CN117825600B - Method for detecting content of each mass component of salvia miltiorrhiza injection intermediate based on one-measurement-multiple-evaluation method - Google Patents
Method for detecting content of each mass component of salvia miltiorrhiza injection intermediate based on one-measurement-multiple-evaluation method Download PDFInfo
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- 238000002347 injection Methods 0.000 title claims abstract description 49
- 239000007924 injection Substances 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000011156 evaluation Methods 0.000 title claims abstract description 18
- 235000011135 Salvia miltiorrhiza Nutrition 0.000 title claims description 14
- 241000304195 Salvia miltiorrhiza Species 0.000 title 1
- IBGBGRVKPALMCQ-UHFFFAOYSA-N 3,4-dihydroxybenzaldehyde Chemical compound OC1=CC=C(C=O)C=C1O IBGBGRVKPALMCQ-UHFFFAOYSA-N 0.000 claims abstract description 134
- 229960003371 protocatechualdehyde Drugs 0.000 claims abstract description 67
- 239000013558 reference substance Substances 0.000 claims abstract description 48
- 239000000243 solution Substances 0.000 claims abstract description 45
- 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 claims abstract description 26
- 238000001514 detection method Methods 0.000 claims abstract description 18
- SNKFFCBZYFGCQN-UHFFFAOYSA-N 2-[3-[3-[1-carboxy-2-(3,4-dihydroxyphenyl)ethoxy]carbonyl-2-(3,4-dihydroxyphenyl)-7-hydroxy-2,3-dihydro-1-benzofuran-4-yl]prop-2-enoyloxy]-3-(3,4-dihydroxyphenyl)propanoic acid Chemical compound C=1C=C(O)C=2OC(C=3C=C(O)C(O)=CC=3)C(C(=O)OC(CC=3C=C(O)C(O)=CC=3)C(O)=O)C=2C=1C=CC(=O)OC(C(=O)O)CC1=CC=C(O)C(O)=C1 SNKFFCBZYFGCQN-UHFFFAOYSA-N 0.000 claims abstract description 13
- SNKFFCBZYFGCQN-VWUOOIFGSA-N Lithospermic acid B Natural products C([C@H](C(=O)O)OC(=O)\C=C\C=1C=2[C@H](C(=O)O[C@H](CC=3C=C(O)C(O)=CC=3)C(O)=O)[C@H](OC=2C(O)=CC=1)C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 SNKFFCBZYFGCQN-VWUOOIFGSA-N 0.000 claims abstract description 13
- 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 claims abstract description 13
- 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 claims abstract description 13
- 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 claims abstract description 13
- STCJJTBMWHMRCD-UHFFFAOYSA-N salvianolic acid B Natural products OC(=O)C(Cc1ccc(O)c(O)c1)OC(=O)C=Cc2cc(O)c(O)c3OC(C(C(=O)OC(Cc4ccc(O)c(O)c4)C(=O)O)c23)c5ccc(O)c(O)c5 STCJJTBMWHMRCD-UHFFFAOYSA-N 0.000 claims abstract description 13
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims abstract description 12
- 229910052708 sodium Inorganic materials 0.000 claims abstract description 12
- 239000011734 sodium Substances 0.000 claims abstract description 12
- 240000007164 Salvia officinalis Species 0.000 claims abstract description 11
- 235000005412 red sage Nutrition 0.000 claims abstract description 11
- PAFLSMZLRSPALU-UHFFFAOYSA-N Salvianic acid A Natural products OC(=O)C(O)CC1=CC=C(O)C(O)=C1 PAFLSMZLRSPALU-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000000523 sample Substances 0.000 claims description 56
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 45
- 239000012488 sample solution Substances 0.000 claims description 19
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- 238000012937 correction Methods 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 13
- 244000132619 red sage Species 0.000 claims description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 229960000583 acetic acid Drugs 0.000 claims description 9
- 239000012362 glacial acetic acid Substances 0.000 claims description 9
- PAFLSMZLRSPALU-MRVPVSSYSA-N (2R)-3-(3,4-dihydroxyphenyl)lactic acid Chemical compound OC(=O)[C@H](O)CC1=CC=C(O)C(O)=C1 PAFLSMZLRSPALU-MRVPVSSYSA-N 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 7
- 239000012088 reference solution Substances 0.000 claims description 7
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 238000011049 filling Methods 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- 229930194268 Salvianic acid Natural products 0.000 abstract 1
- 238000004904 shortening Methods 0.000 abstract 1
- 239000000543 intermediate Substances 0.000 description 26
- 238000003908 quality control method Methods 0.000 description 10
- 238000005259 measurement Methods 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000010812 external standard method Methods 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- YMGFTDKNIWPMGF-QHCPKHFHSA-N Salvianolic acid A Natural products OC(=O)[C@H](Cc1ccc(O)c(O)c1)OC(=O)C=Cc2ccc(O)c(O)c2C=Cc3ccc(O)c(O)c3 YMGFTDKNIWPMGF-QHCPKHFHSA-N 0.000 description 3
- YMGFTDKNIWPMGF-UCPJVGPRSA-N Salvianolic acid A Chemical compound C([C@H](C(=O)O)OC(=O)\C=C\C=1C(=C(O)C(O)=CC=1)\C=C\C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 YMGFTDKNIWPMGF-UCPJVGPRSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
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- 238000004811 liquid chromatography Methods 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
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- 230000000747 cardiac effect Effects 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
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- 238000010828 elution Methods 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
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- 150000002632 lipids Chemical class 0.000 description 1
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- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- ZMMKVDBZTXUHFO-DDWIOCJRSA-M sodium;(2r)-3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate Chemical compound [Na+].[O-]C(=O)[C@H](O)CC1=CC=C(O)C(O)=C1 ZMMKVDBZTXUHFO-DDWIOCJRSA-M 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 208000019553 vascular disease Diseases 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a method for detecting the content of each mass component of an intermediate of a red sage root injection based on a one-test-multiple-evaluation method, which only uses a protocatechuic aldehyde reference substance in the detection process, and simultaneously detects the content of sodium salvianic acid, protocatechuic aldehyde, rosmarinic acid and salvianolic acid B of the intermediate to judge whether the intermediate is qualified, thereby reducing the use amount of the reference substance, reducing the cost of preparing the reference substance solution from more than 3000 yuan to less than 100 yuan once, greatly saving the detection cost, shortening the detection time from 80 minutes to 45 minutes per needle sample injection of optimized chromatographic conditions, and greatly reducing the detection time, thereby reducing the waiting time of the intermediate to the subsequent process.
Description
Technical Field
The invention relates to the technical field of quality evaluation and control of traditional Chinese medicines, in particular to a method for detecting the content of each mass component of an intermediate of a red sage root injection based on a one-test-multiple-evaluation method.
Background
The red sage root injection is prepared with red sage medicine material and through water extraction, alcohol precipitation, purification, encapsulation and disinfection, and is mainly applied to cardiac and cerebral vascular diseases. Clinical practice shows that it has quick and obvious curative effect and wide application in treating acute and severe patients. Modern researches have shown that the red sage root injection has various pharmacological activities of resisting lipid peroxidation, scavenging free radicals, inhibiting platelet adhesion and aggregation, improving microcirculation, improving hemorheology, regulating blood lipid and resisting atherosclerosis. Because of the wide clinical application of the red sage root injection, the quality evaluation and control level of the red sage root injection is more and more concerned.
In the standard preparation method of the salvia miltiorrhiza injection provided by the twenty-first book of the standards issued by the Chinese medicine ministry of the sciences, the pretreatment process of the salvia miltiorrhiza injection is mainly divided into: 1) Extracting Saviae Miltiorrhizae radix for three times, and concentrating; 2) Alcohol precipitation twice and filtering; 3) Recovering ethanol and concentrating; 4) Precipitating with water, adding water, cooling, standing, and filtering; 5) Adjusting pH, boiling, and filtering. The whole process is time-consuming and complex to operate, and the quality control of the process intermediate is worth being focused and studied.
In order to better ensure the stable and controllable quality of the salvia miltiorrhiza injection, the content of intermediates in each stage in the production process is required to be measured and controlled. The method for measuring the content of the finished product of the national medicine standard of the salvia miltiorrhiza injection is directly used for detecting the intermediate, and the content of each component of the intermediate can be measured, but the use amount of the reference substance is more, the detection cost is high, the preparation of the mixed reference substance is complicated, and the detection time is longer, for example, the 731 yuan/branch of the sodium salvianolic acid reference substance, the 36 yuan/branch of protocatechuic aldehyde, the 682 yuan/branch of rosmarinic acid and the 1948 yuan/branch of salvianolic acid B.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for detecting the content of each mass component of the intermediate of the salvia miltiorrhiza injection based on a one-test-multiple-evaluation method, which has the characteristics of simple operation and low detection cost.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the method for detecting the content of each mass component of the intermediate of the salvia miltiorrhiza injection based on a multi-evaluation method is characterized by comprising the following steps of:
S1, preparing a mixed reference substance solution: precisely weighing sodium salvianic acid A reference substance, protocatechuic aldehyde reference substance, rosmarinic acid reference substance and salvianolic acid B reference substance, adding solvent, and mixing to obtain mixed reference substance solution;
s2, preparing a sample solution: precisely sucking the intermediate solution of the radix salviae miltiorrhizae injection, placing the intermediate solution into a volumetric flask, adding 10% (ml/ml) methanol solution to dilute to scale, and shaking uniformly to obtain a sample solution;
s3, measuring relative correction factors: injecting the mixed reference substance solution into a high performance liquid chromatograph to obtain peak areas of each component in the mixed reference substance solution, and calculating relative correction factors f km of sodium salvianic acid A, rosmarinic acid and salvianolic acid B on protocatechuic aldehyde by taking protocatechuic aldehyde as an internal reference substance;
Wherein k is protocatechuic aldehyde; m is other components; a k is the peak area of protocatechuic aldehyde; w k is the sample injection amount of protocatechuic aldehyde; a m is the peak area of the other components; w m is the sample injection amount of other components; f k is the correction factor of protocatechuic aldehyde, f m is the correction factor of other components, and the relative correction factor of each component is f km;
S4, measuring each component in the sample solution: precisely sucking the sample solution, injecting the sample solution into a high performance liquid chromatograph to obtain peak areas of components to be detected in the sample solution, measuring the sample quality of protocatechuic aldehyde in the sample solution by a calibration method except for a protocatechuic aldehyde single reference substance solution, and calculating the sample quality of other components to be detected except for the protocatechuic aldehyde in the sample solution by a formula;
Wherein W m Sample is the sample injection amount of each component in the sample, f km is the relative correction factor of the other components, A m Sample is the peak area of each component in the sample, W k Label (C) is the sample injection amount of protocatechuic aldehyde in the reference, A k Label (C) is the peak area of protocatechuic aldehyde in the reference, C k Label (C) is the concentration of protocatechuic aldehyde in the reference, V Label (C) is the sample injection volume of the reference, C m Sample is the concentration of each component in the sample, V Sample is the sample injection volume of the sample, and V Label (C) and V Sample are equal.
Preferably, in step S1, the solvent is 10% (ml/ml) methanol solution, and the 10% (ml/ml) methanol solution further contains 0.2% (ml/ml) glacial acetic acid.
Preferably, in step S1, the mixed reference solution contains 0.20mg of sodium danshensu, 50 μg of protocatechuic aldehyde, 50 μg of rosmarinic acid and 50 μg of salvianolic acid B per 1ml of the mixed reference solution.
Preferably, in step S2, 0.2% (ml/ml) of glacial acetic acid is also contained in the 10% (ml/ml) methanol solution.
Preferably, in step S3, the test conditions of the high performance liquid chromatograph are: filling a chromatographic column with octadecylsilane chemically bonded silica, taking acetonitrile as a mobile phase A, taking 0.05% (ml/ml) trifluoroacetic acid aqueous solution as a mobile phase B, and enabling the flow rate to be 1ml per minute; column temperature is 40 ℃; the detection wavelength is 288nm, and the theoretical plate number is not less than 20000 calculated according to protocatechuic aldehyde peak.
Preferably, in step S4, the preparation process of the protocatechuic aldehyde single reference substance solution is as follows: precisely weighing protocatechuic aldehyde reference substance, adding solvent, and mixing to obtain protocatechuic aldehyde single reference substance solution.
Preferably, the single reference solution of protocatechuic aldehyde contains 50 μg protocatechuic aldehyde per 1ml solution.
Preferably, the solvent is 10% (ml/ml) methanol solution, and the 10% (ml/ml) methanol solution further contains 0.2% (ml/ml) glacial acetic acid.
Compared with the prior art, the invention has the following beneficial effects:
According to the invention, through one measurement and multiple evaluation, only the protocatechuic aldehyde reference substance is used in the detection process, and meanwhile, the content of sodium salvianic acid A, protocatechuic aldehyde, rosmarinic acid and salvianolic acid B in the intermediate is measured to judge whether the intermediate is qualified, so that the use amount of the reference substance is reduced, the cost of preparing the reference substance solution once is reduced from more than 3000 yuan to less than 100 yuan, the detection cost is greatly saved, and in addition, the detection time of sample injection per needle of the chromatographic condition after optimization is shortened from 80 minutes to 45 minutes, and the detection time is greatly shortened, thereby reducing the waiting time of the intermediate from the follow-up to the process.
Drawings
FIG. 1 is a high performance liquid chromatogram of a mixed control solution;
FIG. 2 is a high performance liquid chromatogram of a protocatechuic aldehyde single control solution;
FIG. 3 is a high performance liquid chromatogram of a test solution.
Detailed Description
The present invention will be described in further detail with reference to the following preferred examples, but the present invention is not limited to the following examples.
Unless otherwise specified, the chemical reagents involved in the present invention are all commercially available.
Examples
A method for detecting the content of each mass component of the intermediate of the salvia miltiorrhiza injection based on a multi-evaluation method comprises the following steps:
(1) Preparation of mixed control solution
Precisely weighing sodium salvianic acid A reference substance, protocatechuic aldehyde reference substance, rosmarinic acid reference substance and salvianolic acid B reference substance, adding 10% (ml/ml) methanol solution (also containing 0.2vol.% glacial acetic acid), and mixing to obtain mixed reference substance solution, wherein each 1ml of mixed solution contains 0.20mg sodium salvianic acid A, 50 μg protocatechuic aldehyde, 50 μg rosmarinic acid and 50 μg salvianolic acid B.
(2) High performance liquid chromatography detection
Respectively sucking 2 μl, 5 μl, 10 μl, 20 μl and 50 μl of the mixed reference solution into liquid chromatograph for detection, wherein the liquid chromatograph has the following test conditions: packing the chromatographic column with octadecylsilane chemically bonded silica gel; gradient elution was performed as specified in table 1 with acetonitrile as mobile phase a and 0.05% (ml/ml) aqueous trifluoroacetic acid as mobile phase B; the flow rate is 1ml per minute; column temperature is 40 ℃; the detection wavelength is 288nm, and the theoretical plate number is not less than 20000 calculated according to protocatechuic aldehyde peak.
TABLE 1
The chromatogram is shown in fig. 1, and the standard curves of four reference substances are obtained by calculation by taking y as the peak area and x as the sample injection amount, as shown in table 2:
TABLE 2
As can be seen from Table 2, the linear relationship of sodium salvianolic acid was good in the range of 0.4016-10.0400. Mu.g, the linear relationship of protocatechuic aldehyde was good in the range of 0.1028-2.5700. Mu.g, the linear relationship of rosmarinic acid was good in the range of 0.1004-2.5100. Mu.g, and the linear relationship of salvianolic acid B was good in the range of 0.1026-2.5650. Mu.g.
(3) Determination of relative correction factors
The relative retention time and relative calibration factor of sodium salvianolic acid, rosmarinic acid and salvianolic acid B are calculated respectively by taking protocatechuic aldehyde as an internal reference and the corresponding peak as an S peak.
Wherein k is protocatechuic aldehyde; m is other components; a k is the peak area of protocatechuic aldehyde; w k is the sample injection amount of protocatechuic aldehyde; a m is the peak area of the other components; w m is the sample injection amount of other components; f k is the correction factor of protocatechuic aldehyde, f m is the correction factor of other components, and the relative correction factor of each component is f km; the results are shown in Table 3:
TABLE 3 Table 3
Relative retention time = peak retention time of other components ++protocatechuic aldehyde peak retention time, mean values of relative retention time and relative calibration factor are shown in table 4 below:
TABLE 4 Table 4
(4) Determination of other Components in a sample
Preparation of protocatechuic aldehyde single reference substance solution: accurately weighing protocatechuic aldehyde reference substance, adding 10vol% methanol solution (containing 0.2vol% glacial acetic acid), and mixing to obtain protocatechuic aldehyde single reference substance solution, wherein each 1ml of solution contains protocatechuic aldehyde 50 μg.
Preparing a test solution: precisely measuring the intermediate, adding 10vol% methanol solution (containing 0.2vol% glacial acetic acid), and mixing to obtain sample solution, wherein each 1ml of sample solution contains 0.05ml of intermediate.
Sucking 5 μl of protocatechuic aldehyde single reference substance solution, and injecting into a liquid chromatograph for detection, wherein the sample injection amount of protocatechuic aldehyde is in a linear range, and the chromatogram is shown in figure 2;
Sucking 5 μl of the sample solution, and injecting into a liquid chromatograph for detection, wherein the sample injection amount of the quality control component is in a linear range, and the chromatogram is shown in figure 3;
the chromatographic test conditions are the same as those described above, so that the peak area of the protocatechuic aldehyde single reference substance and the peak areas of the quality control components of the intermediate test substance are obtained, the content C m Sample of the quality control components of the intermediate test substance is calculated respectively through the participation of relative calibration factors, and the calculation formula is as follows:
Wherein W m Sample is the sample injection amount of each component in the sample, f km is the relative correction factor of the other components, A m Sample is the peak area of each component in the sample, W k Label (C) is the sample injection amount of protocatechuic aldehyde in the reference, A k Label (C) is the peak area of protocatechuic aldehyde in the reference, C k Label (C) is the concentration of protocatechuic aldehyde in the reference, V Label (C) is the sample injection volume of the reference, C m Sample is the concentration of each component in the sample, V Sample is the sample injection volume of the sample, and V Label (C) and V Sample are equal.
(5) Other analytical methods verification terms
According to the rule of the 2020 edition of Chinese pharmacopoeia four department 9101 analysis method verification guidance, the verification work of each analysis method verification item is completed, and each item meets the requirements.
(6) The method of the invention is compared with the external standard method
① Detecting peak areas of each quality control component of the mixed reference substance and the intermediate test substance by using a liquid chromatography method of content measurement items in the national pharmaceutical standard WS3-B-3766-98-2011 of the salvia miltiorrhiza injection, and respectively calculating the content of each quality control component in the intermediate by using an external standard method;
② Measuring the peak area of a protocatechuic aldehyde reference substance and the peak area of each quality control component of an intermediate sample by using a one-measurement-multiple-evaluation-method liquid chromatography condition, and respectively calculating the content of each quality control component in the intermediate by using relative calibration factors;
③ Comparing the measurement results of the intermediate one-measurement multi-evaluation method with the measurement results of the salvia miltiorrhiza injection content measurement method (external standard method), randomly taking a proper amount of n (n is more than or equal to 10) batches of intermediate samples, and respectively measuring and calculating the contents of each component in 4 batches of intermediate A and 6 batches of intermediate B by using the one-measurement multi-evaluation method and the external standard method, wherein the results of the two measurement methods have no obvious difference, and are shown in Table 5:
TABLE 5
Finally, it should be noted that: the above examples are not intended to limit the present invention in any way. Modifications and improvements will readily occur to those skilled in the art upon the basis of the present invention. Accordingly, any modification or improvement made without departing from the spirit of the invention is within the scope of the invention as claimed.
Claims (8)
1. The method for detecting the content of each mass component of the intermediate of the salvia miltiorrhiza injection based on a multi-evaluation method is characterized by comprising the following steps of:
S1, preparing a mixed reference substance solution: precisely weighing sodium salvianic acid A reference substance, protocatechuic aldehyde reference substance, rosmarinic acid reference substance and salvianolic acid B reference substance, adding solvent, and mixing to obtain mixed reference substance solution;
s2, preparing a sample solution: precisely sucking the intermediate solution of the radix salviae miltiorrhizae injection, placing the intermediate solution into a volumetric flask, adding 10% (ml/ml) methanol solution to dilute to scale, and shaking uniformly to obtain a sample solution;
s3, measuring relative correction factors: injecting the mixed reference substance solution into a high performance liquid chromatograph to obtain peak areas of each component in the mixed reference substance solution, and calculating relative correction factors f km of sodium salvianic acid A, rosmarinic acid and salvianolic acid B on protocatechuic aldehyde by taking protocatechuic aldehyde as an internal reference substance;
Wherein k is protocatechuic aldehyde; m is other components; a k is the peak area of protocatechuic aldehyde; w k is the sample injection amount of protocatechuic aldehyde; a m is the peak area of the other components; w m is the sample injection amount of other components; f k is the correction factor of protocatechuic aldehyde, f m is the correction factor of other components, and the relative correction factor of each component is f km;
S4, measuring each component in the sample solution: precisely sucking the sample solution, injecting the sample solution into a high performance liquid chromatograph to obtain peak areas of components to be detected in the sample solution, measuring the sample quality of protocatechuic aldehyde in the sample solution by a calibration method except for a protocatechuic aldehyde single reference substance solution, and calculating the sample quality of other components to be detected except for the protocatechuic aldehyde in the sample solution by a formula;
Wherein W m Sample is the sample injection amount of each component in the sample, f km is the relative correction factor of the other components, A m Sample is the peak area of each component in the sample, W k Label (C) is the sample injection amount of protocatechuic aldehyde in the reference, A k Label (C) is the peak area of protocatechuic aldehyde in the reference, C k Label (C) is the concentration of protocatechuic aldehyde in the reference, V Label (C) is the sample injection volume of the reference, C m Sample is the concentration of each component in the sample, V Sample is the sample injection volume of the sample, and V Label (C) and V Sample are equal.
2. The method according to claim 1, wherein in step S1, the solvent is 10% (ml/ml) methanol solution, and the 10% (ml/ml) methanol solution further contains 0.2% (ml/ml) glacial acetic acid.
3. The method for detecting the content of each mass component of the intermediate of the salvia miltiorrhiza injection based on the one-test-multiple-evaluation method according to claim 1, wherein in the step S1, the mixed reference solution contains 0.20mg of sodium salvianic acid A, 50 mug of protocatechuic aldehyde, 50 mug of rosmarinic acid and 50 mug of salvianolic acid B in each 1ml of the mixed reference solution.
4. The method for detecting the content of each mass component of the intermediate of the red sage root injection based on the one-test-multiple-evaluation method according to claim 1, wherein in the step S2, the 10% (ml/ml) methanol solution further contains 0.2% (ml/ml) glacial acetic acid.
5. The method for detecting the content of each mass component of the intermediate of the red sage root injection based on a multi-evaluation method as set forth in claim 1, wherein in the step S3, the testing conditions of the high performance liquid chromatograph are as follows: filling a chromatographic column with octadecylsilane chemically bonded silica, taking acetonitrile as a mobile phase A, taking 0.05% (ml/ml) trifluoroacetic acid aqueous solution as a mobile phase B, and enabling the flow rate to be 1ml per minute; column temperature is 40 ℃; the detection wavelength is 288nm, and the theoretical plate number is not less than 20000 calculated according to protocatechuic aldehyde peak.
6. The method for detecting the content of each mass component of the intermediate of the salvia miltiorrhiza injection based on a multi-evaluation method according to claim 1, wherein in the step S4, the preparation process of the protocatechuic aldehyde single reference substance solution is as follows: precisely weighing protocatechuic aldehyde reference substance, adding solvent, and mixing to obtain protocatechuic aldehyde single reference substance solution.
7. The method for detecting the content of each mass component of the intermediate of the red sage root injection based on a multi-evaluation method according to claim 6, wherein the protocatechuic aldehyde single reference solution contains 50 mug of protocatechuic aldehyde per 1ml of solution.
8. The method for detecting the content of each mass component of the intermediate of the red sage root injection based on the one-test multi-evaluation method according to claim 6, wherein the solvent is 10% (ml/ml) methanol solution, and the 10% (ml/ml) methanol solution further contains 0.2% (ml/ml) glacial acetic acid.
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| CN110274962A (en) * | 2018-03-13 | 2019-09-24 | 天士力医药集团股份有限公司 | Salvia root polyphenol acid component content measuring method in a kind of QISHEN YIQI DIWAN |
| CN111537641A (en) * | 2020-05-19 | 2020-08-14 | 常熟雷允上制药有限公司 | Quality detection method of red ginseng traditional Chinese medicine decoction pieces |
| CN115429752A (en) * | 2022-09-21 | 2022-12-06 | 常熟雷允上制药有限公司 | Salvia miltiorrhiza injection and preparation method and application thereof |
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| CN103776908A (en) * | 2012-10-17 | 2014-05-07 | 广州白云山和记黄埔中药有限公司 | Detection method of phenolic acid compounds in compound radix salviae miltiorrhizae tablet |
| CN105277643B (en) * | 2014-07-17 | 2018-10-30 | 天士力医药集团股份有限公司 | An a kind of survey for compound danshen dripping pills comments detection method |
| CN111220719B (en) * | 2019-11-11 | 2022-11-25 | 云南生物谷药业股份有限公司 | Method for evaluating quality of ginseng medicinal material by using fingerprint spectrum |
| CN110780004A (en) * | 2019-11-14 | 2020-02-11 | 美国琛蓝营养制品股份有限公司 | Method for establishing fingerprint of traditional Chinese medicine composition or preparation thereof with depression mood regulating effect |
| CN114371234B (en) * | 2021-12-29 | 2023-11-24 | 江西普正制药股份有限公司 | Method for measuring content of red sage root |
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| CN110274962A (en) * | 2018-03-13 | 2019-09-24 | 天士力医药集团股份有限公司 | Salvia root polyphenol acid component content measuring method in a kind of QISHEN YIQI DIWAN |
| CN111537641A (en) * | 2020-05-19 | 2020-08-14 | 常熟雷允上制药有限公司 | Quality detection method of red ginseng traditional Chinese medicine decoction pieces |
| CN115429752A (en) * | 2022-09-21 | 2022-12-06 | 常熟雷允上制药有限公司 | Salvia miltiorrhiza injection and preparation method and application thereof |
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