CN116930350A

CN116930350A - Method for measuring additive in beverage by using ultra-high performance liquid chromatography-mass spectrometer

Info

Publication number: CN116930350A
Application number: CN202310392321.XA
Authority: CN
Inventors: 徐敦明; 吴媛; 袁文萱; 洪煜琛; 潘丽蓉; 王东; 吴俊杰
Original assignee: Xiamen Customs Technical Center
Current assignee: Xiamen Customs Technical Center
Priority date: 2023-04-13
Filing date: 2023-04-13
Publication date: 2023-10-24

Abstract

The invention provides a method for measuring additives in beverages using an ultra-high-performance liquid chromatography-mass spectrometry instrument, and relates to the technical fields of chromatography-mass spectrometry, chromatography-mass spectrometry, and high-resolution chromatography-mass spectrometry. By applying ultra-high performance liquid chromatography-quadrupole-time-of-flight high-resolution mass spectrometry equipment, primary and secondary mass spectrometry data of compounds were obtained, and an initial database was generated based on the mass spectrometry signal characteristics and mass spectrometry information of 40 compounds. The high-throughput data of sample compounds collected through mass spectrometry are compared with the characteristic spectrum data to determine the compounds detected in the sample. Through pattern recognition of mass spectrometry information, sample retention time, mass deviation and isotope peak shape are matched with standard materials. Finally, the mass spectra of 40 kinds of food additives and the detection methods of the additives were constructed to achieve rapid and accurate analysis of 40 kinds of food additives in beverages. The method provided by the invention has good social and economic benefits.

Description

Method for measuring additive in beverage by using ultra-high performance liquid chromatography-mass spectrometer

Technical Field

The invention relates to the technical fields of chromatographic mass spectrometry and chromatographic high-resolution mass spectrometry, in particular to a method for measuring additives in a beverage by an ultra-high performance liquid chromatography-mass spectrometer.

Background

Food additives are a class of natural or synthetic compounds that impart color, flavor, taste and preservative properties to foods. In recent years, the development of food additives in China is rapid, and particularly the development of two types of coloring agents and sweeteners is particularly outstanding, so that the food additives become an indispensable part of the food processing industry. However, most of food additives in the market at present are artificially synthesized substances, and the synthetic raw materials have safety problems, or certain byproducts harmful to human bodies are brought in the process flow, so that excessive eating can cause irreversible harm to the human bodies.

In order to ensure personal safety of consumers, the content of the additive in the food is monitored, and the dosage is controlled with great significance.

The development of simple, rapid and efficient sample pretreatment technology and high-sensitivity analysis and detection methods have become urgent matters for scientists.

The existing detection methods of additives in food mainly comprise Gas Chromatography (GC), gas chromatography-mass spectrometry (GC-MS), gas chromatography-mass spectrometry/mass spectrometry (GC-MS/MS), liquid chromatography (HPLC), liquid chromatography-mass spectrometry/mass spectrometry (GC-MS/MS) and the like. In recent years, high-resolution mass spectrometry has been widely used in high-throughput screening analysis techniques. The high-resolution mass spectrometer can measure the accurate molecular weight of the compound under the ultra-high resolution, can distinguish impurities and co-effluent in complex background, and can reduce the requirement on sample pretreatment.

However, for food samples of complex types, it is difficult to obtain accurate and rapid detection results with existing protocols.

Disclosure of Invention

The invention aims to solve the problems of complex detectable types of additives in the current food, wide research range of food matrixes, complex pretreatment, low detection speed and the like, and provides a method for measuring the additives in the beverage by using an ultra-high performance liquid chromatography-mass spectrometer.

The invention develops a rapid, sensitive and accurate high-throughput screening method based on the technologies of chromatography mass spectrometry, chromatography high-resolution mass spectrometry and the like, performs multidimensional chemical information research and characterization aiming at additives in food, establishes an authoritative additive harmful factor basic database, and develops qualitative and quantitative detection research of the additives; establishing a rapid screening technology of an additive risk factor early warning threshold system; developing a rapid and accurate screening technology suitable for complex food matrixes by utilizing an advanced instrument of liquid chromatography-mass spectrometry; an accurate database of at least 40 common additives is established. The improvement of a food safety guarantee technical system is promoted, and the model and the technical support are provided for the stable driving protection of the body health and society of the masses and the relevant law enforcement departments.

The main content of the invention comprises:

1) The method for accurately detecting the content of the food additive in the typical food matrix is established, a high-selectivity sample pretreatment mode is developed, and the rapid and accurate quantitative detection technical research of the content of the food additive with the rule differentiation in the food is developed by combining the characterization method of the high-resolution mass spectrum and tandem mass spectrum detection technology through the modern chromatographic separation technologies such as gas chromatography, liquid chromatography and multidimensional chromatography, and the national standard of food safety is established, submitted or reported.

2) According to the invention, 4 different types of additives (coloring agents, sweeteners, antioxidants and preservatives) are selected, 40 compounds are taken as research objects, an ultra-high performance liquid chromatography (UPLC) -quadrupole-time-of-flight mass spectrometry (Q-TOF/MS) method of various types of additives in the beverage is established, a primary mass number and secondary mass spectrogram database is established, and qualitative screening can be realized by comparison with the database. The method is simple and quick to operate, and provides reliable technical support for quick screening and quantitative detection of additive residues.

3) The method for measuring the polybrominated diphenyl and polybrominated diphenyl ether substances of the food by using the QuEChERS-gas chromatography-tandem mass spectrometry (GC-MS/MS) method is established. After extraction and concentration of the target with N-hexane or acetonitrile, purification with anhydrous MgSO4, ethylenediamine-N-Propylsilane (PSA) and octadecylsilane chemically bonded silica (C18), separation with DB-5HT column (15 m 0.25mm 0.1 μm) and quantification by external standard method using electron bombardment ionization source.

The invention establishes a database for automatically screening the additive by utilizing high-resolution mass spectrum, takes the beverage as a sample, and based on the establishment of the database, surveys and uses the database to establish, surveys and uses the database to screen and confirm the capability. The invention adopts the high-resolution mass spectrometry technical system to construct the primary and secondary mass spectrometry parameters of 40 additives for the first time, has higher innovation and application, and provides a scientific basis for quick and accurate confirmation of various additives.

The specific scheme is as follows:

a method for determining additives in beverages by ultra performance liquid chromatography-mass spectrometry, comprising the following steps:

1) 40 compound additives are selected, and standard solutions of the additives are prepared and used for establishing a mass spectrum library of the additives;

2) Primary and secondary mass spectrum data of each additive are obtained through scanning of a high-resolution mass spectrometer, and qualitative analysis is performed by utilizing the mass spectrum data;

3) Adding the mass spectrum data of 40 additives into a spectrum library by utilizing a screening data platform established by MassHunter PCDL Manager software, and establishing a mass spectrum library of a target compound;

4) Pretreating the beverage to be measured to obtain a treated sample;

5) Scanning the treated sample in a Full mass mode to obtain mass spectrum information, primarily screening out a compound through accurate molecular weight, and simultaneously taking the corresponding standard solution as a reference; scanning by adopting a tSIM-ddMS2 mode, and carrying out auxiliary qualitative analysis by using the retention time of the standard solution and secondary mass spectrum data so as to screen out target compounds corresponding to 40 food additives;

6) And 5) introducing the mass spectrum information obtained in the step 5) into MassHunter PCDL Manager software, then carrying out component analysis, respectively comparing the target compound obtained by analysis with the accurate mass, the retention time, the accurate mass of fragment ions and the isotope distribution of molecular ions in an information base of 40 food additives in the mass spectrum base, and judging that the beverage contains the standard substance if the retention time deviation is less than 0.1min, the mass deviation is less than 5ppm and the isotope peak shape matching degree is more than 89.5% which means that the measured substance is matched with the standard substance.

The additive standard solution described in method step 1) above: accurately weighing 10.0mg of 40 additive standard substances in a 10mL volumetric flask, dissolving with methanol, fixing volume, preparing into 1.0mg/mL standard stock solution, and storing at-20deg.C in dark place. Taking 1.0mL of each additive single label in a 100mL volumetric flask, and methanol: the volume ratio of pure water is 1:4 to fix the volume, and the additive standard solution with the volume ratio of 10 mug/mL is prepared.

Further, the additive in the step 1) comprises at least one of a preservative, an antioxidant, a colorant and a sweetener, and the additive is 40 compounds: sorbic acid, benzoic acid, methyl parahydroxybenzoate, t-butyl paradiphenol, acesulfame, ethyl parahydroxybenzoate, dehydroacetic acid, sodium cyclamate, propyl parahydroxybenzoate, sodium saccharin, butyl parahydroxybenzoate, propyl gallate, 2, 6-di-t-butyl-p-cresol, sudan I, sudan II, octyl gallate, epicatechin, catechin, aspartame, epigallocatechin, alitame, dodecyl gallate, sudan III, neotame, sudan IV, sucralose, sunset yellow, indigo, epicatechin gallate, allure red, epigallocatechin gallate, acid red, lemon yellow, amaranth, carmine, neored, natamycin, brilliant blue, stevioside, erythrosine.

Further, in the step 2), an ultra-high performance liquid chromatography-four-stage rod-time-of-flight high resolution mass spectrometer is adopted for scanning.

Further, the mass spectral data includes at least 3 of molecular weight, fragment molecular weight, retention time, exact molecular weight, isotope peak shape, secondary mass spectral information CAS, molecular formula.

Preferably, the conditions for mass spectrometry in step 2) are:

a) Ion source: electrospray ion source (ESI), positive and negative ion switching

b) The temperature of the drying gas is 280+/-5 DEG C

c) The velocity of the drying gas is 11-12L/min

d) The temperature of the sheath air is 300+/-10 DEG C

e) The flow rate of sheath gas is 8-12L/min

f) Capillary voltage 4000.+ -.100V

g) Fracture voltage 105V

h) Taper hole voltage 65V

i) The full scanning mass-nuclear ratio range is between 50 and 1000

j) The scanning rate is 1-3 spectra/s

k) And correcting the mass accuracy of the instrument in real time by adopting an internal standard reference solution, wherein the internal standard reference solution comprises reference ions with accurate mass numbers of 121.058, 73 and 922.009 798.

Preferably, the liquid chromatography conditions in step 2) are:

1) Chromatographic column: agilent Eclipse Plus C18, 3.0X105 mm,1.8 μm

2) Sample injection amount: 2.0 to 5.0 mu L

3) Flow rate: 0.3mL/min

4) Column temperature: 40 DEG C

5) Mobile phase a was acetonitrile

6) Mobile phase B is formic acid aqueous solution with volume content of 0.1%

Elution gradients are as follows:

time/min	Mobile phase composition/volume content
		0～1	5% B, the rest is A
1～10	5 to 95 percent of B and the rest is A
		10～15	95% B and the rest A
15～15.1	95-5% of B and the rest of A
		15.1～18	5% B, the rest is A

。

Further, the information obtained by scanning in the step 2) is subjected to preliminary screening with the established TOF/MS database, the retention time error is less than 5%, and the primary parent ion accurate molecular weight error is less than 10 multiplied by 10 ^-11 The compound was confirmed to be the target compound.

Further, the primary screen: the first step: in the full-sweep mode, the standard solution of each additive with the concentration of 500 mug/L is taken out by referring to the accurate molecular weight of the target compound to obtain [ M+H ]] ^- 、[M+H] ⁺ And generating an initial database; and a second step of: in [ M+H ]] ^- 、[M+H] ⁺ The ion information of (2) is a parent ion, and proper collision energy is selected under the condition of Auto-MS/MS to obtain secondary mass spectrum information of the ion information, wherein at least 2 fragment ion peaks with abundance more than 80% are suitable under the condition of retaining excimer ions; and a third step of: inputting the mother ion retention time of the additive and different collision energies at the Targeted MS/MS acquisition interface of the Q-TOF/MS, collecting fragment ion data of the compound under different collision energies, and collecting the collision energies and corresponding fragmentsAnd inputting the piece of ion mass spectrogram information into a database to obtain 40 compound databases.

Further, based on the primary screening results, the parent ion, retention time and different collision energies of the additive were compared with the established library at the MassHunter PCDL acquisition interface of Q-TOF/MS, and the compound was detected with confirmation when at least one fragment ion was in line with the library.

Further, the beverage pretreatment to be tested in step 4): accurately weighing 2.0g of the sample into a 50mL centrifuge tube, diluting the sample to 25mL by the volume ratio of methanol to pure water of 1:4, and performing on-machine analysis by using a 0.22 mu m organic filter membrane.

The beneficial effects are that: the invention optimizes and obtains the accurate molecular weight, retention time, collision energy, fragment ion information and the like of 40 additives by utilizing a high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry, establishes a common automatic screening database of 40 additives, and can realize quick screening of common additives with the accuracy reaching 100 percent.

Drawings

FIG. 1 is an extracted ion flow diagram of a steviol glycoside additive;

FIG. 2 is a first order mass spectrum of steviol glycoside additive;

FIG. 3 is an extracted ion flow diagram of a benzoic acid additive;

FIG. 4 is a first order mass spectrum of a benzoic acid additive;

FIG. 5 is an extracted ion flow diagram of an acesulfame k additive;

FIG. 6 is a first order mass spectrum of an acesulfame k additive;

FIG. 7 is an extracted ion flow diagram of an aspartame additive;

FIG. 8 is a first order mass spectrum of an aspartame additive;

FIG. 9 is an application step 1 of screening a database;

fig. 10 is an application step 2 of the screening database.

Detailed Description

Embodiments of the present invention will be described in more detail below. While the preferred embodiments of the present invention are described below, it should be understood that the present invention may be embodied in various forms and should not be limited to the embodiments set forth herein. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In the examples below, "%" refers to weight percent, unless explicitly stated otherwise.

The solutions used in the examples below were formulated as follows:

preparing a standard solution: accurately weighing 10.0mg of 40 additive standard substances in a 10mL volumetric flask, dissolving with methanol, fixing volume, preparing into 1.0mg/mL standard stock solution, and storing at-20deg.C in dark place. Taking 1.0mL of each additive single label in a 100mL volumetric flask, and methanol: the volume ratio of pure water is 1:4 to fix the volume, and the additive standard intermediate liquid with the volume ratio of 10ug/mL is prepared.

Sample (beverage) treatment to be tested: accurately weighing 2.0g of the sample into a 50mL centrifuge tube, diluting the sample to 25mL by the volume ratio of methanol to pure water of 1:4, and performing on-machine analysis by using a 0.22 mu m organic filter membrane.

The standard used in the invention comprises 40 standard substances, namely sorbic acid, benzoic acid, methyl parahydroxybenzoate, tertiary butyl paradiphenol, acesulfame, ethyl parahydroxybenzoate, dehydroacetic acid, sodium cyclamate, propyl parahydroxybenzoate, sodium saccharin, butyl parahydroxybenzoate, propyl gallate, 2, 6-di-tertiary butyl paracresol, sudan I, sudan II, octyl gallate, epicatechin, catechin, aspartame, epigallocatechin, alitame, dodecyl gallate, sudan III, neotame, sudan IV, sucralose, sunset yellow, indigo, epicatechin gallate, allure, epigallocatechin gallate, acid red, lemon yellow, amaranth, carmine, neotame, natamycin, brilliant blue, stevioside and erythrosine.

The following two beverages are common beverages in the market and are convenient to purchase.

Example 1

In order to examine the screening ability of the constructed spectrum library on actual samples, 2 beverages (protein beverage: milk; carbonated beverage: cola) were selected as examples, and the steps were as follows:

1) The common 4 additives (preservative, antioxidant, colorant and sweetener) are selected to be 40 compounds in total, and 200 mug/L standard solution is prepared and then used for establishing additive mass spectrum library data;

preferably, the scanned information is subjected to preliminary screening with the established TOF/MS database, the retention time error is less than 5%, and the primary parent ion accurate molecular weight error is less than 10 multiplied by 10 ^-11 The result of the preliminary screening was taken as the mass spectrum data.

Preferably, the primary screen comprises:

the first step: in the full-sweep mode, the standard solution of each additive with the concentration of 500 mug/L is taken out by referring to the accurate molecular weight of the target compound to obtain [ M+H ]] ^- 、[M+H] ⁺ And generating an initial database;

and a second step of: in [ M+H ]]-、[M+H] ⁺ The ion information of (2) is a parent ion, and proper collision energy is selected under the condition of Auto-MS/MS to obtain secondary mass spectrum information of the ion information, wherein at least 2 fragment ion peaks with abundance more than 80% are suitable under the condition of retaining excimer ions;

and a third step of: inputting the mother ion retention time and different collision energies of the additives at the Targeted MS/MS acquisition interface of the Q-TOF/MS, collecting fragment ion data under different collision energies by the compound, and inputting the collision energies and the corresponding fragment ion mass spectrum information into a database to obtain the mass spectrum data of 40 additives.

Preferably, based on the primary screening results, the parent ion, retention time, and different collision energies of the additive are compared to the established library at the MassHunter PCDL acquisition interface of Q-TOF/MS, and the compound is detected with confirmation when at least one fragment ion matches the library.

4) Pre-treating the beverage to be assayed: accurately weighing 2.0g of the sample into a 50mL centrifuge tube, diluting the sample to 25mL by the volume ratio of methanol to pure water of 1:4, and performing on-machine analysis by using a 0.22 mu m organic filter membrane.

The 40 compound databases are shown in Table 1.

TABLE 1 database of 40 compounds

And analyzing parameters such as relative deviation of mass numbers in the search result, peak area of the measured compound, signal to noise ratio and the like. The retention time of the obtained target compound is consistent with that of a standard substance, and the deviation of the accurate analysis amount of the primary mass spectrum is less than 5ppm (see figures 1-8).

As can be seen from the comparison of FIG. 1 and FIG. 2, the stevioside has relative deviation of mass numbers, measured compound peak area, signal to noise ratio and other parameters in analysis and search results. Meanwhile, sample injection analysis is carried out under the same condition as that of a standard substance, the retention time of the obtained target compound is consistent, the deviation of the accurate molecular weight of the primary mass spectrum of the standard substance and a sample to be inspected is less than 5ppm, the screening result of a self-built alkaloid spectrum library is further confirmed, and the screening result is met;

as can be seen from the comparison of FIG. 3 and FIG. 4, the relative deviation of the mass numbers of the benzoic acid in the analysis and search results, the peak area of the measured compound, the signal to noise ratio and other parameters. Meanwhile, sample injection analysis is carried out under the same condition as that of a standard substance, the retention time of the obtained target compound is consistent, the deviation of the accurate molecular weight of the primary mass spectrum of the standard substance and a sample to be inspected is less than 5ppm, the screening result of a self-built alkaloid spectrum library is further confirmed, and the screening result is met;

as can be seen from the comparison of fig. 5 and 6, the acesulfame potassium has relative deviation of mass numbers, measured compound peak area, signal to noise ratio and other parameters in analysis and search results. Meanwhile, sample injection analysis is carried out under the same condition as that of a standard substance, the retention time of the obtained target compound is consistent, the deviation of the accurate molecular weight of the primary mass spectrum of the standard substance and a sample to be inspected is less than 5ppm, the screening result of a self-built alkaloid spectrum library is further confirmed, and the screening result is met;

as can be seen from the comparison of FIG. 7 and FIG. 8, the relative deviation of mass numbers, the peak area of the measured compound, the signal to noise ratio and other parameters of the aspartame in the analysis and search result. Meanwhile, sample injection analysis is carried out under the same condition as that of a standard substance, the retention time of the obtained target compound is consistent, the deviation of the accurate molecular weight of the primary mass spectrum of the standard substance and a sample to be inspected is less than 5ppm, the screening result of a self-built alkaloid spectrum library is further confirmed, and the screening result is met;

as can be seen from fig. 9 and 10, massHunter PCDL Manager screens the application steps of the database.

Further corroboration is carried out on the screening result of the self-built additive spectrum library, the screening result is met, and the specific retrieval result is shown in Table 2.

Table 2 beverage sample screening comparison results

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner without contradiction. The various possible combinations of the invention are not described in detail in order to avoid unnecessary repetition.

Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.

Claims

1. The method for determining the additive in the beverage by using the ultra performance liquid chromatography-mass spectrometer is characterized by comprising the following steps of:

4) Pretreating the beverage to be measured to obtain a treated sample;

2. The method according to claim 1, wherein the standard solution of the additive in step 1) comprises: accurately weighing 10.0mg of 40 additive standard substances, dissolving in 10mL volumetric flask, and fixing volume with methanol to obtain standard stock solution of 1.0mg/mL, and storing at-20deg.C in dark place. Taking 1.0mL of each additive single label in a 100mL volumetric flask, and methanol: the volume ratio of pure water is 1:4 to fix the volume, and the additive standard solution with the volume ratio of 10 mug/mL is prepared.

3. The method according to claim 1, wherein the additive in step 1) comprises at least one of a preservative, an antioxidant, a colorant, a sweetener, preferably the additive is 40 compounds: sorbic acid, benzoic acid, methyl parahydroxybenzoate, t-butyl paradiphenol, acesulfame, ethyl parahydroxybenzoate, dehydroacetic acid, sodium cyclamate, propyl parahydroxybenzoate, sodium saccharin, butyl parahydroxybenzoate, propyl gallate, 2, 6-di-t-butyl-p-cresol, sudan I, sudan II, octyl gallate, epicatechin, catechin, aspartame, epigallocatechin, alitame, dodecyl gallate, sudan III, neotame, sudan IV, sucralose, sunset yellow, indigo, epicatechin gallate, allure red, epigallocatechin gallate, acid red, lemon yellow, amaranth, carmine, neored, natamycin, brilliant blue, stevioside, and erythrosine.

4. The method according to claim 1, wherein the scanning is performed in step 2) using an ultra-high performance liquid chromatography-quaternary rod-time-of-flight high resolution mass spectrometer; preferably, the mass spectral data includes at least 3 of molecular weight, fragment molecular weight, retention time, exact molecular weight, isotope peak shape, secondary mass spectral information CAS, molecular formula.

5. The method according to claim 4, wherein the conditions for mass spectrometry in step 2) are: ion source: an electrospray ion source, positive and negative ion switching; the temperature of the drying gas is 280+/-5 ℃; the flow rate of the drying gas is 10-12L/min, the temperature of the sheath gas is 300+/-10 ℃, the flow rate of the sheath gas is 8-12L/min, the capillary voltage is 4000+/-100V, the fragmentation voltage is 105V, and the taper hole voltage is 65V; the full scanning mass-nuclear ratio range is 50-1000 m/z, and the scanning speed is 1-3 spectra/s; and correcting the mass accuracy of the instrument in real time by adopting an internal standard reference solution, wherein the internal standard reference solution comprises reference ions with accurate mass numbers of 121.058, 73 and 922.009 798.

6. The method of claim 4, wherein the liquid chromatography conditions of step 2) are: the column was Agilent Eclipse Plus C, 3.0X105 mm,1.8 μm; sample injection amount: 2.0 to 5.0 mu L; flow rate: 0.3mL/min; column temperature: 40 ℃; mobile phase a is acetonitrile; mobile phase B is formic acid water solution with volume content of 0.1%; elution gradients are as follows:

。

7. The method according to claim 1, wherein the information scanned in step 2) is initially screened from the constructed TOF/MS database with retention time error less than 5% and primary parent ion accurate molecular weight error less than 10 x 10 ^-11 The result of the preliminary screening was taken as the mass spectrum data.

8. The method of claim 7, wherein the preliminary screening in step 2) comprises:

and a second step of: in [ M+H ]] ^- 、[M+H] ⁺ The ion information of (2) is a parent ion, and proper collision energy is selected under the condition of Auto-MS/MS to obtain secondary mass spectrum information of the ion information, wherein at least 2 fragment ion peaks with abundance more than 80% are suitable under the condition of retaining excimer ions;

9. The method of claim 8, wherein the parent ion, retention time, and different collision energies of the additive are compared to the library created at the MassHunter PCDL acquisition interface of Q-TOF/MS based on the primary screening results, and wherein detection of the compound is confirmed when at least one fragment ion matches the library.

10. The method according to claim 1, wherein the beverage pretreatment to be tested in step 4) comprises: accurately weighing 2.0g of the sample into a 50mL centrifuge tube, and adding methanol: the pure water is diluted to 25mL according to the volume ratio of 1:4, and the mixture is subjected to organic filtration membrane analysis of 0.22 mu m.