Determination of anthocyanins in tea by liquid chromatography-mass spectrometry

Abstract

The present invention provides a liquid chromatography-mass spectrometry detection method for anthocyanins in tea leaves, which belongs to the field of quality inspection technology. The method comprises: preparing a system standard working solution: weighing and dissolving the standard products of 6 anthocyanins, and then diluting them step by step to obtain a series of standard working solutions; preparing a sample solution: taking a tea sample to be tested, extracting and centrifuging it, passing the supernatant through an aqueous filter membrane, and dividing the filtered extract into two groups, one group is diluted and the other group is not diluted, and both groups of extracts are sample solutions to be tested; HPLC-MS / MS detection: injecting the series of standard working solutions and the sample solution to be tested into a liquid chromatography-mass spectrometry instrument under the same conditions for analysis, thereby determining the content of each anthocyanin in the sample; the 6 anthocyanins are: pelargonidin, cyanidin, peonidin, delphinidin, morning glory pigment, and malva pigment. This method can accurately detect the content of 6 common anthocyanin monomers in food.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection, and particularly relates to a liquid chromatography-mass spectrometry detection method for anthocyanins in tea. Background Art

[0002] Anthocyanins are a common class of natural plant water-soluble pigments belonging to the flavonoid class. They appear red or purple in acidic environments and blue in alkaline environments. They can make fruits, vegetables, flowers and other plants appear colorful, and the depth of their color is proportional to the anthocyanin content. As a natural food pigment, anthocyanins are abundant, non-toxic and safe, and have high nutritional value and pharmacological effects. They can be used to prevent and treat some diseases. Because tea anthocyanins have a bitter taste and tea made from anthocyanin-rich purple buds and leaves is of poor quality, previous research has tended to reduce anthocyanin accumulation. However, with changes in market demand, tea with high anthocyanin content has gained attention due to its unique quality characteristics and properties, and the development of anthocyanin-rich tea products has become a hot topic.

[0003] With recent advances in scientific research, a variety of methods for the quantitative analysis of anthocyanins have been reported. The most widely used methods include spectrophotometry, HPLC, and HPLC-MS / MS. Spectrophotometry can be used to determine the total amount of anthocyanins, HPLC is suitable for analyzing different anthocyanins in a sample, and HPLC-MS / MS can not only quantitatively detect anthocyanins but also identify their types. However, research on anthocyanin content has mostly focused on well-known plants with high anthocyanin content, such as blueberries, purple sweet potatoes, and black wolfberries. Anthocyanins are also a very important substance in tea, found in 80% of tea leaves, especially purple buds, which have been the subject of much research in recent years. Their anthocyanin content accounts for 0.5% to 1.0% of the dry matter. Therefore, establishing an HPLC-MS / MS method for detecting anthocyanin content in tea is also of great significance. Summary of the Invention

[0004] In view of this, the present invention provides a liquid chromatography-mass spectrometry detection method for anthocyanins in tea. The method of the present invention can accurately detect the contents of six anthocyanin monomers, namely, pelargonidin, cyanidin, paeoniflorin, delphinidin, morning glory pigment, and malvidin, and has the characteristics of simple, rapid and accurate operation.

[0005] The technical solutions of the present invention are as follows:

[0006] The present invention provides a liquid chromatography-mass spectrometry method for detecting anthocyanins in tea leaves, the method comprising:

[0007] S1. Preparation of system standard working solution: Weigh and dissolve the six anthocyanin standards, and then dilute them step by step to obtain a series of standard working solutions;

[0008] S2. Preparation of sample solution: Take the tea sample to be tested, extract and centrifuge it, and then filter the supernatant through an aqueous filter membrane. The filtered extract is divided into two groups, one of which is diluted and the other is not diluted. Both groups of extracts are the sample solutions to be tested;

[0009] S3. HPLC-MS / MS detection: The series of standard working solutions from step S1 are sequentially injected into a liquid chromatography-mass spectrometer, and the corresponding concentrations are subjected to regression analysis using the ion peak area of ​​anthocyanins in the multiple reaction monitoring mode to obtain a standard working curve. Under the same conditions, the sample solution to be tested from step S2 is injected into a liquid chromatography-mass spectrometer to obtain the ion peak area of ​​each anthocyanin in the multiple reaction monitoring mode. Substituting the area into the standard working curve, the content of the six anthocyanins in the sample can be determined;

[0010] The six anthocyanidins are: pelargonidin, cyanidin, peonidin, delphinidin, morning glory pigment and malva pigment.

[0011] The diluted test sample solution was used to determine the content of three anthocyanidins: delphinidin, cyanidin, and pelargonidin, and the undiluted test sample solution was used to determine the content of three anthocyanidins: peonidin, morning glory pigment, and malvidin.

[0012] In one embodiment, in step S1, 10% hydrochloric acid methanol is used in the dissolution process; and in steps S1 and S2, methanol is used in the dilution process.

[0013] Anthocyanins are easily soluble in methanol, ethanol, acetone, water or their mixed solvents. In order to prevent the degradation of non-acylated anthocyanins during the extraction process, a certain concentration of hydrochloric acid or formic acid is usually added to the extraction solvent.

[0014] The preparation of the series of standard solutions is preferably carried out by weighing the standards of 6 anthocyanins, dissolving them in 10% hydrochloric acid methanol solution to prepare single standard stock solutions of 6 anthocyanins, and then taking the single standard stock solutions of 6 anthocyanins to prepare a mixed standard solution, which is then diluted stepwise with methanol to obtain a series of standard working solutions.

[0015] In one specific embodiment, in step S1, the concentrations of the six anthocyanins in the series of standard working solutions are 25 μg / L, 50 μg / L, 100 μg / L, 250 μg / L, 500 μg / L, 1000 μg / L, and 2500 μg / L.

[0016] 1. In one embodiment, in step S2, the specific steps of extracting the tea sample are as follows: adding an acidified ethanol solution to the tea sample, ultrasonicating for 50 minutes, and then water bathing at 100°C for 75 minutes; the volume ratio of ethanol, hydrochloric acid, and water in the acidified ethanol solution is 2:1:1; in step S2, the model of the aqueous phase filter membrane is a 0.22 μm aqueous phase filter membrane.

[0017] In the specific steps of tea extraction, the method further needs to be mixed after adding the acidified ethanol solution, ultrasonicating the solution and water bathing the solution; preferably, the method needs to be mixed by vortexing.

[0018] In one embodiment, the solid-liquid ratio of the tea sample to the acidified ethanol solution is 1:100 (g:mL).

[0019] In one embodiment, in step S2, the centrifugation is performed at 7500 r / min for 3 min.

[0020] In one embodiment, in step S2, the extract is diluted 100 times.

[0021] The dilution multiple of the extract can be adjusted appropriately according to the anthocyanin content in the sample.

[0022] In one embodiment, in step S3, the chromatographic conditions are:

[0023] The chromatographic column model is: Agilent Poroshell 120SB-C18, 2.1 mm × 100 mm, 2.7 μm;

[0024] Column temperature: 40°C;

[0025] Flow rate: 0.35 mL / min;

[0026] Injection volume: 2 μL;

[0027] Mobile phase: Mobile phase A is 1% formic acid in water, mobile phase B is methanol;

[0028] Mobile phase gradient elution program table:

[0029]

[0030] In one embodiment, in step S3, the mass spectrometry conditions are:

[0031] Ion source: electrospray ionization (ESI);

[0032] Scan mode: positive ion scan mode;

[0033] Detection method: multiple reaction monitoring mode (MRM);

[0034] Drying gas: nitrogen;

[0035] Atomizing gas: nitrogen;

[0036] Atomizing gas pressure: 50psi;

[0037] Ion spray voltage: 2000 V;

[0038] Drying gas temperature: 350℃;

[0039] Drying air flow rate: 11L / min;

[0040] The mass spectrometry acquisition parameters are shown in the table:

[0041]

[0042]

[0043] In a specific embodiment, the average spiked recovery of the method ranges from 91.95% to 106.99%, the RSDs are all less than 10%, the detection limit is 0.02 to 17 mg / kg, and the quantification limit is 0.07 to 55 mg / kg.

[0044] The detection limit in the present invention refers to the lowest concentration at which the method can detect the substance. The quantification limit in the present invention refers to the lowest concentration at which the method can accurately quantify the substance.

[0045] Compared with the prior art, the technical effects of the present invention are as follows:

[0046] 1. Compared with the existing high-performance liquid chromatography method that can only perform qualitative and quantitative detection by retention time, the liquid chromatography-mass spectrometry method used in the present invention can simultaneously and more accurately detect the contents of six anthocyanin monomers in the test sample: pelargonidin, cyanidin, paeoniflorin, delphinidin, morning glory pigment, and malvidin.

[0047] 2. The method showed good linearity in the concentration range of 25-2500 μg / L. The average spiked recoveries ranged from 91.95% to 106.99%, with RSDs less than 10%. The limits of detection were 0.02-17 mg / kg, and the limits of quantification were 0.07-55 mg / kg. The stability and precision met the requirements of relevant standards.

[0048] 3. This method is simple to operate, has good sensitivity and high accuracy, and can be applied to related analytical testing work. It can provide a reference for the establishment of standard methods for the detection of anthocyanin content in tea at home and abroad, and can also provide certain technical support for the breeding of purple bud tea trees. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1The effect of the ratio of the sample to be tested and the acidified ethanol solution of the extraction solvent on the response value;

[0050] Figure 2 The effect of the type of extraction solvent on the response value during the extraction of the sample to be tested;

[0051] Figure 3 The effect of the concentration of hydrochloric acid in the acidified ethanol solution used as the extraction solvent for the sample to be tested on the response value;

[0052] Figure 4 The effect of ultrasonic time on the response value during the extraction process of the sample to be tested;

[0053] Figure 5 The effect of water bath time on the response value during the extraction process of the test sample;

[0054] Figure 6 TIC diagrams of anthocyanin standard solutions separated by different types of chromatographic columns;

[0055] Figure 7 The TIC diagrams of the separation of anthocyanins under different mobile phase systems;

[0056] Figure 8 TIC diagram of the separation of anthocyanin standard solution at different column temperatures;

[0057] Figure 9 TIC plots for different elution programs. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0059] General Materials and Methods

[0060] (1) Instruments and equipment

[0061]

[0062] (2) Materials and reagents

[0063] Six anthocyanin standards: pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin (purity ≥ 98%) were purchased from Chengdu Derui Biotechnology Co., Ltd.

[0064] Reagents: chromatographic grade formic acid, high-grade hydrochloric acid, high-grade anhydrous ethanol, and high-grade ammonium formate were purchased from Chengdu Kelong Chemicals Co., Ltd.

[0065] Samples: The tea samples used in the experiment were purchased from tea companies in Sichuan and other provinces.

[0066] The water used in the experiment was super-stored water.

[0067] (3) Preparation of standard working solution

[0068] Accurately weigh 10.00 mg each of pelargonidin, cyanidin, paeoniflorin, delphinidin, morning glory pigment, and malva pigment standards, dissolve them in 10% hydrochloric acid in methanol, and dilute to 10 mL. Mix well to obtain different single standard stock solutions with a concentration of 1000 mg / L. Store at -20°C in the dark until use. The shelf life is 1 month.

[0069] Accurately pipette 100 μL of each of the above 6 anthocyanin single standard stock solutions to prepare 6 mixed standard solutions with a concentration of 10 mg / L, and then dilute them step by step with methanol to prepare a series of mixed standard working solutions with 6 anthocyanin concentrations of 25 μg / L, 50 μg / L, 100 μg / L, 250 μg / L, 500 μg / L, 1000 μg / L, and 2500 μg / L, which are prepared for immediate use.

[0070] (4) Sample pretreatment

[0071] Weigh 0.1000 g of tea powder into a 15 mL centrifuge tube, add 10 mL of acidified ethanol solution (ethanol: hydrochloric acid: water = 2:1:1), vortex mix first, then ultrasonicate for 50 minutes, take out, vortex mix again, then water bath at 100°C for 75 minutes, take out, vortex mix again, then centrifuge at 7500 r / min for 3 minutes, pour the supernatant into a 10 mL volumetric flask, make up to 10 mL, pass through a 0.22 μm aqueous filter membrane, and divide the filtered extract into two groups, one group is diluted 100 times with methanol, and the other group is not diluted. Both groups of extracts are samples to be tested.

[0072] Due to the low detection limit of this method, the detection of three anthocyanidins (delphinidin, cyanidin, and pelargonidin) with high concentrations in tea requires a 100-fold sample dilution. However, no sample dilution is required for the determination of the other three anthocyanidins. The dilution factor can be adjusted appropriately based on the anthocyanin content in the sample.

[0073] (5) Instrumental methods

[0074] Liquid chromatography conditions are:

[0075] The chromatographic column model is: Agilent Poroshell 120SB-C18, 2.1 mm × 100 mm, 2.7 μm;

[0076] Column temperature: 40°C;

[0077] Flow rate: 0.35 mL / min;

[0078] Injection volume: 2 μL;

[0079] Mobile phase: Mobile phase A is 1% formic acid in water, mobile phase B is methanol;

[0080] The mobile phase gradient elution program is shown in Table 1:

[0081] Table 1 Mobile phase gradient elution steps

[0082] Time (min) 1% formic acid water (%) Methanol (%) 0 70 30 5 70 30 10 65 35 20 60 40 20.1 5 95 25 5 95 25.1 70 30 33 70 30

[0083] Mass spectrometry conditions are:

[0084] Ion source: electrospray ionization (ESI);

[0085] Scan mode: positive ion scan mode;

[0086] Detection method: multiple reaction monitoring mode (MRM);

[0087] Drying gas: nitrogen;

[0088] Atomizing gas: nitrogen;

[0089] Atomizing gas pressure: 50psi;

[0090] Ion spray voltage: 2000 V;

[0091] Drying gas temperature: 350℃;

[0092] Drying air flow rate: 11L / min;

[0093] The mass spectrometry acquisition parameters are shown in Table 2.

[0094] Table 2 Mass spectrometry acquisition parameters

[0095]

[0096] Determination:

[0097] The prepared series of mixed standard working solutions were injected into HPLC-MS / MS, and regression analysis was performed using the MRM ion peak areas of the six anthocyanins and their corresponding concentrations to obtain a standard working curve, as shown in Table 3 below. Under the same conditions, the sample solution to be tested was injected into HPLC-MS / MS to obtain the MRM ion peak area of ​​each anthocyanin. Substituting the area into the standard working curve, the content of each anthocyanin in the sample to be tested was determined.

[0098] Table 3 Linear equations, correlation coefficients, linear ranges, and dilution factors of six anthocyanins

[0099]

[0100]

[0101] Example 1 Optimization of sample pretreatment conditions

[0102] 1. Optimization of material-liquid ratio

[0103] To investigate the effect of the solid-liquid ratio on the extraction rate, tea leaves were extracted with a ratio (g:mL) of 1:25, 1:50, 1:75, 1:100, and 1:125 of tea leaves to acidified ethanol solution (ethanol: hydrochloric acid: water = 2:1:1). Each group was tested three times in parallel. The response values ​​(peak areas) of anthocyanins in the test solution under each solid-liquid ratio are shown in Figure 2. Figure 1 .Depend on Figure 1 It can be seen that with the increase of the extraction solution ratio, the extraction efficiency gradually increases. The extraction rate is the highest under the condition of a solid-liquid ratio of 1:100. Further increasing the solution ratio does not significantly improve the extraction efficiency. Therefore, the solid-liquid ratio is selected as 1:100.

[0104] 2. Optimization of extraction solvent type

[0105] To investigate the effect of the type of extraction solvent on the extraction rate, tea leaves were extracted using acidified ethanol solution (ethanol: hydrochloric acid: water = 2:1:1), acidified methanol solution (methanol: hydrochloric acid: water = 2:1:1), acidified acetonitrile solution (acetonitrile: hydrochloric acid: water = 2:1:1), and acidified aqueous solution (hydrochloric acid: water = 1:3) as extraction solvents. Each group was measured in parallel three times. The response value (peak area) of anthocyanins in the test solution under each extraction solvent is shown in Figure 2 .Depend on Figure 2 It can be seen that the extraction efficiency is highest when the acidified ethanol solution (ethanol: hydrochloric acid: water = 2:1:1) is used as the extraction solvent. Therefore, the extraction solvent is selected as the acidified ethanol solution (ethanol: hydrochloric acid: water = 2:1:1).

[0106] 3. Optimization of hydrochloric acid concentration

[0107] In order to investigate the effect of hydrochloric acid concentration in the extraction solvent on the extraction rate, tea leaves were extracted with ethanol aqueous solution (ethanol: water = 2:1) with hydrochloric acid concentrations of 0.5%, 1.0%, 2.0%, 5.0%, 8.0%, 10.0%, 20.0%, 25.0%, and 30.0%, respectively. Each group was measured three times in parallel. The response value (peak area) of anthocyanins in the test solution under various hydrochloric acid concentrations is shown in Figure 3 .Depend on Figure 3As can be seen, the extraction efficiency improved to varying degrees with increasing hydrochloric acid concentration. At 25% and 30% hydrochloric acid concentrations, the extraction efficiency of each analyte was high. Considering the potential damage to the mass spectrometer from excessively high acid concentrations, the hydrochloric acid concentration was not increased. Among the six analytes, the contents of paeoniflorin and malvidin were relatively low, so hydrochloric acid concentrations that resulted in high extraction rates of paeoniflorin and malvidin were prioritized. At 25% hydrochloric acid, the extraction rates of paeoniflorin and malvidin were relatively high, so this hydrochloric acid concentration was selected.

[0108] 4. Optimization of Ultrasound Time

[0109] In order to investigate the effect of ultrasonic time on the extraction rate, tea leaves were extracted with ultrasonic time of 0, 10, 20, 30, 40, 50 and 60 min, and each group was measured three times in parallel. The response value (peak area) of anthocyanins in the test solution under different ultrasonic time conditions is shown in Figure 4 .Depend on Figure 4 It can be seen that the extraction efficiency is highest when the ultrasonic time is 50 minutes, so the ultrasonic time is selected as 50 minutes.

[0110] 5. Optimization of Water Bath Time

[0111] In order to investigate the effect of water bath time on the extraction rate, tea leaves were extracted with water bath time of 30, 45, 60, 75 and 90 min, and each group was measured three times in parallel. The response value (peak area) of anthocyanins in the test solution under different water bath time conditions is shown in Figure 5 .Depend on Figure 5 As can be seen, the extraction efficiency of each analyte was higher when the water bath time was 75 and 90 minutes. Among the six analytes, the contents of peonidin and malvidin were relatively low. Therefore, the water bath time that resulted in the highest extraction rates of peonidin and malvidin should be prioritized. The extraction rates of peonidin and malvidin were relatively high at a water bath time of 75 minutes. Therefore, a water bath time of 75 minutes was selected.

[0112] Example 2 Optimization of liquid chromatography conditions

[0113] 1. Selection of chromatographic columns

[0114] Because anthocyanins are stable only when the pH is less than 3, a low pH resistant column should be selected to ensure the stability of the chromatographic column efficiency. Based on the existing chromatographic columns and the structural characteristics of anthocyanins, the separation effects of two types of chromatographic columns on anthocyanins were investigated. The models and specifications of the chromatographic columns are shown in Table 4. The total ion current (TIC) diagram of the standard solution of anthocyanins separated by different types of chromatographic columns is shown in Table 4. Figure 6 As shown in the figure, 1: delphinium pigment; 2: cyanidin; 3: morning glory pigment; 4: pelargonidin; 5: peonyrin; 6: malva pigment. Figure 6It can be seen that the separation effect and peak shape of the Agilent Poroshell 120EC-C18 column are significantly better than those of the XSELECTTM HSS T3 column. After comprehensive consideration, the Agilent Poroshell 120EC-C18 (2.1mm×100mm, 2.7μm) column was selected as the detection column for this method.

[0115] Table 4 Models, specifications and corresponding TIC graphs of chromatographic columns used for separation of anthocyanins

[0116]

[0117] 2. Optimization of mobile phase type

[0118] After selecting the chromatographic column, the effects of eight different mobile phase systems on the response and separation of anthocyanins were investigated using mixed standard working solutions, as shown in Table 5 and Figure 7 As shown. Figure 7 As can be seen, anthocyanins elute early and exhibit poor separation in the acetonitrile system. Higher acidity in the aqueous phase improves peak separation and shape. Considering the acid resistance of the column, the maximum concentration of formic acid in water was set at 1%. Adding salt to the aqueous phase showed minimal differences in peak elution and shape compared to the absence of salt, so 1% formic acid in water was used directly as the aqueous phase. Overall, the 1% formic acid-methanol system exhibited superior separation, peak shape, and response for each analyte compared to other mobile phase systems. Therefore, 1% formic acid in water-methanol was selected as the mobile phase for this method.

[0119] Table 5 8 different mobile phase types

[0120]

[0121]

[0122] Note: Because the instrument used has low ion response in positive ion mode when simultaneously collecting positive and negative ion modes, the eight mobile phase systems were scanned in positive and negative ion modes respectively.

[0123] 3. Optimization of column temperature

[0124] The temperature of the chromatographic column has different degrees of influence on the separation and response of the analytes. The effects of the chromatographic column at 35℃, 40℃, 45℃ and 50℃ on the separation and response of anthocyanins were investigated. Figure 8 As shown in the figure, 1: delphinium pigment; 2: cyanidin; 3: morning glory pigment; 4: pelargonidin; 5: peonyrin; 6: malvadin; A, B, C, D are column temperatures of 35℃, 40℃, 45℃, and 50℃ respectively. Figure 8As can be seen, increasing the column temperature accelerates the analyte peak elution time, but does not affect the separation between the analytes; the column does not significantly affect the analyte response. To maximize analytical efficiency, reduce column pressure, and extend column life, a column temperature of 40°C was selected after comprehensive consideration.

[0125] 4. Optimization of mobile phase gradient

[0126] In order to ensure good separation and response of anthocyanins, different initial mobile phase ratios were investigated. The effects on the retention time, separation effect and response of anthocyanins are shown in Table 6. Figure 9 From Table 6 and Figure 9 It can be seen that under the isocratic elution condition of low organic phase ratio, the analyte peak time is late and the peak width is wide; under the isocratic elution condition of high organic phase ratio, the analyte peak is fast and the peak separation is poor; under gradient elution, changing the initial proportion of the organic phase and the rate of increase of the organic phase ratio can effectively change the peak time and separation of each analyte; after comprehensive consideration, number 4 was selected as the mobile phase gradient elution program.

[0127] Table 6 Gradient elution program

[0128]

[0129]

[0130] Example 3 Optimization of mass spectrometry conditions

[0131] A single standard working solution of six anthocyanins was prepared using 10% hydrochloric acid in methanol at a concentration of 500 μg / L. This solution was then subjected to primary mass spectrometry scanning in both positive and negative ESI modes to obtain the parent ions of the analytes. Anthocyanins were found to respond better in positive ion mode. The optimal primary mass spectrometry fragmentation voltage for each anthocyanin was then optimized. The molecular ion peaks of each anthocyanin were then analyzed by secondary mass spectrometry to obtain fragment ion information. Finally, the optimal secondary mass spectrometry collision voltage for each anthocyanin was optimized. The optimized MRM information for the anthocyanins is shown in Table 2.

[0132] The effects of drying gas temperature, drying gas flow rate, nebulizer gas pressure, and ion spray voltage on anthocyanin response (peak area) were also investigated within the recommended instrument parameter settings. The results are shown in Tables 7 to 10. As can be seen from the tables, under all experimental conditions, the optimal response conditions for all analytes were concentrated at 350°C, 50 psi, and 2000 V, respectively. Only for the drying gas flow rate parameter, the optimal response conditions for each target analyte were evenly distributed between 10 L / min and 11 L / min. Due to the relatively low response values ​​for delphinidin and malvidin, ion source parameters with higher response values ​​for delphinidin and malvidin were preferred. After comprehensively comparing the response values ​​of each parameter, the final mass spectrometry ion source conditions were determined to be a drying gas temperature of 350°C, a drying gas flow rate of 11 L / min, a nebulizer gas pressure of 50 psi, and an ion spray voltage of 2000 V.

[0133] Table 7 Effects of different drying gas temperatures on the response values ​​of anthocyanins

[0134]

[0135] Table 8 Effects of different drying air flow rates on the response values ​​of anthocyanins

[0136]

[0137] Table 9 Effects of different atomization gas pressures on the response values ​​of anthocyanins

[0138]

[0139] Table 10 Effect of different ion spray voltages on the response values ​​of anthocyanins

[0140]

[0141] Example 4 Performance Evaluation of HPLC-MS / MS Detection Method

[0142] 1. Linearity and sensitivity of the method

[0143] The standard working curves obtained in the "General Materials and Methods" section show good linear relationships for each target compound within the range of 25 to 2500 μg / L, with correlation coefficients R > 0.99. To examine the sensitivity of this method, the detection limit (LOD) was set at a signal-to-noise ratio (S / N) ≥ 3, and the quantification limit (LOQ) was set at a signal-to-noise ratio (S / N) ≥ 10. The results are shown in Table 11. As shown in Table 11, this method demonstrates good sensitivity for the determination of anthocyanins and is well suited for the determination of six anthocyanin compounds in tea.

[0144] Table 11 Linearity and sensitivity experimental results of the method

[0145]

[0146] 2. Accuracy and precision of the method

[0147] Twelve samples of green tea and black tea were accurately weighed. Three of these samples were prepared as test solutions according to the sample pretreatment method described in the "General Materials and Methods." The remaining nine samples were spiked with appropriate amounts of the mixed standard working solution described in the "General Materials and Methods" to produce three different concentration levels of spiked samples: low, medium, and high. The test solutions were then prepared according to the sample pretreatment method described in the "General Materials and Methods." The spiked recovery and precision of this method were investigated under the test conditions described in the "General Materials and Methods." The results are shown in Tables 12 to 14. As shown in the tables, the average recovery of anthocyanins in tea leaves determined by this method ranged from 91.95% to 106.99%, with RSDs less than 10%, meeting the recovery and precision requirements of the relevant standards, indicating that this method is capable of accurately detecting the content of anthocyanins in tea leaves.

[0148] Since this method has a low detection limit, based on the comprehensive consideration of the content in the sample and the linear range of anthocyanins on the instrument, the sample needs to be diluted 100 times when testing the three anthocyanin substances with higher content in tea. At the same time, the matrix effect can be reduced by dilution. It can be seen from the spike recovery results that the matrix effect has indeed been overcome, so there is no need to specifically optimize the matrix effect of the sample.

[0149] Table 12 Green tea sample spike recovery experimental results (n=3)

[0150]

[0151]

[0152] Table 13 Black tea sample spike recovery test results (n=3)

[0153]

[0154] Table 14 Method precision test results

[0155]

[0156]

[0157] 3. Solution stability verification

[0158] 2 μL of black tea and green tea sample solutions and the corresponding low-, medium-, and high-concentration spiked tea solutions were respectively aspirated and injected into liquid chromatography-tandem mass spectrometry for analysis according to the detection conditions described in the “General Materials and Methods” after the black tea and green tea sample solutions were placed for 0 h, 2 h, 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h. After each single standard working solution was placed at -20°C in a dark environment for 7, 15, 30, 45, and 60 days, 2 μL of mixed standard working solutions (prepared using single standard working solutions) with a concentration of 1000 μg / L were respectively aspirated and injected into the liquid chromatography-tandem mass spectrometry according to the detection conditions described in the “General Materials and Methods” to analyze and investigate the stability of the standard working solutions. The specific results are shown in Tables 15 to 17. As can be seen from the table, the sample test solution did not change significantly within 24 hours, and the single standard working solution did not change significantly within 60 days, indicating that the test solution prepared by this method has good stability within 24 hours, and the single standard working solution stored in a light-proof environment at -20°C has good stability within 60 days.

[0159] Table 15 Green tea solution stability test results

[0160]

[0161] Table 16 Black tea solution stability test results

[0162]

[0163] Table 17 Stability test results of single standard working solution

[0164] Substance name RSD of single standard working solution (%) Delphinium pigment 6.40 Cyanidin 4.07 Pelargonium 6.92 Morning glory pigment 8.30 Paeoniflorin 6.15 Malva pigment 8.80

[0165] Example 5

[0166] This example uses the method described in "General Materials and Methods" to determine the contents of six anthocyanins in five tea samples.

[0167] The five tea samples are: Yunnan large-leaf Kung Fu black tea (sample 1), West Lake Longjing green tea (sample 2), Mengding Ganlu green tea (sample 3), Yunlian black tea (sample 4) and Xufu Longya green tea (sample 5). All five tea samples were processed according to the sample pretreatment steps described in the "General Materials and Methods" to obtain the sample solutions to be tested of the five tea samples.

[0168] Under the HPLC-MS / MS operating conditions described in "General Materials and Methods," sample solutions of the five tea samples were injected into the HPLC-MS / MS system to obtain the MRM peak areas for each anthocyanin. These peak areas were then substituted into the standard working curve to determine the content of each anthocyanin in the samples. The assay results are shown in Table 18.

[0169] Table 18 Test results of the samples tested in this embodiment

[0170]

[0171] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A liquid chromatography-mass spectrometry method for detecting anthocyanidins in tea, characterized in that: The method comprises: S1. Preparation of system standard working solution: Weigh and dissolve the six anthocyanin standards, and then dilute them step by step to obtain a series of standard working solutions; S2. Preparation of sample solution: Add acidified ethanol solution to the tea sample, sonicate for 50 minutes, and then incubate in a water bath at 100°C for 75 minutes; the volume ratio of ethanol, hydrochloric acid, and water in the acidified ethanol solution is 2:1:1; In step S2, the aqueous phase filter membrane is a 0.22 μm aqueous phase filter membrane, and the filtered extract is divided into two groups, one group is diluted and the other group is not diluted, and both groups of extracts are the sample solutions to be tested; S3. HPLC-MS / MS detection: The series of standard working solutions from step S1 are sequentially injected into a liquid chromatography-mass spectrometer, and the corresponding concentrations are subjected to regression analysis using the ion peak area of ​​anthocyanins in the multiple reaction monitoring mode to obtain a standard working curve. Under the same conditions, the sample solution to be tested from step S2 is injected into a liquid chromatography-mass spectrometer to obtain the ion peak area of ​​each anthocyanin in the multiple reaction monitoring mode. Substituting the area into the standard working curve, the content of the six anthocyanins in the sample can be determined; The six anthocyanidins are: pelargonidin, cyanidin, peonidin, delphinidin, morning gloryin, and malvadin; In step S3, the chromatographic conditions are: The chromatographic column model is: Agilent Poroshell 120 SB-C18, 2.1 mm × 100 mm, 2.7 μm; Column temperature: 40°C; Flow rate: 0.35 mL / min; Injection volume: 2 μL; Mobile phase: Mobile phase A is 1% formic acid in water, and mobile phase B is methanol; Mobile phase gradient elution program table: The mass spectrometry conditions are: Ion source: electrospray ion source; Scan mode: positive ion scan mode; Detection method: multiple reaction monitoring mode; Drying gas: nitrogen; Atomizing gas: nitrogen; Atomizing gas pressure: 50 psi; Ion spray voltage: 2000 V; Drying gas temperature: 350℃; Drying gas flow rate: 11 L / min; The mass spectrometry acquisition parameters are shown in the table: 。 2. The liquid chromatography-mass spectrometry method for detecting anthocyanins in tea according to claim 1, wherein In step S1, 10% hydrochloric acid methanol is used for the dissolution process; in steps S1 and S2, methanol is used for the dilution process.

3. The liquid chromatography-mass spectrometry method for detecting anthocyanins in tea according to claim 1, wherein In step S1, the concentrations of the six anthocyanins in the series of standard working solutions are 25 μg / L, 50 μg / L, 100 μg / L, 250 μg / L, 500 μg / L, 1000 μg / L, and 2500 μg / L.

4. The liquid chromatography-mass spectrometry method for detecting anthocyanins in tea according to claim 1, wherein The material-to-liquid ratio of tea sample to acidified ethanol solution was 1:100 (g:mL).

5. The liquid chromatography-mass spectrometry method for detecting anthocyanins in tea according to claim 1, wherein In step S2, centrifugation is performed at 7500 r / min for 3 min.

6. The liquid chromatography-mass spectrometry method for detecting anthocyanidins in tea according to claim 1, wherein: In step S2, the extract is diluted 100 times.

7. The liquid chromatography-mass spectrometry method for detecting anthocyanidins in tea according to claim 1, wherein: The average recoveries of the method ranged from 91.95% to 106.99%, with RSDs less than 10%, limits of detection ranging from 0.02 to 17 mg / kg, and limits of quantification ranging from 0.07 to 55 mg / kg.

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