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WO2003038425A1 - Spectrographe de masse a chromatographie en phase liquide - Google Patents

Spectrographe de masse a chromatographie en phase liquide Download PDF

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Publication number
WO2003038425A1
WO2003038425A1 PCT/JP2002/011285 JP0211285W WO03038425A1 WO 2003038425 A1 WO2003038425 A1 WO 2003038425A1 JP 0211285 W JP0211285 W JP 0211285W WO 03038425 A1 WO03038425 A1 WO 03038425A1
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WO
WIPO (PCT)
Prior art keywords
mass spectrometer
solution
liquid
sample
column
Prior art date
Application number
PCT/JP2002/011285
Other languages
English (en)
Japanese (ja)
Inventor
Izumi Ogata
Kisaburo Deguchi
Masako Ishikawa
Takefumi Yokokura
Tadao Mimura
Original Assignee
Hitachi High-Technologies Corporation
Hitachi Science Systems, Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hitachi High-Technologies Corporation, Hitachi Science Systems, Ltd. filed Critical Hitachi High-Technologies Corporation
Priority to JP2003540643A priority Critical patent/JPWO2003038425A1/ja
Publication of WO2003038425A1 publication Critical patent/WO2003038425A1/fr

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Classifications

    • 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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph

Definitions

  • 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.
  • LCZMS liquid chromatograph mass spectrometer
  • MALDI-TOF-MS MALDI-TOF-MS
  • MALDI-TOF-MS 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 ⁇ 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.
  • 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).
  • Q-MS quadrupole mass spectrometer
  • IT-MS ion trap mass spectrometer
  • 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.
  • 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.
  • the MS / 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.
  • 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
  • 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.
  • 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 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.
  • 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.
  • 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 ⁇ mol ZL.
  • 30 ⁇ L of the oligonucleic acid sample was injected from the sample injector 6.
  • the mass number (m) of the 20 base sample is 6096
  • that of the 30 base sample is 9200
  • that of the 40 base sample is 123.60.
  • 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.
  • FIG. 3 shows the mass spectrum at the retention time of each base sample peak detected in FIGS. 2 (a) and (b).
  • 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.
  • 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.
  • FIG. 5 shows a second 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.
  • 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.
  • the mass number of the oligonucleic acid and the mass number of the multiply charged ions to be collected are input in advance.
  • the controller 10 sends a signal to the fraction collector 12 through a signal line.
  • the fraction collector 12 separates the desired oligo nucleic acid sample into a container such as a test tube.
  • 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.
  • 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.
  • the flow path of the hexagonal valve 13 should be switched, for example, every 1 second. .
  • the controller 10 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.
  • 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.
  • 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.
  • 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.
  • the weak base solution 3 methanol or the like can be used.
  • 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.
  • 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.
  • 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 ⁇ F-MS Becomes possible.
  • the efficiency of the analysis using TOF-MS can be greatly increased.
  • 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.
  • 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.

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

L'appareil concerne un appareil destiné l'analyse par spectrographie de masse / chromatographie en phase liquide, utilisé pour analyser les acides oligonucléiques. Un spectrographe de masse à chromatographie en phase liquide comprend une pompe pour alimenter un éluant contenant un agent de couplage d'ions et un sel, un injecteur d'échantillons destiné à injecter un échantillon à analyser dans un canal, une colonne pour fractionner la solution injectée en fonction des composants et un spectrographe de masse destiné à ioniser la solution éluée à partir de la colonne par une source atmosphérique et la détecter; il est caractérisé en plus en ce qu'il possède un système de recombinaison par l'ajout d'une solution faiblement basique entre la colonne et le spectrographe de masse. Grâce à ce système, on peut analyser un acide oligonucléique avec un degré élevé d'efficacité et de sensibilité.
PCT/JP2002/011285 2001-10-31 2002-10-30 Spectrographe de masse a chromatographie en phase liquide WO2003038425A1 (fr)

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JP2003540643A JPWO2003038425A1 (ja) 2001-10-31 2002-10-30 液体クロマトグラフ質量分析装置

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JP2001-333664 2001-10-31

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006292542A (ja) * 2005-04-11 2006-10-26 Hitachi High-Technologies Corp 液体クロマトグラフ質量分析装置
WO2025013534A1 (fr) * 2023-07-07 2025-01-16 株式会社島津製作所 Procédé d'analyse, procédé d'évaluation et programme

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6122252A (ja) * 1984-07-10 1986-01-30 Shimadzu Corp パラコ−ト分析装置
JPH06102251A (ja) * 1992-09-18 1994-04-15 Hitachi Ltd 液体クロマトグラフ直結質量分析計
JPH06194357A (ja) * 1992-12-24 1994-07-15 Kao Corp 液体クロマトグラフ分析法および分析装置
JPH095300A (ja) * 1995-06-22 1997-01-10 Shimadzu Corp 液体クロマトグラフ質量分析装置
JPH11304783A (ja) * 1998-04-20 1999-11-05 Hitachi Ltd 液体クロマトグラフ，質量分析装置及び液体クロマトグラフ質量分析装置

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6122252A (ja) * 1984-07-10 1986-01-30 Shimadzu Corp パラコ−ト分析装置
JPH06102251A (ja) * 1992-09-18 1994-04-15 Hitachi Ltd 液体クロマトグラフ直結質量分析計
JPH06194357A (ja) * 1992-12-24 1994-07-15 Kao Corp 液体クロマトグラフ分析法および分析装置
JPH095300A (ja) * 1995-06-22 1997-01-10 Shimadzu Corp 液体クロマトグラフ質量分析装置
JPH11304783A (ja) * 1998-04-20 1999-11-05 Hitachi Ltd 液体クロマトグラフ，質量分析装置及び液体クロマトグラフ質量分析装置

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006292542A (ja) * 2005-04-11 2006-10-26 Hitachi High-Technologies Corp 液体クロマトグラフ質量分析装置
WO2025013534A1 (fr) * 2023-07-07 2025-01-16 株式会社島津製作所 Procédé d'analyse, procédé d'évaluation et programme

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