WO2003038425A1

WO2003038425A1 - Liquid chromatographic mass spectrograph

Info

Publication number: WO2003038425A1
Application number: PCT/JP2002/011285
Authority: WO
Inventors: Izumi Ogata; Kisaburo Deguchi; Masako Ishikawa; Takefumi Yokokura; Tadao Mimura
Original assignee: Hitachi High-Technologies Corporation; Hitachi Science Systems, Ltd.
Priority date: 2001-10-31
Filing date: 2002-10-30
Publication date: 2003-05-08
Also published as: JPWO2003038425A1

Abstract

It is intended to provide an apparatus for LC/MS analysis to be used in analyzing oligonucleic acids. A liquid chromatographic mass spectrograph which has a pump for feeding an eluent containing an ion pairing agent and a salt, a sample injector for injecting a sample to be analyzed into a channel, a column for fractionating the injected solution depending on components, and a mass spectrograph for ionizing the solution eluted from the column by an atmospheric ion source and detecting the same, characterized by further having a recombining means of adding a weakly basic solution between the column and the mass spectrogram. Owing to the above constitution, an oligonucleic acid can be analyzed at a high efficiency and a high sensitivity.

Description

Specification

Liquid mouth mass spectrometer Technical field

The present invention relates to a liquid chromatograph mass spectrometer (LCZMS) in which a liquid chromatograph (hereinafter referred to as LC) and a mass spectrometer (hereinafter referred to as MS) are combined. It relates to a device for measuring. Background art

At present, in order to purify the synthesized oligonucleic acid, a method of separating the target oligonucleic acid from contaminants using LC is generally used. At this time, in order to separate on a reversed phase or an ion exchange column, Analytical Chemistry, pp. 1320 to 1325, Vol. Chem., 1997, 69, 1320-1325). Appropriate amounts of ion-pairing agents (eg, triethylammonium acetate (Τ-Ε), tributylammonium acetate (TBAΑ), dibutyl acetate) It is necessary to add luanmoyuum (salts such as DB AA) to the eluent.

In order to obtain a mass spectrum of the purified oligonucleic acid, since the mass number of the oligonucleic acid is as large as several thousands to tens of thousands, matrix-assisted laser desorption ionization capable of measuring up to several tens of thousands of masses Time-of-flight mass spectrometer

(MA LD I-TOF-MS). However, when MALDI-TOF-MS was used, ionization was carried out under a high vacuum, so it was necessary to re-prepare samples separated by LC for ionization. In other words, it was not possible to directly guide the sample from LC to MALD I-TOR-MS, and the analysis efficiency using MALD I-T〇F-MS was very poor. Therefore, it was impossible to fractionate the target oligonucleic acid based on the information of the mass spectrum obtained by MALDI-TOF-MS.

On the other hand, MS that can directly introduce a sample from LC and perform mass spectrometry includes a quadrupole mass spectrometer (Q-MS) equipped with an atmospheric pressure ion source and an ion trap mass spectrometer (IT-MS). Are known. Therefore, when the separation of oligonucleic acids and mass spectrometry were to be performed using a device in which the sample from the LC was led to the MS online, that is, LCZMS, the atmospheric pressure ion source as shown above was provided. It is necessary to use MS. As an atmospheric pressure ion source, an electrospray ionization method (ESI), a sonic spray ionization method (SSI), an ion spray (IS), or the like is generally used. Disclosure of the invention

However, the upper limit of the measurable mass number of a Q-MS or an IT-MS equipped with an atmospheric pressure ion source is about several thousand, so that the oligonucleic acid with a large mass number exceeds the upper limit. Therefore, in order to measure oligonucleic acid with Q_MS or IT-MS, multivalent ions must be used.

In addition, if the ion-pairing agent necessary for the separation of oligonucleic acid by LC is mixed into the sample to be introduced into the MS, the generation of ions by an atmospheric pressure ion source such as ESI, SSI, or IS is hindered. (Ion cancellation), causing a significant decrease in MS sensitivity.

To prevent this situation, for example, RAPID

As disclosed in COMMUNICATIONS IN MASS SPECTRMETRY, VOL. 9, 97-102 (1995), it is possible to mix a sample to be introduced into MS with a solution of a weak base such as imidazole solution and measure it with ESI-MS. Proposed.

However, in the above document, the LC and MS were connected online. However, there is no description of a configuration for efficiently mixing a weak base solution with a sample to be introduced into the MS in the apparatus.

Therefore, separation and purification of oligonucleic acid by LCZMS and detection of a mass spectrum with high sensitivity have not been conventionally performed, and it is necessary to perform online separation of oligonucleic acid and mass spectrometry. Was impossible.

An object of the present invention is to provide an apparatus for performing analysis by LC / MS in the analysis of oligonucleic acid.

A characteristic configuration of the present invention is a pump that sends an eluent containing an ion pair agent or a salt, a sample injector that injects a sample to be measured into a flow channel, a column that separates the introduced solution for each component, and the column. A liquid chromatograph mass spectrometer having a mass spectrometer for ionizing and eluting a solution eluted from a solution with an atmospheric pressure ion source, wherein a weak base solution is merged between the column and the mass spectrometer. That is, there is provided a merging means. In the present invention, in the LC / MS in which the eluate from the LC is introduced into the MS online, even if an ion pair agent necessary for the analysis of oligonucleic acid is mixed in the eluate, the MS / MS can be transferred to the MS with high sensitivity. It will be possible to analyze it.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic configuration diagram of the first embodiment.

FIG. 2 is the total ion chromatogram (TIC) obtained by MS9.

FIG. 3 is a mass spectrum at the retention time of each base sample peak in FIG.

FIG. 4 is a mass spectrum of an oligo nucleotide nucleic acid sample of 40 bases.

FIG. 5 is a schematic configuration diagram of the second embodiment. FIG. 6 is a schematic diagram showing the structure of the splitter 11.

FIG. 7 is a schematic configuration diagram of a modified example of the second embodiment.

FIG. 8 is a schematic configuration diagram of the third embodiment.

FIG. 9 is a schematic configuration diagram of a modification of the third embodiment.

FIG. 10 is a schematic configuration diagram of a modified example of the third embodiment. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, examples of the present invention will be described.

(Example 1)

FIG. 1 shows a schematic configuration diagram of the first embodiment. The LC / MS of the present embodiment has a function of selecting a plurality of solutions (organic solvents A and B including a buffer) 1 and '2, and changing the composition of the solution while mixing them over time. A pump 4 with a so-called gradient elution), a sample injector 6 for introducing an oligonucleic acid sample into the flow path, a column 7 for separation of each component, and a weak base solution (eg imidazole solution) 3 It consists of a pump 5 for joining, a mixer (coil or column) 8 for stirring and mixing the combined solution, an MS 9 for ionizing and detecting the eluate introduced from the LC, and a controller 10. The MS 9 used in the present invention uses an atmospheric pressure ion source such as ESI, SSI or IS as the ion source. In addition, an ion trap type, a time-of-flight type, and a quadrupole type device can be used for the mass spectrometer.

The oligonucleic acid sample injected into the channel from the sample injector 6 is separated into a single component by the column 7, and then merges with the weak base solution 3 sent by the pump 5. The combined solution is stirred and mixed by the mixer 8 and introduced into the MS 9 so that mass information of the oligonucleic acid can be obtained. The present invention The weak base solution 3 used in the above method is effective even if a base having a pKb of 5.0 or more, such as a piperidine 'solution, is used in addition to the imidazole solution.

Further, the above pump 4, sample injector 6, pump 5, and MS 9 can all be controlled by the controller 10.

FIG. 2 shows the total ion chromatogram (TIC) obtained by MS9. The TIC in FIG. 2 (a) is a result when the flow rate of the pump 5 is set to 0.0 mL Zmin and the weak base solution 3 is not added, that is, an analysis result under the same conditions as the conventional method. The TIC in FIG. 2 (b) is the result obtained by setting the flow rate of the pump 5 to 1.0 mLZmin and adding the weak base solution 3. The analysis conditions in Fig. 2 are as follows: Solution 1 contains 0.1 mol / L triethylamine acetate; 10% aqueous acetonitril solution (V / V :); Solution 2 contains 0.1 mol / L triethylamine acetate. 20% aqueous solution of acetonitrile (V / V), separation force in column 7 4.6 x 150 mm (5 μm packing material) 0 DS (otatadecylsilane) column, weak base solution 3 0.1 molZL imidazole in acetonitrile solution was used. Pump 4 has a flow rate of 0.5 mL / min, and the composition of solutions 1 and 2 starts from solution 1 solution 2 = 100 0Z 0 and takes 20 minutes to reach solution 1 Ζ solution 2 = 50Ζ50 A variable gradient elution was performed. The oligonucleic acid sample is a mixed aqueous solution of three samples with the number of bases of 20, 30, and 40. The concentrations of the 20 base sample are 24 mol / L and the 30 base sample are 40 mol / L. The L, 40 base sample was 29 μmol ZL. 30 μL of the oligonucleic acid sample was injected from the sample injector 6. By the way, the mass number (m) of the 20 base sample is 6096, that of the 30 base sample is 9200, and that of the 40 base sample is 123.60.

With the TIC in Fig. 2 (a), only the peak of the 20-base sample could be detected. The 30-base sample and the 40-base sample are buried in noise components. No, it cannot be specified on TI c.

In the TIC shown in Fig. 2 (b), it was confirmed that the relative intensity was about 10 times higher than that in Fig. 2 (a), and all oligo nucleic acids contained in the oligo nucleic acid sample were confirmed. Could be detected.

FIG. 3 shows the mass spectrum at the retention time of each base sample peak detected in FIGS. 2 (a) and (b).

In the mass spectrum of Fig. 3 (a) without adding the weak base solution 3, the peak of 20 bases that could be confirmed by TIC was found to have a mass-to-charge ratio (niZz) of 15 Multiply charged ions were provided. The 30-base sample also gives a pentavalent polyvalent ion of mZ z 1839. On the other hand, in the mass spectrum of Fig. 3 (b) when the weak base solution 3 was added, the detection sensitivity was improved for both the 20 base sample and the 30 base sample, and the S / N ratio of the weak base solution 3 was increased. It is about 10 times higher than without. The addition of the weak base solution 3 also increases the valency of the generated ions, and the 20-base sample is a pentavalent ion, m-noz1218, and the 30-base sample is a hexavalent ion. The ions of m / z 1532 and 7-valent mZz1313 were detected, and could be detected on the lower quality number side. In addition, the mass spectrum of the 40-base sample, which was not detected at all when the weak base solution 3 was not added, was also reduced from the 7-valent mZzl 764 to the 10-valent as shown in Fig. 4. It was possible to detect ions up to m / z 1 235 with high sensitivity.

(Example 2)

FIG. 5 shows a second embodiment. In this embodiment, an oligonucleic acid fractionation system in which a fraction collector 12 is connected via a splitter 11 for splitting an eluate from a column 7 to the configuration of the first embodiment is described. This is an example. Fraction collector 1 2 also pump, sample injection It can be controlled by the controller 10 together with the rectifier 6, pump 5, and MS'9.

The splitter 11 has the structure shown in FIG. 6, and has two resistance coils having different lengths inside. Due to the difference in the resistance of this coil, a certain amount of the eluate from column 7 to 1/10 to 1/1000 flows out to the MS 9 side and merges with the weak base solution 3 sent from the pump 5. Let it. In this way, the weak base solution 3 can be combined only with the flow path to the MS 9 _{c. The} combined solution is stirred by the mixer 8 and introduced into the MS 9.

In the controller 10, the mass number of the oligonucleic acid and the mass number of the multiply charged ions to be collected are input in advance. When MS 9 S detects a chromatogram peak corresponding to ions having these mass numbers, the controller 10 sends a signal to the fraction collector 12 through a signal line. Upon receiving this signal, the fraction collector 12 separates the desired oligo nucleic acid sample into a container such as a test tube.

It is also possible to connect a UV detector (not shown) between the splitter 11 and the fraction collector 12 to obtain quantitative information on oligo nucleic acid samples from the peak area of the UV chromatogram. . Further, the fraction collector 12 can sample the sample based on the peak signal detected by the UV detector. In this case, since the MS is performing analysis simultaneously with the fractionation, the fractionated sample can be identified by its mass number information.

In the second embodiment, when the amount of the oligonucleic acid sample is small and it is difficult to detect the sample with MS9, or when it is difficult even to separate the sample solution with the splitter 11 in FIG. As shown in Fig. 7, a hexagonal pulp 13 that alternates between a solid flow path and a dotted flow path at regular time intervals is connected between the column 7 and the MS 9 and the fraction collector 12. The eluate from column 7 is When the hexagonal pulp 13 is switched, it flows out alternately to the MS 9 and the fraction collector 12. As shown in Fig. 2, since the peak width of the one-component oligonucleic acid sample is about 30 seconds, the flow path of the hexagonal valve 13 should be switched, for example, every 1 second. .

When the eluate from the column 7 flows out to the MS 9, it is once held in the sample loop 130 in the hexagonal pulp 13, and then sent to the MS 9 by the weak base solution 3 sent from the pump 5.

In the configuration of FIG. 7 as well, as in the example of FIG. 5, when the MS 9 detects a mouth massogram peak corresponding to an ion having the mass number of the target oligonucleic acid sample, the controller 10 returns to the controller 10. A signal is sent to the fraction collector 12, which collects the target oligonucleic acid sample.

(Example 3)

FIG. 8 shows a third embodiment. This embodiment is an example in which a column is added to the configuration of the first embodiment so that two types of oligonucleic acid samples can be simultaneously analyzed. In addition to columns 15 and mixer 16 as additional flow paths, a splitter 17 for equally dividing weak base solution 3 into two flow paths from columns 7 and 15 and a flow path for columns 7 and 15 A 10-way valve 18 for switching the two flow paths from the outlet to one MS, a transfer solution 19 for sending the eluate reaching the 10-way pulp 18 to the MS 9, and Liquid sending pump 20 is added. The 10-way pulp 18 and the pump 20 can also be controlled by the same controller 10 as the pumps 4 and 14, the sample injector 6, the pump 5, and the MS9. The sample injector 6 can inject a sample into any of the flow paths.

The two types of oligonucleic acid samples injected into the two channels from the sample injector 6 are separated into single components by the respective columns 7 and 15. The weak base solution 3 sent by the pump 5 is divided into two equal volumes by the splitter 17. Each of the divided weak base solutions 3 is combined with the eluate from the columns 7 and 15, respectively, and stirred by the mixers 8 and 16 to be sent to the 10-way pulp 18. The 10-way pulp 18 switches between the solid flow path and the dotted flow path at regular time intervals (for example, 1 second). be introduced. The MS 9 determines, based on the switching time interval of the valve 18, whether the detected signal is due to one of the two flow paths, and processes the two types of signals separately. As a result, mass information of the oligonucleic acid sample dissolved from the two columns 7 and 15 can be obtained by one MS 9.

Here, the transfer solution 19 has a role of sending the eluate from the two flow paths and a role of preventing the mixing of the two eluate in the MS ion source. Therefore, it is preferable that the transfer solution 19 does not affect the analysis in the MS 9, and the use of the weak base solution 3 already added to the eluate may increase the sensitivity of the MS 9 Can do the best. Other than the weak base solution 3, methanol or the like can be used.

9 and 10 show a modification of the present embodiment.

In the example of Fig. 9, the splitters 11 and 21 and the fraction collectors 12 and 22 that can be controlled by one controller 10 are connected, and the oligonucleic acid sample collection according to the number of flow paths is performed. System '. Use the splitters 11 and 21 shown in Fig. 6.

Further, the example of FIG. 10 is a schematic configuration diagram when the amount of the oligonucleic acid sample is small. Without using splitters 11 and 21, weak base solution 3 was combined with all of the eluate from columns 7 and 15 and eluted from columns 7 and 15 by 10-way valve 18. Samples other than those to be introduced into MS 9 A nucleic acid sample is collected in its entirety.

According to the examples shown in FIGS. 9 and 10, even if the sample has a small capacity, it can be collected without leakage except for the purpose of analyzing by MS.

According to each of the above-described examples, highly sensitive analysis of oligonucleic acid can be performed using LCZMS in which eluate from LC is introduced online to MS. Therefore, it is possible to improve the efficiency of the analysis.

Furthermore, the action of a weak base solution facilitates the generation of multiply charged ions, so measurement with an ion trap type or quadrupole type mass spectrometer, which has a lower upper limit of the measurable mass number than T〇F-MS Becomes possible. In addition, the efficiency of the analysis using TOF-MS can be greatly increased.

In addition, since the mass spectrum can be detected with high sensitivity, the oligonucleic acid is fractionated using the mass number of the target oligonucleic acid and the mass number of the polyvalent ion as a signal. Becomes possible. At this time, since the fractionation is performed while taking the mass spectrum, it is possible to record the mass spectrum of the fractionated oligonucleic acid sample simultaneously with the separation as quality data of the sample.

In addition, it enables high-sensitivity analysis and fractionation of multiple oligonucleic acids with less MS while simultaneously performing separation on multiple columns. In addition, when a plurality of oligonucleic acids are analyzed by the MS, the liquid is transferred from the switched pulp to the MS using an independent pump, so that mixing of the samples in the ion source can be prevented.

Claims

The scope of the claims

1. A pump that sends an eluent containing ion-pairing agents and salts, a sample injector that injects the sample to be measured into the flow channel, a power ram that separates the introduced solution for each component, and eluted from the column A liquid chromatograph mass spectrometer having a mass spectrometer for ionizing and detecting a solution with an atmospheric pressure ion source,

Liquid mass chromatograph mass spectrometer characterized by comprising a merging means for merging a weak base solution between the column and the mass spectrometer.

2. In Claim 1,

A liquid mouth mass spectrometer characterized by comprising a mixing means for stirring and mixing the solution in the flow path after the merging.

3. In Claim 2,

A branching means for branching a flow of the solution eluted from the force ram at a predetermined ratio between the force ram and the mixing means, a part of the solution to the mass spectrometer, and the other solution to a fraction collector Liquid mass chromatography mass spectrometer characterized by being introduced.

4. In Claim 3,

A liquid chromatograph mass spectrometer, wherein the merging unit is connected to the branching unit, and mixes the solution of the weak base with a solution introduced into the mass spectrometer.

5. In Claim 3,

The liquid chromatograph mass spectrometer is characterized in that the branching means is a six-way valve, includes a sample loop therein for temporarily holding the solution, and switches a flow path at a predetermined cycle.

6. In Claim 3, The said fraction collector operates according to the signal detected by the said mass spectrometry apparatus, The liquid chromatograph mass spectrometer characterized by the above-mentioned.

7. In Claim 3,

A liquid chromatograph mass spectrometer comprising a UV detector between the branching means and the fraction collector.

8. In claim 1,

The mass spectrometer is an electrospray ionization method, a sonic spray ionization method, and ionization by ion spray.

9. In Claim 1,

The mass spectrometer is a time-of-flight, ion trap, or quadrupole mass spectrometer. Liquid chromatography mass spectrometer (10. Sends eluent containing ion pairing agent or salt) Eluent pump, sample injector for injecting the sample to be measured into the flow channel, multiple columns to separate the introduced solution for each component, and solution eluted from the power ram to be ionized by the atmospheric pressure ion source A liquid chromatograph mass spectrometer having a mass spectrometer for detecting and detecting

Mixing means for stirring and mixing the solution provided in each of the flow paths downstream of the respective force rams;

Merging means for merging a solution of a weak base between the column and the mixing means;

A switching valve through which the solution from each of the mixing means is guided, and which is connected to the mass spectrometer;

And a transfer liquid pump for sending a transfer liquid to the switching pulp. The switching valve is switched by the transfer liquid while switching the switching valve. A liquid chromatograph mass spectrometer, wherein an eluate from each of the force rams led to a lube is alternately introduced into the mass spectrometer.

1 1. In claim 10,

A liquid chromatograph mass spectrometer, wherein the transfer liquid uses a solution of a weak base.

1 2. In claim 10,

The same number of fraction collectors as the number of columns,

In each flow path between the force ram and the mixing means, there is provided a branch means for branching a flow of the solution eluted from the column at a predetermined ratio, and the plurality of branch means are provided with the switching valve and the respective fraction switches. Liquid mass chromatography mass spectrometer characterized by being connected to a collector.

1 3. In claim 12,

A liquid chromatograph mass spectrometer, wherein the merging unit is connected to each of the branching units, and mixes the solution of the weak base with the solution introduced into the switching pulp.

14. In claim 10,

A liquid mouth mass spectrometer which is connected to the switching pulp and has the same number of fraction collectors as the number of the power rams.