Integrated method for high-throughput screening and accurate confirmation of multiple synthetic pigments in Liupao tea based on UHPLC-Q-TOFMS (Ultra High Performance Liquid Chromatography-Quantitative Time of Flight Mass Spectrometry)

Abstract

The invention discloses an integrated method for high-throughput screening and accurate confirmation of multiple synthetic pigments in Liupao tea based on UHPLC-Q-TOFMS, and belongs to the technical field of tea component determination. By means of the method, whether a Liupao tea sample contains 17 synthetic pigments or not can be rapidly subjected to qualitative screening and quantification; the method has the characteristics of simplicity and convenience in operation, high sensitivity and low cost; comprising the following steps: (1) respectively taking 17 synthetic pigment standard substances to prepare pigment standard stock solutions with different numbers and series standard curve solutions, and measuring to obtain spectrum information of pigments with different numbers and spectrum information of series standard curves; (2) collecting data information of each synthetic pigment from different pigment spectrum information, and then establishing a comparison database; (3) taking a Liupao tea sample, and measuring to obtain Liupao tea sample spectrum information; and (4) determining whether the Liupao tea sample contains the synthetic pigments or not after matching by using qualitative software, and calculating the content of the synthetic pigments in the Liupao tea sample.

Description

Technical Field

[0001] This invention relates to the technical field of tea component determination, and more specifically, to an integrated method for high-throughput screening and accurate verification of multiple synthetic pigments in Liubao tea based on UHPLC-Q-TOFMS. Background Technology

[0002] The core processing technique of Liubao tea lies in "piling fermentation." Under specific conditions of high temperature and humidity, polyphenols such as catechins in the tea leaves undergo intense enzymatic and non-enzymatic oxidation, polymerization, and condensation, transforming into theaflavins, thearubigins, and even theabrownins. The combined effect of these natural pigments forms the material basis for the transformation of Liubao tea from the "orange-yellow" color of new tea to the "deep red and bright" color of aged tea. Subsequent aging and fermentation under suitable conditions further stabilizes the tea liquor's color, making it increasingly bright, clear, and mellow in taste. The iconic "red liquor" of Liubao tea is an aesthetic and flavor crystallization formed naturally by the transformation of polyphenols within the tea leaves under the influence of heat, humidity, and microorganisms after a complex "piling fermentation" process and long aging.

[0003] Generally, the "brightness" and "concentration" of the tea liquor are the primary visual indicators for directly judging the age, fermentation process, and value of Liubao tea. A Liubao tea with an attractively bright red liquor often signifies a better degree of fermentation, longer aging time, and a higher market price. However, some unscrupulous merchants, seeking exorbitant profits, sell inferior tea and artificially enhance its color: using cheap raw materials, poorly processed tea, or even recycled "swill tea" (repackaged and sold after processing used tea leaves) as a base, they add synthetic pigments to directly adjust the color of the dry tea or the brewed liquor, instantly creating the illusion of a "deep red," thus passing it off as high-quality or aged tea, infringing on legal rights, and posing food safety risks.

[0004] Currently, the main detection methods for potentially illegally added exogenous synthetic pigments (such as amaranth red and sunset yellow) in Liubao tea are as follows: 1) At the consumer level, consumers can make a preliminary judgment by rubbing the dry tea by hand to observe whether the color fades, or by soaking it in cold water to observe whether the tea liquor color slowly and naturally separates; 2) At the professional rapid screening level, colloidal gold immunochromatographic test strips can be used for on-site qualitative screening of specific pigments; 3) For enforcement and confirmation, GB 31608-2023 "National Food Safety Standard for Tea" explicitly prohibits the addition of any food additives to tea, and synthetic pigments (such as amaranth red and sunset yellow) are "not detectable" in tea. Current methods for detecting synthetic pigments mainly rely on GB 5009.35-2023, "National Food Safety Standard - Determination of Synthetic Colorants in Food," which uses high-performance liquid chromatography (HPLC) to determine 11 synthetic colorants in food (tartrazine, New Red, Amaranth, Indigo, Carmine, Sunset Yellow, Allura Red, Brilliant Blue, Acid Red, Quinoline Yellow, and Erythrosine). While HPLC can effectively separate and quantify common pigments, it is easily affected by interference in complex tea matrices. Using HPLC alone to determine whether Liubao tea contains artificial synthetic pigments is complex, time-consuming, and has low accuracy, making it difficult to meet the industry's needs for rapid screening and quality control. Summary of the Invention

[0005] The purpose of this invention is to provide an integrated method for high-throughput screening and accurate verification of multiple synthetic pigments in Liubao tea based on UHPLC-Q-TOFMS. This method can quickly perform qualitative screening and quantitative analysis of whether Liubao tea samples contain 17 kinds of exogenous synthetic pigments. It is characterized by simple operation, high sensitivity and low cost.

[0006] The technical solution of the present invention is as follows: An integrated method for high-throughput screening and accurate verification of multiple synthetic pigments in Liubao tea based on UHPLC-Q-TOFMS includes the following steps: (1) Take 17 kinds of exogenous added synthetic pigment standards to prepare pigment standard stock solutions and a series of standard curve solutions with different numbers. Put the pigment standard stock solutions and the series of standard curve solutions with different numbers into a liquid phase tandem quadrupole time-of-flight mass spectrometer in sequence to obtain the pigment spectrum information and the spectrum information of the series of standard curves with different numbers. (2) After collecting data information of each exogenously added synthetic pigment from the pigment spectrum information with different numbers obtained in step (1), establish a comparison database and draw standard curves based on the spectrum information of the series of standard curves obtained in step (1). (3) After extracting and purifying the Liubao tea sample, the filtrate was placed in a series liquid chromatography-mass spectrometry instrument to obtain the chromatographic information of the Liubao tea sample; (4) After matching the determination chromatogram information obtained in step (3) with the comparison database established in step (2) using qualitative software, the matching scores of Liubao tea sample and each exogenous synthetic pigment are obtained. Based on the obtained matching scores, it is determined whether these exogenous synthetic pigments are present. The concentration of exogenous synthetic pigments present is determined from the chromatogram information of Liubao tea sample obtained in step (3) from the standard curve obtained in step (2). The content of exogenous synthetic pigments in Liubao tea sample is quantitatively calculated by external standard method, and the determination results are output.

[0007] Furthermore, in step (1), the 17 exogenous synthetic pigment standards are amaranth red, new red, allura red, erythrosine B, carmine, acid red, acid red 87, acid red 95, sunset yellow, quinoline yellow, basic yellow 87, basic light yellow, acid yellow 73, acid yellow 1, acid orange I, golden orange II, and acid orange 3.

[0008] Furthermore, in step (1), the preparation process of preparing pigment standard stock solution by adding each exogenous synthetic pigment standard is as follows: accurately weigh 25 mg of pigment standard, place it in a 25 mL volumetric flask, and dilute to the mark with 10% methanol to obtain standard stock solution. The specific process for preparing a series of standard curve solutions is as follows: Take 100.0 μL of each of the 17 standard stock solutions into a 10.00 mL volumetric flask, dilute to the mark with 10% methanol to obtain a mixed standard solution, and use the mixed standard solution to prepare a series of standard curve solutions with mass concentrations of 10, 20, 50, 80 and 100 ng / mL with 10% methanol.

[0009] Furthermore, in step (2), the established comparison database stores data information of 17 exogenous added synthetic pigments. The data information of each exogenous added synthetic pigment includes the name of the compound, the mass number of the primary mass spectrometer ion, the ion addition form, the retention time, and the mass number of the secondary fragment.

[0010] Furthermore, in step (2), the spectral information of the obtained series of standard curves is plotted with the concentration of the analyte as the abscissa and the peak area of ​​the analyte as the ordinate to obtain the standard curve.

[0011] Furthermore, the extraction and purification process of the Liubao tea sample in step (3) is as follows: Weigh 5 g of the Liubao tea sample into a 50 mL centrifuge tube, add 40 mL of 10% methanol, extract ultrasonically for 5 min, centrifuge at 4000 r / min for 5 min, transfer the supernatant to a 100 mL volumetric flask, repeat the extraction once more, combine the supernatants, and dilute to the mark with 10% methanol. Take 1.5 mL of the supernatant until it contains 25 mg of C. 18The material was placed in a purification tube, vortexed for 1 min, centrifuged at 8000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm PTFE membrane.

[0012] Furthermore, in steps (1) and (3), different numbered pigment standard stock solutions and series of standard solutions or filtrates are placed in a liquid chromatography-tandem quadrupole time-of-flight mass spectrometer for determination, including liquid chromatography determination and mass spectrometry determination: The liquid chromatography determination conditions are as follows: Chromatographic column: Poroshell 120PFP, dimensions 2.1 mm × 150 mm, 1.9 μm; injection volume: 2 μL; column temperature: 30℃; flow rate: 0.3 mL / min; mobile phase A was 5 mmol / L ammonium acetate aqueous solution, and B was methanol; gradient elution mode was used: 0 min: 90% A + 10% B; 0–3 min: 90% A + 10% B; 3–12 min: 20% A + 80% B; 12–15 min: 20% A + 80% B; The mass spectrometry determination conditions are as follows: Drying gas temperature: 200℃; Drying gas flow rate: 14L / min; Nebulizer pressure: 35Psi; Sheath gas temperature: 350℃; Sheath gas flow rate: 11L / min; Capillary voltage: 3500V; Nozzle voltage: 1000V; Pyrolysis voltage: 380V; Scanning mode: Targeted MS / MS mode, negative mode.

[0013] (4.1) The qualitative software extracts data information for each type of exogenously added synthetic pigment from the comparison database established in step (2); (4.2) Based on the mass number of the primary mass spectrometry ion of the exogenous added synthetic pigment obtained in step (4.1), check whether the compound with the mass number of the corresponding primary mass spectrometry ion exists in the determination spectrum information obtained in step (3). If it exists, obtain the other data information of the compound to obtain the data information of the compound in the sample. If it does not exist, set the other information of the compound to zero to obtain the data information of the compound in the sample. (4.3) The qualitative software will compare the data information of the exogenous synthetic pigment extracted from the comparison database with the data information of the compound in the sample obtained in step (4.1) and output the matching score of the exogenous synthetic pigment. (4.4) The qualitative software determines whether the matching of all exogenous added synthetic pigments in the database has been completed. If not, repeat step (4.1). If completed, the matching score of each exogenous added synthetic pigment is judged one by one. If the matching score is greater than or equal to 85, it is determined that the Liubao tea sample contains the exogenous added synthetic pigment and proceeds to the next step. If the matching score is less than 85, it is determined that the sample does not contain the exogenous added synthetic pigment and returns to step (4.1). (4.5) Read the Liubao tea chromatogram information obtained in step (3) from the standard curve obtained in step (2) to determine the concentration of exogenous added synthetic pigments. Quantitatively calculate the content of exogenous added synthetic pigments in the Liubao tea sample using the external standard method and output the measurement results.

[0014] Furthermore, in step (4.5), the content of the exogenous synthetic pigment is calculated using the external standard method. The specific process is as follows: In step (4.5), the content of the exogenous synthetic pigment is quantitatively calculated using the external standard method. The specific process is as follows: The ion peak area corresponding to the exogenous synthetic pigment is obtained from the standard curve obtained in step (2) and the determination spectrum information obtained in step (3). The concentration of the exogenous synthetic pigment contained in the Liubao tea sample is read from the standard curve. The calculation formula is as follows: ; in, X The content (mg / kg) of exogenously added synthetic pigments in the sample; c To obtain the concentration (ng / mL) of exogenously added synthetic pigments from the standard curve; C 0 represents the concentration of the blank sample (ng / mL); v This represents the dilution factor (mL) of the sample solution. m The sample size is in grams (g).

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention presents an integrated method for high-throughput screening and precise verification of multiple synthetic pigments in Liubao tea based on UHPLC-Q-TOFMS. Through rapid extraction, an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS) database of primary and secondary fragment ions for 17 synthetic pigments is established. In the absence of standards, preliminary qualitative screening of samples can be performed quickly, effectively reducing testing costs. When suspicious samples are found, comparisons can be made with standards based on retention time, mass number, and abundance ratio of primary and secondary ions. Targeted MS / MS scanning is then used to qualitatively screen and quantitatively detect whether the suspicious sample contains any of the 17 exogenously added synthetic pigments. The method is simple, rapid, highly sensitive, and low-cost, providing valuable technical support for the regulation of synthetic pigments in Liubao tea. Detailed Implementation

[0016] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but this does not constitute any limitation on the present invention.

[0017] This invention provides an integrated method for high-throughput screening and accurate verification of multiple synthetic pigments in Liubao tea based on UHPLC-Q-TOFMS, comprising the following steps: (1) Take 17 kinds of exogenous added synthetic pigment standards to prepare pigment standard stock solutions and a series of standard curve solutions with different numbers. Put the pigment standard stock solutions and the series of standard curve solutions with different numbers into a liquid phase tandem quadrupole time-of-flight mass spectrometer to measure the pigment spectrum information and the spectrum information of the series of standard curves with different numbers.

[0018] Furthermore, in step (1), the 17 exogenous synthetic pigment standards are amaranth, new red, allura red, erythrosine B, carmine, acid red, acid red 87, acid red 95, sunset yellow, quinoline yellow, basic yellow 87, basic light yellow, acid yellow 73, acid yellow 1, acid orange I, golden orange II, and acid orange 3. The Chinese and English names, CAS numbers, and chemical formulas of these 17 exogenous synthetic pigment standards are shown in Table 1.

[0019] Table 1. Chinese and English names, CAS numbers, and chemical formulas of 117 exogenous synthetic pigments.

[0020] Furthermore, in step (1), the preparation process of preparing pigment standard stock solution for each exogenous added synthetic pigment standard is as follows: accurately weigh 25 mg of pigment standard, place it in a 25 mL volumetric flask, dilute to the mark with 10% methanol to obtain standard stock solution, and store it in the dark at -18 ℃.

[0021] The specific process for preparing a series of standard curve solutions is as follows: Take 100.0 μL of each of the 17 standard stock solutions into a 10.00 mL volumetric flask, dilute to the mark with 10% methanol to obtain a mixed standard solution, and use the mixed standard solution to prepare a series of standard curve solutions with mass concentrations of 10, 20, 50, 80 and 100 ng / mL with 10% methanol, and store at -18 ℃ in the dark.

[0022] (2) After collecting data information of each exogenous added synthetic pigment from the pigment spectrum information with different numbers obtained in step (1), establish a comparison database and draw standard curves based on the spectrum information of the series of standard curves obtained in step (1).

[0023] Furthermore, in step (2), the established comparison database stores data information of 17 exogenous added synthetic pigments. The data information of each exogenous added synthetic pigment includes the name of the compound, the mass number of the primary mass spectrometer ion, the ion addition form, the retention time, and the mass number of the secondary fragments. The specific information is shown in Table 2.

[0024] Table 2. Retention time, primary ion, and secondary ion of 217 exogenous synthetic pigments

[0025] Furthermore, in step (2), the spectral information of the obtained series of standard curves is plotted with the concentration of the analyte as the abscissa and the peak area of ​​the analyte as the ordinate to obtain the standard curve.

[0026] (3) After extracting and purifying the Liubao tea sample, the filtrate was placed in a series liquid chromatography-mass spectrometry instrument to obtain the chromatographic information of the Liubao tea sample.

[0027] Furthermore, the extraction and purification process of the Liubao tea sample in step (3) is as follows: Weigh 5 g of the Liubao tea sample into a 50 mL centrifuge tube, add 40 mL of 10% methanol, extract ultrasonically for 5 min, centrifuge at 4000 r / min for 5 min, transfer the supernatant to a 100 mL volumetric flask, repeat the extraction once more, combine the supernatants, and dilute to the mark with 10% methanol. Take 1.5 mL of the supernatant until it contains 25 mg of C. 18 The material was placed in a purification tube, vortexed for 1 min, centrifuged at 8000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm PTFE membrane.

[0028] Furthermore, in steps (1) and (3), pigment standard stock solutions or filtrates with different numbers are placed in a liquid chromatography-tandem quadrupole time-of-flight mass spectrometer for determination, including liquid chromatography determination and mass spectrometry determination.

[0029] The liquid chromatography determination conditions are as follows: Chromatographic column: Poroshell 120PFP, 2.1mm×150mm, 1.9μm; injection volume: 2μL; column temperature: 30℃; flow rate: 0.3mL / min; mobile phase A was 5mmol / L ammonium acetate aqueous solution, and B was methanol; gradient elution mode was used: 0min: 90%A+10%B; 0~3min: 90%A+10%B; 3~12min: 20%A+80%B; 12~15min: 20%A+80%B, as detailed in Table 3.

[0030] Table 3 Gradient elution conditions for liquid chromatography

[0031] The mass spectrometry determination conditions are as follows: Drying gas temperature: 200℃; Drying gas flow rate: 14L / min; Nebulizer pressure: 35Psi; Sheath gas temperature: 350℃; Sheath gas flow rate: 11L / min; Capillary voltage: 3500V; Nozzle voltage: 1000V; Pyrolysis voltage: 380V; Scanning mode: Targeted MS / MS mode, negative mode.

[0032] (4) After matching the determination chromatogram information obtained in step (3) with the comparison database established in step (2) using qualitative software, the matching scores of Liubao tea sample and each exogenous synthetic pigment are obtained. Based on the obtained matching scores, it is determined whether these exogenous synthetic pigments are present. The concentration of exogenous synthetic pigments present is determined from the chromatogram information of Liubao tea sample obtained in step (3) from the standard curve obtained in step (2). The content of exogenous synthetic pigments in Liubao tea sample is quantitatively calculated by external standard method, and the determination results are output.

[0033] Furthermore, step (4) includes the following steps: (4.1) The qualitative software extracts data information for each type of exogenous added synthetic pigment from the comparison database established in step (2).

[0034] (4.2) Based on the mass number of the primary mass spectrometry ion of the exogenous added synthetic pigment obtained in step (4.1), check whether the compound with the mass number of the corresponding primary mass spectrometry ion exists in the determination spectrum information obtained in step (3). If it exists, obtain the other data information of the compound to obtain the data information of the compound in the sample. If it does not exist, set the other information of the compound to zero to obtain the data information of the compound in the sample.

[0035] (4.3) The qualitative software will compare the data information of the exogenous synthetic pigment extracted from the comparison database with the data information of the compound in the sample obtained in step (4.1) and output the matching score of the exogenous synthetic pigment.

[0036] (4.4) The qualitative software determines whether the matching of all exogenous added synthetic pigments in the database has been completed. If not, repeat step (4.1). If completed, the matching score of each exogenous added synthetic pigment is judged one by one. If the matching score is greater than or equal to 85, it is determined that the Liubao tea sample contains the exogenous added synthetic pigment and proceeds to the next step. If the matching score is less than 85, it is determined that the sample does not contain the exogenous added synthetic pigment and returns to step (4.1).

[0037] (4.5) Read the Liubao tea chromatogram information obtained in step (3) from the standard curve obtained in step (2) to determine the concentration of exogenous added synthetic pigments. Quantitatively calculate the content of exogenous added synthetic pigments in the Liubao tea sample using the external standard method and output the measurement results.

[0038] Furthermore, in step (4.5), the content of the exogenous synthetic pigment is quantitatively calculated using the external standard method. The specific process is as follows: the ion peak area corresponding to the exogenous synthetic pigment is obtained from the standard curve obtained in step (2) and the determination spectrum information obtained in step (3). The concentration of the exogenous synthetic pigment contained in the Liubao tea sample is read from the standard curve. The calculation formula is as follows: ; in, X The content (mg / kg) of exogenously added synthetic pigments in the sample; c To obtain the concentration (ng / mL) of exogenously added synthetic pigments from the standard curve; C 0 represents the concentration of the blank sample (ng / mL); v This represents the dilution factor (mL) of the sample solution. m The sample size is in grams (g).

[0039] This invention presents an integrated method for high-throughput screening and precise verification of multiple synthetic pigments in Liubao tea based on UHPLC-Q-TOFMS. Through rapid extraction, an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS) database of primary and secondary fragment ions for 17 synthetic pigments is established. In the absence of standards, preliminary qualitative screening of samples can be performed quickly, effectively reducing testing costs. When suspicious samples are found, comparisons can be made with standards based on retention time, mass number, and abundance ratio of primary and secondary ions. Targeted MS / MS scanning allows for quantitative analysis to determine the presence of the 17 exogenously added synthetic pigments. The method is simple, rapid, highly sensitive, and low-cost, providing valuable technical support for the regulation of synthetic pigments in Liubao tea.

[0040] To verify the effectiveness of this method, the limits of detection and limits of quantitation for 17 synthetic pigments were calculated using the exogenous addition of synthetic pigments methodology. The specific procedures for each exogenously added synthetic pigment are as follows: Detection limit test: Add 100.0 μL of the standard mixture (10 μg / mL) from step (1) to a 5g Liubao tea matrix sample, add 40 mL of 10% methanol, extract by sonication for 5 min, centrifuge at 4000 r / min for 5 min, transfer the supernatant to a 100 mL volumetric flask, repeat the extraction once more, combine the supernatants, and dilute to the mark with 10% methanol. Take 1.5 mL of the supernatant to a volume containing 25 mg C 18 The material was vortexed for 1 min in a purification tube, centrifuged at 8000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm PTFE membrane to obtain the sample to be tested. The signal-to-noise ratio of each substance was obtained by processing with qualitative software.

[0041] Limit of Quantitation (LOQ) Test: Add 250 μL of the standard mixture (10 μg / mL) from step (1) to a 5 g Liubao tea matrix sample, add 40 mL of 10% methanol, sonicate for 5 min, centrifuge at 4000 r / min for 5 min, transfer the supernatant to a 100 mL volumetric flask, repeat the extraction once more, combine the supernatants, and dilute to the mark with 10% methanol. Take 1.5 mL of the supernatant to a volume containing 25 mg C. 18 The material was vortexed for 1 min in a purification tube, centrifuged at 8000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm PTFE membrane to obtain the sample to be tested. The signal-to-noise ratio of each substance was obtained by processing with qualitative software.

[0042] The 17 different numbered standard mixtures obtained in step (1) were prepared with 10% methanol to form a series of standard curve solutions with mass concentrations of 10, 20, 50, 80, and 100 ng / mL. Standard curves were plotted with the concentration of the analyte on the x-axis and the peak area of ​​the analyte on the y-axis. The limits of detection and limits of quantitation for the 17 exogenously added synthetic pigments were calculated using signal-to-noise ratios of 3 and 10, respectively. The relevant data are detailed in Table 4.

[0043] Table 4. Linear regression equations, correlation coefficients, limits of detection, and limits of quantification for 417 exogenously added synthetic pigments.

[0044] As shown in the table above, the 17 exogenous synthetic pigments exhibited good linearity within the range of 10–100 ng / mL, with a correlation coefficient R0. 2 ≥0.9998. The detection limit is between 0.20 and 0.21 mg / kg, indicating that the instrument used has high sensitivity. The quantitation limit is between 0.48 and 0.57, indicating that the instrument can accurately quantify and ensure the reliability of low concentration data.

[0045] To further verify the effectiveness of this method, the recovery rate and precision of 17 exogenous synthetic pigments in tea matrix were calculated. The specific operation for each exogenous synthetic pigment is as follows: Spiked recovery test: Low level (0.4 mg / kg): Weigh 6 portions of Liubao tea matrix samples, add 200.0 μL of the standard mixture from step (1) to each sample, add 40 mL of 10% methanol, extract ultrasonically for 5 min, centrifuge at 4000 r / min for 5 min, transfer the supernatant to a 100 mL volumetric flask, repeat the extraction once more, combine the supernatants, and dilute to the mark with 10% methanol. Take 1.5 mL of the supernatant to a volume containing 25 mg C 18 The material was vortexed in the purification tube for 1 min, centrifuged at 8000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm PTFE filter membrane to obtain the sample to be tested. The sample was then processed using quantitative software to obtain the sample recovery rate and its relative standard deviation (RSD%) was calculated.

[0046] Medium level (1 mg / kg): Weigh 6 portions of Liubao tea matrix samples, add 200.0 μL of the standard mixture from step (1) to each sample, add 40 mL of 10% methanol, extract by sonication for 5 min, centrifuge at 4000 r / min for 5 min, transfer the supernatant to a 100 mL volumetric flask, repeat the extraction once more, combine the supernatants, and dilute to the mark with 10% methanol. Take 1.5 mL of the supernatant to a volume containing 25 mg C 18The material was placed in a purification tube, vortexed for 1 min, centrifuged at 8000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm PTFE membrane to obtain the sample to be tested. The sample was then processed using quantitative software to obtain the sample recovery rate and calculate its relative standard deviation (RSD%). High level (2 mg / kg): Weigh 6 portions of Liubao tea matrix samples, add 1000 μL of the standard mixture from step (1) to each sample, add 40 mL of 10% methanol, extract by sonication for 5 min, centrifuge at 4000 r / min for 5 min, transfer the supernatant to a 100 mL volumetric flask, repeat the extraction once more, combine the supernatants, and dilute to the mark with 10% methanol. Take 1.5 mL of the supernatant to a volume containing 25 mg C 18 The material was vortexed in the purification tube for 1 min, centrifuged at 8000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm PTFE filter membrane to obtain the sample to be tested. The sample was then processed using quantitative software to obtain the sample recovery rate and its relative standard deviation (RSD%) was calculated. The results are detailed in Table 5.

[0047] Table 5. Recovery rates and relative standard deviations of 17 exogenously added synthetic pigments (n=6)

[0048] As shown in Table 5, the recoveries of the 17 exogenous synthetic pigments in the tea matrix ranged from 75.27% to 114.58%, with relative standard deviations ranging from 0.22% to 4.57%. This indicates that the method possesses good accuracy and precision, meeting the requirements for quantitative analysis.

[0049] The above description is only a preferred embodiment of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An integrated method for high-throughput screening and accurate verification of multiple synthetic pigments in Liubao tea based on UHPLC-Q-TOFMS, characterized in that, Includes the following steps: (1) Take 17 kinds of exogenous added synthetic pigment standards to prepare pigment standard stock solutions and a series of standard curve solutions with different numbers. Put the pigment standard stock solutions and the series of standard curve solutions with different numbers into a liquid phase tandem quadrupole time-of-flight mass spectrometer in sequence to obtain the pigment spectrum information and the spectrum information of the series of standard curves with different numbers. (2) After collecting data information of each exogenously added synthetic pigment from the pigment spectrum information with different numbers obtained in step (1), establish a comparison database and draw standard curves based on the spectrum information of the series of standard curves obtained in step (1). (3) After extracting and purifying the Liubao tea sample, the filtrate was placed in a series liquid chromatography-mass spectrometry instrument to obtain the chromatographic information of the Liubao tea sample; (4) After matching the determination chromatogram information obtained in step (3) with the comparison database established in step (2) using qualitative software, the matching scores of Liubao tea sample and each exogenous synthetic pigment are obtained. Based on the obtained matching scores, it is determined whether these exogenous synthetic pigments are present. The concentration of exogenous synthetic pigments present is determined from the chromatogram information of Liubao tea sample obtained in step (3) from the standard curve obtained in step (2). The content of exogenous synthetic pigments in Liubao tea sample is quantitatively calculated by external standard method, and the determination results are output.

2. The integrated method for high-throughput screening and accurate verification of multiple synthetic pigments in Liubao tea based on UHPLC-Q-TOFMS according to claim 1, characterized in that, In step (1), the 17 exogenous synthetic pigment standards are amaranth, new red, allura red, erythrosine B, carmine, acid red, acid red 87, acid red 95, sunset yellow, quinoline yellow, basic yellow 87, basic light yellow, acid yellow 73, acid yellow 1, acid orange I, golden orange II, and acid orange 3.

3. The integrated method for high-throughput screening and accurate verification of multiple synthetic pigments in Liubao tea based on UHPLC-Q-TOFMS according to claim 2, characterized in that, In step (1), the preparation process of preparing pigment standard stock solution by adding each exogenous synthetic pigment standard is as follows: accurately weigh 25 mg of pigment standard, place it in a 25 mL volumetric flask, and dilute to the mark with 10% methanol to obtain standard stock solution. The specific process for preparing a series of standard curve solutions is as follows: Take 100.0 μL of each of the 17 standard stock solutions into a 10.00 mL volumetric flask, dilute to the mark with 10% methanol to obtain a mixed standard solution, and use the mixed standard solution to prepare a series of standard curve solutions with mass concentrations of 10, 20, 50, 80 and 100 ng / mL with 10% methanol.

4. The integrated method for high-throughput screening and accurate verification of multiple synthetic pigments in Liubao tea based on UHPLC-Q-TOFMS according to claim 1, characterized in that, In step (2), the established comparison database stores data information of 17 exogenous added synthetic pigments. The data information of each exogenous added synthetic pigment includes the name of the compound, the mass number of the primary mass spectrometer ion, the ion addition form, the retention time, and the mass number of the secondary fragment.

5. The integrated method for high-throughput screening and accurate verification of multiple synthetic pigments in Liubao tea based on UHPLC-Q-TOFMS according to claim 1, characterized in that, In step (2), the spectral information of the obtained series of standard curves is plotted with the concentration of the analyte as the abscissa and the peak area of ​​the analyte as the ordinate to obtain the standard curve.

6. The integrated method for high-throughput screening and accurate verification of multiple synthetic pigments in Liubao tea based on UHPLC-Q-TOFMS according to claim 1, characterized in that, The specific steps of extracting and purifying the Liubao tea sample in step (3) are as follows: Weigh 5 g of the Liubao tea sample into a 50 mL centrifuge tube, add 40 mL of 10% methanol, extract ultrasonically for 5 min, centrifuge at 4000 r / min for 5 min, transfer the supernatant to a 100 mL volumetric flask, repeat the extraction once more, combine the supernatants, and dilute to the mark with 10% methanol. Take 1.5 mL of the supernatant until it contains 25 mg of C. 18 The material was placed in a purification tube, vortexed for 1 min, centrifuged at 8000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm PTFE membrane.

7. The integrated method for high-throughput screening and accurate verification of multiple synthetic pigments in Liubao tea based on UHPLC-Q-TOFMS according to claim 1, characterized in that, In steps (1) and (3), different numbered pigment standard stock solutions and series of standard solutions or filtrates are placed in a liquid chromatography-tandem quadrupole time-of-flight mass spectrometer for determination, including liquid chromatography determination and mass spectrometry determination: The liquid chromatography determination conditions are as follows: Chromatographic column: Poroshell 120PFP, dimensions 2.1 mm × 150 mm, 1.9 μm; injection volume: 2 μL; column temperature: 30℃; flow rate: 0.3 mL / min; mobile phase A was 5 mmol / L ammonium acetate aqueous solution, and B was methanol; gradient elution mode was used: 0 min: 90% A + 10% B; 0–3 min: 90% A + 10% B; 3–12 min: 20% A + 80% B; 12–15 min: 20% A + 80% B; The mass spectrometry determination conditions are as follows: Drying gas temperature: 200℃; Drying gas flow rate: 14L / min; Nebulizer pressure: 35Psi; Sheath gas temperature: 350℃; Sheath gas flow rate: 11L / min; Capillary voltage: 3500V; Nozzle voltage: 1000V; Pyrolysis voltage: 380V; Scanning mode: Targeted MS / MS mode, negative mode.

8. The integrated method for high-throughput screening and accurate verification of multiple synthetic pigments in Liubao tea based on UHPLC-Q-TOFMS according to claim 4, characterized in that, Step (4) includes the following steps: (4.1) The qualitative software extracts data information for each type of exogenously added synthetic pigment from the comparison database established in step (2); (4.2) Based on the mass number of the primary mass spectrometry ion of the exogenous added synthetic pigment obtained in step (4.1), check whether the compound with the mass number of the corresponding primary mass spectrometry ion exists in the determination spectrum information obtained in step (3). If it exists, obtain the other data information of the compound to obtain the data information of the compound in the sample. If it does not exist, set the other information of the compound to zero to obtain the data information of the compound in the sample. (4.3) The qualitative software will compare the data information of the exogenous synthetic pigment extracted from the comparison database with the data information of the compound in the sample obtained in step (4.1) and output the matching score of the exogenous synthetic pigment. (4.4) The qualitative software determines whether the matching of all exogenous added synthetic pigments in the database has been completed. If not, repeat step (4.1). If completed, the matching score of each exogenous added synthetic pigment is judged one by one. If the matching score is greater than or equal to 85, it is determined that the Liubao tea sample contains the exogenous added synthetic pigment and proceeds to the next step. If the matching score is less than 85, it is determined that the sample does not contain the exogenous added synthetic pigment and returns to step (4.1). (4.5) Read the Liubao tea chromatogram information obtained in step (3) from the standard curve obtained in step (2) to determine the concentration of exogenous added synthetic pigments. Quantitatively calculate the content of exogenous added synthetic pigments in the Liubao tea sample using the external standard method and output the measurement results.

9. The integrated method for high-throughput screening and accurate verification of multiple synthetic pigments in Liubao tea based on UHPLC-Q-TOFMS according to claim 8, characterized in that, In step (4.5), the content of the exogenous synthetic pigment is quantitatively calculated using the external standard method. The specific process is as follows: the ion peak area corresponding to the exogenous synthetic pigment is obtained from the standard curve obtained in step (2) and the determination spectrum information obtained in step (3). The concentration of the exogenous synthetic pigment contained in the Liubao tea sample is read from the standard curve. The calculation formula is as follows: ； in, X The content of exogenously added synthetic pigments in the sample; c To obtain the concentration of exogenously added synthetic pigments from the standard curve; C 0 represents the concentration of the blank sample; v This is the dilution factor of the sample solution; m This is the sample size.

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