CN115728412A - Method for detecting content of cordycepic acid in fermented cordyceps sinensis powder (Cs-4) - Google Patents
Method for detecting content of cordycepic acid in fermented cordyceps sinensis powder (Cs-4) Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a method for detecting the content of cordycepic acid in fermented cordyceps sinensis (Cs-4). The detection method comprises the following steps: s1, preparation of a reference substance solution: taking a proper amount of cordycepic acid reference substance, precisely weighing, and dissolving with pure water to obtain a reference substance solution; s2, preparing a test solution: adding appropriate amount of fermented Cordyceps powder into conical flask, accurately weighing, adding pure water, weighing, ultrasonically extracting, cooling, weighing, supplementing pure water to reduce weight loss, filtering with 0.22 μm microporous membrane, and collecting filtrate as sample solution; s3, measuring: injecting the reference solution and the test solution into an ultra high performance liquid chromatograph for detection, determining to obtain a chromatogram, and calculating the content of cordycepic acid; chromatographic conditions are as follows: chromatographic column Waters BEH Amide, methanol as phase A and water as phase B. The elution mode of the invention uses gradient elution, compared with isocratic elution, the separation capacity is improved, the analysis period is shortened, the peak type is improved, the tailing of chromatographic peak is reduced, and the sensitivity is increased.
Description
Technical Field
The invention relates to a method for detecting cordycepic acid content, belongs to the technical field of biological sample detection, and particularly relates to a method for detecting cordycepic acid content in fermented cordyceps sinensis powder (Cs-4).
Background
The fermented Cordyceps powder is prepared by deep fermenting and culturing Paecilomyces hepiali (Cs-4) separated from fresh Cordyceps (Cordyceps sinensis (BerK.) Sacc.), filtering the fermentation product, and drying. Modern pharmacological research shows that cordycepic acid has the functions of inhibiting the growth of various germs and preventing and treating cerebral thrombosis, cerebral hemorrhage, myocardial infarction, long-term failure and other diseases, is a good diuretic in clinical application, and has the effects of reducing intracranial pressure, intraocular pressure and the like. The market demand of the fermented cordyceps sinensis powder is increased year by year, the sales of the fermented cordyceps sinensis powder reaches 3089.158 tons in only 2021 years, and the sales of the fermented cordyceps sinensis powder reaches 3.96 hundred million yuan, but the fermented cordyceps sinensis powder only has the national standard of Jiangxi at present and lacks about the detection of cordyceps acid components, so that the quality of the fermented cordyceps sinensis powder on the market is poor.
The fermented Cordyceps powder (Cs-4) contains various bioactive substances such as cordycepic acid, adenosine, ergosterol, amino acids, etc. Cordycepic acid is one of the main effective components of fermented cordyceps sinensis powder, the existing local standard of the fermented cordyceps sinensis powder (Cs-4) only specifies the content ranges of adenosine, guanosine, uridine and ergosterol, and the requirement on the content of the cordycepic acid is lacked.
At present, related documents about the determination research of the content of the active ingredients of the fermented cordyceps sinensis powder are few, only less than 10 documents exist in the last decade, and the determination of the content of polysaccharides, nucleosides and sterols is mainly researched. Cordycepic acid is also called D-mannitol, and mannitol is often mixed with cordycepic acid in the market; however, the research finds that D-mannitol is inconsistent with mannitol in configuration, and a mannitol measuring method cannot be used as a standard for detecting cordycepic acid.
The existing literature has no research on the content determination of cordycepic acid in fermented cordyceps sinensis powder (Cs-4) for a while, and only 3 references relate to the content determination of cordycepic acid isomer mannitol. The existing literature is mainly focused on cordyceps medicinal materials for research on cordyceps acid content determination, and methods for detecting cordyceps acid in fermented cordyceps powder and preparations thereof all use isomer mannitol as a determination target and mainly comprise a titration method, a colorimetric method, an HPLC-RID detection method and the like.
At present, a few published documents report a detection method of cordycepic acid isomer mannitol in fermented cordyceps sinensis powder, such as: determination of mannitol in fermented Cordyceps powder product-professional hygiene and sick and wounded 2005 No. 3, HPLC-RID determination of mannitol content in fermented Cordyceps powder (Cs-4) -drug evaluation 2020, colorimetric determination of mannitol content in fermented Cordyceps powder-Chinese modern application pharmacy 2006 No. 6.
In recent years, there are few documents related to the research on the content measurement of the active ingredients of fermented cordyceps sinensis (Cs-4), and only less than 10 studies on the content measurement of the active ingredients of fermented cordyceps sinensis (Cs-4) are focused on nucleoside ingredients and sterol ingredients. Due to the lack of related research on the determination of cordycepic acid content in the fermented cordyceps sinensis powder (Cs-4), the detection standard of the fermented cordyceps sinensis powder (Cs-4) is not complete. The content detection item of the existing local standard of fermented cordyceps sinensis powder (Cs-4) only specifies the content of adenosine, guanosine, uridine and ergosterol, and only a thin-layer identification item relates to the identification of cordycepic acid isomer mannitol, so that the cordycepic acid cannot be quantified. Cordycepic acid as one of main components of fermented cordyceps sinensis powder (Cs-4) is always lack of related quality standards, so that the quality of the fermented cordyceps sinensis powder (Cs-4) product in the market is unstable.
The existing method for measuring cordycepic acid in traditional Chinese medicinal materials and decoction pieces mainly comprises periodate oxidation method, optical rotation method, colorimetric method, specific gravity method, titration method, HPLC-RID detection method and the like. Although conventional content determination methods such as an optical rotation method, a colorimetric method, a periodate oxidation method and the like are simple and convenient to operate, the result accuracy is not high, the conventional content determination methods are only suitable for determining high-purity samples, and the fermented cordyceps sinensis powder (Cs-4) has complex components, so the conventional content determination methods cannot be used.
The detection methods of the fermented cordyceps sinensis powder and the cordycepic acid in the preparation thereof in the prior literature mainly comprise a titration method, a colorimetric method, an HPLC-RID determination method and the like. Cordycepic acid is polyhydric alcohol containing 6 hydroxyl groups, and the content of cordycepic acid measured by a titration method is inaccurate because the total of titration liquids consumed by components with reduced hydroxyl groups is measured by a redox titration method and is not the actual content of a single mannitol component.
Disclosure of Invention
The invention aims to provide a method for detecting the content of cordycepic acid in fermented cordyceps sinensis powder (Cs-4), wherein the content determination method is an ultra-high performance liquid chromatography-evaporative light detector method, and has the advantages of high sensitivity, high quantitative precision, high detection speed, few samples and the like.
The purpose of the invention and the technical problem to be solved are realized by adopting the following technical scheme. The method for detecting the content of the cordycepic acid in the fermented cordyceps sinensis powder (Cs-4) provided by the invention comprises the following steps of:
s1, preparation of a reference substance solution: taking a proper amount of cordycepic acid reference substance, precisely weighing, and dissolving with pure water to obtain a reference substance solution;
s2, preparing a test solution: adding appropriate amount of fermented Cordyceps powder into conical flask, accurately weighing, adding pure water, weighing, ultrasonically extracting, cooling, weighing, supplementing pure water to reduce weight loss, filtering with 0.22 μm microporous membrane, and collecting filtrate as sample solution;
s3, measuring: injecting the reference solution and the sample solution into an ultra-high performance liquid chromatograph for detection, determining to obtain a chromatogram, and calculating the content of cordycepic acid according to the chromatogram;
wherein, the chromatographic conditions are as follows: the chromatographic column Waters BEH Amide uses methanol as phase A and water as phase B for gradient elution.
Preferably, the method for detecting the content of the cordycepic acid in the fermented cordyceps sinensis powder (Cs-4) comprises the following steps:
s1, preparation of a reference substance solution: taking a proper amount of cordycepic acid reference substance, precisely weighing, and dissolving with pure water to obtain a reference substance solution containing 1-5mg of cordycepic acid per 1 mL.
S2, preparing a test solution: adding a proper amount of fermented cordyceps sinensis powder into a conical flask, precisely weighing, adding 25mL of pure water, weighing, performing ultrasonic or heating reflux extraction for 15-60 min, cooling, weighing, supplementing the weight loss amount with pure water, filtering the filtrate with a 0.22 mu m microporous filter membrane, and taking the subsequent filtrate as a sample solution.
S3, measuring: and injecting the reference solution and the test solution into an ultra-high performance liquid chromatograph for detection, determining to obtain a chromatogram, and calculating the content of cordycepic acid according to the chromatogram.
Preferably, the chromatographic conditions of the method for detecting the content of cordycepic acid in the fermented cordyceps sinensis powder (Cs-4) are as follows:
chromatographic column Waters BEH Amide, the length of the chromatographic column is 100mm, the inner diameter is 2.1mm, the particle size is 1.7 μm, methanol is used as a mobile phase A, water is used as a mobile phase B, the flow rate is 0.20-0.40ml/min, the sample injection amount is 1-2 μ l, the column temperature is 20-40 ℃, the evaporation temperature of an evaporative light detector is 45-80 ℃, and carrier gas (N) is used 2 ) The flow rate is 2.5-2.7L/min.
Preferably, the method for detecting the content of the cordycepic acid in the fermented cordyceps sinensis powder (Cs-4) comprises the following steps:
in S1, preparation of a control solution: taking a proper amount of cordycepic acid reference substance, precisely weighing, and dissolving with pure water to obtain a reference substance solution containing 1mg cordycepic acid per 1 mL;
s2, preparing a test solution: taking 0.1g of fermented cordyceps sinensis powder into a conical flask, precisely weighing, adding 25mL of pure water, weighing, ultrasonically extracting for 30min, cooling, weighing, supplementing with pure water to reduce weight loss, filtering the filtrate with a 0.22 mu m microporous membrane, and taking the subsequent filtrate as a test solution;
s3, measuring: and injecting the reference solution and the test solution into an ultra-high performance liquid chromatograph for detection, determining to obtain a chromatogram, and calculating the content of cordycepic acid according to the chromatogram.
Preferably, the chromatographic conditions of the method for detecting the content of cordycepic acid in the fermented cordyceps sinensis powder (Cs-4) are as follows:
a chromatographic column Waters BEH Amide with a column length of 100mm, an inner diameter of 2.1mm, a particle size of 1.7 μm, methanol as a mobile phase A, water as a mobile phase B, 0-10 min (A: 70% → 90%, B:30% → 10%), 10-15 min (A: 90% → 100%, B:10% → 0%), a flow rate of 0.30ml/min, a sample introduction amount of 1 μ l, a column temperature of 30 ℃, an evaporation temperature of an evaporation photodetector of 65 ℃, and a carrier gas (N: 90% → 100%, B:10% → 0%), a column temperature of 30 ℃, an evaporation temperature of an evaporation photodetector of 65 ℃, and a liquid crystal display 2 ) The flow rate was 2.5L/min.
By means of the technical scheme, the method for detecting the content of the cordycepic acid in the fermented cordyceps sinensis powder (Cs-4) has the following advantages:
1) The method aims at the standard that the fermented cordyceps sinensis powder (Cs-4) lacks cordycepic acid content determination, supplements related research, provides basis for standard formulation, and provides a more favorable, faster and more efficient detection means for production enterprises.
2) The invention provides a method for detecting the content of cordycepic acid in fermented cordyceps sinensis powder (Cs-4), wherein the content determination method is an ultra-high performance liquid chromatography-evaporative light detector method, and the method has the advantages of high sensitivity, high quantitative precision, high detection speed, few samples and the like.
3) The detection method adopts ultra-high performance liquid chromatography for detection, particularly adopts an evaporation light detector, has wide gradient and solvent compatibility, and has no solvent peak interference. Unlike ultraviolet and fluorescence detectors, whose response is independent of the optical properties of the sample, any sample that is less volatile than the mobile phase can be detected without being affected by its functional groups. Compared with a differential detector, the sensitivity is high, and the method can be compatible with gradient elution.
4) The elution mode of the invention uses gradient elution, compared with isocratic elution, the invention improves the separation capability, shortens the analysis period, improves the peak pattern, reduces the tailing of chromatographic peaks and increases the sensitivity.
Drawings
FIG. 1 is a sample chromatogram for a UPLC-ELSD assay;
FIG. 2 is a chromatogram of a test sample of an HPLC-RID assay;
FIG. 3 shows the effect of elution gradient 0-40 min (A: 10% → 100%, B:90% → 0%) on the peaks of the sample chromatogram;
FIG. 4 shows the effect of elution gradient 0-10 min (A: 70% → 90%, B:30% → 10%), 10-15 min (A: 90% → 100%, B:10% → 0%) on the peaks of the sample chromatogram;
FIG. 5 is a graph showing the effect of the evaporation temperature of 45 ℃ on the chromatographic peak of a sample;
FIG. 6 is a graph showing the effect of the evaporation temperature of the detector at 55 ℃ on the chromatographic peaks of the test sample;
FIG. 7 is a graph showing the effect of a detector evaporation temperature of 65 ℃ on the chromatographic peaks of a test sample;
FIG. 8 is a graph showing the effect of the evaporation temperature of 80 ℃ on the chromatographic peak of a sample;
FIG. 9 is a graph showing the effect of a carrier gas flow rate of 2.5L/min on the peak shape and response value of a chromatogram of a test sample;
FIG. 10 is the effect of carrier gas flow rate 2.7L/min on the peak shape and response value of the chromatographic spectrum of the test sample;
FIG. 11 is a chromatogram of a cordycepic acid control of example 1;
FIG. 12 is a chromatogram of a test sample of example 1;
FIG. 13 is a chromatogram of a test sample of comparative example 1.
Detailed Description
A method for detecting cordycepic acid content in fermented cordyceps sinensis powder (Cs-4) comprises the following steps:
step 1, preparation of a reference solution: taking a proper amount of cordycepic acid reference substance, precisely weighing, and dissolving with pure water to obtain reference substance solution.
Step 2, preparation of a test solution: adding appropriate amount of fermented Cordyceps powder into conical flask, precisely weighing, adding pure water, weighing, ultrasonically extracting, cooling, weighing, supplementing pure water, reducing weight loss, filtering with 0.22 μm microporous membrane, and collecting the filtrate as sample solution.
And step 3, determination: injecting the reference solution and the sample solution into an ultra-high performance liquid chromatograph for detection, determining to obtain a chromatogram, and calculating the content of cordycepic acid according to the chromatogram.
Wherein, the chromatographic conditions are as follows:
the chromatographic column Waters BEH Amide uses methanol as phase A and water as phase B for gradient elution.
The detection method of the invention preferably comprises the following steps:
step 1, preparation of a reference solution: taking a proper amount of cordycepic acid reference substance, precisely weighing, and dissolving with pure water to obtain a reference substance solution containing 1-5mg of cordycepic acid per 1 mL.
Step 2, preparation of a test solution: adding a proper amount of fermented cordyceps sinensis powder into a conical flask, precisely weighing, adding 25mL of pure water, weighing, performing ultrasonic or heating reflux extraction for 15-60 min, cooling, weighing, supplementing the weight loss amount with pure water, filtering the filtrate with a 0.22 mu m microporous filter membrane, and taking the subsequent filtrate as a sample solution.
And 3, determining: and injecting the reference solution and the test solution into an ultra-high performance liquid chromatograph for detection, determining to obtain a chromatogram, and calculating the content of cordycepic acid according to the chromatogram.
Wherein, the chromatographic conditions are as follows:
chromatographic column Waters BEH Amide with column length of 100mm, inner diameter of 2.1mm, particle diameter of 1.7 μm, methanol as mobile phase A, water as mobile phase B, 0-40 min (A: 10% → 100%, B:90% → 0%), flow rate of 0.20-0.40ml/min, sample introduction amount of 1-2 μ l, column temperature of 20-40 deg.C, and evaporated lightThe evaporation temperature of the detector is 45-80 ℃, and the carrier gas (N) 2 ) The flow rate is 2.5-2.7L/min.
The detection method of the invention has the most preferable steps as follows:
step 1, preparation of a reference solution: taking a proper amount of cordycepic acid reference substance, precisely weighing, and dissolving with pure water to obtain a reference substance solution containing 1mg of cordycepic acid per 1 mL.
Step 2, preparation of a test solution: taking 0.1g of fermented cordyceps sinensis powder into a conical flask, precisely weighing, adding 25mL of pure water, weighing, ultrasonically extracting for 30min, cooling, weighing, supplementing with pure water to reduce weight loss, filtering the filtrate with a 0.22 mu m microporous membrane, and taking the subsequent filtrate as a test solution.
And 3, determining: injecting the reference solution and the sample solution into an ultra-high performance liquid chromatograph for detection, determining to obtain a chromatogram, and calculating the content of cordycepic acid according to the chromatogram.
Wherein, the chromatographic conditions are as follows:
chromatographic column Waters BEH Amide, chromatographic column length 100mm, inner diameter 2.1mm, particle size 1.7 μm, methanol as mobile phase A, water as mobile phase B, 0-10 min (A: 70% → 90%, B:30% → 10%), 10-15 min (A: 90% → 100%, B:10% → 0%), flow rate 0.30ml/min, sample injection amount 1 μ l, column temperature 30 deg.C, evaporation temperature of evaporation photodetector 65 deg.C, carrier gas (N: 90% → 100%, B:10% → 0%), evaporation temperature of evaporation photodetector 65 deg.C 2 ) The flow rate was 2.5L/min.
The method is obtained by screening, and the screening process is as follows:
1. selection of extraction conditions
1.1 selection of extraction mode
According to the invention, the cordyceps acid in the fermented cordyceps sinensis powder (Cs-4) is extracted by adopting an ultrasonic extraction method and a heating reflux method, and comparison experiment investigation shows that the content difference between the cordyceps acid extracted by the ultrasonic extraction method and the cordyceps acid extracted by the reflux method is not large, and the ultrasonic extraction method is adopted in consideration of the simplicity and convenience of operation and the detection cost.
1.2 selection of extraction vehicle
The extraction effect of different extraction solvents is examined by respectively adding 10% methanol, 30% methanol, 50% methanol, 10% ethanol, 30% ethanol, 50% ethanol, ethanol and water as extraction solvents, and the result shows that the extraction effect of water is the highest, so that water is selected as the extraction solvent.
1.3 selection of extraction time
Respectively carrying out ultrasonic treatment for 15min, 30min and 60min, and inspecting the extraction effects at different extraction times, wherein the results show that the extraction effects are not greatly different when the ultrasonic treatment time is 15min, 30min and 60min, and the ultrasonic treatment time is selected to be 30min in order to ensure sufficient extraction in consideration of the influence of experimental environment.
2. Optimization of chromatographic conditions
2.1 optimization of elution gradient
The gradient elution was carried out for 0 to 40min (a: 10% → 100%, B:90% → 0%) with methanol as the mobile phase a and water as the mobile phase B, and the change ratio of the mobile phases a and B was adjusted at different times based on the results, which revealed that the chromatographic peak shape was the best and the degree of separation from the peaks was the best for 0 to 10min (a: 70% → 90%, B:30% → 10%) and 10 to 15min (a: 90% → 100%, B:10% → 0%) of the elution gradient.
2.2 optimization of the Instrument parameters
The influence of the evaporation temperature of the evaporation photodetector at 45 ℃, 55 ℃, 65 ℃ and 80 ℃ on the peak shape, the response value, the baseline and the separation degree of the chromatographic peak is respectively considered, and the result shows that the peak shape of the chromatographic peak at 65 ℃ is symmetrical, the response value is high, the baseline is smooth and the separation degree is high, so that the evaporation temperature of the evaporation photodetector at 65 ℃ is selected.
The invention improves the prior art, adopts pure water as an extraction solvent, saves the cost and reduces the environmental pollution; the detection is carried out by adopting a UPLC-ELSD method, so that the sensitivity is higher, the peak-appearing time is shorter, and the peak-appearing is more stable; gradient elution is adopted, and chromatographic peak separation degree is better.
Aiming at the condition that the detector in the prior art is greatly influenced by temperature and pressure, the invention purposefully carries out methodology verification, the precision test, the repeatability test and the stability test data obtained by verification are shown in table 1, the RSD is less than 3 percent, and the method has good precision, repeatability and stability and can provide accurate and effective basis for detection.
TABLE 1 precision, stability, repeatability data
The invention has the advantages that the investigation on solvent extraction, extraction mode and extraction time is carried out in a targeted manner, and the optimal solvent extraction, mode and time are obtained through the comparison of experimental data in the table 2, so that the extraction process is simpler and more convenient to operate, the time consumption is shorter, the cost is saved, and the pollution to the environment is reduced.
The experimental data compares 3 kinds of reagents with 4 concentrations to draw a conclusion that pure water is more fully extracted, and the pure water has the advantages of simple preparation process, low cost, no pollution to the environment and the like, so that the experimental cost is reduced, and the pollution to the environment is reduced. By comparing different extraction modes and extraction time, the extraction method which is simpler and more convenient to operate and shorter in detection time is found.
TABLE 2 solvent extraction, extraction mode, extraction time investigation data
Aiming at the condition of long peak time in the prior art, the invention uses the ultra-high performance liquid chromatograph pertinently, shortens the running time from 40min to 15min, and can be obtained by comparing fig. 1 with fig. 2:
compared with the prior art, the chromatographic peak of the cordycepic acid in the chromatogram of the test sample has symmetrical peak shape, good separation degree, no interference of miscellaneous peaks, reduced baseline noise, short peak output time, and reduced peak output time from 27min to 3min, thereby greatly saving the detection time and reducing the detection cost.
As shown in fig. 3-4, the present invention examined the effect of the elution gradient on the chromatographic peak, and the gradient elution was performed with methanol as mobile phase a and water as mobile phase B for 0-40 min (a: 10% → 100%, B:90% → 0%), and the ratio of change in mobile phases a and B was adjusted at different times, indicating that the chromatographic peak shape was optimal, the baseline was stable, and the degree of separation from the peaks was optimal when the elution gradient was 0-10 min (a: 70% → 90%, B:30% → 10%), 10-15 min (a: 90% → 100%, B:10% → 0%).
As shown in FIGS. 5 to 8, the present inventors examined the influence of the evaporation temperature of the evaporation light detector on the peak shape and response value of the chromatographic peak, and compared the influence of the evaporation temperature at 45 deg.C, 55 deg.C, 65 deg.C, 80 deg.C on the peak shape, response value, baseline, and separation degree of the chromatographic peak, and found that the peak shape and response value were the best at 65 deg.C.
As shown in FIGS. 9 to 10, the present inventors examined the influence of the carrier gas flow rate of the evaporative light detector on the peak shape and response value of the chromatogram, and found that the peak shape and response value were optimum when the carrier gas flow rate was 2.5L/min and 2.7L/min, compared with the influence of the carrier gas flow rate on the peak shape, response value, base line, and separation degree of the chromatogram.
The method uses the UPLC-ELSD method, the problems of unstable peak appearance and poor peak shape caused by temperature influence of the HPLC-RID method, high repeatability RSD and long peak appearance time are solved in a targeted manner, the cordyceps acid chromatographic peak and other chromatographic peaks are well separated by utilizing gradient elution, the content determination error caused by interference of miscellaneous peaks is reduced, the content values and the RSD of the two methods are compared according to data in a table 3, and the UPLC-ELSD method is obviously more stable than the HPLC-RID method and has smaller error.
TABLE 3 comparison of UPLC-ELSD assay with HPLC-RID assay
The invention comprises the following steps:
adding a proper amount of fermented cordyceps sinensis powder into a conical flask, precisely weighing, adding 25mL of pure water, weighing, and ultrasonically extracting for 15-60 min.
The invention uses UPLC-ELSD method to detect, chromatographic column Waters BEH Amide, chromatographic column length 100mm, inner diameter2.1mm, particle size of 5 μm, mobile phase of methanol-water 0-10 min (methanol: 70% → 90%, water: 30% → 10%), 10-15 min (methanol: 90% → 100%, water: 10% → 0%), flow rate of 0.20-0.40ml/min, sample amount of 1-2 μ l, column temperature of 20-40 deg.C, evaporation temperature of evaporative light detector of 45-80 deg.C, carrier gas (N) 2 ) The flow rate is 2.5-2.7L/min.
Example 1
1. Preparation of control solutions: taking a proper amount of cordycepic acid reference substance, precisely weighing, and dissolving with pure water to obtain a reference substance solution containing 1mg of cordycepic acid per 1 mL.
2. Preparing a test solution: adding appropriate amount of fermented Cordyceps powder into conical flask, accurately weighing, adding 25mL of pure water, weighing, ultrasonic extracting for 30min, cooling, weighing, supplementing pure water to reduce weight loss, filtering with 0.22 μm microporous membrane, and collecting filtrate as sample solution.
3. Injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph for detection, wherein the chromatographic conditions comprise that: chromatographic column Waters BEH Amide (2.1 mm. Times.100mm, 1.7 μm), methanol as mobile phase A, water as mobile phase B, elution gradient of 0-10 min (A: 70% → 90%, B:30% → 10%), 10-15 min (A: 90% → 100%, B:10% → 0%), flow rate of 0.30mL/min, column temperature of 30 ℃, evaporative light detector drift tube temperature of 65 ℃, carrier gas (N.N.sub. 2 ) The flow rate is 2.5L/min, and the liquid chromatography detection sample injection amount is 1 mu L.
Measuring to obtain chromatogram, and calculating content of cordycepic acid to be 9.19% according to chromatogram.
Example 2
1. Preparation of control solutions: taking a proper amount of cordycepic acid reference substance, precisely weighing, and dissolving with pure water to obtain a reference substance solution containing 1mg cordycepic acid per 1 mL.
2. Preparing a test solution: adding appropriate amount of fermented Cordyceps powder into conical flask, precisely weighing, adding 25mL of pure water, weighing, ultrasonically extracting for 15min, cooling, weighing, supplementing with pure water, reducing weight, filtering, passing the filtrate through 0.22 μm microporous membrane, and collecting the filtrate as sample solution.
3. Injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph for detection, wherein the chromatographic conditions comprise that: chromatographic column Waters BEH Amide (2.1 mm. Times.100mm, 1.7 μm), methanol as mobile phase A, water as mobile phase B, elution gradient of 0-10 min (A: 70% → 90%, B:30% → 10%), 10-15 min (A: 90% → 100%, B:10% → 0%), flow rate of 0.30mL/min, column temperature of 30 ℃, evaporative light detector drift tube temperature of 65 ℃, carrier gas (N.N.sub. 2 ) The flow rate is 2.5L/min, and the sample injection amount is 1 mu L by liquid chromatography detection.
Measuring to obtain chromatogram, and calculating the content of cordycepic acid to be 9.15% according to the chromatogram.
Example 3
1. Preparation of control solutions: taking a proper amount of cordycepic acid reference substance, precisely weighing, and dissolving with pure water to obtain a reference substance solution containing 1mg of cordycepic acid per 1 mL.
2. Preparation of a test solution: adding appropriate amount of fermented Cordyceps powder into conical flask, precisely weighing, adding 25mL of pure water, weighing, ultrasonically extracting for 60min, cooling, weighing, supplementing with pure water, reducing weight, filtering, passing the filtrate through 0.22 μm microporous membrane, and collecting the filtrate as sample solution.
3. Injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph for detection, wherein the chromatographic conditions comprise: chromatographic column Waters BEH Amide (2.1 mm. Times.100mm, 1.7 μm), methanol as mobile phase A, water as mobile phase B, elution gradient of 0-10 min (A: 70% → 90%, B:30% → 10%), 10-15 min (A: 90% → 100%, B:10% → 0%), flow rate of 0.30mL/min, column temperature of 30 ℃, evaporative light detector drift tube temperature of 65 ℃, carrier gas (N.N.sub. 2 ) The flow rate is 2.5L/min, and the sample injection amount is 1 mu L by liquid chromatography detection.
Measuring to obtain chromatogram, and calculating content of cordycepic acid to be 9.07% according to chromatogram.
Comparative example 1
1. Preparation of control solutions: taking a proper amount of cordycepic acid reference substance, precisely weighing, and dissolving with pure water to obtain a reference substance solution containing 1mg cordycepic acid per 1 mL.
2. Preparation of a test solution: adding appropriate amount of fermented Cordyceps powder into conical flask, precisely weighing, adding 25mL of pure water, weighing, ultrasonically extracting for 30min, cooling, weighing, supplementing with pure water, reducing weight, filtering, passing the filtrate through 0.45 μm microporous membrane, and collecting the filtrate as sample solution.
3. Injecting the reference solution and the test solution into a high performance liquid chromatograph for detection, wherein the chromatographic conditions comprise: column Agilent ZORBAX NH2 (4.6 mm. Times.250mm, 5 μm), acetonitrile as mobile phase A, water as mobile phase B, (A: B =80 20), flow rate of 1.0mL/min, column temperature of 40 ℃, differential detector temperature of 35 ℃, liquid chromatography detection sample amount of 10 μ L.
Measuring to obtain chromatogram, and calculating the content of cordycepic acid to be 6.95% according to the chromatogram.
Compared with the traditional HPLC, the speed, the sensitivity and the separation degree of the UPLC are respectively 9 times, 3 times and 1.7 times of the HPLC. The UPLC has the advantages of high resolution, high speed, high sensitivity and the like, and retains the original practicability and principle while comprehensively improving the quality of HPLC, such as detection speed, sensitivity, resolution and the like. The most obvious advantage of using UPLC can shorten the analysis time, improve the detection efficiency, reduce the solvent consumption and reduce the analysis cost, the improvement of the analysis efficiency can also greatly save the detection cost of the technical introduction unit for the production of the related products, improve the production efficiency and improve the production safety management working efficiency and quality of the products of the variety.
The mobile phase a was selected to be methanol rather than acetonitrile, one aspect of methanol over acetonitrile is that it completely dissolves more types of samples, and improved solubility may lead to better overall peak shape. Acetonitrile is easy to form floccule by self-polymerization in the use process to block a chromatographic system, and the price of the chromatographic grade acetonitrile is higher than that of the chromatographic grade methanol, so that the cost is saved by using the methanol. According to toxicological research, acetonitrile has various toxicities such as teratogenicity, carcinogenicity, reproductive toxicity and the like, can cause serious injury to human bodies through skin contact and inhalation, and has relatively less harm to methanol compared with acetonitrile.
The method is developed by using UPLC, and methodology development and research are required according to Chinese pharmacopoeia (9101 analysis method verification guide principle). Firstly, separating chromatographic peaks by using a gradient elution mode, and continuously adjusting according to the peak appearance time, the peak shape and the base line of the chromatographic peaks to obtain the optimal chromatographic conditions of chromatographic peak separation height, symmetrical peak shape and smooth base line. After the optimal chromatographic condition is obtained, the extraction solvent, the extraction mode and the extraction time of the test sample are inspected, and the optimal extraction condition is obtained through comparison of a large amount of experimental data. On the basis, the methodological verification is continuously carried out, the items such as precision, repeatability, stability, intermediate precision, accuracy, durability and the like of the method are investigated, and the feasibility of the method is verified through a plurality of experiments.
As described above, the present invention is only a preferred embodiment, and is not limited in any way, and therefore, any simple modification, equivalent change and modification of the above embodiment according to the technical essence of the present invention will still fall within the scope of the technical solution of the present invention.
Claims (7)
1. A method for detecting the content of cordycepic acid in fermented cordyceps sinensis powder (Cs-4) is characterized by comprising the following steps:
s1, preparation of a reference substance solution: taking a proper amount of cordycepic acid reference substance, precisely weighing, and dissolving with pure water to obtain a reference substance solution;
s2, preparing a test solution: adding appropriate amount of fermented Cordyceps powder into conical flask, precisely weighing, adding pure water, weighing, ultrasonically extracting, cooling, weighing, supplementing pure water, reducing weight loss, filtering with 0.22 μm microporous membrane, and collecting the filtrate as sample solution;
s3, measuring: injecting the reference solution and the test solution into an ultra high performance liquid chromatograph for detection, determining to obtain a chromatogram, and calculating the content of cordycepic acid according to the chromatogram;
wherein, the chromatographic conditions are as follows: the chromatographic column Waters BEH Amide uses methanol as phase A and water as phase B for gradient elution.
2. The detection method according to claim 1, characterized in that: in step S1, preparation of a reference solution: taking a proper amount of cordycepic acid reference substance, precisely weighing, and dissolving with pure water to obtain a reference substance solution containing 1-5mg of cordycepic acid per 1 mL.
3. The detection method according to claim 1, characterized in that:
in step S2, preparing a test solution: taking a proper amount of fermented cordyceps sinensis powder to a conical flask, precisely weighing, adding 25mL of pure water, weighing, performing ultrasonic or heating reflux extraction for 15-60 min, cooling, weighing, supplementing the weight loss by pure water, filtering the filtrate by a 0.22-micron microporous filter membrane, and taking the subsequent filtrate as a test solution.
4. The detection method according to claim 1, characterized in that:
the chromatographic conditions were as follows:
chromatographic column Waters BEH Amide, the length of the chromatographic column is 100mm, the inner diameter is 2.1mm, the particle size is 1.7 μm, methanol is used as a mobile phase A, water is used as a mobile phase B, the flow rate is 0.20-0.40ml/min, the sample injection amount is 1-2 μ l, the column temperature is 20-40 ℃, the evaporation temperature of an evaporative light detector is 45-80 ℃, and carrier gas (N) is used 2 ) The flow rate is 2.5-2.7L/min.
5. The detection method according to claim 2, characterized in that:
in step S1, preparation of a reference solution: taking a proper amount of cordycepic acid reference substance, precisely weighing, and dissolving with pure water to obtain a reference substance solution containing 1mg cordycepic acid per 1 mL.
6. The detection method according to claim 3, characterized in that:
in step S2, preparing a test solution: taking 0.1g of fermented cordyceps sinensis powder into a conical flask, precisely weighing, adding 25mL of pure water, weighing, ultrasonically extracting for 30min, cooling, weighing, supplementing pure water to the reduced weight, filtering the filtrate with a 0.22-micron microporous membrane, and taking the subsequent filtrate as a test solution.
7. The detection method according to claim 4, characterized in that: wherein, the chromatographic conditions are as follows:
a chromatographic column Waters BEH Amide with a column length of 100mm, an inner diameter of 2.1mm, a particle size of 1.7 μm, methanol as a mobile phase A, water as a mobile phase B, 0-10 min (A: 70% → 90%, B:30% → 10%), 10-15 min (A: 90% → 100%, B:10% → 0%), a flow rate of 0.30ml/min, a sample introduction amount of 1 μ l, a column temperature of 30 ℃, an evaporation temperature of an evaporation photodetector of 65 ℃, and a carrier gas (N: 90% → 100%, B:10% → 0%), a column temperature of 30 ℃, an evaporation temperature of an evaporation photodetector of 65 ℃, and a liquid crystal display 2 ) The flow rate was 2.5L/min.
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| US5585473A (en) * | 1994-12-09 | 1996-12-17 | The Biomembrane Institute | Compounds and methods for monosaccharide analysis |
| CN110133129A (en) * | 2019-05-15 | 2019-08-16 | 江西国药有限责任公司 | The detection method of mannitol in a kind of fermentation cordyceps (Cs-4) |
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