JP3341765B1 - Method and apparatus for separating and analyzing anionic compounds - Google Patents

Abstract

Abstract: PROBLEM TO BE SOLVED: To enable stable measurement of an anionic compound. SOLUTION: When separating and analyzing an anionic compound by combining capillary electrophoresis (CE) and mass spectrometry (MS), electroosmosis is performed by using a coating capillary 32 whose inner surface is coated with a cationic. Reverse the flow.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method and an apparatus for separating and analyzing anionic compounds using a capillary electrophoresis / mass spectrometer (CE / MS) combining capillary electrophoresis and mass spectrometry.

[0002]

2. Description of the Related Art Until now, the measurement of anionic compounds has been carried out using a separation analyzer such as ion chromatography (IC), high performance liquid chromatography (HPLC), and capillary electrophoresis (CE). The inventor also has patent 3
No. 038184 proposes a method and an apparatus for analyzing anions, amino acids and saccharides by capillary electrophoresis.

[0003] However, since there are a myriad of anionic compounds (10,000 or more), an extremely large number of analyzers and analytical conditions are required to measure these anionic compounds by an existing measuring method. It was necessary and it was difficult to measure everything in a short time.

In the conventional method, there is almost no selectivity.
The qualitative ability of the anion was poor due to the retention time and the transfer time, so the qualitative ability of the substance was really poor.

On the other hand, in recent years, a method for analyzing ionic substances using a capillary electrophoresis / mass spectrometer (CE / MS) having high sensitivity and high selectivity, which combines capillary electrophoresis and mass spectrometry, has been developed. . The inventor has also disclosed in
-83119 proposes an amino acid analysis method and apparatus using CE / MS.

However, this method using CE / MS is also
This method measures amino acids as cations, and it was impossible to perform simultaneous analysis of anionic compounds including organic acids and sugars.

Normally, in anion measurement by CE / MS, as shown in FIG. 1, a voltage is applied so that the exit (mass detector MS) side of the capillary 10 becomes an anode. In this case, electroosmotic flow (EO) generated when a voltage is applied
F), as indicated by the arrow, flows from the detector to the inlet side of the capillary 10, so that there is a problem that no liquid flows on the detector side and no current flows.

To solve this problem, the electrophoresis buffer introduced into the capillary 10 for separating the sample is
As shown in FIG. 2 (A), a cationic alkylamine such as diethylenetriamine is added, and as shown in FIG. 2 (A), the silanol on the surface of a normally anionically charged capillary (eg, fused silica capillary) 10 is removed. As shown in B), a method of making the EOF in the direction of the detector by making it cationic is used.

[0009] As shown in FIG.
The electrode 12 is attached to the capillary 10 having the tip inserted into 22.
14 and the electrophoresis buffers 22, 26, the high-voltage power supply 1
When a voltage is applied from 6, the alkylamine moves toward the cathode 12 because it is cationic. At this time, in the case of a normal CE as shown in FIG. 3A, the electrophoresis buffer 26 is provided on the anode 14 side (detector side), and the alkylamine is supplied from the outlet side of the capillary 10, so that Stable reversal of the electroosmotic flow becomes possible.

[0010]

However, CE /
When MS is used, as shown in FIG.
Since there is no electrophoresis buffer on the side (detector side), the alkylamine moves to the cathode 12 with time, and the inner surface of the capillary is charged anionic. Therefore, during the analysis, FIG.
As in the case of (A), the flow becomes a normal electroosmotic flow from the outlet side to the inlet side, and there is a problem that a current does not flow during analysis.

Further, in the above method, since the pH of the electrophoresis buffer is 3.0, there is a problem that only anionic compounds that dissociate at pH 3.0 can be measured. FIG. 4 shows pKa and pI of each anionic compound containing an amino acid and a sugar. In the electrophoresis buffer at pH 3.0, anionic compounds having pKa and pI of around 3 can be measured.
Amino acids and sugars having higher pKa and pI could not be measured because they were not ionized.

The present invention has been made to solve the above-mentioned conventional problems, and has as its object to enable stable measurement of an anionic compound.

[0013]

SUMMARY OF THE INVENTION The present invention uses a coating capillary in which the inner surface of a capillary is previously cationically coated when separating and analyzing anionic compounds by combining capillary electrophoresis and mass spectrometry. Thus, the above problem is solved by reversing the electroosmotic flow.

In addition, the inner surface of the capillary is coated with a cation
It is coated with a hydrophilic polymer .

In the above-mentioned capillary electrophoresis, the pH of an electrophoresis buffer such as ammonium acetate, triethylamine, ethanolamine, ammonium carbonate, etc. is adjusted.
High and volatile materials are used .

The present invention also provides a capillary electrophoresis apparatus in which an electroosmotic flow is reversed by using a coating capillary in which the inner surface of a capillary is previously coated with a cation, and the capillary. The object has been achieved by providing a mass spectrometer for analyzing a sample separated by an electrophoresis apparatus.

First, in order to solve the first problem that a current stops flowing during analysis, the present invention employs, for example, An
al. Chem., 70 (1998) 5272, SMIL (S
The electroosmotic flow is reversed using a coating capillary coated with a coating such as uccessive Multiple Ionic-polymer Layer). In these capillaries, since the inner surface of the capillaries is coated with a cationic polymer, the electroosmotic flow is reversed and flows toward the anode (MS detector side). Therefore, even if the alkylamine is not added to the electrophoresis buffer, the current does not stop flowing and the anionic compound can be stably measured by CE / MS.

As the coating capillary, for example, those described in JP-A-10-221305 can be used.

In order to enable simultaneous analysis of anionic compounds, an electrophoresis buffer having a high pH and a high volatility is used. That is, by increasing the pH, most anionic compounds can be dissociated, migrated in the anode direction, and detected by a mass spectrometer. As the volatile migration buffer, for example, ammonium acetate, triethylamine, ethanolamine, ammonium carbonate and the like can be used.

Since the pH of ammonium acetate is 6.8, anionic compounds (most organic acids and amino acids) having a pKa of up to 6.8 can be measured. or,
Since the pH of triethylamine is 12 or more, the use of triethylamine as the electrophoresis buffer makes it possible to simultaneously measure organic acids, amino acids, sugars, and most other anionic compounds.

[0021]

Embodiments of the present invention will be described below in detail with reference to the drawings.

In this embodiment, as shown in FIG. 5, a capillary electrophoresis device (CE) 30 for separating a sample, an electrospray needle 40 as an atomizing device for atomizing the separated sample, an atomizing device, A mass spectrometer (MS) 50 for analyzing an anionic compound from the prepared sample.

The CE 30 has a coating capillary (eg, SMI) whose surface is cationically coated.
An L-capillary) 32, a buffer tank 20 that is introduced into the capillary 32 and stores an electrophoresis buffer 22 for separating a sample, a platinum electrode 12 whose tip is immersed in the electrophoresis buffer 22, A high voltage (for example, −30) is applied to the platinum electrode 12.
kV to +30 kV).

One end of the coating capillary 32 is immersed in the electrophoresis buffer 22, and the other end is connected to the electrospray needle 40.

A sheath liquid 44 stored in a sheath liquid tank 42 is supplied to the electrospray needle 40 at a flow rate suitable for electrospray by a pump 46, and at the same time, fine droplets are generated to ionize the liquid. A facilitating nebulizer gas (eg, nitrogen gas) 48 is provided.

In such an apparatus, when a sample is put into the buffer solution tank 20 and a predetermined high voltage is applied to the platinum electrode 12, the sample and the electrophoresis buffer solution 22 pass through the coating capillary 32 and move through the electrospray needle 40. Move to.

At this time, the anionic compound is separated because the moving speed is different due to the difference in ionic radius and ionicity, and is separated into a band to form the electrospray needle 40.
Electrophoresis. And the electrospray needle 40
And analyzed by MS50.

At this time, since the coating capillary 32 whose inner surface is coated with a cation is used, the electroosmotic flow is reversed and flows toward the anode (MS50). Therefore, even if the alkylamine is not added to the electrophoresis buffer 22, the current does not stop flowing, and the anionic compound can be stably analyzed.

Also, by using a high pH and high volatility material such as ammonium acetate or triethylamine as the electrophoresis buffer 22, it is possible to measure many anionic compounds at once. Become.

[0030]

EXAMPLES Using the above embodiment, organic acids were measured.

As the analysis state of the capillary electrophoresis, a SMIL capillary having an inner diameter of 50 μm, an outer diameter of 350 μm, and a total length of 90 cm was used as the capillary 32. Also, 50 mM ammonium acetate (pH 6.
8) was used. The voltage applied to the platinum electrode 12 by the high voltage power supply 16 is −30 kV, and the temperature of the capillary 32 is 20 ° C.
Was measured. The sample was injected at 50 mbar for 80 seconds using the pressure method.

As an analysis condition of the electrospray ionization mass spectrometer (ESIMS) 40 + 50, MS
The negative voltage is applied to the capillary in the negative ion mode in which anions are selectively introduced into the MS using the negative voltage as the anode. The fragmenter voltage applied to the cone portion 52 was set at 70 V to generate ions of the material. Nitrogen was used as the drying gas 54 used to volatilize the solvent coming from the CE 30, and the temperature of the gas was measured at 300 ° C.

As the sheath liquid 44, an aqueous solution of 10 mM ammonium acetate and 50% methanol was used and fed at a flow rate of 8 μl / min.

FIG. 6 shows an example of measurement of six kinds of organic acids (10 mg / l each) measured under the above conditions. Even though the separation in CE was not complete, the mass chromatogram in MS detected only the organic acid of that mass, so that the organic acid could be measured selectively.

In the above embodiment, the SMIL capillary is used as the coating capillary. However, the type of the coating capillary is not limited to this, and any other type may be used as long as the inner surface is cationically coated. It may be.

The ionization used in the mass spectrometer is not limited to the electrospray method (ESI), but may be an atmospheric pressure chemical ionization method (APCI), a fast atom collision method (FAB), or the like.

Further, the mass spectrometer is not limited to the single-stage quadrupole mass spectrometer shown in FIG.
Other types of mass spectrometers such as time of flight, ion traps, and tandem mass spectrometers (MS / MS, M
S ⁿ ).

[0038]

According to the present invention, even when an alkylamine is not added to the electrophoresis buffer, the current does not stop flowing, and the anionic compound can be measured stably. In particular, it is possible to measure an organic acid at a level of 100 μg / l, which has heretofore been difficult to measure stably by CE / MS.

Further, since it is not necessary to completely separate the sample by CE, the analysis time can be reduced.

Further, by using a high pH and highly volatile electrophoresis buffer, it is possible to measure many anionic compounds at once. In particular, when pH 12 triethylamine is used for the electrophoresis buffer, the CE of all anionic compounds such as organic acids, amino acids, and sugars is reduced.
/ MS can be measured.

[Brief description of the drawings]

FIG. 1 is a cross-sectional view showing the direction of an electroosmotic flow when measuring an anionic compound in a normal capillary.

FIG. 2 is a cross-sectional view showing a state in which an electroosmotic flow is reversed by addition of an alkylamine.

FIG. 3 shows the reason why the conventional CE (A) can stably reverse the electroosmotic flow in the prior art, but the reason why the current stops flowing during the analysis in the case of using CE / MS (B). Schematic diagram shown

FIG. 4 is a table showing pKa and pI of anionic compounds.

FIG. 5 is a schematic diagram illustrating a configuration of a capillary electrophoresis / mass spectrometer according to an embodiment of the present invention.

FIG. 6 is a chromatogram showing a measurement result of an organic acid according to the embodiment.

[Explanation of symbols]

 22: electrophoresis buffer 30: capillary electrophoresis apparatus (CE) 32: coating capillary 40: electrospray needle 50: mass spectrometer (MS)

────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI G01N27 / 62 G01N27 / 26 331G 331Z 331E (56) References JP-A-11-337524 (JP, A) JP-A-8- 54370 (JP, A) JP 10-221305 (JP, A) JP 11-317192 (JP, A) JP 2001-83119 (JP, A) Yoshiho Minoura, Tomoyoshi Soga, Hiroyuki Katayama, Glico Application of capillary electrophoresis / mass spectrometry (CE / MS) for hemoglobin measurement, Chromatography, Japan, 1998, 19/4, 270-271 (58) Fields investigated (Int. Cl. ⁷ , DB name ) G01N 27/447 G01N 27/62 JICST file (JOIS)

Claims (4)

(57) [Claims] 1. When a capillary electrophoresis and mass spectrometry are used in combination to separate and analyze anionic compounds, the electroosmotic flow is reversed by using a coating capillary in which the inner surface of the capillary is previously coated with a cationic coating. A method for separating and analyzing an anionic compound. 2. The method according to claim 1, wherein the inner surface of the capillary is cationic.
The method for separating and analyzing an anionic compound according to claim 1, wherein the method is coated with a polymer . Upon wherein the capillary electrophoresis, as electrophoresis buffer, ammonium acetate, triethylamine, ethanolamine, or the separation method of analyzing anionic compound according to claim 1 which comprises using a carbonic acid ammonium arm . 4. A capillary electrophoresis apparatus in which an electroosmotic flow is reversed by using a coating capillary in which the inner surface of a capillary is previously cationically coated, and a sample separated by the capillary electrophoresis apparatus. And a mass spectrometer for analyzing a compound.