. for 2 minutes. Further, 0.1% TFA aqueous solution (75 ⁇ L) was added and subjected to HPLC.
- the chromatographic conditions used are as follows:
- the first dimension chromatographic separation was performed under the following conditions: Column: ML-1000 (0.53 mm id x 1000 mm) Flow rate: 25 ⁇ L / min Temperature: 45 ° C Mobile phase: 5% MeCN, 0.05% TFA aqueous solution
- FIG. 9A is a chromatogram showing separation of D-form and L-form asparagine
- FIG. 9B is a chromatogram showing separation of D-form and L-form serine
- FIG. 9C is a diagram of D-form and L-form
- FIG. 9D is a chromatogram showing separation of alanine
- FIG. 9D is a chromatogram showing separation of D-form and L-form proline. Asparagine and proline are insufficiently separated from contaminant components that are considered to be derived from biological samples.
- Example 3 In this example, human urine was used as a sample. The same pretreatment as in Comparative Example 3 was performed, and the sample was subjected to HPLC. The chromatogram obtained in the first dimension is shown in FIG. 10A, the chromatogram obtained in the second dimension is shown in FIG. 10B, and the chromatogram obtained in the third dimension is shown in FIG. 10C. As for proline, since the abundance is small, the peak displayed by multiplying the peak by 100 times is also shown.
- the first dimension chromatographic separation was performed under the following conditions: Column: ACR-HT (1.5 mm id x 500 mm) Flow rate: 75 ⁇ L / min Temperature: 45 ° C Mobile phase: 15% MeCN, 0.05% aqueous TFA solution The chromatogram obtained is shown in FIG. 10A.
- the fraction containing asparagine, serine, alanine, and proline was fractionated in the chromatogram obtained from the first dimension chromatography.
- Second dimension chromatography was performed under the following conditions: Column: ML-KSAAAX-002 (1.0 mm id x 150 mm) Flow rate: 100 ⁇ L / min Temperature: 25 ° C. Mobile phase: 0.15% formic acid methanol / acetonitrile (80/20). The obtained chromatogram is shown in FIG. 10B.
- the collected sample was subjected to third-dimensional chromatography.
- the third-dimensional chiral chromatography was performed, for example, under the following conditions: Column: KSAACSP-001S (1.5 mm id x 250 mm) Flow rate: 150 ⁇ L / min Temperature: 25 ° C. Mobile phase: 0.1% formic acid, methanol / acetonitrile (90/10) The obtained chromatogram is shown in FIG. 10C.
- Comparative Example 4 In this example, a method for separating and analyzing D-form and L-form of citrulline, which is an amino acid other than protein-constituting amino acids, is carried out.
- a mouse (C57BL) urine sample was collected and subjected to the same pretreatment as in Comparative Example 3, and the sample was subjected to HPLC.
- the chromatographic conditions used are as follows:
- the first dimension chromatographic separation was performed under the following conditions: Column: ML-1000 (0.53 mm id x 1000 mm) Flow rate: 25 ⁇ L / min Temperature: 45 ° C Mobile phase: 5-25% MeCN, 0.05% aqueous TFA solution The resulting chromatogram is shown in FIG. 11A.
- the second dimensional chiral chromatographic separation was performed under the following conditions: Column: KSAACSP-105S (1.5 mm id x 250 mm) Flow rate: 250 ⁇ L / min Temperature: 25 ° C. Mobile phase: 0.1% formic acid, methanol / acetonitrile (90/10) The resulting chromatogram is shown in FIG. 11B. Contaminating components were present in the vicinity of the D-form and L-form citrulline peaks, and separation was insufficient.
- Example 5 a method for separating and analyzing D-form and L-form of citrulline, which is an amino acid other than protein-constituting amino acids, is carried out.
- a mouse (C57BL) urine sample, a rat (Wistar) urine sample, and a human urine sample were each collected and subjected to the same pretreatment as in Comparative Example 3, and the sample was subjected to HPLC.
- the chromatographic conditions used are as follows:
- the first dimension chromatographic separation was performed under the following conditions: Column: ML-1000 (0.53 mm id x 1000 mm) Flow rate: 25 ⁇ L / min Temperature: 45 ° C Mobile phase: 5% MeCN, 0.05% TFA aqueous solution The obtained chromatograms are shown in FIG. 12A (mouse), FIG. 13A (rat), and FIG. 14A (human).
- Second dimension chromatography was performed under the following conditions: Column: KSAAAX-000 (1.5 mm id x 250 mm) Flow rate: 150 ⁇ L / min Temperature: 10 ° C. Mobile phase: 0.05% formic acid methanol / acetonitrile (90/10). The obtained chromatogram is shown in FIG. 12B (mouse), FIG. 13B (rat), and FIG. 14B (human).
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Abstract
Description
目のクロマトグラフィーで分離されたピークのわずかな重なりにより、2次元目のキラルクロマトグラフィーにおける分離結果に大きく影響を及ぼすことがわかった。そこで、本発明者らが、クロマトグラフィーのシステムについてさらなる検討を行った結果、1次元のクロマトグラフィーにより得られたクロマトグラムにおいて、ベースラインからの立ち上がり開始時及びベースラインへの復帰終了時を厳密に判定し、ベースラインからの立ち上がり開始時及びベースラインへの復帰終了時に複数のピークが含まれる場合、即ちピークの重なりが観察される場合は、その複数成分が含まれる画分を次のクロマトグラフィーに供して、単一成分について完全分離を達成するまでクロマトグラフィー分析を繰り返し行うというシステムを考案した。こうして、目的の分離が達成されたピークに対応する画分を逐次光学異性体の分離を意図したクロマトグラフィー分析(キラルクロマトグラフィー分析)に供することで、生体試料のような複雑なマトリクス中の微量な光学異性体についても精度良く測定することが可能になり、本発明に至った。
[1] クロマトグラフィーによる試料中の複数の成分の光学異性体の分析方法であって、
(i) 複数成分を含む試料を、カラム及び移動相を用いてクロマトグラフィーにより分離及び検出して、クロマトグラムを得る工程、
(ii) 1又は複数のクロマトピークについてベースラインからの立ち上がり開始時及びベースラインへの復帰終了時を決定する工程、
(iii) ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までに対応する画分を別々に逐次分取する工程、
(iv) ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークが単一ピークからなる場合、次の工程に進み、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までに対応する画分が、複数のピークを含む場合、工程(i)~工程(iv)を繰り返す工程、
(v) ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークが単一ピークに対応する画分に対し、キラルカラムクロマトグラフィーにより各光学異性体を分離及び検出して、クロマトグラムを得る工程を含み、
ここで、工程(i)~工程(iv)が繰り返される場合、これまで使用したカラム及び移動相とは異なるカラム及び移動相を用いてクロマトグラフィーを行う、前記分析方法。
[2] ベースラインからの立ち上がり開始時及びベースラインへの復帰終了時が、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までの1又は複数のピークと、他のピークとの間で完全分離が達成された時点である、項目1に記載の分析方法。
[3] 完全分離が、分離度が1.5以上という基準に基づき判定される、項目2に記載の分析方法。
[4] 前記クロマトグラフィーが、高速液体クロマトグラフィーである、項目1~3のいずれか一項に記載の分析方法。
[5] 標準品について予め取得されたクロマトピークにおいて、ベースラインからの立ち上がり開始時及びベースラインへの復帰終了時が決定され、当該開始時及び復帰終了時において画分が取得される、項目1~4のいずれか一項に記載の分析方法。
[6] 前記カラムが、逆相カラム、順相カラム、陽イオン交換カラム、陰イオン交換カラム、キラルカラム、及びそれらのミックスモードカラムからなる群から選ばれる、項目1~5のいずれか一項に記載の分析方法。
[7] 前記分析方法が、さらに以下の:
(vi) 各光学異性体のクロマトグラムから、各成分の各光学異性体を定量する工程
を含む、項目1~6のいずれか一項に記載の分析方法。
[8] 試料が、生物学的試料である、項目1~7のいずれか一項に記載の分析方法。
[9] 分析される成分が、タンパク質を構成するアミノ酸及びその光学異性体である、項目1~8のいずれか一項に記載の分析方法。
[10] カラム、移動相導入部、及び検出部を備える1又は複数のクロマトグラフィーユニットと、キラルカラム、移動相導入部、及び検出部を備えるキラルクロマトグラフィーユニットと、流路と、流路切り替えユニットと、1又は複数の貯液部と、制御部とを備えた、複数成分を分析するシステムであって、
(i) 制御部が、クロマトグラフィーユニットを駆動することにより、試料中の複数の成分を分離及び検出してクロマトグラムを取得し;
(ii) 制御部が、クロマトグラムにおける1又は複数の成分に対応するクロマトピークについてベースラインからの立ち上がり開始時及びベースラインへの復帰終了時を決定し;
(iii) 制御部が、流路切り替えユニットを駆動することにより、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までに対応する画分を1又は複数の貯液部に逐次導入し;
(iv) ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークが、単一ピークからなる場合、次の工程に進み、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークが、複数のピークを含む場合、制御部が、流路切り替えユニットを駆動することにより、1の貯液部から、これまでに用いられたクロマトグラフィーユニットとは異なるクロマトグラフィーユニットに導入し、そして工程(i)~(iv)を繰り返す工程、
(v) 制御部が、流路切り替えユニットを駆動することにより、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までの単一ピークに対応する画分をキラルクロマトグラフィーユニットに導入し;
(vi) 制御部が、キラルクロマトグラフィーユニットを駆動することにより、単一成分の完全分離が達成された画分について、光学異性体を分離・検出して、クロマトグラムを取得する、前記システム。
[11] ベースラインからの立ち上がり開始時及びベースラインへの復帰終了時が、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークと、他のピークとの間で完全分離が達成された時点である、項目10に記載の分析システム。
[12] 完全分離が、分離度が1.5以上という基準に基づき判定される、項目11に記載のシステム。
[13] 標準品について予め取得されたクロマトピークにおいて、ベースラインからの立ち上がり開始時及びベースラインへの復帰終了時が決定され、当該開始時及び復帰終了時において画分が取得される、項目10~12のいずれか一項に記載の分析方法。
[14] 前記工程(ii)において、制御部が、流路切り替えユニットを駆動し、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークに対応する画分をマルチループのループに導入する代わりに、工程(iii)の異なるクロマトグラフィーユニットに導入する、項目10~13のいずれか一項に記載のシステム。
[15] 貯液部が、ループである、項目10~14のいずれか一項に記載のシステム。
[16] これまでに用いられたクロマトグラフィーユニットとは異なるクロマトグラフィーユニットが、これまでに用いられたクロマトグラフィーユニットのカラムと同一カラムを有するが、これまでに用いられたクロマトグラフィーユニットの移動相とは異なる移動相を有する、項目10~15のいずれか一項に記載のシステム。
[17] 前記クロマトグラフィーが、高速液体クロマトグラフィーである、項目10~16のいずれか一項に記載のシステム。
[18] 前記カラムが、逆送カラム、順相カラム陽イオン交換カラム、陰イオン交換カラム、キラルカラム、及びそれらのミックスモードカラムからなる群から選ばれる、項目10~17のいずれか一項に記載のシステム。
[19] さらに(viii)制御部が、各光学異性体のクロマトグラムから各光学異性体を定量する、項目10~18のいずれか一項に記載のシステム。
[20] 試料が、生物学的試料である、項目10~19のいずれか一項に記載のシステム。
[21] 分析される成分が、タンパク質を構成するアミノ酸及びその光学異性体である、項目10~20のいずれか一項に記載のシステム。
(i) 複数成分を含む試料を、カラム及び移動相を用いてクロマトグラフィーにより分離及び検出して、クロマトグラムを得る工程、
(ii) 分析目的の1又は複数成分のクロマトピークについてマトリクス成分を除きベースラインからの立ち上がり開始時及びベースラインへの復帰終了時を決定する工程、
(iii) マトリクス成分を除きベースラインからの立ち上がり開始時からベースラインへの復帰終了時までに対応する画分を別々に逐次分取する工程、
(iv) ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークが単一ピークからなる場合、光学異性体分離の工程に進み、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までに対応する画分が、複数のピークを含む場合、工程(i)~工程(iv)を繰り返す工程、
(v)マトリクス成分を除きベースラインからの立ち上がり開始時からベースラインへの復帰終了時までの単一ピークに対応する画分に対し、キラルクロマトグラフィーにより各光学異性体を分離及び検出して、クロマトグラムを得る工程
を含む。ここで、工程(i)及び工程(ii)が繰り返される場合、これまで使用したカラム及び/又は移動相の組合せとは異なるカラム及び/又は移動相の組合せを用いてクロマトグラフィーを行われることが好ましい。
(i) 制御部が、クロマトグラフィーユニットを駆動することにより、試料中の複数の成分を分離及び検出してクロマトグラムを取得し;
(ii) 制御部が、クロマトグラムにおける1又は複数のクロマトピークについてベースラインからの立ち上がり開始時及びベースラインへの復帰終了時を決定し;
(iii) 制御部が、流路切り替えユニットを駆動することにより、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までに対応する画分を1又は複数の貯液部に逐次導入し;
(iv) ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークが、単一ピークからなる場合、次の工程に進み、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークが、複数のピークを含む場合、制御部が、流路切り替えユニットを駆動することにより、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークに対応する画分を、該画分を含む貯液部から、これまでに用いられたクロマトグラフィーユニットとは異なるクロマトグラフィーユニットに導入し、そして工程(i)~(iv)を繰り返す工程、
(v) 制御部が、流路切り替えユニットを駆動することにより、貯液部からベースラインからの立ち上がり開始時からベースラインへの復帰終了時までの単一ピークに対応する画分をキラルクロマトグラフィーユニットに導入し;
(vi) 制御部が、キラルクロマトグラフィーユニットを駆動することにより、単一成分を含む画分について、光学異性体を分離・検出して、クロマトグラムを取得する
を実行する。
(i) 複数成分を含む試料を、カラム及び移動相を用いてクロマトグラフィーにより分離及び検出して、クロマトグラムを得る工程、
(ii) 1又は複数のクロマトピークについてベースラインからの立ち上がり開始時及びベースラインへの復帰終了時を決定する工程、
(iii) ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までに対応する画分を別々に逐次分取する工程、
(iv) ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までに対応する画分を、これまで使用されたカラムと移動相との組合せとは異なるカラムと移動相との組合せを用いてクロマトグラフィーにより分離して、クロマトグラムを得る工程、
(v) (iv)のクロマトグラム上で1又は複数のクロマトピークについてベースラインからの立ち上がり開始時及びベースラインへの復帰終了時を決定する工程、
(vi) ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までに対応する画分を別々に逐次分取する工程、
(vii) ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークが、単一ピークからなる場合、次の工程に進み、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークが、複数のピークを含む場合、工程(iv)~工程(vii)を繰り返す工程、
(viii) ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までの単一ピークに対応する画分に対し、キラルカラムクロマトグラフィーにより各光学異性体を分離及び検出して、クロマトグラムを得る工程
ここで、工程(iv)~工程(vi)が繰り返される場合、これまで使用したカラム及び移動相とは異なるカラム及び移動相を用いてクロマトグラフィーが行われる。
(i) 制御部が、クロマトグラフィーユニットを駆動することにより、試料中の複数の成分を分離及び検出してクロマトグラムを取得し;
(ii) 制御部が、クロマトグラムにおける1又は複数のクロマトピークについてベースラインからの立ち上がり開始時及びベースラインへの復帰終了時を決定し;
(iii) 制御部が、流路切り替えユニットを駆動することにより、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までに対応する画分を1又は複数の貯液部に逐次導入し;
(iv) 制御部が、流路切り替えユニットを駆動することにより、1の貯液部から、これまでに用いられたクロマトグラフィーユニットとは異なるクロマトグラフィーユニットに導入し;
(v) 制御部が、当該異なるクロマトグラフィーユニットを駆動することにより、1の貯液部に導入された画分をさらに分離・検出してクロマトグラムを取得し;
(vi) 制御部が、クロマトグラムにおける1又は複数のクロマトピークについてベースラインからの立ち上がり開始時及びベースラインへの復帰終了時を決定し;
(vii) 制御部が、流路切り替えユニットを駆動することにより、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までに対応する画分を1又は複数の貯液部に逐次導入し;
(viii) ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークが、単一ピークからなる場合、次の工程に進み、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークが、複数のピークを含む場合、制御部が、流路切り替えユニットを駆動することにより、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークに対応する画分を、該画分を含む貯液部から、これまでに用いられたクロマトグラフィーユニットとは異なるクロマトグラフィーユニットに導入し、工程(v)~(viii)を繰り返す工程、
(ix) 制御部が、流路切り替えユニットを駆動することにより、単一成分を含む画分をキラルクロマトグラフィーユニットに導入し;
(x) 制御部が、キラルクロマトグラフィーユニットを駆動することにより、単一成分を含む画分について、光学異性体を分離・検出して、クロマトグラムを取得する
を実行する。
カラム:ML-1000(0.53mm i.d. × 1000mm)
流速:25μL/分
温度:45℃
移動相:5%MeCN、0.05%TFA水溶液
カラム:ML-KSAAAX-00015250-003(1.5mm i.d. × 250mm)
流速:150μL/分
温度:10℃
移動相:0.15%ギ酸 メタノール/アセトニトリル(85/15)
カラム:KSAACSP-001S15250-070(1.5mm i.d. x 250mm)
流速:150μL/分
温度:25℃
移動相:グルタミン分析用:0.5%ギ酸 メタノール/アセトニトリル(50/50)
セリン分析用:0.6%ギ酸 メタノール/アセトニトリル(40/60)
血漿・血清分離工程;
徐タンパク工程;及び
蛍光誘導体化工程
を含む前処理が行われうる。
本実施例では、本発明の分析方法及びシステムの精度を確認することを目的として、試料として、D:L=1:1のセリン、グルタミンを含む混合水溶液を試料として用いた。
前処理として、この試料に対し、NBD-Fによる蛍光誘導体化処理を行った。
1次元目のクロマトグラフィーは、例えば下記の条件で行った:
カラム:ML-1000(0.53mm i.d. × 1000mm)
流速:25μL/分
温度:45℃
移動相:5%MeCN、0.05%TFA水溶液
得られたクロマトグラムを図5Aに示す。
カラム:ML-KSAAAX-00015250-003(1.5mm i.d. × 250mm)
流速:150μL/分
温度:10℃
移動相:0.15%ギ酸 メタノール/アセトニトリル(85/15)。
得られたクロマトグラムを図5Bに示す。
カラム:KSAACSP-001S15250-070(1.5mm i.d. × 250mm)
流速:150μL/分
温度:25℃
移動相:グルタミン分析用:0.5%ギ酸 メタノール/アセトニトリル(50/50)
セリン分析用:0.6%ギ酸 メタノール/アセトニトリル(40/60)
得られたクロマトグラムを図5Cに示す。
比較例として、逆相クロマトグラフィーと、キラルクロマトグラフィーとを組み合わせた2次元クロマトグラフィーを行った。使用したクロマトグラフィー条件は下記の通りである:
カラム:ML-1000(0.53mm i.d. × 1000mm)
流速:25μL/分
温度:45℃
移動相:5%MeCN、0.05%TFA水溶液
得られたクロマトグラムを図6Aに示す。
カラム:KSAACSP-001S15250-070(1.5mm i.d. × 250mm)
流速:150μL/分
温度:25℃
移動相:グルタミン分析用:0.5%ギ酸 メタノール/アセトニトリル(50/50)
セリン分析用:0.6%ギ酸 メタノール/アセトニトリル(40/60)
得られたクロマトグラムを図6Bに示す。
本実施例では、ヒト血漿中におけるD-セリン及びL-セリン、並びにD-グルタミン及びL-グルタミンの分離分析を行った。前処理として、血漿試料に対し、徐タンパクおよび蛍光誘導体化処理を行った。
カラム:ML-1000(0.53mm i.d. × 1000mm)
流速:25μL/分
温度:45℃
移動相:5%MeCN、0.05%TFA水溶液
得られたクロマトグラムを図7Aに示す。
カラム:ML-KSAAAX-00015250-003(1.5mm i.d. × 250mm)
流速:150μL/分
温度:10℃
移動相:0.15%ギ酸 メタノール/アセトニトリル(85/15)。
得られたクロマトグラムを図7Bに示す。
カラム:KSAACSP-001S15250-070(1.5mm i.d. × 250mm)
流速:150μL/分
温度:25℃
移動相:グルタミン分析用:0.5%ギ酸 メタノール/アセトニトリル(50/50)
セリン分析用:0.6%ギ酸 メタノール/アセトニトリル(40/60)
得られたクロマトグラムを図7Cに示す。
比較例として、逆相クロマトグラフィーと、キラルクロマトグラフィーとを組み合わせた2次元クロマトグラフィーを行った。使用したクロマトグラフィー条件は下記の通りである:
カラム:ML-1000(0.53mm i.d. × 1000mm)
流速:25μL/分
温度:45℃
移動相:5%MeCN、0.05%TFA水溶液
得られたクロマトグラムを図8Aに示す。
カラム:KSAACSP-001S15250-070(1.5mm i.d. × 250mm)
流速:150μL/分
温度:25℃
移動相:グルタミン分析用:0.5%ギ酸 メタノール/アセトニトリル(50/50)
セリン分析用:0.6%ギ酸 メタノール/アセトニトリル(40/60)
得られたクロマトグラムを図8Bに示す。グルタミン、セリンの光学異性体の単一ピークは得られなかった。
比較例として、ヒト尿を試料として、逆相クロマトグラフィーと、キラルクロマトグラフィーとを組み合わせた2次元クロマトグラフィーを行った。前処理として、尿試料に対し、徐タンパクおよび蛍光誘導体化処理を行った。尿試料に20倍量のメタノールを加え、メタノールホモジネートから得た10μLの上清を減圧下で乾燥し、20μLの200mMホウ酸ナトリウム緩衝液(pH8.0)及び5μLの蛍光誘導試薬(40mMの4-フルオロ-7-ニトロ2,1,3-ベンゾオキサジアゾール(NBD-F)の無水MeCN溶液)を加え、60℃で2分間加熱した。さらに0.1%TFA水溶液(75μL)を加え、HPLCに供した。
使用したクロマトグラフィー条件は下記の通りである:
カラム:ML-1000(0.53mm i.d. × 1000mm)
流速:25μL/分
温度:45℃
移動相:5%MeCN、0.05%TFA水溶液
カラム:KSAACSP-001S(1.5mm i.d. × 250mm)
流速:250μL/分
温度:25℃
移動相:0.2%ギ酸 メタノール/アセトニトリル(90/10)
得られたクロマトグラムを図9に示す。
図9Aは、D体及びL体のアスパラギンの分離を示すクロマトグラムであり、図9Bは、D体及びL体のセリンの分離を示すクロマトグラムであり、図9Cは、D体及びL体のアラニンの分離を示すクロマトグラムであり、及び図9Dは、D体及びL体のプロリンの分離を示すクロマトグラムである。アスパラギン、プロリンは生体試料由来と考えられる夾雑成分との分離が不十分である。
本実施例では、ヒト尿を試料としてとして用いた。
比較例3と同様な前処理を実施し、試料をHPLCに供した。一次元目で得られたクロマトグラムを図10Aに示し、2次元目で得られたクロマトグラムを図10Bに示し、そして3次元目で得られたクロマトグラムを図10Cに示す。プロリンについては、存在量が少ないため、ピークを100倍にして表示したピークを併せて示した。
1次元目のクロマトグラフィー分離を下記の条件で行った:
カラム:ACR-HT(1.5mm i.d. × 500mm)
流速:75μL/分
温度:45℃
移動相:15%MeCN、0.05%TFA水溶液
得られたクロマトグラムを図10Aに示す。
カラム:ML-KSAAAX-002(1.0mm i.d. × 150mm)
流速:100μL/分
温度:25℃
移動相:0.15%ギ酸 メタノール/アセトニトリル(80/20)。
得られたクロマトグラムを図10Bに示す。
カラム:KSAACSP-001S(1.5mm i.d. × 250mm)
流速:150μL/分
温度:25℃
移動相:0.1%ギ酸 メタノール/アセトニトリル(90/10)
得られたクロマトグラムを図10Cに示す。
本実施例では、タンパク質構成アミノ酸以外のアミノ酸であるシトルリンについてのD体とL体の分離及び分析方法を実施する。
マウス(C57BL)の尿試料を採取し、比較例3と同様な前処理を実施し、試料をHPLCに供した。使用したクロマトグラフィー条件は下記の通りである:
カラム:ML-1000(0.53mm i.d. × 1000mm)
流速:25μL/分
温度:45℃
移動相:5-25%MeCN、0.05%TFA水溶液
得られたクロマトグラムを図11Aに示す。
カラム:KSAACSP-105S(1.5mm i.d. × 250mm)
流速:250μL/分
温度:25℃
移動相:0.1%ギ酸 メタノール/アセトニトリル(90/10)
得られたクロマトグラムを図11Bに示す。
D体及びL体のシトルリンのピーク近傍に夾雑成分が存在し、分離不十分であった。
本実施例では、タンパク質構成アミノ酸以外のアミノ酸であるシトルリンについてのD体とL体の分離及び分析方法を実施する。マウス(C57BL)の尿試料、ラット(Wistar)尿試料、及びヒト尿試料をそれぞれ採取し、比較例3と同様な前処理を実施し、試料をHPLCに供した。使用したクロマトグラフィー条件は下記の通りである:
カラム:ML-1000(0.53mm i.d. × 1000mm)
流速:25μL/分
温度:45℃
移動相:5%MeCN、0.05%TFA水溶液
得られたクロマトグラムを図12A(マウス)、図13A(ラット)、及び図14A(ヒト)に示す。
カラム:KSAAAX-000(1.5mm i.d. × 250mm)
流速:150μL/分
温度:10℃
移動相:0.05%ギ酸 メタノール/アセトニトリル(90/10)。
得られたクロマトグラムを図12B(マウス)、図13B(ラット)、及び図14B(ヒト)に示す。
分取された試料を、3次元目のクロマトグラフィーに供した。3次元目のクロマトグラフィーを、下記の条件で行った:
カラム:KSAACSP-001S(1.5mm i.d. × 250mm)
流速:200μL/分
温度:25℃
移動相:0.1%ギ酸 メタノール/アセトニトリル(90/10)
得られたクロマトグラムを図12C(マウス)、図13C(ラット)、及び図14C(ヒト)に示す。それぞれの対象において、D体とL体のシトルリンについて分離度1.5以上で完全分離を達成した。
1 分析システム
2 制御部
3 クロマトグラフィーユニット
3-(1) 第一クロマトグラフィーユニット
3-(n) 繰り返しクロマトグラフィーユニット
3-(f) 最終キラルクロマトグラフィーユニット
4 流路切り替えユニット
4-(n) 繰り返し流路切り替えユニット
4-(f) 最終流路切り替えユニット
5 流路
5a,b 流路
10 移動相導入部
10-(1) 第一移動相導入部
10-(n) 繰り返し移動相導入部
10-(f) 最終移動相導入部
11 移動相タンク
12 ポンプ
13 勾配形成ユニット
14 脱気装置
20 試料導入部
30 カラム
30-(1) 第一カラム
30-(n) 繰り返しカラム
30-(f) 最終キラルカラム
35 カラム温度調節器
35-(1) 第一カラム温度調節器
35-(n) 繰り返しカラム温度調節器
35-(f) 最終キラルカラム温度調節器
40 検出器
40-(1) 第一検出器
40-(n) 繰り返し検出器
40-(f) 最終検出器
45 記録計
45-(1) 第一記録計
45-(n) 繰り返し記録計
45-(f) 最終記録計
50 貯液部
50-(n)/20-(n) 繰り返し貯液部
50-(f)/20-(f) 最終貯液部
55 マルチループユニット
56 ループ
57a,b 切り替え手段
S1 スイッチ位置
S2 スイッチ位置
Claims (21)
- クロマトグラフィーによる試料中の複数の成分の光学異性体の分析方法であって、
(i) 複数成分を含む試料を、カラム及び移動相を用いてクロマトグラフィーにより分離及び検出して、クロマトグラムを得る工程、
(ii) 1又は複数のクロマトピークについてベースラインからの立ち上がり開始時及びベースラインへの復帰終了時を決定する工程、
(iii) ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までに対応する画分を別々に逐次分取する工程、
(iv) ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークが単一ピークからなる場合、次の工程に進み、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までに対応する画分が、複数のピークを含む場合、工程(i)~工程(iv)を繰り返す工程、
(v) ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークが単一ピークに対応する画分に対し、キラルカラムクロマトグラフィーにより各光学異性体を分離及び検出して、クロマトグラムを得る工程
を含み、
ここで、工程(i)~工程(iv)が繰り返される場合、これまで使用したカラム及び移動相とは異なるカラム及び移動相を用いてクロマトグラフィーを行う、前記分析方法。 - ベースラインからの立ち上がり開始時及びベースラインへの復帰終了時が、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までの1又は複数のピークと、他のピークとの間で完全分離が達成された時点である、請求項1に記載の分析方法。
- 完全分離が、分離度が1.5以上という基準に基づき判定される、請求項2に記載の分析方法。
- 前記クロマトグラフィーが、高速液体クロマトグラフィーである、請求項1~3のいずれか一項に記載の分析方法。
- 標準品について予め取得されたクロマトピークにおいて、ベースラインからの立ち上がり開始時及びベースラインへの復帰終了時が決定され、当該開始時及び復帰終了時において画分が取得される、請求項1~4のいずれか一項に記載の分析方法。
- 前記カラムが、逆相カラム、順相カラム、陽イオン交換カラム、陰イオン交換カラム、キラルカラム、及びそれらのミックスモードカラムからなる群から選ばれる、請求項1~5のいずれか一項に記載の分析方法。
- 前記分析方法が、さらに以下の:
(vi) 各光学異性体のクロマトグラムから、各成分の各光学異性体を定量する工程
を含む、請求項1~6のいずれか一項に記載の分析方法。 - 試料が、生物学的試料である、請求項1~7のいずれか一項に記載の分析方法。
- 分析される成分が、タンパク質を構成するアミノ酸及びその光学異性体である、請求項1~8のいずれか一項に記載の分析方法。
- カラム、移動相導入部、及び検出部を備える1又は複数のクロマトグラフィーユニットと、キラルカラム、移動相導入部、及び検出部を備えるキラルクロマトグラフィーユニットと、流路と、流路切り替えユニットと、1又は複数の貯液部と、制御部とを備えた、複数成分を分析するシステムであって、
(i) 制御部が、クロマトグラフィーユニットを駆動することにより、試料中の複数の成分を分離及び検出してクロマトグラムを取得し;
(ii) 制御部が、クロマトグラムにおける1又は複数の成分に対応するクロマトピークについてベースラインからの立ち上がり開始時及びベースラインへの復帰終了時を決定し;
(iii) 制御部が、流路切り替えユニットを駆動することにより、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までに対応する画分を1又は複数の貯液部に逐次導入し;
(iv) ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークが、単一ピークからなる場合、次の工程に進み、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークが、複数のピークを含む場合、制御部が、流路切り替えユニットを駆動することにより、1の貯液部から、これまでに用いられたクロマトグラフィーユニットとは異なるクロマトグラフィーユニットに導入し、そして工程(i)~(iv)を繰り返す工程、
(v) 制御部が、流路切り替えユニットを駆動することにより、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までの単一ピークに対応する画分をキラルクロマトグラフィーユニットに導入し;
(vi) 制御部が、キラルクロマトグラフィーユニットを駆動することにより、単一成分の完全分離が達成された画分について、光学異性体を分離・検出して、クロマトグラムを取得する、
前記システム。 - ベースラインからの立ち上がり開始時及びベースラインへの復帰終了時が、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークと、他のピークとの間で完全分離が達成された時点である、請求項10に記載の分析システム。
- 完全分離が、分離度が1.5以上という基準に基づき判定される、請求項11に記載のシステム。
- 標準品について予め取得されたクロマトピークにおいて、ベースラインからの立ち上がり開始時及びベースラインへの復帰終了時が決定され、当該開始時及び復帰終了時において画分が取得される、請求項10~12のいずれか一項に記載の分析方法。
- 前記工程(ii)において、制御部が、流路切り替えユニットを駆動し、ベースラインからの立ち上がり開始時からベースラインへの復帰終了時までのピークに対応する画分をマルチループのループに導入する代わりに、工程(iii)の異なるクロマトグラフィーユニットに導入する、請求項10~13のいずれか一項に記載のシステム。
- 貯液部が、ループである、請求項10~14のいずれか一項に記載のシステム。
- これまでに用いられたクロマトグラフィーユニットとは異なるクロマトグラフィーユニットが、これまでに用いられたクロマトグラフィーユニットのカラムと同一カラムを有するが、これまでに用いられたクロマトグラフィーユニットの移動相とは異なる移動相を有する、請求項10~15のいずれか一項に記載のシステム。
- 前記クロマトグラフィーが、高速液体クロマトグラフィーである、請求項10~16のいずれか一項に記載のシステム。
- 前記カラムが、逆送カラム、順相カラム陽イオン交換カラム、陰イオン交換カラム、キラルカラム、及びそれらのミックスモードカラムからなる群から選ばれる、請求項10~17のいずれか一項に記載のシステム。
- さらに(viii)制御部が、各光学異性体のクロマトグラムから各光学異性体を定量する、請求項10~18のいずれか一項に記載のシステム。
- 試料が、生物学的試料である、請求項10~19のいずれか一項に記載のシステム。
- 分析される成分が、タンパク質を構成するアミノ酸及びその光学異性体である、請求項10~20のいずれか一項に記載のシステム。
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