CN105588885B - Salvianolic acid extract fingerprint spectrum and content determination method of related components - Google Patents
Salvianolic acid extract fingerprint spectrum and content determination method of related components Download PDFInfo
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Abstract
The invention relates to a method for establishing a salvianolic acid extract fingerprint spectrum and measuring the content of related components, which comprises the following steps: step 1: establishing a digital quantitative fingerprint spectrum of the salvianolate extract by adopting an ultra-high performance liquid phase multi-wavelength visible ultraviolet detection method; step 2: identifying peaks in the fingerprint spectrum in the step 1 by using a mass spectrum, and determining fingerprint peaks shared by ultraviolet detection; and step 3: and (3) determining the contents of the three index components which are identified in the step (2) and have larger contents, more obvious activities and definite structures by adopting a high performance liquid chromatography variable wavelength detection method.
Description
The technical field is as follows:
the invention relates to a detection method of a traditional Chinese medicine extract, in particular to a method for establishing a salvianolic acid extract fingerprint spectrum and measuring contents of related components
Background art:
the Saviae Miltiorrhizae radix is a plant of Salvia of Labiatae. Also named: salvia miltiorrhiza, red root, sanguisorba officinalis, clovershrub, and the like. Salvia miltiorrhiza, which is medicinal in root, is bitter in taste and slightly cold in nature. Has effects in promoting blood circulation, regulating menstruation, dispelling blood stasis, promoting tissue regeneration, tranquilizing mind, cooling blood, eliminating carbuncle, and relieving swelling and pain. Can be used for treating menoxenia, dysmenorrhea, puerperal abdominal pain due to blood stasis, joint pain, neurasthenia, insomnia, palpitation, carbuncle, swelling, and skin ulcer. The modern medical clinical proves that the salvia miltiorrhiza has the functions of expanding blood vessels and increasing the blood flow of coronary arteries, is used for treating diseases such as coronary heart disease, angina, myocardial infarction, tachycardia and the like, and has obvious curative effect; it also has good therapeutic effect on chronic hepatitis and early liver cirrhosis.
The Saviae Miltiorrhizae radix contains two main effective components, namely liposoluble tanshinone and water soluble salvianolic acid. Modern medicine believes that the salvianolic acid (salvianolie acids) can reduce the range of myocardial infarction, relieve the state of illness, protect myocardial ischemia and reperfusion injury of rats, obviously inhibit platelet aggregation, anticoagulation and fibrinolysis, reduce blood fat and resist atherosclerosis, and has great clinical medicinal value. The compounds mainly comprise salvianolic acid, tanshinol, protocatechualdehyde, caffeic acid and the like, wherein the salvianolic acid B is a main effective component in water-soluble components of the salvia miltiorrhiza.
The salvianolate injection is a product which is already on the market, and the active substance of the salvianolate injection is called salvianolate. There are several methods for extracting salvianolic acid, such as: taking salvia miltiorrhiza decoction pieces, decocting and extracting the salvia miltiorrhiza decoction pieces for three times by using purified water, each time lasting for 0.5-1 hour, combining three extracting solutions, cooling, adjusting the extracting solutions to be acidic by using hydrochloric acid solution, standing for 4-8 hours, filtering to obtain clear liquid medicine, purifying the liquid medicine by using a polyamide column chromatography process, washing the liquid medicine by using purified water after sample loading, eluting by using sodium bicarbonate solution, collecting eluent, adjusting the eluent to be acidic by using hydrochloric acid solution, purifying the liquid medicine by using a macroporous resin column chromatography process, washing the liquid medicine by using purified water after sample loading, eluting by using ethanol solution, collecting the eluent, decompressing and concentrating the eluent to recover ethanol, refrigerating the obtained concentrated solution for 12-24 hours, filtering, adjusting the pH value of the filtrate to be 5.3-6.0 by using sodium hydroxide solution, and freeze. Then mixing the salvianolate extract with auxiliary materials to prepare various preparations. For example, mixing with mannitol solution, adjusting pH to 5.5-6.0, diluting to constant volume, and freeze drying to obtain salvianolic acid injection.
As the active ingredient of the salvianolic acid injection, the quality control items are not comprehensive in the current quality standard situation of the salvianolic acid extract and the preparation, and the content of the salvianolic acid is generally determined by adopting an ultraviolet-visible spectrophotometry. The method calculates the content of the salvianolic acid by taking the salvianolic acid B as a reference, and has poor specificity and accuracy. In addition, for the traditional Chinese medicine preparation with complex components, only one component is used for controlling the content index, and the identification of the complex components is not sufficient while the integrity is lacked.
Therefore, the above-mentioned complicated components are not sufficiently identified and the quantitative determination is not accurate. The invention develops a UPLC chromatographic fingerprint method, and utilizes liquid quality to carry out peak identification, thereby determining fingerprint peaks shared by 23 ultraviolet detections. And meanwhile, an HPLC content determination method is established for the identified three index components with larger content, more obvious activity and definite structure.
The invention aims to comprehensively characterize the quality of a traditional Chinese medicine preparation, provides a method for identifying 23 salvianolic acid compounds by establishing a digital quantitative fingerprint spectrum of a salvianolic acid extract by using an UPLC-PDA (ultra-high performance liquid phase multi-wavelength visible ultraviolet detection) method, identifying the consistency and stability of multiple batches of salvianolic acid extract samples by using a two-stage system fingerprint quantitative method, detecting 39 chemical components together, and combining multi-stage mass spectrum information and a reference substance. Considering the cost of applying UPLC to large-scale detection and the wide applicability of HPLC, the contents of the three index components of rosmarinic acid, alkannic acid and salvianolic acid B are simultaneously measured by an HPLC-VWD method (high performance liquid chromatography variable wavelength detection method). The method ensures the accuracy and the effectiveness, considers the principle of cost saving, is more suitable for expanded production, and improves the quality control standard of the existing salvianolic acid for injection.
The invention content is as follows:
the invention provides a salvianolic acid extract fingerprint spectrum and a content determination method, which are characterized by comprising the following steps:
step 1: establishing a digital quantitative fingerprint spectrum of the salvianolate extract by adopting an ultra-high performance liquid phase multi-wavelength visible ultraviolet detection method;
step 2: identifying peaks in the fingerprint spectrum in the step 1 by using a mass spectrum, and determining fingerprint peaks shared by ultraviolet detection;
and step 3: and (3) determining the contents of the three index components which are identified in the step (2) and have larger contents, more obvious activities and definite structures by adopting a high performance liquid chromatography variable wavelength detection method.
Further, the specific steps of step 1 are as follows:
(1) preparation of control solutions: precisely weighing reference substances including danshensu sodium, protocatechualdehyde, salvianolic acid D, salvianolic acid A, salvianolic acid-LM, rosmarinic acid, alkannic acid and salvianolic acid B, adding ultrapure water for dissolving, and fixing the volume until the final concentrations of the danshensu sodium, protocatechualdehyde, salvianolic acid D, salvianolic acid A and salvianolic acid-LM are respectively 500 mu g/ml in 200-plus type, the final concentrations of the rosmarinic acid and the alkannic acid are respectively 600 mu g/ml in 300-plus type, and the final concentration of the salvianolic acid B is 1500 mu g/ml in 500-plus type;
(2) preparation of a test solution: precisely weighing 10-50 batches of salvianolic acid extracts respectively, dissolving the samples in ultrapure water, filtering the solution after dissolution, and diluting the solution into a solution with the concentration of 1mg/mL-5 mg/mL;
(3) determination of fingerprint spectrum: precisely absorbing 1-5 mu L of test solution, injecting into an ultra-high performance liquid chromatograph, and recording chromatogram;
wherein, the chromatographic conditions are as follows:
a chromatographic column: a UPLC C18 column or a T3 column;
mobile phase: solvent A: water, containing 0.1% -0.5% formic acid; solvent B: acetonitrile containing 1% -5% methanol;
gradient elution:
0-3min,12-18％B；3-15min,18％B；15-17min,18-20％B；17-25min,20％B；25-28min,20-23％B；28-35min,23％B；35-40min,23-40％B；40-45min,40-95％B；45-50min,95％B；
temperature: 30-40 ℃;
flow rate: 0.2-0.3 ml/min;
chromatogram spectrum acquisition range: 190-400 nm.
Preferably, in step 1,
the chromatographic column preferably comprises: a UPLC C18 column;
the mobile phase is preferably:
solvent A: water, 0.2% formic acid; solvent B: acetonitrile, 3% methanol; or
Solvent A: water, 0.1% formic acid; solvent B: acetonitrile, 2% methanol.
The chromatographic conditions of the UPLC are obtained by screening:
1. selection of chromatography columns
The invention compares the chromatographic columns of three different brands which are widely used at present: (A) fortis C181.7μm，2.1x 50mm；(B)Agilent Eclipse Plus C18，1.8μm，2.1x 150mm；(C)Waters HSS T3C181.7 μm,2.1 × 150 mm. It can be seen from the liquid phase comparison spectrograms of the three columns that the peak emergence time of the component to be analyzed on the A-type chromatographic column is late, the separation effect is poor, and the chromatographic column A is excluded in consideration of the cost of expanding the production and the accuracy of the final component identification result; then comparing the spectrums of the salvianolic acid sample in the chromatographic column B and the chromatographic column C, the separation effect of the chromatographic column B is obviously better than that of the chromatographic column C, and the number of chromatographic peaks in the B picture is obviously more than that in the C picture, which is beneficial to the comprehensiveness of component research. Repeated experiments show that Eclipse has a good separation effect on phenolic acid components with high polarity, so that the Eclipse C18 chromatographic column is optimized and selected. The chromatogram is shown in FIG. 1.
2. Screening of elution gradient:
(1) adopting a solvent A: water, containing 0.1% formic acid, solvent B: acetonitrile, system optimization, results show that the more polar chromatogram is difficult to separate from neighboring impurities. The following are recorded:
(A)0-3min:12-20％B,3-10min:20-22％B,10-22min:22％B,22-27min:22-35％B,27-29min:35％-50％B；
(B)0-3min:12-18％B,3-10min:18-22％B,10-12min:22％B,12-22min,22-25％B；22-27min:25-35％B,27-29min:35％-50％B；
(C)0-2min:12-18％B,2-5min:18-22％B,5-15min:22-25％B,15-20min:25-45％B,20-25min:45％-95％B.
the chromatogram is shown in FIG. 2.
(2) The mobile phase was optimized as solvent a: water, containing 0.2% formic acid, solvent B: acetonitrile containing 2% methanol, improved the separation details significantly, separated in 5-10 minutes. Further optimization, the high polar phenolic acid was separated in 18% organic phase isocratic elution (fig. 3, 13 min). The following are recorded:
(A)0-3min:12-18％B,3-10min:18-20％B,10-20min:20％B；
(B)0-3min:12-17％B,3-20min:17％B,20-23min:17-20％B；
(C)0-3min:12-18％B,3-15min:18％B,15-17min:18-20％B,17-25min:20％B.
the chromatogram is shown in FIG. 3 and FIG. 4 (FIG. 3 is an enlarged view).
3. Methodology investigation
(1) Precision test
The sample solution (lot 20130202) was continuously injected 6 times, and the relative retention time and the relative peak area of the common peak were examined with the alkannic acid as a reference. The RSD of each common peak relative to the retention time is less than 0.5 percent, and the RSD of the relative peak area is less than 3.0 percent, which indicates that the precision of the instrument is good.
(2) Repeatability test
6 portions of a sample (batch No. 20130202) were sampled, and a sample solution was prepared and examined by the method described in the above step 1, and the relative retention time and the relative peak area of the common peak were examined with alkannic acid as a reference. RSD of each common peak relative retention time is less than 0.5%, and RSD of the relative peak area is less than 3.5%, indicating that the method has good repeatability.
(3) Stability test
Taking a test sample solution (batch No. 130301), injecting samples for analysis for 0, 2, 4, 8, 12 and 24 hours respectively, and taking alkannic acid as a reference to respectively investigate the relative retention time and the relative peak area of the common peak.
The results show that the RSD of each common peak relative to the retention time is less than 0.5%, and the RSD of the relative peak area is less than 3.0%. The test solution was shown to be stable at room temperature for 24 h.
UPLC chromatogram (280nm) of salvianolic acid extract sample is shown in FIG. 5.
By utilizing the method in the step 1, UPLC fingerprint spectrum analysis can be performed on a plurality of salvianolic acid extract samples, and 23 common peak ultraviolet spectrums of each sample are compared and analyzed, so that the consistency and the stability of the plurality of salvianolic acid extract samples are identified.
For the comparison and analysis of the similarity of the fingerprint spectrums of different batches, various software sold in the market can be adopted for carrying out the comparison and analysis.
The invention preferably adopts software of 'Chinese medicine chromatogram fingerprint super information characteristic digital evaluation system 4.0' to carry out secondary quantitative fingerprint analysis on the result signal, determines the comparison fingerprint, and calculates the similarity of each batch of fingerprints by taking the comparison fingerprint as reference to obtain the final fingerprint quality results of a plurality of batches of samples.
The system fingerprint quantitative method objectively and accurately describes the contribution of each chemical substance to the chemical fingerprint of the compound preparation in a quantitative manner. Taking UPLC fingerprint of salvianolic acid extract as a whole, and monitoring the chemical fingerprint quantity and distribution ratio of salvianolic acid extract from the whole by using macro qualitative similarity (Sm); quantifying similarity by macros
(Pm) monitoring the integral content of the salvianolic acid extract by chemical fingerprint; and limiting the UPLC fingerprint proportion variation range of the salvianolic acid extract by using the absolute value of the relative deviation of the sample fingerprint homogenization variation coefficient (alpha), wherein Sm, Pm and alpha are calculated according to a formula method.
TABLE 1 systematic fingerprint quantitative method for classifying quality grade standard of Chinese medicine
The quality grade of the traditional Chinese medicine is identified by Sm, Pm and alpha together by adopting the system fingerprint quantitative method standard shown in Table 1, and the consistency and the stability of a plurality of batches of samples are identified by the grade division and the grade lower than the quality V as unqualified, so that the quality is evaluated.
Further, the mass spectrum conditions in step 2 are as follows:
the quadrupole-time-of-flight mass spectrometry (qTOF) adopts a (-) -ESI ion source, the atomized gas is high-purity nitrogen, and the collision gas is high-purity helium;
scanning range: m/z is 100-1200;
collision energy: 6 eV;
MSE gradient cleavage energy: 20-40 eV.
As can be seen from the total ion flow diagram of the salvianolic acid extract (figure 6), the peaks of the components have higher response under the condition of negative ions and correspond to the ultraviolet chromatogram.
From the exact relative molecular masses measured by time-of-flight mass spectrometry, the possible molecular composition (error < 5X 100) was calculated using high resolution mass spectrometry data analysis (e.g., MassLynx mass spectrometry software)-6) And combining the fragment information of the secondary mass spectrum of each component peak of the ion trap mass spectrometer and the literature report of known compounds of salvia miltiorrhiza, analyzing chromatographic peaks, and identifying 23 corroborable compounds from a test sample. See table below.
TABLE 2 Salvianolic acid extract chemical ingredient identification List
Further, the three index components in step 3 are rosmarinic acid, lithospermic acid and salvianolic acid B respectively.
The method comprises the following steps of adopting a high performance liquid chromatography variable wavelength detection method to detect the contents of three index components of rosmarinic acid, alkannic acid and salvianolic acid B:
(1) preparing a test solution: precisely weighing salvianolic acid extract, adding initial mobile phase for dissolving, fixing volume to final concentration of 0.6-1.2mg/mL, and shaking;
(2) preparation of mixed control solution: accurately weighing appropriate amounts of rosmarinic acid, lithospermic acid and salvianolic acid B reference substances respectively, and adding an initial mobile phase to prepare a mixed reference substance solution to obtain a mixed reference substance solution with the mass concentration of rosmarinic acid of 100-150 mu g/mL, the mass concentration of lithospermic acid of 100-150 mu g/mL and the mass concentration of salvianolic acid B of 1.0-2.5 mg/mL;
(3) precisely sucking 1-5 μ L of the test solution, injecting into a high performance liquid chromatograph, and simultaneously determining the content of rosmarinic acid, lithospermic acid and salvianolic acid B in the test solution by adopting an external standard peak area method;
wherein, the chromatographic conditions are as follows:
a chromatographic column: HPLC C18 column;
mobile phase: phase A: water, containing 0.3 to 0.6 percent of formic acid; phase B: acetonitrile, 2-4% of methanol and 0.3-0.6% of formic acid;
gradient elution: 0-40 min, 20% B; 40-50 min, 20-70% B;
flow rate: 0.9-1.1 mL/min;
column temperature: 30-35 ℃;
detection wavelength: 288 nm.
Wherein the initial mobile phase in the steps (1) and (2) refers to a mixed solution of 80% of A mobile phase and 20% of B mobile phase.
Preferably, in step 3,
the mobile phase is preferably:
phase A: water, 0.5% formic acid; phase B: acetonitrile, 2% methanol and 0.5% formic acid; or
Phase A: water, 0.3% formic acid; phase B: acetonitrile, 3% methanol and 0.3% formic acid;
the flow rate is preferably 1 mL/min;
the column temperature is preferably 30 ℃.
The HPLC chromatographic conditions of the invention are obtained by groping:
1. selection of mobile phase:
in the search for the mobile phase, different proportions of acetonitrile-water, methanol-water, acetonitrile-trifluoroacetic acid water, acetonitrile-phosphoric acid water, acetonitrile-formic acid water, and acetonitrile-methanol-trifluoroacetic acid water, acetonitrile-methanol-formic acid water were examined, and finally, it was found that, as a phase a: water (containing 0.3 to 0.6 percent of formic acid); phase B: the separation effect of the target peak is better when acetonitrile (containing 2-4% of methanol and 0.3-0.6% of formic acid) is eluted in a gradient way, particularly phase A: water (0.5% formic acid); phase B: acetonitrile (containing 3% methanol and 0.5% formic acid) has the best separation effect, and is mainly characterized in that rosmarinic acid and trace substances on the left and right sides can achieve baseline separation, and the map is shown in fig. 7.
Under the condition of HPLC chromatogram, the chromatographic peaks of rosmarinic acid, alkannic acid and salvianolic acid B in the test solution can be well separated from the peaks of other components. As shown in fig. 8.
2. Selection of acquisition wavelength of DAD detector:
the maximum absorption of salvianolic acid B is about 288nm, and the maximum absorption of lithospermic acid is 250 nm. Comparing the control product spectrograms under 250, 280, 330 and 360nm, selecting 280nm as the absorption wavelength of a typical fingerprint, and using 330nm for the comparison of the separation degree in the condition optimization process. The liquid chromatogram of the mixed reference of salvianolic acids at different detection wavelengths is shown in FIG. 9.
3. Investigation of linear relationships
(1) Accurately weighing 0.4 mL, 1mL, 2mL, 4 mL, 8 mL and 10mL of the mixed reference substance solution respectively, placing in a 10mL measuring flask, metering to a scale with the initial mobile phase, and shaking up to obtain a series of standard solutions.
(2) Separately, 10. mu.L of each sample was injected, and the peak area A was recorded as the peak area A against the mass concentration C (. mu.g.L)-1) And performing linear regression to draw a standard curve.
The linear regression equation A of the rosmarinic acid is 23.787C-0.1082, R2 is 1.0000, and the linear range is 4.82-120.50 mu g.L-1；
The lithospermic acid linear regression equation A is 15.580C-1.8253, R2 is 1.0000, and the linear range is 4.84-121.10 mu g.L-1；
The linear regression equation A of salvianolic acid B is 12.963C-5.4623, R2 is 1.0000, and the linear range is 60.80-1520.00 mu g.L-1。
4. Methodology investigation
(1) Precision test
Accurately sucking sample solution, continuously feeding sample for 6 times with sample amount of 10 μ L according to the above HPLC chromatographic conditions, and measuring the peak areas of rosmarinic acid, lithospermic acid, and salvianolic acid B.
Measuring the RSD of the rosmarinic acid peak area to be 0.44 percent; RSD of the area of the alkannic acid peak is 0.30 percent; RSD of salvianolic acid B peak area is 0.29%.
The results show that the precision of the instrument is good.
(2) Repeatability test
And precisely weighing 6 parts of extract samples respectively, measuring according to a given test sample preparation method and HPLC chromatographic conditions, recording peak areas, and calculating the content of each measured component respectively.
Measuring the RSD of the content of the rosmarinic acid to be 0.86 percent; RSD of alkannic acid content is 0.70%; the content of salvianolic acid B is RSD 0.61%.
The results show that the precision of the method is good.
(3) Intermediate precision test
Taking the same batch of salvianolic acid, preparing a test solution according to a given test preparation method, respectively carrying out sample injection measurement on the same instrument by different people on different days by the same person, and calculating the RSD of each measured component content of different people on different days.
RSD of rosmarinic acid content 0.34% was measured by the same person over three days; RSD of alkannic acid content is 0.74%; RSD of salvianolic acid B content is 0.37%;
the RSD of the rosmarinic acid content is measured by three persons in the same instrument to be 0.68 percent; RSD 1.14% of alkannic acid content; the content of salvianolic acid B is RSD 0.86%.
(4) Stability test
And (3) taking the newly prepared mixed reference substance and the test solution, and performing chromatographic analysis on the mixed reference substance and the test solution according to the chromatographic conditions at 0, 3.5, 7, 10.5, 14, 18 and 24 hours respectively.
Measuring the RSD of the rosmarinic acid peak area in the mixed reference substance to be 1.14 percent; RSD of the area of the alkannic acid peak is 1.09 percent; RSD of the peak area of the salvianolic acid B is 1.17 percent.
RSD of rosmarinic acid peak area in the test sample is 0.52%; RSD of the area of the alkannic acid peak is 0.70 percent; RSD of salvianolic acid B peak area is 0.47%.
The results show that the control and the sample have good stability within 24 hours.
(5) Sample application recovery test
Preparing a sample solution according to a given sample preparation method, precisely measuring 5ml into a 10ml volumetric flask, precisely adding a proper amount of a mixed reference substance solution respectively to prepare solutions with the concentrations of 70%, 100% and 130%, wherein each concentration is 3 parts and 9 parts in total, then using an initial mobile phase to fix the volume, using the initial mobile phase as the sample solution with the concentrations of low, medium and high, and calculating the recovery rate and the RSD value.
The average recovery rate of the rosmarinic acid is 100.88 percent and the RSD is 0.80 percent; the average recovery rate of the alkannic acid is 103.03 percent, and the RSD is 0.99 percent; the average recovery rate of the salvianolic acid B is 101.92 percent, and the RSD is 1.16 percent. See tables 4-6.
TABLE 3 rosmarinic acid sample recovery
TABLE 4 recovery rate of alkannic acid sample
TABLE 5 recovery rate of alkannic acid sample
The salvianolate extract can be prepared by any suitable method in the prior art, and can also be any preparation of salvianolate.
Preferably, the preparation method of the salvianolate extract comprises the following steps: taking salvia miltiorrhiza decoction pieces, decocting and extracting the salvia miltiorrhiza decoction pieces for three times by using purified water, each time lasting for 0.5-1 hour, combining three extracting solutions, cooling, adjusting the extracting solutions to be acidic by using hydrochloric acid solution, standing for 4-8 hours, filtering to obtain clear liquid medicine, purifying the liquid medicine by using a polyamide column chromatography process, washing the liquid medicine by using purified water after sample loading, eluting by using sodium bicarbonate solution, collecting eluent, adjusting the eluent to be acidic by using hydrochloric acid solution, purifying the liquid medicine by using a macroporous resin column chromatography process, washing the liquid medicine by using purified water after sample loading, eluting by using ethanol solution, collecting the eluent, decompressing and concentrating the eluent to recover ethanol, refrigerating the obtained concentrated solution for 12-24 hours, filtering, adjusting the pH value of the filtrate to be 5.3-6.0 by using sodium hydroxide solution, and freeze.
The salvianolic acid extract can also be injection, and the preparation method comprises the following steps: dissolving the salvianolate extract and mannitol in a weight ratio of 1: 0.1-0.5 in 10-20 times of water for injection respectively; and uniformly mixing the two solutions, adding medicinal carbon, heating, stirring, filtering, cooling the liquid medicine, performing ultrafiltration, adjusting the pH value of the filtrate to 5.5-6.0, adding injection water to a constant volume, uniformly stirring, filtering the liquid medicine by using a microporous membrane, filling into a penicillin bottle, and freeze-drying to obtain the salvianolate for injection.
At present, the research on fingerprint spectra in the quality standards of salvianolic acid extracts and preparations (such as injections) is very incomplete, and the quality of the preparations is not completely characterized by the contents of several components in consideration of the specificity and clinical safety of Chinese medicinal preparations. The UPLC chromatographic fingerprint method is combined with liquid quality to perform peak identification, 23 ultraviolet detection common fingerprint peaks are determined, the integrity and the complexity of the traditional Chinese medicine preparation are fully represented, and meanwhile, two ultraviolet detection wavelengths 288 and 330 are selected on the aspect of detector selection considering that the preparation contains components with weak ultraviolet absorption. In data processing, software of ' Chinese medicine chromatogram fingerprint super information characteristic digital evaluation system 4.0 ' developed by the Sun's auspicious professor and the like is selected, and compared with fingerprint software released by the Chinese pharmacopoeia, the software has obvious advantages and can reflect the consistency and stability of preparation batches. The research has certain guiding significance for the basic research, the quality control and the clinical medication of the salvianolate extract and the medicinal substances of the preparation.
According to the invention, the quality control standard is improved from the whole aspect and the key component aspect in a quantitative manner by means of UPLC fingerprint qualitative and specific effect components (rosmarinic acid, lithospermic acid and salvianolic acid B are three components with larger content in known components and have definite structures, wherein the salvianolic acid B is a main active component in salvianolic acid for injection and has the effects of resisting platelet aggregation, resisting thrombus, resisting lipid peroxidation of brain microsomes and scavenging free radicals). Compared with the prior art, the quality controllability of the product is determined comprehensively and specifically, and due to the introduction of the UPLC technology, the existing determination time generally adopting the HPLC technology is greatly shortened, so that the complexity characterization of the fingerprint spectrum of the whole product is realized. Meanwhile, a combined measuring mode combining HPLC is selected for specific evidence components, so that the product quality is fully ensured, and the performance of the product is qualitatively determined while the measuring cost is saved. The detection result has good reproducibility and stability, is beneficial to industrial popularization, and is suitable for large-scale implementation as a product quality control standard.
Drawings
FIG. 1 liquid chromatogram of different chromatographic columns for separating salvianolic acid extract
FIG. 2 is a liquid chromatogram of a sample of a salvianolic acid extract with different elution gradients (detection wavelength of 330 nm; reference is ACN ratio)
FIG. 3 is a liquid chromatogram of a sample of a salvianolic acid extract with different elution gradients (detection wavelength of 330 nm; drawing is an organic phase ratio)
FIG. 4 is a liquid chromatogram of a sample of a salvianolic acid extract with different elution gradients (partially enlarged in FIG. 3, detection wavelength 330nm)
FIG. 5 UPLC chromatogram of salvianolic acid extract sample (280nm)
FIG. 6 QTOF-MS total ion flow diagram of Salvia miltiorrhiza polyphenol acid extract sample
FIG. 7 separation chromatogram (288nm) of salvianolic acid extract under the final chromatographic conditions
FIG. 8 HPLC chromatogram of control and salvianolic acid sample (1. rosmarinic acid; 2. lithospermic acid; 3. salvianolic acid B)
FIG. 9 is a liquid chromatogram of a mixed control of salvianolic acids at different detection wavelengths, which is (A)250nm, (B)280nm, (C)330nm, (D)360nm from top to bottom
FIG. 1021 batch UPLC/MS UV spectrogram overlap of Salvianolic acid extract (280nm)
FIG. 113 HPLC overlay chart for content determination of salvianolic acid extract batches
Detailed Description
Example 1
Step 1: establishing a digital quantitative fingerprint spectrum of the salvianolate extract by adopting an ultra-high performance liquid phase multi-wavelength visible ultraviolet detection method;
(1) instruments and reagents: waters UPLC system, tandem Xevo G2qTOF mass spectrometer. Salvianolic acid extract is provided by Tianshili proud pharmaceutical Co. Acetonitrile and methanol were chromatographically pure (merck), formic acid was chromatographically pure (sigma), ultra pure water. The reference substances, danshensu sodium, protocatechualdehyde, salvianolic acid B and rosmarinic acid, were obtained from the institute of food and drug testing, alkannic acid (Tianjin Mark Biotechnology), salvianolic acid D (Tianjin Tech science and technology, Ltd.), salvianolic acid A (Nantong Feiyu) and salvianolic acid-LM (Tianshili Co., Ltd.).
(2) Preparation of control solutions:
respectively weighing about 5mg salvianolic acid B as reference; rosmarinic acid and lithospermic acid controls were each about 3 mg; adding about 2mg each of sodium danshensu, protocatechualdehyde, salvianolic acid D, salvianolic acid A, and salvianolic acid-LM reference substance into 5mL volumetric flask, adding ultrapure water to desired volume, shaking, and filtering with microporous membrane (0.22 μm) to obtain filtrate.
(3) Preparation of a test solution: precisely weighing 21 batches of salvianolic acid extracts respectively, dissolving the samples in ultrapure water, dissolving the samples in a 10mL volumetric flask, filtering the dissolved samples through a 0.22 mu m filter membrane, and diluting the dissolved samples into a solution with the concentration of 1 mg/mL;
(4) determination of fingerprint spectrum: precisely absorbing 2 mu L of the test solution, injecting into an ultra-high performance liquid chromatograph, and recording the chromatogram;
wherein, the chromatographic conditions are as follows:
a chromatographic column: agilent Eclipse Plus C18,1.8 μm,2.1 × 150 mm;
mobile phase: solvent A: water, 0.2% formic acid; solvent B: acetonitrile, 3% methanol;
gradient elution:
0-3min,12-18％B；3-15min,18％B；15-17min,18-20％B；17-25min,20％B；25-28min,20-23％B；28-35min,23％B；35-40min,23-40％B；40-45min,40-95％B；45-50min,95％B；
temperature: 30 ℃;
flow rate: 0.2 ml/min;
chromatogram spectrum acquisition range: 190-400 nm.
Step 2: identifying peaks in the fingerprint spectrum in the step 1 by using a mass spectrum, and determining fingerprint peaks shared by ultraviolet detection;
the mass spectrometry conditions were as follows:
qTOF adopts (-) -ESI ion source, the atomization gas is high-purity nitrogen, and the collision gas is high-purity helium;
scanning range: m/z is 100-1200;
collision energy: 6 eV;
MSE gradient cleavage energy: 20-40 eV;
performing UPLC fingerprint analysis on 21 batches of radix Salviae Miltiorrhizae polyphenol acid extract samples under the above chromatographic conditions. Comparative analysis was performed on the 23 common peak uv spectra of each sample. The obtained ultraviolet spectrum is shown in FIG. 10.
The similarity of the fingerprints of each batch is calculated by using software of a Chinese medicine chromatogram fingerprint super-information characteristic digital evaluation system 4.0.
The quality of 21 batches of samples was evaluated using the systematic fingerprint quantitation standard shown in table 1. The results are shown in Table 6:
TABLE 6 calculation of the 2 nd level evaluation results of the traditional Chinese medicine system fingerprint quantitative method
| Para. | Sm | P2％ | α | Grade | Quality of |
| S1 | 0.966 | 63.8 | 0.015 | Ⅵ | In general |
| S2 | 0.443 | 2.1 | 1.243 | Ⅷ | Bad quality |
| S3 | 0.957 | 45.3 | 0.038 | Ⅷ | Bad quality |
| S4 | 0.969 | 70 | 0.031 | Ⅵ | In general |
| S5 | 0.965 | 62.2 | 0.039 | Ⅵ | In general |
| S6 | 0.987 | 65 | 0.012 | Ⅵ | In general |
| S7 | 0.982 | 64 | 0.035 | Ⅵ | In general |
| S8 | 0.955 | 98.7 | 0.009 | Ⅰ | Is excellent in |
| S9 | 0.944 | 96.4 | 0.015 | Ⅱ | Is very good |
| S10 | 0.986 | 100.6 | 0.021 | Ⅰ | Is excellent in |
| S11 | 0.992 | 104.3 | 0.007 | Ⅰ | Is excellent in |
| S12 | 0.994 | 107.9 | 0.011 | Ⅱ | Is very good |
| S13 | 0.992 | 109.3 | 0.019 | Ⅱ | Is very good |
| S14 | 0.969 | 106.9 | 0.019 | Ⅱ | Is very good |
| S15 | 0.991 | 103.7 | 0.006 | Ⅰ | Is excellent in |
| S16 | 0.992 | 106.3 | 0 | Ⅱ | Is very good |
| S17 | 0.986 | 104.3 | 0.015 | Ⅰ | Is excellent in |
| S18 | 0.994 | 94.3 | 0.012 | Ⅱ | Is very good |
| S19 | 0.988 | 106.9 | 0.001 | Ⅱ | Is very good |
| S20 | 0.973 | 94.3 | 0.031 | Ⅱ | Is very good |
| S21 | 0.979 | 94.8 | 0.005 | Ⅱ | Is very good |
And (4) analyzing results: the unqualified quality of the batches S1-S7 is matched with the seven overdue products which are produced in earlier batches, which shows that the method can better identify the quality of the medicines, well distinguish the overdue products which have larger quality difference and are produced in earlier batches, and can be used for inspecting the quality of the produced medicines.
And step 3: and (3) determining the contents of the three index components which are identified in the step (2) and have larger contents, more obvious activities and definite structures by adopting a high performance liquid chromatography variable wavelength detection method.
(1) Instruments and reagents: an Agilent 1100 high performance liquid chromatograph (Agilent Technologies, Germany) equipped with a quaternary pump, an autosampler, a column oven, and a VWD detector; METTLER TOLEDO electronic balance (type XS); 5 batches of salvianolic acid (available from Tianjin Tianshili proud pharmaceutical Co., Ltd.); salvianolic acid B control (USP, 95% purity, lot: F0M 013); rosmarinic acid control (USP, 99.4% purity, lot: F0M 076); lithospermic acid control (provided by Tianjin Tianshili Chinese medicine, purity 95.37%, batch number: 12092801); methanol (chromatographically pure, Merck); acetonitrile (chromatographically pure, Merck); ultrapure water; formic acid (reagent grade, Sigma-Aldrich).
(2) Preparation of a test solution: precisely weighing 15mg of each of the 3 batches of salvianolate extracts, adding the initial mobile phase for dissolving, fixing the volume to 25mL, and shaking up to obtain the final product.
(3) Preparation of mixed control solution: accurately weighing appropriate amount of rosmarinic acid, lithospermic acid and salvianolic acid B reference substances respectively, and adding initial mobile phase to prepare mixed reference substance solution to obtain mixed reference substance solution with rosmarinic acid mass concentration of 120.50 μ g/mL, lithospermic acid mass concentration of 121.10 μ g/mL and salvianolic acid B mass concentration of 1520.00 μ g/mL.
(4) Precisely sucking 2 mu L of the test solution, injecting into a high performance liquid chromatograph, and simultaneously determining the content of rosmarinic acid, lithospermic acid and salvianolic acid B in the test solution by adopting an external standard peak area method;
wherein, the chromatographic conditions are as follows:
a chromatographic column: agilent ZORBAX Eclipse Plus C18 (4.6X 250mm,5 μm);
mobile phase: phase A: water, 0.5% formic acid; phase B: 2% methanol and 0.5% formic acid;
gradient elution: 0-40 min, 20% B; 40-50 min, 20-70% B;
flow rate: 1mL min-1;
column temperature: 30 ℃;
the detection wavelength is 288 nm;
the results are shown in Table 7 and the HPLC overlay is shown in FIG. 11.
TABLE 7.3 the contents of the components in the Salvianolic acid extracts of batches
Example 2
Step 1: establishing a digital quantitative fingerprint spectrum of the salvianolate extract by adopting an ultra-high performance liquid phase multi-wavelength visible ultraviolet detection method;
(1) preparation of control solutions:
weighing about 2.5mg salvianolic acid B as reference; rosmarinic acid and lithospermic acid controls were each about 1.5 mg; adding about 1mg each of sodium danshensu, protocatechualdehyde, salvianolic acid D, salvianolic acid A, and salvianolic acid-LM reference substance into 5mL volumetric flask, adding ultrapure water to desired volume, shaking, and filtering with microporous membrane (0.22 μm) to obtain filtrate.
(2) Preparation of a test solution: precisely weighing 10 batches of salvianolic acid extracts respectively, dissolving the samples in ultrapure water, dissolving the samples in a 10mL volumetric flask, filtering the dissolved samples through a 0.22 mu m filter membrane, and diluting the dissolved samples into a solution with the concentration of 5 mg/mL;
(3) determination of fingerprint spectrum: precisely absorbing 1 mu L of test solution, injecting into an ultra-high performance liquid chromatograph, and recording a chromatogram map to obtain the test solution;
wherein, the chromatographic conditions are as follows:
a chromatographic column: waters HSS T3C18，1.7μm，2.1x 150mm；
Mobile phase: solvent A: water, 0.1% formic acid; solvent B: acetonitrile, 2% methanol;
gradient elution:
0-3min,12-18％B；3-15min,18％B；15-17min,18-20％B；17-25min,20％B；25-28min,20-23％B；28-35min,23％B；35-40min,23-40％B；40-45min,40-95％ B；45-50min,95％B；
temperature: 40 ℃;
flow rate: 0.3 ml/min;
chromatogram spectrum acquisition range: 190-400 nm.
Step 2: identifying peaks in the fingerprint spectrum in the step 1 by using a mass spectrum, and determining fingerprint peaks shared by ultraviolet detection;
the mass spectrometry conditions were as follows:
qTOF adopts (-) -ESI ion source, the atomization gas is high-purity nitrogen, and the collision gas is high-purity helium;
scanning range: m/z is 100-1200;
collision energy: 6 eV;
MSE gradient cleavage energy: 20-40 eV;
performing UPLC fingerprint analysis on 10 batches of salvianolic acid extract samples under the chromatographic conditions. Comparative analysis was performed on the 23 common peak uv spectra of each sample.
The similarity of the fingerprints of each batch is calculated by using software of a Chinese medicine chromatogram fingerprint super-information characteristic digital evaluation system 4.0.
The quality evaluation was performed on 10 batches of samples using the systematic fingerprint quantitation standard shown in table 1.
And step 3: and (3) determining the contents of the three index components which are identified in the step (2) and have larger contents, more obvious activities and definite structures by adopting a high performance liquid chromatography variable wavelength detection method.
(1) Preparation of a test solution: precisely weighing salvianolic acid extract, adding initial mobile phase for dissolving, and fixing volume to 1.2mg/mL, and shaking.
(2) Preparation of mixed control solution: accurately weighing appropriate amount of rosmarinic acid, alkannic acid, and salvianolic acid B reference, and adding initial mobile phase to obtain mixed reference solution with rosmarinic acid mass concentration of 100 μ g/ml, alkannic acid mass concentration of 150 μ g/ml, and salvianolic acid B mass concentration of 1.0 mg/ml.
(4) Precisely absorbing 5 mu L of the test solution, injecting into a high performance liquid chromatograph, and simultaneously determining the content of rosmarinic acid, lithospermic acid and salvianolic acid B in the test solution by adopting an external standard peak area method;
wherein, the chromatographic conditions are as follows:
a chromatographic column: agilent ZORBAX Eclipse Plus C18 (4.6X 250mm,5 μm);
mobile phase: phase A: water, 0.3% formic acid; phase B: 3% methanol and 0.3% formic acid;
gradient elution: 0-40 min, 20% B; 40-50 min, 20-70% B;
flow rate: 0.9 mL/min;
column temperature: 35 ℃;
the detection wavelength is 288 nm;
example 3
Step 1: establishing a digital quantitative fingerprint spectrum of the salvianolate extract by adopting an ultra-high performance liquid phase multi-wavelength visible ultraviolet detection method;
(1) preparation of control solutions:
weighing about 7.5mg salvianolic acid B as reference; rosmarinic acid and lithospermic acid controls were each about 2 mg; adding about 2.5mg each of sodium danshensu, protocatechualdehyde, salvianolic acid D, salvianolic acid A, and salvianolic acid-LM reference substance into 5mL volumetric flask, adding ultrapure water to desired volume, shaking, and filtering with microporous membrane (0.22 μm) to obtain filtrate.
(2) Preparation of a test solution: precisely weighing 50 batches of salvianolic acid extracts respectively, dissolving the samples in ultrapure water, dissolving the samples in a 10mL volumetric flask, filtering the dissolved samples through a 0.22 mu m filter membrane, and diluting the dissolved samples into a solution with the concentration of 3 mg/mL;
(3) determination of fingerprint spectrum: precisely absorbing 5 mu L of test solution, injecting into an ultra-high performance liquid chromatograph, and recording a chromatogram map to obtain the test solution;
wherein, the chromatographic conditions are as follows:
a chromatographic column: agilent Eclipse Plus C18,1.8 μm,2.1 × 150 mm;
mobile phase: solvent A: water, 0.5% formic acid; solvent B: acetonitrile, 5% methanol;
gradient elution:
0-3min,12-18％B；3-15min,18％B；15-17min,18-20％B；17-25min,20％B；25-28min,20-23％B；28-35min,23％B；35-40min,23-40％B；40-45min,40-95％B；45-50min,95％B；
temperature: 35 ℃;
flow rate: 0.2 ml/min;
chromatogram spectrum acquisition range: 190-400 nm.
Step 2: identifying peaks in the fingerprint spectrum in the step 1 by using a mass spectrum, and determining fingerprint peaks shared by ultraviolet detection;
the mass spectrometry conditions were as follows:
qTOF adopts (-) -ESI ion source, the atomization gas is high-purity nitrogen, and the collision gas is high-purity helium;
scanning range: m/z is 100-1200;
collision energy: 6 eV;
MSE gradient cleavage energy: 20-40 eV;
performing UPLC fingerprint analysis on 50 batches of salvianolic acid extract samples under the chromatographic conditions. Comparative analysis was performed on the 23 common peak uv spectra of each sample.
The similarity of the fingerprints of each batch is calculated by using software of a Chinese medicine chromatogram fingerprint super-information characteristic digital evaluation system 4.0.
The quality evaluation was performed on 50 batches of samples using the systematic fingerprint quantitation standard shown in table 1.
And step 3: and (3) determining the contents of the three index components which are identified in the step (2) and have larger contents, more obvious activities and definite structures by adopting a high performance liquid chromatography variable wavelength detection method.
(1) Preparation of a test solution: precisely weighing salvianolic acid extract, adding initial mobile phase for dissolving, and fixing volume to 1.0mg/mL, and shaking.
(2) Preparation of mixed control solution: accurately weighing appropriate amount of rosmarinic acid, alkannic acid, and salvianolic acid B reference, and adding initial mobile phase to obtain mixed reference solution with rosmarinic acid mass concentration of 150 μ g/ml, alkannic acid mass concentration of 100 μ g/ml, and salvianolic acid B mass concentration of 2.5 mg/ml.
(4) Precisely sucking 1 μ L of the test solution, injecting into a high performance liquid chromatograph, and simultaneously determining the content of rosmarinic acid, lithospermic acid and salvianolic acid B in the test solution by adopting an external standard peak area method;
wherein, the chromatographic conditions are as follows:
a chromatographic column: agilent ZORBAX Eclipse Plus C18 (4.6X 250mm,5 μm);
mobile phase: phase A: water, 0.6% formic acid; phase B: 4% methanol and 0.6% formic acid;
gradient elution: 0-40 min, 20% B; 40-50 min, 20-70% B;
flow rate: 1.1 mL/min;
column temperature: 30 ℃;
the detection wavelength was 288 nm.
Claims (7)
1. A method for measuring the fingerprint spectrum and the content of a salvianolic acid extract is characterized by comprising the following steps:
step 1: establishing a digital quantitative fingerprint spectrum of the salvianolate extract by adopting an ultra-high performance liquid phase multi-wavelength visible ultraviolet detection method;
step 2: identifying peaks in the fingerprint spectrum in the step 1 by using a mass spectrum, and determining fingerprint peaks shared by ultraviolet detection;
and step 3: measuring the contents of the three index components which are identified in the step 2 and have larger contents, more obvious activities and definite structures by adopting a high performance liquid chromatography variable wavelength detection method;
wherein, the specific steps of the step 1 are as follows:
(1) preparation of control solutions: precisely weighing reference substances including danshensu sodium, protocatechualdehyde, salvianolic acid D, salvianolic acid A, salvianolic acid-LM, rosmarinic acid, alkannic acid and salvianolic acid B, adding ultrapure water for dissolving, and fixing the volume until the final concentrations of the danshensu sodium, protocatechualdehyde, salvianolic acid D, salvianolic acid A and salvianolic acid-LM are respectively 500 mu g/mL in 200-plus type, the final concentrations of the rosmarinic acid and the alkannic acid are respectively 600 mu g/mL in 300-plus type, and the final concentration of the salvianolic acid B is 1500 mu g/mL in 500-plus type;
(2) preparation of a test solution: precisely weighing 10-50 batches of salvianolic acid extracts respectively, dissolving the samples in ultrapure water, filtering after dissolving, and diluting into a solution with the concentration of 1mg/mL-5 mg/mL;
(3) determination of fingerprint spectrum: precisely absorbing 1-5 mu L of test solution, injecting into an ultra-high performance liquid chromatograph, and recording chromatogram;
wherein, the chromatographic conditions are as follows:
a chromatographic column: a UPLC C18 column;
mobile phase: solvent A: water, containing 0.1% -0.5% formic acid; solvent B: acetonitrile containing 1% -5% methanol;
gradient elution:
0-3min,12-18％B；3-15min,18％B；15-17min,18-20％B；17-25min,20％B；25-28min,20-23％B；28-35min,23％B；35-40min,23-40％B；40-45min,40-95％B；45-50min,95％B；
temperature: 30-40 ℃;
flow rate: 0.2-0.3 ml/min;
chromatogram spectrum acquisition range: 190-400 nm;
the specific steps of step 3 are as follows:
(1) preparing a test solution: precisely weighing salvianolic acid extract, adding initial mobile phase for dissolving, fixing volume to final concentration of 0.6-1.2mg/mL, and shaking;
(2) preparation of mixed control solution: accurately weighing appropriate amounts of rosmarinic acid, lithospermic acid and salvianolic acid B reference substances respectively, and adding an initial mobile phase to prepare a mixed reference substance solution to obtain a mixed reference substance solution with the mass concentration of rosmarinic acid of 100-150 mu g/mL, the mass concentration of lithospermic acid of 100-150 mu g/mL and the mass concentration of salvianolic acid B of 1.0-2.5 mg/mL;
(3) precisely sucking 1-5 μ L of the test solution, injecting into a high performance liquid chromatograph, and simultaneously determining the content of rosmarinic acid, lithospermic acid and salvianolic acid B in the test solution by adopting an external standard peak area method;
wherein, the chromatographic conditions are as follows:
a chromatographic column: HPLC C18 column;
mobile phase: phase A: water, containing 0.3 to 0.6 percent of formic acid; phase B: acetonitrile, 2-4% of methanol and 0.3-0.6% of formic acid;
gradient elution: 0-40 min, 20% B; 40-50 min, 20-70% B;
flow rate: 0.9-1.1 mL/min;
column temperature: 30-35 ℃;
detection wavelength: 288 nm;
wherein, the initial mobile phase in the steps (1) and (2) refers to a mixed solution of 80% of A mobile phase and 20% of B mobile phase.
2. The method of claim 1, wherein, in step 1,
the chromatographic column preferably comprises: a UPLC C18 column;
the mobile phase is preferably:
solvent A: water, 0.2% formic acid; solvent B: acetonitrile, 3% methanol; or
Solvent A: water, 0.1% formic acid; solvent B: acetonitrile, 2% methanol.
3. The method of claim 1, wherein the mass spectrometry conditions in step 2 are as follows:
the quadrupole-time-of-flight mass spectrometry adopts a (-) -ESI ion source, the atomization gas is high-purity nitrogen, and the collision gas is high-purity helium;
scanning range: m/z is 100-1200;
collision energy: 6 eV;
MSE gradient cleavage energy: 20-40 eV.
4. The method of claim 1, wherein, in step 3,
the mobile phase is preferably:
phase A: water, 0.5% formic acid; phase B: acetonitrile, 2% methanol and 0.5% formic acid; or
Phase A: water, 0.3% formic acid; phase B: acetonitrile, 3% methanol and 0.3% formic acid;
the flow rate is preferably 1 mL/min;
the column temperature is preferably 30 ℃.
5. The method according to any one of claims 1 to 4, wherein the salvianolic acid extract is prepared by any suitable method in the art, or prepared into any preparation.
6. The method of claim 5, wherein the salvianolic acid extract is prepared by: taking salvia miltiorrhiza decoction pieces, decocting and extracting the salvia miltiorrhiza decoction pieces for three times by using purified water, each time lasting for 0.5-1 hour, combining three extracting solutions, cooling, adjusting the extracting solutions to be acidic by using hydrochloric acid solution, standing for 4-8 hours, filtering to obtain clear liquid medicine, purifying the liquid medicine by using a polyamide column chromatography process, washing the liquid medicine by using purified water after sample loading, eluting by using sodium bicarbonate solution, collecting eluent, adjusting the eluent to be acidic by using hydrochloric acid solution, purifying the liquid medicine by using a macroporous resin column chromatography process, washing the liquid medicine by using purified water after sample loading, eluting by using ethanol solution, collecting the eluent, decompressing and concentrating the eluent to recover ethanol, refrigerating the obtained concentrated solution for 12-24 hours, filtering, adjusting the pH value of the filtrate to be 5.3-6.0 by using sodium hydroxide solution, and freeze.
7. The method of claim 5, wherein the salvianolic acid extract is an injection prepared by: dissolving the salvianolate extract and mannitol in a weight ratio of 1: 0.1-0.5 in 10-20 times of water for injection respectively; and uniformly mixing the two solutions, adding medicinal carbon, heating, stirring, filtering, cooling the liquid medicine, performing ultrafiltration, adjusting the pH value of the filtrate to 5.5-6.0, adding injection water to a constant volume, uniformly stirring, filtering the liquid medicine by using a microporous membrane, filling into a penicillin bottle, and freeze-drying to obtain the salvianolate for injection.
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| CN110274962B (en) * | 2018-03-13 | 2022-10-18 | 天士力医药集团股份有限公司 | Method for measuring content of salvianolic acid component in astragalus-ginseng qi-tonifying dropping pills |
| CN108982703B (en) * | 2018-08-23 | 2021-05-14 | 中国检验检疫科学研究院 | Liquid chromatography-mass spectrometry detection method for polyphenol substances |
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