CN107843665A - A kind of assay method of adenosine content and its application - Google Patents
A kind of assay method of adenosine content and its application Download PDFInfo
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Abstract
The invention belongs to analytical chemistry detection field, more particularly to a kind of assay method of adenosine content and its application.The invention provides a kind of assay method of adenosine content, the assay method is：Dissolving, purification, filtering and efficient liquid phase measure.Present invention also offers a kind of application of assay method including described in any of the above one in chlorogenic acid content detection.Present invention also offers a kind of application of assay method including described in any of the above one in rutin content detection.It can be obtained through measuring, technical scheme provided by the invention, Accurate Determining can be realized to adenosine, chlorogenic acid and rutin content in testing sample, further, effectively increase the detection efficiency and detection accuracy of adenosine in soft capsule, chlorogenic acid and rutin content.
Description
Technical field
The invention belongs to analytical chemistry detection field, more particularly to a kind of assay method of adenosine content and its application.
Background technology
Light network plain (Fruitflow) is a kind of water soluble tomato concentrate, is that first science of passing through proves, can promote blood flow
The natural solution of health.Light network element is a kind of breakthrough antithrombotic composition, platelet aggregation can be kept, so as to contribute to
Maintain cardiovascular system health.A kind of light network element dispensing natural as safety, will not destroy required coagulation process after injury；
The clinical research delivered shows, light network element can make 97% subject, and platelet aggregation reduces in 1.5 hours upon intake, work(
Effect can be held up to 18 hours；Meanwhile found in 10 clinical researches, the plain long-term use of no side effects of light network, allergy are anti-
Should or with the incompatible phenomenon of other drugs.
Wherein, adenosine, chlorogenic acid and rutin are the significant functional components of light network element, determine adenosine, chlorogenic acid and rutin
Content provide foundation for the quality standard containing light network element class preparation.In the prior art in published document detection method,
Three adenosine, chlorogenic acid and rutin compositions can not be detected simultaneously；Moreover, pre-treating method of the document for sample, can not also reach
To simultaneously extract adenosine, chlorogenic acid and the effect of rutin.
Therefore, assay method and its application of a kind of adenosine content are developed, can not be simultaneously for solving in the prior art
The technological deficiency of adenosine, chlorogenic acid and rutin content, becomes those skilled in the art and urgently solves in accurate detection soft capsule
Certainly the problem of.
The content of the invention
In view of this, the invention provides a kind of assay method of adenosine content and its application, for solving prior art
In, the technological deficiency of adenosine, chlorogenic acid and rutin content in soft capsule can not be accurately detected simultaneously, effectively increase soft capsule
Middle adenosine, the detection efficiency of chlorogenic acid and rutin content and detection accuracy.
The invention provides a kind of assay method of adenosine content, the assay method is：
Step 1: dissolving：It is ultrasonic to obtain the first product after testing sample mixes with the first solvent, the second solvent；
Step 2: purification：After the first product centrifugation, upper liquid is discarded；Obtain the second product；
Step 3: filtering：Second product is dissolved in the second solvent, and organic phase filter membrane filters to obtain prepare liquid；
Step 4: efficient liquid phase determines：Prepare liquid determines peak area through high performance liquid chromatography, with standard curve control, obtains
Determinand content；
First solvent is selected from：One or more in petroleum ether, n-hexane and ether, the second solvent choosing
From：One or more in methanol solution, 10% acetonitrile solution and 10% ethanol solution that volumetric concentration is 20%.
Preferably, in step 1, the volume ratio of the first solvent and the second solvent is 1：(2~3).
Preferably, in step 2, the rotating speed of the centrifugation is 5000~8000r/min, time of the centrifugation for 1~
3min。
Preferably, in step 3, the volume ratio of second product and second solvent is 1：(1~3).
Preferably, in step 3, the aperture of the organic phase filter membrane is 0.22 μm.
Preferably, in step 4, in the measure of high performance liquid chromatography, mobile phase A is trifluoroacetic acid solution, and Mobile phase B is
Methanol, it is measured by the way of gradient elution.
Preferably, in step 4, in the measure of high performance liquid chromatography, flow velocity is 0.8~1.2ml/min, column temperature is 25~
40℃。
Preferably, in step 4, in the measure of high performance liquid chromatography, Detection wavelength 254nm.
Present invention also offers a kind of assay method including described in any of the above one in chlorogenic acid content detection
Using.
Present invention also offers a kind of assay method including described in any of the above one in rutin content detection should
With.
In summary, the invention provides a kind of assay method of adenosine content, the assay method to be：It is Step 1: molten
Solution：It is ultrasonic to obtain the first product after testing sample mixes with the first solvent, the second solvent；Step 2: purification：First product
After centrifugation, upper liquid is discarded, obtains the second product；Step 3: filtering：Second product is dissolved in the second solvent, organic phase filter
Membrane filtration obtains prepare liquid；Step 4: efficient liquid phase determines：Prepare liquid determines peak area through high performance liquid chromatography, with standard curve
Control, obtains determinand content；First solvent is selected from：One or more in petroleum ether, n-hexane and ether, described
Two solvents are selected from：Volumetric concentration is 20% methanol solution, 10% acetonitrile solution and one kind or more in 10% ethanol solution
Kind.Present invention also offers a kind of application of assay method including described in any of the above one in chlorogenic acid content detection.
Present invention also offers a kind of application of assay method including described in any of the above one in rutin content detection.Through experiment
Measure can obtain, technical scheme provided by the invention, can realize accurate survey to adenosine, chlorogenic acid and rutin content in testing sample
It is fixed, further, effectively increase the detection efficiency and detection accuracy of adenosine in soft capsule, chlorogenic acid and rutin content.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
The embodiment of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can also basis
The accompanying drawing of offer obtains other accompanying drawings.
Fig. 1 is the standard working curve of adenosine；
Fig. 2 is the standard working curve of chlorogenic acid；
Fig. 3 is the standard working curve of rutin.
Embodiment
The embodiments of the invention provide a kind of assay method of adenosine content and its application, for solving in the prior art,
The technological deficiency of adenosine, chlorogenic acid and rutin content in soft capsule can not be accurately detected simultaneously, effectively increased in soft capsule
Adenosine, the detection efficiency of chlorogenic acid and rutin content and detection accuracy.
The technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described implementation
Example only part of the embodiment of the present invention, rather than whole embodiments.It is common based on the embodiment in the present invention, this area
The every other embodiment that technical staff is obtained under the premise of creative work is not made, belong to the model that the present invention protects
Enclose.
In order to which the present invention is described in more detail, with reference to embodiment to a kind of measure side of adenosine content provided by the invention
Method and its application, are specifically described.
Embodiment 1
The present embodiment is the specific embodiment for preparing prepare liquid.
In the present embodiment, selected testing sample is certain commercially available light network element auxiliary blood fat reducing soft capsule.
After 0.25g testing samples mix with the first solvents of 10ml 1, the second solvents of 25ml 1, with 37Hz frequency ultrasound
1min, obtain the first product 1.After first product 1 centrifuges 2min under 8000r/min rotating speed, upper liquid is discarded, obtains the second product
1.The second products of 25ml 1 are dissolved in the second solvents of 25ml 1, are shaken up after being settled to 50ml, are filtered through 0.22 μm of organic phase filter membrane,
Obtain prepare liquid 1.During this is prepared, the first solvent 1 is petroleum ether, and the second solvent 1 is 20% methanol solution.
After 0.25g testing samples mix with the first solvents of 17ml 2, the second solvents of 17ml 2, with 37Hz frequency ultrasound
1min, obtain the first product 2.After first product 2 centrifuges 3min under 6000r/min rotating speed, upper liquid is discarded, obtains the second product
2.The second products of 20ml 2 are dissolved in the second solvents of 30ml 2, are shaken up after being settled to 50ml, are filtered through 0.22 μm of organic phase filter membrane,
Obtain prepare liquid 2.During this is prepared, the first solvent 2 is n-hexane, and the second solvent 2 is 10% acetonitrile solution.
After 0.25g testing samples mix with the first solvents of 9ml 3, the second solvents of 27ml 3, with 37Hz frequency ultrasound 1min,
Obtain the first product 3.After first product 3 centrifuges 1min under 5000r/min rotating speed, upper liquid is discarded, obtains the second product 3.
The second products of 12.5ml 3 are dissolved in the second solvents of 37.5ml 3, are shaken up after being settled to 50ml, through 0.22 μm of organic phase filter membrane mistake
Filter, obtains prepare liquid 3.During this is prepared, the first solvent 3 is ether, and the second solvent 3 is 10% ethanol solution.
Embodiment 2
The present embodiment is the specific embodiment for preparing standard solution.
In the present embodiment, adenosine standard items are purchased from SIGMA, and its lot number is WXBC1570V, purity 99%；Chlorogenic acid mark
Quasi- product are purchased from National Institute for Food and Drugs Control, and its lot number is 110753-201415, purity 96.2%；Rutin standard items
Purchased from National Institute for Food and Drugs Control, its lot number is 100080-201409, purity 91.9%.
Precision weighs chlorogenic acid standard items 5.92mg, is placed in 10ml brown volumetric flasks, adds 50%V/V methanol dissolving
Afterwards, constant volume, shake up, obtain chlorogenic acid storing solution, stored for future use in 4 DEG C of refrigerators.
Precision weighs chlorogenic acid reference substance 5.92mg, is placed in 10ml brown volumetric flasks, adds 50% methanol and dissolves and determine
Hold to scale, shake up, produce chlorogenic acid storing solution.Storing solution is placed in 4 DEG C of refrigerator and stored for future use.
Precision weighs adenosine reference substance 8.44mg, control substance of Rutin 4.49mg, is placed in 100ml brown volumetric flasks, adds
The of short duration ultrasound of 20ml methanol, adds the of short duration ultrasonic dissolution of 50ml water, and precision adds 1.00ml chlorogenic acid storing solutions, adds water constant volume
To scale, shake up, produce.Contrast solution is placed in 4 DEG C of refrigerator and stored for future use.
Embodiment 3
The specific embodiment that the present embodiment determines for the range of linearity of detection method provided by the invention.
3.1 chromatographic condition
Mobile phase is：A phases：0.05% (0.025%-0.1%) trifluoroacetic acid solution, B phase methanol；Gradient elution；Specifically
Type of elution refer to table 1.
Table 1
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0.0 | 95 | 5 |
| 3.0 | 90 | 10 |
| 15.0 | 80 | 20 |
| 18.0 | 77 | 23 |
| 23.0 | 50 | 50 |
| 28.0 | 25 | 75 |
| 28.1 | 95 | 5 |
| 32.0 | 95 | 5 |
Flow velocity：1.0ml/min；Detection wavelength：Adenosine (254nm), chlorogenic acid (325nm) and rutin (325nm)；Column temperature：
30℃；Chromatographic column：Phenomenex Luna, C18 (2), 100 × 4.6mm, 3 μm.3.2 draw standard curve
By standard solution made from embodiment 2, μ l of sample introduction 2,5 μ l, 10 μ l, 20 μ l, 30 μ l are distinguished, under the conditions of 3.1,
Analysis measure is carried out, draws standard working curve.
3.3 measure prepare liquids
By standard solution made from embodiment 2 under the conditions of 3.1, analysis measure is carried out.
The computational methods of acquired results are：X=C × V/M × K；In formula：X-sample adenosine (chlorogenic acid/rutin) contains
Amount, mg/g；The quality of M-sample, g；K-unit conversion factor (K=1)；The dilution volume of V-sample, ml；C-sample is molten
The concentration of adenosine (chlorogenic acid/rutin), mg/ml in liquid.3.4 test data
By above-mentioned calculating, in measured standard solution, adenosine, chlorogenic acid, rutin content respectively refering to table 2~
Table 4.
Table 2
Table 3
Table 4
It can be drawn from 2~table of table 4, testing result obtained by detection method provided by the invention is accurate, with theoretical content phase
It symbol, can truly reflect the content of sample, the purpose of quality control is played to sample.
3.5 standard working curve figures
Using concentration as abscissa, peak area is ordinate, draws standard working curve.The explanation of specific works curve please join
Read brief description of the drawings.
It can be drawn from Fig. 1 to Fig. 3, detection method provided by the invention, its linear concentration is wide, is adapted to various concentrations sample
Measure, be well positioned to meet the demand of detection.
Can be drawn by the present embodiment, adenosine, chlorogenic acid and the coefficient R of rutin are respectively 1.00000,1.00000,
1.00000, so it is 0.0167112mg/ml to 0.250668mg/ml to determine adenosine in concentration with this method, chlorogenic acid is dense
Spend for 0.001139008mg/ml to 0.01708512mg/ml, rutin concentration be 0.00825262mg/ml extremely
Present good linear between 0.1237893mg/ml, meet GB/T27404-2008《Good Laboratory controls specification》Requirement
【GB/T27404-2008 requires coefficient R >=0.99】.
Embodiment 4
The present embodiment is the specific embodiment of detection method replica test provided by the invention.
6 parts of same samples are taken, after carrying out sample treatment according to the preparation method of three kinds of prepare liquids in embodiment 2 respectively,
Prepare liquid 1, unemployed liquid 2 and prepare liquid 3 are repeated twice sample size detection respectively, calculate its RSD (%), acquired results refer to
5~table of table 7.
Table 5
Table 6
Table 7
Can be drawn from 5~table of table 7, the RSD (%) of adenosine, chlorogenic acid and rutin content is respectively 3.3%, 2.3%,
3.4%, show that detection method provided by the invention has preferable repeatability, meet GB/T27404-2008《Good Laboratory control
Specification processed》Requirement【GB/T27404-2008 requires RSD (%)≤3.8%】.
Embodiment 5
The present embodiment is the specific embodiment of detection method stability test provided by the invention.
After reference substance solution made from embodiment 2 is placed into 0h, 1h, 2h, 4h, 8h, 12h at room temperature respectively, by 3.1 colors
Spectral condition determines peak area, calculates its RSD (%).Acquired results refer to 8~table of table 10.
Table 8
Table 9
Table 10
Can be drawn by 8~table of table 10, adenosine, chlorogenic acid and control substance of Rutin solution place at room temperature respectively 0h, 1h,
After 2h, 4h, 8h, 12h, RSD (%) is respectively 0.1%, 0.5%, 0.2%, shows adenosine, chlorogenic acid and control substance of Rutin solution
Having good stability in 12 hours at room temperature.
Further, to assay method provided by the invention, precision, blank, detection limit, quantitative limit, the rate of recovery are carried out
Experiment, meets GB/T27404-2008《Good Laboratory controls specification》Requirement, it was demonstrated that content assaying method is scientific and effective,
The purpose of quality control can be played to adenosine, chlorogenic acid and the rutin content of light network element auxiliary blood fat reducing soft capsule.
In summary, the invention provides a kind of assay method of adenosine content, the assay method to be：It is Step 1: molten
Solution：It is ultrasonic to obtain the first product after testing sample mixes with the first solvent, the second solvent；Step 2: purification：First product
After centrifugation, upper liquid is discarded, obtains the second product；Step 3: filtering：Second product is dissolved in the second solvent, organic phase filter
Membrane filtration obtains prepare liquid；Step 4: efficient liquid phase determines：Prepare liquid determines peak area through high performance liquid chromatography, with standard curve
Control, obtains determinand content；First solvent is selected from：One or more in petroleum ether, n-hexane and ether, described
Two solvents are selected from：Volumetric concentration is 20% methanol solution, 10% acetonitrile solution and one kind or more in 10% ethanol solution
Kind.Present invention also offers a kind of application of assay method including described in any of the above one in chlorogenic acid content detection.
Present invention also offers a kind of application of assay method including described in any of the above one in rutin content detection.Through experiment
Measure can obtain, technical scheme provided by the invention, can realize accurate survey to adenosine, chlorogenic acid and rutin content in testing sample
It is fixed, further, effectively increase the detection efficiency and detection accuracy of adenosine in soft capsule, chlorogenic acid and rutin content.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of assay method of adenosine content, it is characterised in that the assay method is：
Step 1: dissolving：It is ultrasonic to obtain the first product after testing sample mixes with the first solvent, the second solvent；
Step 2: purification：After the first product centrifugation, upper liquid is discarded；Obtain the second product；
Step 3: filtering：Second product is dissolved in the second solvent, and organic phase filter membrane filters to obtain prepare liquid；
Step 4: efficient liquid phase determines：Prepare liquid determines peak area through high performance liquid chromatography, with standard curve control, obtains to be measured
Thing content；
First solvent is selected from：One or more in petroleum ether, n-hexane and ether, second solvent are selected from：Body
Product concentration is the one or more in 20% methanol solution, 10% acetonitrile solution and 10% ethanol solution.
2. the assay method of adenosine content according to claim 1, it is characterised in that in step 1, the first solvent and
The volume ratio of two solvents is 1：(2~3).
3. the assay method of adenosine content according to claim 1, it is characterised in that in step 2, the centrifugation turns
Speed is 5000~8000r/min, and the time of the centrifugation is 1~3min.
4. the assay method of adenosine content according to claim 1, it is characterised in that in step 3, second product
Volume ratio with second solvent is 1：(1~3).
5. the assay method of adenosine content according to claim 1, it is characterised in that in step 3, the organic phase filter
The aperture of film is 0.22 μm.
6. the assay method of adenosine content according to claim 1, it is characterised in that in step 4, high performance liquid chromatography
Measure in, mobile phase A is trifluoroacetic acid solution, and Mobile phase B is methanol, is measured by the way of gradient elution.
7. the assay method of adenosine content according to claim 1, it is characterised in that in step 4, high performance liquid chromatography
Measure in, flow velocity is 0.8~1.2ml/min, and column temperature is 25~40 DEG C.
8. the assay method of adenosine content according to claim 1, it is characterised in that in step 4, high performance liquid chromatography
Measure in, Detection wavelength 254nm.
A kind of 9. application of assay method including described in claim 1 to 7 any one in chlorogenic acid content detection.
A kind of 10. application of assay method including described in claim 1 to 7 any one in rutin content detection.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111044632A (en) * | 2019-12-27 | 2020-04-21 | 江西省肿瘤医院(江西省癌症中心) | Method for detecting adenosine content in urine and application thereof |
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Application publication date: 20180327 |