CN110426475A - A kind of detection method of health-care cold tea - Google Patents
A kind of detection method of health-care cold tea Download PDFInfo
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- CN110426475A CN110426475A CN201910739799.9A CN201910739799A CN110426475A CN 110426475 A CN110426475 A CN 110426475A CN 201910739799 A CN201910739799 A CN 201910739799A CN 110426475 A CN110426475 A CN 110426475A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
Aiming at the problem that existing health-care cold tea can not carry out quality monitoring, the present invention provides a kind of detection methods of health-care cold tea, the detection method of the health-care cold tea includes: to detect chlorogenic acid in health-care cold tea, rutin, liquiritin and the step of Determination of Quercitrin the step of detecting the stevioside content in health-care cold tea.The detection method is reproducible, and precision is high, and accuracy is high.
Description
Technical field
The invention belongs to health-care cold tea detection fields, specifically, being related to a kind of detection method of health-care cold tea.
Background technique
It is " a kind of health-care cold tea and preparation method thereof ", application that applicant, which had submitted patent name on 06 25th, 2018,
Number be 201810663543X, the invention provides a kind of health-care cold tea, health-care cold tea of the invention includes that the following raw material is prepared into
It arrives: common rabdosia leaf leaf, STEVIA REBAUDIANA, Radix Glycyrrhizae and FLOS CHRYSANTHEMI from Hangzhou of China.Wherein, common rabdosia leaf leaf contains a variety of flavones such as rutin, quercitin, in Radix Glycyrrhizae
Liquiritin is mainly contained, stevioside is contained in STEVIA REBAUDIANA, contains chlorogenic acid in FLOS CHRYSANTHEMI from Hangzhou of China.In the production of the health-care cold tea, such as
What carries out quality monitoring just at a problem to the health-care cold tea.Therefore it is badly in need of developing a kind of reproducible, precision height, it is quasi-
The high detection method of exactness.
Summary of the invention
1, it to solve the problems, such as
Aiming at the problem that existing health-care cold tea can not carry out quality monitoring, the present invention provides a kind of detections of health-care cold tea
Method, the detection method of the health-care cold tea include: the step of detecting the stevioside content in health-care cold tea, to detect health-care cold tea
In chlorogenic acid, rutin, liquiritin and the step of Determination of Quercitrin.The detection method is reproducible, and precision is high, accuracy
It is high.
2, technical solution
To solve the above problems, the present invention adopts the following technical scheme that.
A kind of detection method of health-care cold tea, the health-care cold tea include that the following raw material is prepared: common rabdosia leaf leaf, sweetleaf
The detection method of chrysanthemum, Radix Glycyrrhizae and FLOS CHRYSANTHEMI from Hangzhou of China, the health-care cold tea includes:
The step of detecting the stevioside content in health-care cold tea:
Test solution and stevioside reference substance solution are provided；
Using octadecylsilane chemically bonded silica as filler, using acetonitrile-water as mobile phase, Isocratic clution, in liquid phase color
The test solution and stevioside reference substance solution are detected in spectrometer, external standard method is analyzed to obtain the stevia rebaudianum in the health-care cold tea
Glycosides content；
Detect chlorogenic acid in health-care cold tea, rutin, liquiritin and the step of Determination of Quercitrin:
Test solution, chlorogenic acid reference substance solution, control substance of Rutin solution, liquiritin reference substance solution and quercitrin are provided
Glycosides reference substance solution；
Using octadecylsilane chemically bonded silica as filler, using acetonitrile as mobile phase A, using acetic acid water as Mobile phase B, gradient
Elution, detects the test solution, chlorogenic acid reference substance solution, control substance of Rutin solution, liquiritin in liquid chromatograph
Reference substance solution and quercitin reference substance solution, external standard method analyze to obtain chlorogenic acid, rutin, liquiritin in the health-care cold tea
And Determination of Quercitrin.
Preferably, in the step of stevioside content detected in health-care cold tea, the volume of acetonitrile in the mobile phase
The step of volume ratio with water is 75:25, the chlorogenic acid detected in health-care cold tea, rutin, liquiritin and Determination of Quercitrin
In, the acetic acid water is the acetic acid water of 0.5% volume.
Preferably, in the step of stevioside content in the detection health-care cold tea, the flow velocity of the mobile phase is 0.8~
In the step of 1.0mL/min, the chlorogenic acid detected in health-care cold tea, rutin, liquiritin and Determination of Quercitrin, the flowing
The flow velocity of phase is 0.6~1.0mL/min.
Preferably, in the step of stevioside content detected in health-care cold tea, the flow velocity of the mobile phase is
In the step of 0.8mL/min, the chlorogenic acid detected in health-care cold tea, rutin, liquiritin and Determination of Quercitrin, the flowing
The flow velocity of phase is 0.9mL/min.
Preferably, in the step of stevioside content detected in health-care cold tea, the column temperature of the detection is 30~40
DEG C, chlorogenic acid, rutin, liquiritin in the detection health-care cold tea and the step of Determination of Quercitrin in, the column temperature of the detection
It is 30~40 DEG C.
Preferably, in the step of stevioside content detected in health-care cold tea, the column temperature of the detection is 30 DEG C, institute
In the step of stating the chlorogenic acid detected in health-care cold tea, rutin, liquiritin and Determination of Quercitrin, the column temperature of the detection is 30
℃。
Preferably, it is described detection health-care cold tea in stevioside content the step of in, the Detection wavelength be 200~
In the step of 400nm, the chlorogenic acid detected in health-care cold tea, rutin, liquiritin and Determination of Quercitrin, the Detection wavelength
For 200~400nm.
Preferably, in the step of stevioside content detected in health-care cold tea, the Detection wavelength is 210nm, institute
In the step of stating the chlorogenic acid detected in health-care cold tea, rutin, liquiritin and Determination of Quercitrin, the Detection wavelength is 330nm.
Preferably, in the step of chlorogenic acid in the detection health-care cold tea, rutin, liquiritin and Determination of Quercitrin, second
The initial volume ratio of nitrile and the acetic acid water of 0.5% volume is 15:85.
Preferably, in the step of chlorogenic acid in the detection health-care cold tea, rutin, liquiritin and Determination of Quercitrin, institute
State gradient elution program are as follows:
0~10 minute, mobile phase A was 15~20%, and Mobile phase B is 85~77%；
10~35 minutes, mobile phase A was 20~50%, and Mobile phase B is 77~75%；
35~45 minutes, mobile phase A was 50~85%, and Mobile phase B is 75~76%；
45~55 minutes, mobile phase A was 85~15%, and Mobile phase B is 76~85%；
55~60 minutes, mobile phase A was 15~15%, and Mobile phase B is 85~85%.
3, beneficial effect
Compared with the prior art, the invention has the benefit that
(1) using detection method of the invention can by health-care cold tea chlorogenic acid, rutin, liquiritin, quercitin and
Stevioside is quantitative to be detected, and precision is high, reproducible, and accuracy is high；
It (2) can be quickly through corresponding active component content be detected, to reach identification using detection method of the invention
With guarantee health-care cold tea quality standard, production efficiency is improved.
Detailed description of the invention
Fig. 1 is health-care cold tea pictorial diagram；
Fig. 2 is the constant liquid of remaining condition when the mobile phase in the chromatographic condition of 2.3.2 is changed to -0.5% acetic acid of methanol
Phase map；
Fig. 3 is using the health-care cold tea chromatograms under the chromatographic condition of 2.3.2；
Fig. 4 is health-care cold tea sample and standard chromatogram, wherein
4A is four kinds of hybrid standard product solution chromatograms, using 2.3.2 chromatographic condition；
4B is the health-care cold tea solution chromatogram under 2.3.2 chromatographic condition, detects 20180426 batches；
4C is stevioside standard chromatogram, using 2.3.1 chromatographic condition；
4D is the health-care cold tea sample chromatogram figure under 2.3.1 chromatographic condition, detects 20180426 batches；
The corresponding component in each peak in figure are as follows: 1. chlorogenic acids；2. rutin；3. liquiritin；4. quercitin；5. stevioside.
Fig. 5 is sample chromatogram figure under the conditions of different column temperatures, wherein
5A column temperature is 25 DEG C；
5B column temperature is 30 DEG C；
5C column temperature is 35 DEG C；
5D column temperature is 40 DEG C
The corresponding component in each peak in figure are as follows: 1. chlorogenic acids；2. rutin；3. liquiritin；4. quercitin；5. stevioside.
Sample chromatogram figure under the conditions of Fig. 6 difference overall flow rate, wherein
6A overall flow rate is 0.6mL/min-1；
6B overall flow rate is 0.8mL/min-1；
6C overall flow rate is 1.0mL/min-1；
The corresponding component in each peak in figure are as follows: 1. chlorogenic acids；2. rutin；3. liquiritin；4. quercitin.
Sample chromatogram figure under the conditions of Fig. 7 difference Detection wavelength, wherein
7A Detection wavelength is 210nm；
7B Detection wavelength is 260nm；
7C Detection wavelength is 3300nm；
7D Detection wavelength is 360nm；
The corresponding component in each peak in figure are as follows: 1. chlorogenic acids；2. rutin；3. liquiritin；4. quercitin；
Fig. 8 is stevioside standard chromatogram under 2.3.2 chromatographic condition；
Fig. 9 is the process flow chart of health-care cold tea.
Specific embodiment
Present invention will now be described in detail with reference to the accompanying drawings..
Embodiment 1
1 experimental material and instrument
1.1 materials and reagent
1.2 instrument
2 experimental methods
The preparation of 2.1 health-care cold teas
2.1.1 feedstock processing
Common rabdosia leaf leaf, STEVIA REBAUDIANA, Hang Ju are purchased from Suzhou Lvyuan Chinese Medicine Technology Co., Ltd. and the pharmacy Suzhou City Zhong Jing
The raw materials such as colored and Radix Glycyrrhizae.Desktop material in dry cleansing is spread out, and the impurity screening such as residue therein, foreign matter is rejected,
It is sealed in a cool and dry place with pure water cleaning, drying postposition.
2.1.2 extracting solution is prepared
(1) common rabdosia leaf leaf extract: weighing common rabdosia leaf leaf and clean in right amount, puts into the 60-70 times of quality pure water boiled,
Temperature is turned down, and one to two hours are decocted under the conditions of slightly boiled, are filtered under diminished pressure, and abandons residue, collects common rabdosia leaf leaf leach cooking liquid, and use is pure
Water purification dilutes constant volume, must clarify common rabdosia leaf leaf extract.
(2) STEVIA REBAUDIANA extracting solution: weighing STEVIA REBAUDIANA and clean in right amount, puts into the 60-70 times of quality pure water boiled, temperature
It suitably turns down, decocts one or two hour, filter dilution with common rabdosia leaf leaf extract.
(3) FLOS CHRYSANTHEMI from Hangzhou of China extracting solution: appropriate FLOS CHRYSANTHEMI from Hangzhou of China cleaning is weighed, is put into the 60-70 times of quality pure water boiled, temperature
It reduces, decocts one or two hour, filter dilution with common rabdosia leaf leaf extract.
(4) it licorice extract: is put into the 30-40 times of quality pure water boiled after weighing a small amount of Radix Glycyrrhizae cleaning, temperature is suitable
It when turning down, decocts one or two hour, filters dilution with common rabdosia leaf leaf extract.
2.1.3 process flow
It is specific as shown in Figure 9.
2.1.4 operating procedure
Selection: to select that not damaged, aroma of pure is strong, the raw material with respective clear feature.
Cleaning and drying: will wash away that dirt is husky, soil with pure water after impurity removing, rinsing 2-3 times pays attention to as far as possible to clean
Keep the integrality of raw material；Low temperature drying in an oven after draining, avoids the loss of effective component in raw material.
Decoct: the accurate pure water for measuring certain mass reduces temperature in beaker, after boiling and is decocted, former decocting
It is suitably stirred during material with glass bar, makes to extract abundant.
It is filtered under diminished pressure: well-done raw material will be decocted and filtered while hot respectively, bottle,suction adapter tube is first removed after filtering, it is rear to close pumping
Pump.
Dilution: leach cooking liquid is put into 250mL volumetric flask, is settled to scale with the pure water dilution being cooled to room temperature after boiling
Line.
Allotment: each raw material extracting solution is repeatedly matched according to a certain percentage, is mixed, health-care cold tea is obtained.
It is filling: vial after deployed health-care cold tea is sterilized aseptically rapid filling and sealing；Health care
Herbal tea pouring process requires sterile working, is strictly sealed.
Sterilization and cooling: canned health-care cold tea is sterilized 20min in 100 DEG C of boiling water bath；It is naturally cold after sterilizing
But to room temperature.The glassware used in health-care cold tea preparation is all in advance by cleaning and sterilization.
2.2 health-care cold tea formulation optimizations
2.2.1 orthogonal test factor level determines
Single factor exploration such as amount of water for each raw material extracting solution boil temperature raising degree, boil and mention the time in preliminary experiment early period
It has probed into and has determined optimum extraction condition, carried out health-care cold tea formulation optimization on this basis: using four parts of raw material extracting solutions as shadow
The factor of sound, health-care cold tea sensory evaluation scores are interpretational criteria, carry out L9(34) orthogonal test, determine best allocating technology.Factor level
It is shown in Table 1.
1 health-care cold tea of table is formulated orthogonal test factor and level set
1 health-care cold tea of table is formulated orthogonal test factor and level set
2.2.2 subjective appreciation method
Experienced 10 people of the person of judging is organized, respectively from the mouthfeel of health-care cold tea, fragrance, color, tissue morphology etc. four
Aspect carries out subjective appreciation, and full marks are 100 points.Evaluation criterion is shown in Table 2.
The evaluation of 2 health-care cold tea sense organ fiducial mark of table
Functional component quantitative analysis in 2.3 health-care cold teas
Health-care cold tea is detected using SPD-20A high performance liquid chromatograph, with rutin, chlorogenic acid, liquiritin, quercitin and sweet tea
This five kinds of ingredients of chrysanthemum glycosides measure content of each ingredient in health-care cold tea as health-care cold tea quality control index.Due to each object
Matter property is different, cannot separate well each ingredient under same chromatographic condition, i.e., cannot be simultaneously by five on a map
Kind substance is shown well, in order to obtain better separating effect, specifies each component content in health-care cold tea, and it is suitable to be badly in need of
Chromatographic condition to health-care cold tea carry out quality control.
Quantitative analysis is carried out to health-care cold tea functional components, probes into chlorogenic acid, stevioside, rutin, liquiritin, quercitin
Content in health-care cold tea, since stevioside sugar amount is high, soluble easily in water and polarity is big, when analyzing health-care cold tea sample to more
The conditions such as kind elution program, mobile phase and Detection wavelength carry out test and probe into, and find in chlorogenic acid and rutin, liquiritin, quercitrin
Glycosides ingredient, which separates, cannot separate stevioside in good situation, therefore use two kinds of elution process by health-care cold tea five
Kind functional component, which is presented on two chromatograms, carries out quantitative analysis.
The stevioside standard chromatogram under 2.3.2 chromatographic condition, as shown in figure 8, stevioside standard items do not have appearance.
Containing five kinds of functional components such as chlorogenic acid, stevioside, rutin, liquiritin and quercitin in health-care cold tea, content compared with
It is low, but it is all immune etc. with for example anti-inflammatory antibacterial adjusting of respective physiological function in health-care cold tea, improve health-care cold tea health care
Effect.
2.3.1 the chromatographic condition of stevioside in health-care cold tea is detected
Chromatographic column: Shim Pack ODS C18 column (250mm × 4.6mm, 5 μm), mobile phase: acetonitrile (A)-water (B) (body
Product is than being 75: 25), Isocratic clution, overall flow rate: 0.8mL/min, column temperature: 30 DEG C, Detection wavelength: 210nm.
2.3.2 the chromatographic condition of health-care cold tea Content of Chlorogenic Acid, rutin, liquiritin and Determination of Quercitrin is detected
Chromatographic column: Shim Pack ODS C18 column (250mm × 4.6mm, 5 μm), mobile phase: -0.5% vinegar of acetonitrile (A)
Sour water (B) (initial volume ratio is 15: 85), flow velocity: 0.9mL/min, column temperature: 30 DEG C, Detection wavelength: 330nm.Elution requirement is shown in
Table 3.
- 0.5% acetic acid water gradient elution table of 3 acetonitrile of table
2.3.3 the preparation of standard solution
Accurate weighing chlorogenic acid, rutin, liquiritin, quercitin and stevioside standard items, quality be followed successively by 0.58mg,
0.28mg, 1.08mg, 2.00mg, 12.75mg are respectively placed in 25mL volumetric flask, and it is fixed that stevioside standard items ultrapure water dilutes
Hold, another four kinds of standard items are diluted constant volume with methanol respectively, are inverted rock uniformly back and forth, be kept in dark place in 4 DEG C of refrigerators.System
It obtains standard items mass concentration and is respectively as follows: chlorogenic acid 0.0232mg/mL, rutin 0.0112mg/mL, liquiritin 0.0432mg/mL, Mongolian oak
Skin glycosides 0.0800mg/mL and stevioside 0.5028mg/mL.
The analysis of 2.4 health-care cold tea physicochemical and microbial indexes
Health-care cold tea is completed by the preparation of above-mentioned process, is measured with abbe's refractometer to health-care cold tea at 20 DEG C, can
It directly reads or conversion obtains soluble solid content in health-care cold tea.Health-care cold tea pH value measures reading with pH meter
Number.
3 results and analysis
3.1 optimization of orthogonal test health-care cold teas are formulated result and analysis
Designed by 1 factor level of table and carry out L9 (34) orthogonal test, it is comprehensive to test data intuitively and variance analysis, come true
Determine the optimum formula of health-care cold tea.The raw material proportioning optimum results of health-care herbal tea beverage are as shown in table 4.
4 health-care cold tea ratio optimization test results and analysis of table
5 analysis of variance table of table
Note: F0.1(2,2)=9.00, F0.05(2,2)=19.00, F0.01(2,2)=99.00
Orthogonal experiment data is analyzed using Minitab software, obtains table 4 and table 5, can intuitively be analyzed by table 4
Influence each factor peci-order of health-care cold tea sensory evaluation scores are as follows: STEVIA REBAUDIANA extracting solution (B) > common rabdosia leaf extracting solution (A) > Radix Glycyrrhizae mentions
Take liquid (C) > FLOS CHRYSANTHEMI from Hangzhou of China extracting solution (D)；The results of analysis of variance shows that factor A, B, C have conspicuousness, and D factor does not have significantly
Property.Comprehensively consider, obtaining health-care cold tea optimum proportion scheme is A2B2C3D1, i.e. common rabdosia leaf extracting solution 10mL, STEVIA REBAUDIANA are extracted
Liquid 5mL, licorice extract 5mL, FLOS CHRYSANTHEMI from Hangzhou of China extracting solution 10mL.Health-care cold tea product filling amount is 330mL, and wherein common rabdosia leaf extracts
Liquid and FLOS CHRYSANTHEMI from Hangzhou of China extracting solution respectively account for 33.33%, and STEVIA REBAUDIANA extracting solution and licorice extract respectively account for 16.67%.By process flow system
It is in kind to obtain health-care cold tea product, sees Fig. 1.
The foundation of functional component quantitative analysis method in 3.2 health-care cold teas
3.2.1 the selection of mobile phase
When detecting stevioside in health-care cold tea, the screening of mobile phase is all the condition using 2.3.1, constant in other conditions
In the case where, mobile phase is screened；
When detecting health-care cold tea Content of Chlorogenic Acid, rutin, liquiritin and quercitin, the screening of mobile phase is all using 2.3.2
Condition mobile phase is screened in the case where other conditions are constant；
In order to reflect the presence of the substances such as rutin in health-care cold tea, chlorogenic acid, liquiritin, quercitin and stevioside completely
And content, Experimental Research, the separating effect of more each mobile phase are carried out under other chromatographic condition same cases.
For the special nature of stevioside, selective flow phase: methanol (A)-water (B), acetonitrile-water；
To remaining four kinds of ingredients (chlorogenic acid, rutin, liquiritin and quercitin) selective flow phase: -0.5% acetic acid of methanol,
- 0.5% acetic acid of acetonitrile is made comparisons respectively.Experimental result discovery: to stevioside, methanol can shorten its retention time and isolate
Peak peak shape symmetry is bad, therefore selects acetonitrile-water；To rutin, chlorogenic acid, liquiritin, quercitin, separation of the acetonitrile than methanol
Effect is more preferable, and chromatographic peak peak shape is preferable.Acid concentration is excessively high to damage chromatographic column, and too low then separating effect is bad,
Therefore 0.5% acetic acid water is optimum concentration；If acetic acid ratio height can make each ingredient retention time too long in mobile phase, too it is low not
Conducive to chlorogenic acid separation.Therefore, -0.5% acetic acid of acetonitrile is selected, using volume ratio 15:85 as gradient elution original ratio.Separation effect
Fruit is as shown in Figure 2 and Figure 3.1.5 are all larger than in the separating degree in Fig. 3 between adjacent peak, can reach and be kept completely separate.
This experiment is probed by repeatedly different elution programs, determines best elution program: 0~10min 85%-77%B；
10~35min 77%-75%B；35~45min 75%-76%B；45~55min 76%-85%B；55~60min
85%B.Under this elution requirement, can making health-care cold tea Content of Chlorogenic Acid and three kinds of flavones, (three kinds of flavones refer to rutin, Radix Glycyrrhizae
Glycosides, quercitin) etc. ingredient to be measured be kept completely separate, peak shape is more sharp symmetrical.
The chromatogram of each health-care cold tea sample and standard items is shown in Fig. 4 A, Fig. 4 B, Fig. 4 C, Fig. 4 D,
Wherein, Fig. 4 A is the four kinds of hybrid standard product solution chromatograms obtained using 2.3.2 chromatographic condition sample introduction, described four
Kind hybrid standard product solution is chlorogenic acid standard solution, rutin standard solution, liquiritin standard solution and quercitin mark
Quasi- product solution is mixed to get；
Fig. 4 B is using 2.3.2 chromatographic condition, the health-care cold tea solution chromatogram of detection 20180426 batches；
Fig. 4 C is using 2.3.2 chromatographic condition, stevioside standard chromatogram；
Fig. 4 D is to detect 20180426 batches of health-care cold tea sample chromatogram figures using 2.3.1 chromatographic condition.
3.2.2 the selection of chromatographic column column temperature
When detecting stevioside in health-care cold tea, the screening of column temperature is all the condition using 2.3.1, constant in other conditions
In the case of, column temperature is screened；
When detecting health-care cold tea Content of Chlorogenic Acid, rutin, liquiritin and quercitin, the screening of column temperature is all using 2.3.2
Condition screens column temperature in the case where other conditions are constant；
Test compare chromatogram column temperature under the conditions of 25 DEG C, 30 DEG C, 35 DEG C and 40 DEG C this 4 temperature chlorogenic acid, rutin,
The separation situation of various functional component chromatographic peaks in liquiritin, quercitin, stevioside standard items and health-care cold tea.As a result it sends out
Existing, in the 4 temperature conditions investigated, under the conditions of 25 DEG C, chlorogenic acid, which is unable to get, to be efficiently separated, 30 DEG C, 35 DEG C, 40 DEG C of items
Separating effect is preferable under part.It is 25 DEG C that stevioside separating resulting, which is column temperature, in sample, and stevioside is connected with other peaks, 30 DEG C, 35
DEG C, then separating degree is greater than 1.5 under the conditions of 40 DEG C, good separation.It, can the lower item of preferred energy consumption on the basis of analog result
Part, thus can be preferably chromatographic column temperature be 30 DEG C.The chromatogram of each column temperature is as shown in Fig. 5 A, Fig. 5 B, Fig. 5 C, Fig. 5 D.
3.2.3 the selection of flow rate of mobile phase
When detecting stevioside in health-care cold tea, the screening of flow rate of mobile phase is all the condition using 2.3.1, in other conditions
In the case where constant, flow rate of mobile phase is screened；
When detecting health-care cold tea Content of Chlorogenic Acid, rutin, liquiritin and quercitin, the screening of flow rate of mobile phase is all to use
2.3.2 condition screens flow rate of mobile phase in the case where other conditions are constant；
Test compares flow rate of mobile phase in 0.6~1.0mLmin-1Interior chlorogenic acid, rutin, liquiritin, quercitin, sweet tea
The separation situation of each functional component chromatographic peak in chrysanthemum glycosides standard items and health-care cold tea.As a result, it has been found that flow velocity 0.6mLmin-1
When, the substance peak of stevioside cannot be efficiently separated, and flow velocity is greater than 0.8mLmin-1When start the substance peak point of stevioside
It is greater than 1.5 from degree, good separation；Flow velocity is 0.6mLmin-1When, chlorogenic acid, rutin, liquiritin, quercitin separation
Degree is preferable, 0.8~1.0mLmin of overall flow rate-1Separating effect is more excellent.It is acetonitrile-water, flow velocity it is advantageous to mobile phase
0.8mL·min-1When detect stevioside, preferable mobile phase is -0.5% acetic acid of acetonitrile, flow velocity 0.9mLmin-1When detect green original
Acid, rutin, liquiritin, quercitin.Each chromatogram such as Fig. 6 A, Fig. 6 B under different in flow rate, shown in Fig. 6 C.
3.2.4 the selection of Detection wavelength
When detecting stevioside in health-care cold tea, the screening of wavelength is all the condition using 2.3.1, constant in other conditions
In the case of, wavelength flow velocity is screened；
When detecting health-care cold tea Content of Chlorogenic Acid, rutin, liquiritin and quercitin, the screening of wavelength is all using 2.3.2
Condition screens wavelength in the case where other conditions are constant；
Chlorogenic acid, rutin, liquiritin, quercitin, stevioside standard items are swept in 200~400nm wave-length coverage
It retouches, show that each mark according to the ultraviolet absorption peak of product, is concentrated mainly on 210,260, tetra- 330,360nm wavelength.In this four wavelength
Under hybrid standard product are detected, discovery stevioside at 210nm has a maximum absorption band, and chlorogenic acid, rutin, liquiritin,
Quercitin has maximum absorption band at 330nm, and comprehensive various aspects consideration can divide 2 wavelength to be detected, i.e., stevioside detects
Wavelength is 210nm, and chlorogenic acid, rutin, liquiritin, quercitin Detection wavelength are 330nm.Chromatogram under the conditions of different wave length is such as
Fig. 7 A, Fig. 7 B, Fig. 7 C, shown in Fig. 7 D.
The investigation of 3.3HPLC quantitative analysis method
3.3.1 system suitability test
It is accurate respectively to draw 20 μ l injecting chromatograph of health-care cold tea test solution, by chromatographic condition under " 2.3.2 " item into
Sample, calculate separately chlorogenic acid, rutin, liquiritin and quercitin theoretical cam curve, tailing factor and separating degree, data are such as
Under:
6 system of table adapts to data
Precision draws 20 μ l injecting chromatograph of health-care cold tea test solution, by chromatographic condition sample introduction under " 2.3.1 " item, warp
Calculate stevioside theoretical cam curve, tailing factor and separating degree, data are as follows:
The above results are analyzed it is found that the tailing factor of each compound chromatographic peak is respectively less than in health-care cold tea detected
2.0%, reach good baseline separation, the rule that the theoretical cam curve of each compound peaks chromatographic peak is met the quality standard with impurity peaks
Fixed, in 60min, the separating degree between each compound chromatographic peak and adjacent peak is all larger than 1.5, and symmetry is good, can meet a variety of
It is separated while compound.
3.3.2 linear relationship is investigated
It accurately draws each titer 1mL, 2mL, 3mL, 4mL, 5mL with pipette to move into respectively in 10mL volumetric flask, green original
Sour and rutin, liquiritin, quercitin standard items are settled to graduation mark with methanol respectively；Stevioside standard items are settled to ultrapure water
Graduation mark.Corresponding to 20 μ L of chromatographic condition sequence sample introduction by each substance, (its Content of Chlorogenic Acid and rutin, liquiritin, quercitin are by 2.3.2
Chromatographic condition sample introduction, stevioside press 2.3.1 chromatographic condition sample introduction), obtain the peak area of each concentration standards.In Excel table
In, each standard concentration and corresponding peak area are inputted, using concentration as horizontal axis, corresponding peak area is that the longitudinal axis draws the standard items mark
Directrix curve obtains 5 standard curves, related coefficient and the range of linearity, is shown in Table 7.In the linear range, each ingredient in health-care cold tea
Concentration and corresponding peak area there is good linear relationship.
7 standard curve of table, related coefficient and the range of linearity
3.3.3 precision test
Accurate absorption 0.0232mg/mL chlorogenic acid respectively, 0.0112mg/mL rutin, 0.0432mg/mL liquiritin,
0.0800mg/mL quercitin is distinguished continuous sample introduction 4 times by chromatographic condition under " 2.3.1 " item, measures respective peak area, according to
Measurement result calculates relative standard deviation RSD.Calculate the corresponding RSD of chlorogenic acid, rutin, liquiritin, quercitin is respectively
0.86%, 1.23%, 0.607%, 0.549%.It is accurate again to draw 20 μ l of 0.5028mg/mL stevioside reference substance solution, it presses
Chromatographic condition sample introduction under " 2.3.2 " item, measures its peak area, calculates relative standard deviation RSD according to measurement result, calculates to obtain sweet tea
It is 0.93% that chrysanthemum glycosides, which corresponds to RSD result,.All Compound RS D are respectively less than 2.0%, show that this method is accurate under two kinds of testing conditions
It spends good.
3.3.4 stability test
Respectively at 0,2,4,8,16,18,20 μ l injecting chromatograph of health-care cold tea test solution is drawn for 24 hours, is first pressed
" 2.3.2 " item chromatographic condition sample introduction, measure peak area, retention time, calculate chlorogenic acid relative peak area RSD be 1.01%,
Relative retention time RSD is 0.98%；Rutin relative peak area RSD is 0.69%, and relative retention time RSD is 0.74%；It is sweet
Careless glycosides Mongolian oak relative peak area RSD is 0.95%, and relative retention time RSD is 0.89%；Quercitin relative peak area RSD is
0.66%, relative retention time RSD are 0.85%.According still further to " 2.3.1 " item chromatographic condition sample introduction is first pressed, investigates stevioside and stablize
Property, its corresponding peak area and retention time are calculated, as a result obtaining stevioside relative peak area RSD is 1.12%, relative retention time
RSD is 0.93%.The above result shows that health-care cold tea test solution is good in internal stability for 24 hours.
3.3.5 repetitive test
Precision weighs each 5 parts of same batch common rabdosia leaf, STEVIA REBAUDIANA, FLOS CHRYSANTHEMI from Hangzhou of China, Radix Glycyrrhizae, prepares 5 in parallel by under the conditions of " 2.1 "
Part health-care cold tea sample detects first by sample introduction is repeated under " 2.3.2 " chromatographic condition and chlorogenic acid relative peak area is calculated
RSD is 0.84%, and relative retention time RSD is 0.93%；Rutin relative peak area RSD is 0.54%, relative retention time RSD
It is 0.67%；Quercitin relative peak area RSD is 0.43%, and relative retention time RSD is 0.77%.Further according to " 2.3.1 " color
Spectral condition repeats sample introduction, and obtaining stevioside relative peak area RSD is 0.72%, and relative retention time RSD is 0.88%.Two kinds of items
Under part, all Compound RS D are respectively less than 2.0%, show that the repeatability of the measuring method is good.
3.3.6 sample recovery rate is tested
The medicinal material sample for accurately weighing known content is prepared into corresponding sample solution according to 2.1.2 method, and precision is added
The respective standard product of known content, calculate the corresponding rate of recovery, the results are shown in Table 8 accordingly.
8 sample recovery rate experimental result of table
Each component content in 3.4 measurement health-care cold teas
Health-care cold tea accurately is prepared according to process flow, is placed on 2mL sample with 0.45 μm of filtering with microporous membrane after preparing
In product bottle, according to the chromatographic condition in 2.3, respectively into 20 μ L same samples, wherein 20180426 batches of health-care cold tea sample introduction results
As shown in Fig. 4 B, 4D, Fig. 4 A is using 2.3.2 chromatographic condition sample introduction four kinds of hybrid standard product (i.e. chlorogenic acid, rutin, Radix Glycyrrhizae
4 kinds of glycosides, quercitin standard items) solution chromatogram, Fig. 4 C is using 2.3.1 chromatographic condition sample introduction stevioside standard chromatogram.
Different batches health-care cold tea sample is measured with same elution program, it is bent according to health-care cold tea sample chromatogram figure and standard
Line is calculated each component content, is shown in Table 8.
Stevioside is measured using the chromatographic condition of 2.3.1 in table 8；
8 Content of Chlorogenic Acid of table, rutin, liquiritin, quercitin are measured using the chromatographic condition of 2.3.2.
The content of each ingredient in 9 health-care cold tea sample of table
It is shown by table 9, each component content floating range very little, illustrates the guarantor mixed every time in three batch health-care cold teas
Strong each stable components of herbal tea, it is excellent to embody health-care cold tea preparation process, and batch is suitble to repeatedly to carry out health-care cold tea production.It measures
Each component content in health-care cold tea: 6.16 ± 0.15 μ g/mL of chlorogenic acid, 3.41 ± 0.16 μ g/mL of rutin, liquiritin 13.24 ±
0.22 μ g/mL, 24.11 ± 0.14 μ g/mL of quercitin, 140.1 ± 0.22 μ g/mL of stevioside.
The detection of 3.5 health-care cold tea physicochemical and microbial indexes
To being cooked, deployed, filling and after 100 DEG C of water dip sterilization 20min health-care cold tea according to process flow, with
Machine extracts the herbal tea sample that lot number is 20180501 and carries out index of refraction measurement at 20 DEG C, and reading is 1.3348 ± 0.23, conversion
Obtaining soluble solid content afterwards is 1.28 ± 0.15%；Health-care cold tea pH is 5.80 ± 0.21.Health-care cold tea preparation whole process exists
It is operated under aseptic condition, total plate count≤1CFU/mL, no pathogenic bacteria.
4 conclusions
Quality control is carried out to health-care cold tea by high performance liquid chromatography, experimental result is shown: in the range of linearity: green original
Sour 2.32-11.60 μ g/mL, rutin 1.12-5.60 μ g/mL, liquiritin 4.32-21.60 μ g/mL, quercitin 8.00-40.00 μ
In g/mL, stevioside 50.28-251.40 μ g/mL, the concentration of each ingredient has good with corresponding peak area in health-care cold tea
Linear relationship.Measure each component content in health-care cold tea: 6.16 ± 0.15 μ g/mL of chlorogenic acid, 3.41 ± 0.16 μ g/mL of rutin,
13.24 ± 0.22 μ g/mL of liquiritin, 24.11 ± 0.14 μ g/mL of quercitin, 140.1 ± 0.22 μ g/mL of stevioside.
Claims (10)
1. a kind of detection method of health-care cold tea, the health-care cold tea includes that the following raw material is prepared: common rabdosia leaf leaf, sweetleaf
The detection method of chrysanthemum, Radix Glycyrrhizae and FLOS CHRYSANTHEMI from Hangzhou of China, the health-care cold tea includes:
The step of detecting the stevioside content in health-care cold tea:
Test solution and stevioside reference substance solution are provided；
Using octadecylsilane chemically bonded silica as filler, using acetonitrile-water as mobile phase, Isocratic clution, in liquid chromatograph
The middle detection test solution and stevioside reference substance solution, the stevioside that external standard method is analyzed to obtain in the health-care cold tea contain
Amount；
Detect chlorogenic acid in health-care cold tea, rutin, liquiritin and the step of Determination of Quercitrin:
Test solution, chlorogenic acid reference substance solution, control substance of Rutin solution, liquiritin reference substance solution and quercitin pair are provided
According to product solution；
Using octadecylsilane chemically bonded silica as filler, using acetonitrile as mobile phase A, using acetic acid water as Mobile phase B, gradient is washed
It is de-, the test solution, chlorogenic acid reference substance solution, control substance of Rutin solution, liquiritin pair are detected in liquid chromatograph
According to product solution and quercitin reference substance solution, external standard method analyze to obtain chlorogenic acid in the health-care cold tea, rutin, liquiritin and
Determination of Quercitrin.
2. the detection method of health-care cold tea according to claim 1, stevioside content in the detection health-care cold tea
In step, the volume of acetonitrile and the volume ratio of water are 75:25 in the mobile phase, the chlorogenic acid detected in health-care cold tea,
In the step of rutin, liquiritin and Determination of Quercitrin, the acetic acid water is the acetic acid water of 0.5% volume.
3. the detection method of health-care cold tea according to claim 2, stevioside content in the detection health-care cold tea
In step, the flow velocity of the mobile phase is 0.8~1.0mL/min, chlorogenic acid, rutin, Radix Glycyrrhizae in the detection health-care cold tea
In the step of glycosides and Determination of Quercitrin, the flow velocity of the mobile phase is 0.6~1.0mL/min.
4. the detection method of health-care cold tea according to claim 3, stevioside content in the detection health-care cold tea
In step, the flow velocity of the mobile phase is 0.8mL/min, chlorogenic acid, rutin, liquiritin and the Mongolian oak in the detection health-care cold tea
In the step of skin glycosides content, the flow velocity of the mobile phase is 0.9mL/min.
5. the detection method of health-care cold tea according to claim 1, stevioside content in the detection health-care cold tea
In step, the column temperature of the detection is 30~40 DEG C, chlorogenic acid, rutin, liquiritin and the quercitrin in the detection health-care cold tea
In the step of glycosides content, the column temperature of the detection is 30~40 DEG C.
6. the detection method of health-care cold tea according to claim 5, stevioside content in the detection health-care cold tea
In step, the column temperature of the detection is 30 DEG C, and chlorogenic acid, rutin, liquiritin and the quercitin in the detection health-care cold tea contain
In the step of amount, the column temperature of the detection is 30 DEG C.
7. the detection method of health-care cold tea according to claim 1, stevioside content in the detection health-care cold tea
In step, the Detection wavelength is 200~400nm, chlorogenic acid, rutin, liquiritin and the quercitrin in the detection health-care cold tea
In the step of glycosides content, the Detection wavelength is 200~400nm.
8. the detection method of health-care cold tea according to claim 7, stevioside content in the detection health-care cold tea
In step, the Detection wavelength is 210nm, chlorogenic acid, rutin, liquiritin and the Determination of Quercitrin in the detection health-care cold tea
The step of in, the Detection wavelength be 330nm.
It is green in the detection health-care cold tea 9. the detection method of health-care cold tea according to any one of claims 1 to 8
In the step of ortho acid, rutin, liquiritin and Determination of Quercitrin, the initial volume ratio of acetonitrile and 0.5% acetic acid water is 15:85.
It is green in the detection health-care cold tea 10. the detection method of health-care cold tea described in -9 any one according to claim 1
In the step of ortho acid, rutin, liquiritin and Determination of Quercitrin, the gradient elution program are as follows:
0~10 minute, mobile phase A was 15~20%, and Mobile phase B is 85~77%；
10~35 minutes, mobile phase A was 20~50%, and Mobile phase B is 77~75%；
35~45 minutes, mobile phase A was 50~85%, and Mobile phase B is 75~76%；
45~55 minutes, mobile phase A was 85~15%, and Mobile phase B is 76~85%；
55~60 minutes, mobile phase A was 15~15%, and Mobile phase B is 85~85%.
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