JP2667576B2 - Mass spectrometry of organic polar compounds - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to mass spectrometry of organic polar compounds by chemical ionization, and more particularly to mass spectrometry of organic polar compounds such as sugars and alcohols.

[Prior Art] Carbohydrates such as sugars are widely present in cells of plants and animals and play important roles in biochemistry, and these analyzes are extremely important in biochemistry.

However, the analyzer of the compound generally has many problems because it is very troublesome and the measurement sensitivity is insufficient. Therefore, a simple and highly sensitive analysis method has been required. GC (gas chromatography), GC / MS (mass spectrometry), H
FAB and LC / MS methods using PLC (high pressure liquid chromatography) and mass spectrometry have been developed.

Since sugars and alcohols are thermally unstable and have poor volatility as they are, analysis by GC, GC / MS or the like cannot be performed without derivatization treatment that increases volatility and increases thermal stability. On the other hand, HPLC does not necessarily require a derivatization treatment. However, sugars and carbohydrates generally do not have absorption in the ultraviolet or visible range. The use of the near-ultraviolet (） 200 nm) region would allow the detection of these compounds, but in practice it would be impossible to measure due to interference from the buffer used for HPLC. Therefore, also in HPLC, these compounds are subjected to pre- or post-column labeling (derivatization) in order to give absorption in the ultraviolet region.

Honda and colleagues report that high-sensitivity analysis of saccharides can be performed by post-column labeling of boric acid complexes using anion exchange chromatography [Analytical
Biochemistry: Anal. Biochem. Vol.281, pp340 (198
3)].

[Problems to be Solved by the Invention] However, HPLC merely gives a retention time and cannot obtain information on the structure or molecular weight.

In mass spectrometry, saccharides are exclusively subjected to soft ionization, for example, SIMS (Secondary Ion Mass Spectrometry), FD (Field Desorption; electrolytic desorption ionization) or LD (Lesar Desorption; laser desorption ionization). Has been analyzed. All of these soft ionizations provided enough analytical information. However, since saccharides present in a living body are generally present in a mixture, strict separation and purification of the sample is required prior to the above-described analysis method.

HPLC / MS, or LC / MS, has emerged as a powerful weapon in the analysis of biological materials. This is because they can achieve the separation and ionization of nonvolatile and thermally unstable substances without derivatization. Some saccharides were measured by thermospray LC / MS. Many fragment ions and pseudo-molecular ions (M +
H) ⁺ and (M + NH ₄ ) ⁺ appear. But,
This thermospray LC / MS method also failed to measure trisaccharides or higher polysaccharides.

In these compounds, the glycosyl bond is attacked by the reagent ion, so that the fragment ion becomes dominant. For this reason, pseudo-molecular ions almost disappear. In addition, the mass spectrum becomes so complicated that it is difficult to analyze due to the generation of by-products such as thermal acetylation instead of simple chemical ionization. Sakairi and Kamihara succeeded in giving molecular ions of many saccharides by the atmospheric pressure spray method, which is one of the ion vaporization methods [Analytical Chemistry: Anal. Chem. Vol. 61 pp. 1159 (1989)]. Alkali metal adducts could be given simply by spraying at 500 ° C or higher. However, even in this atmospheric pressure spray method,
Eluents based on organic solvents did not always give good results.

Atmospheric pressure chemical ionization (APCI), which performs chemical ionization at atmospheric pressure, did not give information on molecular weight for trisaccharides or higher, as in the thermospray method.

An object of the present invention is to provide a mass spectrometric method for an organic polar compound, in which a compound such as a saccharide existing as a mixture is separated without modification and mass spectrometric analysis is performed.

[Means for Solving the Problems] The gist of the present invention for solving the problems is as follows.

In a mass spectrometric method in which a polar organic solvent and an organic polar compound sample are introduced into an ion source of a measuring device under atmospheric pressure, the sample molecule is ionized by atmospheric pressure chemical ionization, and a mass spectrum is measured. The compound sample is an organic polar compound having a hydroxyl group or a carboxyl group, and the polar organic solvent is selected from polar organic solvents that form negative ions, and a halogen-containing compound that generates a halogen ion is added to the organic solvent and the organic polar compound sample. After mixing in an ion source, by ionizing by an atmospheric pressure chemical ionization method, a halogen negative ion is generated by an ion molecule reaction between the organic solvent ion and the halogen-containing compound, and the halogen negative ion and the organic polar compound Ionization of the sample molecule by an addition reaction with the sample molecule for mass spectrometry. In mass spectrometry of the organic polar compound to symptoms. Chemical ionization includes positive and negative ion chemical ionization. The following are known as main ionization processes in negative ion chemical ionization.

(1) Proton transfer reaction (2) Negative ion addition reaction (3) Nucleophilic reaction (4) Charge transfer reaction Especially, proton transfer reaction and negative ion addition reaction are practical TSP method (thermospray method) and APCI method ( This is an important reaction in atmospheric pressure chemical ionization. F ^- ion or
Cl ^- ion has been used as an important reagent ion to give a negative ion CI mass spectrum. In this case, a mixed gas of a halogenated hydrocarbon and methane is used as a reagent gas. When a halogen negative ion (for example, Cl ⁻ ) collides with an organic polar compound having a hydroxyl group, a carboxyl group, or the like in the gas phase, an intermediate having an excessive kinetic energy is once produced. This intermediate subsequently loses excess kinetic energy due to collisions with other molecules and becomes a stable adduct. A compound having a halogen negative ion and a hydroxyl group or a carboxyl group generates a stable addition ion by hydrogen bonding. Under atmospheric pressure to several tens Pa, collisions between molecules frequently occur, and a large number of negatively added ions are stably generated. Although the collision of halogen negative ions and neutral molecules is a possibility that ionization by exchange of protons (proton transfer reaction) occurs, halogen negative ion conjugate acid (e.g. Cl ^- HCl for negative ions) is neutral for the strong acid molecular Cl ^- proton transfer to does not occur.

The halogen negative ion can be obtained by, for example, mixing hydrochloric acid or the like into the LC mobile phase. However, strong acids such as hydrochloric acid generally cannot be used because they corrode LC pumps and other equipment.

Further, since the pH of the solution greatly affects the separation of LC, hydrochloric acid cannot be freely mixed due to the ionization. A salt such as ammonium chloride can also be mixed, but in this case, the polarity of the mobile phase is increased, which affects retention of a highly polar substance. Therefore, it is preferable that the solvent does not dissociate halogen ions and the like in the state of being in the LC pump, column, detector, etc., and produces halogen ions in the chemical ionization ion source of LC / MS to form additional ions.

Examples of such a halogen-containing compound include methylene chloride, chloroform, carbon tetrachloride, trichloroethylene, and the like. Among them, chloroform is most preferable because it easily produces chlorine ions by chemical ionization.

[Operation] The operation of the present invention will be described with reference to the drawings.

Figure 2 shows the negative ion-atmospheric pressure chemical ionization of methanol.
3 shows a mass spectrum (N.A., NI-APCI).

Each peak appearing in the mass spectrum can be interpreted as follows.

m / z = 31,63, ... _{[CH 3 O · (CH 3 OH} ) n ] ^{- n = 0,1 m / z =} 32,50, ... [O ₂ · (H ₂ On] ^- n = 0, 1,2 m / z = 32,64, ... _{[O 2 · (CH 3 OH)} n ] ^- n = 0, 1, 2 where, O ₂ and H ₂ O is incorporated in the spray in the air Probably due to oxygen and water.

FIG. 3 is an NI-APCI mass spectrum obtained by introducing a 95: 5 mixed solution of methanol: chloroform into an APCI ion source.

Two ion species groups are observed in this mass spectrum.

m / z = 35,67,99 ... [Cl · (CH ₃ OH) n] ^{- n = 0,1,2 ... m / z} = 53,85,117 ... _{[Cl · H 2 O · (CH} 3 OH) n ] ^- was demonstrated to generate ions ^- n = 0, 1, 2 ... methanol and chloroform Cl by introducing into the APCI ion source.

Halogenated hydrocarbons (particularly chloroform) is a strong base (eg OH ^- etc. ^- or CH ₃ O) has been known to produce halide anions react with. Here, CH ₃ generated by corona discharge from methanol O ^- has decreased and demonstrated to produce ions ^- base such as negative ions attacking Cl chloroform molecule. This reaction is a well-known reaction in a solution.

The generated halogen negative ion collides with a compound having a hydroxyl group or the like, and generates a halogen addition ion. Generally, the added ions after collision stabilization are stable, and no further fragmentation occurs. Therefore, molecular weight information can be easily obtained. The Cl as halogen ion ^- another advantage of using the resulting. It is a stable isotope of Cl element ( ³⁵ Cl: ³² Cl
= 3: 1), the Cl ^- adduct exhibits a Cl element isotope ratio (3: 1), which simplifies identification.

In FIG. 4, methanol: chloroform (95: 5) was used as the LC mobile phase and dissolved in a solution having the same composition as the mobile phase.
3 shows a mass spectrum obtained by introducing 1 μg of sucrose into an APCI ion source.

Quasi-molecular ion m / z377 (M + Cl] ^-. Is summer and the reference peak other weak peak [M + Cl + CH ₃ OH] ^- m / z 409 _{[M + Cl + H 2 O +} CH 3 OH ] ^- m / z 427 [M-H] ^- m / z341 is observed.

Some low intensity fragment ions are observed at m / z 150 to 320. As a result, a chlorine ion adduct ion of an organic polar compound having a hydroxyl group was produced by introducing a mixed solution of chloroform: methanol and corona discharge APCI.

FIG. 5 shows the results of studying which solvent is superior as a source of Cl ^- ions.

Dissolve Sucrose in water: methanol (50:50), 1μg
Of sucrose was introduced while changing three mixed solvents.
The three mixed solvents are carbon tetrachloride, methylene chloride and chloroform, each a 5% methanol solution. Of these three systems, the chloroform-methanol system gave the highest sensitivity. Methylene chloride: 8 times that of methanol system and 1000 times that of carbon tetrachloride / methanol system. This is in good agreement with the tendency for reactivity to bases in solution. From this, it can be seen that chloroform is the most suitable among these three chlorine-containing solvents.

FIG. 6A shows the results of a study on how much the concentration of chloroform can be used to analyze saccharides.

Three types of chloroform-acetonitrile mixed solutions having different concentrations were prepared, and sucrose solutions having sequentially changed concentrations were injected. In FIG. 6A, the vertical axis is Sucrose's [M + Cl] ⁻
The ion current value and the horizontal axis represent the amount of sucrose introduced. As shown in Fig. 6A, chloroform concentration is 0.5%,
It can be seen that the response can be up to about 0 μg or more. If it is about 1% with respect to methanol, acetonitrile, water or the like, chloroform is sufficiently dissolved.

FIG. 6B shows the response of [M + Cl] ⁻ ion when the kind of sugar was changed using 0.5% chloroform / acetonitrile. Every individual sugar has a different response
A linear relationship is obtained from 1 ng to 100 ng.

Embodiment An embodiment of the present invention will be described below with reference to FIG.

Here, an example of an atmospheric pressure chemical ionization mass spectrometer (LC / APCI-MS) directly connected to a liquid chromatograph (LC) is shown.

The methanol and chloroform stored in the solvent storage bottles 1 and 2 are sent out by pumps 3 and 4, mixed by a mixer 5, and sent to a column 7 via an injector 6. The sample solution is introduced from the injector 6, separated by the column 7, and sent to the atmospheric pressure chemical ionization (APCI) interface by the plastic tube 15. The APCI interface includes an atomizer 9, a desolvation chamber 10, an APCI ion source 16, a differential exhaust system 17, and the like. Atomizer 9 and solvent removal chamber are cartridge heaters
12 allows independent heating control. In the atomizer, a metal capillary 8 having an inner diameter of 0.1 mmφ is welded to a heat block.

The solution that has flowed out of the LC reaches the atomizer and gushes from the tip of the capillary at once. Atomizer temperature is 20
It is set between 0 ° C and 300 ° C. The generated mist enters a desolvation chamber heated to 400 ° C., which has a hole with an inner diameter of 5 mm, and vaporization of the mist is promoted. The vaporized solvent molecules are ionized by corona discharge generated by the high voltage (3 kV to 5 kV) applied to the needle electrode 11.

CH ₃ O ⁻ ions and the like generated from methanol attack the chloroform molecule CHCl ₃ and generate Cl ⁻ ions as a result. The Cl ⁻ ion further collides with a molecule M such as a sugar to form an additional ion [M + Cl] ⁻ . The generated additional ions enter the differential pumping system through the first pores 13, and neutral solvent molecules and the like are removed by a vacuum pump. The solvent that has not participated in the reaction in the APCI ion source unit 16 is discharged out of the interface from the outlet. The adduct ions further enter the mass spectrometer via the second pore 14 and provide a mass spectrum.

In this embodiment, two kinds of liquids (methanol and chloroform) are used, but two or more kinds may be used. Also,
Chloroform may be mixed in advance with a solvent such as methanol. That is, the halogen-containing compound (solvent) and the polar solvent may be mixed by any means.

FIG. 7 is a configuration diagram of a post column for mixing chloroform before introduction into the APCI interface after separation by LC.

Methanol is pumped out by pump 3 and the
The mixture reaches the mixer 5 via the column 7. Chloroform is sent out by the pump 4, mixed with the methanol solution in the mixer 5, and sent to the APCI interface. In this case, a system in which chloroform does not flow through the column can be realized.

[Effects of the Invention] According to the present invention, it is possible to prepare LC conditions in which halogen ions do not exist in a solution state, corrosion of a pump, a column and the like is prevented, and LC separation is not impaired. Further, since a halogen ion-added ion can be easily generated by the ion source, highly reliable analysis can be performed.

[Brief description of the drawings]

FIG. 1 is a block diagram of the present invention, FIG. 2 is a negative ion atmospheric pressure chemical ionization (NI-APCI) mass spectrum of methanol, and FIG. 3 is a NI-AP of a chloroform: methanol (5:95) solution.
CI mass spectrum, Fig. 4 shows NI-APCI mass spectrum of chloroform: methanol (5:95) solution containing 1 µg of Sucrose, and Fig. 5 shows 5% carbon tetrachloride, methylene chloride and chloroform-methanol as solvents. When used
Sucrose [M + Cl] ^- mass spectrum shows the response of ions, the FIG. 6 A 0.05%, 0.5%, 5 % chloroform - Sucrose for acetonitrile [M + Cl] ^- responding,
FIG. 6 is a curve diagram showing the response of [M + Cl] ⁻ of a sugar to a 0.5% chloroform-acetonitrile solution, and FIG. 7 is a structural diagram showing an example of a post column. 1 ... solvent storage bottle, 2 ... solvent storage bottle, 3 ... pump, 4 ... pump, 5 ... mixer, 6 ... injector, 7 ... column, 8 ... capillary, 9 ... atomizer , 10 ... desolvation chamber, 11 ... needle electrode, 12 ... cartridge heater, 13 ... first pore, 14 ... second pore, 15
... Plastic tube, 16 ... Atmospheric pressure chemical ion source, 17 ... Differential exhaust system.

Claims (4)

(57) [Claims] 1. A mass spectrometry method in which a polar organic solvent and a sample of an organic polar compound are introduced into an ion source of a measuring device under atmospheric pressure, and the sample molecule is ionized by atmospheric pressure chemical ionization to measure a mass spectrum. The organic polar compound sample is an organic polar compound having a hydroxyl group or a carboxyl group, and the polar organic solvent is selected from polar organic solvents that form negative ions, and the halogen-containing compound that generates a halogen ion is combined with the organic solvent and the organic solvent. After mixing the polar compound sample in the ion source, by ionizing by an atmospheric pressure chemical ionization method, a halogen negative ion is generated by an ion molecule reaction between the organic solvent ion and the halogen-containing compound, and the halogen negative ion The sample molecules are ionized by an addition reaction of Mass spectrometry of the organic polar compounds, characterized by. 2. The method for mass spectrometry of an organic polar compound according to claim 1, wherein corona discharge is used for ionization in said ion source. 3. A polar organic solvent introduced into the ion source,
3. The mass spectrometric method for an organic polar compound according to claim 1, wherein the organic polar compound sample and the halogen-containing compound are supplied via a liquid chromatograph. 4. The method for mass spectrometry of an organic polar compound according to claim 1, wherein the halogen-containing compound producing the halogen ion is chloroform.