CN114720595A - Method for determining content of rosmarinic acid in Xinhuang tablets - Google Patents
Method for determining content of rosmarinic acid in Xinhuang tablets Download PDFInfo
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- DOUMFZQKYFQNTF-WUTVXBCWSA-N (R)-rosmarinic acid Chemical compound C([C@H](C(=O)O)OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-WUTVXBCWSA-N 0.000 title claims abstract description 150
- 239000009291 xinhuang Substances 0.000 title claims abstract description 79
- ZZAFFYPNLYCDEP-HNNXBMFYSA-N Rosmarinsaeure Natural products OC(=O)[C@H](Cc1cccc(O)c1O)OC(=O)C=Cc2ccc(O)c(O)c2 ZZAFFYPNLYCDEP-HNNXBMFYSA-N 0.000 title claims abstract description 75
- DOUMFZQKYFQNTF-MRXNPFEDSA-N rosemarinic acid Natural products C([C@H](C(=O)O)OC(=O)C=CC=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-MRXNPFEDSA-N 0.000 title claims abstract description 75
- TVHVQJFBWRLYOD-UHFFFAOYSA-N rosmarinic acid Natural products OC(=O)C(Cc1ccc(O)c(O)c1)OC(=Cc2ccc(O)c(O)c2)C=O TVHVQJFBWRLYOD-UHFFFAOYSA-N 0.000 title claims abstract description 75
- 238000000034 method Methods 0.000 title claims abstract description 45
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 15
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 7
- 238000001514 detection method Methods 0.000 claims abstract description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 51
- 239000000243 solution Substances 0.000 claims description 29
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- 239000000706 filtrate Substances 0.000 claims description 7
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- 238000004587 chromatography analysis Methods 0.000 description 2
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- CGIGDMFJXJATDK-UHFFFAOYSA-N indomethacin Chemical compound CC1=C(CC(O)=O)C2=CC(OC)=CC=C2N1C(=O)C1=CC=C(Cl)C=C1 CGIGDMFJXJATDK-UHFFFAOYSA-N 0.000 description 2
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- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 description 1
- 239000004380 Cholic acid Substances 0.000 description 1
- DGABKXLVXPYZII-UHFFFAOYSA-N Hyodeoxycholic acid Natural products C1C(O)C2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 DGABKXLVXPYZII-UHFFFAOYSA-N 0.000 description 1
- HOEVRHHMDJKUMZ-UHFFFAOYSA-N Isofraxidin Chemical compound C1=CC(=O)OC2=C1C=C(OC)C(O)=C2OC HOEVRHHMDJKUMZ-UHFFFAOYSA-N 0.000 description 1
- ANCHXLMTFNOVDK-UHFFFAOYSA-N Isofraxidin Natural products COC1=C(O)C(OC)=CC2=C1OC=CC2=O ANCHXLMTFNOVDK-UHFFFAOYSA-N 0.000 description 1
- 240000004274 Sarcandra glabra Species 0.000 description 1
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- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 1
- 229960002471 cholic acid Drugs 0.000 description 1
- 235000019416 cholic acid Nutrition 0.000 description 1
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- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- DGABKXLVXPYZII-SIBKNCMHSA-N hyodeoxycholic acid Chemical compound C([C@H]1[C@@H](O)C2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 DGABKXLVXPYZII-SIBKNCMHSA-N 0.000 description 1
- 229960000905 indomethacin Drugs 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
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- 239000007924 injection Substances 0.000 description 1
- 238000001474 liquid chromatography-evaporative light scattering detection Methods 0.000 description 1
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- 238000004809 thin layer chromatography Methods 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 238000002137 ultrasound extraction Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention provides a method for measuring the content of rosmarinic acid in Xinhuang tablets. The method is carried out by adopting a high performance liquid chromatography, and the chromatographic conditions are as follows: a chromatographic column: welch AQ-C18, 4.6X 250mm,5 μm, mobile phase: acetonitrile: 0.1% aqueous phosphoric acid solution ═ 19:81, flow rate: 0.8mL/min, detection wavelength: 342nm, column temperature: at 30 ℃. The method is reliable, simple and convenient, has strong specificity and good repeatability, stability and precision, can be used as a method for measuring the content of Xinhuang tablets, provides technical support for measuring the content of rosmarinic acid in the Xinhuang tablets, and is used for improving the product quality of the Xinhuang tablets.
Description
Technical Field
The invention belongs to the field of detection of traditional Chinese medicines, and particularly relates to a method for determining the content of rosmarinic acid in Xinhuang tablets.
Background
Xinhuang tablet of Xiamen Chinese medicine factory, ltd, has effects of clearing heat and removing toxicity, promoting blood circulation by removing blood stasis, and relieving swelling and pain. With the annual increase of the sales of Xinhuang tablets, the improvement of the product quality standard has a vital significance on the stability and the sustainable development of the product quality.
In recent years, the research on chemical components and quality control of the Xinhuang tablets has been advanced. The ingredients such as cholic acid and hyodeoxycholic acid in Xinhuang tablets are identified by thin-layer chromatography in 2020 edition pharmacopoeia of China, and the content of isofraxidin and indomethacin is measured by using an HPLC method (Guanbin. high performance liquid chromatography-evaporative light scattering detection method (HPLC-ELSD) fingerprint spectrum of Xinhuang tablets is established [ J ]. Haixian pharmacy 2020,32(09): 72-75.). However, no method for measuring the content of rosmarinic acid (rosmarinic acid) in Xinhuang tablets exists at present.
Rosmarinic acid is an effective component in Xinhuang tablets, and the content of rosmarinic acid has important influence on the quality of the Xinhuang tablets. It is a polyphenolic acid with the chemical formula C18H16O8The chemical structure is as follows:
the establishment of the determination method of the rosmarinic acid content in the Xinhuang tablets is a key item for improving the quality of the Xinhuang tablets, and has important significance for the stability and the sustainable development of the quality of medicines.
Disclosure of Invention
The invention aims to provide an accurate and reliable determination method for the content of rosmarinic acid in Xinhuang tablets, provide technical support for determination of the content of rosmarinic acid in the Xinhuang tablets, and effectively control the quality of the Xinhuang tablets.
Therefore, the invention provides a method for measuring the content of rosmarinic acid in Xinhuang tablets, which is carried out by adopting high performance liquid chromatography, wherein the chromatographic conditions are as follows:
a chromatographic column: welch AQ-C18, 4.6X 250mm,5 μm,
mobile phase: acetonitrile: 0.1% aqueous phosphoric acid solution ═ 19:81
Flow rate: 0.8mL/min of the water-soluble polymer,
detection wavelength: at the wavelength of 342nm, the particle size of the nano-particle,
column temperature: at 30 ℃.
In a specific embodiment, the assay method of the invention comprises the steps of:
s1, preparation of control solutions: preparing 1ml of solution containing 10 mug of rosmarinic acid reference substance by using methanol;
s2, preparation of a test solution: collecting 0.5g XINHUANG tablet powder; precisely adding 25ml of methanol; sealing and weighing; heating and refluxing; cooling, weighing, and supplementing the lost weight with methanol; shaking, filtering, and collecting the filtrate;
s3, the control solution prepared in S1 and the sample solution prepared in S2 were injected into a liquid chromatograph under the following chromatographic conditions:
the column was Welch AQ-C18 (4.6X 250mm,5 μm), and the mobile phase was acetonitrile: 0.1% aqueous phosphoric acid solution ═ 19:81, the flow rate is 0.8mL/min, the sample amount is 5 mul, the detection wavelength is 342nm, the column temperature is 30 ℃,
and S4, calculating the content of rosmarinic acid in the Xinhuang tablets according to the measurement result. In the method S1 of the present invention, the rosmarinic acid control can be a commercially available product, for example, a product of the chinese institute for food and drug assay (e.g., 11871-202007).
Preferably, in the method S2, the powder of the xinhuang pian is a powder sieved by the fourth pharmacopoeia. The pharmacopoeia sieve refers to a standard sieve specified by Chinese pharmacopoeia.
Preferably, in the process S2 according to the invention, the heating reflux time is from 0.5 to 1 hour, preferably 0.5 hour.
Preferably, in the method S3 of the present invention, Agilent1260 high performance chromatograph is used.
In the method S4 of the present invention, the calculation of the rosmarinic acid content in the xinhuang pian is not particularly limited, and may be performed according to a conventional method in the art. For example, a standard curve can be prepared according to the measurement result of the reference solution, and then the content of rosmarinic acid in the test solution and the Xinhuang tablets can be calculated according to the standard curve. In one embodiment, a linear expression of the peak area and the sample amount of the rosmarinic acid reference substance is obtained according to the measurement result of the reference substance solution, then the content of rosmarinic acid in the test substance solution is calculated by the linear expression, and then the content of rosmarinic acid in the Xinhuang tablets is converted.
In the invention, qualitative and quantitative analysis is carried out on the rosmarinic acid in the Xinhuang tablets, the specificity, linearity, precision, accuracy, range and the like of the rosmarinic acid component in the Xinhuang tablets are verified by a content determination methodology, and the extraction mode, extraction time, flow and the like are researched, so that the method is used as an experimental research basis for establishing a method for determining the content of the rosmarinic acid in the Xinhuang tablets, and finally the method for determining the content of the rosmarinic acid in the Xinhuang tablets is established.
The present invention has been described in detail hereinabove, but the above embodiments are merely illustrative in nature and are not intended to limit the present invention. Furthermore, there is no intention to be bound by any theory presented in the preceding prior art or the summary or the following examples.
Further, the present invention may be variously modified and have various forms. It will be apparent to those skilled in the art in view of this disclosure that many modifications can be made to the method without departing from the spirit and scope of the invention. Accordingly, those skilled in the art will appreciate that the invention includes all modifications, equivalents, and alternatives falling within the spirit and technical scope of the invention.
Unless expressly stated otherwise, a numerical range throughout this specification includes any sub-range therein and any numerical value incremented by the smallest sub-unit within a given value. Unless expressly stated otherwise, numerical values throughout this specification represent approximate measures or limitations to the extent that such deviations from the given values, as well as embodiments having approximately the stated values and having the exact values stated, are included. Other than in the operating examples provided at the end of the detailed description, all numbers expressing quantities or conditions of parameters (e.g., quantities or conditions) used in the specification (including the appended claims) are to be understood as being modified in all instances by the term "about" whether or not "about" actually appears before the number. "about" means that the numerical value so stated is allowed to be somewhat imprecise (with some approach to exactness in that value; about or reasonably close to that value; approximately). If the imprecision provided by "about" is not otherwise understood in the art with this ordinary meaning, then "about" as used herein indicates at least the variations that can be produced by the ordinary methods of measuring and using these parameters. For example, "about" can include variations of less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, or less than or equal to 0.5%.
Drawings
Fig. 1 is an HPLC chromatogram of a negative control sample, a rosmarinic acid control, and xinhuang tablets in the systematic adaptability and specificity test in the examples.
FIG. 2 is a standard rosmarinic acid curve of the linear relationship test in the examples.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Examples
1. Materials and methods
1.1 reagents and drugs
TABLE 1 reagents and drugs
1.2 Main instruments
TABLE 2 Main instruments
2 Experimental methods
2.1 high Performance liquid chromatography conditions
Unless otherwise stated, the high performance liquid chromatography conditions are as shown in table 3 below.
TABLE 3 high Performance liquid chromatography conditions
2.2 preparation of the solution
Unless otherwise stated, solutions were formulated as follows.
2.2.1 preparation of control solutions:
taking a proper amount of rosmarinic acid reference substance; precisely weighing; methanol was added to make 1ml of a solution containing 10. mu.g.
2.2.2 preparation of test solutions:
collecting 0.5g XINHUANG tablet powder (sieve IV); precisely adding 25ml of methanol; sealing and weighing; heating and refluxing for 0.5 hour; cooling, weighing, and supplementing the weight loss with methanol; shaking, filtering, and collecting the filtrate.
2.2.3 preparation of negative sample solutions:
except for not using the sarcandra glabra (containing rosmarinic acid), the Xinhuang tablet is prepared according to the Xinhuang tablet prescription, and negative sample Xinhuang tablet powder lacking the rosmarinic acid is obtained.
Taking 0.5g negative sample Xinhuang pian powder (No. four sieve); precisely adding 25ml of methanol; sealing and weighing; heating and refluxing for 0.5 hour; cooling, weighing, and supplementing the lost weight with methanol; shaking, filtering, and collecting the filtrate.
3. Examination of extraction conditions
The experiment researches the extraction time, the extraction mode and whether the rosmarinic acid is extracted by soaking overnight.
3.1 Effect of extraction time on the extraction Effect of Rosmarinic acid
Taking 0.5g Xinhuang tablet powder (four sieves) in two parts; precisely adding 25ml of methanol; sealing and weighing; heating and refluxing for 0.5 hour and 1 hour respectively; cooling, weighing, and supplementing the lost weight with methanol; shaking, filtering, and collecting the filtrate.
The results show that the content of the rosmarinic acid is not obviously different, the two extraction times of 30min and 60min are not obviously different from the extraction effect of the rosmarinic acid, and the 30min heating reflux is simpler and more convenient.
3.2 Effect of extraction mode on the extraction Effect of Rosmarinic acid
Taking 0.5g Xinhuang tablet powder (four sieves) in two parts; precisely adding 25ml of methanol; sealing and weighing; respectively heating and refluxing for 0.5 hour and performing ultrasonic extraction for 0.5 hour; cooling, weighing, and supplementing the lost weight with methanol; shaking, filtering, and collecting the filtrate.
The results show that the content of the rosmarinic acid extracted by heating and refluxing is 0.3345%, and the content of the rosmarinic acid extracted by ultrasonic is 0.3201%, which shows that the content of the rosmarinic acid extracted by heating and refluxing in the Xinhuang tablets is slightly higher than that of the rosmarinic acid extracted by ultrasonic.
3.3 Effect of overnight soaking on the extraction Effect of Rosmarinic acid
Taking 0.5g Xinhuang tablet powder (four sieves) in two parts; precisely adding 25ml of methanol; sealing and weighing; one part is soaked overnight, heated and refluxed for 0.5 hour, and the other part is directly heated and refluxed for 0.5 hour; cooling, weighing, and supplementing the lost weight with methanol; shaking, filtering, and collecting the filtrate.
The results show that the contents of the two are almost consistent, which indicates that whether the rosmarinic acid is soaked overnight has no obvious difference on the extraction effect of the rosmarinic acid.
In conclusion, the extraction conditions of no need of soaking overnight, heating reflux extraction and extraction time of 30min are selected.
3.4. Examination of stability of test solution
The Xinhuang pian solution is prepared according to 2.2.2, and is injected into a liquid chromatograph for determination according to 2.1 chromatographic conditions after being placed for 2 hours, 8 hours, 16 hours, 20 hours and 24 hours.
As a result, as shown in table 4, the peak area average value of rosmarinic acid in the xinhuang pian was 111.045, RSD was 1.14%, and RSD was < 2.00% within 24 hours, demonstrating that the stability of rosmarinic acid in the xinhuang pian was good within 24 hours.
TABLE 4 stability test results
4. Selection of chromatographic conditions
4.1 selection of the Mobile phase
Acetonitrile-0.1% phosphoric acid aqueous solution was prepared at mobile phase ratios of (15:85), (19:81), (20:80), (23:77) and (25:75), respectively, and experiments were performed.
The results showed that the degrees of separation were 172, 1.98, 1.82, 1.69, 1.57 respectively,
the results show that the separation of the mobile phase (19:81) is the best, the symmetry factor is the best, the results are stable and the reproducibility is good.
4.2 selection of chromatography columns
Except that three different brands of columns were selected: experiments were carried out with chromatographic conditions of 2.1, except for medium spectrum Red AQ-C18, WelchAQ-C18, Agilent TC-C18.
The results show that the separation degree of Welch AQ-C18 is slightly better, but the content results of the three methods have no obvious difference, and the results are shown in Table 5, so that a Welch AQ-C18 chromatographic column can be preferentially selected.
4.3 selection of column temperature
Experiments were carried out under chromatography conditions of 2.1, except that the column temperatures were 25 ℃, 30 ℃ and 35 ℃, respectively.
The results show that the column temperature at 30 ℃ is slightly better than the other column temperatures, but the results for the three methods are not significantly different, see table 5, so 30 ℃ can be selected as the preferred method.
4.4 selection of flow Rate
Experiments were performed under chromatographic conditions of 2.1, except that three different flow rates were selected of 0.6ml/min, 0.8ml/min, 1.0ml/min, respectively.
The results show that the degree of separation at a flow rate of 0.8ml/min is slightly better than that of the other methods, but the results of the contents of the three methods are not significantly different, and the results are shown in Table 5, so that a flow rate of 0.8ml/min can be selected preferentially.
Table 5 column conditions selection results
As shown in table 5, when the same xinhuang pian test sample is used for measuring rosmarinic acid under different column temperatures, RSD is 0.52%; rosmarinic acid was measured at different flow rates with an RSD of 0.88%; the rosmarinic acid is measured by replacing different chromatographic columns, and the RSD is 0.80 percent and is less than 2.00 percent.
The result shows that the determination result of the rosmarinic acid is not obviously influenced by slightly changing the chromatographic column condition.
4.5 Final defined chromatographic conditions
Combining the above experimental results, the finally determined chromatographic conditions were as follows: a chromatographic column: welch AQ-C18 (4.6X 250mm,5 μm); the mobile phase is acetonitrile-0.1% phosphoric acid (19: 81); the detection wavelength is 342 nm; the flow rate is 0.8 ml/min; the sample amount is 5 mul; the column temperature was 30 ℃.
5. Methodology validation
5.1 System Adaptation Observation and specificity test
Preparing a rosmarinic acid reference solution, a Xinhuang tablet test solution and a Xinhuang tablet negative sample solution according to 2.2, and injecting into a liquid chromatograph under 2.1 chromatographic conditions for determination.
The result is shown in fig. 1, at the same retention time position of the chromatogram peak of the rosmarinic acid reference substance, the Xinhuang tablet negative sample has no chromatogram peak, the Xinhuang tablet has corresponding chromatogram peak, and the separation degree between chromatogram peaks is good. The method has good system applicability and specificity.
5.2 Linear relationship test
Preparing rosmarinic acid reference solution according to 2.2.1, precisely sucking 0 μ l, 2 μ l, 5 μ l, 8 μ l and 10 μ l respectively according to 2.1 chromatographic conditions, and injecting into liquid chromatograph for determination.
As shown in fig. 2, the linear expression of the peak area and the sample amount of the rosmarinic acid control is: 3062.9x +0.9099, R20.999. The rosmarinic acid is in the range of 0.0921mg, and the rosmarinic acid peak area has a better linear relation with the sample amount.
5.3 precision test
Preparing Xinhuang tablet solution according to 2.2.2, and continuously injecting 5-needle sample according to 2.1 chromatographic conditions to be measured by a liquid chromatograph.
As shown in table 6, the peak area of rosmarinic acid in 5 th bolus injection of xinhuang pian was 110.937, RSD was 1.04%, and RSD was < 2.00%, which proved that the precision of the instrument was good.
TABLE 6 results of precision test
5.4 repeatability test
Preparing 6 parts of the Xinhuang tablet solution of the same batch according to 2.2.2, and injecting the solution into a liquid chromatograph for determination according to 2.1 chromatographic conditions.
As shown in table 7, the average value of the peak area of rosmarinic acid in 6 Xinhuang tablets of the same batch is 111.063, the RSD is 1.49%, and the RSD is less than 2.00%, which proves that the method has good repeatability.
TABLE 7 results of the repeatability tests
5.5 sample application recovery test
Preparing Xinhuang tablet solution with known rosmarinic acid content according to 2.2.2, adding rosmarinic acid reference substance solution with known content of 0.8 times, 1.0 times and 1.2 times of the sample amount, and injecting into liquid chromatograph for determination according to 2.1 chromatographic condition.
As shown in table 8, the average recovery rate of rosmarinic acid in the xinhuang pian was 103%, RSD was 1.56%, and RSD was < 2.00%, demonstrating the good recovery rate of the method.
TABLE 8 recovery test results
5.6 repeatability Range examination
Weighing three parts of Xinhuang tablet powder 0.25g (for preparing a low-concentration test sample) and Xinhuang tablet powder 0.95g (for preparing a high-concentration test sample), preparing Xinhuang tablet solution according to 2.2.2, and injecting the Xinhuang tablet solution into a liquid chromatograph for determination according to 2.1 chromatographic conditions.
As shown in Table 9-1, the average rosmarinic acid content of the Xinhuang tablets (0.25 g at low concentration) and the average rosmarinic acid content of the Xinhuang tablets (0.95 g at high concentration) are 0.03341, RSD is 0.66% and RSD is less than 2.00%, thus proving that the method has good repeatability at low and high concentrations.
TABLE 9-1 repeatability Range examination (Low concentration samples Nos. 1-3 and high concentration samples Nos. 4-6)
5.7 Range of accuracy survey
Weighing 0.2g of Xinhuang tablets with known rosmarinic acid content and 0.8g of Xinhuang tablets, preparing Xinhuang tablet solutions according to 2.2.2, respectively adding about 1 time of the rosmarinic acid reference substance solution with known content, and injecting the Xinhuang tablets into a liquid chromatograph for determination according to 2.1 chromatographic conditions.
As shown in table 9-2, the average recovery rates of rosmarinic acid in 0.2g and 0.8g of xinhuang pian were 104.7%, RSD was 1.24%, and RSD was < 2.00%, demonstrating that the method has good accuracy at low and high concentrations.
TABLE 9-2 accuracy Range examination (Low concentration samples Nos. 1-3 and high concentration samples Nos. 4-6)
5.8 sample assay
Taking ten samples of Xinhuang pian according to 2.2.2 to prepare Xinhuang pian solution, and continuously injecting two samples into a liquid chromatograph for determination according to 2.1 chromatographic conditions.
As shown in Table 10, the content determination results of ten Xinhuang pian samples (210201 and 210210):
TABLE 10 determination of sample content
The comprehensive methodological verification results show that the method for determining the content of the rosmarinic acid established by the invention is reliable, simple and convenient, has strong specificity, good repeatability, stability and precision, can be used as a method for determining the content of the rosmarinic acid in the Xinhuang tablets, provides technical support for determining the content of the rosmarinic acid in the Xinhuang tablets, and is used for improving the product quality of the Xinhuang tablets.
Claims (8)
1. The method for measuring the content of rosmarinic acid in Xinhuang tablets is carried out by adopting high performance liquid chromatography, and the chromatographic conditions are as follows:
a chromatographic column: welch AQ-C18, 4.6X 250mm,5 μm,
mobile phase: acetonitrile: 0.1% aqueous phosphoric acid solution ═ 19:81,
flow rate: 0.8mL/min of the water-soluble polymer,
detection wavelength: at the wavelength of 342nm, the particle size of the nano-particle,
column temperature: at 30 ℃.
2. The method for determining the content of rosmarinic acid in Xinhuang pian as claimed in claim 1, wherein the method comprises the following steps:
s1, preparation of control solutions: preparing 1ml of solution containing 10 mug of rosmarinic acid reference substance by using methanol;
s2, preparation of a test solution: collecting 0.5g XINHUANG tablet powder; precisely adding 25ml of methanol; sealing and weighing; heating and refluxing; cooling, weighing, and supplementing the lost weight with methanol; shaking, filtering, and collecting the filtrate;
s3, the control solution prepared in S1 and the sample solution prepared in S2 were injected into a liquid chromatograph under the following chromatographic conditions:
the column was Welch AQ-C18 (4.6X 250mm,5 μm), and the mobile phase was acetonitrile: 0.1% aqueous phosphoric acid solution ═ 19:81, the flow rate is 0.8mL/min, the sample amount is 5 mul, the detection wavelength is 342nm, the column temperature is 30 ℃,
and S4, calculating the content of rosmarinic acid in the Xinhuang tablets according to the measurement result.
3. The assay method according to claim 2, wherein the Xinhuang pian powder is powder screened by the fourth pharmacopoeia in S2.
4. The method according to claim 2, wherein the heating reflux time in S2 is 0.5 to 1 hour.
5. The method according to claim 4, wherein the heating reflux time is 0.5 hour.
6. The method according to claim 2, wherein in S3 Agilent1260 high performance chromatograph is used.
7. The assay method according to claim 2, wherein in S4, a calibration curve is prepared based on the assay result of the control solution, and then the content of rosmarinic acid in the test solution and the xinhuang pian is calculated based on the calibration curve.
8. The assay method according to claim 7, wherein a linear expression of the peak area and the sample amount of the rosmarinic acid control is obtained from the assay result of the control solution, and then the content of rosmarinic acid in the test solution is calculated from the linear expression, and further converted into the content of rosmarinic acid in the Xinhuang tablets.
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