CN111077245A - UPLC characteristic spectrum establishing method and detection method of radix semiaquilegiae medicinal material - Google Patents
UPLC characteristic spectrum establishing method and detection method of radix semiaquilegiae medicinal material Download PDFInfo
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Abstract
The invention relates to a method for establishing and detecting an UPLC (ultra performance liquid chromatography) characteristic spectrum of a muskroot-like semiaquilegia root medicinal material. The establishing method comprises the following steps: preparation of control solutions: dissolving a geldanolide reference substance, a lithospermum cyanoside reference substance and a magnoflorine reference substance with a solvent to obtain a reference substance solution; preparation of a test solution: taking semiaquilegia root medicinal material powder, adding a solvent for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a test solution; and injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph for detection, wherein a mobile phase A adopted by the ultra-high performance liquid chromatograph is methanol, a mobile phase B is a phosphoric acid aqueous solution with the volume fraction of 0.03-0.07%, and the elution mode is gradient elution. The establishing method can obtain the characteristic spectrum of the semiaquilegia root medicinal material, wherein the characteristic spectrum comprises 12 characteristic peaks. In addition, the content of gelsoline and magnoflorine in the radix semiaquilegiae medicinal material can be simultaneously measured, the quality of the radix semiaquilegiae medicinal material can be comprehensively reflected, and the method is accurate and reliable, simple and convenient to operate and good in reproducibility.
Description
Technical Field
The invention relates to quality detection of traditional Chinese medicinal materials, in particular to a UPLC (ultra performance liquid chromatography) characteristic spectrum establishing method and a detection method of a semiaquilegia root medicinal material.
Background
Semiaquilegia adoxoides (DC.) Makino, which is collected in early summer, washed, dried and fibrous root removed, is collected in Chinese pharmacopoeia 2015 edition and is derived from dried root tuber of Semiaquilegia adoxoides (DC.) Makino, which is a plant belonging to Ranunculaceae. The original plant semiaquilegia is named as semiaquilegia, herba euphorbiae lathyris, herba muricariae or gynura bicolor, and the semiaquilegia is named as thousand-year semiaquilegia, herba euphorbiae scopariae, fructus geriae scabrae, radix peruviae, xianzao or gynura bicolor root. Tian Ma Zi is known as Qiannian rat feces and Zi Sha Tian Ku Gen from Ben Cao gang mu Shi Yi of Zhao Ming in Qing Dynasty.
The semiaquilegia root mainly comprises lactones, nitryl, cyanides, alkaloids and the like as chemical components, has the effects of clearing heat and removing toxicity, relieving swelling and eliminating stagnation and the like, and is clinically commonly used for carbuncle and furuncle, acute mastitis, scrofula and snake and insect bite. The muskroot-like semiaquilegia root has rich resources and wide distribution, and can be seen in provinces such as Henan, Jiangsu, Anhui, Zhejiang, Jiangxi, Fujian, Hubei, Hunan, Guangdong, Guangxi, Shaanxi, Sichuan, Guizhou and the like. Because the Chinese pharmacopoeia 2015 edition lacks quantitative indexes of the radix semiaquilegiae medicinal material and a method for integrally controlling the quality of the radix semiaquilegiae medicinal material, and the research reports on the quality control standard of the radix semiaquilegiae are few at present, it is necessary to establish a comprehensive and reliable detection method for evaluating the quality of the radix semiaquilegiae medicinal material.
Disclosure of Invention
Therefore, a UPLC characteristic spectrum establishing method of the semiaquilegia root medicinal material is needed. The establishing method can establish the characteristic spectrum of the radix semiaquilegiae medicinal material, wherein the characteristic spectrum contains 12 characteristic peaks, and can simultaneously determine the content of the geldanolide and the magnoflorine in the radix semiaquilegiae medicinal material, the quality of the radix semiaquilegiae medicinal material can be comprehensively reflected, and the method is accurate and reliable, simple and convenient to operate and good in reproducibility.
A UPLC characteristic spectrum establishing method of radix semiaquilegiae medicinal materials comprises the following steps:
preparation of control solutions: dissolving a geldanolide reference substance, a lithospermum cyanoside reference substance and a magnoflorine reference substance with a solvent to obtain a reference substance solution;
preparation of a test solution: taking semiaquilegia root medicinal material powder, adding a solvent for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a test solution;
and injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph for detection, wherein a mobile phase A adopted by the ultra-high performance liquid chromatograph is methanol, a mobile phase B is a phosphoric acid aqueous solution with the volume fraction of 0.03-0.07%, and the elution mode is gradient elution.
In one embodiment, the gradient elution comprises the following procedure:
the volume percentage of the mobile phase A is increased from 0% to 6% in 0-12 min;
the volume percentage of the mobile phase A is increased from 6% to 80% within 12-60 min.
In one embodiment, the conditions of the ultra high performance liquid chromatograph include:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 255-265 nm; flow rate: 0.2-0.3 mL/min; column temperature: 12 to 18 ℃.
In one embodiment, in the preparation of the test solution, the solvent for extraction is 65-75% methanol aqueous solution by volume fraction, the extraction method is reflux extraction, and the extraction time is 25-35 min.
In one embodiment, the characteristic spectrum comprises 12 common peaks, and the 12 common peaks comprise characteristic peaks of geldanolide, lithospermic cyanidin and magnoflorine.
The invention also provides a method for detecting the content of the index components of the semiaquilegia root medicinal material, which comprises the following steps:
preparation of index component reference solution: taking a geldanolide reference substance and a magnoflorine reference substance, adding a solvent for dissolving, and preparing index reference substance solutions with different concentrations;
preparation of a test solution: taking muskroot-like semiaquilegia root medicinal material powder to be detected, adding a solvent for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a sample solution;
respectively injecting the index component reference substance solutions with different concentrations into an ultra-high performance liquid chromatograph for detection, and constructing a concentration-peak area standard curve of the index component; injecting the test solution into an ultra-high performance liquid chromatograph for detection, and comparing a detection map with a concentration-peak area standard curve of the index component;
the mobile phase A adopted by the ultra-high performance liquid chromatograph is methanol, the mobile phase B is a phosphoric acid aqueous solution with the volume fraction of 0.03-0.07%, and the elution mode is gradient elution.
In one embodiment, the gradient elution comprises the following procedure:
the volume percentage of the mobile phase A is increased from 0% to 6% in 0-12 min;
the volume percentage of the mobile phase A is increased from 6% to 80% within 12-60 min.
In one embodiment, the conditions of the ultra high performance liquid chromatograph include:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 255-265 nm; flow rate: 0.2-0.3 mL/min; column temperature: 12 to 18 ℃.
In one embodiment, in the preparation of the test solution, the solvent for extraction is 65-75% methanol aqueous solution by volume fraction, the extraction method is reflux extraction, and the extraction time is 25-35 min.
The invention also provides a method for synchronously detecting the characteristic spectrum and the index component content of the semiaquilegia root medicinal material, which comprises the following steps:
constructing a characteristic spectrum of the semiaquilegia root medicinal material according to the establishing method;
constructing a concentration-peak area standard curve of the index components of the semiaquilegia root medicinal material according to the index component content detection method;
preparing a sample solution to be tested: taking muskroot-like semiaquilegia root medicinal material powder to be detected, adding a solvent for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a sample solution;
injecting the sample solution to be detected into an ultra-high performance liquid chromatograph for detection, and comparing a detection spectrum with a characteristic spectrum of the semiaquilegia root medicinal material and a concentration-peak area standard curve of index components;
the mobile phase A adopted by the ultra-high performance liquid chromatograph is methanol, the mobile phase B is a phosphoric acid aqueous solution with the volume fraction of 0.03-0.07%, and the elution mode is gradient elution.
In one embodiment, the gradient elution comprises the following procedure:
the volume percentage of the mobile phase A is increased from 0% to 6% in 0-12 min;
the volume percentage of the mobile phase A is increased from 6% to 80% within 12-60 min.
In one embodiment, the conditions of the ultra high performance liquid chromatograph include:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 255-265 nm; flow rate: 0.2-0.3 mL/min; column temperature: 12 to 18 ℃.
In one embodiment, in the preparation of the sample solution to be tested, the solvent for extraction is a methanol aqueous solution with a volume fraction of 65-75%, the extraction method is reflux extraction, and the extraction time is 25-35 min.
Compared with the prior art, the invention has the following beneficial effects:
the establishing method can establish the characteristic spectrum of the muskroot-like semiaquilegia root medicinal material by adopting proper UPLC chromatographic conditions, fills the blank of the prior art for detecting the quality of the muskroot-like semiaquilegia root, contains 12 characteristic peaks in the characteristic spectrum, can comprehensively reflect the quality of the muskroot-like semiaquilegia root medicinal material, is stable and strong in specificity, accurate and reliable, simple and convenient to operate and good in reproducibility, and provides a scientific and new method for controlling the quality of the muskroot-like semiaquilegia root medicinal material.
The index component content detection method provided by the invention can accurately quantify the index components of gelonin and magnoflorine in the semiaquilegia root medicinal material by adopting a proper UPLC chromatographic condition, and has the advantages of reliable result and simplicity and convenience in operation.
Meanwhile, the invention can establish and obtain the characteristic spectrum of the radix semiaquilegiae medicinal material by adopting a proper UPLC chromatographic condition, wherein the characteristic spectrum contains 12 characteristic peaks, and can simultaneously measure the contents of two index components of gelonin and magnoflorine in the radix semiaquilegiae medicinal material, and synchronously realize the detection of the characteristic spectrum and the content of double index components of the radix semiaquilegiae medicinal material, so that the quality information of the radix semiaquilegiae medicinal material can be comprehensively reflected, the quality control and evaluation of the radix semiaquilegiae medicinal material are facilitated, a powerful reference is provided for the improvement of the quality standard of the radix semiaquilegiae medicinal material, and the detection method is simple and convenient to operate, high in precision, high in accuracy, good in stability, good in reproducibility and convenient to popularize and apply.
Drawings
FIG. 1 is a characteristic spectrum of 15 batches of radix semiaquilegiae medicinal materials in one embodiment of the present invention;
FIG. 2 is a characteristic diagram of a semiaquilegia root medicinal material established in an embodiment of the invention (Peak 3: geldanolide; Peak 5: lithospermum cyanoside; Peak 10: magnoflorine);
FIG. 3 is a special investigation map of the above feature map of radix Semiaquilegiae;
FIG. 4 is a special investigation map for measuring index component content of radix Semiaquilegiae;
FIG. 5 is a linear inspection map of index composition of radix semiaquilegiae for columbaria;
FIG. 6 is a linear investigation map of index components of radix semiaquilegiae, magnoflorine.
Detailed Description
The method for establishing the UPLC characteristic spectrum of the semiaquilegia root medicinal material and the detection method thereof are further described in detail with reference to specific examples.
The instruments and reagents used in the examples of the invention were as follows:
1. instrument for measuring the position of a moving object
Ultra-high performance liquid chromatograph: thermo Vanqish UPLC.
An electronic balance: ME203E (METTLER TOLEDO Corp.).
An ultra-pure water machine: MiliQ Direct 8 (Merck Millipore corporation).
A chromatographic column: waters ACQUITY HSS T3(2.1 mm. times.150 mm, 1.8 μm).
2. Reagent
Acetonitrile and phosphoric acid are chromatographically pure, and water is ultrapure water; the other reagents are analytically pure.
Griffonia simplicifolia reference (batch No. 18091205; source: Doppel Biometrics Ltd.; content%: 98.29); the reference product of arnebia cyanhydrin (batch number: 63492-69-3; source: Shanghai Shidande Standard technical service Co., Ltd.; content%: 99.6); magnoflorine reference (batch No. wkq 17063003; source: Sichuan Weickqi Biotech Co., Ltd.; content: 98).
The percentages in the examples of the present invention are all volume percentages unless otherwise specified.
Example 1
The embodiment is a UPLC characteristic spectrum establishing method of a semiaquilegia root medicinal material.
Radix semiaquilegiae medicinal materials: collecting 15 batches of radix Semiaquilegiae medicinal materials from Guangdong, Guangxi, Hunan, Hubei, Chongqing and other production places, wherein the 15 batches of radix Semiaquilegiae medicinal materials meet the regulation of the traditional Chinese pharmacopoeia in 2015.
The establishment method comprises the following steps:
1. chromatographic conditions and System suitability test
Octadecylsilane chemically bonded silica was used as a filler (Waters ACQUITY HSS T3, column length 150mm, inner diameter 2.1mm, particle diameter 1.8 μm); gradient elution was performed as specified in table 1 with methanol as mobile phase a and 0.05% phosphoric acid solution as mobile phase B; flow rate 0.25ml per minute; the column temperature is 15 ℃; the detection wavelength was 260 nm. The number of theoretical plates should not be less than 30000 calculated by the Grignard peak.
TABLE 1 gradient elution Table
2. Preparation of control solutions
Taking appropriate amount of geldanolide reference substance, radix Arnebiae cyanogenic glycoside reference substance, and magnoflorine reference substance, precisely weighing, and adding 70% methanol to obtain solution containing 40 μ g per 1ml as reference substance solution.
3. Preparation of test solution
Taking radix semiaquilegiae as a medicinal material, preparing into powder (sieving by a No. two sieve), taking about 1.0g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, weighing, heating and refluxing for 30 minutes, cooling, weighing again, complementing the weight loss by 70% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the traditional Chinese medicine.
4. Measurement of
Precisely sucking 1 μ l of each of the reference solution and the sample solution, injecting into a ultra-high performance liquid chromatograph, and measuring.
5. Measurement results
Adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system to carry out common peak matching on the characteristic spectrums of 15 batches of muskroot-like semiaquilegia root medicinal materials, generating a comparison characteristic spectrum, shown in figure 2, determining 12 characteristic peaks in total, carrying out identification on chemical components of the 12 common peaks, confirming that the peak 3 is a gelsolin peak, the peak 5 is a lithospermum cyanogen glycoside peak, the peak 10 is magnoflorine, and the peak 5 is a reference peak S, preparing reference data of the characteristic spectrum analysis method of the muskroot-like semiaquilegia root medicinal materials, selecting the characteristic peaks from the specific characteristic spectrum angle of the muskroot-like semiaquilegia root medicinal materials, calculating the relative retention time and the relative peak area of each characteristic peak, and forming the full appearance of the characteristic spectrum of the muskroot-like semiaquilegia root medicinal materials. Specifically, the arnebia euchroma glycoside peak of peak 5 is taken as a reference peak, the relative retention time and the relative peak area of each remaining characteristic peak are calculated, and the experimental results are shown in tables 2 and 3.
TABLE 215 relative retention times of characteristic spectra of radix Semiaquilegiae
Table 315 relative peak areas of characteristic spectra of radix semiaquilegiae
The result shows that 15 batches of radix semiaquilegiae medicinal materials have 12 common peaks in the characteristic spectrum with good separation degree and stability. Wherein 3 peaks respectively correspond to retention time of corresponding reference substance peaks of the reference substance, the peak corresponding to the reference substance of the lithospermum cyanogen glycoside is S peak, relative retention time of each characteristic peak and the S peak is calculated, the relative retention time is within +/-10% of a specified value, and the specified values are 0.56 (peak 1), 0.66 (peak 2), 0.97 (peak 4), 1.15 (peak 6), 1.40 (peak 7), 1.45 (peak 8), 1.62 (peak 9), 1.96 (peak 11) and 2.43 (peak 12).
5. Methodology validation
(1) Specificity test
Taking the sample solution, blank solvent and reference solution prepared in the same batch, detecting under the liquid chromatography condition, injecting 1 μ l of sample each time, and recording chromatogram. As a result, as shown in FIG. 3, no chromatographic peak was observed at the retention time position corresponding to the characteristic peak in the blank solvent chromatogram, indicating that the extraction solvent had no interference with the measurement of each characteristic peak and had good specificity.
(2) Precision test
And taking the same sample solution, repeatedly injecting samples for 6 times under the liquid chromatography condition, injecting 1 mu l of sample each time, taking the lithospermum cyanogenic glycoside peak as a reference peak S, calculating the relative retention time and the relative peak area of each characteristic peak and the S peak, and calculating the RSD value. The RSD of the relative retention time of each characteristic peak is less than 2%, the RSD of the relative peak area is less than 3%, and the result shows that the precision of the instrument is good.
(3) Repeatability test
Taking the same batch of samples, preparing 6 parts of sample solution according to the sample preparation method, carrying out sample injection measurement under the liquid chromatography condition, carrying out sample injection for 1 mu l each time, taking the lithospermum cyanogenic glycoside peak as a reference peak S, calculating the relative retention time and the relative peak area of each characteristic peak and the S peak, and calculating the RSD value. The RSD of the relative retention time of each characteristic peak is less than 2%, the RSD of the relative peak area is less than 3%, and the result shows that the analysis method is good in repeatability.
(4) Stability test
Taking the test solution, placing at room temperature, detecting under the liquid chromatography conditions for 0 hour, 2 hours, 9 hours, 16 hours, 22 hours and 24 hours respectively, feeding 1 mul of the test solution each time, taking the arnebia cyanhydrin peak as a reference peak S, calculating the relative retention time and the relative peak area of each characteristic peak and the S peak, and calculating the RSD value. The RSD of the relative retention time of each characteristic peak is less than 2%, the RSD of the relative peak area is less than 3%, and the result shows that the stability of the test solution is good within 24 hours.
Example 2
The embodiment is a method for detecting the content of index components of a semiaquilegia root medicinal material.
Radix semiaquilegiae has the effects of clearing away heat and toxic materials, relieving swelling and dissipating stagnation and the like, and is clinically commonly used for carbuncle and furuncle, acute mastitis, scrofula and snake and insect bite. Wherein, the lactones, the nitryl and the cyano have biological activities of antibiosis, anti-inflammation, antioxidation and the like, and the alkaloid components are the effective pharmacological components for resisting tumors. The invention evaluates the semiaquilegia root medicinal material by taking the geldanolide and the magnoflorine as content measurement indexes.
The detection method comprises the following steps:
1. chromatographic conditions and System suitability test
Octadecylsilane chemically bonded silica was used as a filler (Waters ACQUITY HSS T3, column length 150mm, inner diameter 2.1mm, particle diameter 1.8 μm); gradient elution was performed as specified in table 4 using methanol as mobile phase a and 0.05% phosphoric acid solution as mobile phase B; flow rate 0.25ml per minute; the column temperature is 15 ℃; the detection wavelength was 260 nm. The number of theoretical plates should not be less than 30000 calculated by the Grignard peak.
TABLE 4 gradient elution Table
2. Preparation of reference substance solution of index component
Taking a proper amount of a geldanolide reference substance and a magnoflorine reference substance, precisely weighing, and adding 70% methanol to prepare a mixed reference substance solution containing 2 mug, 20 mug, 80 mug, 160 mug, 240 mug and 400 mug of geldanolide and containing 2 mug, 50 mug, 100 mug, 200 mug, 300 mug and 500 mug of magnoflorine in each 1ml, so as to obtain the drug.
3. Preparation of test solution
Taking radix semiaquilegiae as a medicinal material, preparing into powder (sieving by a No. two sieve), taking about 1.0g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, weighing, heating and refluxing for 30 minutes, cooling, weighing again, complementing the weight loss by 70% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the traditional Chinese medicine.
4. Measurement of
Precisely sucking 1 μ l of each of the reference solution and the sample solution, injecting into a ultra-high performance liquid chromatograph, and measuring.
5. Measurement results
And (3) taking the index component reference substance solution, detecting according to the liquid phase condition, feeding 1 mul of sample each time, and establishing a standard curve by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate. The standard curve equation for geldanolide is: y is 0.4186x-0.2392, and the standard curve equation of magnoflorine is as follows: 0.0994 x-0.0553. And calculating the contents of the gelonin and the magnoflorine in 15 batches of radix semiaquilegiae medicinal materials according to the standard curve equation of the index components.
The results of measuring the content of 15 batches of radix semiaquilegiae medicinal materials are shown in the following table 5:
TABLE 515 measurement results of radix Semiaquilegiae crude drug
6. Methodology validation
(1) Specialization inspection
Taking the prepared sample solution, index component reference solution and blank solvent of the same batch, detecting under the liquid chromatography condition, injecting 1 μ l of sample each time, and recording chromatogram. The results are shown in FIG. 4. The result shows that no chromatographic peak exists in the blank solvent chromatogram at the retention time positions corresponding to the guragliflorin and the magnoflorine, and the result shows that the extraction solvent has no interference to the content measurement of the guragliflorin and the magnoflorine and has good specificity.
(2) Investigation of linear relationships
① Grignard lactone Linearity examination
Preparing a reference substance solution stock solution of the glitazone: accurately weighing 5.339mg of glatiramer reference, placing into a 10ml measuring flask, and adding 70% methanol to obtain reference stock solution containing 524.77031 μ g of glatiramer per 1 ml.
Respectively and precisely measuring 0.1ml, 0.5ml, 1ml and 3ml of the reference substance storage solution, respectively placing the reference substance storage solution into 100ml, 10ml, 5ml and 5ml measuring bottles, adding 70% methanol to fix the volume to scale, respectively preparing a series of reference substance solutions containing 0.52477 mu g of geldanamycin, 2.6239 mu g of geldanamycin, 52.4770 mu g of geldanamycin, 104.9541 mu g of geldanamycin and 314.8622 mu g of geldanamycin per 1ml, respectively detecting under the liquid chromatography condition, carrying out linear regression calculation by taking the concentration of the geldanamycin as a horizontal coordinate and the measured peak area as a vertical coordinate, drawing a standard curve, wherein the regression equation of the geldanamycin is as follows: y is 0.4186x-0.2392, the correlation coefficient r is 1.0000, and the linear relation between the injection concentration and the peak area is good when the injection concentration of the geldanolide is in the range of 0.525 mu g/ml to 524.770 mu g/ml. The results are shown in FIG. 5.
② magnoflorine linearity study
Preparation of a magnoflorine reference solution stock solution: precisely weighing the magnoflorine reference substance 5.246mg, placing into a 10ml measuring flask, and adding 70% methanol to prepare a reference substance stock solution containing 514.108 μ g magnoflorine per 1ml magnoflorine.
Respectively and precisely measuring 0.5ml, 1ml, 2ml and 3ml of the reference substance storage solution, respectively placing the reference substance storage solution into 100ml, 10ml, 5ml and 5ml volumetric flasks, adding 70% methanol to fix the volume to the scale, respectively preparing a series of reference substance solutions each containing 2.5705 mu g of magnoflorine, 51.4108 mu g of magnoflorine, 102.8216 mu g of magnoflorine, 205.6432 mu g of magnoflorine and 308.4648 mu g of magnoflorine into each 1ml of the reference substance storage solution, respectively, detecting the reference substance storage solution under the liquid chromatography condition, carrying out linear regression calculation by taking the concentration of the magnoflorine as a horizontal coordinate and the measured peak area as a vertical coordinate, and drawing a standard curve, wherein the regression equation of the magnoflorine is as follows: y is 0.0994x-0.0553, the correlation coefficient r is 0.9997, and the linear relation between the injection concentration and the peak area is good when the injection concentration of the magnoflorine is in the range of 2.571 mu g/ml-514.108 mu g/ml. The results are shown in FIG. 6.
(3) Precision test
Taking the reference substance with the same index component, repeating sample injection for 6 times under the above liquid chromatogram condition, and calculating peak area RSD value with sample injection of 1 μ l each time. The result shows that the RSD value of the peak area of the Grifola frondosa lactone is 0.41 percent, and the RSD value of the peak area of the magnoflorine is 0.62 percent, which indicates that the precision of the instrument is good.
(4) Repeatability test
Taking the same batch of samples, preparing 6 parts of sample solution according to the sample preparation method, and performing sample injection measurement under the condition of the liquid chromatography, wherein the sample injection amount is 1 mu l each time. The result shows that the mean value of the content of the geldanolide is 0.22mg/g, and the RSD value is 2.24 percent; the mean value of the magnoflorine content is 0.61mg/g, and the RSD value is 2.77%, which shows that the analysis method has good repeatability.
(5) Accuracy test
And (3) adopting a reference substance to carry out sample adding recovery rate determination, wherein the ratio of the addition amount of the geldanolide reference substance and the magnoflorine reference substance to the content of the to-be-detected component geldanolide and magnoflorine in the test solution is 0.5: 1,1: 1,1.5: 1, designing 3 groups of experiments, wherein each group is divided into 3 parts in parallel, precisely transferring appropriate amount of Griffonia simplicifolia reference substance storage solution and magnoflorine reference substance storage solution, and drying under a nitrogen blowing instrument. Taking about 0.5g of a same batch of samples with known content, precisely weighing, placing in 3 groups of conical flasks, wherein each group is parallel to 3 parts, preparing a sample solution according to a sample preparation method, carrying out sample injection measurement under the liquid chromatography condition, carrying out sample injection of 1 microliter each time, and respectively calculating the accuracy, see tables 6 and 7, wherein the results show that the recovery rate of the gelsoline content measurement analysis method in the radix semiaquilegiae medicinal material is in the range of 92.26-98.34%, and the recovery rate of the magnoflorine content measurement analysis method is in the range of 89.71-95.81%, and meet the requirement of verification guidance principle of the quality standard analysis method of drugs of 9101 version 2015 in Chinese pharmacopoeia.
TABLE 6 Geliellan content determination accuracy survey of radix Semiaquilegiae medicinal materials
TABLE 7 Limonium wrightii content determination accuracy survey in Limonium wrightii medicinal materials
(6) Stability test
Taking the test solution, placing at room temperature, detecting under the above liquid chromatography conditions at 0 hr, 2 hr, 9 hr, 16 hr, 22 hr and 24 hr, respectively, injecting 1 μ l of sample each time, recording peak area, and calculating RSD value. The result shows that the RSD value of the content of the glitazone is 1.32 percent when the injection is determined within 24 hours; the RSD value of the magnoflorine content is 0.97%, which shows that the stability of the test solution is better within 24 hours.
Meanwhile, the UPLC spectrums of the 15 batches of semiaquilegia root medicinal materials can be respectively compared with the comparison characteristic spectrum of the semiaquilegia root established in the embodiment 1, the characteristic spectrum detection result of the semiaquilegia root medicinal materials in the corresponding batch and the content result of the columnadenlactone and magnoflorine in the index component can be synchronously obtained, and the evaluation of the semiaquilegia root medicinal materials can be comprehensively carried out.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (13)
1. A UPLC characteristic spectrum establishing method of a radix semiaquilegiae medicinal material is characterized by comprising the following steps:
preparation of control solutions: dissolving a geldanolide reference substance, a lithospermum cyanoside reference substance and a magnoflorine reference substance with a solvent to obtain a reference substance solution;
preparation of a test solution: taking semiaquilegia root medicinal material powder, adding a solvent for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a test solution;
and injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph for detection, wherein a mobile phase A adopted by the ultra-high performance liquid chromatograph is methanol, a mobile phase B is a phosphoric acid aqueous solution with the volume fraction of 0.03-0.07%, and the elution mode is gradient elution.
2. The UPLC feature spectrum establishment method of semiaquilegia root medicinal material according to claim 1, wherein the gradient elution comprises the following procedures:
the volume percentage of the mobile phase A is increased from 0% to 6% in 0-12 min;
the volume percentage of the mobile phase A is increased from 6% to 80% within 12-60 min.
3. The method for establishing the UPLC feature map of the semiaquilegia root medicinal material according to claim 1, wherein the conditions of the ultra-high performance liquid chromatograph include:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 255-265 nm; flow rate: 0.2-0.3 mL/min; column temperature: 12 to 18 ℃.
4. The method for establishing the UPLC feature map of the semiaquilegia root medicinal material according to claim 1, wherein in the preparation of the test solution, the solvent for extraction is a methanol aqueous solution with a volume fraction of 65-75%, the extraction method is reflux extraction, and the extraction time is 25-35 min.
5. The method for establishing the UPLC characteristic spectrum of the semiaquilegia root medicinal material according to any one of claims 1 to 4, wherein the characteristic spectrum comprises 12 common peaks, and the 12 common peaks comprise characteristic peaks of geldanolide, lithospermic cyanidin and magnoflorine.
6. A method for detecting the content of index components of a semiaquilegia root medicinal material is characterized by comprising the following steps:
preparation of index component reference solution: taking a geldanolide reference substance and a magnoflorine reference substance, adding a solvent for dissolving, and preparing index reference substance solutions with different concentrations;
preparation of a test solution: taking muskroot-like semiaquilegia root medicinal material powder to be detected, adding a solvent for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a sample solution;
respectively injecting the index component reference substance solutions with different concentrations into an ultra-high performance liquid chromatograph for detection, and constructing a concentration-peak area standard curve of the index component; injecting the test solution into an ultra-high performance liquid chromatograph for detection, and comparing a detection map with a concentration-peak area standard curve of the index component;
the mobile phase A adopted by the ultra-high performance liquid chromatograph is methanol, the mobile phase B is a phosphoric acid aqueous solution with the volume fraction of 0.03-0.07%, and the elution mode is gradient elution.
7. The method for detecting the content of the index components of the semiaquilegia root medicinal material according to claim 6, wherein the gradient elution comprises the following steps:
the volume percentage of the mobile phase A is increased from 0% to 6% in 0-12 min;
the volume percentage of the mobile phase A is increased from 6% to 80% within 12-60 min.
8. The method for detecting the content of the index components in the semiaquilegia root crude drug according to claim 6, wherein the conditions of the ultra-high performance liquid chromatograph include:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 255-265 nm; flow rate: 0.2-0.3 mL/min; column temperature: 12 to 18 ℃.
9. The method for detecting the content of the index components in the semiaquilegia root crude drug according to any one of claims 6 to 8, wherein in the preparation of the test solution, the solvent for extraction is a methanol aqueous solution with a volume fraction of 65 to 75%, the extraction method is reflux extraction, and the extraction time is 25 to 35 min.
10. A method for synchronously detecting a characteristic spectrum and index component content of a semiaquilegia root medicinal material is characterized by comprising the following steps:
constructing a characteristic spectrum of the radix semiaquilegiae medicinal material according to the establishing method of any one of claims 1 to 5;
constructing a concentration-peak area standard curve of the index components of the semiaquilegia root medicinal material according to the index component content detection method of any one of claims 6 to 8;
preparing a sample solution to be tested: taking muskroot-like semiaquilegia root medicinal material powder to be detected, adding a solvent for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a sample solution;
injecting the sample solution to be detected into an ultra-high performance liquid chromatograph for detection, and comparing a detection spectrum with a characteristic spectrum of the semiaquilegia root medicinal material and a concentration-peak area standard curve of index components;
the mobile phase A adopted by the ultra-high performance liquid chromatograph is methanol, the mobile phase B is a phosphoric acid aqueous solution with the volume fraction of 0.03-0.07%, and the elution mode is gradient elution.
11. The method for synchronously detecting the characteristic spectrum and the index component content of the semiaquilegia root medicinal material according to claim 10, wherein the gradient elution comprises the following procedures:
the volume percentage of the mobile phase A is increased from 0% to 6% in 0-12 min;
the volume percentage of the mobile phase A is increased from 6% to 80% within 12-60 min.
12. The method for synchronously detecting the characteristic spectrum and the index component content of the semiaquilegia root medicinal material according to claim 10, wherein the conditions of the ultra-high performance liquid chromatograph include:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 255-265 nm; flow rate: 0.2-0.3 mL/min; column temperature: 12 to 18 ℃.
13. The method for synchronously detecting the characteristic spectrum and the index component content of the semiaquilegia root medicinal material according to any one of claims 10 to 12, wherein in the preparation of the sample solution to be detected, the extracted solvent is a methanol aqueous solution with a volume fraction of 65-75%, the extraction method is reflux extraction, and the extraction time is 25-35 min.
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