CN109541104B - Method for identifying pinellia ternate in pinellia ternate syrup - Google Patents
Method for identifying pinellia ternate in pinellia ternate syrup Download PDFInfo
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- CN109541104B CN109541104B CN201811423876.1A CN201811423876A CN109541104B CN 109541104 B CN109541104 B CN 109541104B CN 201811423876 A CN201811423876 A CN 201811423876A CN 109541104 B CN109541104 B CN 109541104B
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention relates to the technical field of drug detection in chemical analysis, in particular to a method for identifying pinellia ternate in pinellia ternate syrup, which comprises the following steps: extracting protein from a sample to be detected, performing enzymolysis treatment by trypsin, and centrifuging to obtain a supernatant; and injecting the supernatant into a liquid chromatogram-mass spectrometer for multi-reaction monitoring. The method has strong specificity, simple operation and easy popularization and use. The method can greatly improve quality control level of rhizoma Pinelliae and other Chinese patent medicines containing rhizoma Pinelliae, and ensure safety and effectiveness of clinical application of Chinese medicine.
Description
Technical Field
The invention relates to the technical field of drug detection in chemical analysis, in particular to a method for identifying pinellia ternate in pinellia ternate syrup.
Background
At present, the authenticity qualitative research on the pinellia ternata in the Chinese patent medicine is very few, and the specificity of the method has serious problems. The traditional Chinese medicine containing pinellia tuber has more than four hundred, but only a few of the traditional Chinese medicines such as baohe tablets, pinellia tuber injection and the like adopt the thin-layer chromatography to qualitatively identify the pinellia tuber, and mainly aim at performing the thin-layer identification on amino acid or sitosterol chemical components contained in the pinellia tuber, the components generally exist in animal and plant medicines, have no specificity, and can not accurately and qualitatively identify the pinellia tuber in the traditional Chinese medicine at all. The prior art also describes the qualitative and quantitative identification and quality control of pinellia ternata by using a high performance liquid chromatography method, and the specificity is not strict enough for the reasons.
The production enterprises of the pinellia ternate syrup have about 80 families in China, have huge annual production capacity, are clinically common Chinese patent medicines for relieving cough and reducing sputum, consist of eight medicines such as raw pinellia ternate, and have only two physicochemical identification items of volatile oil and amino acid and a conventional inspection item of syrup in the standard, so the standard is very laggard. Especially, pinellia tuber is a monarch drug in the prescription, is the main efficacy drug of the product for relieving cough and reducing sputum, but has no quality control item, and cannot effectively control the quality of the drug. Pinellia ternata contains starch, protein, amino acid, alkaloid, organic acid, sitosterol, adenosine and other components, contains very complex chemical components, has relatively low content of other components except starch, cannot completely determine the active ingredients of the pinellia ternata so far, belongs to a precious and toxic traditional Chinese medicinal material, so that the quality control of the pinellia ternata is both important and difficult in the pinellia ternata traditional Chinese medicinal material and the Chinese patent medicine containing the pinellia ternata, and the research on the quality control of the pinellia ternata is very necessary. Under the condition that effective ingredients of pinellia ternate are not completely clear, the qualitative identification of the pinellia ternate in the quality control of the pinellia ternate syrup is far superior to the effect of content measurement by taking one or two chemical ingredients as indexes, the components such as succinic acid, citric acid, guanosine, ephedrine, sitosterol and the like in the content measurement of the pinellia ternate at present do not have specificity, and particularly, the components in compound preparations such as the pinellia ternate syrup and the like do not have specificity at all, so that the qualitative identification quality control method of the special pinellia ternate in the pinellia ternate syrup is urgently needed to carry out effective quality control on the feeding and extraction use conditions of the monarch drug pinellia ternate in the other party.
Disclosure of Invention
In order to solve the problem of poor specificity in pinellia ternate identification in the prior art, the application discloses a method for identifying pinellia ternate in pinellia ternate syrup with high specificity.
Considering the difference between the protein contained in the pinellia ternata and the protein contained in other traditional Chinese medicines, the protein can be subjected to enzymolysis by using a proper enzyme, the difference peptide segment of the pinellia ternata relative to other traditional Chinese medicines can be found out by a method of sequence comparison and the like with the known protein in a database, the ionization mode of the mass spectrum of the characteristic peptide segment is deduced by analyzing the structure of the difference peptide segment, the parent ion and the daughter ion of the characteristic peptide segment are determined and determined, and the detection method aiming at the difference peptide segment is finally established by carrying out experimental method verification on a plurality of batches of pinellia ternata samples and pinellia ternata syrup samples.
The invention is obtained by the following steps:
a method for identifying pinellia ternate in pinellia ternate syrup comprises the following steps:
(1) extracting protein from a sample to be detected, performing enzymolysis treatment by trypsin, and centrifuging to obtain a supernatant;
(2) injecting the supernatant into a liquid chromatography-mass spectrometer, carrying out multi-reaction monitoring in an electrospray ionization and positive ion mode, selecting m/z double charges 642.32 → 708.38 and 642.32 → 878.48 as a detection ion pair of pinellia ternate, indicating that a sample to be detected contains the pinellia ternate at a retention time of 6.24 +/-0.2 min, and indicating that no spectral peak is present at the retention time of 6.24 +/-0.2 min, and indicating that the sample to be detected does not contain the pinellia ternate.
The identification method preferably collects mass spectrum signals for 3-10 min.
According to the identification method, preferably, a liquid sample to be detected is subjected to protein enrichment by alcohol precipitation, absolute ethyl alcohol is used for alcohol precipitation, and the volume ratio of the absolute ethyl alcohol to the liquid sample to be detected is 5: 1.
the identification method preferably adopts the following detection conditions of liquid phase and mass spectrum in the liquid chromatogram-mass spectrometer:
liquid phase conditions: the chromatographic column is ACQUITY UPLC® BEH C18Gradient elution is carried out at the column temperature of 35 ℃ and the flow rate of 0.3mL/min, wherein the column temperature is 2.1 multiplied by 100mm and the column diameter is 1.7 mu m, the mobile phase A is 0.1 percent formic acid solution, and the mobile phase B is 0.1 percent formic acid acetonitrile solution;
mass spectrum conditions: performing multi-reaction monitoring by adopting a mass spectrum detector in an electrospray ionization and positive ion mode, wherein the flow rate of sheath gas is 46L/hr; auxiliary gas flow rate, 542L/hr; spray voltage, 3.6 KV; the ion source temperature is 150 ℃; auxiliary gas temperature, 400 ℃.
The identification method preferably comprises the following operation of protein extraction and trypsin enzymolysis treatment:
taking 2ml of liquid sample to be detected, adding 10ml of absolute ethyl alcohol, shaking uniformly, standing for 1h, centrifuging, drying precipitate at 60 ℃, adding 1ml of 1% NH4HCO3Dissolving the solution and 20 μ L of 0.50M dithiothreitol solution, treating at 100 deg.C for 10min, cooling to room temperature, adding 50 μ L of 0.55M iodoacetamide solution, reacting in dark for 30min, mixing, and centrifuging; adding 10 μ L10 mg/ml bovine trypsin solution, and performing enzymolysis at 37 deg.C for 15 min; then the mixture is treated at 100 ℃ for 5min, taken out, cooled to room temperature and centrifuged to obtain supernatant.
The identification method preferably comprises the following operation of protein extraction and trypsin enzymolysis treatment:
taking 20mg of solid sample to be detected, adding 1ml of 1% NH4HCO3Shaking the solution and 20 μ L of 0.50M dithiothreitol solution, treating at 100 deg.C for 10min, cooling to room temperature, adding 50 μ L of 0.55M iodoacetamide solution, reacting in dark for 30min, mixing, and centrifuging; adding 10 μ L10 mg/ml bovine trypsin solution, and performing enzymolysis at 37 deg.C for 15 min; then the mixture is treated at 100 ℃ for 5min, taken out, cooled to room temperature and centrifuged to obtain supernatant.
The identification method preferably comprises a gradient elution procedure: 0 → 3min, mobile phase A95%, mobile phase B5%; 3 → 8min, mobile phase A95% → 70%, mobile phase B5% → 30%; 8 → 8.1min, mobile phase A70% → 0%, mobile phase B30% → 100%; 8.1 → 10min, mobile phase A0%, mobile phase B100%.
The invention has the beneficial effects that:
the method carries out qualitative identification on the semi-summer by carrying out mass spectrum detection on the peptide segments with the difference characteristics contained in the species, has very strong specificity, is simple to operate and is easy to popularize and use. The method can greatly improve quality control level of rhizoma Pinelliae and Chinese patent medicine containing rhizoma Pinelliae, and ensure safety and effectiveness of clinical application of Chinese medicine.
Drawings
FIG. 1 shows the chromatogram of pinellia ternata (No. 1) (extracted 642.32 → 708.38 and 642.32 → 878.48 ions),
FIG. 2 is a chromatogram of the control drug of pinellia ternata (No. 6) (extracted 642.32 → 708.38 and 642.32 → 878.48 ions),
FIG. 3 is a chromatogram of pinellia ternate syrup (No. 7) (extraction 642.32 → 708.38 and 642.32 → 878.48 ions),
FIG. 4 is a chromatogram of pinellia ternate syrup (No. 10) (extraction 642.32 → 708.38 and 642.32 → 878.48 ions),
FIG. 5 is a chromatogram of the negative control (No. 15) (extraction 642.32 → 708.38 and 642.32 → 878.48 ions).
Detailed Description
The invention is further illustrated by the following specific examples:
preparation of relevant solutions in the experiment:
DTT solution: weighing appropriate amount of DTT (dithiothreitol), and dissolving with water to obtain (concentration of 0.50M);
IAA solution: weighing an appropriate amount of IAA (iodoacetamide), and dissolving with water to obtain (the concentration is 0.55M, and the preparation is carried out on site);
ammonium bicarbonate solution: weighing a proper amount of ammonium bicarbonate, and dissolving with water to obtain the ammonium bicarbonate (the concentration is 1.0%);
bovine trypsin solution: weighing appropriate amount of bovine trypsin, dissolving with acetic acid solution to obtain (concentration of 10 mg/ml), subpackaging into small parts, and storing at-20 deg.C for use.
Example 1
1 Instrument, reagent and sample
The instrument comprises the following steps: waters Quattro Premier XE high performance liquid chromatography-mass spectrometer; sartorius CP225D electronic balance.
Reagent: trypsin (manufactured by Sigma, lot number: SLBS 8956), Dithiothreitol (DTT), Iodoacetamide (IAA), ammonium bicarbonate, and acetic acid were all analytical grade.
Sample preparation: pinellia ternata syrup (from market samples); pinellia ternata (collected on the market); pinellia ternata control drug (provided by the Central Hospital, batch No.: 121272-201605).
2 liquid phase and Mass Spectrometry conditions
Liquid phase conditions: the chromatographic column is ACQUITY UPLC® BEH C18(2.1X 100mm, 1.7 μm), column temperature 35 deg.C, flow rate 0.3mL/min, mobile phase A0.1% formic acid solution, B0.1% formic acid acetonitrile solution, gradient elution, see Table below.
Epigradient elution procedure
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 95 | 5 |
| 3 | 95 | 5 |
| 8 | 70 | 30 |
| 8.1 | 0 | 100 |
| 10 | 0 | 100 |
Mass spectrum conditions: performing multi-reaction monitoring by adopting a mass spectrum detector, electrospray ionization (ESI) and a positive ion mode, wherein the flow rate of sheath gas is 46L/hr; auxiliary gas flow rate, 542L/hr; spray voltage, 3.6 KV; the ion source temperature is 150 ℃; auxiliary gas temperature, 400 ℃. M/z (double charge) 642.32 → 708.38 and 642.32 → 878.48 are selected as detection ion pairs of pinellia ternata, and the injection volume is 5 μ l.
3 preparation of the solution
3.1 preparation of test solutions
Precisely measuring 2ml of rhizoma Pinelliae syrup, adding 10ml of anhydrous ethanol, shaking, standing for 1h, centrifuging (12000 rpm, 10 min), oven drying at 60 deg.C, precisely adding 1ml of 1% NH4HCO3Fully dissolving the solution and 20 mu L of DTT solution, treating at 100 ℃ for 10min, taking out, cooling to room temperature, precisely adding 50 mu L of IAA solution, reacting for 30min in a dark place, uniformly mixing, and centrifuging (12000 rpm, 10 min); adding 10 μ L of bovine trypsin solution precisely, and performing enzymolysis at 37 deg.C for 15 min; then treating at 100 deg.C for 5min, taking out, cooling to room temperature, centrifuging (14000 rpm, 15 min), and collecting supernatant.
3.2 preparation of medicinal solution
Weighing rhizoma Pinelliae and control materials 20mg respectively, precisely weighing, and precisely adding 1ml1% NH4HCO3Shaking the solution and 20 μ L DTT solution, treating at 100 deg.C for 10min, taking out, cooling to room temperature, adding 50 μ L IAA solution precisely, reacting in dark for 30min, mixing, and centrifuging (12000 rpm, 10 min); adding 10 μ L of bovine trypsin solution precisely, and performing enzymolysis at 37 deg.C for 15 min; treating at 100 deg.C for 5min, cooling to room temperature, centrifuging (14000 rpm for 15 min), and collecting supernatant to obtain control solution.
3.3 preparation of negative control solution
Weighing corresponding medicinal materials according to the prescription proportion of the pinellia ternata syrup, and preparing the negative control solution without pinellia ternata according to the preparation process of the pinellia ternata syrup. Precisely measuring negative control solution 2ml, adding anhydrous ethanol 10ml, shaking, standing for 1 hr, centrifuging (12000 rpm, 10 min), and precipitatingDrying at 60 deg.C, and precisely adding 1ml of 1% NH4HCO3Shaking the solution and 20 μ L DTT solution, treating at 100 deg.C for 10min, taking out, cooling to room temperature, adding 50 μ L IAA solution precisely, reacting in dark for 30min, mixing, and centrifuging (12000 rpm, 10 min); adding 10 μ L of bovine trypsin solution precisely, and performing enzymolysis at 37 deg.C for 15 min; then treating at 100 deg.C for 5min, taking out, cooling to room temperature, centrifuging (14000 rpm, 15 min), and collecting supernatant.
4 measurement of the sample
And (3) respectively injecting 5 mul of the test solution, the pinellia ternate medicinal material, the control medicinal material solution and the negative control solution into a liquid chromatography-mass spectrometer, carrying out multi-reaction monitoring in an electrospray ionization (ESI) and positive ion mode, only collecting mass spectrum signals for 3-10 min in order to reduce the pollution of the sample to the mass spectrometer, and directly introducing the mobile phase carrying the sample flowing out of the chromatographic column into a waste liquid pipe in other time periods. M/z (double charge) 642.32 → 708.38 and 642.32 → 878.48 were selected as the detector ion pair for analysis.
5 results and analysis
5.1 results
The different samples collected were tested according to the method described above. Wherein 8 batches of the pinellia ternata syrup are inspected according to the existing quality standard and are all qualified. The results are as follows.
Results of the examination of the Table samples
| Numbering | Name (R) | Producing area or unit | Whether the ion pair/retention time (min) of pinellia ternata is detected | |
| 1 | Pinellia ternata (Thunb.) Breit | Shandong (mountain east) | Is/6.24 | Detecting pinellia ternata |
| 2 | Pinellia ternata (Thunb.) Breit | Sichuan | Is/6.24 | Detecting pinellia ternata |
| 3 | Pinellia ternata (Thunb.) Breit | (Anhui) | Is/6.23 | Detecting pinellia ternata |
| 4 | Pinellia ternata (Thunb.) Breit | North of a lake | Is/6.24 | Detecting pinellia ternata |
| 5 | Pinellia ternata (Thunb.) Breit | Gansu | Is/6.23 | Detecting pinellia ternata |
| 6 | Rhizoma Pinelliae reference drug | Inspection yard | Is/6.23 | Detecting pinellia ternata |
| 7 | Pinellia ternata syrup | Shandong pharmaceutical Co Ltd | Is/6.23 | Detecting pinellia ternata |
| 8 | Pinellia ternata syrup | Guangdong pharmaceutical Co Ltd | Is/6.24 | Detecting pinellia ternata |
| 9 | Pinellia ternata syrup | Guangxi pharmaceutical Co Ltd | Is/6.23 | Detecting pinellia ternata |
| 10 | Pinellia ternata syrup | Gansu pharmaceutical Co Ltd | Whether or not | Not detected pinellia ternata |
| 11 | Pinellia ternata syrup | Guangzhou pharmaceutical Co Ltd | Is/6.23 | Detecting pinellia ternata |
| 12 | Pinellia ternata syrup | Henan some pharmaceutical Co Ltd | Is/6.24 | Detecting pinellia ternata |
| 13 | Pinellia ternata syrup | Jiangsu pharmaceutical company | Is/6.23 | Detecting pinellia ternata |
| 14 | Pinellia ternata syrup | Jiangxi pharmaceutical Co Ltd | Is/6.23 | Detecting pinellia ternata |
| 15 | Negative control | Laboratory self-control | Whether or not | Not detected pinellia ternata |
5.2 analysis
From the above results, it can be seen that the chromatogram peaks of the pinellia ternate drug, the pinellia ternate control drug and the 7 batches of the pinellia ternate syrup occurred around the retention time of 6.24min (the chromatogram of the extracted ion 642.32 → 708.38 and the chromatogram of the extracted ion 642.32 → 878.48), and the chromatogram peaks of the negative control containing no pinellia ternate did not occur around the retention time of 6.24min (the chromatogram of the extracted ion 642.32 → 708.38 and the chromatogram of the extracted ion 642.32 → 878.48). Only 1 batch of pinellia ternate syrup (number 10) has no detected pinellia ternate, the sampling amount of the batch of pinellia ternate syrup is adjusted to 10ml, 50ml of ethanol is added for treatment, other preparation methods are not changed, the result can detect the pinellia ternate, the pinellia ternate protein content in the pinellia ternate syrup of the enterprise is very low compared with other enterprise products, the result is negative under the test condition, and the fact that the pinellia ternate is not detected can be judged and can be related to the quality or the feeding amount of the pinellia ternate of the enterprise. The 8 batches of pinellia ternata syrup samples are qualified through the current standard test, and 1 batch of the samples which are not qualified is detected by adopting the method, so that the quality of the pinellia ternata syrup can be distinguished, and the method can be used for quality control of pinellia ternata in the pinellia ternata syrup. In order to ensure the accuracy of the test result, the chromatogram extracted ions 642.32 → 708.38 and 642.32 → 878.48 of the sample to be tested must have obvious chromatographic peak simultaneously, and the pinellia ternata can be judged to be detected.
In order to ensure the accuracy of the test result, the chromatogram of the extracted ions 642.32 → 708.38 and 642.32 → 878.48 of the pinellia ternata control medicinal material under the test condition must have obvious chromatographic peak simultaneously for testing, otherwise, the state of the instrument or the preparation method of the sample should be checked until the test requirement is met.
In the experiment, the preparation method of the test solution is considered, and the pinellia ternate syrup sample is directly subjected to enzymolysis without alcohol precipitation, so that the characteristic ion pair cannot be detected due to serious interference of other components in the sample. Because the pinellia ternate syrup granules contain a large amount of micromolecular interference substances such as sucrose and the like, and the pinellia ternate protein is a macromolecular substance, the pinellia ternate protein can be enriched and purified by using an alcohol precipitation mode, the use amount of ethanol is considered during alcohol precipitation, 1, 3, 5 and 7 times of the sampling volume of the pinellia ternate syrup are compared respectively, 5 times of ethanol and 7 times of ethanol are added to obtain a result, the signal response strength is similar, 1 time of the signal response strength is weaker, and 5 times of ethanol is added selectively. The sampling amount of the pinellia ternate syrup is inspected, 1ml, 2ml and 3ml of the samples are respectively compared, and as a result, the signal response intensity of 1ml of the sampling amount is weaker, the signal response intensity of 2ml of the samples is moderate, and the sampling amount is 2ml in order to reduce the pollution to a mass spectrometer.
The technology can be used for the specificity identification of pinellia ternate medicinal materials, pinellia ternate syrup and other Chinese patent medicines containing pinellia ternate partially, the quality control level of the pinellia ternate medicinal materials, decoction pieces and the Chinese patent medicines containing pinellia ternate partially can be improved by the method, the problem of the specificity identification of the pinellia ternate is well solved, and the method has important significance for ensuring the medication safety and the clinical curative effect of the Chinese medicines.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.
Claims (4)
1. The method for identifying pinellia ternate in pinellia ternate syrup is characterized by comprising the following steps of:
(1) extracting protein from a sample to be detected, performing enzymolysis treatment by trypsin, and centrifuging to obtain a supernatant;
(2) injecting the supernatant into a liquid chromatogram-mass spectrometer, carrying out multi-reaction monitoring in an electrospray ionization and positive ion mode, selecting m/z double charges 642.32 → 708.38 and 642.32 → 878.48 as a detection ion pair of pinellia ternate, and indicating that a sample to be detected contains the pinellia ternate and no chromatographic peak exists in the retention time of 6.24 +/-0.2 min, and indicating that the sample to be detected does not contain the pinellia ternate, wherein the chromatographic peak is present in the retention time of 6.24 +/-0.2 min;
the detection conditions of liquid phase and mass spectrum in the liquid chromatogram-mass spectrometer are as follows:
liquid phase conditions: the chromatographic column is ACQUITY UPLC® BEH C18Gradient elution is carried out at the column temperature of 35 ℃ and the flow rate of 0.3mL/min, wherein the column temperature is 2.1 multiplied by 100mm and the column diameter is 1.7 mu m, the mobile phase A is 0.1 percent formic acid solution, and the mobile phase B is 0.1 percent formic acid acetonitrile solution; gradient elution procedure: 0 → 3min, mobile phase A95%, mobile phase B5%; 3 → 8min, mobile phase A95% → 70%, mobile phase B5% → 30%; 8 → 8.1min, mobile phase A70% → 0%, mobile phase B30% → 100%; 8.1 → 10min, mobile phase A0%, mobile phase B100%;
mass spectrum conditions: performing multi-reaction monitoring by adopting a mass spectrum detector in an electrospray ionization and positive ion mode, wherein the flow rate of sheath gas is 46L/hr; auxiliary gas flow rate, 542L/hr; spray voltage, 3.6 KV; the ion source temperature is 150 ℃; auxiliary gas temperature, 400 ℃.
2. The identification method according to claim 1, wherein the mass spectrum signal is collected for 3 to 10 min.
3. The identification method according to claim 1 or 2, wherein the protein is enriched by subjecting the liquid sample to be tested to alcohol precipitation, and the alcohol precipitation is performed using absolute ethanol, wherein the volume ratio of absolute ethanol to the liquid sample to be tested is 5: 1.
4. the identification method according to claim 1 or 2, characterized in that the protein extraction and trypsin enzymatic treatment are carried out as follows:
taking 2ml of liquid sample to be detected, adding 10ml of absolute ethyl alcohol, shaking uniformly, standing for 1h, centrifuging, drying precipitate at 60 ℃, adding 1ml of 1% NH4HCO3Dissolving the solution and 20 μ L of 0.50M dithiothreitol solution, treating at 100 deg.C for 10min, cooling to room temperature, adding 50 μ L of 0.55M iodoacetamide solution, reacting in dark for 30min, mixing, and centrifuging; adding 10 μ L10 mg/ml bovine trypsin solution, and performing enzymolysis at 37 deg.C for 15 min; then the mixture is treated at 100 ℃ for 5min, taken out, cooled to room temperature and centrifuged to obtain supernatant.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388446A (en) * | 2014-10-23 | 2015-03-04 | 江苏师范大学 | Pinellia ternate squalene synthase gene as well as coded protein and application thereof |
| CN106749599A (en) * | 2016-12-13 | 2017-05-31 | 山东省食品药品检验研究院 | A kind of application flow of keratin feature peptide in antelope's horn detection of adulterations |
| CN108318597A (en) * | 2018-01-29 | 2018-07-24 | 山东省食品药品检验研究院 | It is a kind of at the same differentiate in sample whether the method containing antelope's horn and cornu bubali |
| CN108333287A (en) * | 2018-01-29 | 2018-07-27 | 山东省食品药品检验研究院 | A kind of quantitative detecting method of Cornu Saigae Tataricae powder |
| CN108517322A (en) * | 2018-04-24 | 2018-09-11 | 河北省农林科学院 | A kind of pinellia pedatisecta Schott trypsin inhibitor gene, the albumen of its coding and pest-resistant application |
-
2018
- 2018-11-27 CN CN201811423876.1A patent/CN109541104B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388446A (en) * | 2014-10-23 | 2015-03-04 | 江苏师范大学 | Pinellia ternate squalene synthase gene as well as coded protein and application thereof |
| CN106749599A (en) * | 2016-12-13 | 2017-05-31 | 山东省食品药品检验研究院 | A kind of application flow of keratin feature peptide in antelope's horn detection of adulterations |
| CN108318597A (en) * | 2018-01-29 | 2018-07-24 | 山东省食品药品检验研究院 | It is a kind of at the same differentiate in sample whether the method containing antelope's horn and cornu bubali |
| CN108333287A (en) * | 2018-01-29 | 2018-07-27 | 山东省食品药品检验研究院 | A kind of quantitative detecting method of Cornu Saigae Tataricae powder |
| CN108517322A (en) * | 2018-04-24 | 2018-09-11 | 河北省农林科学院 | A kind of pinellia pedatisecta Schott trypsin inhibitor gene, the albumen of its coding and pest-resistant application |
Non-Patent Citations (6)
| Title |
|---|
| UPLC－QTOF－MS结合主成分分析法用于角类药材的鉴别研究;程显隆 等;《中华中医药学会第七次中药分析学术交流会论文集》;20141107;第8-11页 * |
| 半夏及掌叶半夏毒针晶中共性毒蛋白的研究;郁红礼等;《中华中医药杂志》;20110501(第05期);第1037-1042页 * |
| 周法东 等.高效液相色谱串联质谱法检测阿胶枣中的阿胶成分.《河北省科学院学报》.2017,第34卷(第3期), * |
| 特征肽段检测技术用于胶类药材专属性鉴别方法研究;程显隆 等;《中国药学杂志》;20150131;第50卷(第2期);第104-108页 * |
| 纤维素酶产生菌的筛选及产酶条件优化;李洋;《中国优秀硕士学位论文全文数据库（工程科技Ⅰ辑）》;20160315(第3期);第B018-113页 * |
| 高效液相色谱串联质谱法检测阿胶枣中的阿胶成分;周法东 等;《河北省科学院学报》;20170930;第34卷(第3期);第52-57页 * |
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