CN1911273A - Fingerprint atlas detection method for powder injection contg. high content of tanshin polyphenolic acid salts - Google Patents
Fingerprint atlas detection method for powder injection contg. high content of tanshin polyphenolic acid salts Download PDFInfo
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Abstract
A fingerprint test method for the high-content powder injection of polydanshinolate includes testing red sage root, testing polydanshinolate and testing the powder injection to ensure high quality of product.
Description
Technical field:
The present invention relates to technical field of Chinese medicines.Be specifically related to a kind of high content salvianolate injectable powder fingerprint atlas detection method.
Background technology:
Radix Salviae Miltiorrhizae (Salvia miltiorrhiza Bunge) is one of traditional blood-activating and stasis-removing, and secular clinical practice basis is arranged.Radix Salviae Miltiorrhizae Injection is the common drug for the treatment of diseases such as coronary heart disease, angina pectoris, myocardial infarction, cerebral infarction clinically, but existing injection is indeterminate because of effective ingredient, the not strict clinical efficacy instability that causes of quality control index, and untoward reaction occurs often.The appearance of these problems, key are that the effective ingredient of Radix Salviae Miltiorrhizae fails to illustrate fully.
The inventor has carried out the systematic study of the water soluble ingredient of Radix Salviae Miltiorrhizae at these problems, and the aqueous soluble active constituent of finding Radix Salviae Miltiorrhizae is that the form with salt is present in the raw medicinal herbs, and these compositions are mainly Radix Salviae Miltiorrhizae acetate magnesium and homologue etc.Find by screening active ingredients and pharmaceutical research: Radix Salviae Miltiorrhizae acetate magnesium and its homologue are active component, and wherein the strongest with the Radix Salviae Miltiorrhizae acetate magnesium pharmacological action, it is the main active of Radix Salviae Miltiorrhizae
The Radix Salviae Miltiorrhizae acetate magnesium structural formula
And to have illustrated first with the salvianolate injectable powder be that the polyphenol hydrochlorate of main component is most important effective active position in the Radix Salviae Miltiorrhizae.
The inventor successfully provides the novel formulation of Radix Salviae Miltiorrhizae on this basis, and said preparation is a kind of high content salvianolate injectable powder, and wherein Radix Salviae Miltiorrhizae acetate magnesium content surpasses 80%, and all the other compositions are alkannic acid magnesium, rosmarinic acid sodium and a small amount of danshensu.
Summary of the invention:
Technical problem to be solved by this invention is research design high content salvianolate injectable powder fingerprint atlas detection method.
The present invention carries and has encircleed a kind of high content salvianolate injectable powder fingerprint atlas detection method through big quantity research.
Fingerprint atlas detection method of the present invention comprises three parts:
(1) red rooted salvia fingerprint atlas detection method;
(2) salvianolate fingerprint atlas detection method;
(3) salvianolate injectable powder fingerprint atlas detection method.
Specifically describe the test of detection method of the present invention and this method of formulation below:
1, the medicinal materials fingerprint detection method of salvianolate injectable powder:
Method of the present invention is based on following test:
The used crude drug of salvianolate injectable powder is a Radix Salviae Miltiorrhizae, and the Chinese phonetic alphabet is Danshen.
(1) source
Radix Salviae Miltiorrhizae is a labiate, and it draws in formal name used at school is Savia miltiorrhiza Bge, and medicinal part has rhizome for root.Totally 10 batches of medical materials, the place of production are respectively Anhui Province's Bozhou City five horses (No.), Anhui Province's Bozhou City five horses (two horses), the Zhang Dian of Anhui Province (No.), the Zhang Dian of Anhui Province (No. two), the Bozhou City Da Yang of Anhui Province (No.), the Bozhou City Da Yang of Anhui Province (No. two), Anhui Province's Bozhou City 19 li (No. one), Anhui Province's Bozhou City 19 li (No. two), Bozhou City cross rivers, Anhui Province, Anhui Province's Bozhou City justice door.Numbering is respectively A, B, C, D, E, F, G, H, I, J.
(2) preparation of test sample
Poly phenolic acid of Radix Salviae Miltiorrhizae is a water soluble ingredient, therefore adopt the water logging bubble after, the method for reflux, extract, can extract most poly phenolic acid of Radix Salviae Miltiorrhizae constituents wherein.Regulate pH and make it become acidity, use ethyl acetate extraction then, be used for liquid-phase chromatographic analysis behind the extract standardize solution, the result proves that this extracting method is feasible, and the dependency between intermediate and finished product finger printing is good.
(3) preparation of object of reference
Red rooted salvia is analyzed through HPLC and a plurality of chromatographic peaks to be occurred after said method extracts, but be its main component with Radix Salviae Miltiorrhizae acetic acid, and the integral area of Radix Salviae Miltiorrhizae acetic acid is shared large percentage and more stable in finger printing, so we select is object of reference.Its preparation method is for directly being dissolved in mobile phase analysis with Radix Salviae Miltiorrhizae acetate magnesium salt reference substance, and the result proves that this preparation method is feasible.
(4) mobile phase determines
The mensuration of herbal extract has been selected 5 kinds of flow phase system altogether: (1) methanol-0.1% glacial acetic acid (5: 95); (2) methanol-5mol/L potassium dihydrogen phosphate solution (30: 70); (3) methanol-5mol/L potassium dihydrogen phosphate solution (25: 75); (4) methanol-acetonitrile-water (41.5: 1: 48.5, transfer pH to 2.20) with formic acid; (5) methanol-acetonitrile-water-formic acid is (42: 1: 59: 0.2).The result shows, (42: 1: 59: 0.2) system was the best with methanol-acetonitrile-water-formic acid in the various mobile phases, each chromatographic peak (254nm) separating degree is better on the spectrogram, and retention time is suitable, therefore selects the mobile phase of this flow phase system as the HPLC finger printing detection of Radix Salviae Miltiorrhizae Injection.
(5) selection of detection wavelength
Selected 254nm, 290nm2 kind wavelength altogether, in the HPLC spectrogram under these two kinds of wavelength a plurality of chromatographic peaks have been arranged all, but chromatographic peak is more and the peak type is better when detecting wavelength and be 254nm, takes all factors into consideration, and selects 254nm for making the mensuration wavelength of finger printing.
(6) stability and precision test
Preparation sample with the medical material that is numbered C is a test sample, respectively at 0 day, 10 days and 30 days sample introductions, write down each total chromatographic peak retention time and integral area, retention time and integral area with Radix Salviae Miltiorrhizae acetate magnesium are reference, converse the relative retention time and the relative integral area at each total peak, the result shows having good stability of test sample.Be numbered the preparation sample continuous sample introduction 6 times of the medical material of C in addition, write down each total chromatographic peak retention time and integral area, retention time and integral area with Radix Salviae Miltiorrhizae acetate magnesium are reference, converse the relative retention time and the relative integral area at each total peak, the results are shown in Table 1.By table 1 as seen, (relatively) retention time at each total peak and (relative) integral area basically identical, relative standard deviation is all less than 3.0%.
The test of table 1 salvianolate injectable powder medicinal materials fingerprint precision
| Total peak numbering | Relative retention time (RSD, %) | Relative peak area (RSD, %) |
| 1 | 1.03 | 1.24 |
| 2 | 0.94 | 2.35 |
| 3 | 1.09 | 2.01 |
| 4 | 0.92 | 2.45 |
| 5 | 0.51 | 1.56 |
| 6 | 1.63 | 1.12 |
| 7(S) | 1.39 | 2.15 |
Annotate: " S " represents object of reference
(7) repeatability test
Get 5 parts of test samples that are numbered C, be prepared as stated above and detect, investigate the concordance of each chromatographic peak relative retention time, peak area ratio, the results are shown in Table 2.The relative retention time at 7 total peaks being investigated (wherein be numbered 1 and 2 chromatographic peak and be the group peak) and the relative standard deviation of relative peak area are all less than 3%.
The test of table 2 poly phenolic acid of Radix Salviae Miltiorrhizae injectable powder medicinal materials fingerprint repeatability
| The chromatographic peak numbering | Relative retention time (RSD, %) | Relative peak area (RSD, %) |
| 1 2 3 4 5 6 7(S) | 1.2 1.4 1.9 2.1 2.0 1.9 1.8 | 2.5 2.6 2.8 2.7 2.8 3.0 2.9 |
Notes S is an object of reference
(8) finger printing and technical parameter
1) finger printing
According to 10 batches of given relevant parameters of test sample HPLC collection of illustrative plates, red number medical material all occurs within 60min by the chromatographic peak that sample preparation methods is prepared the gained test sample; 2 hours chromatogram shows 1 hour and does not occur any chromatographic peak later on.Relatively the chromatogram of each batch sample finds that it is that each batch sample is common that 7 chromatographic peaks are wherein arranged, and numbering is respectively 1,2,3,4,5,6,7, and wherein 1 and 2 are the group peak.
2) ratio of total fingerprint peaks area and retention time
10 batches of medical material test samples are object of reference with the Radix Salviae Miltiorrhizae acetate magnesium, the integral area of each fingerprint peaks and integral area, and retention time and relative retention time see Table 3; Table 4 is depicted as ratio, the difference that each total fingerprint peaks accounts for total peak area, and the repeatability of visible each batch test sample of result is better, also meets " specification requirement of Chinese medicine finger printing research " (provisional).Wherein have the mean value calculation of the retention time of peak 1 and 2 by each chromatographic peak retention time of group peak, peak area is by each the chromatographic peak area sum calculating of group peak.
The ratio of total fingerprint peaks area of table 3 salvianolate injectable powder medical material and retention time
| The fingerprint peaks numbering | The fingerprint peaks relative area | The fingerprint peaks relative retention time |
| 1 2 3 4 5 6 7(S) | 0.109 0.172 0.121 0.020 0.035 0.126 1.0 | 0.22 0.32 0.47 0.55 0.60 0.77 1.0( *18.63) |
Annotate: S is an object of reference,
*Be the absolute retention time of object of reference
The ratio and the difference thereof of the shared total peak area of each total fingerprint peaks of table 4 medical material
| Total fingerprint peaks numbering | The area percentage at shared total peak (%) | Standard difference (%) | Actual measurement peak area value (%) | |
| Minima | Maximum | |||
| 1 2 3 4 5 6 7 | 6.35(＜10％) 9.5(＜10％) 7.06(＜10％) 1.16(＜10％) 2.07(＜10％) 7.35(＜10％) 58.38(20％) | - - - - - - 12.1 | - - - - - - 49.87 | - - - - - - 66.67 |
3) non-total peak area
10 batch samples of the test ratio of the shared total peak area of non-total peak area, the table 5 as a result of letting it pass of falling into a trap.
By table 5 as seen, the non-total peak area of each batch medical material accounts for total peak area all less than 10%, meets " specification requirement of Chinese medicine finger printing research " (provisional), and also promptly the used medical material of explanation production poly phenolic acid of Radix Salviae Miltiorrhizae salt injection injectable powder meets the requirements.
The percentage ratio of the non-total stricture of vagina peak area of table 5 salvianolate injectable powder HPLC finger printing
| Mission Number | The percentage ratio of non-total fingerprint peaks area (%) |
| A | 7.94 |
| B | 5.39 |
| C | 9.18 |
| D | 6.13 |
| E | 3.57 |
| F | 7.97 |
| G | 7.35 |
| H | 6.9 |
| I | 9.1 |
| J | 8.74 |
On the basis of above-mentioned a large amount of tests, determined high content salvianolate red rooted salvia stricture of vagina figure spectrum detection method.
The invention provides the red rooted salvia fingerprint atlas detection method of high content salvianolate injectable powder, this method comprises the following steps:
Radix Salviae Miltiorrhizae (Danshen) latin name Rakix Salviae Miltiorrhizae
(1) source
Derive from the dry root and rhizome of labiate Radix Salviae Miltiorrhizae Salviae miltiorrhizae Bge.
(2) preparation of test sample
Medical material is crossed 40 mesh sieves after pulverizer is pulverized, get each batch medicinal powder 500mg, the accurate title, decide, place 100ml tool plug triangular flask, respectively add the 30ml distilled water immersion and spend the night, be transferred in the round-bottomed flask, reuse 30ml distillation cooling is back with 10% hydrochloric acid adjust pH to 2.0 (measuring with acidometer), reuse ethyl acetate extraction 3 times, each 80ml.Use the mobile phase ultrasonic dissolution behind the extract concentrating under reduced pressure, and be settled to 10ml, as need testing solution with volumetric flask.HPLC uses filtering with microporous membrane before analyzing.
(3) preparation of object of reference solution
Radix Salviae Miltiorrhizae acetic acid accurately takes by weighing 4.0mg as object of reference, is settled to 5ml with mobile phase, as object of reference solution.
(4) detection method
Assay method is high performance liquid chromatography (HPLC).
1) instrument and reagent
Agilent1100 high performance liquid chromatograph and Chemstation chromatographic work station, ZorbaxC
18(4.6mm * 25cm, 5 μ m) chromatography post.Used methanol is chromatographically pure, and water is redistilled water, and formic acid is analytical pure.
2) condition determination and assay method
The a assay method
Mobile phase be methanol-acetonitrile-water-formic acid (42: 1: 59: 0.2), the detection wavelength was 254nm, and column temperature is 30 ℃, slope 2, peak width 0.05, the smallest peaks area is 50, minimum peak height is 2, flow velocity is 0.7ml/min;
B. assay method
Get each batch medical material need testing solution 5 μ L respectively, inject high performance liquid chromatograph and carry out chromatography, write down the retention time and the integral area at each peak.
(5) salvianolate injectable powder medicinal materials fingerprint and technical parameter
1) demarcation of finger printing and total fingerprint peaks
According to 10 batches of given relevant parameters of test sample HPLC collection of illustrative plates, it is that each batch sample is common that 7 chromatographic peaks are arranged in the chromatogram of red rooted salvia, the percentage ratio that its peak area summation accounts for the total peak area sum is 91.87%, numbering is respectively 1,2,3,4,5,6,7, wherein 1 and 2 are the group peak, and the ratio of each peak relative retention time is 1: 2: 3: 4: 5: 6: 7=0.22: 0.32: 0.47: 0.55: 0.60: 0.77: 1.00 (seeing the HPLC collection of illustrative plates).Fig. 1
2) ratio of total fingerprint peaks area
The ratio of 7 specified total peak-to-peak areas is 1: 2: 3 in the 10 batches of test samples: 4: 5: 6: 7=0.109: 0.172: 0.121: 0.020: 0.035: 0.126: 1.00.The percentage ratio meansigma methods of each shared total peak area in total peak is respectively group peak 1,6.35%; Group peak 2,9.50%; Peak 3,7.06%; Peak 4,1.16%; Peak 5,2.07%; Peak 6,7.35%; Peak 7 (object of reference), 58.38%.This shows, have only the relative peak area at peak 7 to surpass 10% in 7 total peaks, and the relative peak area at other 6 total peaks is all below 10%, therefore only require the existence of its chromatographic peak, and not requiring the concordance of its peak area ratio, it is 46.28%-70.48% that the relative peak area at peak 7 allows excursion.
2, salvianolate fingerprint atlas detection method:
Method of the present invention is based on following test:
(1) preparation of test sample
Salvianolate is totally 10 batches of intermediate product of the present invention.
(2) preparation of object of reference (reference substance) solution
Owing to also be that Radix Salviae Miltiorrhizae acetic acid is its main component in the salvianolate, consider its peak area shared large percentage and more stable in finger printing, so we select be object of reference.Also for directly being dissolved in the mobile phase, method of proof is feasible as a result, and is respond well for preparation method.
(3) mobile phase determines
5 kinds of flow phase system have been selected in test altogether: (1) methanol-0.1% glacial acetic acid (5: 95); (2) methanol-5mol/L potassium dihydrogen phosphate solution (30: 70); (3) methanol-5mol/L potassium dihydrogen phosphate solution (25: 75); (4) methanol-acetonitrile-water (41.5: 1: 48.5, transfer pH to 2.20) with formic acid; (5) methanol-acetonitrile-water-formic acid is (42: 1: 59: 0.2).The result shows, (42: 1: 59: 0.2) system was the best with methanol-acetonitrile-water-formic acid in the various mobile phases, each chromatographic peak (254nm) separating degree is better on the spectrogram, and retention time is suitable, therefore selects the mobile phase of this flow phase system as the HPLC finger printing detection of Radix Salviae Miltiorrhizae Injection.
(4) selection of detection wavelength
254nm, 290nm2 kind wavelength have been selected in the process of the test altogether, in the HPLC spectrogram under these two kinds of wavelength a plurality of chromatographic peaks are arranged all, but chromatographic peak is more and the peak type is better when detecting wavelength and be 254nm, takes all factors into consideration, and selects 254nm for making the mensuration wavelength of finger printing.
(5) stability and precision test
With lot number is that 000717 salvianolate is a test sample, respectively at 0 day, and 10 days and 30 days sample introductions, results sample is stable in 30 days.Sample thief continuous sample introduction 6 times, write down each total chromatographic peak retention time and integral area, retention time and integral area with Radix Salviae Miltiorrhizae acetate magnesium are reference, converse the relative retention time and the relative integral area at each total peak, result's (table 1) shows (relatively) retention time and the integral area basically identical at each total peak of salvianolate, and relative standard deviation is all less than 3%.
The precision test of table 1 salvianolate HPLC chromatography
| Total peak numbering | The relative standard deviation of relative retention time and relative peak area (RSD, %) | |
| Relative retention time | Relative peak area | |
| 1 | 242 | 2.65 |
| 3 | 2.61 | 2.43 |
| 4(S) | 2.52 | 0.62 |
Annotate: S represents object of reference
(6) repeatability test
Get lot number and be 6 parts of 000717 Radix Salviae Miltiorrhizae test samples, be prepared as stated above and detect, investigate the concordance of each chromatographic peak relative retention time, peak area ratio, result's (table 2) shows that the relative standard deviation of the relative retention time at 3 total peaks of 6 parts of test samples and relative peak area is all less than 3%.
Table 2 salvianolate HPLC analyzes the repeatability test
| The chromatographic peak numbering | Relative retention time (RSD, %) | Relative peak area (RSD, %) |
| 1 | 2.21 | 1.64 |
| 3 | 2.52 | 1.35 |
| 4(S) | 2.46 | 1.24 |
4, salvianolate HPLC finger printing and technical parameter
(1) demarcation of finger printing and total fingerprint peaks
According to 10 batches of given relevant parameters of test sample HPLC collection of illustrative plates, the chromatographic peak of all the components of salvianolate all occurs within 60min, and 4 chromatographic peaks are all arranged in each batch sample.Consider that 3 total peaks only appear in some batch sample in the finished product, and these three total peaks all occur in salvianolate, and three peak area sums are greater than 95%, therefore determine with finished product in identical three peaks be the total fingerprint peaks of salvianolate finger printing.
(2) ratio of total fingerprint peaks area and retention time
10 batches of intermediate test samples are object of reference with the Radix Salviae Miltiorrhizae acetate magnesium, the integral area of each total fingerprint peaks and relative integral area, and retention time and relative retention time see Table 3; Total fingerprint peaks accounts for ratio, the difference of total peak area in shown in the table 4, and the repeatability of visible each batch test sample of result is better, also meets " specification requirement of Chinese medicine finger printing research " (provisional).
The ratio of total fingerprint peaks area of table 3 poly phenolic acid of Radix Salviae Miltiorrhizae salt injection injectable powder and retention time
| The fingerprint peaks numbering | The fingerprint peaks relative area | The fingerprint peaks relative retention time |
| 1 | 0.02 | 0.77 |
| 3 | 0.07 | 0.93 |
| 4(S) | 1.0 | 1.0(18.31±0.56 *) |
Annotate:
*Be the absolute retention time of object of reference
On above-mentioned a large amount of experimental basis, the invention provides the high content salvianolate fingerprint atlas detection method.
The invention provides the salvianolate fingerprint atlas detection method, this method comprises the following steps:
1, the preparation of need testing solution
Get salvianolate 5.0mg respectively, precision is weighed, put in the 5ml volumetric flask, usefulness mobile phase methanol-acetonitrile-water formic acid (42: 1: 59: 0.2) standardize solution, draw 5 μ L injection performance liquid chromatographic column and analyze.
2, the preparation of object of reference (reference substance) solution
Accurately get Radix Salviae Miltiorrhizae acetate magnesium photograph product 4.0mg, precision is weighed, and puts in the 5ml volumetric flask, uses mobile phase methanol-acetonitrile-water-formic acid (42: 1: 59: 0.2) standardize solution, the object of reference (reference substance) of usefulness salvianolate finger printing.
3, detection method
Assay method is high performance liquid chromatography (HPLC).
4, the appointment of finger printing and total fingerprint peaks
4 chromatographic peaks all appear in (1) 10 batch of salvianolate test sample HPLC collection of illustrative plates, determine peak 1 (relative retention time 0.77), peak 3 (relative retention time 0.93) and peak 4 (relative retention time 1.00) are total fingerprint peaks, and promptly this 3 total peaks all should appear in each batch sample.(seeing the HPLC collection of illustrative plates) Fig. 2
(2) ratio of total fingerprint peaks area
10 batches of test samples are object of reference with the Radix Salviae Miltiorrhizae acetate magnesium, and the ratio of integral area that surpasses the total fingerprint peaks of total peak area 5% is peak 3: peak 4=0.07: 1.00, and wherein the permission excursion at peak 3 is 4.33%-8.05%, peak 4 is an object of reference.
3, the fingerprint atlas detection method of high content salvianolate injectable powder:
Method of the present invention is based on following test:
(1) preparation of test sample
The salvianolate injectable powder is totally 10 batches of products of the present invention.These product are freeze-dried powder, consider that wherein composition is single relatively, therefore directly dissolve as need testing solution with mobile phase, solvent peak occurs to avoid using other solvents.The result proves that preparation method is feasible.
(2) preparation of object of reference
Because Radix Salviae Miltiorrhizae acetic acid is its main component in the salvianolate injectable powder, 4 chromatographic peaks appear at most in 10 batches of injectable powder behind efficient liquid phase chromatographic analysis, the integral area of Radix Salviae Miltiorrhizae acetic acid is shared large percentage and more stable in finger printing, thus we select be object of reference.Its preparation method also is that the preparation method with reference to test sample directly is dissolved in the analysis of mobile phase row with Radix Salviae Miltiorrhizae acetate magnesium salt reference substance.
(3) mobile phase determines
Process of the test has been selected 5 kinds of flow phase system altogether: (1) methanol-0.1% glacial acetic acid (5: 95); (2) methanol-5mol/L potassium dihydrogen phosphate solution (30: 70); (3) methanol-5mol/L potassium dihydrogen phosphate solution (25: 75); (4) methanol-acetonitrile-water (41.5: 1: 48.5, transfer pH to 2.20) with formic acid; (5) methanol-acetonitrile-water-formic acid is (42: 1: 59: 0.2).The result shows, (42: 1: 59: 0.2) system was the best with methanol-acetonitrile-water-formic acid in the various mobile phases, each chromatographic peak (254nm) separating degree is better on the spectrogram, and retention time is suitable, therefore selects the mobile phase of this flow phase system as the HPLC finger printing detection of Radix Salviae Miltiorrhizae Injection.
(4) selection of detection wavelength
254nm, 290nm2 kind wavelength have been selected in the process of the test altogether, in the HPLC spectrogram under these two kinds of wavelength 3 chromatographic peaks are arranged all, in addition when analyzing medical material, chromatographic peak is more and the peak type is better when detecting wavelength and being 254nm, take all factors into consideration, select 254nm for making the mensuration wavelength of finger printing.
(5) stability
With lot number is that 0001216 injectable powder is a test sample, respectively at 0 day, 10 days and 30 days sample introductions, record respectively advances to have chromatographic peak retention time and integral area, retention time and integral area with Radix Salviae Miltiorrhizae acetate magnesium are reference, converse the relative retention time and the relative integral area at each total peak, the result shows having good stability of test sample.
(6) precision test
With lot number is that 001216 sample is test sample continuous sample introduction 6 times, write down each total chromatographic peak retention time and integral area, retention time and integral area with Radix Salviae Miltiorrhizae acetate magnesium are reference, converse the relative retention time and the relative integral area at each total peak, result's (table 1) shows (relatively) retention time and (relative) integral area unanimity at the total peak of salviol hydrochlorate injectable powder, and relative standard deviation is all less than 1.5%.The result shows that the precision of instrument is good.
The test of table 1 salvianolate injectable powder HPLC analytical precision
| Total peak numbering | The relative standard deviation of relative retention time and relative peak area (RSD, %) |
| Relative retention time | Relative peak area | |
| 1 2 3(S) | 0.45 0.51 0.55 | 1.12 0.52 1.41 |
Annotate: S is with reference to the phase thing
(7) repeatability test
Get 5 parts of the test samples of lot number 001216, row preparation and detecting in the time of as stated above, investigate the concordance of each chromatographic peak relative retention time, peak area ratio, result's (table 2) shows that 5 parts of test samples all have 4 chromatographic peaks, wherein 3 are total peak, the phase distich standard deviation of its relative retention time and relative peak area shows that all less than 3% its repeatability is good.
Table 2 salvianolate injectable powder HPLC analyzes the repeatability test
| The chromatographic peak numbering | Relative retention time (RSD, %) | Relative peak area (RSD, %) |
| 2 (non-total peaks) 3,1 (total peak) (total peak) 4 (total peak, S) | 1.9 2.1 1.7 1.8 | 1.7 0.8 1.6 0.8 |
Annotate: S is an object of reference
4, finger printing and technical parameter
(1) finger printing
Under specified chromatographic condition, investigated eluting 1 hour and 2 hours chromatogram go out the peak situation, all chromatographic peaks are all complete with interior eluting at 25 minutes as a result.The relative retention time and the peak area at 10 each total peak of batch sample see Table 3.
Table 3 pellet is with total fingerprint peaks area of polyphenol hydrochlorate injectable powder and retention time ratio
| The fingerprint peaks numbering | The ratio of fingerprint peaks relative area | Relative retention time ratio |
| 1 2 | 0.017 0.051 | 0.78 0.93 |
| 3(S) | 1.0 | 1.0(18.13±0.65 *) |
Annotate: S is an object of reference,
*Be the absolute retention time of object of reference
(2) demarcation of total fingerprint peaks
By the fingerprint pattern technology requirement, the peak area sum of total fingerprint peaks should be more than 95%, test result according to 10 batch samples, 4 chromatographic peaks appear in 5 batch samples, and 3 chromatographic peaks appear in other 5 batch samples, these 3 chromatographic peaks are chromatographic peaks common in 10 batch samples, and its peak area sum-total average is 99.69%, therefore select these 3 peaks as total fingerprint peaks.The ratio of the shared total peak area in total peak and the maximum and the minima of surveying each batch sample peak area see Table 4.
On above-mentioned a large amount of experimental basis, determined high-load salviol hydrochlorate injectable powder fingerprint image detection method.
The invention provides high content salvianolate injectable powder fingerprint atlas detection method, this method comprises the following steps:
(1) preparation of need testing solution
Get injectable powder 5.0mg respectively, precision is weighed, put in the 5mL volumetric flask, usefulness mobile phase methanol-acetonitrile-water formic acid (42: 1: 59: 0.2) standardize solution, draw 5 μ L injection performance liquid chromatographic column and analyze.
(2) preparation of object of reference solution
Accurately get Radix Salviae Miltiorrhizae acetate magnesium reference substance 4.0mg, precision is weighed, and puts in the 5mL volumetric flask, and ((42: 1: 59: 0.2)) standardize solution is used for the object of reference (reference substance) of salvianolate injectable powder finger printing with mobile phase methanol-acetonitrile-water formic acid.
(3) detection method
Assay method is high performance liquid chromatography (HPLC).
1) instrument and reagent
Agilent1100 high performance liquid chromatograph and Chemstation chromatographic work station, ZorbaxC
18(4.6mm * 25cm, 5 μ m) chromatography post.Used methanol is chromatographically pure, and water is redistilled water, and formic acid is analytical pure.
2) condition determination and assay method
A. condition determination
Mobile phase be methanol-acetonitrile-water-formic acid (42: 1: 59: 0.2), the detection wavelength was 254nm, and column temperature is 30 ℃, slope 2, peak width 0.05, the smallest peaks area is 20, minimum peak height is 1, flow velocity is 0.7ml/min;
B. assay method
Get each batch injection need testing solution 5 μ L respectively, inject high performance liquid chromatograph and analyze, write down the retention time and the integral area at each peak.
(4) finger printing and technical parameter
1) demarcation of finger printing and total fingerprint peaks
According to 10 batches of given relevant parameters of test sample HPLC collection of illustrative plates, the chromatographic peak of salvianolate injectable powder all the components all occurs within 60min, and determines that (peak 1: retention time is 14.10min to 3 chromatographic peaks; Peak 2: retention time is 17.00min; Peak 3: retention time is 18.25min) for having fingerprint peaks.(seeing the HPLC finger printing) Fig. 3
2) ratio of total fingerprint peaks area and retention time
The ratio of the relative retention time of each total fingerprint peaks is: 1/ peak, peak, 2/ peak 3=0.78/0.93/1.00.The percentage ratio meansigma methods of peak 1 shared total peak area is 1.75%, the meansigma methods that peak 2 accounts for the percentage ratio of total peak area is 4.97%, the meansigma methods of the percentage ratio of peak 3 shared total peak areas is 92.97%, therefore having only peak 3 in 3 total peaks is that the peak area of object of reference surpasses 5%, can't calculate its ratio by the fingerprint pattern technology requirement, the theoretic permission excursion in this peak is 74.38%-111.56%.
Method of the present invention has realized at same chromatographic condition (methanol-acetonitrile-water-formic acid (42: 1: 59: 0.2, detect wavelength 254nm) under, direct injected just can be finished the test of finger printing, inspection through many batches of finished products, intermediate and medical material, the result shows that this method is stable, reliable, easy, can control effectively red can polyphenol hydrochlorate injectable powder in the quality of main component (Radix Salviae Miltiorrhizae acetate magnesium) and other component, show that simultaneously finished product, intermediate, the stability of medical material, the repeatability of different lot numbers is good.
The quality standard that the invention provides salvianolate is as follows:
[method for making] gets Radix Salviae Miltiorrhizae, decocts with water three times, decocts 2 hours at every turn.Merge decoction liquor, filter, filtrate is by macroporous resin, water, 20% alcoholic solution, 50% alcoholic solution eluting successively.Collect the eluent of 50% alcoholic solution, be evaporated to 1/10 of Radix Salviae Miltiorrhizae amount, slowly add ethanol down and make solution contain the alcohol amount to reach 90%, leave standstill, get supernatant and filter in stirring, the filtrate reconcentration is to 1/20 of the Radix Salviae Miltiorrhizae amount, slowly add dehydrated alcohol down and make solution contain the alcohol amount to reach 95%, leave standstill, get supernatant and filter in stirring, filtrate decompression is concentrated into dried, pulverizes promptly.
[character] this product is a light brown powder, and mildly bitter flavor is little puckery.
This product 0.2g is got in [discriminating] (1), after the blazing ashing, adds a small amount of 6% acetum and makes dissolving, adds water to 10ml, filters, and filtrate should show the identification (appendix IV of Chinese Pharmacopoeia version in 2000) of magnesium salt.
(2) in the chromatogram that writes down under the assay item, test sample is answered tool and the consistent chromatographic peak of Radix Salviae Miltiorrhizae acetate magnesium reference substance retention time.
[inspection] acidity is got this product, adds water and makes the solution that every 1ml contains 5mg, measures (an appendix VII of Chinese Pharmacopoeia version in 2000 G) in accordance with the law and measures, and pH value should be 4.0-6.0.
Moisture is got this product, measures according to aquametry (an appendix IX of Chinese Pharmacopoeia version in 2000 the H three therapeutic methods of traditional Chinese medicine), must not cross 5.0%.
Protein is got this product 50mg, adds water 10ml and makes dissolving.Get 1ml, add 30% sulfosalicylic acid solution 1ml of new preparation, mix, placed 5 minutes, muddiness must not occur.
Tannin is got this product 50mg, add water 10ml and make dissolving, filter, hold back tannin with the ultrafilter membrane of molecular cut off 3000, filter membrane and filter wash secondary with water, each 10ml takes out filter membrane, water 10ml washing, get washing liquid 1ml, the normal saline 5ml that contains 1% Ovum Gallus domesticus album that adds new preparation placed 10 minutes, muddiness or precipitation must not occur.
Resin is got this product 50mg, adds water 10ml and makes dissolving.Get 5ml, add 1 of hydrochloric acid, placed 30 minutes, should not have floccule and separate out.
Oxalates is got this product 50mg, adds water 10ml and makes dissolving, gets 2ml, adds 3% calcium chloride solution 2-3 and drips, and places 10 minutes, muddiness or precipitation must not occur.
Residue on ignition is got this product 0.2g, checks in accordance with the law and leaves over (an appendix IX of Chinese Pharmacopoeia version in 2000 J) residue and should be 4.5-7.5%.
Heavy metal is got the residue of leaving under the residue on ignition item, checks to contain (appendix IXE second method of Chinese Pharmacopoeia version in 2000) heavy metal and must not cross 10/1000000ths in accordance with the law.
Arsenic salt is got this product 1.0g, add 2% magnesium nitrate alcoholic solution 5ml, light, in the afterburnt, burning with little heated earlier makes carbonization, blazing to ashing fully at 500-600 ℃ again, put cold, add hydrochloric acid 5ml and water 21ml makes dissolving, check (an appendix IX of Chinese Pharmacopoeia version in 2000 F first method) in accordance with the law, arsenic content must not cross 2/1000000ths.
The potassium ion precision takes by weighing this product 20mg, checks (an appendix IX of Chinese Pharmacopoeia version in 2000 S) in accordance with the law, should be up to specification.
The macroporous adsorbent resin organic residue is measured according to gas chromatography (two appendix VIII of Chinese Pharmacopoeia version in 2000 P, second method).
Macroporous adsorbent resin organic residue normal hexane, benzene, toluene, xylol, o-Dimethylbenzene, styrene and 1,2-diethylbenzene precision takes by weighing normal hexane, benzene, toluene, xylol, o-Dimethylbenzene, styrene and 1, the 2-diethylbenzene is an amount of, add 0.6% sodium dodecyl sulfate solution and make the solution that contains 15 μ g, 10 μ g, 12 μ g, 12 μ g, 13 μ g, 16 μ g and 13 μ g among every 1ml respectively, shake up, precision is measured 5ml, put in the 20ml head space sampling bottle, in contrast solution; Other gets the about 0.2g of this product, and accurate the title decides, and puts in the 20ml head space sampling bottle, and the accurate 0.6% sodium dodecyl sulfate solution 5ml that adds shakes up, as need testing solution.Measure according to Determination of Residual Organic Solvents algoscopy (two appendix VIII of Chinese Pharmacopoeia version in 2000 P, second method), with cross-linked polyethylene glycol capillary chromatographic column (30m * 0.25mm, thick coating 0.25 μ m), 50 ℃ of column temperatures; Theoretical cam curve is calculated with the toluene peak should be not less than 7000, and the peak-to-peak separating degree of each composition should be up to specification.Get contrast solution and need testing solution after 15 minutes, measure in accordance with the law, the record chromatogram 120 ℃ of heating.In the chromatogram of need testing solution as show and the corresponding chromatographic peak of contrast solution, press external standard method with calculated by peak area, contain normal hexane, toluene, xylol, o-Dimethylbenzene, styrene and 1, the 2-diethylbenzene all must not cross 0.002%, contains benzene and must not cross 0.0002%.
The preparation of haemolysis 2% Sanguis Leporis seu oryctolagi red blood cell suspension
Get the rabbit cardiac blood, be equipped with in the container of pearl glass, jolting number minute, remove Fibrinogen, make to become to take off fine blood, add the physiology sodium chloride solution, shake up, centrifugal, the supernatant that inclines, sedimentary erythrocyte reuse physiological sodium chloride solution washing 3-4 time is till the apparent redness of centrifugal back supernatant, press the gained erythrocyte volume then, be diluted to 2% suspension with physiological sodium chloride solution.Used the same day, and the time spent shakes up.
The preparation of need testing solution
Get this product, add physiological sodium chloride solution and make the solution that every 1ml contains 5mg.
It is some that test method is got test tube, every batch of test sample is established 5 groups, every group 2 pipe, other establishes negative and each 2 pipe of positive control, and the according to the form below proportional quantity adds 2% red blood cell suspension, physiological sodium chloride solution and distilled water successively, after shaking up, in 37 ℃ of waters bath with thermostatic control, kept 30 minutes, add not commensurability need testing solution then respectively, after shaking up, put in 37 ℃ of waters bath with thermostatic control.Begin to observe once in per 15 minutes, observed once in per 1 hour after 1 hour, observed altogether 4 hours.The positive control pipe should be transparent redness, and negative control pipe and 0.3ml must not have haemolysis and coacervation for test tube.
| Test sample | Negative control | Positive control | |||||
| 1 | 2 | 3 | 4 | 5 | |||
| 2% red blood cell suspension (ml) | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 |
| Physiological sodium chloride solution (ml) | 2.0 | 2.1 | 2.2 | 2.3 | 2.4 | 2.5 | |
| Distilled water (ml) | 2.5 | ||||||
| Test sample (ml) | 0.5 | 0.4 | 0.3 | 0.2 | 0.1 |
[finger printing] measured according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).
Chromatographic condition and system suitability test octadecylsilane chemically bonded silica are filler; (42: 1: 59: 0.2) be mobile phase, flow velocity was per minute 0.7ml with methanol-acetonitrile-water-formic acid; The detection wavelength is 254nm, 30 ℃ of column temperatures.Theoretical cam curve is calculated by the Radix Salviae Miltiorrhizae acetate magnesium peak should be not less than 7000.
It is an amount of that the preparation of object of reference solution takes by weighing the Radix Salviae Miltiorrhizae acetate magnesium reference substance, and accurate the title decides, and adds mobile phase and makes the solution that every 1ml contains 0.8mg, promptly.
It is an amount of that this product is got in the preparation of need testing solution, and accurate the title decides, and puts in the 5ml measuring bottle, adds mobile phase and makes the solution that every 1ml contains 1.0mg, promptly.
Accurate respectively object of reference solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
With peak identical with the object of reference retention time in the test sample finger printing is the S peak, calculates relative retention time and peak area ratio, should meet the following requirements:
The test sample finger printing should be similar to standard finger-print;
Finger printing should have 3 total peaks;
Relative retention time 0.77 (1), 0.93 (2), 1.00 (s), its relative deviation must not mistake ± 10%;
Peak area ratio: with S peak-to-peak area ratio is 1.00, and peak area ratio must not be greater than 0.088 (No. 2 peaks)
Non-total peak area must not be greater than 5% of total peak area
Standard finger-print Fig. 4
Instrument, chromatographic column and integral parameter: Agilent 1100 series of high efficiency chromatograph of liquid, the Chemstation chromatographic work station.Chromatographic column is Zorbax C
18(4.6mm * 25cm, 5 μ m); 30 ℃ of column temperatures.Slope: 2, peak width: 0.05; The smallest peaks area is 20, and minimum peak height is 1.
[assay]
Radix Salviae Miltiorrhizae acetate magnesium is measured according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).
Chromatographic condition and system suitability test octadecylsilane chemically bonded silica are filler, are mobile phase with methanol-5mmol/L potassium dihydrogen phosphate (30: 70); The detection wavelength is 290nm.Number of theoretical plate calculates by the Radix Salviae Miltiorrhizae acetate magnesium peak should be not less than 1500, and the separating degree of Radix Salviae Miltiorrhizae acetate magnesium peak and adjacent peak should be greater than 2.0.
It is an amount of that the Radix Salviae Miltiorrhizae acetate magnesium reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds mobile phase and makes the solution that every 1ml contains 30 μ g, promptly.
This product 20mg is got in the preparation of need testing solution, accurate claims surely, puts in the 50ml measuring bottle, with the mobile phase dissolving and be diluted to scale, shakes up, and precision is measured 2ml, puts in the 20ml measuring bottle, is diluted to scale with mobile phase, shakes up, promptly.
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
This product contains the Radix Salviae Miltiorrhizae acetate magnesium amount and should be 80～90%.
[function cures mainly] invigorates blood circulation, blood stasis dispelling, promote blood circulation.Be used for the coronary heart disease stable angina pectoris, be classified as I, II level, angina pectoris symptom shows as gently, moderate, and Chinese medical discrimination is the heart blood silt person, and disease is seen chest pain, uncomfortable in chest, cardiopalmus.
[usage and dosage] intravenous drip.A 200mg uses with 5% glucose injection 250ml-500ml dissolving back.1 time on the one.2 weeks of the course of treatment.
[untoward reaction]
1. dizzy, dizzy, distending pain in the head take place in small number of patients.
2. idol has the patient to cause slight headache soon because of quiet speed in transfusion.
3. the blood Glutamate pyruvate transaminase rises is arranged once in a while, after drug withdrawal, disappear.
[points for attention]
1. the careful usefulness of bleeding tendency person is arranged.
2. anemia of pregnant woman, the careful usefulness of women breast-feeding their children.
3. still there is not sufficient drug interaction research data at present.
[storage] sealing is kept in Dark Place.
[specification] every packed 1kg (containing Radix Salviae Miltiorrhizae acetate magnesium 0.8kg).Every packed 3kg (containing Radix Salviae Miltiorrhizae acetate magnesium 2.4kg).Every bottled 5kg (containing Radix Salviae Miltiorrhizae acetate magnesium 4.0kg).
[packing] foil laminated film bag.
[preparation] Radix Salviae Miltiorrhizae for injection polyphenol hydrochlorate
[effect duration] tentative 18 months.
The present invention also provides the quality standard of Radix Salviae Miltiorrhizae for injection polyphenol hydrochlorate as follows:
This product is that salvianolate is made sterilized powder.
[method for making] gets salvianolate 50g, 100g or 200g, and with water for injection dissolving, through Entkeimung, be distributed into 1000 bottles, lyophilization promptly.
[character] this product is the loose block of light brown, and mildly bitter flavor is little puckery.
This product 0.2g is got in [discriminating] (1), after the blazing ashing, adds a small amount of 6% acetum and makes dissolving, adds water to 10ml, filters, and filtrate should show the identification (appendix IV of Chinese Pharmacopoeia version in 2000) of magnesium salt.
(2) in the chromatogram that writes down under the assay item, test sample is answered tool and the consistent chromatographic peak of Radix Salviae Miltiorrhizae acetate magnesium reference substance retention time.
[inspection] clarity is operated in super-clean bench.Get 5 of clean tool plug nessler colorimetric tubes, add filterable in advance water for injection 10ml respectively, by " clarity test detailed rules and regulations and criterion " injectable sterile powder item, rotate gently in umbrella canopy edge, make solvent form eddy flow, look with visual inspection immediately, the record hair is counted, as blank, add 1 bottle of test sample then respectively, make dissolving fully, in the horizontal observation in umbrella canopy edge, rotate gently or swing, look, the record hair, count with visual inspection, deduction is blank, promptly.
Every bottle of contained hair and the white point of 200-500 μ m, white piece and color dot sum that is shorter than 0.5cm of this product must not be above 10.
Particulate matter is got the content of 1 bottle of this product, with the 125ml dissolving of purifying waste water, makes blank with purifying waste water, and measures (an appendix IX of Chinese Pharmacopoeia version in 2000 R light blockage method) in accordance with the law, should be up to specification.
Acidity is got this product, adds water and makes the solution that every 1ml contains 5mg, measures (an appendix VII of Chinese Pharmacopoeia version in 2000 G) in accordance with the law, and pH value should be 4.0-6.0.
Moisture is got this product, measures according to aquametry (an appendix IX of Chinese Pharmacopoeia version in 2000 the H three therapeutic methods of traditional Chinese medicine), must not cross 5.0%.
Protein is got this product 50mg, adds water 10ml and makes dissolving.Get 1ml, add 30% sulfosalicylic acid solution 1ml of new preparation, mix, placed 5 minutes, muddiness must not occur.
Tannin is got this product 50mg, add water 10ml and make dissolving, filter, hold back tannin with the ultrafilter membrane of molecular cut off 3000, filter membrane and filter wash secondary with water, each 10ml takes out filter membrane, water 10ml washing, get washing liquid 1ml, the normal saline 5ml that contains 1% Ovum Gallus domesticus album that adds new preparation placed 10 minutes, muddiness or precipitation must not occur.
Resin is got this product 50mg, adds water 10ml and makes dissolving.Get 5ml, add 1 of hydrochloric acid, placed 30 minutes, should not have floccule and separate out.
Oxalates is got this product 50mg, adds water 10ml and makes dissolving, gets 2ml, adds 3% calcium chloride solution 2-3 and drips, and places 10 minutes, muddiness or precipitation must not occur.
Residue on ignition is got this product 0.2g, checks in accordance with the law and leaves over (an appendix IX of Chinese Pharmacopoeia version in 2000 J) residue and should be 4.5-7.5%.
Heavy metal is got this product 1.0g, checks that in accordance with the law (an appendix IX of Chinese Pharmacopoeia version in 2000 E second method) contains heavy metal and must not cross 10/1000000ths.
Arsenic salt is got this product 1.0g, add 2% magnesium nitrate alcoholic solution 5ml, light, afterburnt, burning with little heated earlier makes carbonization, 500-600 ℃ of blazing ashing extremely fully, puts cold again, add hydrochloric acid 5ml and water 21ml makes dissolving, check that in accordance with the law (an appendix IX of Chinese Pharmacopoeia version in 2000 F first method) arsenic content must not cross 2/1000000ths.
Pyrogen is got this product, adds sterilized water for injection and makes the solution that every 1ml contains 5mg, checks (an appendix XIII of Chinese Pharmacopoeia version in 2000 A) in accordance with the law, and dosage should be up to specification by the every 1Kg injection of rabbit body weight 2ml.
The preparation of haemolysis 2% Sanguis Leporis seu oryctolagi red blood cell suspension
Get the rabbit cardiac blood, be equipped with in the container of pearl glass, jolting number minute, remove Fibrinogen, make to become to take off fine blood, add the physiology sodium chloride solution, shake up, centrifugal, the supernatant that inclines, sedimentary erythrocyte reuse physiological sodium chloride solution washing 3-4 time is till the apparent redness of centrifugal back supernatant, press the gained erythrocyte volume then, be diluted to 2% suspension with physiological sodium chloride solution.Used the same day, and the time spent shakes up.
The preparation of need testing solution
Get this product, add physiological sodium chloride solution and make the solution that every 1ml contains 5mg.
It is some that test method is got test tube, every batch of test sample is established 5 groups, every group 2 pipe, other establishes negative and each 2 pipe of positive control, and the according to the form below proportional quantity adds 2% red blood cell suspension, physiological sodium chloride solution and distilled water successively, after shaking up, in 37 ℃ of waters bath with thermostatic control, kept 30 minutes, add not commensurability need testing solution then respectively, after shaking up, put in 37 ℃ of waters bath with thermostatic control.Begin to observe once in per 15 minutes, observed once in per 1 hour after 1 hour, observed altogether 4 hours.The positive control pipe should be transparent redness, and negative control pipe and 0.3ml must not have haemolysis and coacervation for test tube.
| Test sample | Negative control | Positive control |
| 1 | 2 | 3 | 4 | 5 | |||
| 2% red blood cell suspension (ml) | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 |
| Physiological sodium chloride solution (ml) | 2.0 | 2.1 | 2.2 | 2.3 | 2.4 | 2.5 | |
| Distilled water (ml) | 2.5 | ||||||
| Test sample (ml) | 0.5 | 0.4 | 0.3 | 0.2 | 0.1 |
[finger printing] measured according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).
Chromatographic condition and system suitability test octadecylsilane chemically bonded silica are filler: (42: 1: 59: 0.2) be mobile phase, flow velocity was per minute 0.7ml with methanol-acetonitrile-water-formic acid; The detection wavelength is 254nm, 30 ℃ of column temperatures.Theoretical cam curve is calculated by the Radix Salviae Miltiorrhizae acetate magnesium peak should be not less than 7000.
It is an amount of that the preparation of object of reference solution takes by weighing the Radix Salviae Miltiorrhizae acetate magnesium reference substance, and accurate the title decides, and adds mobile phase and makes the solution that every 1ml contains 0.8mg, promptly.
It is an amount of that this product is got in the preparation of need testing solution, and accurate the title decides, and puts in the 5ml measuring bottle, adds mobile phase and makes the solution that every 1ml contains 1.0mg, promptly.
Accurate respectively object of reference and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
With peak identical with the object of reference retention time in the test sample finger printing is the S peak, calculates relative retention time and peak area ratio, should meet the following requirements.
The test sample finger printing should be similar to standard finger-print;
Finger printing should have 3 total peaks;
Relative retention time 0.78 (1), 0.93 (2), 1.00 (s), its relative deviation must not mistake ± 10%:
Non-total peak area must not be greater than 5% of total honeybee area
Standard finger-print
Instrument, chromatographic column and integral parameter: HP 1100 series of high efficiency chromatograph of liquid, the Chemstation chromatographic work station.Chromatographic column Zorbax C
18(4.6mm * 25cm, 5 μ m); 30 ℃ of column temperatures.Slope: 2, peak width: 0.05; The smallest peaks area is 20, and minimum peak height is 1.
[assay]
Radix Salviae Miltiorrhizae acetate magnesium is measured according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).
Chromatographic condition and system suitability test octadecylsilane chemically bonded silica are filler, are mobile phase with methanol-5mmol/L potassium dihydrogen phosphate (30: 70); The detection wavelength is 290nm.Number of theoretical plate calculates by the Radix Salviae Miltiorrhizae acetate magnesium peak should be not less than 1500, and the separating degree of Radix Salviae Miltiorrhizae acetate magnesium peak and adjacent peak should be greater than 2.0.
It is an amount of that the Radix Salviae Miltiorrhizae acetate magnesium reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds mobile phase and makes the solution that every 1ml contains 30 μ g, in contrast product solution.
The content under the content uniformity item is got in the preparation of need testing solution, and mixing is got 20mg, and accurate the title decides, put in the 50ml measuring bottle, with mobile phase dissolving and be diluted to scale, shake up, precision is measured 2ml, put in the 20ml measuring bottle, be diluted to scale, shake up, promptly with mobile phase.
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
This product contains the 90-110% that the Radix Salviae Miltiorrhizae acetate magnesium amount should be labelled amount for every bottle.
[function cures mainly] invigorates blood circulation, blood stasis dispelling, promote blood circulation.Be used for the coronary heart disease stable angina pectoris, be classified as I, II level, angina pectoris symptom shows as gently, moderate, and Chinese medical discrimination is the heart blood silt person, and disease is seen chest pain, uncomfortable in chest, cardiopalmus.
[usage and dosage] intravenous drip.A 200mg uses with 5% glucose injection 250ml-500ml dissolving back.1 time on the one.2 weeks of the course of treatment.
[untoward reaction]
4. dizzy, dizzy, distending pain in the head take place in small number of patients.
5. idol has the patient to cause slight headache soon because of quiet speed in transfusion.
6. the blood Glutamate pyruvate transaminase rises is arranged once in a while, after drug withdrawal, disappear.
[points for attention]
4. the careful usefulness of bleeding tendency person is arranged.
5. anemia of pregnant woman, the careful usefulness of women breast-feeding their children.
6. still there is not sufficient drug interaction research data at present.
[specification] every bottled 50mg (containing Radix Salviae Miltiorrhizae acetate magnesium 40mg).Every bottled 100mg (containing Radix Salviae Miltiorrhizae acetate magnesium 80mg).Every bottled 200mg (containing Radix Salviae Miltiorrhizae acetate magnesium 160mg).
[storage] sealing is kept in Dark Place.
[packing] cillin bottle packing.
[effect duration] tentative 18 months.
Description of drawings:
Fig. 1 red rooted salvia HPLC figure.
Fig. 2 salvianolate HPLC figure.
Fig. 3 high content salvianolate injectable powder HPLC figure.
The specific embodiment
Embodiment 1:
The red rooted salvia finger printing detects
[finger printing] measured according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).
Chromatographic condition and system suitability test octadecylsilane chemically bonded silica are filler; (42: 1: 59: 0.2) be mobile phase, flow velocity was per minute 0.7ml with methanol-acetonitrile-water-formic acid; The detection wavelength is 254nm, 30 ℃ of column temperatures.Theoretical cam curve is calculated by the Radix Salviae Miltiorrhizae acetate magnesium peak and is not less than 7000.
Radix Salviae Miltiorrhizae acetate magnesium reference substance 4.1mg decided in the accurate title of the preparation of object of reference solution, adds mobile phase and be diluted to the 5ml measuring bottle, promptly.
The preparation of need testing solution is got Radix Salviae Miltiorrhizae and is pulverized, and crosses the medicine sieve No. 3, and precision takes by weighing 0.5002g, put in the 100ml iodine flask, add water 30ml soaked overnight, transfer in the round-bottomed flask, water 30ml cleans iodine flask, cleanout fluid is incorporated round-bottomed flask into, reflux 2 hours filters while hot, and pH value to 2.0 (measuring with acidometer) is regulated with 10% hydrochloric acid solution in filtrate cooling back, with ethyl acetate extraction 3 times, each 80ml.The extract decompression and solvent recovery adds the mobile phase ultrasonic dissolution, is transferred in the 10ml measuring bottle, and is diluted to scale with mobile phase, shakes up, and with the microporous filter membrane filtration of 0.45 μ m, gets subsequent filtrate promptly.
Accurate respectively object of reference and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
With peak identical with the object of reference retention time in the test sample finger printing is the S peak, calculates relative retention time and peak area ratio.
The test sample finger printing has 7 total peaks;
Relative retention time: 0.20 0.23 (1), 0.31 0.34 (2), 0.47 (3), 0.55 (4), 0.60 (5), 0.78 (6), 1.00 (s), its relative deviation be no more than ± and 10%;
Peak area: 821.2 (1), 915.4 (2), 1468.9 (3), 657.8 (4), 1165.5 (5), 2359.6 (6), 15441.2 (s)
Non-total peak area percentage ratio is 7.31%, less than 10% of total honeybee area
The test sample finger printing is consistent with the red rooted salvia standard finger-print, requirement up to specification.
Embodiment 2:
The salvianolate finger printing detects
[finger printing] measured according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).
Chromatographic condition and system suitability test octadecylsilane chemically bonded silica are filler; (42: 1: 59: 0.2) be mobile phase, flow velocity was per minute 0.7ml with methanol-acetonitrile-water-formic acid; The detection wavelength is 254nm, 30 ℃ of column temperatures.Theoretical cam curve is calculated by the Radix Salviae Miltiorrhizae acetate magnesium peak and is not less than 7000.
Radix Salviae Miltiorrhizae acetate magnesium reference substance 4.8mg decided in the accurate title of the preparation of object of reference solution, adds mobile phase and be diluted to the 5ml measuring bottle, promptly.
This product 5.5mg is got in the preparation of need testing solution, puts in the 5ml measuring bottle, adds mobile phase and is diluted to the 5ml measuring bottle, promptly.
Accurate respectively object of reference and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
With peak identical with the object of reference retention time in the test sample finger printing is the S peak, calculates relative retention time and peak area ratio.
The test sample finger printing has 3 total peaks;
Relative retention time: 0.76 (1), 0.89 (2), 1.00 (s), its relative deviation are no more than ± and 10%;
Peak area: 154.38 (1), 359.80 (2), 7697.9 (s)
Non-total peak area percentage ratio is 1.93%, less than 5% of total honeybee area
The test sample finger printing is consistent with the salvianolate standard finger-print, requirement up to specification.
Embodiment 3:
Salvianolate injectable powder finger printing detects
[finger printing] measured according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).
Chromatographic condition and system suitability test octadecylsilane chemically bonded silica are filler; (42: 1: 59: 0.2) be mobile phase, flow velocity was per minute 0.7ml with methanol-acetonitrile-water-formic acid; The detection wavelength is 254nm, 30 ℃ of column temperatures.Theoretical cam curve is calculated by the Radix Salviae Miltiorrhizae acetate magnesium peak and is not less than 7000.
Radix Salviae Miltiorrhizae acetate magnesium reference substance 4.3mg decided in the accurate title of the preparation of object of reference solution, adds mobile phase and be diluted to the 5ml measuring bottle, promptly.
This product 5.0mg is got in the preparation of need testing solution, puts in the 5ml measuring bottle, adds mobile phase and is diluted to the 5ml measuring bottle, promptly.
Accurate respectively object of reference and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
With peak identical with the object of reference retention time in the test sample finger printing is the S peak, calculates relative retention time and peak area ratio.
The test sample finger printing has 3 total peaks;
Relative retention time: 0.79 (1), 0.94 (2), 1.00 (s), its relative deviation are no more than ± and 10%;
Peak area: 526.20 (1), 1072.74 (2), 21240 (s)
Non-total peak area percentage ratio is 2.62%, less than 5% of total honeybee area
The test sample finger printing is consistent with salvianolate injectable powder standard finger-print, requirement up to specification.
Claims (4)
1, a kind of high content salvianolate injectable powder fingerprint atlas detection method is characterized in that this detection method comprises:
(1) red rooted salvia fingerprint atlas detection method;
(2) high content salvianolate fingerprint atlas detection method;
(3) high content salvianolate injectable powder fingerprint atlas detection method.
2, a kind of high content salvianolate injectable powder fingerprint atlas detection method according to claim 1 is characterized in that wherein said red rooted salvia fingerprint atlas detection method comprises the following steps:
(1) preparation of Radix Salviae Miltiorrhizae test sample
Medical material is crossed 40 mesh sieves after pulverizer is pulverized, get each batch medicinal powder 500mg, the accurate title, decide, place 100ml tool plug triangular flask, respectively add the 30ml distilled water immersion and spend the night, be transferred in the round-bottomed flask, reuse 30ml distilled water cleans triangular flask, cleanout fluid changes in the round-bottomed flask in the lump, reflux, extract, 2h.Filtered while hot, the filtrate cooling is back with 10% hydrochloric acid adjust pH to 2.0 (measuring with acidometer), reuse ethyl acetate extraction 3 times, each 80ml.Use the mobile phase ultrasonic dissolution behind the extract concentrating under reduced pressure, and be settled to 10ml, as need testing solution with volumetric flask.HPLC uses filtering with microporous membrane before analyzing;
(2) preparation of object of reference solution
Radix Salviae Miltiorrhizae acetate magnesium accurately takes by weighing 4.0mg as object of reference, is settled to 5ml with mobile phase, as object of reference solution;
(3) detection method
Assay method is a high performance liquid chromatography:
1) instrument and reagent
Agilent1100 high performance liquid chromatograph and Chemstation chromatographic work station, ZorbaxC
18(4.6mm * 25cm, 5 μ m) chromatography post.Used methanol is chromatographically pure, and water is redistilled water, and formic acid is analytical pure.
2) condition determination and assay method
The a assay method
Mobile phase be methanol-acetonitrile-water-formic acid (42: 1: 59: 0.2), the detection wavelength was 254nm, and column temperature is 30 ℃, slope 2, peak width 0.05, the smallest peaks area is 50, minimum peak height is 2, flow velocity is 0.7ml/min;
B. assay method
Get each batch medical material need testing solution 5 μ l respectively, inject high performance liquid chromatograph and carry out chromatography, write down the retention time and the integral area at each peak;
(4) salvianolate injectable powder medicinal materials fingerprint and technical parameter
1) demarcation of finger printing and total fingerprint peaks
According to 10 batches of given relevant parameters of test sample HPLC collection of illustrative plates, it is that each batch sample is common that 7 chromatographic peaks are arranged in the chromatogram of red rooted salvia, the percentage ratio that its peak area summation accounts for the total peak area sum is 91.87%, numbering is respectively 1,2,3,4,5,6,7, wherein 1 and 2 are the group peak, and the ratio of each peak relative retention time is 1: 2: 3: 4: 5: 6: 7=0.22: 0.32: 0.47: 0.55: 0.60: 0.77: 1.00;
2) ratio of total fingerprint peaks area
The ratio of 7 specified total peak-to-peak areas is 1: 2: 3 in the 10 batches of test samples: 4: 5: 6: 7=0.109: 0.172: 0.121: 0.020: 0.035: 0.126: 1.00.The percentage ratio meansigma methods of each shared total peak area in total peak is respectively group peak 1,6.35%; Group peak 2,9.50%; Peak 3,7.06%; Peak 4,1.16%; Peak 5,2.07%; Peak 6,7.35%; Peak 7 (object of reference), 58.38%.This shows, have only the relative peak area at peak 7 to surpass 10% in 7 total peaks, and the relative peak area at other 6 total peaks is all below 10%, therefore only require the existence of its chromatographic peak, and not requiring the concordance of its peak area ratio, it is 46.28%-70.48% that the relative peak area at peak 7 allows excursion.
3, a kind of high content salvianolate injectable powder fingerprint atlas detection method according to claim 1 is characterized in that wherein said salvianolate fingerprint atlas detection method comprises the following steps:
(1), the preparation of need testing solution
Get salvianolate 5.0mg respectively, precision is weighed, put in the 5ml volumetric flask, usefulness mobile phase methanol-acetonitrile-water-formic acid (42: 1: 59: 0.2) standardize solution, draw 5 μ L injection performance liquid chromatographic column and analyze;
(2), the preparation of reference substance solution
Accurately get Radix Salviae Miltiorrhizae acetate magnesium according to product 4.0mg, precision is weighed, and puts in the 5ml volumetric flask, and (42: 1: 59: 0.2) standardize solution was used for the reference substance of salvianolate finger printing to usefulness mobile phase methanol-acetonitrile-water-formic acid;
(3), detection method
Assay method is a high performance liquid chromatography:
1) instrument and reagent
Agilent1100 high performance liquid chromatograph and Chemstation chromatographic work station, ZorbaxC
18(4.6mm * 25cm, 5 μ m) chromatography post.Used methanol is chromatographically pure, and water is redistilled water, and formic acid is analytical pure.
2) condition determination and assay method
A. condition determination
Mobile phase be methanol-acetonitrile-water-formic acid (42: 1: 59: 0.2), the detection wavelength was 254nm, and column temperature is 30 ℃, slope 2, peak width 0.05, the smallest peaks area is 20, minimum peak height is 1, flow velocity is 0.7ml/min;
B. assay method
Get each batch injection need testing solution 5 μ L respectively, inject high performance liquid chromatograph and analyze, write down the retention time and the integral area at each peak;
(4) finger printing and technical parameter
1) demarcation of finger printing and total fingerprint peaks
According to 10 batches of given relevant parameters of test sample HPLC collection of illustrative plates, the chromatographic peak of salvianolate all the components all occurs within 60min, and determines 3 chromatograph peak-to-peaks 1: retention time is 14.10min; Peak 2: retention time is 17.00min; Peak 3: retention time is that 18.25min is total fingerprint peaks;
2) ratio of total fingerprint peaks area and retention time
The ratio of the relative retention time of each total fingerprint peaks is: 1/ peak, peak, 2/ peak 3=0.78/0.93/1.00.The percentage ratio meansigma methods of peak 1 shared total peak area is 1.75%, the meansigma methods that peak 2 accounts for the percentage ratio of total peak area is 4.97%, the meansigma methods of the percentage ratio of peak 3 shared total peak areas is 92.97%, therefore having only peak 3 in 3 total peaks is that the peak area of reference substance surpasses 5%, can't calculate its ratio by the fingerprint pattern technology requirement, the theoretic permission excursion in this peak is 74.38%-111%.
(5), the appointment of finger printing and total fingerprint peaks
1) 4 chromatographic peaks all appear in 10 batches of salvianolate test sample HPLC collection of illustrative plates, determine peak 1 relative retention time 0.77, and peak 3 relative retention time 0.93 and peak 4 relative retention time 1.00 are total fingerprint peaks, and promptly this 3 total peaks all should appear in each batch sample;
2) ratio of total fingerprint peaks area
10 batches of test samples are object of reference with the Radix Salviae Miltiorrhizae acetate magnesium, and the ratio of integral area that surpasses the total fingerprint peaks of total peak area 5% is peak 3: peak 4=0.07: 1.00, and wherein the permission excursion at peak 3 is 4.33%-8.05%, peak 4 is a reference substance.
4, a kind of high-load poly phenolic acid of Radix Salviae Miltiorrhizae injectable powder fingerprint atlas detection method according to claim 1 is characterized in that wherein said salvianolate injectable powder fingerprint atlas detection method comprises the following steps:
(1) preparation of need testing solution
Get injectable powder 5.0mg respectively, precision is weighed, put in the 5mL volumetric flask, usefulness mobile phase methanol-acetonitrile-water formic acid (42: 1: 59: 0.2) standardize solution, draw 5 μ L injection performance liquid chromatographic column and analyze;
(2) preparation of object of reference solution
Accurately get Radix Salviae Miltiorrhizae acetate magnesium reference substance 4.0mg, precision is weighed, and puts in the 5mL volumetric flask, and (42: 1: 59: 0.2) standardize solution was used for the reference substance of salvianolate injectable powder finger printing with mobile phase methanol-acetonitrile-water-formic acid;
(3) detection method
Assay method is a high performance liquid chromatography:
1) instrument and reagent
Agilent1100 high performance liquid chromatograph and Chemstation chromatographic work station, ZorbaxC
18(4.6mm * 25cm, 5 μ m) chromatography post.Used methanol is chromatographically pure, and water is redistilled water, and formic acid is analytical pure.
2) condition determination and assay method
A. condition determination
Mobile phase be methanol-acetonitrile-water-formic acid (42: 1: 59: 0.2), the detection wavelength was 254nm, and column temperature is 30 ℃, slope 2, peak width 0.05, the smallest peaks area is 20, minimum peak height is 1, flow velocity is 0.7ml/min;
B. assay method
Get each batch injection need testing solution 5 μ L respectively, inject high performance liquid chromatograph and analyze, write down the retention time and the integral area at each peak;
(4) finger printing and technical parameter
1) demarcation of finger printing and total fingerprint peaks
According to 10 batches of given relevant parameters of test sample HPLC collection of illustrative plates, the chromatographic peak of salvianolate injectable powder all the components all occurs within 60min, and determines 3 chromatograph peak-to-peaks 1: retention time is 14.10min; Peak 2: retention time is 17.00min; Peak 3: retention time is that 18.25min is total fingerprint peaks;
2) ratio of total fingerprint peaks area and retention time
The ratio of the relative retention time of each total fingerprint peaks is: 1/ peak, peak, 2/ peak 3=0.78/0.93/1.00.The percentage ratio meansigma methods of peak 1 shared total peak area is 1.75%, the meansigma methods that peak 2 accounts for the percentage ratio of total peak area is 4.97%, the meansigma methods of the percentage ratio of peak 3 shared total peak areas is 92.97%, therefore having only peak 3 in 3 total peaks is that the peak area of reference substance surpasses 5%, can't calculate its ratio by the fingerprint pattern technology requirement, the theoretic permission excursion in this peak is 74.38%-111%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717384B (en) * | 2009-11-24 | 2011-10-12 | 正大青春宝药业有限公司 | Compound in salvia miltiorrhiza bungered sage root injection and application thereof in curing treating cardiovascular disease |
| CN103149310A (en) * | 2013-01-21 | 2013-06-12 | 贵州景峰注射剂有限公司 | Fingerprint building method and quality control method of Shenxiong glucose injection raw material, Shenxiong glucose injection midbody and Shenxiong glucose injection preparation |
| CN103245687A (en) * | 2013-05-07 | 2013-08-14 | 江苏省中医药研究院 | Component-structure-based quality control and detection method for danshen injection |
| CN103980236A (en) * | 2009-11-17 | 2014-08-13 | 中国科学院上海药物研究所 | Salvianolate, preparation method and application thereof |
-
2005
- 2005-08-12 CN CNB2005100287411A patent/CN100522191C/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103980236A (en) * | 2009-11-17 | 2014-08-13 | 中国科学院上海药物研究所 | Salvianolate, preparation method and application thereof |
| CN103980236B (en) * | 2009-11-17 | 2017-10-03 | 中国科学院上海药物研究所 | Polydanshinolate and its production and use |
| CN101717384B (en) * | 2009-11-24 | 2011-10-12 | 正大青春宝药业有限公司 | Compound in salvia miltiorrhiza bungered sage root injection and application thereof in curing treating cardiovascular disease |
| CN103149310A (en) * | 2013-01-21 | 2013-06-12 | 贵州景峰注射剂有限公司 | Fingerprint building method and quality control method of Shenxiong glucose injection raw material, Shenxiong glucose injection midbody and Shenxiong glucose injection preparation |
| CN103149310B (en) * | 2013-01-21 | 2015-05-20 | 贵州景峰注射剂有限公司 | Fingerprint building method of Shenxiong glucose injection preparation |
| CN103245687A (en) * | 2013-05-07 | 2013-08-14 | 江苏省中医药研究院 | Component-structure-based quality control and detection method for danshen injection |
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