JPH08236063A - Liquid chromatographic mass spectrometer and mass spectrometry - Google Patents

Abstract

PURPOSE: To perform mass spectrometry with high sensitivity by effectively using a trace quantity of sample. CONSTITUTION: Frit 16 is formed on one end of a capillary column 15, and a filler 18 is filled there. This is used as a mass spectrometric column, and a separative component oozing out from the frit 16 is ionized by dynamic FAB and dynamic LSIMS. Therefore, after a sample is injected, it is not split. Since it is ionized immediately after the component is separated, a peak does not expand.

Description

Detailed Description of the Invention

[0001]

The present invention relates to pharmaceuticals, biochemistry, medicine,
Liquid chromatograph / mass spectrometer (LC / MS) used for analysis of trace components in fields such as general chemical industry and environment
Regarding

[0002]

2. Description of the Related Art In a chromatograph / mass spectroscope, components separated in a chromatograph section are ionized by an appropriate interface and mass spectrometric analysis is carried out in the mass spectrometric section. Liquid Chromatograph / Mass Spectrometer (LC / M
Among the interfaces used in S), dynamic FAB (Fast Atom Bombardment) and dynamic LSIMS (Liquid Secondary Ion Mass Spectros).
copy). Dynamic FAB exudes the components separated by liquid chromatography from the tip of a thin tube,
High-speed neutral molecules such as Xe collide there and are ionized. At this time, a porous frit, a screen or the like may be provided at the tip of the thin tube. In addition, dynamic LSIMS is attached to the tip of a thin tube (not a neutral molecule).
Dynamic F except that ions such as Cs + collide
Similar to AB.

LC / M using these interfaces
An example of S is shown in FIG. The operation is as follows. The mobile phase is sent to the analytical column 43 by the pump 41, the mobile phase leaving the analytical column 43 is split by the splitter 44, and only a part is sent to the capillary column 45. When the sample is injected into the mobile phase from the injector 42 in this state, the sample is separated in the analytical column 43,
Each component is exuded from the frit 46 provided at the tip of the capillary column 45. The tip portion of the capillary column 45 is enlarged and shown in FIG. In this figure,
Reference numeral 48 is a cap for holding the frit 46, and reference numeral 49 is a septum for holding the cap 48. By irradiating the frit 46 with high-speed neutral molecules or ions, the sample exuded from the frit 46 is ionized and introduced into the mass spectrometer 50 for mass spectrometry.

[0004]

Since dynamic FAB and dynamic LSIMS are ionization methods as described above, the flow rate of the liquid sample must be extremely small. Therefore, as shown in FIG. 4A, the liquid sample after being separated by the analysis column 43 is divided into about 20: 1 to 1000: 1 by the splitter 44, and 1 / the column flow rate is used.
Only 20 to 1/1000 (usually 1 to 10 μL / min) is allowed to flow through these interfaces.

Therefore, when the amount of the sample is small, the amount of the sample to be subjected to the mass analysis due to the split is further reduced, and even if the mass spectrometer 50 has high sensitivity, it is below the detection limit thereof. It often goes undetected.
In addition, the large amount side (19
Even if an attempt is made to collect the sample by concentrating the column effluent (from / 20 to 999/1000), there is a problem that denaturation or decomposition occurs due to the concentration operation.

Next, when the component of interest in the biological sample has an extremely low concentration, it is necessary to repeat the cleanup and concentration many times, but this operation causes impurities to be contaminated.
May cause column clogging. In order to remove such impurities, it is possible to use column switching (switching the flow path of the sample after being separated by the column and guiding only the target component to the mass spectrometer). At a small flow rate, dead volume or adsorption that occurs at the valve or its connection causes an analysis error. Further, when the impurity is a protein, its multiply charged ions have the same [mass / charge] (m / z) as that of the peptide, which may mask the peptide to be analyzed in mass spectrometry. Therefore, it is necessary to remove such impurities as much as possible when analyzing an unknown sample.

The present invention has been made to solve these problems, and an object of the present invention is to provide a liquid chromatograph mass spectrometer capable of effectively utilizing a small amount of sample and performing highly sensitive analysis. With the equipment
Further, it is to provide an effective method for removing impurities in advance in such an apparatus.

[0008]

Means for Solving the Problems, Actions and Effects The liquid chromatograph mass spectrometer according to the present invention made to solve the above problems comprises: a) a pump for delivering a mobile phase to a liquid delivery path; ) An analytical capillary column, one end of which is connected to the liquid feed path, at least a portion of which is filled with a component separating filler, and the other end of which is a frit fixed, and c) a sample is injected into the liquid feed path. An injector, and d) particle irradiation means for irradiating the frit with ions or neutral molecules to ionize the liquid exuding from the frit.

The mobile phase sent to the liquid feed path by the pump is sent to the analytical capillary column and exudes from the frit at the "other end". In addition, this pump has
A small flow rate for a micro liquid chromatograph may be used, or it may be sent by an ordinary pump and then split by a splitter to send only a small amount to an analytical capillary column. When the sample is injected from the injector in this state, the sample is separated by the component separation packing material packed in the analytical capillary column, and the separated components are sequentially exuded from the frit, as in a normal liquid chromatograph.
The components are ionized by irradiating the frit with high-speed neutral molecules or ions from the particle irradiating means, and mass analysis is performed in the mass spectrometric section at the subsequent stage.

As described above, in the liquid chromatograph mass spectrometer according to the present invention, all the samples separated in the column are exuded from the frit without being split. Therefore, even a small amount of sample can be effectively used without waste and sensitivity can be improved. High-performance analysis can be performed. Further, since the sample consumption amount is small as described above, it is possible to use the sample for other analysis without performing the work of concentrating and collecting the liquid discharged in the split line as in the conventional case. There is also. Further, since there is no connecting portion on the downstream side of the column, there is no dead volume, and there is an effect that the spread of the peak after the column is prevented.

In order to minimize the spread of the peaks after separation of the components, it is desirable to fill the frit side end of the packing material as much as possible in the analytical capillary column.

Next, in the case of using the above-described capillary column according to the present invention, the method of effectively removing impurities is as follows: a) Size exclusion to a capillary column having the same inner diameter as the above analytical capillary column. Prepare a pre-column packed with a packing material for chromatography, inject a sample into it, and b) remove a high-molecular weight or higher polymer impurity by flowing a predetermined amount of mobile phase into the pre-column, and c) use the above-mentioned pre-stage. Connecting the column to the analytical capillary column, and d) sending a mobile phase to the preceding column to deliver the target low-molecular component having the predetermined molecular weight or less to the analytical capillary column. Is.

The packing material for size exclusion chromatography is
It has the characteristic that components of various molecular weights are adsorbed in advance and the mobile phase is allowed to flow therethrough, and then the components with higher molecular weight elute in order, and the relationship between the total flow rate and the maximum molecular weight of the eluted components is It is almost uniquely determined by the filler, the size of the capillary, and the like. Therefore, as described above, the sample is injected into the pre-column and held in the packing material, and the mobile phase is allowed to flow therethrough by a predetermined amount determined according to the dimensions of the pre-column, so that the molecular weight above the analytical target component is Only the impurities that it has can be discharged from the preceding column. After removing impurities in this way, the former column is connected to the analytical capillary column, and when the mobile phase is further flown from the former column, the low-molecular components that are the object of analysis elute from the packing material for size exclusion chromatography of the former column, and the analytical capillary Sent to the column. Since the former column and the analytical capillary column have the same inner diameter, the dead volume between them can be made zero by appropriately connecting them. In the analytical capillary column, the components are separated as described above, and all the separated components are eluted from the frit and ionized without being split.

As described above, in the method according to the present invention, the polymer compound is removed before the analytical capillary column without performing column switching, so that the analytical capillary column is prevented from being clogged and the life is extended. Also,
When a biological sample is analyzed, the possibility of masking an unknown target component by polyvalent ions of a protein is greatly reduced, so that highly accurate analysis can be performed.

[0015]

EXAMPLE A liquid chromatograph mass spectrometer (LC / MS) apparatus which is an example of the present invention will be described with reference to FIGS. FIG. 1 shows only a portion before the mass spectrometric section (50 in FIG. 4A) of the LC / MS apparatus of the present embodiment, and includes a liquid feed pump 11, a splitter 14, an injector 12, and a capillary column 15. Are arranged in this order. In the LC / MS apparatus of the present embodiment, the capillary column 15 is filled with the packing material 18 for sample separation, and the components are separated there. Therefore, the capillary column 15 is a conventional analytical column (see FIG. 4A). 43). Then, the injector 12 is placed after the splitter 14, and all the injected sample is sent to the capillary column 15. Although FIG. 1 shows an example in which the normal pump 11 and the splitter 14 are used, a small flow pump for a micro liquid chromatograph may be used instead of them.

The tip of the capillary column 15 is enlarged and shown in FIG. The capillary column 15 used in this example is made of fused silica and has an inner diameter of 75 μm and a length of 100 c.
m. The packing 18 is filled only in the tip portion of the capillary column 15 (for example, about 20 cm), and the frit 16 is packed at the tip of the capillary column 15. In FIG. 2 (a), symbol 20 indicates a protective outer tube, 2
1 is a septum, 22 is a residual liquid absorbing material made of a porous metal, 2
Reference numeral 3 is a reinforcing ring.

The method of manufacturing the capillary column 15 is as follows. First, one end of a fused silica tube is slightly inserted into a vial containing particles such as limosorb, and the particles are put into the tube. This is sintered with a high temperature micro burner to form the frit 16. Next, the filler for separating the components is put in a solvent such as hexane and sufficiently stirred. A predetermined amount of the high pressure pump is fed into the fused silica tube, and only the solvent is exuded from the tip to fill the filler 18 in the tip portion. Then, as shown in FIG. 2A, a septum 21, a residual liquid absorbent 22, and a reinforcing ring 23 are added and put in the protective tube 20.

In the LC / MS of this embodiment, the injector 1
Since the sample injected in 2 is not split, almost all the mass can be analyzed by mass spectrometry, and highly sensitive analysis can be performed. In addition, the packing material 18 of the capillary column 15
Since there is no dead volume between the frit 16 and the frit 16, there is no peak spread, and highly accurate analysis can be performed. Furthermore, a conventional capillary column (see FIG.
In (b), since the frit 46 is larger than the cross section of the column 45, the exudate accumulates in the frit 46, which causes a so-called memory effect and lowers the degree of separation. Column 15
The memory effect is minimized because it exists only within the tube of the. The residual liquid absorbent 22 provided around the tip of the capillary column 15 also absorbs the liquid exuded from the frit 16 to further reduce the memory effect.

High energy (for example, 20 kV or more) is required to perform highly sensitive analysis with dynamic LSIMS.
If you want to use the Cs ⁺ ion beam of
As shown in (b), a metal mesh 19 may be added to the tip of the capillary column 15. As a result, damage to the frit 16 can be suppressed and the life of the capillary column 15 can be extended.

When a biological sample is analyzed, the target component is often in an extremely small amount, and in order to extract it, the cleanup operation needs to be repeated many times. For example,
The CLASS-I peptides, which are a group of peptides involved in the immune reaction existing on the HLA of human T cells, are within one fraction even if they are fractionated by LC before analysis by LC / MS. There are nearly 1,000 species. Therefore, many of them can obtain only a few fmol (femtomol) in total even if 10 ¹⁰ T cells are treated. When the clean-up operation is repeated for extracting such a trace component, high molecular weight impurities are contaminated, and the packing material 18 of the capillary column 15 is clogged. Therefore, when analyzing such a sample, it is desirable to perform the method as shown in FIG.

First, as shown in FIG. 3A, a capillary (previous column) 31 filled with a packing material 32 for size exclusion chromatography is prepared (a frit 33 for preventing the packing material 32 from flowing out is provided at the tip. ), A pump (not shown) and an injector 26. Then, the mobile phase is caused to flow by the pump, the sample is injected from the injector 26, and the sample is filled with the packing material 32 for size exclusion chromatography.
To hold. Next, a predetermined amount of mobile phase is caused to flow through the pre-column 31 by the pump. The amount of mobile phase to flow at this time is determined as follows. The size exclusion chromatography packing material 32 is provided with a calibration curve as shown in FIG. From this calibration curve, an amount capable of removing impurities having a molecular weight larger than the molecular weight of the target component to be subjected to mass spectrometry is set in advance as the above-mentioned predetermined amount. For example, in FIG.
In the case of the calibration curve of (c), by flowing about 9 ml of mobile phase, impurity components (mainly proteins) with a molecular weight of about 3000 or more flow out to the drain, and components below it (CLASS-
Only the molecular weight of the I peptide is about 1000) and the size exclusion chromatography packing material 32 remains and remains.

In this way, the impurities are removed, and the pre-column 31 holding only the target component having a predetermined molecular weight or less is then connected to the capillary column 15. In the example of FIG. 3B, the pre-column 31 and the capillary column 15 are connected by a Teflon tube 34, and the connection portion is further fixed by a zero dead volume connector 35 from above. In addition, if you prepare a slightly tight Teflon tube, warm it and make the diameter a little larger, connect both columns 31, 15 and then cool, you can use the zero dead volume connector 35 up to about 300 psi. However, reliable connection is possible. After connecting both columns 31 and 15 in this manner, the mobile phase is again flowed to the preceding column 31 by the pump. As a result, as shown in the calibration curve of FIG. 3C, lower molecular weight components including the analysis target component are separated from the size exclusion chromatography packing material 32 and are sent to the capillary column 15. At this time, since the capillary column 15 is in reverse phase, the analysis target component is stopped at the inlet of the packing section and concentrated, and only the salts pass through the packing material 18. After removing the salts by this, the usual analytical conditions (0.5% acetic acid aqueous solution:
When CH ₃ CN = 100: 0 → 40: 60), the CLASS-I peptide as the target component is separated by the filler 18 and is exuded from the frit 16 and ionized.

In the above method, the packing material 32 for size exclusion chromatography was used to remove the polymer component, but if it is sufficient to remove only the larger impurities, that is, the packing material, a reverse phase column is used instead. It can also be used as the front-stage column 31.

The impurity removal method shown in FIG. 3 is applied to the dynamic FAB and dynamic LSIM described in the above embodiment.
Not only S, but also the ESI (Electro Spray) method is effective.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram of a part up to a mass spectrometric section of a liquid chromatograph mass spectrometer (LC / MS) which is an embodiment of the present invention.

FIG. 2 is an enlarged cross-sectional view of the tip portion of the LC / MS capillary column of the example.

FIG. 3 is an explanatory diagram showing a method for effectively removing impurities when using the LC / MS of the embodiment.

FIG. 4 is a schematic configuration diagram of a conventional LC / MS.

[Explanation of symbols]

 11 ... Liquid-sending pump 12 ... Injector 14 ... Splitter 15 ... Capillary column 16 ... Frit 18 ... Component separation packing 50 ... Mass spectrometer

Claims (2)

[Claims] 1. A pump for delivering a mobile phase to a liquid feed passage, b) one end connected to the liquid feed passage, at least a part of which is filled with a component separating filler, and the other end of which is a frit. A) an analytical capillary column in which is fixed, c) an injector for injecting a sample into the liquid feeding path, and d) a particle irradiation means for irradiating the frit with ions or neutral molecules to ionize the liquid exuded from the frit, A liquid chromatograph mass spectrometer, comprising: 2. A) a preparatory column in which a capillary column having the same inner diameter as that of the analytical capillary column according to claim 1 is packed with a packing material for size exclusion chromatography, and a sample is injected into the preparative column; and b) the preparatory column. High molecular weight or higher molecular weight impurities are removed by flowing a predetermined amount of mobile phase into the column, c) the pre-column is connected to the analytical capillary column according to claim 1, and d) the mobile phase is further passed into the pre-column. Thus, the liquid chromatograph mass spectrometric method comprising the steps of sending the target low-molecular component having the predetermined molecular weight or less to the analytical capillary column.