RU2469314C2 - Method of identifying organic compounds based on high-performance liquid chromatography and mass spectrometry - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method of identifying organic compounds based on high-performance liquid chromatography and mass spectrometry involves detection of separated components in positive and negative ion detection mode after chromatographic separation of substances and ionisation by electrostatic spraying at atmospheric pressure and selecting the detection mode based on maximum information content. The first polarity mass spectrum is then measured and the probable molecular weight is determined by identifying adducts based on possible isotopic composition of atoms in the molecule. Further, the mass spectra of daughter ions are measured. Identification coefficients are calculated based on the first polarity mass spectrum and the daughter mass spectra. Chromatographic parameters and results of mass spectrometer measurements are then compared with data from a library of mass spectra of known substances.

EFFECT: high reliability of identification, broader functional capabilities of the mass spectrometric technique.

6 tbl, 2 dwg

Description

The invention relates to the study or analysis of materials by determining their chemical or physical properties, specifically by dividing into components (components) using adsorption and their mass spectrometric studies.

The invention can be used to identify organic compounds using the method of high performance liquid chromatography and mass spectrometric detection.

Modern mass spectrometry is an effective method for studying the composition, structure, thermodynamic and kinetic properties of substances in any state of aggregation. The high sensitivity, the large amount of information received, the almost universal applicability led to an extremely rapid growth in the breadth and diversity of applications of mass spectrometric methods.

Chromatography-mass spectrometric methods are widely used for qualitative and quantitative analysis of mixtures of various organic compounds. Chromatography-mass spectrometry is based on gas column chromatography (GC-MS) or liquid chromatography (HPLC-MS) and mass spectrometry. Using chromatographic methods, the mixture is divided into separate components, and using the mass spectral method, quantitative analysis, identification and determination of the structure of substances are carried out.

Despite the commonality of the methods underlying the chromatography-mass spectrometry, and solved in the analysis of problems, there are fundamental differences in GC-MS and HPLC-MS.

In the GC-MS method, the stream flowing from the capillary column of a gas-liquid chromatograph (eluate) consists of a carrier gas (usually helium) and vapors of separated substances. The eluate can be directly introduced into the ion source of the mass spectrometer, since the gas flow rate in the capillary columns is low (1 ... 2 cm ³ / min) and the vacuum pump manages to pump out the incoming gas, preventing the creation of increased pressure in the source. Given the high resolution of capillary column chromatography, it will become clear why it, in combination with mass spectrometry, has been one of the most effective methods for studying small amounts of mixtures of organic compounds for half a century.

The eluate leaving the column of a high performance liquid chromatograph is a solution of separable substances in the eluent. Such a solution cannot be introduced into an ion source without a sharp increase in pressure. The problem of combining a liquid chromatograph with a mass spectrometer has long hindered the widespread use of HPLC-MS. The solution to this problem, in particular, was the use of a different ionization method compared to the ionization method that is traditionally used in the GC-MS method.

We are talking about the most common method of ionization by electron impact (EI) in the GC-MS, which consists in exposing the analyte to an electron beam (usually at a pressure of 1 · 10 ^-6 mm Hg). The source of electrons is a heated cathode filament; electrons are accelerated by an electric field with a potential difference of 0 to 100 V.

An alternative method is the method of "soft" ionization (chemical ionization at atmospheric pressure and electrostatic atomization at atmospheric pressure). With “soft” ionization, the mass spectra contain fewer fragmentation ions than with electron impact ionization. As a result, “soft” ionization makes it possible to detect molecular ions (or protonated molecular ions) that are absent in mass spectra with electron impact ionization (D. Brown, A. Floyd, M. Sainsbury. Spectroscopy of organic substances. Translation from English A.A. Kiryushkina [Text] / M .: Mir, 1992 - 300 p.).

Extensive reference material on the mass spectra of organic compounds has been accumulated in hard ionization mass spectrometry (electron impact) mass spectrometry for more than half a century of history of the method, which allows reliable identification of almost any unknown compound by the set of peaks and the ratio of their intensities.

To a large extent this was facilitated by the increasingly active use of computer technology over the past 20 ... 25 years for the registration and processing of mass spectrometric data. Due to its versatility and reproducibility, it was precisely the electron ionization spectra that became the basis of computer libraries of mass spectra. The most efficient currently is the NIST / EPA / NIH Mass Spectral Library, the next version of which NIST08 was released in 2008. It includes 220,460 spectra for 192,108 compounds. In addition, it contains 224,038 published and estimated gas chromatographic retention indices for 21,847 electron-ionization library compounds, as well as 5308 MS / MS spectra for 5308 precursor ions (A. Lebedev, K. S. Lebedev, B. F. Myasoedov , Rybalchenko IV, Sigeykin GI, Suvorkin VN Mass spectrometric identification of highly toxic alkyl fluorophosphonates. Mass spectrometry. Vol. 3, No. 4 (2006). Internet resource: www.sisweb.com/nist .).

Various variations of the identification method based on GC-MS are known, for example, a method involving chromatographic and mass spectrometric analysis and generation of at least one elution profile in three-dimensional measurement, the coordinates of which are the retention time of the compound (chromatographic parameter), m / z ratio (mass spectrometric parameter) and signal intensity. The method also provides for the automatic assignment of elution profiles (Method and system for chromatographic and mass spectrometric analysis. G01N 30/86. Patent No. WO 2005015199 A1, priority dated July 6, 2004. Inventions of the world (Review journal) 2006, No. 6) .

A method for the identification of organic compounds based on GC-MS electronic ionization is the closest of the analogues of the present invention.

Despite the undoubted advantages of the method of identifying organic compounds by gas chromatography in combination with mass spectrometric detection with electron impact ionization, the solution of a number of analytical problems requires the use of high performance liquid chromatography.

The ability to analyze high molecular weight organic compounds, the absence of temperature effects on the analyzed sample, mass spectrometric detection with "soft" ionization, for example electrostatic spraying at atmospheric pressure, create the conditions for the predominant use of this method for both quantitative and qualitative analysis. Moreover, in the HPLC-MS method, only two parameters are used for identification: retention time and mass of the pseudomolecular ion.

The detection of molecular ions (or protonated pseudomolecular ions) during “soft” ionization, which are absent in electron impact ionization mass spectra, on the one hand, facilitates the identification of the compound, especially when the alleged chemical formula of the substance is known. On the other hand, in the absence of any data on the unknown connection of these two parameters, it is not enough for reliable and reliable identification.

The objective of the present invention is to improve the method of identification of organic compounds based on the method of high performance liquid chromatography and mass spectrometry.

The solution of this problem involves a technical result, which consists in increasing the reliability and objectivity of identification, expanding the functionality of the mass spectrometric method. Based on the results obtained, in the future it seems possible to implement a software product for the identification of organic compounds.

The problem is solved in that in the method for identifying organic compounds based on the method of high performance liquid chromatography and mass spectrometry according to the proposed solution, after chromatographic separation of substances and ionization by electrostatic spraying at atmospheric pressure, the separated components are detected in the registration mode of positive and negative ions and the mode selected detection for maximum information content; measuring the mass spectrum of the first polarity and determining the probable molar mass by recognizing adducts taking into account the possible isotopic composition of the atoms included in the molecule; measure the mass spectra of daughter ions; based on the mass spectrum of the first polarity and daughter spectra, the identification coefficients are calculated; The chromatographic parameters and the results of mass spectrometric measurements are compared with the data of the library of mass spectra of known substances.

The objects of this study are pesticides belonging to various classes of organic substances. Table 1 shows the objects of research and their characteristics.

Considering the fact that most of the compounds under study are polar substances with several possible ionizable chemical centers, we can assume the possibility of their detection under conditions of “soft” ionization due to electrostatic sputtering and, at the same time, expect a simpler picture in terms of the number of fragmented ions compared to the conditions hard ionization by electron impact.

At the first stage of the identification of organic compounds by high performance liquid chromatography with mass spectrometric detection, a comparative analysis of a solution of one of the studied compounds in methanol (a solution of prometrine at a concentration of 1 mg / ml) was carried out by HPLC-MS and GC-MS. In figures 1, 2 shows the chromatograms and mass spectra of promethrin obtained by HPLC-MS and GC-MS.

The analysis of the given spectral data allows us to draw the following conclusions:

the spectrogram of promethrin obtained by the HPLC-MS method contains only three characteristic peaks, while the spectrogram obtained by the GC-MS method contains a whole spectrum of peaks of different intensities;

identification of the test compound using the HPLC-MS method is facilitated by the fact that of the three ions, the most intense ion of the first polarity is the pseudo-molecular ion [M + H] ⁺ with a mass per unit greater molecular weight of the compound;

on the other hand, in hard ionization mass spectrometry (electron impact), the use of a well-known software device allows reliable identification of an unknown compound by the set of peaks and the ratio of their intensities (in this case, the use of the NIST08 mass spectra library confirmed the promethrin structure with a high degree of probability).

At the next stage of work, all the research objects presented in Table 1 were subjected to HPLC analysis with mass-selective detection in the mode of registration of positive or negative ions. It was found that compounds detected in the registration mode of positive ions are not detected in the registration mode of negative ions. Some compounds are not detected in either mode; it is probably necessary to use the chemical ionization mode for their detection.

To increase the reliability of identification of organic compounds using "soft" ionization (HPLC-MS method), studies were conducted to study the ratios of the resulting pseudomolecular ions.

An analysis of the organic compounds studied by HPLC with mass selective detection by positive ions made it possible to isolate in the mass spectrum, in addition to the main pseudomolecular ion with a mass of [M + H] ^+, at the same retention time, daughter pseudomolecular ions with a mass of [M + 2] ⁺ and [ M + 3] ⁺ , corresponding to the molecular weights of the compound [M + 1] and [M + 2]. Their appearance is due to the isotopic composition of carbon and hydrogen atoms, primarily those entering the molecule of the compound.

When working with negative ions, the main detectable ion is [M-H] ^- . The formation of a daughter negative ion [M + e] ^{- is} possible. Moreover, in accordance with Table 1, the existence of pseudomolecular ions with several masses is possible due to the presence in nature of several isotopes of atoms that make up the molecule of the organic compound.

Tables 2-4 show the results of measurements of the mass spectra of methanol solutions with a concentration in the range from 1.0 · 10 ^-2 to 1.0 mg / ml of prometrin and dipolatan (detection in the registration mode of positive ions) and bromacil detected by negative ions .

An analysis of the data in Tables 2 and 4 during detection in the registration mode of positive ions and comparison of the areas of the ions of the first polarity and daughter ions belonging to the same compound allowed us to conclude that the ratio of these values is an individual characteristic of the compound under consideration.

So, for promethrin (table 2), the retention time of which under the conditions of analysis by HPLC with mass-selective detection in the registration mode of positive ions is 4.6 minutes, the following coefficients are characteristic: K ₁ = 7.6; K ₂ = 19.0.

where K ₁ and K ₂ are relative coefficients,

S _max - the maximum area of the ion, conv. units (in the detection mode of positive ions, this is the area of the ion of the first polarity, the pseudomolecular ion [M + H] ⁺ ),

S ₂ and S ₃ are the areas of daughter ions with a mass of [M + 2] ⁺ and [M + 3] ^+, respectively, conv. units

The found coefficients (identification coefficients) of promethrin retain their value in the range of solution concentrations from 1.0 · 10 ^-2 to 1.0 mg / ml with a relative standard deviation not exceeding 6.8% (number of determinations 12).

In the case of dipolatane (Table 4), the picture is complicated by the presence of several possible ions of the first polarity due to the different isotopic composition of the atoms of the molecule containing four chlorine atoms, sulfur and nitrogen atoms. The pseudo-molecular ions of the first polarity and their daughter ions formed by the addition of the adduct (NH ₄ ) ⁺ , the presence of which is possible when using ammonium acetate as a component of the mobile phase, are present in the mass spectrum of dipholatane. In this case, the absence of daughter ions for the ion of the first polarity with a mass of 347 is characteristic.

When working with negative ions, the ratio of the areas of ions determines the identification coefficient (K ^- ) for the corresponding molecular weight:

where (K ^- ) is the identification coefficient for the corresponding molecular mass during detection in the registration mode of negative ions;

S _[MH] ^- and S _{[M + e]} ^- - respectively, the peak area of the ion with mass [M-H] ^- and [M + e] ^- .

For bromacyl (table 3), the values of the identification coefficient for two possible masses, taking into account the isotopic composition of the molecule, are 9.2 and 9.7 and retain their value in a wide concentration range.

Analyzing the results shown in tables 2-4, we can conclude that not only the values of the coefficients, but also the presence or absence of the second coefficient are individual characteristics of the compound, depending on its chemical composition and structure.

In a similar manner, other representatives of pesticides selected as objects of this work were investigated. Of these, in addition to promethrin and dipholatane, forat, karbofos and its admixture, linurol, planavin, and dactal are detected in the mode of recording positive ions. However, for these compounds no formation of negative ions was observed.

For negative ions, it was possible to detect, in addition to bromacil, banvel, 2,4-D, dinoserba, piclorama. Some compounds (from the class of polycyclic halogen derivatives — dildrin; from thiourea derivatives — dodine) are not detected in either mode.

Table 5 shows the results of calculations of the identification coefficients K ₁ and K ₂ for the studied pesticides during detection in the detection mode of positive ions.

Table 5 The identification coefficients of the studied pesticides during detection in the registration mode of positive ions Connection Name The mass of the pseudomolecular ion, [M + H] ⁺ , a. eat. Retention time, min Identification Averages The number of determinations in calculating the average value Maximum standard deviation,% K ₁ K ₂ Forat 261 7.7 10.5 6.2 8 5,6 Karbofos 331 4.8 8.4 10.8 9 3.9 Kalbofos admixture 285 4.8 10.3 9.1 5 6.2 Linurol 249 3.9 10.5 - 5 9.2 251 9.9 6.3 Prometrine 242 4.6 7.6 19.0 8 4.4 Planavin 346 5.8 6.1 15.0 7 2.5 Difolatan 348 4.8 - - 9 9.1 350 * 14.2 2.1 365 ** 7.8 - 367 ** 7.8 - 369 *** 3.8 4.0 372 **** 9,4 2.5 Dactal 331 6.8 Impossibility of detection, low sensitivity Note: * - in accordance with table 1, there may be four masses: 349, 351, 353 and 354 in the presence of one, two, three or four isotopes of chlorine Cl ³⁷ in the molecule;
** - ions [M + NH ₄ ] ⁺ , corresponding to molecular weights 347 and 349, the formation of which is possible when using ammonium acetate as one of the components of the mobile phase;
*** - ion [M + NH ₄ ] ⁺ , corresponding to a molecular weight of 351, the formation of which is possible using ammonium acetate as one of the components of the mobile phase;
**** - ion [M + NH ₄ ] ⁺ , corresponding to a molecular weight of 354 when taking into account the isotopic composition, the formation of which is possible using ammonium acetate as one of the components of the mobile phase.

An analysis of the data in Table 5 shows that forat, karbofos and its impurities, promethrin and planavin are characterized by the presence of one pseudomolecular ion [M + H] ⁺ and its daughter ions. All these compounds do not contain halogen atoms (table 1).

In the case of linurol, as in the case of dipholatane, an ion spectrum is observed. Both of these compounds contain chlorine atoms, for which two isotopes exist in nature, differing in mass by 2 amu, which is consistent with the basic concepts of ionization of halogen-containing compounds (D. Brown, A. Floyd, M Sainsbury, Spectroscopy of Organic Substances, Translated from English by A. A. Kiryushkin [Text] / M.: Mir, 1992. - 300 p.). All this confirms the fact that the picture of "soft" ionization by electrospray at atmospheric pressure is unique for each compound and reflects its individuality.

Table 6 shows the results of calculations of identification coefficients (K ^- ) for the studied pesticides during detection in the mode of registration of negative ions.

Table 6 The values of the identification coefficient (K ^- ) of the studied pesticides during detection in the registration mode of negative ions Connection Name The mass of the pseudomolecular ion, [M-H] ^- , a. eat. Retention time, min The average values of the coefficient (K ^- ) The number of determinations in calculating the average value The maximum relative standard deviation,% Dinoseb 239 1,2 8.3 12 5.7 2,4-D 219 1.4 11,2 9 3.9 221 11.0 9 1.9 223 - - - Banvel D 219 1,1 13,2 6 2.2 221 - - - 223 - - - Bromacil 259 2,3 9.2 7 1.7 261 9.7 7 5,6 Picloram 239 1,1 16,0 5 10.1 241 14.5 5 9.7 243 - - - Note: "-" in the mass spectrum there is no daughter ion [M + e] ^-

Analysis of the detection results of the studied pesticides in the mode of registration of negative ions allows us to draw the following conclusions:

identification coefficient (K ^- ) is an individual characteristic of the compound and retains its value in the concentration range from 1.0 · 10 ^-2 to 1.0 mg / ml;

for those compounds for which the existence of several molecular masses is possible, the closeness of the coefficient values (K ^- ) determined for each of the masses is observed;

as in the case of detection in the registration mode of positive ions, the combination of parameters: mass number, retention time, and identification coefficient (K ^- ) will improve the reliability of identification of the studied compounds.

An example of the method

1. When analyzing by HPLC with mass-selective detection (the analysis conditions are shown in figure 1) a mixture of pesticides of unknown composition was found chromatographic peak with a retention time equal to 1.1 ... 1.3 minutes The measured mass spectrum of the first polarity allows the molecular weight of the compound to be determined. This value is 240 amu

Compounds with a similar molecular weight are dinoseb, picloram and promethrin. Identification only by the chromatographic parameter and the mass of the main pseudomolecular ion allows us to make the assumption that this peak cannot belong to prometrin, since its retention time is 4.6 min (table 2). An additional confirmation of the correctness of the assumption made is the fact that its detection is carried out in the mode of registration of positive ions, while the unknown compound was detected by negative ions, such as dinoseb and piclorams.

After comparing the chromatographic parameters, ions of the first polarity and the detection mode, the next stage of identification is the measurement of daughter mass spectra and determination of identification coefficients. In the case of detection by negative ions, we are talking about the coefficient (K ^- ). According to table 6, dinoseb has only one coefficient equal to 8.3 for a molecular weight of 240. Pikloram is characterized by two coefficient values: for a mass of 240 it is 16.0, and for a mass of 242 it is 14.5 (table 6). Significant differences in the value of the coefficient and in the number of coefficients make it possible to distinguish piclorams from dinoseb with high reliability.

2. A more complex case of recognition of two compounds is the case of 2,4-D and banvel: both of them have the same molecular weight equal to 220; the same retention time under chromatographic conditions by HPLC with mass-selective detection, equal to 1.1 ... 1.4 min; both compounds are detected by negative ions. Therefore, the use of an identification coefficient (K ^- ) is the only way to reliably distinguish between these two compounds: a banvel is characterized by the presence of only one coefficient value for mass 220, equal to 13.2; 2,4-D is characterized by the presence of two close values of the coefficient for the masses (220 and 222) equal to 11.2 and 11.0 (table 6).

3. An HPLC analysis with mass selective detection in the positive-ion registration mode revealed an unknown compound with a first-mass ion mass of 348 and a retention time of 4.3 minutes. By the mass of the ion of the first polarity and the retention time, the closest compound is dipholatane with a retention time of 4.8 minutes (tables 4, 5).

As is known (Wigderhaus M.S. Calculations in gas chromatography [Text] / M.S. Wigderhaus. - M .: Chemistry, 1978. - 248 p.), During chromatographic analysis, a decrease in the concentration of a compound in a solution can lead to an increase in retention time . In this case, it is quite possible to increase the retention time from 4.3 minutes to (4.7 ... 4.8) minutes, which can lead to an error in identification. The use of identification coefficients improves the reliability of identification.

So, in the given example, the unknown compound was characterized by the presence of only one first coefficient, equal to 3.6. Difolatan, on the contrary, is characterized by the absence of the first coefficient for mass equal to 347, but there are coefficients K ₁ and K ₂ for mass 349 equal to 14.2 and 2.1, respectively, and coefficients calculated for masses 347 and 349 and daughter ions formed by the addition of an adduct (table 4).

All this confirms the fact that the picture of "soft" ionization by electrospray at atmospheric pressure is unique for each compound and reflects its individuality. Not only the coefficient values themselves, but also the presence or absence of the second coefficient are individual characteristics of an organic compound, depending on its chemical composition and structure. The use of additional information along with the mass of the studied ion and the retention parameter will increase the reliability and reliability of the identification of organic compounds and expand the functionality of the mass spectrometric method.

Claims (1)

A method for identifying organic compounds based on the method of high performance liquid chromatography and mass spectrometry, characterized in that after chromatographic separation of substances and ionization by electrostatic spraying at atmospheric pressure, the separated components are detected in the registration mode of positive and negative ions and the detection mode is selected for maximum information content; measuring the mass spectrum of the first polarity and determining the probable molecular weight by recognizing adducts taking into account the possible isotopic composition of the atoms included in the molecule; measure the mass spectra of daughter ions; based on the mass spectrum of the first polarity and daughter mass spectra, the identification coefficients are calculated; The chromatographic parameters and the results of mass spectrometric measurements are compared with the data of the library of mass spectra of known substances.

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