JP3575663B2 - Automatic analyzer - Google Patents

Description

[0001]
[Industrial applications]
The present invention relates to a system for simultaneously purifying, separating, and identifying various compounds contained in a combinatorial library.
[0002]
BACKGROUND OF THE INVENTION AND PRIOR ART
In recent years, combinatorial chemistry has been actively used in the field of fine chemistry, including pharmaceutical companies. Combinatorial chemistry is a method for synthesizing various compounds by combining various raw materials and reactions, and searching for useful drugs and the like in a short period of time by examining the physiological activities and the like. In particular, in drug development, combinatorial chemistry has a great advantage because tens of thousands of compounds must be found to be effective and highly safe for specific diseases.
On the other hand, separation and identification of a small amount of sample are performed simultaneously by combining high performance liquid chromatography and a mass spectrometer. In this case, it is necessary to add a matrix such as glycerin to assist ionization. However, in order to sufficiently mix the added matrix with the mobile phase of high performance liquid chromatography, a part of the T-tube is concentric. A T-shaped tube is used, which is characterized by providing a filter near the end point of the inner tube (Japanese Patent Application Laid-Open No. Hei 8-211019). With such a contrivance, it has become possible to quickly and accurately separate and identify a very small amount of sample.
[0003]
[Problems to be solved by the invention]
In order to provide a combinatorially synthesized compound for screening for various pharmacological activities, it is essential to purify the compound and confirm the structure, but this purification and confirmation of the structure require much time and effort. In order to fully utilize the advantages of combinatorial synthesis, it is necessary not only to separate and identify a conventional trace sample, but also to quickly purify the sample and confirm its structure.
[0004]
[Means for Solving the Problems]
The present invention provides a liquid sending pump for sending a mobile phase, and a sample in a combinatorial library is injected into a high performance liquid chromatography by an autoinjector, separated into components in the sample by a column, and then the eluate is splitter. , One of the channels reaches the fraction collector via a detector and is separated for each component. The other channel is mixed with the matrix liquid by the matrix addition system, and then the mass spectrometer is used. Is a system for simultaneously purifying, fractionating, and identifying compounds in a combinatorial library.
[0005]
The combinatorial library in the present invention is one of the components of the combinatorial chemistry technology, and is a kind of a database of chemical substances that are candidates for pharmaceuticals and the like. In the present invention, purification, fractionation, and identification of many compounds contained in a combinatorial library can be performed at the same time, and the construction of this system is an object of the present invention.
According to the system according to the present invention, not only the compounds contained in the combinatorial library, but also the medicinal component itself, impurities or degradation products in the medicinal component, metabolites, purification and separation of unknown compounds such as natural products and biologically active substances, etc. And identification.
The high performance liquid chromatography in the present invention is a commonly used high performance liquid chromatography, and includes a liquid sending pump, an injector, a column, a detector, and the like. In the present invention, to inject into a high-performance liquid chromatography means to inject a sample into the device according to the present invention using an auto injector. The time interval of the sample injection and the injection amount can be arbitrarily determined.
[0006]
The column in the present invention is not particularly limited as long as it is a column that can be connected to high-performance liquid chromatography, and any column for analysis or preparative separation can be used, and either a normal phase or reverse phase column can be used. However, it is preferable to use a preparative column in order to obtain a large amount of the compound in the subsequent operation.
Although one or more mobile phases can be used for high-performance liquid chromatography, the mixing ratio of two or more solvents must be adjusted in order to separate many compounds contained in the combinatorial library in a short time. It is preferable to use a so-called gradient method in which the temperature is gradually changed.
[0007]
The splitter in the present invention is a device that divides the mobile phase flowing out of the column into two channels, and the flow rates of the two channels can be adjusted arbitrarily. In the present invention, one of the flow paths is connected to the fractionation device, the other is connected to the mass spectrometry system, the ratio of the flow rate of both, if the flow rate of the flow path to the fractionation device is 1000, The flow rate of the flow path leading to the mass spectrometer is 0.1 to 10, preferably 0.5 to 5.
[0008]
The detector in the present invention is an apparatus for detecting a compound contained in a combinatorial library separated by a column, and the detecting means is an ultraviolet-visible spectrophotometer detector, a fluorescence detector, a differential refractive index detector Or a light scattering detector, and a means capable of detecting the expected compound with the highest sensitivity can be appropriately selected. Usually, an ultraviolet-visible spectrophotometer is most often used. The detector is usually connected to a recorder that records that the compound has been detected, and may be connected to an integrator if necessary. Of course, the integrator can be connected alone.
[0009]
The fraction collector in the present invention is an apparatus for collecting a compound separated by a column into a test tube or the like. Generally, a fraction collector separates an effluent by moving a collection tube such as a test tube at regular intervals, but in the present invention, in order to efficiently capture the components detected by the detector, the fraction collector is used. Can be controlled by a computer so that the preparative tube moves in conjunction with the electric signal.
[0010]
The matrix addition system connected to the other flow path in the present invention is an apparatus for adding a matrix liquid sent from a syringe pump to a mobile phase through a T-shaped tube. In order to sufficiently mix the matrix solution with the mobile phase, it is preferable to use a T-shaped tube disclosed in JP-A-8-211019. The matrix liquid is used for facilitating ionization of a sample in a FAB (Fast Atom Bombardment) ionization method of a mass spectrometer, and its components are determined by the analysis conditions of the mass spectrometer. Although different, glycerol, nitrobenzyl alcohol, triethanolamine and the like are usually used. The concentration of the matrix is usually 1 to 5 v / v%, preferably 2 to 3 v / v% based on the mobile phase. The flow rate of the syringe pump is appropriately adjusted so that the matrix has the above-mentioned concentration, and is usually 2 μL / min.
[0011]
The mass spectrometer according to the present invention includes FAB (Fast Atom Bombardment: fast atom bombardment), ESI (Electrospray Ionization: electrospray ionization), APCI (Atmospheric Pressure Chemical Ionization: MADI-Mixed Atomization) An apparatus capable of performing laser desorption (matrix assisted laser desorption) or the like can be used, but an apparatus equipped with a Frit-FAB interface is preferable. The Frit-FAB interface is a device for extruding a sample sent by high-performance liquid chromatography onto a metal called a frit, and ionizing the sample by colliding FAB gas therewith.
[0012]
【Example】
Hereinafter, the present invention will be described in detail with reference to the embodiment shown in FIG.
Reference numeral 1 denotes an auto-injector to which liquid sending pumps 2 and 3 for high performance liquid chromatography are connected. Different mobile phases 4, 5 flow through the pumps 2, 3, respectively, and the mixing ratio of 4, 5 can be arbitrarily changed by a computer incorporated in the auto injector. 1 stores a sample solution of a combinatorial library, and an arbitrary amount of liquid is automatically injected at an arbitrarily set time interval.
[0013]
The auto injector 1 is connected to a column 6, which is further connected to a splitter 7. The flow path is divided into two systems by 7, one of which is connected to the detector 8 and further connected to the fraction collector 9. A recorder 10 and an integrator 11 are electrically connected to 8. Reference numeral 10 is also electrically connected to 9, so that the sampling time of the fraction collector can be controlled by the electric signal obtained by the detector. The compounds of the combinatorial library separated by the column 6 are fractionated by the fraction collector 9.
[0014]
The other flow path divided by the splitter 7 is connected to a T-tube 12. A syringe pump 13 is connected to the T-shaped tube, and the matrix solution is mixed with the mobile phase at a constant speed. The T-tube is further connected to a mass spectrometer 14, where the matrix mixed with the mobile phase is injected into the mass spectrometer. The flow ratio of the two flow paths divided by 7 is (flow rate of the flow path leading to the fraction collector) :( flow rate of the flow path leading to the mass spectrometer) = 2995: 5. The analysis conditions and the like used in Example 1 are as follows.
[0015]
Auto injector: LC-10A, manufactured by Shimadzu Corporation
Liquid sending pump: LC-6AD, manufactured by Shimadzu Corporation
Column: GL Science, Inertzil-PrepODS (inner diameter 10 mm, length 250 mm)
Detector: Shimadzu SPD-6A (UV-visible spectrophotometer detector)
Integrator: CR-4AX, manufactured by Shimadzu Corporation
Recorder: Pantos, UniorderU228
Fraction connector: Gilson, FC204B
Mass spectrometer: JMS-SX102A manufactured by JEOL
[0016]
High-performance liquid chromatography condition detection: UV 210 nm
Column temperature: room temperature Mobile phase A: trifluoroacetic acid: methanol: water = 1: 100: 900
Mobile phase B: trifluoroacetic acid: methanol: water = 1: 900: 100
Gradient (mixing ratio and speed of mobile phases A and B): 0 to 100% (0 to 25 min)
Injection volume: 300 μL
Sample concentration: 1 to 10 mg / mL
Mobile phase flow rate: 3.0 mL / min
Mass spectrometry interface matrix: 2% glycerol syringe pump flow rate: 2 μL / min
[0017]
Mass spectrometry conditions Ion mode: FAB +
Scan range: 10-1000
Flow rate: 5 μL / min
Scan speed: 10.0 sec
Cyclic time: 5.78 sec
Resolution: 1000
[0018]
Fraction collector condition mode: time (1 fraction / min, 15 to 35 min)
[0019]
【The invention's effect】
FIG. 2 shows a mass chromatogram, a UV spectrum and a mass spectrum of a combinatorially synthesized compound (C ₂₅ H ₂₇ N ₃ O ₄ , [M + H] ⁺ = 434) obtained by the system according to the present invention. Since each device was connected online, all operations were performed automatically, and the measurement time was 47 minutes per sample. The purity of the fractionated compound was 99% or more.
By using the system according to the present invention, it became possible to analyze 30 compounds per day fully automatically. In addition, in addition to (M + H) ⁺ in the mass spectrum, a fragment ion peculiar to the structure of each compound could be confirmed at the same time, and more accurate structural information could be obtained. Furthermore, since the fractionated compound was highly pure, it could be immediately subjected to screening such as bioassay.
[0020]
[Brief description of the drawings]
FIG. 1 is an explanatory diagram of a system showing an embodiment of the present invention.
FIG. 2 is a compound obtained by combinatorial synthesis from above, a UV chromatogram, a mass chromatogram, and a mass spectrogram obtained by the system according to the present invention.

Claims (4)

A liquid sending pump for sending the mobile phase, an auto-injector for injecting the sample in the combinatorial library into the high performance liquid chromatography, a column for separating the components in the sample, and two eluates from the column. It consists of a splitter that divides the system into flow paths, a detector that detects the components, a fraction collector that separates the components, a matrix addition system that injects the matrix, and a mass spectrometer.The detector and the fraction collector are separated by the splitter. A system for simultaneously purifying, separating, and identifying compounds in a combinatorial library that is connected to one of the divided flow paths, and the matrix addition system and the mass spectrometer are connected to the other flow path separated by a splitter. The system according to claim 1, wherein the mobile phase used for high performance liquid chromatography is two kinds of solvents. The system according to claim 1, wherein the detector is an ultraviolet-visible spectrophotometer detector, a fluorescence detector, a differential refractive index detector or a light scattering detector to which a recorder and / or an integrator is connected. The system according to claim 1, wherein the matrix addition system is a system for adding a matrix liquid sent by a syringe pump to the eluate through a T-tube.

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