CN103018317A - Novel non-standard-dependence quantitative analysis method based on study on homologous/similar compound structure-mass-spectrum response relationship - Google Patents

Abstract

The invention belongs to the field of analysis, relates to a quantitative analysis method for homologous/similar compounds, and particularly relates to a quantitative compound analysis method for a complicated matrix sample which does not contain a standard substance. The method comprises the following steps of: (1) selecting a series of homologous/similar compounds, and carrying out mass-spectrum quantitative methodological study and textual research on the homologous/similar compounds; (2) carrying out zero-intercept linear fitting according to established standard curves of all the compounds; (3) carrying out structural optimization on the compounds by using molecular simulation software, and calculating related molecular descriptors; (4) carrying out establishment and verification on the relationship between the structures and mass-spectrum responses of the compounds by using the molecular simulation software; (5) carrying out qualitative analysis on the complicated matrix sample by employing related mass spectrometry technologies; (6) calculating the slope coefficients of linear fit standard curves of the verified series compounds according to a structure-mass-spectrum response relationship equation established previously; and (7) obtaining the concentrations of the compounds in the complicated matrix sample by using the fit standard curves of the series compounds. Thus, the non-standard-dependence quantitative analysis is realized.

Description

A new standard-independent quantitative analysis method based on the study of the structure-mass response relationship of homologous/similar compounds

technical field

The technical invention belongs to the field of analysis, and relates to the quantitative analysis of homologous/similar compounds, especially the quantitative analysis method of compounds that do not have standard substances in complex matrix samples.

Background technique

In complex matrix research fields such as herbal medicine, environment, food and pharmaceuticals, multi-component simultaneous quantification can reveal the content of key substances or potential active compounds, which is very important for overall quality control and effect/toxicity assessment. However, compared with qualitative analysis, quantitative analysis faces more problems and challenges. For example, there are many non-target analysis strategies that can predict the structure of unknown compounds without relying on standards, but for quantitative analysis, the lack of standards has become a very big shackle because of the different responses of each compound; in addition, For quantitative analysis, systematic methodological research is also more stringent than qualitative analysis, and a series of items such as linear range, accuracy, precision, stability, extraction recovery rate, and medium effect need to be investigated.

In complex systems, homologous/similar compounds are often the main form of organic compounds, and these compounds may exert biological effects in a holistic or synergistic form. In previous studies, one or some existing standard compounds with high content in the system were often used as evaluation indicators to represent homologous compounds for quantitative analysis, quality evaluation or further pharmacokinetic research, but this idea There are obvious limitations, since some compounds or metabolites may still produce biological effects or toxic reactions at low concentrations, and thus cannot be simply ignored.

In recent years, the development of analytical instruments, especially high-resolution mass spectrometry, has facilitated the analysis of compounds in complex systems. At the same time, some semi-quantitative or relative quantitative methodologies have also been proposed to overcome the difficulty of standard products in the quantification of complex systems. get the bottleneck. For example, someone proposes to use the standard curve of a compound in the same series/series of compounds as a reference to quantify the remaining compounds directly or through molecular weight calibration; someone first uses a series of compounds to study the relative relationship of their responses in a specific instrument, and then later In quantification, only one compound can be used to achieve absolute quantification of these compounds; the concept of "extract standard curve" has also been proposed to evaluate the pharmacokinetic process of homologous/serial compounds in vivo. However, these methods are limited to the estimation of relative content after all, and cannot accurately extrapolate the actual concentration of compounds without standard substances, and cannot really get rid of the limitation of standard substances.

In addition to the progress in the field of analysis, some computer-aided cheminformatics strategies have also been applied to research in the fields of pharmaceuticals and the environment, such as the discovery of drug leads, the study of the structure-activity relationship of a series of compounds in complex systems, etc. Among these computer-aided methods, the most widely used ones are molecular docking/pharmacophore analysis and quantitative structure-activity relationship/structure retention time relationship research. However, these techniques have no application examples in the quantitative research of complex system series of compounds. The reason may be that there are many factors affecting the quantification of compounds, and no suitable quantitative indicators have been found to characterize the instrument response of compounds.

The technical invention aims to solve the problem of lack of standard products in the quantitative process of homologous/serial compounds. By selecting an appropriate quantitative index that can characterize the mass spectrometry response of the compound, the relationship between the intrinsic physical and chemical structure properties of the compound and the mass spectrometry response is established. Through the establishment and verification of the model, it can explain the key physical and chemical parameters that affect the response of homologous/serial compounds when mass spectrometry is used to quantify them, and predict the response of homologous/serial compounds in mass spectrometry, so as to realize complex matrices that do not depend on standards Quantification of homologous/serial compounds in

Contents of the invention

The purpose of this technical invention is to provide a new quantitative analysis method based on the study of homologous/similar compound structure-mass spectrum response relationship that does not depend on standard substances, and is completed by the following steps:

1. Select a series of homologous/similar compounds for mass spectrometric quantitative methodological research and research:

Select as many and easy-to-obtain standards as possible to establish a mass spectrometry quantitative method, determine its quantitative range, and conduct systematic methodological research on accuracy, precision, stability, medium effect, extraction recovery and other items.

2. According to the standard curve of each compound established within a certain concentration range, perform linear fitting with zero intercept:

Considering that the standard curve of the compound contains two items of slope and intercept, the slope reflects the response strength of the compound, and the existence of the intercept is mainly to correct the accuracy of low-concentration samples. Ideally, the standard curve of the compound should cross the zero point, so A linear fit with zero intercept was performed, and the slope of the fit was used as an index to characterize the compound response.

3. Use molecular simulation software to optimize the structure of the compound, and calculate the molecular descriptors related to its physical and chemical properties:

Use molecular simulation software to process the structure of the standard compound, calculate its optimal conformation, and calculate the physical and chemical parameter descriptors of the compound under the optimal conformation conditions, such as molecular weight, polarity, energy, electrical properties, etc.

4. Use molecular simulation software to establish and verify the relationship between the compound structure (including various physical and chemical parameter descriptors, parameters related to the mass spectrometry response in the analysis conditions) and the mass spectrometry response (the slope of the linear fitting standard curve):

For the existing compounds, select a part as the training set, establish the relationship between the compound structure and the mass spectrometry response, which can be expressed in the form of multiple linear regression equation (MLR), artificial neural network (BNN), etc., and use the rest of the compounds as the prediction set , to verify the equation. It is necessary to ensure that the established equation has a good degree of fitting and interpretation, the included independent variables have certain practical significance, and the overfitting of the equation must be prevented.

5. Apply relevant mass spectrometry technology to qualitatively analyze complex matrix samples, find and determine the main ion peaks from the total ion current spectrum, and identify the structure of the series of compounds screened through mass spectrometry information such as accurate molecular mass and fragmentation pathway:

In the invention of this technology, the TripleTOF 5600 (LC/MS-Q-TOF) system produced by the Canadian ABI company was used to search and identify the organic acid compounds in the traditional Chinese medicine compound injection Mailuoning injection. Firstly, screening is carried out according to the characteristic fragment ions and fragmentation pathways of such compounds, and then structural identification is carried out according to the molecular formula and fragment information of the screened compounds.

6. According to the previously established structure-mass spectrum response relationship equation, calculate the slope of the linear fitting standard curve of the identified series of compounds, that is, predict the standard curve of the series of compounds:

The molecular simulation software was used to optimize the structure of the identified compounds, and the molecular descriptors were calculated, which were substituted into the previously established structure-mass spectrum response relationship equation, and the fitting slope value was calculated to obtain the fitting standard curve.

7. Use the fitting standard curve of this series of compounds to perform quantitative analysis on these compounds, and obtain their concentrations in complex matrix samples, that is, to achieve quantitative analysis independent of standards:

Substitute the ratio of the peak area of the compound to the internal standard into the fitted standard curve to obtain its estimated concentration. If the estimated concentration is within the quantitative range established by the previous methodology, the estimation has high accuracy.

The technical invention has the following advantages:

1. For a series of homologous/similar compounds, the key physical and chemical parameters that affect the mass spectrometry response can be obtained, which can be used to predict the level of the mass spectrometry response of the compound;

2. Through the established model of the relationship between compound structure and mass spectrometry response, other homologous/similar compounds in complex systems that do not actually exist in standard products can be more accurately quantified;

3. The present invention is practicable and has universal applicability, and is not limited to the analysis of a certain type of homologous/similar compounds in complex matrix samples.

Description of drawings

Figure 1: Total ion chromatograms of organic acid standard (a), blank excipient preparation (b), Mailuoning injection (c) in negative ion mode.

Figure 2; The comparison between the predicted value and the actual value in the constructed compound structure-mass spectrum response relationship.

Detailed ways

Embodiment 1; Quantitative Analysis of Organic Acids Components in Mailuoning Injection

1. Select a series of homologous/similar compounds for mass spectrometric quantitative methodological research and research;

Select benzoic acid (A1), 4-methylbenzoic acid (A2), salicylic acid (A3), 3-hydroxybenzoic acid (A4), 4-hydroxybenzoic acid (A5), cinnamic acid (A6), 2- Hydroxy-3-methylbenzoic acid (A7), protocatechuic acid (A8), 2,4-dihydroxybenzoic acid (A9), 4-methylcinnamic acid (A10), coumaric acid (A11), vanilla acid (A12), isovanillic acid (A13), gallic acid (A14), 4-methoxycinnamic acid (A15), 2-methoxycinnamic acid (A16), caffeic acid (A17), 3,4-di Methoxybenzoic acid (A18), ferulic acid (A19), syringic acid (A20), 3,4,5-trimethoxycinnamic acid (A21), chlorogenic acid (A22), 1,5-dioxo Caffeoylquinic acid (A23), 3,4-dioxocaffeoylquinic acid (A24), and 3,5-dioxocaffeoylquinic acid (A25) were used for quantitative methodological establishment of 25 organic acid standards And investigation, internal standard selection 2-hydroxyhippuric acid. The TripleTOF 5600 (LC/MS-Q-TOF) system produced by Canada ABI Company was used to establish a mass spectrometry quantitative method for a series of organic acids, and the methodological research was carried out on linear range, accuracy, precision, stability and other items. The total ion chromatograms of organic acid standards under specific analysis conditions are shown in Figure 1(a).

2. According to the standard curve of each compound established within a certain concentration range, perform linear fitting with zero intercept;

In the quantitative range of 50ng/ml-10ug/ml concentration, carry out linear fitting with zero intercept, and examine its quantitative accuracy. The results are shown in Table 1.

3. Use molecular simulation software to optimize the structure of the compound, and calculate the molecular descriptors related to its physical and chemical properties;

Use Sybyl-X 1.2 (Tripos Inc., St.Louis, USA) software to process the structure of the standard compound, and calculate its optimal conformation; use Discovery Studio 2.5 (Accelrys Inc., San Diego, USA) software to calculate the optimal conformation Physicochemical parameter descriptors of compounds under conditions, such as molecular weight, polarity, energy, electrical properties, etc.

4. Use molecular simulation software to establish and verify the relationship between the compound structure (including various physical and chemical parameter descriptors, parameters related to the mass spectrometry response in the analysis conditions) and the mass spectrometry response (the slope of the linear fitting standard curve);

In order to avoid the negative value of the fitting slope, the natural logarithmic value (LN(SlopeRate)) of the slope of the linear fitting standard curve of the compound was selected as a quantitative parameter to characterize its mass spectral response. For the existing 25 compounds, 20 of them were selected as the training set, and the relationship between the structure and the mass spectrometry response was established in the form of a multiple regression equation (MLR), and the remaining 5 compounds were used as the prediction set. The MLR equation is verified. The fitting results are shown in Table 2(a), Table 2(b) and Figure 2.

5. Apply relevant mass spectrometry technology to qualitatively analyze complex matrix samples, find and determine the main ion peaks from the total ion current spectrum, and identify the structure of the series of compounds screened through mass spectrometry information such as accurate molecular mass and fragmentation pathway;

Using the TripleTOF 5600 (LC/MS-Q-TOF) system produced by the Canadian ABI company, the organic acid compounds in the traditional Chinese medicine compound injection Mailuoning injection were searched and identified. The injection diluted 100 times with methanol was directly Sample injection, the method is the same as before. The total ion current chromatograms of the blank excipient preparation and Mailuoning injection are shown in Figure 1(b) and 1(c). The drug-derived compounds in the injection were selected by comparison with the spectra of the blank excipient preparations, screened according to the characteristic fragment ions and fragmentation pathways of the compounds, and the organic acid compounds were selected, and then the compounds were selected according to the Molecular formula and fragment information for further structural identification. A total of 34 organic acid compounds were identified from Mailuoning injection, and the results are shown in Table 3.

6. According to the previously established structure-mass spectrum response relationship equation, calculate the slope of the linear fitting standard curve of the identified series of compounds, that is, predict the standard curve of the series of compounds;

The structure of the organic acid compounds identified in Mailuoning injection was optimized with Sybyl-X 1.2 software, and the molecular descriptors were calculated with Discovery Studio 2.5 software, and substituted into the previously established structure-mass spectrum response relationship equation, Calculate the fitting slope value of each compound to obtain its fitting standard curve.

7. Use the fitting standard curve of this series of compounds to conduct quantitative analysis on these compounds, and obtain their concentration in complex matrix samples, that is, to achieve quantitative analysis independent of standards;

Substitute the ratio of the peak area of each organic acid compound to the peak area of the internal standard into the fitting standard curve to obtain its estimated concentration. For the compounds with standard substances, the absolute quantification was carried out at the same time, and the absolute quantification was compared with the results obtained by this method. The results are shown in Table 4.

It can be seen from Table 4 that among the 34 compounds identified in Mailuoning Injection, 16 compounds have standards. By comparing the results of absolute quantification and fitting quantification of these 16 compounds, it is found that the fitting result has a high prediction accuracy, and can be more accurate for organic acid compounds with concentrations in the range of 50ng/ml-10ug/ml Quantitative. The invention is practicable, has the characteristics of universal applicability, and can also be used for quantitative analysis of other types of homologous/similar compounds in complex matrix samples.

Table 2(a) Regression and statistical investigation of the MLR equation

Table 2(b) Parameters and coefficients in the MLR equation

Table 3 Qualitative information of the compounds identified in Mailuoning Injection

Quantitative results and comparison of compounds identified in Table 4 Mailuoning Injection

Claims (6)

1. A new quantitative analysis method independent of standard substance based on the research of homologous/similar compound structure-mass spectrum response relationship, characterized in that it consists of the following steps: a: Select a series of homologous/similar compounds for mass spectrometric quantitative methodological research and research; b: According to the standard curve of each compound established within a certain concentration range, perform linear fitting with zero intercept; c: Use molecular simulation software to optimize the structure of the compound, and calculate the molecular descriptors related to its various physical and chemical properties; d: Use molecular simulation software to establish and verify the relationship between the compound structure (including various physical and chemical parameter descriptors, parameters related to the mass spectrometry response in the analysis conditions) and the mass spectrometry response (the slope of the linear fitting standard curve); e: Apply relevant mass spectrometry technology to qualitative analysis of complex matrix samples, find and determine the main ion peaks from the total ion current spectrum, and identify the structure of the series of compounds screened through mass spectrometry information such as accurate molecular mass and fragmentation pathway; f: According to the previously established structure-mass spectrum response relationship equation, calculate the slope of the linear fitting standard curve of the identified series of compounds, that is, predict the standard curve of the series of compounds; g: Use the fitting standard curve of this series of compounds to perform quantitative analysis on these compounds, and obtain their concentrations in complex matrix samples, that is, to achieve quantitative analysis independent of standards. 2. A series of homologous/similar compounds as claimed in claim 1a, characterized in that they have similar or identical structural nuclei or physical and chemical properties, such as alkaloid compounds, ginsenoside compounds, polycyclic aromatic hydrocarbon compounds, insecticidal Agent compounds, etc., the technical invention will be specifically described by taking organic acid compounds as an example. 3. The molecular simulation software as claimed in claims 1c and 1d, characterized in that it provides a platform for molecular processing, such as realizing functions such as energy minimization of compounds and quantitative structure-activity relationship analysis. 4. The mass spectrum response as claimed in claim 1d is characterized in that the intensity of the response of the characterization compound in the quantitative analysis instrument includes the slope of the standard curve and the slope after mathematical formula calculation processing or fitting processing, the technical invention The natural logarithm of the slope of the linear fitting standard curve is used as an example for specific illustration. 5. The related mass spectrometry technology as claimed in claim 1e is characterized in that it can separate and analyze the structure of the sample, including various liquid-mass spectrometry, gas-mass spectrometry and other technologies. The invention of this technology uses the Canadian ABI The TripleTOF5600 (LC/MS-Q-TOF) system produced by the company. 6. Complex matrix samples as claimed in claim 1e, including and not limited to plant extracts, Chinese herbal medicine preparations, biological samples, environmental samples, food samples, etc., the invention of the present technology consists of honeysuckle, Scrophulariaceae, Achyranthes bidentata, Dendrobium The traditional Chinese medicine compound injection Mailuoning Injection prepared from four kinds of medicinal materials is taken as an example for specific illustration.

Images