CN109001365B - Method for detecting salvia miltiorrhiza - Google Patents
Method for detecting salvia miltiorrhiza Download PDFInfo
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Abstract
The invention provides a method for detecting salvia miltiorrhiza. The detection method comprises the following steps: thin-layer chromatography detection, tanshinone substance content detection, salvianolic acid B content detection, and fingerprint detection. In the detection method, the thin-layer chromatography developing agent and the chromatographic conditions are optimized, so that the separation effect of each impurity peak is better, and the detection result is more accurate; meanwhile, the detection method has better tolerance, reproducibility and specificity.
Description
Technical Field
The invention relates to the field of traditional Chinese medicine quality detection, and particularly relates to a method for detecting salvia miltiorrhiza.
Background
Salvia miltiorrhiza, which is the dried root and rhizome of Salvia miltiorrhiza Bge (Salvia miliiorrhiza Bge.) belonging to the family Labiatae, is distributed in most areas of China. It has effects of promoting blood circulation, removing blood stasis, dredging channels, relieving pain, clearing heart fire, relieving restlessness, cooling blood, and resolving carbuncle, and can be used for treating cardiovascular diseases in clinic, such as compound Saviae Miltiorrhizae radix tablet, compound Saviae Miltiorrhizae radix dripping pill, DANQI tablet, GUANXINDANSHEN tablet, etc., which are common clinical preparations.
For quality standard detection of Salvia miltiorrhiza, various corresponding methods are provided in standard methods such as pharmacopoeia and the like. However, in the conventional standard detection method, although the standard requirement can be met, the deviation is large, and the accuracy and precision of the detection result still need to be improved.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a method for detecting salvia miltiorrhiza, which has good tolerance, reproducibility and specificity and accurate detection result.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a method for detecting salvia miltiorrhiza comprises the following steps: (a) preparing Saviae Miltiorrhizae radix reference medicinal material into reference medicinal material solution; collecting tanshinone IIA and salvianolic acid B to obtain corresponding reference solutions;
detecting a salvia miltiorrhiza test sample solution by adopting a thin-layer chromatography method by taking ethyl acetate-butanone-glacial acetic acid as a developing agent;
(b) detecting the content of tanshinone in Saviae Miltiorrhizae radix by high performance liquid chromatography; wherein, in the high performance liquid chromatography, gradient elution is carried out by adopting the following conditions:
wherein, the mobile phase A is acetonitrile; the mobile phase B is a mixed solution of buffer solution with pH of 3.2-3.6 and acetonitrile, and the ratio of the buffer solution to the acetonitrile is 93-95: 5-7;
(c) detecting the content of salvianolic acid B in Saviae Miltiorrhizae radix by high performance liquid chromatography, and performing gradient elution under the following conditions:
wherein the mobile phase A is acetonitrile, and the mobile phase B is a buffer solution with pH3.0-4.0.
Compared with the prior art, the invention has the beneficial effects that:
in the detection method, the separation effect of each impurity peak is better and the detection result is more accurate by optimizing the chromatographic conditions; meanwhile, the detection method has better tolerance, reproducibility and specificity.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a diagram showing the results of thin layer chromatography control tests in example 1 of the present invention;
FIG. 2 is a linear calibration curve according to example 2 of the present invention;
FIG. 3 is a linear calibration curve according to example 3 of the present invention;
FIG. 4 is a matching chart of the precision similarity evaluation system in example 4 of the present invention;
FIG. 5 is a matching chart of the repeatability similarity evaluation system in embodiment 4 of the present invention;
fig. 6 is a matching diagram of the stability similarity evaluation system in embodiment 4 of the present invention;
FIG. 7 is a matching diagram of a system for evaluating similarity of 15 batches of Salvia miltiorrhiza Bunge in example 4 of the present invention;
fig. 8 is a reference fingerprint of salvia miltiorrhiza bunge in embodiment 4 of the invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The salvia miltiorrhiza detection method provided by the invention is optimized on the basis of standard detection methods such as pharmacopoeia and the like, and the separation effect of impurities is improved and the accuracy of a detection result is improved by adjusting and optimizing a developing agent used in thin-layer chromatography separation, an eluent used in high-performance liquid chromatography and an elution method.
In the detection method, related substances in the salvia miltiorrhiza are mainly detected by thin layer chromatography and high performance liquid chromatography.
Specifically, the method comprises the following steps:
(1) thin layer chromatography detection
The thin-layer chromatography can analyze a plurality of samples on a thin-layer plate at the same time, and the color image of the chromatography has strong characteristics, is easy to observe, identify and compare in parallel and is one of the common methods for identifying traditional Chinese medicines.
In the existing standard method, the method mainly adopts a method that ethanol extract of salvia miltiorrhiza is used as centrifugal supernatant liquid of a test sample solution, chloroform-toluene-ethyl acetate-methanol-formic acid (6: 4: 8:1: 4) is used as a developing agent and is expanded to about 4cm, the supernatant liquid is taken out and dried, petroleum ether (60-90 ℃) and ethyl acetate (4: 1) are used as a developing agent and are expanded to about 8cm, the supernatant liquid is taken out and dried, and the detection is carried out by respectively inspecting under sunlight and an ultraviolet lamp (365 nm). The method is complicated to operate, needs to be developed in different developing agents for multiple times in sequence, and needs to be monitored for a long time.
In the invention, after the raw material salvia miltiorrhiza to be detected is crushed, the powder is sieved by a third sieve to obtain salvia miltiorrhiza powder; then, adding the salvia miltiorrhiza powder into ether for ultrasonic treatment for 6-10 min, filtering, removing filter residues, evaporating the obtained filtrate to dryness, and dissolving with ethanol to obtain a salvia miltiorrhiza test sample solution;
meanwhile, preparing the salvia miltiorrhiza reference medicinal material into a salvia miltiorrhiza reference medicinal material solution according to the same method of the salvia miltiorrhiza test sample solution;
adding ethanol into tanshinone IIA and salvianolic acid B, and making into corresponding reference solutions.
Then, respectively developing a salvia miltiorrhiza test sample solution, a salvia miltiorrhiza reference medicinal material solution, a tanshinone IIA reference solution and a salvianolic acid B reference solution by using a mixed solution of ethyl acetate, butanone and glacial acetic acid in a volume ratio of 8:1:2 under the same environmental conditions; spreading for about 8cm, taking out, air drying, and performing contrast inspection under sunlight and ultraviolet lamp (365 nm).
As can be seen from the thin layer chromatography method of the invention, the detection method of the invention does not need to be developed in different solutions for many times, and the used solvent does not contain organic solvents such as benzene and toluene which have great harm to human bodies, so the safety is good.
(2) Detection of tanshinone substance content in salvia miltiorrhiza sample by high performance liquid chromatography
The reference solution was prepared as follows: accurately weighing appropriate amount of tanshinone IIA reference substance, placing into a measuring flask, adding methanol to obtain solution containing 20 μ g per 1 ml;
the preparation method of the test solution refers to the following steps: placing 0.3g of radix Salviae Miltiorrhizae powder to be detected (sieved by a third sieve) in a conical flask, adding 50ml of methanol precisely, weighing, ultrasonically treating (power 140W and frequency 42kHz) for 30min, cooling, weighing again, supplementing the lost weight with methanol, shaking, filtering, and collecting the filtrate;
the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; the column temperature is 20 ℃; the detection wavelength is 270 nm; the number of theoretical plates should not be lower than 60000 calculated according to tanshinone IIA peak;
acetonitrile is used as a mobile phase A; taking a mixed solution of citric acid-sodium citrate buffer solution with pH value of 3.2-3.6 and acetonitrile in a volume ratio of (93-95) to (5-7) as a mobile phase B, and performing gradient elution according to the following conditions:
0-5 min, mobile phase A10 → 20%, mobile phase B90 → 80% (volume ratio, the sum of the two is 100%, the same below); 5-22 min, mobile phase A20 → 60%, mobile phase B80 → 40%; 22-22.5 min, mobile phase A60 → 10%, mobile phase B40 → 90%; 22.5-25 min, 10% of mobile phase A and 90% of mobile phase B.
Compared with the elution methods disclosed in the prior art such as pharmacopoeia and the like, the detection method disclosed by the invention can further improve the impurity separation effect and improve the detection precision and accuracy by adjusting the eluent and the elution method.
(3) Detection of salvianolic acid B content in salvia miltiorrhiza sample by high performance liquid chromatography
The control solution was prepared as follows: accurately weighing appropriate amount of salvianolic acid B reference, placing into a measuring flask, and adding methanol-water (8:2) mixed solution to obtain solution containing 0.10mg per 1 ml.
The preparation method of the test solution is as follows: placing the salvia miltiorrhiza powder to be detected (passing through a third sieve) in a conical bottle with a plug, taking about 0.15g of salvia miltiorrhiza sample powder to be detected (passing through the third sieve), precisely weighing, placing in the conical bottle with the plug, precisely adding 50ml of methanol-water (8:2) mixed solution, sealing the plug, weighing, ultrasonically treating (power 140W, frequency 42kHz) for 30 minutes, cooling, weighing again, complementing the lost weight with the methanol-water (8:2) mixed solution, shaking up, filtering, precisely weighing 5ml of subsequent filtrate, transferring to a 10ml measuring flask, adding the methanol-water (8:2) mixed solution to dilute to a scale, shaking up, filtering, and taking the subsequent filtrate to obtain the salvia miltiorrhiza product.
The chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; the column temperature is 20 ℃; the flow rate is 1.2ml per minute, and the detection wavelength is 286 nm; the number of theoretical plates is not less than 6000 calculated according to the salvianolic acid B peak;
acetonitrile is used as a mobile phase A, a disodium hydrogen phosphate-sodium citrate buffer solution with the pH value of 3.0-4.0 is used as a mobile phase B, and elution is carried out according to the following conditions:
0-15 min, mobile phase A10 → 15%, mobile phase B90 → 85% (volume ratio, the sum of the two is 100%, the same below); 15-30 min, mobile phase A15 → 35%, mobile phase B85 → 65%; 30-40 min, mobile phase A35 → 10%, mobile phase B65 → 90%; 40-45 min, 10% of mobile phase A and 90% of mobile phase B.
Different from isocratic elution methods disclosed in pharmacopoeia and other prior arts, in the invention, a gradient elution mode is adopted to detect salvianolic acid B. Similarly, the separation effect of impurities can be further improved and the detection precision and accuracy can be improved by adjusting the eluent and the elution method.
(4) Furthermore, the detection method of the invention can further comprise a step of detecting at least one index of impurities, water, total ash, acid insoluble ash, metals and harmful elements, and extracts, thereby realizing the overall detection of the quality of the salvia miltiorrhiza.
Example 1 thin layer chromatography of Salvia miltiorrhiza samples
Taking 1g of Saviae Miltiorrhizae radix (screened by a third sieve), adding diethyl ether about 10ml, performing ultrasonic treatment for 8min, filtering, removing residue, evaporating the filtrate, and dissolving with 3ml ethanol to obtain Saviae Miltiorrhizae radix sample solution;
taking 1g of Saviae Miltiorrhizae radix reference medicinal material, and preparing into Saviae Miltiorrhizae radix reference medicinal material solution according to the same method;
collecting tanshinone IIA control, adding ethanol to obtain tanshinone IIA control solution with content of 0.5 mg/ml;
adding ethanol into salvianolic acid B reference to obtain 1.5mg/ml salvianolic acid reference solution.
Then, according to thin layer chromatography, sucking 5 μ l of each of the four solutions, respectively dropping on the same silica gel G thin layer plate to form strips, developing with ethyl acetate, butanone and glacial acetic acid (8:1:2) as developing agents to about 8cm, taking out, air drying, and respectively inspecting under sunlight and ultraviolet lamp (365 nm);
wherein, the amount of the developing solvent is as follows: 10ml of each; the unfolding mode is as follows: a double-groove spreading cylinder (10 multiplied by 20 cm); and (3) span extension: 8 cm; color development: inspecting under an ultraviolet lamp (365 nm); temperature: 25 ℃; humidity: 55 percent of
In the chromatogram of the test solution, spots or fluorescent spots of the same color appear at the positions corresponding to the chromatograms of the reference material and the reference solution, and the result is shown in FIG. 1.
Example 2 detection of the content of tanshinone in Salvia miltiorrhiza samples
Preparation of a reference solution:
accurately weighing appropriate amount of tanshinone IIA reference substance, placing into a measuring bottle, adding methanol to obtain solution containing 20 μ g per 1 ml.
Preparing a test solution:
taking about 0.3g of salvia miltiorrhiza sample powder to be detected (passing through a third sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of methanol, weighing, carrying out ultrasonic treatment (power 140W and frequency 42kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss by using methanol, shaking up, filtering, and taking a subsequent filtrate.
③ chromatographic conditions and system applicability test
Octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is used as a mobile phase A; taking a mixed solution of a sodium citrate-citric acid buffer solution with a volume ratio of 95:5 and pH value of 3.2 and acetonitrile as a mobile phase B, and carrying out gradient elution according to the following conditions:
| time (min) | Mobile phase A (v/v%) | Mobile phase B (v/v%) |
| 0～5 | 10→20 | 90→80 |
| 5～22 | 20→60 | 80→40 |
| 22～22.5 | 60→10 | 40→90 |
| 22.5～25 | 10 | 90 |
The column temperature is 20 ℃; the detection wavelength was 270 nm. The number of theoretical plates should not be lower than 60000 calculated according to tanshinone IIA peak.
The determination method comprises the following steps: respectively sucking 10ul of each of the reference solution and the test solution. Injecting into liquid chromatograph, and measuring. Calculating the corresponding retention time of cryptotanshinone and tanshinone I with tanshinone IIA reference and corresponding peak as S peak, wherein the relative retention time is within + -5% of the specified value. The corresponding retention times and correction factors are shown in the following table:
| component to be measured (Peak) | Relative retention(s) | Time correction factor |
| Cryptotanshinone | 0.75 | 1.18 |
| Tanshinone I | 0.79 | 1.31 |
| Tanshinone IIA | 1.00 | 1.00 |
Linear test
Collecting tanshinone IIA control solution (23.02 μ g/ml). 2, 4, 8, 12, 16 and 20 mul are precisely measured and injected into a chromatograph, and the results are shown in the following table according to the text chromatographic condition test:
| numbering | 1 | 2 | 3 | 4 | 5 | 6 |
| Sample volume (ug) | 0.04604 | 0.09208 | 0.18416 | 0.27624 | 0.36832 | 0.4604 |
| Peak area (A) | 318.9614 | 642.0988 | 1288.8495 | 1932.5615 | 2576.9949 | 3223.0073 |
The peak area is used as the ordinate (Y) and the sample amount is used as the abscissa (X) to draw a standard curve, and the resolution is shown in FIG. 1. The regression equation is: 7006.6x-2.9372, correlation coefficient R 21, the linear relation of tanshinone IIA in the range of 0.04604-0.4604 ug is good;
instrument precision test
Taking a reference substance solution prepared under the item I, repeatedly injecting samples for 6 times under the chromatographic condition, determining the peak area of tanshinone IIA, calculating the RSD (tanshinone IIA) value to be 0.24 percent, indicating that the precision of the instrument is good, and determining results are shown in the following table:
| numbering | 1 | 2 | 3 | 4 | 5 | 6 | Mean value of | RSD％ |
| Area (A) | 1611.9132 | 1611.9429 | 1617.2340 | 1618.1431 | 1618.9027 | 1621.7645 | 1616.650067 | 0.24％ |
Sixth repeatability test
Precisely weighing 6 parts of the same batch of salvia miltiorrhiza samples, and measuring the content of the tanshinone in the samples according to the method, wherein the average content is 0.30 percent, and the RSD value is 1.74 percent, which shows that the method for measuring the tanshinone has good repeatability; the results are shown in the following table:
stability test
Preparing one part of test solution according to the method below, standing at room temperature, injecting samples for 0, 2, 4, 6, 8 and 10 hours respectively according to the proposed chromatographic condition, and measuring the peak area. The RSD% is calculated to be 0.19%, which shows that the cryptotanshinone, the tanshinone I and the tanshinone IIA are relatively stable within 10 hours. The stability test results are shown in the following table:
| numbering | 0h | 2h | 4h | 6h | 8h | 10h | Mean value of | RSD(％) |
| Total peak area (A) | 1342.5241 | 1342.8654 | 1345.2145 | 1347.1201 | 1341.0213 | 1340.2689 | 1343.16915 | 0.19％ |
Recovery test
And (4) adopting a sample recovery method. Taking 6 parts of sample with known content (content of tanshinone IIA): 1.8mg/g), precisely weighing 0.15g each part, adding 5ml of tanshinone IIA reference solution (50 μ g/ml) respectively, and preparing according to the preparation method below. The average recovery was 98.98% and the RSD% value was 0.95% as determined under the chromatographic conditions described above, with the results shown in the following table:
ninthly, content determination of sample
The ten samples were subjected to assay as described above and the results are shown in the following table:
as can be seen from the above data, the salvia miltiorrhiza bunge detected in the invention contains tanshinone IIA (C)19H18O3) Cryptotanshinone (C)19H20O3) And tanshinone I (C)18H12O3) The average of the total amount was 0.43%.
Example 3 detection of Salvianolic acid B content in Salvia miltiorrhiza Bunge samples
Preparation of a reference solution:
accurately weighing appropriate amount of salvianolic acid B reference, placing into a measuring flask, and adding methanol-water (8:2) mixed solution to obtain solution containing 0.10mg per 1 ml.
② preparation of test solution
Taking about 0.15g of salvia miltiorrhiza powder to be detected (passing through a third sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of methanol-water (8:2) mixed solution, sealing the plug, weighing, carrying out ultrasonic treatment (power 140W and frequency 42kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with the methanol-water (8:2) mixed solution, shaking up, filtering, precisely weighing 5ml of subsequent filtrate, transferring to a 10ml measuring flask, adding the methanol-water (8:2) mixed solution to dilute to scale, shaking up, filtering, and taking the subsequent filtrate to obtain the salvia miltiorrhiza powder.
③ chromatographic conditions and system applicability test
Octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is taken as a mobile phase A, and disodium hydrogen phosphate-sodium citrate buffer solution with the pH value of 3.2 is taken as a mobile phase B; the elution was carried out with a linear gradient according to the following conditions:
the column temperature is 20 ℃; the flow rate was 1.2ml per minute and the detection wavelength was 286 nm. The number of theoretical plates should not be less than 6000 calculated according to the salvianolic acid B peak.
The determination method comprises the following steps: respectively sucking 10ul of reference solution and test solution precisely; injecting into liquid chromatograph, and measuring.
Linear test
Taking salvianolic acid B control solution (0.105881 mg/ml). Precisely measuring 2, 4, 8, 12 and 16 μ l respectively, injecting into a chromatograph, and testing according to the text chromatographic conditions, wherein the results are shown in the following table:
| numbering | 1 | 2 | 3 | 4 | 5 |
| Sample volume (ug) | 0.211762 | 0.423524 | 0.847048 | 1.270572 | 1.694096 |
| Peak area (A) | 334.7132 | 673.6862 | 1342.6668 | 2012.1085 | 2679.7456 |
Drawing a standard curve by taking the peak area as a vertical coordinate (Y) and the sample amount as a horizontal coordinate (X), wherein the standard curve is shown in figure 2;
the regression equation is: 1581.3x +2.2111, coefficient of correlation R 21, the linear relationship of salvianolic acid B is good in the range of 0.211762-2.11762 ug.
Instrument precision test
Taking the reference substance solution prepared under the item of the first step, repeating sample injection for 6 times under the chromatographic conditions, measuring the area of the danshinolic acid B peak, and calculating the RSD% (danshinolic acid B) value to be 0.70%, which indicates that the precision of the instrument is good, and the measuring results are shown in the following table:
| numbering | 1 | 2 | 3 | 4 | 5 | 6 | Mean value of | RSD％ |
| Area (A) | 1119.0043 | 1115.1924 | 1112.9785 | 1097.9058 | 1109.6002 | 1103.6553 | 1109.72275 | 0.70％ |
Sixth repeatability test
6 parts of the same batch of samples (batch No. 20161101) are precisely weighed, the content of the salvianolic acid B in the samples is measured according to the method, the average content is 7.0 percent, and the RSD value is 0, which shows that the method for measuring the salvianolic acid B has good repeatability; the results of the measurements are shown in the following table:
stability test
Preparing one part of test solution according to the method below, standing at room temperature, injecting samples for 0, 2, 4, 6, 8 and 10 hours respectively according to the proposed chromatographic condition, and measuring the peak area. The calculated RSD% was 0.19%, indicating that salvianolic acid B is relatively stable over 10 h. The stability test results are shown in the following table:
recovery test
And (4) adopting a sample recovery method. 6 parts of a sample with a known content (salvianolic acid B): 70 mg/. g) are precisely weighed, 0.075g of the sample is accurately weighed, 50.0mg of the salvianolic acid B control is respectively added, and the preparation method is carried out according to the preparation method under the item 1.3.2. The average recovery was 98.75% and the RSD% value was 0.98, as measured under the chromatographic conditions described above, with the results shown in the following table:
ninthly, content determination of sample
The ten samples were subjected to content measurement according to the method of text loading, and the results are shown in the following table:
as can be seen from the above data, the Salvia miltiorrhiza Bunge detected by the invention contains salvianolic acid B (C)36H30O16) The average value of the amount of (B) was 5.0%.
Example 4 fingerprint
(1) Instrument and reagent
The instrument comprises the following steps: high performance liquid chromatograph: an agent 1260; a chromatographic workstation: an Agilent 1260 workstation; a chromatographic column: ZORBAX SB-C18(250 mm. times.4.6, 5 μm); an electronic analytical balance.
Reagent: acetonitrile is chromatographic pure, and water is ultrapure water; citric acid, sodium citrate and other reagents are analytically pure.
Sample preparation: collecting Saviae Miltiorrhizae radix in the culture base
Comparison products: rosmarinic acid reference substance, and salvianolic acid B reference substance.
(2) Method of producing a composite material
Chromatographic conditions and system applicability test with octadecylsilane chemically bonded silica as filler (column length 25cm, inner diameter 4.6mm, particle size 5 μm); performing gradient elution according to the method for detecting the content of salvianolic acid B in the salvia miltiorrhiza sample in the example 3; the detection wavelength is 286 nm; the column temperature is 20 ℃; the flow rate was 1.0ml per minute.
Preparation of reference solution A proper amount of rosmarinic acid reference substance and salvianolic acid B reference substance are precisely weighed, and methanol is added to prepare solutions each containing 0.2mg per 1 ml.
Preparation of test solution about 0.2g of the powder (sieved by a third sieve) is precisely weighed, 50ml of methanol is precisely added, and ultrasonic treatment is carried out for 20min to obtain the test solution.
The determination method comprises precisely sucking reference solution and sample solution 10 μ 1 respectively, injecting into liquid chromatograph, determining, and recording chromatogram of 75 min.
Methodology validation
Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica was used as a filler (column length 25cm, inner diameter 4.6mm, particle diameter 5 μm); performing gradient elution according to the method for detecting the content of salvianolic acid B in the salvia miltiorrhiza sample in the example 3; the detection wavelength is 286 nm; the column temperature is 30 ℃; the flow rate was 1.0ml per minute. The number of theoretical plates should not be less than 20000 calculated according to the peak of rosmarinic acid.
The specificity is as follows: respectively preparing a test solution (2 parts of parallel sample) according to the preparation method of the test solution, precisely sucking 10 mu l of each of the reference solution, the test solution (2 parts of parallel sample), the blank solution and the prepared solvent, injecting into a liquid chromatograph, measuring, and recording a chromatogram.
Precision: the sample solution prepared according to the preparation method of the sample solution is taken, the same sample solution is repeatedly injected for 6 times, no obvious difference exists in the overall appearance of the fingerprint spectrum through visual observation, the similarity is calculated by adopting a traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation system (2004A edition), and the matching graph is shown in figure 4.
The results shown in fig. 4 show that the similarity of the chromatographic fingerprint measured by the same instrument is 1.000, and the total similarity is more than or equal to 0.950, which indicates that the instrument has good precision and meets the fingerprint detection requirements.
Repeatability: the sample solution prepared according to the preparation method of the sample solution is operated for 6 times in parallel, under the chromatographic conditions, the chromatogram is recorded, the similarity is calculated by adopting a traditional Chinese medicine fingerprint similarity evaluation system (2004 edition A), and the matching graph is shown in figure 5.
As can be seen from the results shown in FIG. 5, the similarity of the chromatogram fingerprints measured by the same instrument is respectively 0.997, 0.999, 0.951, 0.997, 0.999 and 1.000, and the similarity is more than or equal to 0.95, which indicates that the method has good repeatability and meets the requirement of fingerprint detection.
Stability: the same sample is taken and detected at 6 time points of 0, 2, 4, 6, 8, 10h and the like respectively, the whole appearance of the fingerprint is observed visually without obvious change, the similarity is calculated by a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2004A edition), and a matched graph is shown as figure 6.
From the results of fig. 6, it can be seen that the similarity of the chromatographic fingerprint measured in the same instrument is 0.961, 0.994, 1.000, 0.994, 1.000, 1.000, respectively, and the similarity is greater than or equal to 0.950, which indicates that the components of the sample solution are stable in this time and meet the fingerprint detection requirements.
Taking 15 batches of salvia miltiorrhiza medicinal materials, carrying out tests according to the treatment of the test products in the method (2), and recording chromatograms. Wherein, the fingerprint numbers of 15 batches of salvia are shown in the following table:
| numbering | Batch number |
| S1 | 20141101 |
| S2 | 20141102 |
| S3 | 20141103 |
| S4 | 20141104 |
| S5 | 20141105 |
| S6 | 20151101 |
| S7 | 20151102 |
| S8 | 20151103 |
| S9 | 20151104 |
| S10 | 20151105 |
| S11 | 20161101 |
| S12 | 20161102 |
| S13 | 20161103 |
| S14 | 20161104 |
| S15 | 20161105 |
Analyzing by a traditional Chinese medicine fingerprint similarity evaluation system (2004 version A), taking a chromatogram of an S1 origin sample as a reference map, performing a median generation method, selecting a time width of 0.10min, and performing multi-point correction to generate a salvia miltiorrhiza medicinal material fingerprint common mode. The similarity is shown in the following table:
as can be seen from the data in the table above, the overall similarity of the fingerprint spectra of fifteen batches of the salvia miltiorrhiza medicinal materials is 0.954-1 (greater than 0.95, meeting the standard requirement). The matching chart of the fifteen batches of salvia miltiorrhiza medicinal material similarity evaluation system is shown in fig. 7. Meanwhile, the reference finger-prints generated by fifteen batches of salvia miltiorrhiza medicinal materials are shown in figure 8; in fig. 8, peak 2 is a characteristic peak of rosmarinic acid; peak 4 is characteristic of salvianolic acid B.
Comparative example 1
According to the method disclosed in the prior art, the tanshinone substance detection is carried out on the salvia miltiorrhiza in the same batch in the embodiment 2, and the specific method is as follows:
octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is taken as a mobile phase A, 0.02 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table;
| time (minutes) | Mobile phase a (acetonitrile) | Mobile phase B (0.1% phosphoric acid aqueous solution) |
| 0 | 10 | 90 |
| 15 | 20 | 80 |
| 35 | 25 | 75 |
| 45 | 30 | 70 |
| 55 | 90 | 10 |
| 70 | 90 | 10 |
The column temperature is 20 ℃; the detection wavelength was 270 nm. The number of theoretical plates should not be lower than 60000 calculated according to tanshinone IIA peak.
The control solution and the selection of the specific assay method and related parameters were the same as in example 2.
With reference to the method of example 2, respectively, a fifth-ninthly test was performed with the following results:
comparative example 2
According to the method disclosed in the prior art, the salvianolic acid detection is carried out on the salvia miltiorrhiza bunge of the same batch in the embodiment 2;
chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.1% phosphoric acid solution (22: 78) is used as a mobile phase; the column temperature is 20 ℃; the flow rate was 1.2ml per minute; the detection wavelength was 286 nm. The number of theoretical plates should not be less than 6000 calculated according to the salvianolic acid B peak.
The control solution and the selection of the specific assay and related parameters were the same as in example 3.
With reference to the method of example 3, respectively, a fifth-ninthly test was performed with the following results:
while particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (4)
1. A method for detecting salvia miltiorrhiza is characterized by comprising the following steps:
（a）
preparing Saviae Miltiorrhizae radix reference medicinal material into reference medicinal material solution; collecting tanshinone IIA and salvianolic acid B to obtain corresponding reference solutions;
detecting a salvia miltiorrhiza test sample solution by adopting a thin-layer chromatography method by taking ethyl acetate-butanone-glacial acetic acid as a developing agent;
（b）
detecting the content of tanshinone in Saviae Miltiorrhizae radix by high performance liquid chromatography;
in the high performance liquid chromatography, octadecylsilane chemically bonded silica is used as a filling agent, and gradient elution is carried out under the following conditions:
wherein, the mobile phase A is acetonitrile; the mobile phase B is a mixed solution of buffer solution with pH of 3.2-3.6 and acetonitrile, and the ratio of the buffer solution to the acetonitrile is 93-95: 5-7; the buffer solution is sodium citrate-citric acid buffer solution;
（c）
detecting the content of salvianolic acid B in Saviae Miltiorrhizae radix by high performance liquid chromatography with octadecylsilane chemically bonded silica as filler, and performing gradient elution under the following conditions:
wherein the mobile phase A is acetonitrile, and the mobile phase B is a buffer solution with the pH value of 3.0-4.0; the buffer solution is disodium hydrogen phosphate-sodium citrate buffer solution;
in the step (a), the volume ratio of ethyl acetate, butanone and glacial acetic acid in the developing solvent is (7-10): (1-3): (1-3);
in the step (a), the preparation of the salvia miltiorrhiza test solution comprises the following steps:
adding diethyl ether into Saviae Miltiorrhizae radix powder, ultrasonic treating, filtering, removing residue, evaporating the filtrate, and dissolving with ethanol to obtain Saviae Miltiorrhizae radix sample solution.
2. The method for detecting salvia miltiorrhiza according to claim 1, wherein in the step (a), the volume ratio of ethyl acetate, butanone and glacial acetic acid in the developing solvent is 8:1: 2.
3. The detection method according to claim 1, wherein the time of the ultrasonic treatment is 6 to 10 min.
4. The method according to claim 1, further comprising a step of detecting at least one index selected from the group consisting of impurities, water, total ash, acid-insoluble ash, heavy metals, harmful elements, and leachate.
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