CN108169356A - A kind of method that ASE-HPLC methods measure spinosin content in spina date seed - Google Patents
A kind of method that ASE-HPLC methods measure spinosin content in spina date seed Download PDFInfo
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- CN108169356A CN108169356A CN201711365339.1A CN201711365339A CN108169356A CN 108169356 A CN108169356 A CN 108169356A CN 201711365339 A CN201711365339 A CN 201711365339A CN 108169356 A CN108169356 A CN 108169356A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The invention discloses a kind of methods that ASE HPLC methods measure spinosin content in spina date seed, belong to field of chemical detection.It is intended to provide a kind of method for taking ASE extractions that are short and can reach ideal effect and being combined with HPLC detection methods.This method extracts Spine Date Seed using ASE methods, wherein the ASE methods include first using n-hexane, then extracted with ethyl alcohol, and collect alcohol extraction liquid；Using HPLC methods to the spinosin content in alcohol extraction liquid.The present invention can replace assay of the official method to spinosin in spina date seed and the content monitoring to the spinosin in spina date seed.
Description
Technical field
The invention belongs to field of chemical detection, especially a kind of ASE-HPLC methods measure spinosin content in spina date seed
Method.
Background technology
《Chinese Pharmacopoeia》Version one in 2015 prepares test sample method to take this product powder (crossing No. four sieves) about 1g, and precision claims
It is fixed, it puts in Soxhlet extractor, adds petroleum ether (60~90 DEG C) in right amount, be heated to reflux 4 hours, discard petroleum ether liquid, the dregs of a decoction are flung to
Solvent is transferred in conical flask, is added in 70% ethyl alcohol 20ml, is heated to reflux 2 hours, is filtered, and filter residue is washed with 70% ethyl alcohol 5ml
It washs, merges washing lotion and filtrate, for recycling design to doing, residue adds ethyl alcohol to dissolve, and is transferred in 5ml measuring bottles, and ethyl alcohol is added to be shaken to scale
It is even, filtration, take subsequent filtrate to get.
This method return time is long, more demanding to the professional standards of technical staff.
Invention content
Against the above deficiency, the present invention is intended to provide a kind of take ASE-HPLC methods measure that is short and can reaching effect same
The method of spinosin content in spina date seed.
In order to solve the above technical problem, the present invention provides technical solution be such：A kind of ASE-HPLC methods measure
The method of spinosin content, includes the following steps successively in spina date seed：
Step 1：Spine Date Seed is extracted using ASE methods, wherein the ASE methods include first using n-hexane, then
It is extracted with ethyl alcohol, and collects alcohol extraction liquid；
Step 2：Using HPLC methods to the spinosin content in alcohol extraction liquid.
Preferably, the step 1 includes following sub-steps：
Step S1：By spina date seed sample comminution, sieving takes 1g to be uniformly mixed with 1g diatomite；
Step S2：By the mixture obtained by step 1 loaded on being placed in the 10ml ASE abstraction pools of filter membrane, add diatomite to
Pond mouthful is parallel；
Step S3：It is cleaned with 25ml n-hexane extractions, then is extracted with 25ml ethyl alcohol, 50ml is settled to ethyl alcohol.
Preferably, the parameter of the removal step described in step S3 is：Temperature is 80 DEG C, time 8min, and number is 1 time,
Flush volume is 60%, purge time 60s.
Preferably, the parameter of the extraction step described in step S3 is：Temperature is 100 DEG C, time 8min, and number is 1 time,
Flush volume is 60%, purge time 60s.
Preferably, the detection parameters of the HPLC methods described in step 1 are：Chromatographic column is Thermo AQ；Column temperature is 40 DEG C；Stream
Speed is 0.3mL/min；Mobile phase is acetonitrile-water；Detection wavelength is 335nm.
Preferably, the specification of the chromatographic column is 1.7 μm, 50 × 2.1mm.
Preferably, the mobile phase is added in using the method for gradient elution, wherein specific parameter is as follows：
Elution total time is 30min；
0~10min, the volume ratio of acetonitrile-water is 12~19 in mobile phase：88~81；
10~16min, the volume ratio of acetonitrile-water is 19~20 in mobile phase：81~80；
16~22min, the volume ratio of acetonitrile-water is 20~100 in mobile phase：80~0；
22~30min, the volume ratio of acetonitrile-water is 100 in mobile phase：0.
The present invention has the following advantages compared with conventional method：
1st, the present invention, which improves temperature, reduces the viscosity of solvent, reduces the prevention that solvent enters sample matrices, increases molten
Agent enters the diffusion of sample matrices, reduces the surface tension between solvent and sample matrices, makes the capacity of solvent dissolving determinand
Increase；
2nd, since liquid is much larger than solvability of the gas to solute to the solvability of solute, so the boiling of extraction liquids
Point is improved with pressure rise, so as to which solvent be made to remain at liquid at high temperature under high pressure；
3rd, the present invention once investigated the solvent that Accelerate solvent extraction uses, and has paper to mention and is extracted using 70% ethyl alcohol
Spinosin relative amount is higher, and impurity is less, therefore to ethyl alcohol and 70% ethyl alcohol is used to carry out solvent investigation, as a result show second
Alcohol is consistent with 70% ethyl alcohol extraction spinosin content, and impurity peaks are essentially identical, therefore uses ethyl alcohol Extraction solvent；
4th, the main influence for investigating extraction times to result of the present invention, with the method that ASE singles extract to sample in experiment
It repeats extraction three times, receives each sample liquid respectively, analysis is extracted as a result, the result shows that extraction is primary almost
Number uses 1 time to save the time；
5th, the repeatability of ASE extracting methods of the present invention is good.
Specific embodiment
With reference to embodiment, claims of the present invention is described in further detail, but do not formed
Any limitation of the invention, any limited number of time modification made in the claims in the present invention protection domain, people is in the present invention
Claims within.
Embodiment 1
1st, instrument and equipment and reagent
1.1 instrument：Electronic analytical balance (XA205DU), ASE350 Accelerate solvent extractions instrument (DIONEX companies of the U.S.),
Thermo U3000UHPLC liquid chromatographs.
1.2 reagent：
Water：Meet level-one water as defined in GB/T 6682；
Acetonitrile：Chromatographically pure；
N-hexane, ethyl alcohol：It analyzes pure.
2nd, method
The preparation of 2.1 reference substance solutions：
Take spinosin reference substance appropriate, it is accurately weighed, add ethyl alcohol be made solution of every 1ml containing 0.2mg to get.
The preparation of 2.2 test solutions
2.2.1《Chinese Pharmacopoeia》Version one prepares test sample method within 2015：
This product powder (crossing No. four sieves) about 1g is taken, it is accurately weighed, it puts in Soxhlet extractor, adds (60~90 DEG C) of petroleum ether suitable
Amount, is heated to reflux 4 hours, discards petroleum ether liquid, and the dregs of a decoction fling to solvent, are transferred in conical flask, adds in 70% ethyl alcohol 20ml, adds
Heat reflux 2 hours, filtration, filter residue is washed with 70% ethyl alcohol 5ml, merges washing lotion and filtrate, for recycling design to doing, residue adds ethyl alcohol
Dissolving, is transferred in 5ml measuring bottles, and ethyl alcohol is added to shake up to scale, filters, take subsequent filtrate to get.
2.2.2 Accelerate solvent extraction method (ASE) prepares test sample method：
Step S1：By spina date seed sample comminution, sieving takes 1g to be uniformly mixed with 1g diatomite；
Step S2：By the mixture obtained by step 1 loaded on being placed in the 10ml ASE abstraction pools of filter membrane, add diatomite to
Pond mouthful is parallel；
Step S3：It is cleaned with 25ml n-hexane extractions, then is extracted with 25ml ethyl alcohol, 50ml is settled to ethyl alcohol.
2.3 ASE extraction conditions
The parameter of the removal step is：Temperature is 80 DEG C, time 8min, and number is 1 time, and flush volume is
60%, purge time 60s；
The parameter of the extraction step is：Temperature is 100 DEG C, time 8min, and number is 1 time, and flush volume is
60%, purge time 60s.
2.4 chromatographic conditions and system suitability
Using octadecylsilane chemically bonded silica as filler；Using octadecylsilane chemically bonded silica as filler.
The detection parameters of HPLC methods are：Chromatographic column is Thermo AQ, 1.7 μm, 50 × 2.1mm；Column temperature is 40 DEG C；Flow velocity
For 0.3mL/min；Mobile phase is acetonitrile-water；Detection wavelength is 335nm.
Preferably, the mobile phase is added in using the method for gradient elution, wherein specific parameter is as follows：
Elution total time is 30min；
0~10min, the volume ratio of acetonitrile-water is 12~19 in mobile phase：88~81；
10~16min, the volume ratio of acetonitrile-water is 19~20 in mobile phase：81~80；
16~22min, the volume ratio of acetonitrile-water is 20~100 in mobile phase：80~0；
22~30min, the volume ratio of acetonitrile-water is 100 in mobile phase：0.
2.5 measuring method
It is measured according to high performance liquid chromatography (general rule 0512)；
It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, inject liquid chromatograph, measure to get.
The requirement of 2.6 standard limited values
This product is calculated by dry product, containing spinosin (C28H32O15) 0.080% must not be less than.
2.7 calculate (external standard method)
In formula：CR-reference substance solution concentration, unit are micro- gram per liter (mg/L)；The peak area of AX-test sample；AR—
Reference substance peak area.
Pay attention to：
It should be dismantled and cleaned out using preceding extraction bottom of pond portion, otherwise easily cause pressure instability；
Otherwise the filter paper of abstraction pool bottom should cause leakage in sealing ring；
Abstraction pool fills elastic moderate during sample, and too loose to be easy to cause extracting solution excessive；
Check whether gas cylinder air pressure reaches 1Mpa before booting；
It is cleaned out after use, abstraction pool will be dried and (be got rusty easily) in time.
3rd, result
3.1 linear relationship
Step SS1：Spinosin reference substance is taken, it is accurately weighed, add ethyl alcohol that solution of every 1ml containing about 0.2mg is made, i.e.,
；
Step SS2：Then accurate 0.1 μ l of the solution, 0.5 μ l, 1 μ l, 1.5 μ l, the 2 μ l of drawing enter HPLC measure respectively,
And measured according to the above method, the results detailed in Table 1；
Step SS3：Linear regression is carried out with concentration (mg)-peak area；
Step SS4：Acquire regression equation：Y=6685.2191x-7.7407, R2=0.9999, i.e. spinosin exists
It is in good linear relationship in the range of 0.01439~0.2878mg.
The linear test result of 1 spinosin of table
3.2 repetitive test
Take the sample (lot number of identical lot number：1508015) 1g, it is totally 4 parts, accurately weighed, it is carried by above-mentioned ASE extracting methods
Test solution is taken, sample size is 1 μ L, and with above-mentioned chromatographic condition parallel laboratory test, the content for measuring spinosin in sample is shown in
Table 2, RSD 0.8%, the results showed that ASE extracting methods repeatability is good.
Table 2 measures the content of spinosin in sample
3.3 precision and stability test
Spinosin reference substance solution (0.1439mg/ml) is taken, continuous sample introduction 6 times records peak area, and peak area RSD is
0.7%, show that instrument precision is good.
Same test solution is taken, is placed at room temperature, is measured in accordance with the law respectively at 0,2,4,8,12h.
As a result the RSD for showing spinosin peak area is 0.8%, shows that test solution is stablized in 12h.
3.4 sample difference instruments extract result and with official method results contrast (3 batches)
3 sample of table uses the extraction Comparative result of different ASE instrument respectively
4 sample of table is respectively using ASE methods and the extraction Comparative result of official method
4 discuss：
The selection of 4.1 ASE Extraction solvents：
The present invention once investigated the solvent that Accelerate solvent extraction uses, and has paper to mention and extracts this using 70% ethyl alcohol
Skin promise element relative amount is higher, and impurity is less, therefore to ethyl alcohol and 70% ethyl alcohol is used to carry out solvent investigation, as a result show ethyl alcohol
It is consistent with 70% ethyl alcohol extraction spinosin content, and impurity peaks are essentially identical, therefore use ethyl alcohol Extraction solvent.
The optimization of 4.2 ASE extraction conditions
The main influence for investigating extraction times to result of the invention, with the method that ASE singles extract to sample weight in experiment
Multiple extraction three times, receives each sample liquid respectively, the results are shown in Table 5, and analysis is as a result, the result shows that extraction is primary almost complete
Entirely, therefore extraction times use 1 time to save the time.
Table 5
| Ethyl alcohol extracts | As a result % |
| Extraction 1 time | 0.1311 |
| Extraction 2 times | 0.0002 |
| Extraction 3 times | 0.0008 |
Above-described is only presently preferred embodiments of the present invention, all timess made in the range of the spirit and principles in the present invention
What modifications, equivalent substitutions and improvements etc., should be included within the scope of the present invention.
Claims (7)
1. a kind of method that ASE-HPLC methods measure spinosin content in spina date seed, it is characterised in that, successively including following steps
Suddenly:
Step 1:Spine Date Seed is extracted using ASE methods, wherein the ASE methods include first using n-hexane, then use second
Alcohol extracts, and collects alcohol extraction liquid；
Step 2：Using HPLC methods to the spinosin content in alcohol extraction liquid.
2. the method that a kind of ASE-HPLC methods according to claim 1 measure spinosin content in spina date seed, feature
It is, the step 1 includes following sub-steps：
Step S1：By spina date seed sample comminution, sieving takes 1g to be uniformly mixed with 1g diatomite；
Step S2：By the mixture obtained by step 1 loaded on being placed in the 10ml ASE abstraction pools of filter membrane, add diatomite to and Chi Kou
It is parallel；
Step S3：It is cleaned with 25ml n-hexane extractions, then is extracted with 25ml ethyl alcohol, 50ml is settled to ethyl alcohol.
3. the method that a kind of ASE-HPLC methods according to claim 2 measure spinosin content in spina date seed, feature
It is, the parameter of the removal step described in step S3 is：Temperature is 80 DEG C, time 8min, and number is 1 time, and flush volume is
60%, purge time 60s.
4. the method that a kind of ASE-HPLC methods according to claim 2 measure spinosin content in spina date seed, feature
It is, the parameter of the extraction step described in step S3 is：Temperature is 100 DEG C, time 8min, and number is 1 time, and flush volume is
60%, purge time 60s.
5. the method that a kind of ASE-HPLC methods according to claim 1 measure spinosin content in spina date seed, feature
It is, the detection parameters of the HPLC methods described in step 1 are：Chromatographic column is Thermo AQ；Column temperature is 40 DEG C；Flow velocity is 0.3mL/
min；Mobile phase is acetonitrile-water；Detection wavelength is 335nm.
6. the method that a kind of ASE-HPLC methods according to claim 5 measure spinosin content in spina date seed, feature
It is, the specification of the chromatographic column is 1.7 μm, 50 × 2.1mm.
7. the method that a kind of ASE-HPLC methods according to claim 5 measure spinosin content in spina date seed, feature
It is, the mobile phase is added in using the method for gradient elution, wherein specific parameter is as follows：
Elution total time is 30min；
0~10min, the volume ratio of acetonitrile-water is 12~19 in mobile phase：88~81；
10~16min, the volume ratio of acetonitrile-water is 19~20 in mobile phase：81~80；
16~22min, the volume ratio of acetonitrile-water is 20~100 in mobile phase：80~0；
22~30min, the volume ratio of acetonitrile-water is 100 in mobile phase：0.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109239224A (en) * | 2018-10-17 | 2019-01-18 | 山西大学 | 9 kinds of method for quantitatively determining while enter blood component in semen ziziphi spinosae water extract |
| CN109324126A (en) * | 2018-09-21 | 2019-02-12 | 山西中医药大学 | A method of 9 kinds of chemical components in semen ziziphi spinosae are measured simultaneously using UPLC-MS/MS |
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- 2017-12-18 CN CN201711365339.1A patent/CN108169356A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109324126A (en) * | 2018-09-21 | 2019-02-12 | 山西中医药大学 | A method of 9 kinds of chemical components in semen ziziphi spinosae are measured simultaneously using UPLC-MS/MS |
| CN109239224A (en) * | 2018-10-17 | 2019-01-18 | 山西大学 | 9 kinds of method for quantitatively determining while enter blood component in semen ziziphi spinosae water extract |
| CN109239224B (en) * | 2018-10-17 | 2021-07-02 | 山西大学 | Method for simultaneously and quantitatively measuring 9 blood-entering components in spina date seed water extract |
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Application publication date: 20180615 |