CN112285223A

CN112285223A - Liquid chromatography-mass spectrometry combined analysis model and construction method thereof

Info

Publication number: CN112285223A
Application number: CN202011058773.7A
Authority: CN
Inventors: 韩立峰; 刘二伟; 于海洋; 张祎; 王涛; 窦志英; 高秀梅
Original assignee: Tianjin University of Traditional Chinese Medicine
Current assignee: Tianjin University of Traditional Chinese Medicine
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2021-01-29

Abstract

The invention provides a liquid chromatography-mass spectrometry combined analysis model and a construction method thereof, relating to the field of traditional Chinese medicine component identification, wherein the flow rate is 0.15-0.25mL min < -1 >; the column temperature is 25-35 ℃; the voltage of the positive ion spray is 3.0-3.5 kV; the negative ion spraying voltage is 2.3-3.0 kV; the temperature of the capillary tube is 180 ℃ and 220 ℃; the heating temperature of the auxiliary device is 370-.

Description

Liquid chromatography-mass spectrometry combined analysis model and construction method thereof

Technical Field

The invention relates to the field of traditional Chinese medicine component identification, in particular to a liquid chromatography-mass spectrometry combined analysis model and a construction method thereof.

Background

Chinese herbal medicine has been used for thousands of years and has been explored for a long time in the field of natural product research. The identification of natural products has been a difficult task in long-term research efforts. The chemical components of natural products are often very complex, and many of the components with good drug effect are often only in a small proportion in the extract, and the trace components can determine the application of a natural product [1 ]. In many areas where life is closely related, such as environmental quality, food safety, biomedicine and clinical diagnosis, low concentrations of compounds are also frequently involved. Therefore, the ability to detect low concentrations of substances, particularly low concentrations of compounds in a complex background (complex matrix), is a very important functional indicator for analytical instruments. Today, the most prominent and popular technique in this field of research is undoubtedly mass spectrometry [2 ]. At present, mass spectrometers are the most important analytical instruments for analytical chemistry because of their detection capabilities, which are far superior to other analytical instruments.

The mass spectrometer can be divided into a low-resolution mass spectrometer and a high-resolution mass spectrometer, wherein the low-resolution mass spectrometer comprises a quadrupole mass spectrometer and an ion trap mass spectrometer [3-4], and the high-resolution mass spectrometer comprises a time-of-flight mass spectrometer [5], an electrostatic orbitrap mass spectrometer, a Fourier transform ion cyclotron resonance mass spectrometer [6] and a magnetic mass spectrum. The mass spectrometer with low resolution has simple and small structure, lighter mass and easy cleaning, can not obtain accurate mass number, has slow scanning speed and low sensitivity, has low fragmentation degree of parent ions, and is not beneficial to structure analysis. The high-resolution mass spectrometer has high resolution, good peak separation effect, high scanning speed and low detection limit, and can obtain accurate mass number. The mass spectrometer has high quality precision, high resolution and wide dynamic range, can provide large-range qualitative and quantitative analysis, and overcomes the defects of difficult maintenance and complex operation of the high-resolution mass spectrometer.

The high-resolution mass spectrum (HR-MS) has extremely high detection sensitivity and specificity, can provide a mass-to-charge ratio with high resolution and accuracy, can acquire abundant structural information such as excimer ions and fragment ions of chromatographic peaks, is favorable for identification and attribution of chromatographic peaks, has become a powerful tool for representing complex natural products, is one of the most effective analysis means for the research of active ingredients of traditional Chinese medicines, and is widely applied to the research of traditional Chinese medicines. The flavonoid and benzophenone natural products are similar in structure, the mass spectrum molecular weight is close, and the cracking path is similar. At present, the identification work of two types of compounds based on UPLC-HR-MS has greater difficulty

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a liquid chromatography-mass spectrometry combined analysis model and a construction method thereof.

The invention is realized by the following technical scheme: a liquid chromatography-mass spectrometry analysis model comprises a flow rate of 0.15-0.25mL min-1; the column temperature is 25-35 ℃; the voltage of the positive ion spray is 3.0-3.5 kV; the negative ion spraying voltage is 2.3-3.0 kV; the temperature of the capillary tube is 180 ℃ and 220 ℃; the assist device heating temperature is 370 ℃ and 420 ℃.

According to the technical scheme, the flow rate is preferably 0.2mL min < -1 >; the column temperature is 30 ℃; the positive ion spray voltage is 3.2 kV; the negative ion spraying voltage is 2.8 kV; the capillary temperature is 200 ℃; the temperature of the auxiliary heater is 400 ℃.

A method for constructing a liquid chromatography-mass spectrometry combined analysis model comprises the following steps;

(1) analyzing a plurality of groups of standard substances by adopting a UHPLC-Q-Orbitrap MS technology and combining with a response surface experimental design to obtain related data;

(2) collecting the related data obtained in the step (1) by using Xcailbur software;

(3) according to the total area of chromatographic peaks, a correlation coefficient R2 and a standard deviation RSD, screening a stationary phase and optimizing and screening a mobile phase;

(4) according to the experimental design of the response surface, the optimization experiment is carried out on the flow rate, the column temperature, the positive ion spray voltage, the negative ion spray voltage, the capillary temperature and the heating temperature of the auxiliary device.

According to the technical scheme, preferably, 50 groups of standard substances are selected in the step (1), wherein the standard substances comprise 25 groups of flavonoid compounds and 25 groups of benzophenone compounds.

According to the technical scheme, preferably, the UHPLC-Q-Orbitrap MS technology in the step (1) adopts a positive and negative switching ion scanning method of Full-MS/dd-MS2, and simultaneously turns on the Inclusion function, the source spray voltage (spray voltage) positive and negative ions are respectively 3.2kV and 2.8kV, the capillary temperature (capillary temperature) is 200 ℃, the auxiliary heating temperature (auxiliary heating temperature) is 400 ℃, the sheath gas (sheath gas, N2) is 35, the auxiliary gas (auxiliary gas, N2) is 10, the collision energy is 20V, 40V, 60V, the scanning range is m/z 100-1500, and the resolution is 70000.

According to the technical scheme, preferably, the data acquisition of the Xcailbur software in the step (2) comprises chromatographic peak extraction and automatic integration, the obtained related information of mass-to-charge ratio, retention time and peak area is recorded through Excel to obtain a table matrix, and the total area of the chromatographic peaks, the standard deviation RSD and the related coefficient R2 are calculated.

The invention has the beneficial effects that: the constructed liquid chromatogram-mass spectrum combined analysis model can ensure that the total area of chromatographic peaks is larger, and the peak areas of the standard substances are uniformly distributed, thereby effectively providing precondition for mass spectrum data required by a subsequent deep neural network model.

Drawings

FIG. 1 is a heat map of the response of a chromatographic column separation mixed standard in positive ion scan mode;

FIG. 2 is a heat map of the response of a chromatographic column separation mixed standard in negative ion scan mode;

FIG. 3 is a heat map of the response of each mobile phase separated mixed standard in positive ion scan mode;

FIG. 4 thermal map of mobile phase in negative ion scan mode;

FIG. 5 is a scattergram showing the correlation between the experimental data of the peak area (a) and the correlation coefficient R2(b) and the predicted value;

FIG. 6 is a contour plot and a response plot of flow rate, negative ion spray voltage, capillary temperature, assist device heating temperature versus peak area;

FIG. 7 is a contour plot and a response plot of capillary temperature and helper heating temperature versus correlation coefficient R2;

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it is obvious that the described embodiments are some, not all embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

As shown in the figure, the invention provides a liquid chromatography-mass spectrometry combined analysis model, which comprises a flow rate of 0.15-0.25mL min < -1 >; the column temperature is 25-35 ℃; the voltage of the positive ion spray is 3.0-3.5 kV; the negative ion spraying voltage is 2.3-3.0 kV; the temperature of the capillary tube is 180 ℃ and 220 ℃; the assist device heating temperature is 370 ℃ and 420 ℃.

The specific method comprises the following steps:

1 materials and methods

1.1 Experimental materials

1.1.1 Experimental drugs and reagents

Chromatographically pure acetonitrile was obtained from feishel corporation, usa; ultrapure water (Watson Company Limited); chromatographically pure methanol (Fisher, Fair lawn, NJ, USA); formic acid (ACS, Wilmington, USA); acetic acid (ACS, Wilmington, USA). The total of 50 reference substances are divided into 25 flavonoids compounds and 25 benzophenone compounds.

The 25 flavonoids are: apigenin (1), kaempferol (2), 3-methylkaempferol (3), leontodeflavonoid A (4), floramanoid B (5), floramanoid C (6), floramanoid E (7), floramanoid F (8), floramanoid A (9), floraseafvoside D (10), leontodeflavonoid C (11), (2S) -4',5, 7-trihydroxyflavanone (12), quercetin (13), nanaoside (14), luteolin-4' -O-beta-D-glucopyranoside (15), floraseaflavoside A (16), kaempferol-3-O-rutinoside (17), (-) -epicatechin (18), daidzin (19), formononetin (20), genistin (21), hirsisisisin (22), (-) -catechin C (23), epigallocatechin C (23), isoquercitrin (25).

The 25 benzophenone compounds are: gentisatexatone A2(26), 1,3,5, 8-tetrahydroxypyrapone (27), gentisatexatone C4(28), 3,7, 8-trioxyphenoxy-1-O-beta-D-glucopyranoside (29), gentisatexatone C (30), gentisatexatone A1(31), 1,7-dihydroxy-3,4-dimethoxyxanthone (32), 1,3,8-trihydroxy-4,5-dimethoxyxanthone (33), 1, 7-dihydroxyl-3, 4, 8-dimethoxyxanthone (34), gentisatexatone A1(35), gentisatexatone C1(36), homomangiferin (37), gentisatexatone A → 82, 1- [ beta-xylopyranone ] -1-O-beta-6-D-1- [ 1-O-6-D → 7-D (39-dimethoxypyranone), xylopyranone C3639-D-6-D-O-1-3, 8-dimethoxypyranone (39), xylopyranone C3639-D-6-D-xylopyranone (39, 3-D → 7-D → 8-xylopyranone (39-D → 8-D → 7-xylopyranone (3, 7-D → 8-xylopyranone (3, beta-L → 7-L → 1, L → 7-D → 8- Pyranose ] -7-hydroxy-3, 7, 8-trimethoxyphthalimide (40), 1-O- [ β -D-glucopyranosyl (1 → 6) - β -D-glucopyranosyl ] -3,8-dihydroxy-4, 5-dimethoxyxanthosine (41), foliamangirose C1(42), foliamangirose C3(43), irifoilophenone-3-C- β -glucoside (44), foliamangirose A4(45), foliamangirose B (46), foliamangirose A1(47), foliamangirose C2(48), foliamangirose A3(49), and foliamangirose C4 (50).

1.1.2 Experimental instruments

Ultimate 3000 ultra-high performance liquid systems (Thermo Fisher Scientific, San Jose, CA, USA); high resolution mass spectrometer Q-Orbitrap MS (Thermo Fisher Scientific, Bremen, Germany); d2012 high speed centrifuge (da long xing chu laboratory instruments ltd, beijing, china); KQ-250E ultrasonic cleaning apparatus (kunshan ultrasonic instruments ltd., jiangsu, china); AX205 ten-thousandth of a balance (Mettler Toledo, Switzerland); XW-80A vortex mixer (Shanghai, China, Shanghai, China). Here, 18 different types of chromatography columns are used in common for the screening of the stationary phase.

Table 118 alternative column information

1.2 Experimental methods

1.2.1 preparation of test solutions

Precisely weighing 1mg of 50 reference substances, respectively placing in 10mL volumetric flasks, adding methanol for dissolving and diluting to scale, ultrasonically extracting for 10min, centrifuging the extract solution 14000 r.min < -1 > for 10min, taking supernatant, adding methanol for diluting the obtained supernatant to 1 mu g.ml < -1 >, swirling for 2min, taking 100 mu L, and placing in a sample injection vial for later use.

1.2.2 chromatographic conditions

A chromatographic system: thermo Fisher U3000 ultra performance liquid chromatography system, column: waters ACQUITY UPLC HSS T3 analytical column (2.1X 100mm, 1.8 μm), Volter, USA, sample volume 1 μ L, flow rate 0.2mL min-1, column temperature: 30 ℃, sample chamber temperature: 15 ℃; mobile phase: phase A is 0.1% formic acid water, phase B is acetonitrile; gradient elution conditions: 0min, 5% B; 15min, 95% B; 16min, 95% B; 17min, 5% B; 20min, 5% B.

1.2.3 Mass Spectrometry conditions

Mass spectrometry was performed on a Thermo Fisher Q-Orbitrap MS (Q-atmospheric) system using a high energy electrospray ion source (HESI source). The positive and negative switching ion scanning method of Full-MS/dd-MS2 was applied while the Inclusion function was turned on, the Inclusion list including the positive and negative ion information of 50 of the 1.1.1 controls. Source parameters: the source spray voltage (spray voltage) positive and negative ions are respectively 3.2kV and 2.8kV, the capillary temperature (capillary temperature) is 200 ℃, the auxiliary heating temperature (auxiliary heating temperature) is 400 ℃, the sheath gas (N2) is 35, the auxiliary gas (N2) is 10, the collision energy is 20V, 40V, 60V, the scanning range is m/z 100-1500, and the resolution is 70000.

1.2.4 data processing and analysis

The method comprises the steps of firstly extracting chromatographic peaks of liquid original data through Xcaliibur 4.0 software, automatically integrating the chromatographic peaks, recording obtained information such as mass-to-charge ratio, retention time, peak area and the like through Excel to obtain a table matrix, and calculating the total area of the chromatographic peaks, the relative standard deviation RSD and a correlation coefficient R2. 2 results of the experiment

2.1 stationary phase screening

Reverse phase chromatography is the most widely used chromatographic separation mode with relatively mature development. It has the characteristics of wide application range, high selectivity, high separation degree, good reproducibility and the like. Under the same conditions, the total area of chromatographic peaks, the correlation coefficient R2 and the relative standard deviation RSD of 50 standards detected in a positive and negative ion mode are compared through 18 reverse phase chromatographic columns. The larger the total area of chromatographic peaks is, the more suitable the chromatographic column is, the smaller the correlation coefficient R2 is, the larger the relative standard deviation RSD is, the more uniform the peak area dispersion of the chromatographic peaks is, and the larger the peak area of a single chromatographic peak is not, so that the larger the total area of the chromatographic peaks is, the better the chromatographic column screening chromatographic peak of the experiment is, the smaller the correlation coefficient R2 is, the larger the relative standard deviation RSD is, the better the chromatographic column screening chromatographic peak of the experiment is. The results of the experiments on 18 chromatographic columns are shown in tables 2 and 3. As can be seen from tables 2 and 3, in the positive ion scanning mode, the total areas of the chromatographic peaks are sorted in the order from large to small, and the first 5 chromatographic columns are: HSS T3(8026224604) > Luna Omega 1.6u Polar C18(7769890545) > Zorbax SB-AQ (7294470783) > BEH C18(6938588903) > HSS C18 SB (6927061756). In the positive ion scanning mode, the correlation coefficients R2 are sorted in order from small to large, and the first 5 chromatographic columns are: CSH Cyano (0.0996) < CSH Fluoro-Phenyl (0.1025) < HSS C18 SB (0.1029) < HSS T3(0.1071) < Zorbax Eclipse Plus C18 (0.1122). In the positive ion scanning mode, the relative standard deviation RSD is sorted in the order from large to small, and the first 5 chromatographic columns are: CSH Cyano (2.74) > HSS C18 SB (2.73) > CSH Fluoro-Phenyl (2.72) > HSS T3(2.65) > Zorbax SB-C18 (2.56).

TABLE 2 Experimental results of chromatographic column separation mixed reference substance in positive ion mode

Through the comparison, the chromatographic columns HSS T3, CSH Cyano, CSH Fluoro-Phenyl and HSS C18 SB are better in comprehensive performance in the positive ion scanning mode.

In the negative ion scanning mode, the total areas of chromatographic peaks are sorted from large to small, and the first 5 chromatographic columns are: HSS T3(28756128263) > Kinetex 1.7u Biphenyl

(23353477042) > Zorbax SB-C18(18478731653) > BEH C18(16439335917) > HSS C18 SB (16215277029). In the negative ion scanning mode, the correlation coefficients R2 are sorted from small to large, and the first 5 chromatographic columns are: HSS T3(0.0387) < Kinetex 1.7u EVO C18(0.032) < CORTECS C18(0.0488) < Luna Omega 1.6u Polar C18(0.0516) < Zorbax SB-AQ (0.0568). In the negative ion scanning mode, the relative standard deviation RSD is sorted from big to small, and the first 5 chromatographic columns are: zorbax SB-AQ (1.66) > CSH C18(1.65) > CSH Fluoro-Phenyl (1.64) > Luna Omega 1.6u Polar C18(1.62) > HSS C18 SB (1.61). Through the comparison, the chromatographic columns HSS T3, Kinetex 1.7u Biphenyl, Luna Omega 1.6u Polar C18 and Zorbax SB-AQ have better comprehensive performances in the negative ion scanning mode. In combination with the performance of the chromatographic column in the positive ion scanning mode, the chromatographic column HSST 3 has better performance in both the positive and negative scanning modes. Then using MeV4.9.0 software to make a heat map according to the chromatographic peak areas of 50 reference substances in 18 chromatographic columns, and through the heat map, we can see that the chromatographic column HSST 3 with the serial number of 12 has the most uniform color distribution under a positive and negative scanning mode, and further illustrate that the analysis is carried out by using the chromatographic column HSST 3, the total area of the chromatographic peak is not only larger, but also the peak area of each reference substance is larger, and the result that the total area of the chromatographic peak is larger because the peak area of each reference substance is larger does not meet the requirements of the chromatographic column.

TABLE 3 experiment results of various chromatographic column separation mixed reference substances in anion mode

2.2 Mobile phase screening

The mobile phase is one of the main factors affecting chromatographic separation. Six mobile phases, A: H2O + 0.1% Formic Acid (FA) and B: methanol (MeOH), A: H2O + 0.1% formic acid and B: Acetonitrile (ACN), A: H2O + 0.05% FA and B: ACN, A: H2O + 0.1% AA and B: ACN, A: H2O + 0.05% AA and B: ACN, A: H2O and B: ACN, were examined separately in this experiment, and the total area of chromatographic peaks, correlation coefficient R2, relative standard deviation RSD and thermogram distribution under different mobile phases were compared. Suitable organic phases were first found by comparing A: H2O + 0.1% FA with B: MeOH, A: H2O + 0.1% FA with B: ACN, unifying the A phase, and the results are shown in Table 4, Table 5. As can be seen from the table, when acetonitrile is used as the organic phase, the total area of the chromatographic peaks is the largest in both the positive ion mode and the negative ion mode, the correlation coefficient R2 is small, and the relative standard deviation RSD is large, and therefore, acetonitrile is determined to be the organic phase. From table 6, table 7 we can see that, except for a: 0.05% FA and B: acetonitrile mobile phase, and other four chromatographic conditions have small difference. In positive ion mode, a: 0.05% AA and B: acetonitrile chromatogram peak total area is largest, whereas a: H2O and B: the correlation coefficient R2 for acetonitrile is the smallest and the relative standard deviation RSD the largest. In the negative ion mode, a: 0.1% AA and B: acetonitrile has the largest total area of chromatographic peaks, whereas a: 0.1% FA and B: the correlation coefficient R2 of acetonitrile is the smallest, and the relative standard deviation RSD is the largest for a: 0.1% AA and B: and (3) an acetonitrile system. We further identified phase a by heat map, fig. 3, fig. 4. From the heatmap, it can be seen that a: 0.1% FA and B: the acetonitrile distribution was most uniform, so the mobile phase was finally determined to be a: 0.1% FA and B: and (3) acetonitrile.

TABLE 4 optimization of organic phases in positive ion scanning mode

TABLE 5 optimization of organic phases in negative ion scanning mode

2.3 response surface design

2.3.1 response surface Experimental design

Design-Expert 10.0 software was used for response surface test Design [12-13 ]. A six-factor two-level experiment is combined with a Box-Behnken center combination design, the influence of X1 (flow rate), X2 (column temperature), X3 (positive ion spray voltage), X4 (negative ion spray voltage), X5 (capillary temperature) and X6 (auxiliary device heating temperature) on the peak area and the correlation coefficient R2 is considered by taking the peak area and the correlation coefficient R2 as indexes, and the optimal liquid quality condition is determined. Table 8 lists the range of the independent variables and their levels. To reduce the effect of extrinsic factors on the experimental process, they were randomly grouped and included five center points to calculate the repeatability of the method.

2.3.2 data analysis and model evaluation

To reduce the effect of extrinsic factors on the experimental process, they were randomly grouped, including five center points, to calculate the repeatability of the method. Table 9 lists the protocol and results of 53 runs using the Box-Behnken design. Fig. 5 is a scatter diagram of the corresponding relationship between the experimental data of the total area of the chromatographic peak and the correlation coefficient R2 and the predicted value, and it can be seen from the scatter diagram that the scatter points are close to both sides of the same straight line. As can be seen from both table 9 and fig. 5, there is close agreement between the experimental data and the predicted values.

According to the experimental Design and results of the response surface of table 9, variance analysis is performed on the peak area and the correlation coefficient R2 by using Design-Expert 10.0 software, and the coefficients of the polynomial model equation are shown in table 10 and table 11. The analysis of variance is also called F test, the significance of the polynomial model equation is evaluated through the F test, the larger the F value is, the more significant the equation is represented, and the better the fitting degree is. The importance of each coefficient also needs to be determined by the p-value. A p value of <0.05 indicates significant differences in the coefficients, and a p value of <0.0001 indicates very significant differences. As can be seen from table 10, F is 150.06 in the polynomial model obtained by response surface design, and a p value of <0.0001 indicates that the response value in the polynomial model has a significant linear relationship with each factor, and the reliability of the response surface model is high. The p value of the mismatching term is not significant, namely 0.0717>0.05, which shows that the influence of unknown factors on the experimental result is small, and the experimental model is fully fitted to the experimental data, so that the equation is feasible. The correlation coefficient R2 is 0.983, which indicates that the agreement between the experimental peak area value and the predicted peak area value of the response surface is relatively high and the error is relatively small. The influence of each factor on the peak area is in the following order from large to small: x5 (capillary temperature) > X1 (flow rate) > X4 (negative ion spray voltage) > X2 (column temperature) > X3 (positive ion spray voltage) > X6 (assist device heating temperature). Wherein, the influence of X1 (flow rate) and X5 (capillary temperature) on the peak area reaches an extremely significant level, X4 (negative ion spray voltage) is a significant level, and the rest three items are not significant.

Table 10 analysis of variance of polynomial model of peak area

From table 10, quadratic polynomial model equations between the peak area and X1 (flow rate), X2 (column temperature), X3 (positive ion spray voltage), X4 (negative ion spray voltage), X5 (capillary temperature), and X6 (assist device heating temperature) can be obtained:

Y＝4.88E+010-4.44E+011*X1+9.15E+009*X2-3.04E+010*X3-3.25E+010*X4-1.75E+010*X5+5.69E+010*X6+3.02E+008*X1*X2-1.17E+010*X1*X3-1.05E+010*X1*X4+1.26E+009*X1*X5+1.10E+007*X1*X6+9.14E+007*X2*X3-1.35E+008*X2*X4-9.82E+004*X2*X5-3.03E+007*X2*X6+5.65E+009*X3*X4-1.21E+007*X3*X5-7.77E+005*X3*X6-2.02E+007*X4*X5+1.72E+007*X4*X6-2.62E+005*X5*X6+5.69E+011*X12-1.39E+008*X22+2.90E+009*X32+4.68E+009*X42-2.08E+004*X52-8.23E+004*X62-1.49E+009*X12*X5+4.59E+005*X22*X6(1)。

as can be seen from table 11, F of the polynomial model obtained by designing the response surface is 13.69, and the p value is less than 0.0001, which indicates that the response value of the polynomial model has a significant linear relationship with each factor, and the reliability of the response surface model is high. The correlation coefficient R2 is 0.9366, which indicates that the experimental peak area value and the predicted peak area value of the response surface are relatively high in agreement and have relatively small errors. The influence of each factor on the peak area is in the following order from large to small: x5 (capillary temperature) > X6 (assist device heating temperature) > X2 (column temperature) > X3 (positive ion spray voltage) > X4 (negative ion spray voltage) > X1 (flow rate). Among them, the influence of X5 (capillary temperature) on the peak area reached a very significant level.

TABLE 11 correlation coefficient R₂Analysis of variance of polynomial model

Note: significant (p value < 0.0001); significant (p value <0.05)

A quadratic polynomial model equation between the correlation coefficient R2 and X1 (flow rate), X2 (column temperature), X3 (positive ion spray voltage), X4 (negative ion spray voltage), X5 (capillary temperature), and X6 (assist device heating temperature) can be obtained from table 11:

Y＝-2.55E+01+4.52E+002*X1-5.48E+003*X2-8.09E+004*X3+1.68E+001*X4+1.81E+003*X5+2.86E+005*X6+6.22E+003*X1*X2-8.60E+002*X1*X3+7.73E+002*X1*X4-3.25E+005*X1*X5+3.25E+005*X1*X6+2.28E+003*X2*X3-1.02E+003*X2*X4-4.19E+006*X2*X5-4.62E+006*X2*X6-6.40E+003*X3*X4-8.60E+005*X3*X5-1.60E+005*X3*X6-1.38E+004*X4*X5+9.30E+005*X4*X6-4.11E+007*X5*X6-2.90E+01*X12+3.04E+005*X22+8.58E+004*X32-2。17E+002*X42-1.98E+006*X52-5.89E+009*X62 (2)。

the response surface graph is a curved surface graph of a three-dimensional space formed by the response values to the test factors, and the optimal parameters and the interaction among the parameters can be visually seen from the response surface analysis graph. As can be seen from table 10, X1 (flow rate), X4 (negative ion spray voltage), and X5 (capillary temperature) p values of less than 0.05 are significant terms, so contour plots and three-dimensional surface plots of the 3-factor interaction quadratic terms X1 (flow rate) × 4 (negative ion spray voltage), X1 (flow rate) × 5 (capillary temperature), and X4 (negative ion spray voltage) × 5 (capillary temperature) are shown in fig. 6, and since the quadratic term X5 (capillary temperature) × 6 (assist device heating temperature) is also a significant term, it is also shown in fig. 6. Fig. 7 is a contour diagram and a response surface diagram of X5 (capillary temperature) × X6 (assist device heating temperature) term of the correlation coefficient R2.

2.3.3 validation of predictive models

The optimal conditions for liquid phase mass spectrometry were determined by Design-Expert 10.0 software analysis as follows: the flow rate is 0.2mL min < -1 >; the column temperature is 30 ℃; the positive ion spray voltage is 3.2 kV; the negative ion spraying voltage is 2.8 kV; the capillary temperature is 200 ℃; the temperature of the auxiliary heater is 400 ℃. Under the condition, the theoretical value calculated according to the formula is as follows: the peak area was 49269454339, and the correlation coefficient R2 was 0.138. Three groups of verification tests are carried out according to the obtained data, and the obtained actual values are as follows: the peak area value was 52678326874, and the correlation coefficient R2 was 0.142. The experimental value is not obviously different from the theoretical value, and the result is proved to be reasonable and reliable.

In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of describing the invention and simplifying the description, but do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

In the description of the invention, it is to be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meaning of the above terms in the present invention can be understood in specific cases to those skilled in the art.

Finally, it should be noted that: the above embodiments are only used for illustrating the technical solution of the invention, and not for limiting the same; while the invention has been described in detail and with reference to the foregoing embodiments, those skilled in the art will appreciate that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A liquid chromatography-mass spectrometry combined analysis model is characterized by comprising a flow rate of 0.15-0.25mL min < -1 >; the column temperature is 25-35 ℃; the voltage of the positive ion spray is 3.0-3.5 kV; the negative ion spraying voltage is 2.3-3.0 kV; the temperature of the capillary tube is 180 ℃ and 220 ℃; the assist device heating temperature is 370 ℃ and 420 ℃.

2. The LC-MS combination assay model of claim 1, comprising a flow rate of 0.2mL min "1; the column temperature is 30 ℃; the positive ion spray voltage is 3.2 kV; the negative ion spraying voltage is 2.8 kV; the capillary temperature is 200 ℃; the temperature of the auxiliary heater is 400 ℃.

3. A method for constructing a liquid chromatography-mass spectrometry analysis model used for constructing the liquid chromatography-mass spectrometry analysis model of claim 1 or 2, comprising the steps of;

4. The method for constructing a liquid chromatography-mass spectrometry combined analysis model according to claim 3, wherein 50 sets of standard substances are selected in step (1), wherein the standard substances comprise 25 sets of flavonoids and 25 sets of benzophenones.

5. The method for constructing a liquid chromatography-mass spectrometry combined analysis model as claimed in claim 4, wherein the UHPLC-Q-Orbitrap MS technology of step (1) adopts a positive and negative switching ion scanning method of Full-MS/dd-MS2, and simultaneously turns on the Inclusion function, the source spray voltage (spray voltage) positive and negative ions are 3.2kV and 2.8kV respectively, the capillary temperature (capillary temperature) is 200 ℃, the assist device heating temperature (assist heating temperature) is 400 ℃, the sheath gas (sheath gas, N2) is 35, the assist gas (assist gas, N2) is 10, the collision energy is 20V, 40V, 60V, the scanning range is m/z 100 7001500, and the resolution is 00.

6. The method for constructing the liquid chromatography-mass spectrometry combined analysis model according to claim 5, wherein the data acquisition of the Xcailbur software in the step (2) comprises chromatographic peak extraction and automatic integration, the obtained related information of mass-to-charge ratio, retention time and peak area is recorded through Excel to obtain a table matrix, and the total area of chromatographic peaks, the standard deviation RSD and the related coefficient R2 are calculated.