CN110426475B - Detection method of health-care herbal tea - Google Patents
Detection method of health-care herbal tea Download PDFInfo
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Abstract
Aiming at the problem that the quality of the existing health-care herbal tea cannot be monitored, the invention provides a detection method of the health-care herbal tea, which comprises the following steps: detecting the stevioside content in the health-care herbal tea, and detecting the chlorogenic acid, rutin, liquiritin and quercitrin content in the health-care herbal tea. The detection method has the advantages of good repeatability, high precision and high accuracy.
Description
Technical Field
The invention belongs to the field of detection of health-care herbal tea, and particularly relates to a detection method of health-care herbal tea.
Background
The applicant submits a patent name of 'a health-care herbal tea and a preparation method thereof' on 25.06.2018, with the application number of 201810663543X, and the health-care herbal tea provided by the invention is prepared from the following raw materials: folium Jujubae, stevia rebaudiana Bertoni, radix Glycyrrhizae, and flos Chrysanthemi. Wherein the folium Jujubae contains rutin, quercetin, etc., and Glycyrrhrizae radix mainly contains glycyrrhizin, stevia contains stevioside, and flos Chrysanthemi contains chlorogenic acid. In the production of the health-care herbal tea, how to monitor the quality of the health-care herbal tea becomes a difficult problem. Therefore, it is urgently needed to develop a detection method with good repeatability, high precision and high accuracy.
Disclosure of Invention
1. Problems to be solved
Aiming at the problem that the quality of the existing health-care herbal tea cannot be monitored, the invention provides a detection method of the health-care herbal tea, which comprises the following steps: detecting the stevioside content in the health-care herbal tea, and detecting the chlorogenic acid, rutin, liquiritin and quercitrin content in the health-care herbal tea. The detection method has the advantages of good repeatability, high precision and high accuracy.
2. Technical scheme
In order to solve the above problems, the present invention adopts the following technical solutions.
A detection method of health-care herbal tea comprises the following steps: the detection method of the health-care herbal tea comprises the following steps:
detecting the stevioside content in the health-care herbal tea:
providing a test solution and a stevioside control solution;
using octadecylsilane chemically bonded silica as a filler, using acetonitrile-water as a mobile phase, eluting at equal concentration, detecting the test solution and stevioside reference solution in a liquid chromatograph, and analyzing by an external standard method to obtain the stevioside content in the health herbal tea;
the method comprises the following steps of detecting the contents of chlorogenic acid, rutin, liquiritin and quercitrin in the health-care herbal tea:
providing a test solution, a chlorogenic acid reference substance solution, a rutin reference substance solution, a liquiritin reference substance solution and a quercitrin reference substance solution;
using octadecylsilane chemically bonded silica as a filling agent, acetonitrile as a mobile phase A, and acetic acid water as a mobile phase B, carrying out gradient elution, detecting the test sample solution, the chlorogenic acid reference substance solution, the rutin reference substance solution, the liquiritin reference substance solution and the quercitrin reference substance solution in a liquid chromatograph, and analyzing by an external standard method to obtain the contents of chlorogenic acid, rutin, liquiritin and quercitrin in the health-care herbal tea.
Preferably, in the step of detecting the stevioside content in the health herbal tea, the volume ratio of acetonitrile to water in the mobile phase is 75:25, and in the step of detecting the chlorogenic acid, rutin, glycyrrhizin and quercetin content in the health herbal tea, the acetic acid water is 0.5% of acetic acid water by volume.
Preferably, in the step of detecting the stevioside content in the health herbal tea, the flow rate of the mobile phase is 0.8-1.0 mL/min, and in the step of detecting the chlorogenic acid, rutin, glycyrrhizin and quercetin content in the health herbal tea, the flow rate of the mobile phase is 0.6-1.0 mL/min.
Preferably, in the step of detecting the stevioside content in the health herbal tea, the flow rate of the mobile phase is 0.8mL/min, and in the step of detecting the chlorogenic acid, rutin, liquiritin and quercetin content in the health herbal tea, the flow rate of the mobile phase is 0.9 mL/min.
Preferably, in the step of detecting the stevioside content in the health-care herbal tea, the column temperature is 30-40 ℃, and in the step of detecting the chlorogenic acid, rutin, liquiritin and quercitrin content in the health-care herbal tea, the column temperature is 30-40 ℃.
Preferably, in the step of detecting the stevioside content in the health herbal tea, the column temperature of the detection is 30 ℃, and in the step of detecting the chlorogenic acid, rutin, liquiritin and quercitrin content in the health herbal tea, the column temperature of the detection is 30 ℃.
Preferably, in the step of detecting the stevioside content in the health herbal tea, the detection wavelength is 200-400 nm, and in the step of detecting the chlorogenic acid, rutin, liquiritin and quercitrin content in the health herbal tea, the detection wavelength is 200-400 nm.
Preferably, in the step of detecting the stevioside content in the health herbal tea, the detection wavelength is 210nm, and in the step of detecting the chlorogenic acid, rutin, liquiritin and quercetin content in the health herbal tea, the detection wavelength is 330 nm.
Preferably, in the step of detecting the contents of chlorogenic acid, rutin, liquiritin and quercitrin in the health herbal tea, the initial volume ratio of acetonitrile to 0.5% by volume of acetic acid water is 15: 85.
Preferably, in the step of detecting the contents of chlorogenic acid, rutin, liquiritin and quercitrin in the health-care herbal tea, the gradient elution procedure is as follows:
for 0-10 minutes, the content of the mobile phase A is 15-20%, and the content of the mobile phase B is 85-77%;
10-35 minutes, wherein the mobile phase A is 20-50%, and the mobile phase B is 77-75%;
35-45 minutes, wherein the mobile phase A is 50-85%, and the mobile phase B is 75-76%;
45-55 minutes, wherein the mobile phase A is 85-15%, and the mobile phase B is 76-85%;
the time is 55-60 minutes, the mobile phase A is 15-15%, and the mobile phase B is 85-85%.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) by adopting the detection method, the chlorogenic acid, the rutin, the liquiritin, the quercitrin and the stevioside in the health-care herbal tea can be quantitatively detected, and the detection method has the advantages of high precision, good repeatability and high accuracy;
(2) by adopting the detection method, the content of the corresponding active ingredients can be quickly detected, so that the quality standard of the health-care herbal tea is identified and ensured, and the production efficiency is improved.
Drawings
FIG. 1 is a diagram of a health herbal tea;
FIG. 2 is a liquid chromatogram under 2.3.2 chromatographic conditions when the mobile phase is changed to methanol-0.5% acetic acid, with the remaining conditions unchanged;
FIG. 3 is a liquid chromatogram of a health herbal tea under 2.3.2 chromatographic conditions;
fig. 4 is a chromatogram of a sample and a standard of health herbal tea, wherein,
4A is a chromatogram of four mixed standard solution, and 2.3.2 chromatographic conditions are adopted;
4B is a chromatogram of the health-care herbal tea solution under the chromatographic condition of 2.3.2, and 20180426 batches of detection are carried out;
4C is a chromatogram of stevioside standard substance, and adopts 2.3.1 chromatographic conditions;
4D is a chromatogram of the health-care herbal tea sample under the chromatographic condition of 2.3.1, and 20180426 batches of detection are carried out;
the components corresponding to each peak in the figure are: 1. chlorogenic acid; 2. rutin; 3. liquiritin; 4. quercetin; 5. stevioside.
FIG. 5 is a sample chromatogram under different column temperature conditions, wherein,
the column temperature of 5A is 25 ℃;
the column temperature of 5B is 30 ℃;
the column temperature of 5C is 35 ℃;
the column temperature of 5D was 40 DEG C
The components corresponding to each peak in the figure are: 1. chlorogenic acid; 2. rutin; 3. liquiritin; 4. quercetin; 5. stevioside.
FIG. 6 sample chromatograms at different total flow rates, wherein
The total flow rate of 6A is 0.6mL/min-1；
The total flow rate of 6B is 0.8mL/min-1；
The total flow rate of 6C is 1.0mL/min-1；
The components corresponding to each peak in the figure are: 1. chlorogenic acid; 2. rutin; 3. liquiritin; 4. quercetin.
FIG. 7 is a sample chromatogram under different detection wavelength conditions, wherein,
the detection wavelength of 7A is 210 nm;
the detection wavelength of 7B is 260 nm;
the detection wavelength of 7C is 3300 nm;
the 7D detection wavelength is 360 nm;
the components corresponding to each peak in the figure are: 1. chlorogenic acid; 2. rutin; 3. liquiritin; 4. quercetin;
FIG. 8 is a chromatogram of a stevioside standard under 2.3.2 chromatographic conditions;
fig. 9 is a process flow chart of health herbal tea.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings.
Example 1
1 Experimental materials and instruments
1.1 materials and reagents
1.2 instruments
2 method of experiment
2.1 preparation of health-promoting herbal tea
2.1.1 treatment of the raw materials
The method comprises purchasing folium Jujubae, sweet stevia, flos Chrysanthemi and Glycyrrhrizae radix from Suzhou Luyuan Chinese medicinal science and technology limited company and Zhongjing pharmacy. Spreading the materials on a dry and clean table, screening and removing impurities such as residue and foreign matters, cleaning with purified water, oven drying, and sealing in a cool and dry place.
2.1.2 preparation of the extract
(1) Extracting the leaves of the Wangzao date: weighing a proper amount of the Wangzao date leaves, cleaning, putting into boiled purified water with the mass of 60-70 times, adjusting the temperature to be low, decocting for one to two hours under the condition of slight boiling, filtering under reduced pressure, removing residues, collecting the Wangzao date leaf decoction, diluting with purified water to a constant volume, and obtaining the clarified Wangzao date leaf decoction.
(2) Stevia extract: weighing stevia rebaudiana Bertoni, cleaning, putting into boiled purified water 60-70 times of the stevia rebaudiana Bertoni, properly adjusting the temperature, decocting for one to two hours, and filtering and diluting the mixture in the same process as the Wangzao jujube leaf extract.
(3) And D, chrysanthemum morifolium extract: weighing an appropriate amount of flos Chrysanthemi, cleaning, placing into boiled 60-70 times of pure water, cooling, decocting for one to two hours, filtering and diluting the decoction as the folium Jujubae extract.
(4) Licorice root extract: weighing a small amount of liquorice, cleaning, putting into boiled purified water with the mass of 30-40 times, properly adjusting the temperature, decocting for one to two hours, and filtering and diluting the mixture in the same way as the Wangzao jujube leaf extract.
2.1.3 Process flow
As shown in particular in fig. 9.
2.1.4 working steps
Selecting materials: raw materials which are not damaged, have pure and rich fragrance and have respective clear characteristics need to be selected.
Cleaning and drying: removing impurities from the raw materials, washing with purified water to remove dust and soil, rinsing for 2-3 times until the raw materials are clean, and keeping the integrity of the raw materials as much as possible; after draining, the raw materials are dried in a drying oven at low temperature, so that the loss of effective components in the raw materials is avoided.
Decocting: accurately weighing a certain amount of purified water in a beaker, boiling, then decocting at a reduced temperature, and properly stirring with a glass rod during the process of decocting the raw materials to fully extract the raw materials.
And (3) reduced pressure filtration: filtering the decocted materials respectively while hot, taking down the connection tube of the filtration bottle after filtration, and then closing the air pump.
Diluting: the boiling extract is put into a 250mL volumetric flask, and is diluted to constant volume to the scale mark by purified water which is cooled to room temperature after being boiled.
Blending: mixing the above extractive solutions at a certain ratio for several times, and mixing to obtain health herbal tea.
Filling: rapidly filling and sealing the prepared health-care herbal tea in a sterilized glass bottle under an aseptic condition; the filling process of the health-care herbal tea requires aseptic operation and strict sealing.
Sterilization and cooling: sterilizing the health herbal tea in 100 deg.C boiling water bath for 20 min; and naturally cooling to room temperature after sterilization. Glassware used in the preparation of the health-care herbal tea is cleaned and sterilized in advance.
2.2 optimization of health-care herbal tea formula
2.2.1 orthogonal test factor level determination
The optimal extraction conditions are explored and determined in early-stage preliminary experiments on single factor investigation, such as water addition amount, boiling temperature and boiling time, of each raw material extracting solution, and the formula of the health-care herbal tea is optimized on the basis that: taking four parts of raw material extract as influencing factors and sensory evaluation of health-care herbal tea as evaluation criteria, and carrying out L9(34) And performing orthogonal test to determine the optimal blending process. The factor levels are shown in table 1.
TABLE 1 health herbal tea formulation orthogonal test factors and level settings
TABLE 1 health herbal tea formulation orthogonal test factors and level settings
2.2.2 sensory evaluation method
10 persons with an experienced organization evaluate the health-care herbal tea in four aspects of taste, aroma, color, tissue form and the like, and the full score is 100. The evaluation criteria are shown in Table 2.
TABLE 2 sensory Standard evaluation of health herbal tea
2.3 quantitative analysis of functional components in health-care herbal tea
The SPD-20A high performance liquid chromatograph is adopted to detect the health herbal tea, and the five components of rutin, chlorogenic acid, liquiritin, quercetin and stevioside are taken as the quality control indexes of the health herbal tea, and the content of each component in the health herbal tea is measured. Due to different properties of various substances, the components cannot be well separated under the same chromatographic condition, namely five substances cannot be well shown on one map, and in order to obtain a better separation effect and determine the content of each component in the health-care herbal tea, the quality control of the health-care herbal tea under the appropriate chromatographic condition is urgently needed.
The method is characterized in that the functional components of the health-care herbal tea are quantitatively analyzed, the content of chlorogenic acid, stevioside, rutin, liquiritin and quercitrin in the health-care herbal tea is researched, and due to the fact that stevioside is high in sugar content, easy to dissolve in water and high in polarity, when a health-care herbal tea sample is analyzed, conditions such as various elution programs, a mobile phase, detection wavelengths and the like are tested and researched, the stevioside cannot be separated under the condition that the chlorogenic acid, the rutin, the liquiritin and the quercitrin are well separated, and therefore the five functional components in the health-care herbal tea are quantitatively analyzed on two chromatograms by adopting two elution methods.
Under the chromatographic conditions of 2.3.2, the stevioside standard chromatogram shows that no peak is produced in the stevioside standard chromatogram as shown in figure 8.
The health-care herbal tea contains five functional components such as chlorogenic acid, stevioside, rutin, liquiritin, quercitrin and the like, and the content is low, but the health-care herbal tea has respective physiological functions such as anti-inflammation, bacteriostasis, immunity regulation and the like, and the health-care efficacy of the health-care herbal tea is improved.
2.3.1 chromatographic conditions for detecting stevioside in health-care herbal tea
A chromatographic column: shim Pack ODS C18 column (250 mm. times.4.6 mm, 5 μm), mobile phase: acetonitrile (a) -water (B) (volume ratio 75: 25), isoconcentrated elution, total flow rate: 0.8mL/min, column temperature: 30 ℃, detection wavelength: 210 nm.
2.3.2 chromatographic conditions for detecting the contents of chlorogenic acid, rutin, liquiritin and quercitrin in the health-care herbal tea
A chromatographic column: shim Pack ODS C18 column (250 mm. times.4.6 mm, 5 μm), mobile phase: acetonitrile (a) -0.5% acetic acid water (B) (starting volume ratio 15: 85), flow rate: 0.9mL/min, column temperature: 30 ℃, detection wavelength: 330 nm. The elution conditions are shown in Table 3.
TABLE 3 acetonitrile-0.5% acetic acid water gradient elution table
2.3.3 preparation of Standard solution
Precisely weighing chlorogenic acid, rutin, liquiritin, quercetin and stevioside standard substances, wherein the mass is 0.58mg, 0.28mg, 1.08mg, 2.00mg and 12.75mg in sequence, respectively placing the substances into 25mL volumetric flasks, diluting the stevioside standard substance with ultrapure water to a constant volume, diluting the other four standard substances with methanol to a constant volume, uniformly shaking the substances in a back-and-forth inverted mode, and storing the substances in a refrigerator at 4 ℃ in a dark place. The prepared standard quality and concentration are respectively as follows: chlorogenic acid 0.0232mg/mL, rutin 0.0112mg/mL, glycyrrhizin 0.0432mg/mL, quercetin 0.0800mg/mL and stevioside 0.5028 mg/mL.
2.4 physicochemical and microbiological indicators analysis of herbal tea
The health-care herbal tea is prepared according to the process, the health-care herbal tea is measured by an Abbe refractometer at the temperature of 20 ℃, and the content of soluble solids in the health-care herbal tea can be directly read out or converted. The pH of the health herb tea is read by a pH meter.
3 results and analysis
3.1 orthogonal test optimization of health herbal tea formulation results and analysis
An L9(34) orthogonal test is carried out according to the factor level design of the table 1, and the optimal formula of the health-care herbal tea is determined by comprehensive visual and variance analysis of test data. The raw material ratio optimization results of the health herbal tea beverage are shown in table 4.
TABLE 4 health herbal tea proportion optimization test results and analysis
TABLE 5 ANOVA TABLE
Note: f0.1(2,2)＝9.00,F0.05(2,2)＝19.00,F0.01(2,2)＝99.00
Analyzing the orthogonal test data by utilizing Minitab software to obtain tables 4 and 5, wherein the strength sequence of all factors influencing the sensory evaluation of the health-care herbal tea can be visually analyzed from the table 4 as follows: stevia rebaudiana extractive solution (B), Wangzao jujube extractive solution (A), liquorice extractive solution (C) and Hangzhou chrysanthemum extractive solution (D); the analysis of variance results show that the factors A, B, C are all significant, and the factor D is not significant. Comprehensively considering, the optimal proportioning scheme of the obtained health-care herbal tea is A2B2C3D1, namely 10mL of Wangzao date seed extract, 5mL of stevia extract, 5mL of liquorice extract and 10mL of Hangzhou chrysanthemum extract. The filling amount of the health-care herbal tea product is 330mL, wherein the Wangzao jujube extract and the Hangzhou chrysanthemum extract respectively account for 33.33 percent, and the stevia extract and the liquorice extract respectively account for 16.67 percent. A health herbal tea product is prepared according to the process flow, and is shown in figure 1.
3.2 establishment of quantitative analysis method for functional components in health-care herbal tea
3.2.1 selection of Mobile phase
When stevioside in the health-care herbal tea is detected, the mobile phase is screened under the condition of 2.3.1, and under the condition that other conditions are not changed, the mobile phase is screened;
when chlorogenic acid, rutin, liquiritin and quercitrin in the health-care herbal tea are detected, the mobile phase is screened under the condition of 2.3.2, and under the condition that other conditions are not changed, the mobile phase is screened;
in order to completely reflect the existence and content of rutin, chlorogenic acid, liquiritin, quercetin, stevioside and other substances in the health-care herbal tea, experimental research is carried out under the same other chromatographic conditions, and the separation effect of each mobile phase is compared.
Aiming at the special properties of stevioside, a mobile phase is selected: methanol (a) -water (B), acetonitrile-water;
selecting mobile phases for the rest four components (chlorogenic acid, rutin, liquiritin and quercitrin): methanol-0.5% acetic acid, acetonitrile-0.5% acetic acid were compared, respectively. The experimental result shows that: for stevioside, methanol can shorten the retention time of the stevioside, and the separated peak has poor shape symmetry, so acetonitrile-water is selected; the separation effect of rutin, chlorogenic acid, liquiritin and quercitrin in acetonitrile is better than that of methanol, and the chromatographic peak shape is better. The chromatographic column is damaged when the acid concentration is too high, and the separation effect is poor when the acid concentration is too low, so that 0.5% acetic acid water is the optimal concentration; if the ratio of acetic acid in the mobile phase is high, the retention time of each component is too long, and if the ratio is too low, the separation of chlorogenic acid is not facilitated. Therefore, acetonitrile-0.5% acetic acid is selected, and the initial ratio of elution is gradient with the volume ratio of 15: 85. The separation effect is shown in fig. 2 and 3. The degrees of separation between adjacent peaks in fig. 3 are all greater than 1.5, enabling complete separation.
The experiment is researched by a plurality of different elution programs, and the optimal elution program is determined: 85% -77% of B in 0-10 min; 77-75% of B in 10-35 min; 75% -76% of B in 35-45 min; 76% -85% B in 45-55 min; 55-60 min 85% B. Under the elution condition, chlorogenic acid and three flavones (rutin, liquiritin and quercitrin) in the health-care herbal tea can be completely separated after the components are detected, and the peak shapes are sharp and symmetrical.
Chromatograms of the health herbal tea sample and the standard product are shown in FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D,
wherein, fig. 4A is a chromatogram of four mixed standard solutions obtained by sampling under 2.3.2 chromatographic conditions, and the four mixed standard solutions are obtained by mixing a chlorogenic acid standard solution, a rutin standard solution, a liquiritin standard solution and a quercitrin standard solution;
FIG. 4B is a chromatogram of a solution of 20180426 batches of herbal tea tested under 2.3.2 chromatographic conditions;
FIG. 4C is a chromatogram of a stevioside standard using 2.3.2 chromatographic conditions;
FIG. 4D is a chromatogram of 20180426 batches of herbal tea samples tested under 2.3.1 chromatographic conditions.
3.2.2 selection of column temperature of chromatographic column
When stevioside in the health-care herbal tea is detected, the column temperature is screened under the condition of 2.3.1, and under the condition that other conditions are not changed;
when chlorogenic acid, rutin, liquiritin and quercitrin in the health-care herbal tea are detected, the column temperature is screened under the condition of 2.3.2, and under the condition that other conditions are not changed;
the test compares the separation conditions of chromatographic peaks of various functional components in chlorogenic acid, rutin, liquiritin, quercetin, stevioside standard products and health-care herbal tea under the condition that the chromatographic column temperature is 4 temperatures of 25 ℃, 30 ℃, 35 ℃ and 40 ℃. As a result, it was found that chlorogenic acid could not be effectively separated under 25 ℃ and the separation effect was better under 30 ℃, 35 ℃ and 40 ℃ among the 4 temperature conditions examined. The separation result of stevioside in the sample is that the column temperature is 25 ℃, stevioside is connected with other peaks, the separation degree is more than 1.5 under the conditions of 30 ℃, 35 ℃ and 40 ℃, and the separation effect is good. On the basis of similar results, conditions with lower energy consumption may be preferred, and therefore a column temperature of 30 ℃ may be preferred. The chromatograms at each column temperature are shown in fig. 5A, 5B, 5C, and 5D.
3.2.3 selection of flow rates of the mobile phase
When stevioside in the health-care herbal tea is detected, the flow velocity of the mobile phase is screened under the condition of 2.3.1, and the flow velocity of the mobile phase is screened under the condition that other conditions are not changed;
when chlorogenic acid, rutin, liquiritin and quercitrin in the health-care herbal tea are detected, the flow velocity of the mobile phase is screened under the condition of 2.3.2, and the flow velocity of the mobile phase is screened under the condition that other conditions are not changed;
the test compares that the flow velocity of the mobile phase is 0.6-1.0 mL/min-1Separating chromatographic peaks of functional components in the chlorogenic acid, rutin, liquiritin, quercetin, stevioside standard product and health-care herbal tea. As a result, it was found that the flow rate was 0.6 mL. multidot.min-1When the flow rate is higher than 0.8mL/min, the stevioside peak can not be effectively separated-1The separation degree of the stevioside substance peak is more than 1.5, and the separation effect is good; the flow rate was 0.6 mL. min-1When in use, the separation degree of chlorogenic acid, rutin, liquiritin and quercitrin is better, and the total flow rate is 0.8-1.0 mL/min-1The separation effect is better. Therefore, it is preferable that the mobile phase is acetonitrile-water at a flow rate of 0.8 mL. min-1Detecting stevioside, preferably acetonitrile-0.5% acetic acid as mobile phase, with flow rate of 0.9mL/min-1Chlorogenic acid, rutin, liquiritin and quercitrin are detected. The chromatograms at different flow rates are shown in fig. 6A, 6B, and 6C.
3.2.4 selection of detection wavelength
When stevioside in the health-care herbal tea is detected, the wavelength screening adopts the condition of 2.3.1, and under the condition that other conditions are not changed, the wavelength flow rate is screened;
when detecting chlorogenic acid, rutin, liquiritin and quercitrin in the health-care herbal tea, the wavelength is screened under the condition of 2.3.2, and under the condition that other conditions are not changed;
scanning the chlorogenic acid, rutin, liquiritin, quercetin and stevioside standard products within a wavelength range of 200-400 nm to obtain ultraviolet absorption peaks of the standard products, wherein the ultraviolet absorption peaks are mainly concentrated at four wavelengths of 210nm, 260nm, 330nm and 360 nm. The mixed standard substance is detected under the four wavelengths, and the stevioside has a maximum absorption peak at 210nm, while the chlorogenic acid, the rutin, the liquiritin and the quercitrin have a maximum absorption peak at 330nm, and the detection can be carried out by 2 wavelengths in all aspects, namely the stevioside detection wavelength is 210nm, and the chlorogenic acid, the rutin, the liquiritin and the quercitrin detection wavelength is 330 nm. Chromatograms under different wavelength conditions are shown in fig. 7A, 7B, 7C, and 7D.
3.3 investigation of HPLC quantitative analysis methodology
3.3.1 systematic adaptability test
Precisely absorbing 20 mul of the test solution of the health-care herbal tea respectively, injecting into a chromatograph, injecting according to the chromatographic condition under the item of 2.3.2, and respectively calculating the theoretical plate number, tailing factor and separation degree of chlorogenic acid, rutin, liquiritin and quercitrin, wherein the data is as follows:
TABLE 6 System Adaptation data
Precisely absorbing 20 mul of the health herbal tea sample solution, injecting into a chromatograph, injecting sample according to the chromatographic condition under the item of 2.3.1, and calculating to obtain the theoretical plate number, tailing factor and separation degree of stevioside, wherein the data are as follows:
the analysis of the results shows that the tailing factors of the chromatographic peaks of the compounds in the detected health-care herbal tea are less than 2.0 percent, the tailing factors are well separated from impurity peaks, the theoretical plate number of the chromatographic peaks of the compounds meets the specification of quality standards, the separation degree between each chromatographic peak of the compounds and adjacent peaks is greater than 1.5 within 60min, the symmetry is good, and the simultaneous separation of various compounds can be met.
3.3.2 Linear relationship investigation
Accurately sucking 1mL, 2mL, 3mL, 4mL and 5mL of each standard solution by a pipette, respectively transferring into a 10mL volumetric flask, and respectively metering the volume of the chlorogenic acid, the rutin, the liquiritin and the quercitrin standard substance to scale marks by using methanol; and (4) metering the stevioside standard substance to a scale mark by using ultrapure water. And sequentially injecting 20 μ L of chlorogenic acid, rutin, liquiritin and quercitrin according to the chromatographic conditions corresponding to the substances (wherein the chlorogenic acid, the rutin, the liquiritin and the quercitrin are injected according to the chromatographic conditions of 2.3.2, and the stevioside is injected according to the chromatographic conditions of 2.3.1), and obtaining the peak area of the standard substance with each concentration. In the Excel table, the concentrations of the respective standards and the corresponding peak areas were input, and the standard curves of the standards were plotted with the concentrations as the horizontal axis and the corresponding peak areas as the vertical axis, to obtain 5 standard curves, correlation coefficients and linear ranges, as shown in table 7. In a linear range, the concentrations of all the components in the health-care herbal tea have a good linear relation with the corresponding peak areas.
TABLE 7 Standard Curve, correlation coefficient and Linear Range
3.3.3 precision test
Precisely absorbing 0.0232mg/mL chlorogenic acid, 0.0112mg/mL rutin, 0.0432mg/mL liquiritin and 0.0800mg/mL quercitrin respectively, continuously injecting samples for 4 times respectively according to the chromatographic condition under the item of '2.3.1', measuring the respective peak areas, and calculating the relative standard deviation RSD according to the measuring result. Calculated to obtain the RSD corresponding to chlorogenic acid, rutin, liquiritin and quercitrin which are respectively 0.86%, 1.23%, 0.607% and 0.549%. Precisely sucking 0.5028mg/mL stevioside control solution 20 μ l, injecting sample under the chromatographic condition of '2.3.2', measuring its peak area, calculating relative standard deviation RSD according to the measurement result, and calculating to obtain stevioside corresponding RSD result of 0.93%. All compounds RSD were less than 2.0%, indicating good precision of the method under both assay conditions.
3.3.4 stability test
Respectively sucking 20 μ l of the health herbal tea sample solution at 0, 2, 4, 8, 16, 18, and 24 hr, injecting into chromatograph, injecting sample according to "2.3.2" chromatographic condition, measuring peak area and retention time, calculating to obtain chlorogenic acid relative peak area RSD of 1.01% and relative retention time RSD of 0.98%; the relative peak area RSD of rutin is 0.69 percent, and the relative retention time RSD is 0.74 percent; the relative peak area RSD of liquiritin is 0.95 percent, and the relative retention time RSD is 0.89 percent; the relative peak area RSD of the quercitrin is 0.66 percent, and the relative retention time RSD is 0.85 percent. And then according to the chromatographic condition of '2.3.1' item, sampling, investigating stevioside stability, calculating the corresponding peak area and retention time, and obtaining the result that the relative peak area RSD of stevioside is 1.12% and the relative retention time RSD is 0.93%. The results show that the health-care herbal tea test solution has good stability within 24 hours.
3.3.5 repeatability test
Precisely weighing 5 parts of each of the same batch of the Wangzao date, the stevia rebaudiana, the Hangzhou chrysanthemum and the liquorice, preparing 5 parts of health-care herbal tea samples in parallel under the condition of 2.1, repeatedly injecting samples under the chromatographic condition of 2.3.2, detecting and calculating to obtain a relative peak area RSD of chlorogenic acid of 0.84 percent and a relative retention time RSD of 0.93 percent; the relative peak area RSD of rutin is 0.54 percent, and the relative retention time RSD is 0.67 percent; the relative peak area RSD of the quercitrin is 0.43 percent, and the relative retention time RSD is 0.77 percent. And then repeatedly injecting samples according to the chromatographic condition of 2.3.1 to obtain the stevioside with the relative peak area RSD of 0.72 percent and the relative retention time RSD of 0.88 percent. Under both conditions, all compounds had RSD less than 2.0%, indicating good reproducibility of the assay.
3.3.6 sample recovery test
Accurately weighing medicinal material samples with known content, preparing corresponding sample solution according to 2.1.2 method, precisely adding corresponding standard samples with known content, calculating corresponding recovery rate, and finding corresponding results in Table 8.
TABLE 8 sample recovery test results
3.4 determination of the content of each component in the health-care herbal tea
The health herbal tea is accurately prepared according to the process flow, the prepared health herbal tea is filtered by a 0.45 mu m microporous filter membrane and then placed in a 2mL sample bottle, 20 mu L of the same sample is respectively added according to the chromatographic conditions in 2.3, wherein, the sample injection results of 20180426 batches of the health herbal tea are shown in figures 4B and 4D, the chromatogram of the four mixed standard products (namely chlorogenic acid, rutin, liquiritin and 4 standard products of quercitrin) solution is injected by adopting 2.3.2 chromatographic conditions in figure 4A, and the chromatogram of the stevioside standard product is injected by adopting 2.3.1 chromatographic conditions in figure 4C. The same elution program is used for measuring the health-care herbal tea samples of different batches, and the contents of all the components are calculated according to the chromatogram and the standard curve of the health-care herbal tea samples, which is shown in table 8.
Stevioside in Table 8 was determined using chromatographic conditions of 2.3.1;
table 8 chlorogenic acid, rutin, liquiritin, and quercitrin were measured using 2.3.2 chromatography.
TABLE 9 content of each component in the health herbal tea sample
Table 9 shows that the floating range of the contents of the components in the three batches of health-care herbal tea is very small, which indicates that the components of the health-care herbal tea prepared each time are stable, shows that the preparation process of the health-care herbal tea is excellent, and is suitable for the production of the health-care herbal tea in batches for many times. The content of each component in the health-care herbal tea is measured as follows: chlorogenic acid 6.16 +/-0.15 mu g/mL, rutin 3.41 +/-0.16 mu g/mL, liquiritin 13.24 +/-0.22 mu g/mL, quercetin 24.11 +/-0.14 mu g/mL and stevioside 140.1 +/-0.22 mu g/mL.
3.5 physicochemical and microbiological indicators detection of health-care herbal tea
Randomly selecting 20180501 batch herbal tea samples to measure refractive index at 20 deg.C, reading to 1.3348 + -0.23, and converting to obtain soluble solid content of 1.28 + -0.15%; the pH value of the health-care herbal tea is 5.80 +/-0.21. The whole preparation process of the health-care herbal tea is operated under the aseptic condition, the total number of colonies is less than or equal to 1CFU/mL, and no pathogenic bacteria exist.
4 conclusion
The quality control of the health-care herbal tea is carried out by high performance liquid chromatography, and the experimental result shows that: in the linear range: chlorogenic acid 2.32-11.60 mu g/mL, rutin 1.12-5.60 mu g/mL, liquiritin 4.32-21.60 mu g/mL, quercitrin 8.00-40.00 mu g/mL, stevioside 50.28-251.40 mu g/mL, and the concentration of each component in the health-care herbal tea has a good linear relationship with the corresponding peak area. The content of each component in the health-care herbal tea is measured as follows: chlorogenic acid 6.16 +/-0.15 mu g/mL, rutin 3.41 +/-0.16 mu g/mL, liquiritin 13.24 +/-0.22 mu g/mL, quercetin 24.11 +/-0.14 mu g/mL and stevioside 140.1 +/-0.22 mu g/mL.
Claims (7)
1. A detection method of health-care herbal tea comprises the following steps: the detection method of the health-care herbal tea comprises the following steps:
detecting the stevioside content in the health-care herbal tea:
providing a test solution and a stevioside control solution;
using octadecylsilane chemically bonded silica as a filler, using acetonitrile-water as a mobile phase, eluting at equal concentration, detecting the test solution and stevioside reference solution in a liquid chromatograph, and analyzing by an external standard method to obtain the stevioside content in the health herbal tea;
the method comprises the following steps of detecting the contents of chlorogenic acid, rutin, liquiritin and quercitrin in the health-care herbal tea:
providing a test solution, a chlorogenic acid reference substance solution, a rutin reference substance solution, a liquiritin reference substance solution and a quercitrin reference substance solution;
using octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A, and acetic acid water as a mobile phase B, carrying out gradient elution, detecting the test sample solution, the chlorogenic acid reference substance solution, the rutin reference substance solution, the liquiritin reference substance solution and the quercitrin reference substance solution in a liquid chromatograph, and analyzing by an external standard method to obtain the contents of chlorogenic acid, rutin, liquiritin and quercitrin in the health-care herbal tea;
in the step of detecting the stevioside content in the health-care herbal tea, the volume ratio of acetonitrile to water in the mobile phase is 75:25, and in the step of detecting the chlorogenic acid, rutin, liquiritin and quercitrin content in the health-care herbal tea, the acetic acid water is 0.5% acetic acid water by volume;
elution procedure: 85-77% of B in 0-10 min; 77-75% of B in 10-35 min; 75-76% B in 35-45 min; 76-85% B in 45-55 min; 85% B in 55-60 min;
a chromatographic column: shim Pack ODS C18 column, 250X 4.6mm, 5 μm.
2. The method for detecting the content of the stevioside in the health-care herbal tea as claimed in claim 1, wherein the flow rate of the mobile phase in the step of detecting the content of the chlorogenic acid, the rutin, the liquiritin and the quercetin in the health-care herbal tea is 0.8-1.0 mL/min, and the flow rate of the mobile phase in the step of detecting the content of the chlorogenic acid, the rutin, the liquiritin and the quercetin in the health-care herbal tea is 0.6-1.0 mL/min.
3. The method for detecting the content of the stevioside in the health herbal tea as claimed in claim 2, wherein the flow rate of the mobile phase in the step of detecting the content of the chlorogenic acid, the rutin, the liquiritin and the quercetin in the health herbal tea is 0.8mL/min, and the flow rate of the mobile phase in the step of detecting the content of the chlorogenic acid, the rutin, the liquiritin and the quercetin in the health herbal tea is 0.9 mL/min.
4. The method for detecting the content of the stevioside in the health-care herbal tea as claimed in claim 1, wherein the column temperature in the step of detecting the content of the chlorogenic acid, the rutin, the liquiritin and the quercetin in the health-care herbal tea is 30-40 ℃.
5. The method for detecting the content of the stevioside in the health-care herbal tea as claimed in claim 4, wherein the column temperature of the detection in the step of detecting the content of the chlorogenic acid, the rutin, the liquiritin and the quercitrin in the health-care herbal tea is 30 ℃.
6. The method for detecting the content of the stevioside in the health-care herbal tea as claimed in claim 1, wherein in the step of detecting the content of the stevioside in the health-care herbal tea, the detection wavelength is 200-400 nm, and in the step of detecting the content of the chlorogenic acid, the rutin, the liquiritin and the quercitrin in the health-care herbal tea, the detection wavelength is 200-400 nm.
7. The method for detecting the content of the stevioside in the health-care herbal tea as claimed in claim 6, wherein the detection wavelength is 210nm, and the detection wavelength is 330nm in the step of detecting the content of the chlorogenic acid, the rutin, the liquiritin and the quercitrin in the health-care herbal tea.
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