RU2599330C1 - Method of mass-spectrometric analysis of chemical compounds - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to methods of laser desorption-ionization, can be used for mass-spectrometric analysis and identification of chemical compounds in liquid and gaseous samples. Method for mass-spectrometric analysis of chemical compounds includes application of molecules of chemical compounds onto the surface of a solid material by adsorption or deposition, laser desorption-ionization by pulse laser radiation affecting the material and detecting ions of the chemical compounds in a mass analyzer. Herewith the laser desorption-ionization is carried out in presence of a reagent gas selected from a group of compounds of general formula C_nH_2nR, where n=1÷4, R=OH, CN, I.

EFFECT: technical result is higher accuracy and reliability of identification of chemical compounds, in particular, due to extraction in the mass spectrum of a peak characterizing the molecular weight of the compounds while reducing concentration of the analyzed compounds.

12 cl, 6 dwg

Description

The invention relates to methods of laser desorption-ionization and can be used for mass spectrometric analysis and identification of chemical compounds in liquid and gaseous samples. In particular, the inventive method can be used in analytical practice to determine the composition of chemical compounds, in medicine and pharmacology to identify biologically active components of drugs, in safety systems for determining the presence of narcotic and explosive substances in real time.

Laser desorption-ionization methods are widely used in mass spectrometric analysis. Laser desorption-ionization technology is based on applying the analyzed sample to the surface of the substrate and exposure to pulsed laser radiation on the substrate. As a result of this effect, ions of chemical compounds are formed in the gas phase, which are detected in the mass analyzer.

A known method of matrix-activated laser desorption-ionization (MALDI). In the known method, in addition to the analyzed sample, a special matrix is also deposited on the surface of the substrate, which absorbs laser radiation well. The matrix performs two main functions - the translation of analyte molecules into the gas phase and their ionization. The action of laser radiation leads to pulsed evaporation (ablation) of the matrix and the analyte being crystallized with it. A region of relatively high pressure is formed above the surface of the sample, in which ionization reactions proceed [US Patent No. 5118937, class. G01N 1/00; G01N 27/447; G01N 27/62; G01N 27/64; G01N 30/72; H01J 49/10; H01J 49/16, publ. 1992].

The method is applicable for the determination of high molecular weight compounds with a molecular weight of up to several hundred thousand amu Pulse laser radiation with a wavelength of 300 nm and higher is used, in particular laser radiation of an N ₂ laser, a CO ₂ laser, an Er laser, an Er-YAG laser. It is proposed to use benzoic, nicotinic, carboxylic acids, their derivatives, glycerin, and a number of other chemical compounds as matrices that absorb laser radiation.

The known method of ionization of chemical compounds has the following disadvantages:

- the method is of little use for determining chemical and biochemical compounds with small molecular masses due to ionization processes and strong fragmentation of the matrix molecules that create intense chemical noise in the mass range up to about 600 amu;

- the method allows to determine only the qualitative composition of the studied samples;

- The organic matrices used in MALDI are not universal. Each of them can be used to analyze a very limited number of classes of compounds, and, in particular, to select a matrix, it is necessary to know the approximate composition of the studied samples;

- the method is not applicable for the analysis of substances in a gaseous or liquid state of aggregation;

- the joint crystallization of the matrix and the analyzed biochemical compounds, which is a necessary condition of the known method, can change the physicochemical properties of the compounds (which in their natural state exist in the form of solutions), and in some cases the known method can give incorrect information about their composition and structure.

The closest technical solution to the proposed one is the method of mass spectrometric determination of chemical compounds, including the deposition of molecules of chemical compounds on the surface of a solid-state material by adsorption or deposition, laser desorption-ionization by exposing the material to pulsed laser radiation and detecting ions of chemical compounds in a mass analyzer [ U.S. Patent No. 6288390, cl. H01J 49/04; H01J 49/16, publ. 2001].

In the method, the analyzed solution is applied to the surface of a substrate made of porous silicon. After evaporation of the solvent, the substrate with the analyte deposited on its surface is placed in the ion source of the mass spectrometer and exposed to pulsed UV laser radiation, which is completely absorbed in the porous silicon layer. As a result of laser desorption-ionization in the gas phase, protonated analyte molecules are formed, which are detected in a mass spectrometer.

The known method of ionization of chemical compounds is characterized by the absence of intense chemical noise and therefore allows the detection of chemical and biochemical compounds with small molecular weights. In addition, the active layer made of porous silicon is more versatile than matrices in the MALDI method.

The disadvantage of the prototype is the lack of the possibility of unambiguous identification of the defined chemical compounds (analytes) under the conditions of interference (overlapping) peaks of analyte ions with peaks of substrate ions or related components and / or due to fragmentation of analyte ions.

The objective of the invention is to increase the reliability and reliability of identification of chemical compounds, in particular, due to the selection in the mass spectrum of a peak characterizing the molecular weight of the compound, while reducing the concentration of the analyzed compounds.

This problem is solved by the fact that in the method of mass spectrometric determination of chemical compounds, including applying molecules of chemical compounds to the surface of a solid-state material by adsorption or deposition, laser desorption-ionization by exposing the material to pulsed laser radiation and detecting ions of chemical compounds in a mass analyzer, laser desorption-ionization is carried out in the presence of a reagent gas selected from the group of compounds of the general formula C _n H _2n R, where n = 1 ÷ 4, R = OH, CN, I.

It is preferable to use a reagent gas stream directed to the surface of the solid-state material.

It is advisable to use a single compound or a mixture of compounds, such as alcohols, for example methanol, and nitriles, for example acetonitrile, as well as isotopically enriched compounds, for example, deuterated methanol or deuterated acetonitrile, as a reagent gas.

It is advisable to use porous, nanocrystalline or amorphous semiconductor materials such as silicon, germanium, zinc oxide, titanium oxide, cadmium selenide as a semiconductor material.

It is preferable to use pulsed laser radiation with a wavelength selected from the condition of its absorption in the solid-state material, with an intensity lower than the melting threshold of the solid-state material.

It is advisable to carry out laser desorption-ionization in the presence of a reactant gas in a vacuum, at pressures generated by the reactant gas not exceeding 10 ^-4 mm Hg. or at atmospheric pressure, and use a mass spectrometer or ion mobility spectrometer as a mass analyzer.

The invention consists in the following. In the process of laser desorption-ionization, in particular desorption-ionization on the surface of porous silicon, protonated molecules of chemical compounds are formed, previously precipitated from solution or adsorbed from the gas phase. Protonation is the main ionization channel in these methods and is due to the laser-induced proton transfer process. In this case, only chemical compounds with high values of proton affinity are ionized. The action of radiation on the substrate also leads to its heating and desorption of analyte molecules and ions. In known methods, the resulting protonated molecules of chemical compounds (analytes) are immediately directed and detected in a mass analyzer. In the inventive method, the ions formed in the process of laser desorption-ionization, then react with the molecules of the reactant gas. In the interaction of protonated molecules (MN) ⁺ with compounds having the formula C _n H _{2n + 1} R (where n = 1 ÷ 4, R = OH, CN, SH, I), this kind of reaction can be written in the form:

(MH) ⁺ + C _n H _{2n + 1} R → (MC _n H _{2n + 1} ) ⁺ + HR.

For example, when exposed to methanol molecules, methylation reactions occur:

(MH) ⁺ + CH ₃ OH → (SCH ₃ ) ⁺ + H ₂ O.

As a result, ions characterizing the analyte are introduced into the mass analyzer, but having a different mass than the protonated molecules and having other chemical properties. This approach has several advantages compared to known methods, as illustrated by the examples below.

The probability of the formation of ions (MC _n H _{2n + 1} ) ⁺ during the interaction of protonated analyte molecules with reagent gas molecules C _n H _{2n + 1} R decreases with increasing n, so it is advisable to use compounds with a low n value as a reagent gas, and namely, n≤4.

If necessary, to ensure high reproducibility of the analysis results, laser desorption-ionization of chemical compounds is usually carried out in vacuum. In this case, the solid-state material is in vacuum under the influence of stripping laser radiation with an intensity lower than the threshold for its destruction, which ensures the invariance of the physicochemical composition of the surface of the material, complete desorption of all chemical compounds on its surface and, therefore, high reproducibility of the analysis results. The addition of a reactant gas leads to an increase in pressure, i.e. to worsening vacuum. Moreover, on the one hand, an increase in the pressure created by the reactant gas increases the probability of the formation of ions (MC _n H _{2n + 1} ) ⁺ due to an increase in the number of collisions of protonated analyte molecules with the reactant gas molecules, but, on the other hand, a relatively high pressure degrades the quality of the mass spectra. In addition, the possibility of increasing the pressure of the reactant gas is limited by the performance of the vacuum pumping systems. As a consequence of these factors, under vacuum conditions, it is advisable to carry out laser desorption-ionization in the presence of a reagent gas that creates a pressure not exceeding 10 ^-4 mm Hg. At values greater than 10 ^-4 mm Hg, the noise level begins to increase, the mass resolution and the quality of the mass spectra deteriorate, while the magnitude of the ion peak itself (MC _n H _{2n + 1} ) ^{+ is} already weakly dependent on pressure . For quantitative analysis, it is necessary that the flow of the reactant gas remains constant during the analysis and in the sequential analysis of various samples.

For a qualitative analysis, laser desorption-ionization of chemical compounds can be carried out at atmospheric pressure. In this case, there are no restrictions on the magnitude of the pressure generated by the regent gas, with the exception of those due to the design of the sample inlet into the mass analyzer.

In FIG. 1 presents a schematic diagram of an installation for implementing the proposed method.

In FIG. 2 - mass spectrum of diphenhydramine obtained by the prototype.

In FIG. 3 - mass spectrum of diphenhydramine obtained by the present method.

In FIG. 4 - mass spectrum of atmospheric air obtained by the present method (blank experiment).

In FIG. 5 is a mass spectrum of atmospheric air containing pyrrolidine vapors obtained by the present method.

In FIG. 6 is a mass spectrum of valine and leucine obtained by the present method, under conditions of laser desorption-ionization at atmospheric pressure.

The method is implemented as follows.

In FIG. 1 presents a General installation diagram for implementing the proposed method. A substrate 1 of a solid-state material with detectable compounds deposited on its surface is placed in the ion source of the mass spectrometer using a gateway system for quickly replacing the ion emitter 2. In the analysis of volatile compounds, the latter can be deposited on the surface of the substrate by adsorption from the gas phase as in an ion source, so and without it. The radiation of a pulsed laser 3 is focused on the surface of the substrate. A high voltage is applied to the accelerating grid of the ion source, which is used to accelerate the ions. The metered reagent gas is introduced using system 4, consisting of a capillary located in the vacuum chamber of the mass spectrometer, a gas flow regulator, and a source of reagent gas vapors located outside the device.

Example 1

In a comparative example, the determination of diphenhydramine in a solution without using a reagent gas and using it is presented.

On the surface of a porous silicon substrate, 5 μl of a solution of diphenhydramine (the active ingredient of the drug "diphenhydramine) in water with a concentration of 1 μM is applied. The applied solution is dried in an atmosphere of air, after which a porous silicon substrate is placed in the ion source of a linear time-of-flight mass analyzer. The residual pressure of the gas (air) in the ion source is 5 × 10 ^-7 mm Hg. The surface of the substrate with the deposited substance is exposed to radiation from a nitrogen laser (laser wavelength 337 nm) with a laser pulse repetition rate of 1 Hz, and a laser pulse duration of 3 ns. The intensity of the laser radiation is chosen equal to 30 mJ / cm ² . The registration of ions is carried out in a positive ionization mode. The result is a mass spectrum in the region of protonated molecules shown in FIG. 2. It is seen that in the mass spectrum there is a peak corresponding to protonated diphenhydramine molecules with a mass to charge ratio of m / z 256 (the ion structure is shown in Fig. 2), however, the amplitude (or area) of the peak is small and approximately corresponds to the detection threshold. At lower analyte concentrations in the analyzed sample, this peak is not recorded.

Then, a stream of methanol vapor is directed onto the surface of the substrate, which creates a pressure of 2 × 10 ⁻⁵ mm Hg, the radiation of a nitrogen laser is focused on another part of the surface of the substrate coated with analyte, and a new mass spectrum is recorded. The resulting mass spectrum is shown in FIG. 3. It can be seen that a new peak with m / z 270 is recorded in the mass spectrum, which corresponds to methylated diphenhydramine molecules (the ion structure is shown in Fig. 3). The peak amplitude exceeds the background signal by more than 20 times. The molecular weight of the compound is determined from the measured value of m / z as a value equal to (m / z-15).

Example 2

The example presents the determination of pyrrolidine in air.

The substrate of amorphous silicon obtained by the standard method of radio frequency sputtering is kept in the laboratory air for 30 seconds and placed in the ion source of a linear time-of-flight mass spectrometer. A laser beam of a Nd: YAG laser with a wavelength of 355 nm (the third harmonic of the main radiation) is focused on the surface of amorphous silicon using a lens. The laser pulse repetition rate is 50 Hz, and the laser pulse duration is 0.5 ns. The intensity of the laser radiation is chosen equal to 25 mJ / cm ² . For registration of ions using a positive ionization mode. Then, a vapor stream of acetonitrile and ethanol is directed onto the surface of the substrate. The pressure created by the pairs of acetonitrile and ethanol is 1 × 10 ^-4 mm Hg. The result is a blank mass spectrum as depicted in FIG. four.

Then, the amorphous silicon substrate is placed in the same atmosphere, but additionally containing pyrrolidine vapors with a concentration of 10 ppb. The substrate is kept in the atmosphere for 30 seconds and placed in the ion source of a time-of-flight mass spectrometer. Then pairs of acetonitrile and ethanol are fed into the ion source. The pressure created by the pairs of acetonitrile and ethanol is 1 × 10 ^-4 mm Hg. For laser desorption of ionization, the same laser parameters are used as in the blank experiment. As a result, the mass spectrum shown in FIG. 5.

From the data of FIG. Figure 4 shows that in a blank experiment, a number of peaks are present in the mass spectrum that can interfere with the determination and identification of analytes. In particular, a peak with m / z 72 is recorded in the mass spectrum, which interferes with the peak of protonated pyrrolidine (Fig. 5) and, therefore, distorts the metrological results of the analysis. However, when using a vapor flow of acetonitrile and ethanol on a substrate, two peaks corresponding to methylated (M + CH ₃ ) ⁺ and ethylated (M + C ₂ H ₅ ) ⁺ pyrrolidine ions are recorded in the mass spectrum along with the protonated pyrrolidine vapor. These peaks are in the area of the mass spectrum, free from interference.

Example 3

The example presents the simultaneous determination of valine and leucine in solution.

On the surface of a nanocrystalline zinc oxide ZnO substrate, 2 μl of an aqueous solution containing amino acids valine (V) and leucine (L) with concentrations of 10 μM is applied. After evaporation of the solvent, the substrate is placed at a distance of 2 cm from the intake mass analyzer device. The surface of the substrate with the deposited substance is exposed to the radiation of a nitrogen laser (laser wavelength 337 nm) with a laser pulse repetition rate of 5 Hz, and a laser pulse duration of 3 ns. The intensity of the laser radiation is chosen equal to 40 mJ / cm ² . Laser desorption-ionization is carried out at atmospheric pressure. On the surface of the substrate is directed vapor stream deuterated iodomethane CD ₃ I. flux density CD ₃ I (of the substance into per unit surface area per unit time) is 0.1 mg / (cm ² × s). A quadrupole-time-of-flight mass spectrometer is used as a mass analyzer, in which ions are transported to the vacuum chamber from the ionization region through a high-frequency gas-filled transport quadrupole, and ions are detected using a time-of-flight reflectron. As a result, the mass spectrum depicted in FIG. 6.

Valine and leucine are homologues; therefore, the peak of methylated valine interferes with the peak of protonated leucine. In order to avoid interference while simultaneously determining homologues, it is advisable to use isotopically enriched compounds as a reagent gas. From the data of FIG. Figure 6 shows that when using deuterated methyl iodide, peaks of analytes (M + CD ₃ ) ⁺ are recorded in the mass spectrum that do not interfere with the peaks of protonated molecules, which avoids errors in the determination and identification of the compounds being determined.

The presence in the mass spectrum of a peak characterizing the molecular weight of a chemical compound is an important factor in the determination and identification of the compound, since it allows us to establish its gross formula. One of the interfering factors in determining a number of compounds is the strong fragmentation of the resulting analyte ions. For example, protonated diphenhydramine formed under conditions of laser desorption-ionization on the surface of porous silicon (according to the prototype method) is an unstable compound that fragmentes with the formation of low molecular weight, and therefore uninformative, ions. The degree of fragmentation (the ratio of the number of fragment ions to the total number of ions of a compound) is approximately 95%. In turn, the methylated diphenhydramine ion formed by the claimed method (Example 1) is a stable compound and does not fragment under conditions of laser desorption-ionization. Such a significant difference in resistance is due to the following: diphenhydramine, like a number of other drugs, contains a terminal amino group. Compounds of chemical classes such as diphenhydramine, when attached to a proton, stabilize their structure by partial proton transfer to the electronegative oxygen atom. Such charge transfer leads to a weakening of the oxygen – carbon bond and a significant decrease in the energy of its dissociation, which, in turn, leads to intense fragmentation under conditions of laser desorption – ionization. In turn, when substituting hydrogen in the amino group for methyl (i.e., as a result of methylation), proton transfer is not possible. Therefore, methylated diphenhydramine, as well as similar compounds, does not decompose under conditions of laser desorption-ionization. Thus, the use of the proposed method can improve the reliability of identification of diphenhydramine in the analysis of solutions with concentrations of this compound less than 1 μm.

Another interfering factor is the interference of analyte ions with peaks of the substrate ions and / or associated sample components having the same m / z value as the analyte ions. Using the proposed method allows you to shift the peak characterizing the molecular weight of the analyte to a region free of interference, thereby ensuring the reliability of the analysis. It should be noted that the reagent gases used in the inventive method have a low proton affinity energy and therefore do not ionize under conditions of laser desorption-ionization and do not interfere with analysis. Studies have also shown that under constant analysis conditions, the yield of ions of the form (MC _n H _{2n + 1} ) ⁺ remains constant, which allows quantitative measurements.

Claims (12)

1. The method of mass spectrometric determination of chemical compounds, including the deposition of molecules of chemical compounds on the surface of a solid-state material by adsorption or deposition, laser desorption-ionization by exposing the material to pulsed laser radiation and detecting ions of chemical compounds in a mass analyzer, characterized in that the laser desorption-ionization is carried out in the presence of a reagent gas selected from the group of compounds of the general formula C _n H _2n R, where n = 1 ÷ 4, R = OH, CN, I. 2. The method according to p. 1, characterized in that they use a stream of reagent gas directed to the surface of the solid-state material. 3. The method according to p. 1, characterized in that as the reagent gas use one compound or a mixture of compounds. 4. The method according to p. 1, characterized in that as the reagent gas use alcohols, for example methanol, and nitriles, for example acetonitrile. 5. The method according to PP. 1 and 4, characterized in that the isotopically enriched compounds, for example deuterated methanol or deuterated acetonitrile, are used as a reagent gas. 6. The method according to p. 1, characterized in that as a solid-state material using semiconductor materials, such as silicon, germanium, zinc oxide, titanium oxide, cadmium selenide. 7. The method according to PP. 1 and 6, characterized in that as the semiconductor material using porous, nanocrystalline or amorphous semiconductor materials. 8. The method according to p. 1, characterized in that they use pulsed laser radiation with a wavelength selected from the conditions for its absorption in a solid-state material. 9. The method according to p. 1, characterized in that they use pulsed laser radiation with an intensity lower than the melting threshold of solid state material. 10. The method according to p. 1, characterized in that the laser desorption-ionization in the presence of a reactant gas is carried out in vacuum, at pressures generated by the reactant gas not exceeding 10 ^-4 mm Hg 11. The method according to p. 1, characterized in that the laser desorption-ionization in the presence of a reagent gas is carried out at atmospheric pressure. 12. The method according to p. 1, characterized in that as a mass analyzer using a mass spectrometer or ion mobility spectrometer.

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