JPWO2003038425A1 - Liquid chromatograph mass spectrometer - Google Patents

Abstract

An object of the present invention is to provide an apparatus for performing analysis by LC / MS in oligonucleic acid analysis. Specifically, a pump for feeding an eluent containing an ion-pairing agent and a salt, a sample injector for injecting a sample to be measured into a flow path, a column for separating the introduced solution into each component, and elution from the column A liquid chromatograph mass spectrometer having a mass spectrometer for ionizing and detecting a solution with an atmospheric pressure ion source, comprising a merging means for merging a weak base solution between the column and the mass spectrometer. It is characterized by. This configuration enables highly efficient and highly sensitive analysis of oligonucleic acids.

Description

TECHNICAL FIELD The present invention relates to a liquid chromatograph mass spectrometer (LC / MS) 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 In order to purify synthesized oligonucleic acid, a method of separating a target oligonucleic acid and impurities by using LC is generally used. In this case, for separation on a reverse phase or ion exchange column, it is discussed in Analytical Chemistry, 1997, 69, pages 1320 to 1325 (Anal. Chem., 1997, 69, 1320-1325). As described above, an appropriate amount of ion-pairing agent (for example, a salt of triethylammonium acetate (TEAA), tributylammonium acetate (TBAA), dibutylammonium acetate (DBAA), etc.) needs to be added to the eluent.
In addition, to obtain a mass spectrum of purified oligonucleic acid, the mass number of oligonucleic acid can be as large as several thousand to several tens of thousands. It was measured with a device (MALDI-TOF-MS). However, when MALDI-TOF-MS is used, since ionization is performed at a high vacuum, it is necessary to prepare a sample separated by LC again for ionization. That is, it was impossible to introduce a sample from LC directly to MALDI-TOF-MS, and the analytical efficiency using MALDI-TOF-MS was very poor. Therefore, it was impossible to sort out the target oligonucleic acid based on the mass spectrum information obtained by MALDI-TOF-MS.
On the other hand, as MS capable of mass spectrometry by directly introducing a sample from LC, there are a quadrupole mass spectrometer (Q-MS) and an ion trap mass spectrometer (IT-MS) equipped with an atmospheric pressure ion source. Are known. Therefore, when the sample from the LC is led online to the MS, that is, when the separation of the oligonucleic acid and the mass analysis are performed by the LC / MS, it is necessary to use the MS having the atmospheric pressure ion source as described above. There is. As the 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, since Q-MS and IT-MS provided with an atmospheric pressure ion source have a measurable mass number upper limit of about several thousand, an oligonucleic acid having a large mass number exceeds the upper limit. Therefore, in order to measure oligonucleic acid by Q-MS or IT-MS, multivalent ions must be used.
In addition, if the ion pair agent required for LC separation of oligonucleic acid is mixed in the sample introduced into the MS, the generation of ions in an atmospheric pressure ion source such as ESI, SSI or IS is hindered (ion suppression) ), The sensitivity of the MS is significantly reduced.
As a method for preventing such a situation, for example, RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 9, 97-102 (1995), it is proposed to mix a sample introduced into MS with a weak base solution such as an imidazole solution and measure by ESI-MS.
However, the above document does not show any configuration for efficiently mixing a weak base solution with a sample introduced into MS in an apparatus in which LC and MS are connected online.
Therefore, separation and purification of oligonucleic acid by LC / MS and detection of mass spectrum with high sensitivity have not been conventionally performed, and it has been impossible to perform oligonucleic acid separation and mass spectrometry online. .
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 includes a pump for feeding an eluent containing an ion-pairing agent and a salt, a sample injector for injecting a sample to be measured into a flow path, a column for separating the introduced solution into components, and the column. A liquid chromatograph mass spectrometer having a mass spectrometer for ionizing and detecting a solution eluted from an atmospheric pressure ion source, and a merging means for joining a weak base solution between the column and the mass spectrometer It is to have.
In the present invention, in the LC / MS in which the eluate from the LC is introduced into the MS online, even when the ion pair agent necessary for the analysis of the oligonucleic acid is mixed in the eluent, the MS is sensitive. It can be analyzed.
BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below.
(Example 1)
FIG. 1 shows a schematic configuration diagram of the first embodiment. The LC / MS of this embodiment selects a plurality of solutions (organic solvents A and B including buffers) 1 and 2 and changes the composition of the solution while mixing them over time (so-called gradient). Elution) pump 4, sample injector 6 for introducing an oligonucleic acid sample into the flow path 6, column 7 for separating each component, and pump 5 for merging a weak base solution (for example, imidazole solution) 3, merging It comprises a mixer (coil or column) 8 for stirring and mixing the prepared 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 an ion source. Moreover, each device of an ion trap type, a time-of-flight type, and a quadrupole type can be used for the mass spectrometer.
The oligonucleic acid sample injected into the flow path from the sample injector 6 is separated into single components 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 to obtain mass information of the oligonucleic acid. The weak base solution 3 used in the present invention is effective even when a base having a pKb of 5.0 or higher, such as a piperidine solution, is used in addition to the imidazole solution.
The pump 4, the sample injector 6, the pump 5, and the MS 9 can all be controlled by the controller 10.
FIG. 2 is an example of a total ion chromatogram (TIC) obtained by MS9. The TIC in FIG. 2 (a) is the result when the flow rate of the pump 5 is 0.0 mL / min and the weak base solution 3 is not added, that is, the analysis result under the same conditions as in the conventional method. The TIC in FIG. 2 (b) is the result obtained by adding the weak base solution 3 with the flow rate of the pump 5 being 1.0 mL / min.
The analysis conditions in FIG. 2 were as follows: Solution 1 was 0.1 mol / L triethylamine acetate-containing 10% acetonitrile aqueous solution (v / v), Solution 2 was 0.1 mol / L triethylamine acetate-containing 20% acetonitrile aqueous solution (v / v). The separation column 7 was an ODS (octadecylsilane) column having a column size of 4.6 × 150 mm (5 μm packing material), and the weak base solution 3 was an acetonitrile solution of 0.1 mol / L imidazole. The flow rate of the pump 4 is 0.5 mL / min, and the composition of the solutions 1 and 2 starts from solution 1 / solution 2 = 100/0 and changes to solution 1 / solution 2 = 50/50 over 20 minutes. Elution was performed. The oligonucleic acid sample was a mixed aqueous solution of 3 samples with 20, 30, and 40 bases, each concentration being 24 μmol / L for the 20 base sample, 40 μmol / L for the 30 base sample, and 29 μmol / L for the 40 base sample. . 30 μL of this oligonucleic acid sample was injected from the sample injector 6. Incidentally, the mass number (m) of the 20 base sample is 6096, the 30 base sample is 9200, and the 40 base sample is 12360.
In the TIC of 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 and cannot be specified on the TIC.
In the TIC of FIG. 2 (b), it is possible to confirm an improvement in sensitivity of about 10 times relative intensity compared to the case of FIG. 2 (a). I was able to detect it.
FIG. 3 shows the mass spectrum of each base sample peak detected in FIGS. 2 (a) and 2 (b).
In the mass spectrum of FIG. 3 (a) when the weak base solution 3 is not added, a 20-base sample whose peak was confirmed by TIC had a tetravalent multivalent ion at a mass-to-charge ratio (m / z) 1523. Gave. A 30 base sample also gave a pentavalent polyvalent ion of m / z 1839. On the other hand, in the mass spectrum of FIG. 3 (b) when the weak base solution 3 is added, the detection sensitivity is improved for both the 20 base sample and the 30 base sample, and the S / N ratio is when the weak base solution 3 is not added. Compared to about 10 times. Further, the addition of the weak base solution 3 increases the valence of the ions to be generated. In the 20 base sample, a pentavalent m / z 1218 ion, which is a pentavalent ion, and in a 30 base sample, a hexavalent m / z 1532 and a 7 valent ion. The ion of m / z 1313 was detected and could be detected on the lower mass number side. Furthermore, as shown in FIG. 4, the mass spectrum of a 40 base sample that could not be detected at all when no weak base solution 3 was added was sensitive from 7 valent m / z 1764 to 10 valent m / z 1235 ions. We were able to detect well.
(Example 2)
FIG. 5 shows a second embodiment. This embodiment is an example of an oligonucleic acid sorting system in which a fraction collector 12 is connected to the configuration of the first embodiment via a splitter 11 that splits the eluate from the column 7. The fraction collector 12 can also be controlled by the controller 10 together with the pump 4, sample injector 6, pump 5, and MS9.
The splitter 11 has the structure shown in FIG. 6 and includes two resistance coils having different lengths. Due to the difference in the resistance value of the coil, a fixed amount of about 1/10 to 1/100 of the eluate from the column 7 is caused to flow out to the MS 9 side and merged with the weak base solution 3 sent from the pump 5. In this way, the weak base solution 3 can be merged only in the flow path to the MS 9. The combined solution is stirred by the mixer 8 and introduced into the MS 9.
The mass number of oligonucleic acid and the mass number of multivalent ions to be sorted are input to the controller 10 in advance. When the MS 9 detects chromatogram peaks corresponding to ions having these mass numbers, the controller 10 sends a signal to the fraction collector 12 through a signal line. In response to this signal, the fraction collector 12 sorts the target oligonucleic 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 and obtain quantitative information of the oligonucleic acid sample from the peak area value of the UV chromatogram. Further, the fraction collector 12 can sort the sample based on the peak signal detected by the UV detector. In this case, since MS is analyzing simultaneously with fractionation, the fractionated sample can be identified by its mass number information.
Further, in the second embodiment, when the amount of oligonucleic acid sample is small and detection by MS9 is difficult, or when it is difficult to separate the sample solution by splitter 11 in FIG. 6, as shown in FIG. In addition, a hexagonal valve 13 that alternately switches between a solid line flow path and a dotted line flow path at regular time intervals is connected between the column 7 and the MS 9 and the fraction collector 12. The eluate from the column 7 alternately flows out to the MS 9 and the fraction collector 12 by switching the hexagonal valve 13. As shown in FIG. 2, since the peak width of the single-component oligonucleic acid sample is about 30 seconds, the flow path of the hexagonal valve 13 is switched at intervals of 1 second, for example.
When the eluate from the column 7 flows out to the MS 9, the eluate is once held in the sample loop 130 in the six-way valve 13 and then sent to the MS 9 by the weak base solution 3 sent by the pump 5.
Also in the configuration of FIG. 7, as in the example of FIG. 5, when the MS 9 detects a chromatogram peak corresponding to an ion having the mass number of the target oligonucleic acid sample, the controller 10 sends a signal to the fraction collector 12. Then, the fraction collector 12 sorts out the target oligonucleic acid sample.
(Example 3)
FIG. 8 shows a third embodiment. This example is an example in which a column is added to the configuration of the first example so that two types of oligonucleic acid samples can be analyzed simultaneously. In addition to the column 15 and the mixer 16 which are additional flow paths, two flow paths from the outlets of the splitter 17 and the columns 7 and 15 for equally dividing the weak base solution 3 into the two flow paths from the columns 7 and 15 are provided. A ten-way valve 18 for switching for introduction into one MS, a transfer liquid 19 for sending the eluate reaching the ten-way valve 18 to the MS 9, and a liquid feed pump 20 are added. The ten-way valve 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 MS 9. The sample injector 6 can inject a sample into any flow path.
Two kinds of oligonucleic acid samples injected into the two flow paths 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 equally divided into two flow paths by the splitter 17. The divided weak base solutions 3 merge with the eluates from the columns 7 and 15, and are stirred by the mixers 8 and 16 and sent to the ten-way valve 18. The ten-way valve 18 switches between the solid line flow path and the dotted line flow path at regular time intervals (for example, 1 second), and the eluate from the two columns is alternately introduced into the MS 9 by the transfer liquid 19 sent by the pump 20. Based on the switching time interval of the valve 18, the MS 9 determines which of the two flow paths is the detected signal and processes the two types of signals separately. Thereby, the mass information of the oligonucleic acid sample eluted from the two columns 7 and 15 with one MS 9 can be obtained.
Here, the transfer liquid 19 has a role of preventing the mixing of the two eluates in the ion source of the MS, as well as a role of sending the eluates from the two flow paths. Therefore, the transfer liquid 19 should be one that does not affect the analysis in the MS 9, and the use of the weak base solution 3 already added to the eluent can increase the sensitivity of the MS 9 and give the best result. Other than the weak base solution 3, methanol or the like can be used.
9 and 10 show a modification of this embodiment.
The example of FIG. 9 realizes an oligonucleic acid sample sorting system according to the number of flow paths by connecting splitters 11 and 21 and fraction collectors 12 and 22 that can be controlled by one controller 10. The splitters 11 and 21 used are those shown in FIG.
Moreover, the example of FIG. 10 is a schematic block diagram when the amount of oligonucleic acid sample is small. Other than the sample introduced into the MS 9 among the samples eluted from the columns 7 and 15 by the ten-way valve 18 by combining the weak base solution 3 with the total amount of the eluate from the columns 7 and 15 without using the splitters 11 and 21. The whole amount of the oligonucleic acid sample is collected.
According to the examples of FIGS. 9 and 10, even in the case of a sample having a small volume, it can be collected without omission except for the purpose of analysis by MS.
According to each Example shown above, highly sensitive analysis of oligonucleic acid is attained using LC / MS in which the eluate from LC is introduced on-line into MS. Therefore, the efficiency of analysis can be improved.
Furthermore, the action of the weak base solution facilitates the generation of multivalent ions, enabling measurement with an ion trap type or quadrupole type mass spectrometer having a lower upper limit of the measurable mass number than TOF-MS. Become. In addition, the efficiency of analysis can be greatly increased in analysis using TOF-MS.
In addition, since mass spectra can be detected with high sensitivity, oligonucleic acid can be fractionated using the mass number of the target oligonucleic acid and the mass number of the polyvalent ions as signals. At this time, since the fractionation is performed while taking the mass spectrum, it is possible to record the mass spectrum of the sorted oligonucleic acid sample simultaneously with the separation as the quality data of the sample.
Furthermore, it is possible to perform high-sensitivity analysis and fractionation of a plurality of oligonucleic acids by a smaller number of MSs while simultaneously performing separation by a plurality of columns. In addition, when analyzing a plurality of oligonucleic acids by MS, liquid feeding from the switching valve to the MS is performed by an independent pump, so that mixing of samples in the ion source can be prevented.
[Brief description of the drawings]
FIG. 1 is a schematic block diagram of the first embodiment.
FIG. 2 is a total ion chromatogram (TIC) obtained by MS9.
FIG. 3 is a mass spectrum at the retention time of each base sample peak of FIG.
FIG. 4 is a mass spectrum of a 40 base oligonucleic acid sample.
FIG. 5 is a schematic block 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 modification 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.

Claims (14)

A pump that delivers an eluent containing an ion-pairing agent and salt, a sample injector that injects the sample to be measured into the flow path, a column that separates the introduced solution into components, and a solution that has eluted from the column is ionized at atmospheric pressure A liquid chromatograph mass spectrometer having a mass spectrometer ionized and detected by a source,
A liquid chromatograph mass spectrometer comprising a merging means for merging a weak base solution between the column and the mass spectrometer. In claim 1,
A liquid chromatograph mass spectrometer comprising mixing means for stirring and mixing the solution in the flow path after the merge. In claim 2,
Branch means for branching the flow of the solution eluted from the column at a predetermined ratio is provided between the column and the mixing means, and a part of the solution is introduced into the mass spectrometer and the other solution is introduced into the fraction collector. A liquid chromatograph mass spectrometer characterized by being configured as described above. In claim 3,
The merging means is connected to the branching means, and the weak base solution is mixed with the solution to be introduced into the mass spectrometer. In claim 3,
The liquid chromatograph mass spectrometer is characterized in that the branching means is a hexagonal valve, has a sample loop that temporarily holds the solution therein, and switches the flow path at a predetermined cycle. In claim 3,
The liquid chromatograph mass spectrometer is characterized in that the fraction collector operates in accordance with a signal detected by the mass spectrometer. In claim 3,
A liquid chromatograph mass spectrometer comprising a UV detector between the branching means and the fraction collector. In claim 1,
A mass spectrometer is an ionization method using an electrospray ionization method, a sonic spray ionization method, and an ion spray. In claim 1,
The mass spectrometer is a time-of-flight type, ion trap type, or quadrupole type mass spectrometer. Eluent pump that delivers an eluent containing ion-pairing agent and salt, sample injector that injects the sample to be measured into the flow path, multiple columns that separate the introduced solution for each component, and elution from the column A liquid chromatograph mass spectrometer having a mass spectrometer for ionizing and detecting a solution with an atmospheric pressure ion source,
Mixing means for stirring and mixing the solution provided in each of the downstream flow paths of the columns;
Merging means for merging a weak base solution between the column and the mixing means;
A switching valve through which a solution from each mixing means is guided and connected to the mass spectrometer;
A transfer liquid pump for supplying transfer liquid to the switching valve;
A liquid chromatograph mass spectrometer that alternately introduces the output liquid from each column guided to the switching valve by the transfer liquid while switching the switching valve. In claim 10,
A liquid chromatograph mass spectrometer characterized in that a weak base solution is used as the transfer liquid. In claim 10,
With the same number of fraction collectors as the number of columns,
Branching means for branching the flow of the solution eluted from the column at a predetermined rate in each flow path between the column and the mixing means,
The liquid chromatograph mass spectrometer is characterized in that the plurality of branching means are connected to the switching valve and each fraction collector. In claim 12,
The merging means is connected to each of the branching means, and the weak base solution is mixed with the solution introduced into the switching valve. In claim 10,
A liquid chromatograph mass spectrometer having the same number of fraction collectors as the number of columns connected to the switching valve.

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