Method for determining diacetoxyl butyl and derivatives thereof in tea beverage through high-resolution mass spectrometry

Abstract

The method comprises the following steps: sucking a tea beverage sample, placing the tea beverage sample in a volumetric flask, adding an acetic acid methanol solution, carrying out ultrasonic extraction, cooling to room temperature, adding an acetic acid acetonitrile solution, fixing the volume to a scale, and uniformly shaking to obtain a to-be-detected solution A; freezing and centrifuging the to-be-detected solution A, and filtering by using an organic filter membrane to obtain a to-be-detected solution B; and taking the to-be-detected liquid B, determining the to-be-detected liquid B by adopting a liquid-phase series quadrupole rod time-of-flight mass spectrometry, comparing the obtained determination result with standard data in the standard database, and judging whether the tea beverage sample contains diacetate butyl, dipropofol butyl, dibutyl phenol butyl, dipentanol butyl, diheptanol butyl, dioctanol butyl, 4-chlorodiacetate butyl and dicyclohexyl formyl phenol butyl or not. The method can be used for rapidly and accurately determining diacerein butyl and the derivative thereof in the tea beverage, and provides technical support for monitoring and guaranteeing food safety.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and in particular relates to a method for determining diacetyl and its derivatives in tea beverages by high-resolution mass spectrometry. Background Art

[0002] Tea beverages refer to beverages made from tea extract, tea powder, and concentrated liquid as the main raw materials. They have the unique flavor of tea, contain natural tea polyphenols, caffeine and other effective ingredients of tea, and have both nutritional and health benefits. They are multifunctional beverages that are refreshing and thirst-quenching.

[0003] Diacetaminophen is a chemical substance that gradually decomposes into acetic acid and phenol after oral administration in the intestine when it encounters alkaline intestinal fluid. The irritation of phenol to the intestinal mucosa is dozens of times stronger than that of phenol peptide, so its catharsis effect is stronger. This chemical substance is not included in the Chinese Pharmacopoeia and is not a clinical drug. The structure of the diacetaminophen molecule is modified, and the groups on its structure can be changed to form a diacetaminophen derivative.

[0004] Therefore, in order to ensure food safety, it is necessary to establish a high-resolution mass spectrometry method that can simultaneously detect bisacetaminophen and its derivatives in tea beverages, which can quickly and accurately determine bisacetaminophen, bispropanol, bisbutamol, bispentamol, bisheptamol, bisoctinol, 4-chlorobisacetaminophen, and dicyclohexylphenol in tea beverages. Summary of the invention

[0005] In view of the above shortcomings, the present invention discloses a method for determining diacetyl and its derivatives in tea beverages by high-resolution mass spectrometry, which can quickly and accurately determine diacetyl, propanol, butylphenol, bisbutylphenol, bispentanol, bisheptanol, bisoctinol, 4-chlorodiacetylphenol and dicyclohexylphenol in tea beverages.

[0006] The present invention is achieved by adopting the following technical solutions: A method for determining bisacetyl butyl and its derivatives in tea beverages by high-resolution mass spectrometry comprises the following steps: S1. Pipette 1.00 mL of the tea beverage sample into a 25.00 mL volumetric flask, then add 15 mL of acetic acid methanol solution, then perform ultrasonic extraction for 25 min, cool to room temperature, add acetic acid acetonitrile solution to the mark, then shake well to obtain test solution A; S2, placing the test solution A obtained in step S1 at a temperature of 5° C. and a speed of 9000-10000 r / min for refrigerated centrifugation for 5 min, and then filtering with a 0.2 μm organic filter membrane to obtain the test solution B; S3. Take the test solution B obtained in step S2 and measure it by liquid phase tandem quadrupole time-of-flight mass spectrometry. The chromatographic parameters are as follows: an octadecylsilane bonded silica gel chromatographic column with a size of 2.1×50 mm and 1.8 um is used, the injection volume is 3 μL, the column oven temperature is 45°C, and the mobile phase is composed of a 0.1% volume fraction of formic acid aqueous solution and acetonitrile; the mass spectrometry parameters are as follows: the drying gas temperature is 210°C, the drying gas flow rate is 14 L / min, the nebulizer pressure is 35 Psi, the sheath gas temperature is 345°C, the sheath gas flow rate is 11 L / min, the capillary voltage is 3500 V, the nozzle voltage is 1000 V, the pyrolysis voltage is 380 V, and the scanning mode is the targeted secondary acquisition mode (Targeted MS / MS), and includes positive mode and / or negative mode, the scanning range of level 1 is 105-1000m / z, the scanning range of level 2 is 110-575m / z, and the collision energy is 20V, 40V, 45V; S4. Compare the determination result obtained in step S3 with the standard data in the standard database to determine whether the tea beverage sample contains bisacetphenidyl, bispropanol, bisbutamol, bispentamol, bisheptamol, bisoctinol, 4-chlorobisacetphenidyl, and biscyclohexylphenol.

[0007] The method of the present invention uses high-resolution mass spectrometry (liquid phase tandem quadrupole time-of-flight mass spectrometry) to determine bisacetaminophen, bispropanol, bisbutamol, bispentamol, bisheptamol, bisoctinol, 4-chlorobisacetaminophen and biscyclohexylphenol in tea beverages. The method has the advantage that high-resolution mass spectrometry (liquid phase tandem quadrupole time-of-flight mass spectrometry) has a good anti-interference effect on the determination of target components in complex matrices.

[0008] The qualitative analysis of bisacetaminophen and its derivatives in liquid phase requires the use of standard substances to quantify the retention time of chromatographic peaks. It is difficult to separate the target peaks and impurity peaks in the complex matrix of tea beverages. The determination is easily interfered by co-eluting substances, and it takes a long time to separate these co-eluting substances from the target substances. Due to the certain differences in the matrix of the tested samples, when using the liquid phase method, it is necessary to conduct certain preliminary experiments on the tested samples first, and then determine the gradient method before formally carrying out the test.

[0009] High-resolution mass spectrometry (liquid phase tandem quadrupole time-of-flight mass spectrometry) monitors the mass-to-charge ratio of the substance being tested. It can also perform qualitative analysis through the mass-to-charge ratio without separating the target substance and impurity peaks in the sample. The measurement mode used in this method is the targeted secondary detection mode (Targeted MS / MS). The instrument can automatically perform secondary mass spectrometry monitoring on the mass-to-charge ratio within the monitoring time, so that the collected data can obtain primary and secondary mass spectrometry results at the same time. The data obtained through the secondary mass spectrometry is combined with the established database, and the measured components are qualitatively analyzed according to the mass spectrometry qualitative quarter principle, and the qualitative analysis of the target substance in a complex matrix can achieve good accuracy.

[0010] This method can simultaneously determine eight types of bisacetaminophen and their analogues within 8 minutes. The existing food supplement inspection standard BJS 202209 only detects bisacetaminophen, and the instrument used is a low-resolution mass spectrometer with a detection time of 20 minutes. This method takes less time than the supplementary inspection standard, and the speed-up of the test time saves the mobile phase and instrument running time.

[0011] The instrument used in this method is a high-resolution mass spectrometer (liquid phase tandem quadrupole time-of-flight mass spectrometer). The precise mass-to-charge ratio (the mass-to-charge ratio can be read to the fourth decimal place) is used in qualitative analysis. Compared with the low-resolution mass spectrometer (which can only read the mass-to-charge ratio to the first decimal place), the resolution of the instrument used in this method has obvious advantages.

[0012] Furthermore, the acetic acid-methanol solution in step S1 is obtained by mixing acetic acid and methanol in a volume ratio of (0.2-1):100. By controlling the usage ratio of acetic acid and methanol, an acetic acid-methanol solution suitable for extracting diacetylbutyric acid and its derivatives is obtained, ensuring that diacetylbutyric acid and its derivatives in the tea beverage can be effectively and completely extracted, thereby improving the determination accuracy.

[0013] Furthermore, the acetic acid acetonitrile solution in step S1 is obtained by mixing acetic acid and acetonitrile in a volume ratio of (0.3-0.8):100. By controlling the usage ratio of acetic acid and acetonitrile, an acetic acid acetonitrile solution suitable for extracting diacetyl butyrate and its derivatives is obtained, ensuring that diacetyl butyrate and its derivatives in the tea beverage can be effectively and completely extracted, thereby improving the determination accuracy.

[0014] Further, in step S2, the test solution A obtained in step S1 is treated with a cation exchange resin, and then placed in a temperature of 5°C and a speed of 9000-10000 r / min for refrigerated centrifugation for 5 minutes, and then filtered using a 0.2 μm organic filter membrane to obtain the test solution B. Since tea beverages contain a certain amount of tea polyphenols, and tea polyphenols also have good solubility in acetic acid, methanol and acetonitrile, in order to reduce the interference of tea polyphenols and other substances on the determination, the present invention uses a cation exchange resin to effectively adsorb and remove tea polyphenols and other substances in the test solution to reduce the interference of the determination.

[0015] Furthermore, the cation exchange resin is a modified cation exchange resin. The preparation method of the modified cation exchange resin is as follows: take deionized water 2 to 3 times the mass of the cation exchange resin, then add the cation exchange resin, chitosan and glutaraldehyde in sequence, then heat to 60 to 80°C and stir to react for 20 to 30 minutes, then add nano hydroxyapatite and pectin and ultrasonically treat for 10 to 15 minutes, then continue to stir and react at 60 to 70°C for 2 to 3 hours, filter to obtain a precipitate, and then dry the precipitate to obtain the modified cation exchange resin; the mass ratio of the cation exchange resin, chitosan, nano hydroxyapatite, pectin and glutaraldehyde is 100: (20 to 30): (15 to 20): (5 to 10): (3 to 5). The present invention adds chitosan, pectin, nano-hydroxyapatite and other materials to the cation exchange resin material for cross-linking reaction, thereby achieving the effect of modifying the cation exchange resin material, further improving the thermal stability and adsorption performance of the cation exchange resin, facilitating the adsorption and removal of tea polyphenols and other substances in the test solution, reducing interference with the determination of diacetyl and its derivatives, and improving the determination accuracy.

[0016] Furthermore, in the determination of chromatographic parameters in step S3, the mobile phase gradient is:

[0017] Furthermore, the standard database in step S4 is prepared by mixing standard samples of bisacetphenidyl, bispropanol, bisbutamol, bispentamol, bisheptamol, bisoctinol, 4-chlorobisacetphenidyl and biscyclohexylphenol to prepare a series of standard solutions of concentrations, and then measuring the standard solutions according to the methods described in steps S1 to S3, and establishing the standard database according to the obtained measurement results.

[0018] Compared with the prior art, this technical solution has the following beneficial effects: The technical solution of the present invention adopts liquid phase tandem quadrupole time-of-flight mass spectrometry to measure tea beverages, which can effectively identify whether unauthorized bisacetphenol tincture and its derivatives (bispropanol, bisbutamol, bispentamol, bisheptamol, bisoctinol, 4-chlorobisacetphenol and biscyclohexylphenol) are added to the tea beverages. In addition, the method of the present invention is simple to operate and has high accuracy. It can also simultaneously measure bisacetphenol tincture and its derivatives, greatly shortening the measurement time, saving the amount of mobile phase reagents, and improving the measurement and analysis efficiency. It is suitable for detecting and analyzing tea beverage samples in large quantities. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the chromatogram of the negative sample determined by the method described in Example 1.

[0020] Figure 2 It is a chromatogram of the positive sample determined by the method described in Example 1, wherein A to H are diacetylphenol, dipropenol, dibutenol, diheptylphenol, dioctylphenol, 4-chlorodiacetylphenol, and dicyclohexylphenol, respectively.

[0021] Figure 3 It is a chromatogram of the standard solution measured by the method described in Example 4, wherein A to H are diacetylphenol, dipropenol, dibutenol, diheptylphenol, dioctylphenol, 4-chlorodiacetylphenol, and dicyclohexylphenol.

[0022] Figure 4 It is the positive mode secondary mass spectrum of bisacetin in the standard database described in Example 4.

[0023] Figure 5 It is the secondary mass spectrum of the positive mode of Biprofen in the standard database described in Example 4.

[0024] Figure 6 It is the positive mode secondary mass spectrum of butylphenol in the standard database described in Example 4.

[0025] Figure 7 It is the positive mode secondary mass spectrum of bispentanol in the standard database described in Example 4.

[0026] Figure 8 This is the positive mode secondary mass spectrum of diheptylbutanol in the standard database described in Example 4.

[0027] Fig. 9 It is the secondary mass spectrum of the positive mode of bisoctin in the standard database described in Example 4.

[0028] Fig.10 It is the secondary mass spectrum of the positive mode of 4-chlorobisacetphenol tincture in the standard database described in Example 4.

[0029] Fig.11 It is the positive mode secondary mass spectrum of biscyclohexylphenol in the standard database described in Example 4.

[0030] Fig.12 It is the negative mode secondary mass spectrum of bisacetin in the standard database described in Example 4.

[0031] Fig.13 It is the negative mode secondary mass spectrum of Biprofen in the standard database described in Example 4.

[0032] Fig.14 It is the negative mode secondary mass spectrum of bisbutamol in the standard database described in Example 4.

[0033] Fig.15 It is the negative mode secondary mass spectrum of bispentanol in the standard database described in Example 4.

[0034] Fig.16 It is the negative mode secondary mass spectrum of diheptylbutanol in the standard database described in Example 4.

[0035] Fig.17 It is the negative mode secondary mass spectrum of bisoctin in the standard database described in Example 4.

[0036] Fig.18 It is the negative mode secondary mass spectrum of 4-chlorobisacetol tincture in the standard database described in Example 4.

[0037] Fig.19 It is the negative mode secondary mass spectrum of biscyclohexylphenol in the standard database described in Example 4. DETAILED DESCRIPTION

[0038] The present invention is further described by the following examples, but is not intended to be limiting of the present invention. Specific experimental conditions and methods not specified in the following examples are conventional methods commonly known to those skilled in the art.

[0039] Example 1: A method for determining bisacetyl butyl and its derivatives in tea beverages by high-resolution mass spectrometry, comprising the following steps: S1. 1.00 mL of the tea beverage sample is taken and placed in a 25.00 mL volumetric flask, followed by adding 15 mL of acetic acid methanol solution, followed by ultrasonic extraction for 25 min, and then cooled to room temperature and added with acetic acid acetonitrile solution to the scale, followed by shaking to obtain a test solution A; the acetic acid methanol solution is obtained by mixing acetic acid and methanol in a volume ratio of 0.5:100; the acetic acid acetonitrile solution is obtained by mixing acetic acid and acetonitrile in a volume ratio of 0.5:100; S2, treating the test solution A obtained in step S1 with a cation exchange resin, and then subjecting the solution to refrigerated centrifugation at a temperature of 5° C. and a speed of 9000 r / min for 5 min, and then filtering the solution with a 0.2 μm organic filter membrane to obtain the test solution B; The cation exchange resin is a modified cation exchange resin. The preparation method of the modified cation exchange resin is as follows: take deionized water 3 times the mass of the cation exchange resin, then add the cation exchange resin, chitosan and glutaraldehyde in sequence, then heat to 70°C and stir for reaction for 25 minutes, then add nano hydroxyapatite and pectin and ultrasonic treatment for 15 minutes, then continue to stir for reaction at 65°C for 2.5 hours, filter to obtain a precipitate, and then dry the precipitate to obtain the modified cation exchange resin; the mass ratio of the cation exchange resin, chitosan, nano hydroxyapatite, pectin and glutaraldehyde is 100:25:16:8:2; S3. Take the test solution B obtained in step S2 and measure it by liquid phase tandem quadrupole time-of-flight mass spectrometry. The chromatographic parameters are as follows: an octadecylsilane bonded silica gel chromatographic column with a size of 2.1×50 mm and 1.8 um is used, the injection volume is 3 μL, the column oven temperature is 45°C, and the mobile phase is composed of a 0.1% volume fraction of formic acid aqueous solution and acetonitrile; the mass spectrometry parameters are as follows: the drying gas temperature is 210°C, the drying gas flow rate is 14 L / min, the nebulizer pressure is 35 Psi, the sheath gas temperature is 345°C, the sheath gas flow rate is 11 L / min, the capillary voltage is 3500 V, the nozzle voltage is 1000 V, the pyrolysis voltage is 380 V, and the scanning mode is the targeted secondary acquisition mode (Targeted MS / MS), and includes positive mode and / or negative mode, the scanning range of level 1 is 105-1000m / z, the scanning range of level 2 is 110-575m / z, and the collision energy is 20V, 40V, 45V; The mobile phase gradient is: S4, comparing the determination result obtained in step S3 with the standard data in the standard database to determine whether the tea beverage sample contains bisacetphenidyl, bispropanol, bisbutamol, bispentamol, bisheptamol, bisoctanoylphenol, 4-chlorobisacetphenidyl, and biscyclohexylphenol; The detection chromatogram of the negative sample is as follows Figure 1 As shown, the detection chromatogram of the positive sample is as follows Figure 2 shown.

[0040] The results of the sample determination are matched with the database. When the deviation of the precise molecular weight of the substance calculated by the instrument workstation is less than 5ppm, and the deviation of the secondary mass spectrometry fragment ion is less than 10ppm, that is, the number of identification points is greater than or equal to 4 (calculated according to Table 5 of the EU Non-mandatory Regulation 2002-657-EC), it can be confirmed that the sample contains this compound; The identification point number is calculated as follows: the identification point number of the parent ion is 2, and the identification point number of each product ion (daughter ion) obtained by the parent ion conversion is 2.5. According to the qualitative requirements, the high-resolution mass spectrometer (liquid phase tandem quadrupole time-of-flight mass spectrometer) contains at least one parent ion and one product ion (daughter ion) converted from the parent ion; The method can be used to determine the substances of bisacetaminophen, bispropanol, bisbutanol, bispentanol, bisheptanol, bisoctinol, 4-chlorobisacetaminophen and dicyclohexylphenol in tea beverages. The deviation of the accurate mass number of the primary mass spectrum of each substance is less than 5ppm through database matching, and the deviation of the secondary mass spectrum product ion is less than 10ppm, which can meet the four qualitative identification points. The content of the compound can be calculated by using the corresponding standard for the identified compound, and the specificity is strong. The quantitative method is to use each reference substance to quantify the identified bisacetphenbutamol, bispropanol, bisbutamol, bispentamol, bisheptamol, bisoctinol, 4-chlorobisacetphenbutamol, and biscyclohexylphenol by the external standard method. The quantitative ion is the daughter ion, and the qualitative ion is the parent ion corresponding to the compound. The accurate mass-to-charge ratio of the parent ion and the daughter ion is used for further qualitative analysis of the compound.

[0041] Example 2: A method for determining bisacetaminophen and its derivatives in tea beverages by high-resolution mass spectrometry, comprising the following steps: S1. 1.00 mL of the tea beverage sample is taken and placed in a 25.00 mL volumetric flask, followed by adding 15 mL of acetic acid methanol solution, followed by ultrasonic extraction for 25 min, and then cooled to room temperature and added with acetic acid acetonitrile solution to the scale, followed by shaking to obtain a test solution A; the acetic acid methanol solution is obtained by mixing acetic acid and methanol in a volume ratio of 0.2:100; the acetic acid acetonitrile solution is obtained by mixing acetic acid and acetonitrile in a volume ratio of 0.3:100; S2, treating the test solution A obtained in step S1 with a cation exchange resin, then subjecting the solution to refrigerated centrifugation at a temperature of 5° C. and a speed of 9500 r / min for 5 min, and then filtering the solution with a 0.2 μm organic filter membrane to obtain the test solution B; The cation exchange resin is a modified cation exchange resin. The preparation method of the modified cation exchange resin is as follows: taking deionized water twice the mass of the cation exchange resin, then sequentially adding the cation exchange resin, chitosan and glutaraldehyde, then heating to 60° C. and stirring for reaction for 30 minutes, then adding nano-hydroxyapatite and pectin and ultrasonic treatment for 10 minutes, then continuing to stir and react at 60° C. for 3 hours, filtering to obtain a precipitate, and then drying the precipitate to obtain the modified cation exchange resin; the mass ratio of the cation exchange resin, chitosan, nano-hydroxyapatite, pectin and glutaraldehyde is 100:20:15:5:3; S3. Take the test solution B obtained in step S2 and measure it by liquid phase tandem quadrupole time-of-flight mass spectrometry. The chromatographic parameters are as follows: an octadecylsilane bonded silica gel chromatographic column with a size of 2.1×50 mm and 1.8 um is used, the injection volume is 3 μL, the column oven temperature is 45°C, and the mobile phase is composed of a 0.1% volume fraction of formic acid aqueous solution and acetonitrile; the mass spectrometry parameters are as follows: the drying gas temperature is 210°C, the drying gas flow rate is 14 L / min, the nebulizer pressure is 35 Psi, the sheath gas temperature is 345°C, the sheath gas flow rate is 11 L / min, the capillary voltage is 3500 V, the nozzle voltage is 1000 V, the pyrolysis voltage is 380 V, and the scanning mode is the targeted secondary acquisition mode (Targeted MS / MS), and includes positive mode and / or negative mode, the scanning range of level 1 is 105-1000m / z, the scanning range of level 2 is 110-575m / z, and the collision energy is 20V, 40V, 45V; The mobile phase gradient is: S4. Compare the determination result obtained in step S3 with the standard data in the standard database to determine whether the tea beverage sample contains bisacetphenidyl, bispropanol, bisbutamol, bispentamol, bisheptamol, bisoctinol, 4-chlorobisacetphenidyl, and biscyclohexylphenol.

[0042] Example 3: A method for determining bisacetyl and its derivatives in tea beverages by high-resolution mass spectrometry, comprising the following steps: S1. 1.00 mL of the tea beverage sample is taken and placed in a 25.00 mL volumetric flask, followed by adding 15 mL of acetic acid methanol solution, followed by ultrasonic extraction for 25 min, and then cooled to room temperature and added with acetic acid acetonitrile solution to the scale, followed by shaking to obtain a test solution A; the acetic acid methanol solution is obtained by mixing acetic acid and methanol in a volume ratio of 1:100; the acetic acid acetonitrile solution is obtained by mixing acetic acid and acetonitrile in a volume ratio of 0.8:100; S2, treating the test solution A obtained in step S1 with a cation exchange resin, then subjecting the solution to refrigerated centrifugation at a temperature of 5° C. and a speed of 10,000 r / min for 5 min, and then filtering the solution with a 0.2 μm organic filter membrane to obtain the test solution B; The cation exchange resin is a modified cation exchange resin. The preparation method of the modified cation exchange resin is as follows: deionized water 3 times the mass of the cation exchange resin is taken, and then the cation exchange resin, chitosan and glutaraldehyde are added in sequence, and then the mixture is heated to 80° C. and stirred for reaction for 20 minutes, and then nano-hydroxyapatite and pectin are added and ultrasonically treated for 10 minutes, and then the mixture is stirred and reacted at 70° C. for 2 hours, and a precipitate is obtained by suction filtration, and then the precipitate is dried to obtain the modified cation exchange resin; the mass ratio of the cation exchange resin, chitosan, nano-hydroxyapatite, pectin and glutaraldehyde is 100:30:20:10:5; S3. Take the test solution B obtained in step S2 and measure it by liquid phase tandem quadrupole time-of-flight mass spectrometry. The chromatographic parameters are as follows: an octadecylsilane bonded silica gel chromatographic column with a size of 2.1×50 mm and 1.8 um is used, the injection volume is 3 μL, the column oven temperature is 45°C, and the mobile phase is composed of a 0.1% volume fraction of formic acid aqueous solution and acetonitrile; the mass spectrometry parameters are as follows: the drying gas temperature is 210°C, the drying gas flow rate is 14 L / min, the nebulizer pressure is 35 Psi, the sheath gas temperature is 345°C, the sheath gas flow rate is 11 L / min, the capillary voltage is 3500 V, the nozzle voltage is 1000 V, the pyrolysis voltage is 380 V, and the scanning mode is the targeted secondary acquisition mode (Targeted MS / MS), and includes positive mode and / or negative mode, the scanning range of level 1 is 105-1000 m / z, the scanning range of level 2 is 110-575 m / z, and the collision energy is 20V, 40V, 45V; The mobile phase gradient is: S4. Compare the determination result obtained in step S3 with the standard data in the standard database to determine whether the tea beverage sample contains bisacetphenidyl, bispropanol, bisbutamol, bispentamol, bisheptamol, bisoctinol, 4-chlorobisacetphenidyl, and biscyclohexylphenol.

[0043] Example 4: A standard database is established by using the method for determining bisacetaminophen and its derivatives in tea beverages using high-resolution mass spectrometry, which comprises the following steps: S1, take standard products of bisacetphenbutamol, bispropanol, bisbutamol, bispentanol, bisheptanol, bisoctinol, 4-chlorobisacetphenbutamol and biscyclohexylphenol butamol to prepare a series of standard solutions of concentration, and use the standard solutions as samples for detection, draw 1.00 mL of the sample and place it in a 25.00 mL volumetric flask, then add 15 mL of acetic acid methanol solution, then ultrasonically extract for 25 min, cool to room temperature, add acetic acid acetonitrile solution to the scale, and then shake to obtain a test solution A; the acetic acid methanol solution is obtained by mixing acetic acid and methanol in a volume ratio of 0.5:100; the acetic acid acetonitrile solution is obtained by mixing acetic acid and acetonitrile in a volume ratio of 0.5:100; S2, treating the test solution A obtained in step S1 with a cation exchange resin, and then subjecting the solution to refrigerated centrifugation at a temperature of 5° C. and a speed of 9000 r / min for 5 min, and then filtering the solution with a 0.2 μm organic filter membrane to obtain the test solution B; The cation exchange resin is a modified cation exchange resin. The preparation method of the modified cation exchange resin is as follows: take deionized water 3 times the mass of the cation exchange resin, then add the cation exchange resin, chitosan and glutaraldehyde in sequence, then heat to 70°C and stir for reaction for 25 minutes, then add nano hydroxyapatite and pectin and ultrasonic treatment for 15 minutes, then continue to stir for reaction at 65°C for 2.5 hours, filter to obtain a precipitate, and then dry the precipitate to obtain the modified cation exchange resin; the mass ratio of the cation exchange resin, chitosan, nano hydroxyapatite, pectin and glutaraldehyde is 100:25:16:8:2; S3. Take the test solution B obtained in step S2 and measure it by liquid phase tandem quadrupole time-of-flight mass spectrometry. The chromatographic parameters are as follows: an octadecylsilane bonded silica gel chromatographic column with a size of 2.1×50 mm and 1.8 um is used, the injection volume is 3 μL, the column oven temperature is 45°C, and the mobile phase is composed of a 0.1% volume fraction of formic acid aqueous solution and acetonitrile; the mass spectrometry parameters are as follows: the drying gas temperature is 210°C, the drying gas flow rate is 14 L / min, the nebulizer pressure is 35 Psi, the sheath gas temperature is 345°C, the sheath gas flow rate is 11 L / min, the capillary voltage is 3500 V, the nozzle voltage is 1000 V, the pyrolysis voltage is 380 V, and the scanning mode is the targeted secondary acquisition mode (Targeted MS / MS), and includes positive mode and / or negative mode, the scanning range of level 1 is 105-1000m / z, the scanning range of level 2 is 110-575m / z, and the collision energy is 20V, 40V, 45V; The mobile phase gradient is: S4. A standard database is established based on the obtained measurement results, wherein the standard database includes the following information data corresponding to bisacetphenbutamol, bispropanol, bisbutamol, bispentamol, bisheptamol, bisoctinol, 4-chlorobisacetphenbutamol and biscyclohexylphenol: CAS number, molecular formula, accurate mass-to-charge ratio of parent ion (m / z), accurate mass-to-charge ratio of daughter ion (m / z), polarity mode, secondary mass spectrometry trigger time (min), secondary mass spectrometry trigger time range (min); the measurement results of some standard solutions are shown in Figure 3 The secondary mass spectra of each compound in the positive mode and negative mode in the established standard database are shown in Tables 1 and 2 and Figures 4 to 19 .

[0044] Table 1:

[0045] Table 2:

[0046] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for determining bisacetyl butyl and its derivatives in tea beverages by high resolution mass spectrometry, characterized in that: The following steps are involved: S1. Pipette 1.00 mL of the tea beverage sample into a 25.00 mL volumetric flask, then add 15 mL of acetic acid methanol solution, then perform ultrasonic extraction for 25 min, cool to room temperature, add acetic acid acetonitrile solution to the mark, then shake well to obtain test solution A; S2, placing the test solution A obtained in step S1 at a temperature of 5° C. and a speed of 9000-10000 r / min for refrigerated centrifugation for 5 min, and then filtering with a 0.2 μm organic filter membrane to obtain the test solution B; S3, taking the test liquid B obtained in step S2 and measuring it by liquid phase tandem quadrupole time-of-flight mass spectrometry, the chromatographic parameters are: using an octadecylsilane bonded silica gel chromatographic column with a size of 2.1×50mm and 1.8um, the injection volume is 3μL, the column oven temperature is 45°C, and the mobile phase is composed of a 0.1% volume fraction of formic acid aqueous solution and acetonitrile; the mass spectrometry parameters are: the drying gas temperature is 210°C, the drying gas flow rate is 14L / min, the nebulizer pressure is 35Psi, the sheath gas temperature is 345°C, the sheath gas flow rate is 11L / min, the capillary voltage is 3500V, the nozzle voltage is 1000V, the pyrolysis voltage is 380V, the scanning mode is the target secondary acquisition mode, and includes positive mode and / or negative mode, the scanning range of level 1 is 105-1000m / z, the scanning range of level 2 is 110-575m / z, and the collision energy is 20V, 40V, 45V; S4. Compare the determination result obtained in step S3 with the standard data in the standard database to determine whether the tea beverage sample contains bisacetphenidyl, bispropanol, bisbutamol, bispentamol, bisheptamol, bisoctinol, 4-chlorobisacetphenidyl, and biscyclohexylphenol.

2. The method for determining bisacetyl butyl and its derivatives in tea beverages by high resolution mass spectrometry according to claim 1, characterized in that: The acetic acid-methanol solution in step S1 is obtained by mixing acetic acid and methanol in a volume ratio of (0.2-1):

100.

3. The method for determining bisacetyl and its derivatives in tea beverages by high resolution mass spectrometry according to claim 1, characterized in that: The acetic acid-acetonitrile solution in step S1 is obtained by mixing acetic acid and acetonitrile in a volume ratio of (0.3-0.8):

100.

4. The method for determining bisacetyl and its derivatives in tea beverages by high resolution mass spectrometry according to claim 1, characterized in that: In step S2, the test solution A obtained in step S1 is treated with a cation exchange resin, then subjected to refrigerated centrifugation at a temperature of 5°C and a speed of 9000-10000 r / min for 5 minutes, and then filtered using a 0.2 μm organic filter membrane to obtain the test solution B.

5. The method for determining bisacetyl butyl and its derivatives in tea beverages by high resolution mass spectrometry according to claim 1, characterized in that: The cation exchange resin is a modified cation exchange resin. The preparation method of the modified cation exchange resin is as follows: take deionized water 2 to 3 times the mass of the cation exchange resin, then add the cation exchange resin, chitosan and glutaraldehyde in sequence, then heat to 60 to 80°C and stir to react for 20 to 30 minutes, then add nano hydroxyapatite and pectin and ultrasonically treat for 10 to 15 minutes, then continue to stir and react at 60 to 70°C for 2 to 3 hours, filter to obtain a precipitate, and then dry the precipitate to obtain the modified cation exchange resin; the mass ratio of the cation exchange resin, chitosan, nano hydroxyapatite, pectin and glutaraldehyde is 100: (20 to 30): (15 to 20): (5 to 10): (3 to 5).

6. The method for determining bisacetyl butyl and its derivatives in tea beverages by high resolution mass spectrometry according to claim 1, characterized in that: In the determination chromatographic parameters in step S3, the mobile phase gradient is: 。 7. The method for determining bisacetyl and its derivatives in tea beverages by high resolution mass spectrometry according to claim 1, characterized in that: The standard database in step S4 is prepared by mixing standard products of bisacetphenidine, bispropanol, bisbutamol, bispentamol, bisheptamol, bisoctinol, 4-chlorobisacetphenidine and biscyclohexylphenol to prepare a series of standard solutions of concentrations, and then measuring the standard solutions according to the methods described in steps S1 to S3, and establishing the standard database according to the obtained measurement results.

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