CN113588808B - Content determination method for simultaneously detecting multiple active ingredients in Xueping capsules - Google Patents
Content determination method for simultaneously detecting multiple active ingredients in Xueping capsules Download PDFInfo
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Abstract
The invention relates to a method for simultaneously detecting contents of various active ingredients in Xueping capsules, which adopts an HPLC method to simultaneously detect ginsenoside Rg of pseudo-ginseng and radix rehmanniae in Xueping capsules 1 Ginsenoside Rb and ginsenoside Rb 1 Notoginsenoside R 1 The content of the digitoxin D is that octadecylsilane chemically bonded silica is used as a filling agent; taking methanol as phase A and phosphoric acid aqueous solution with volume concentration of 0.01-1.0% as phase B, and performing gradient elution; an ultraviolet detector or a diode array detector is adopted; the detection method can make up the defects of the existing method, can simultaneously detect a plurality of effective components of monarch drug and precious drug in the Xueping capsule, effectively control the quality of the drugs, improve the detection efficiency, reduce the detection cost, and correlate the quality of the drugs with the effective components of clinical treatment, thereby achieving the stable, controllable, efficient and safe standard, further better meeting the medical requirements and providing a basis for improving the quality standard of the variety.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for simultaneously detecting the content of multiple effective components of radix rehmanniae and pseudo-ginseng in Xueping capsules.
Background
Metrorrhagia and metrostaxis is the general term for vaginal bleeding during non-menstruation period of women. The clinical manifestation is vaginal bleeding. Metrorrhagia due to sudden onset of bleeding and excessive amount of bleeding; the amount of bleeding is small or dribbling. Functional uterine bleeding in Western medicine, vaginal bleeding caused by female genital inflammation, tumor, etc. all belong to the category of metrorrhagia and metrostaxis. Metrorrhagia and metrostaxis is a more serious and complex symptom in female menstrual diseases. This disease is common in adolescent women and climacteric women. It is usually caused by blood heat, qi deficiency, liver and kidney yin deficiency, blood stasis, qi stagnation and other damage to the thoroughfare and conception vessels, and failure of the thoroughfare and conception vessels to control qi deficiency. For metrorrhagia, bleeding should be treated first to prevent dizziness and collapse, and after blood deficiency or blood stasis, the principle of treating the primary aspect can be examined. Metrorrhagia is a common sudden bleeding with urgent arrival and excessive blood volume; dripping blood, slow arrival and small blood volume. Although the bleeding of metrorrhagia and metrostaxis are different, their pathogenesis is consistent, and they are often transformed into each other in the course of disease development, such as metrorrhagia is often called clinically because metrostaxis may become metrostaxis due to long-term consumption of qi and blood, and may become metrostaxis due to long-term metrostaxis. Just as "Jisheng Fang": the disease of metrorrhagia is called "metrostaxis" because of the same syndrome, and mild cases are called "metrostaxis". The disease belongs to common diseases, and is usually caused by alternation of collapse and leakage and causal coherence, so that the disease is lingering and difficult to cure, and becomes a difficult and serious gynecological disease. This disease is equivalent to anovulatory dysfunctional uterine bleeding disease in western medicine. Genital inflammation and irregular vaginal bleeding caused by some genital tumors can also be treated according to the syndrome differentiation.
The Xueping capsule is collected in the 'standard department of gynecology of Chinese patent medicine of State drug administration' and is produced by the company Limited in the Xian Qian standing grain pharmaceutical industry, and has been on the market for many years, has good effect on metrorrhagia and metrostaxis caused by blood heat with blood stasis, and comprises the following main components: rehmannia root, prepared rhubarb, pseudo-ginseng, garden burnet root, cuttlebone, india madder root and cattail pollen (fried). Has the effects of clearing away heat, dispelling blood stasis, stopping bleeding, and regulating menstruation. Can be used for treating metrorrhagia and metrostaxis due to blood heat and blood stasis. The symptoms include irregular menstrual cycle, irregular menstruation, increased menstruation volume, or dripping, deep red color, thick texture, blood clots, vexation, dry mouth and constipation. The tongue is red and the pulse is smooth and rapid. However, in the existing detection method of Xueping capsules, only thin-layer identification is carried out on the ministerial drug of cooked rhubarb and the conductant drug of pseudo-ginseng, and the content determination of emodin is carried out on the ministerial drug of cooked rhubarb; the content of monarch drug and other drugs in the prescription is not measured, the specificity of the existing quality control standard is poor, the quality of the drug cannot be comprehensively reflected, and the clinical curative effect index cannot be controlled, so that a detection method of the product is necessary to be further perfected, and the control index is associated with the clinical curative effect, so that the quality and the curative effect of the product are further ensured.
The rehmannia root mainly contains iridoid glycoside chemical components, and pharmacological research shows that the iridoid glycoside components are main active components. The existing literature mostly measures catalpol component in rehmannia glutinosa, but researches show that the thermal stability is poor, and the catalpol content is reduced to 1/10 of the original catalpol content when rehmannia glutinosa and prepared rehmannia glutinosa are processed from fresh products. The digitonin D is stable, has high contents of radix rehmanniae and radix rehmanniae Preparata, and hardly decomposes during processing; the experimental research on the effects of nourishing yin, enriching blood and reducing blood sugar of the digitonin D shows that: the rehmannia root glycoside D can obviously increase the weight of a yin deficiency model mouse, and obviously reduce the cAMP content in plasma; the rehmannia root glycoside D can obviously increase the white blood cell number, the platelet number, the reticulocyte number, the bone marrow DNA content and the body weight of a blood deficiency model mouse; diluteoside D has a tendency to lower blood glucose in diabetic model mice. The pharmacodynamic action of the digitonin D is consistent with that of traditional Chinese medicine, the content is higher and more stable, the digitonin D is more suitable to be used as a quality control standard substance of rehmannia root in medicinal materials and preparations of the rehmannia root, and meanwhile, the component has a larger medicinal value and a better application prospect. So far, no literature report related to a quality evaluation method of the digitoxin D in the Xueping capsules is found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a content determination method for simultaneously detecting various active ingredients in a Xueping capsule, which is used for detecting the active ingredient of the rehmannia root, i.e. the digitoxin D in the monarch drug and the ginsenoside Rg in the panax notoginseng, i.e. the noble drug 1 Ginsenoside Rb 1 Notoginsenoside R 1 The detection method can make up the defects of the prior method, can simultaneously detect a plurality of effective components of the monarch drug and the precious drug in the Xueping capsule only by preparing, mixing and contrasting, effectively controls the quality of the drugs, improves the detection efficiency, reduces the detection cost, and correlates the quality of the drugs with the effective components of clinical treatment, thereby achieving the purposes of stability,Controllable, efficient and safe standards, further better meets medical needs, and provides basis for improving the quality standard of the variety.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a content determination method for simultaneously detecting various active ingredients in a Xueping capsule is disclosed, wherein the pharmaceutical composition is prepared from the following seven medicinal materials: rehmannia root, cooked rhubarb, garden burnet root, pseudo-ginseng, cuttlebone, madder and cattail pollen (fried); determining digitonin D and ginsenoside Rg by liquid chromatography 1 Ginsenoside Rb and ginsenoside Rb 1 Notoginsenoside R 1 Comprises the following steps:
(1) Chromatographic conditions and System suitability test
Octadecylsilane chemically bonded silica is used as a filling agent; gradient elution is carried out by taking an organic solvent as an A phase and a dilute acid aqueous solution with a certain concentration as a B phase, wherein the organic solvent is selected from methanol, acetonitrile or acetonitrile-methanol mixed solution, and the dilute acid aqueous solution is selected from a phosphoric acid aqueous solution with a volume percentage concentration of 0.01-1.0% or an acetic acid aqueous solution with a volume percentage concentration of 0.01-0.5%; the column temperature is 20-40 ℃; an ultraviolet detector or a diode array detector is adopted to detect the wavelength of 200-300nm; the flow rate is 0.8ml/min-1.2ml/min; the number of theoretical plates is not less than 3000 calculated by the digitoxin D;
preferably, the phase A is methanol, and the phase B is phosphoric acid aqueous solution with the volume percentage concentration of 0.05 percent; the flow rate is 1.0ml/min; the column temperature is 30 ℃; the detection wavelength was 203nm.
Preferably, when gradient elution is performed, the gradient elution procedure is as follows: 0 to 10min,5% by weight of A;10 to 15min,5 to 10% A; 15-25min, 10% by weight of A; 25-28min, 8% by weight of A.
The gradient elution procedure is as follows:
| time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0～10 | 5 | 95 |
| 10～15 | 5→10 | 95→90 |
| 15～25 | 10 | 90 |
| 25～28 | 8 | 92 |
(2) Preparation of control solutions
Taking reference substance ginsenoside Rg 1 Ginsenoside Rb 1 Notoginsenoside R 1 Precisely weighing digitoxin D, and adding methanol to obtain ginsenoside Rg containing 0.3 mg/lml 1 Ginsenoside Rb and ginsenoside Rb 1 Notoginsenoside R 1 And 30 mu g of a solution of digitoxin D;
(3) Preparation of test solution
Grinding the content of the Heptaculum Capsule, weighing 0.2g, precisely weighing, placing into a conical flask with a plug, precisely adding 5-50ml of extraction solvent, weighing, heating under reflux or ultrasonic extracting, cooling, adding diatomaceous earth or not, shaking, centrifuging at high speed, filtering supernatant, and collecting filtrate.
Preferably, the extraction solvent is methanol; the mass volume ratio of the test sample to the extraction solvent is 1g.
Preferably, the ultrasonic extraction is carried out for 10-60min, and further preferably, the ultrasonic extraction time is 50min.
Preferably, the added purifying agent is diatomite; further preferred diatomaceous earth has a mass of 10g.
Preferably, the high-speed centrifugation frequency is 2000-5000r/min, and the centrifugation time is 20-60min; more preferably, the centrifugation frequency is 4500r/min, and the centrifugation time is 40min.
Preferably, the filtering method is filter head filtering, and the pore size of the filter head is 0.22 μm.
(4) Measurement of
Respectively and precisely sucking the reference solution and the test solution, injecting into a high performance liquid chromatograph, and measuring to obtain a measurement result.
Preferably, precisely sucking 10-mul of each of the reference substance and the test solution; further preferably, the control solution and the test solution are each precisely pipetted at 10. Mu.l.
The prescription of the blood balancing capsule comprises: 450g of rehmannia root, 300g of madder, 450g of garden burnet root, 300g of cattail pollen (fried), 300g of cuttlebone, 180g of pseudo-ginseng and 180g of cooked rhubarb. In the formula, rehmannia root, radix rehmanniae is used as a monarch drug of the formula for clearing heat and cooling blood, nourishing yin and promoting the production of body fluid; the prepared rhubarb has the effects of clearing heat, cooling blood and stopping bleeding, removing blood stasis, resolving masses and activating stagnancy, the garden burnet root has the effects of cooling blood and stopping bleeding, and astringing the taste, and the two medicines are matched to help monarch as the minister; the madder root has the effects of cooling blood and stopping bleeding, activating blood and removing stasis, is commonly used for various kinds of bleeding caused by blood heat, the cuttle bone can astringe and stop bleeding, and is good at treating metrorrhagia and metrostaxis, the fried cattail pollen can astringe and stop bleeding, promote blood circulation and remove blood stasis, three medicines are used together to assist monarch and minister medicines to strengthen the functions of stopping bleeding, removing blood stasis, cooling blood and regulating menstruation, and the three medicines are used as adjuvant medicines together; notoginseng, with warm nature, can stop bleeding without retaining stasis, and is used as a guiding drug in the recipe in combination with other drugs. The medicines have the effects of clearing heat and dissolving stasis, stopping bleeding and regulating menstruation, and are used for treating metrorrhagia and metrostaxis caused by blood heat with blood stasis.
Compared with the prior art, the invention has the following beneficial technical effects:
in the content determination method of the Xueping capsule in the prior art, only thin-layer identification is carried out on the ministerial drug rhubarb and the conductant drug pseudo-ginseng, and the content determination of the emodin is carried out on the ministerial drug rhubarb; is not right toThe monarch drug and other ministerial drugs in the prescription are subjected to content determination, the specificity of the existing quality control standard is poor, the quality of the drug cannot be comprehensively reflected, and the clinical curative effect index cannot be controlled, so that a detection method of the product is necessary to be further perfected, and the control index is associated with the clinical curative effect, so that the quality and the curative effect of the product are further ensured. The invention relates to a content determination method for simultaneously detecting various effective components in Xueping capsules, which is used for detecting the effective component of digitoside D in the monarch drug dried rehamnnia root and the ginsenoside Rg in the precious drug pseudo-ginseng 1 Ginsenoside Rb 1 Notoginsenoside R 1 The detection method is sensitive, has strong specificity, is accurate and has good reproducibility, can make up the defects of the prior method, can simultaneously detect a plurality of effective components of the monarch drug and the precious drug in the Xueping capsule only by preparing and mixing contrast, effectively controls the quality of the drug, improves the detection efficiency, reduces the detection cost, associates the drug quality with the effective components of clinical treatment, thereby achieving the standards of stability, controllability, high efficiency and safety, further better meeting the medical requirements and providing a basis for improving the quality standard of the variety.
Drawings
The invention is further described below with reference to the accompanying drawings:
fig. 1 and fig. 2 are specific test chromatograms of the invention under chromatographic conditions, wherein a is a blank solution chromatogram, b is a reference solution chromatogram, c is a negative sample solution chromatogram, and d is a test sample solution chromatogram.
FIG. 3 is a chromatogram of a portion of a mobile phase of a selective assay of a mobile phase under chromatographic conditions in accordance with the present invention; in the figure, a is a chromatogram of mobile phase No. 1 in table 1 (acetonitrile-water or methanol-water 5).
FIG. 4 is instrument precision chromatogram for the first 3 experiments under chromatographic conditions in accordance with the present invention;
figure 5 is an instrument precision chromatogram for the last 3 experiments under chromatographic conditions in accordance with the present invention.
FIG. 6 is a graph showing the linear relationship of xanthosine D under chromatographic conditions in accordance with the present invention.
FIG. 7 shows notoginsenoside R under chromatographic conditions in accordance with the present invention 1 A linear relationship graph.
FIG. 8 shows ginsenoside Rb under chromatographic conditions in accordance with the present invention 1 And (4) a linear relation graph.
FIG. 9 shows ginsenoside Rg under chromatographic conditions in the present invention 1 And (4) a linear relation graph.
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way. The experimental procedures in the following examples are all conventional ones unless otherwise specified. The raw materials, reagents and materials used in the following examples are all commercially available products unless otherwise specified.
Example 1: prescription of Xueping capsule and its preparation method
Prescription: rubia cordifolia 300g sanguisorba officinalis 450g cattail pollen (parched) 300g rehmannia glutinosa 450g
Cuttlebone 300g pseudo-ginseng 180g cooked rhubarb 180g
The preparation method comprises the following steps: taking 90g of pseudo-ginseng from the seven raw materials, and crushing the pseudo-ginseng into fine powder for later use. Crushing the rest Notoginseng radix, mixing with radix et rhizoma Rhei preparata and rehmanniae radix, adding 80% ethanol, heating and reflux-extracting for 2 times, each for 1 hr, mixing the ethanol extractive solutions, and filtering to obtain filtrate. Decocting garden burnet root, cuttlebone, madder root, cattail pollen (fried) and dregs after alcohol extraction in water for 3 times, each time for 1 hour, merging decoction liquids, filtering, concentrating into clear paste with the relative density of 1.10-1.15 (60 ℃), adding ethanol until the alcohol content reaches 50%, stirring evenly, refrigerating for 24 hours at 5-10 ℃, taking supernate and alcohol extraction liquid to merge, recovering ethanol, and concentrating into extract with the relative density of 1.25-1.30 (60 ℃) under reduced pressure. Adding the fine powder of the pseudo-ginseng, mixing uniformly, drying at 80 ℃, crushing into fine powder, adding a proper amount of starch if necessary, placing the mixture into a mixer, mixing uniformly, granulating, drying at 70-80 ℃, and encapsulating to prepare 1000 granules.
Example 2: selection of content determination wavelength and mobile phase in Xueping capsule
2.1 instruments and materials
A Saimer fly Ultimate3000 type high performance liquid chromatograph; AS5150A ultrasonic cleaner, kunshan ultrasonic instruments ltd; GB204 electronic balance, AT201 electronic balance, mettler-toledo; LD4-2 electric centrifuge, tan City laboratory Instrument factory. Methanol/acetonitrile (chromatographically pure) was purchased from semer fisher technologies; the other reagents are purchased from analytical purifiers and purchased from Tianjin Daimao chemical reagent factories; diatomaceous earth (for chromatography) was purchased from national chemical group, chemical reagents, ltd. The Heipin capsules used in this experiment were all produced by the company Siranqian Heiyao pharmaceutical industry Co. The reference substances are purchased from Shanxi general medicine science and technology Limited and produced by the verification of Chinese medicine biological products.
2.2 selection of wavelength
The digitoxin D reference substance solution is subjected to wavelength scanning in the range of 200-500nm, the result shows that the maximum absorption is generated at 203nm and 275nm, and the 203nm with less interference is selected as the detection wavelength considering that the absorbance of other components in the prescription is higher at about 275nm and more interference exists. Mixing ginsenoside Rg 1 Ginsenoside Rb 1 Notoginsenoside R 1 The solution is scanned at 200-500nm, and the result shows that the absorption is maximum at 203nm and 213nm, and the 203nm with less interference is selected as the detection wavelength considering that the absorption of other components in the prescription is larger at about 213nm and the interference is more.
2.3 selection of the Mobile phase
Preliminary experiments revealed that the experiments were carried out using mobile phases such as acetonitrile-water, methanol-water, acetonitrile-1.0% aqueous phosphoric acid, acetonitrile-0.2% aqueous hac, methanol-0.1% phosphoric acid, methanol-0.05% phosphoric acid, etc. The results show that when organic solvents and dilute acid solutions with different proportions are tested at equal degrees, the peak shapes or the separation degrees of the four effective components are poor; the separation effect is best when methanol-0.05 percent phosphoric acid aqueous solution is used, and the baseline is the most stable. And further optimizing a methanol-0.05 percent phosphoric acid aqueous solution mobile phase system.
To make the digitoxin D and ginsenoside Rg in the sample 1 Ginsenoside, ginsenosideRb 1 Notoginsenoside R 1 The method can obtain effective separation while the peak shape, the theoretical plate number and the like meet the requirements, particularly the digitonin D chromatographic peak and the impurity peak are difficult to separate, through a large number of experiments, different solvents and different elution ratios are adopted in the experiment investigation, the test map selected by part of mobile phase is shown in figure 3, the result shows that the mobile phase system only consists of methanol-0.05% phosphoric acid water solution, the gradient separation effect is best under the condition of gradient elution, the base line is most stable, 4 component chromatographic peaks can be completely separated, 4 target components present better peak-out time, peak type and separation degree, and the condition can meet the relevant requirements of analysis and determination. See in particular table 1 below.
Table 1 mobile phase selection test results
Example 3: research on specialization
3.1 preparation of negative samples:
prescription: 300g of madder, 450g of garden burnet root, 300g of cattail pollen (fried), 300g of cuttlebone and 180g of cooked rhubarb
The preparation method comprises the following steps: mixing radix et rhizoma Rhei preparata and rehmanniae radix, adding 80% ethanol, heating and reflux-extracting for 2 times, each for 1 hr, mixing the ethanol extractive solutions, and filtering to obtain filtrate. Decocting sanguisorba officinalis, cuttlebone, madder, cattail pollen (fried) and dregs after alcohol extraction for 3 times with water, each time for 1 hour, combining decoction liquids, filtering, concentrating into clear paste with the relative density of 1.10-1.15 (60 ℃), adding ethanol until the ethanol content reaches 50%, stirring evenly, refrigerating for 24 hours at the temperature of 5-10 ℃, taking supernate and ethanol extract liquid to be combined, recovering ethanol, concentrating into extract with the relative density of 1.25-1.30 (60 ℃), drying at the temperature of 80 ℃, crushing into fine powder, adding a proper amount of starch if necessary, uniformly mixing in a mixer, granulating, drying at the temperature of 70-80 ℃, encapsulating, and preparing into 1000 granules.
3.2 preparation of negative sample solution:
taking negative sample content, grinding, weighing 0.2g, accurately weighing, placing in a conical flask with a plug, accurately weighing 20ml of methanol, ultrasonically extracting for 50min, weighing to make up for loss, cooling, adding 10g of diatomite, shaking up, centrifuging at 4500r/min for 40min, filtering supernatant, and taking subsequent filtrate.
3.3 preparation of test solution:
the test method comprises the following steps: taking the content of the same batch of the Heipin capsules, grinding, weighing 0.2g, precisely weighing, placing in a conical flask with a plug, precisely adding 5-50ml of an extraction solvent, weighing, heating, refluxing or ultrasonically extracting, cooling, adding or not adding kieselguhr, shaking up, centrifuging at high speed, filtering supernatant, and taking a subsequent filtrate to obtain the Heipin capsule; respectively sucking 10 μ l of each of the reference solution and the sample solution, injecting into high performance liquid chromatograph, and measuring to obtain measurement result. The results are detailed in table 2.
TABLE 2 selection test results of test article preparation methods
The results show that the complete effective components can be fully extracted by adding methanol for ultrasonic extraction for 50min and heating reflux for 3 hours, but the problems that the filtering is difficult to be carried out by adopting a 0.22-micron filter head for direct filtering, the filtering can be carried out only after the pressure is increased, the filter membrane is frequently damaged when the pressure is increased are found, and the problems that the filtering is basically difficult to be carried out smoothly by adopting the 0.22-micron filter membrane for heating reflux and the filtering is directly carried out by adopting the 0.45-micron filter head can be improved. In the experiment of sample preparation, the problem that the extracting solution is difficult to directly filter can be successfully solved by adopting the diatomite as the purifying agent and a high-speed centrifugation measure, and the content measurement is not influenced. Therefore, the preparation method for determining the best test sample comprises the following steps: taking the content of the Heptamine capsule, grinding, weighing 0.2g, precisely weighing, placing in a conical flask with a plug, precisely adding 20ml of methanol, weighing, ultrasonically extracting for 50min or refluxing for 3 h, weighing to make up for loss, cooling, adding 10g of diatomite, shaking, centrifuging at 4500r/min for 40min, filtering the supernatant, and taking the subsequent filtrate.
3.4 chromatographic conditions and System suitability test
Octadecylsilane chemically bonded silica is used as a filling agent; according to the chromatographic conditions described in example 2 and No. 4 of Table 1, the number of theoretical plates should be not less than 3000 calculated as digitoxin D;
preparation of control solutions: taking reference substance ginsenoside Rg 1 Ginsenoside Rb 1 Notoginsenoside R 1 DIHUANGGAND, precisely weighing, and adding squama Manis to obtain a mixture containing ginsenoside Rg 0.3 mg/lml 1 Ginsenoside Rb 1 Notoginsenoside R 1 And 30 mu g of a solution of the digitoxin D;
preparation of a blank solution: methanol was taken directly as a blank solution.
And (3) determination: precisely sucking 10 μ l each of the negative sample solution, the reference solution, the sample solution and the blank solution, injecting into high performance liquid chromatograph, and measuring.
The results show that: in the chromatogram of the sample solution, ginsenoside Rg is detected 1 Ginsenoside Rb 1 Notoginsenoside R 1 The same chromatographic peak is arranged at the corresponding position of the chromatographic peak of the digitoxin D reference substance; negative and blank reagents are not interfered, which shows that the chromatographic condition has good specificity. See in particular figures 1-2 below.
Example 4: repeatability test
4.1 chromatographic conditions and System suitability test
Octadecylsilane chemically bonded silica is used as a filling agent; according to the chromatographic conditions described in example 2 and No. 4 of Table 1, the number of theoretical plates should be not less than 3000 calculated as digitoxin D;
4.2 precisely absorbing 10 mul of the same sample solution, injecting the sample solution into a liquid chromatograph, repeatedly injecting the sample for 6 times, and calculating the corresponding total peak area RSD.
4.3 the results show that the RSD values of the peak areas are 0.6%,2.9%,0.7%,1.0%, less than 3.0%; indicating good precision. The results are shown in Table 3 below and FIGS. 4-5.
TABLE 3 precision test data sheet
| Number of times | For the first time | For the second time | The third time | Fourth time | Fifth time | The sixth time | RSD |
| Retention time of Dihuang glycoside D | 19.59 | 19.36 | 18.97 | 18.85 | 18.95 | 19.31 | 1.5％ |
| Notoginseng radix saponin R 1 Retention time | 21.73 | 21.49 | 21.09 | 20.91 | 21.02 | 21.47 | 1.5％ |
| Ginsenoside Rg 1 Retention time | 24.08 | 23.79 | 23.32 | 23.14 | 23.26 | 23.82 | 1.6％ |
| Ginsenoside Rb 1 Retention time | 26.96 | 26.63 | 26.15 | 26.03 | 26.07 | 26.69 | 1.5％ |
| Area of the peak of the Dihuang glycoside D | 181.740 | 179.163 | 181.838 | 181.255 | 180.591 | 180.458 | 0.6％ |
| Notoginseng radix saponin R 1 Peak area | 284.717 | 295.866 | 297.855 | 296.814 | 292.832 | 276.803 | 2.9％ |
| Ginsenoside Rg 1 Peak area | 1155.727 | 1176.202 | 1177.021 | 1171.992 | 1168.560 | 1179.552 | 0.7％ |
| Ginsenoside Rb 1 Peak area | 844.168 | 850.017 | 853.672 | 831.610 | 843.045 | 851.845 | 1.0％ |
Example 5: linearity, range
5.1 chromatographic conditions and System suitability test
Octadecylsilane chemically bonded silica is used as a filling agent; according to the chromatographic conditions described in example 2 and in Table 1 No. 4, the number of theoretical plates should be not less than 3000 calculated as digitoxin D.
5.2 suction of ginsenoside Rg 1 Ginsenoside Rb 1 Notoginsenoside R 1 And the concentration of each reference substance of the mixed solution of the digitoxin D reference substances is shown in the following table, 10 mu l of each reference substance is injected into a liquid chromatograph, a chromatogram is recorded, the concentration (x) is subjected to linear regression analysis by using a peak area (y), a linear relation graph is drawn, and a linear equation is reported. (see FIGS. 6-9 for details)
TABLE 4 Linear relationship test data sheet
5.3 results show that under the condition of the sample amount of 10 mul, the digitonin D is 17.22 to 103.32 mu g/ml and the notoginsenoside R is 1 Ginsenoside Rb is added in the amount of 0.0854mg/ml to 1.3672mg/ml 1 0.0872 mg/ml-1.3956 mg/ml, ginsenoside Rg 1 0.0994 mg/ml-1.5912 mg/ml, and has good linear relation with the peak area within the concentration range.
Example 6: accuracy (recovery)
6.1 chromatographic conditions and System suitability test
Octadecylsilane chemically bonded silica is used as a filling agent; according to the chromatographic conditions described in example 2 and No. 4 of Table 1, the number of theoretical plates should be not less than 3000 calculated as digitoxin D;
6.2 preparation of control solutions: taking a digitonin D reference substance, precisely weighing, and adding methanol to prepare a reference substance solution with the concentration of 34.44 mu g/ml;
6.3 preparation of test solution: taking the content of the Heiping capsule, grinding, weighing 1.0g of the content and 9 parts of the content respectively, precisely weighing, placing in a conical flask with a plug, then weighing 9 parts of the digitoxin D reference substance respectively, sequentially adding into the conical flask after sample addition, precisely adding 20ml of methanol, weighing, ultrasonically extracting for 50min, weighing for complementing loss, cooling, adding 10g of diatomite, shaking uniformly, centrifuging at 4500r/min for 40min, filtering the supernatant, and taking the subsequent filtrate to obtain the capsule.
Percent recovery = (C-A)/B × 100%
Wherein A is the amount of the component to be tested contained in the test sample; b is the amount of the added reference substance; c is an observed value.
TABLE 5 recovery test data sheet
The standard requires that: the recovery rate is 85-110%, and the RSD is less than 4.0%; experimental results show that the method has good accuracy.
Example 7: intermediate precision
7.1 chromatographic conditions and System suitability test
Octadecylsilane chemically bonded silica is used as a filling agent; according to the chromatographic conditions described in example 2 and No. 4 of Table 1, the number of theoretical plates should be not less than 3000 calculated as digitoxin D;
7.2 in the same laboratory, different experimenters A and B perform experiments on different dates by using different chromatographs, recording chromatograms and calculating the content. (the detailed map is shown in FIGS. 4-5)
TABLE 6 precision test data sheet
7.3 the result shows that the invented patented method is good in precision.
Example 8: durability
8.1 chromatographic conditions and System suitability test
Octadecylsilane chemically bonded silica is used as a filling agent; according to the chromatographic conditions described in example 2 and No. 4 of Table 1, the number of theoretical plates should be not less than 3000 calculated as digitoxin D;
8.2 taking the same sample solution, standing at room temperature for 0, 4, 8, 12, 24 and 36 hours respectively, and precisely sucking 10 mul to inject into a high performance liquid chromatograph;
TABLE 7 stability test data Table for Dihuang glycoside D
| Sample introduction time point | Retention time | Peak area |
| 0h | 18.95 | 180.591 |
| 4h | 18.88 | 181.233 |
| 8h | 18.78 | 184.345 |
| 12h | 18.97 | 179.544 |
| 24h | 19.02 | 181.235 |
| 36h | 18.85 | 182.214 |
| Mean value of | 18.91 | 181.530 |
| RSD | 0.5％ | 1.0％ |
TABLE 8 notoginsenoside R 1 Stability test data sheet
| Sample introduction time point | Retention time | Peak area |
| 0h | 21.02 | 292.832 |
| 4h | 21.54 | 296.786 |
| 8h | 21.04 | 291.445 |
| 12h | 21.03 | 292.456 |
| 24h | 21.12 | 292.987 |
| 36h | 21.05 | 293.441 |
| Mean value of | 21.13 | 293.320 |
| RSD | 1.0％ | 0.7％ |
TABLE 9 ginsenoside Rg 1 Stability test data sheet
| Sample introduction time point | Retention time | Peak area |
| 0h | 23.34 | 1168.544 |
| 4h | 23.45 | 1172.456 |
| 8h | 23.29 | 1169.542 |
| 12h | 23.23 | 1189.456 |
| 24h | 23.47 | 1171.221 |
| 36h | 23.44 | 1156.654 |
| Mean value of | 23.37 | 1171.310 |
| RSD | 0.42％ | 0.90％ |
TABLE 10 ginsenoside Rb 1 Stability test data sheet
| Sample introduction time point | Retention time | Peak area |
| 0h | 26.07 | 843.045 |
| 4h | 25.89 | 848.456 |
| 8h | 26.12 | 839.264 |
| 12h | 26.45 | 832.421 |
| 24h | 25.95 | 845.487 |
| 36h | 26.24 | 841.023 |
| Mean value of | 26.12 | 841.620 |
| RSD | 0.8％ | 0.7％ |
8.3, the results show that the RSD value of the retention time of the test solution within 24 hours and the RSD value of the peak area are both less than 2.0 percent; the test solution was stable for 36h at room temperature.
Example 9: ginsenoside Rg in Xueping capsule 1 Ginsenoside Rb 1 Notoginsenoside R 1 Content determination of digitonin D and stability test thereof
Samples from large-scale production of Hepial capsules (lot Nos. T0200026, T0200027, T0200028, manufactured by Seikaga Kappaphysa, K.K.) were subjected to accelerated test and room temperature standing test. Accelerated testing: placing the sample in a closed environment with the relative humidity of 75% +/-5% and the temperature of 40 +/-2 ℃ for 6 months, sampling in 0, 1, 2, 3 and 6 months for character observation and microorganism limit detection; room temperature standing experiment: the sample was placed in a closed environment with a relative humidity of 60% +/-5% and a temperature of 25 deg.C + -2 deg.C for 3 months, and sampled and examined at 0, 3, 6, 12, and 18 months, and the test results are shown in Table 10.
TABLE 11 Capsule stability test data sheet
The experimental results show that the ginsenoside Rg of the invention can be placed for 6 months and 18 months at room temperature in an accelerated test 1 Ginsenoside Rb 1 Notoginsenoside R 1 The contents of the digitonin D and the ginsenoside Rg are not changed significantly and meet the requirements, which indicates that the application of the ginsenoside Rg 1 Ginsenoside Rb 1 Notoginsenoside R 1 The method for detecting the content of the digitoxin D is used for more effectively controlling the quality of the medicine and is taken into consideration in the quality standard in the next step.
In summary, the following steps: the ginsenoside Rg in the Xueping capsule 1 Ginsenoside Rb 1 Notoginsenoside R 1 The content determination method of the digitoxin D determines the scientificity of the method from the aspects of a repetitive chromatographic expression chart, a test solution stability chart, instrument precision, recovery rate, linearity, specificity and the like. The content determination of monarch drug and precious drug of the preparation is increased, the detection cost is reduced, the detection efficiency is improved, the safety and effectiveness of the preparation are ensured, the defects of the prior art are overcome, and meanwhile, the detection accuracy is further improved.
The embodiments given above are preferable examples for implementing the present invention, and the present invention is not limited to the above-described embodiments. Any non-essential addition and replacement made by the technical characteristics of the technical scheme of the invention by a person skilled in the art belong to the protection scope of the invention.
Claims (5)
1. A method for simultaneously detecting the contents of multiple effective components in Xueping capsule features that the liquid chromatography is used to detect the contents of the effective components of rehmannia root, rehmannia root and ginsenoside Rg in notoginseng 1 Ginsenoside Rb 1 Notoginsenoside R 1 The content of (b) is measured simultaneously, specifically comprising the steps of:
s1 chromatographic conditions and System suitability test
Octadecylsilane chemically bonded silica is used as a filling agent; taking methanol as phase A and phosphoric acid aqueous solution with the volume percentage concentration of 0.05% as phase B, and performing gradient elution, wherein the gradient elution procedure is as follows: 0 to 10min,5% by weight of A;10 to 15min,5 to 10% A; 15-25min, 10% by weight of A; 25-28min, 8% by weight A; the column temperature is 20-40 ℃; an ultraviolet detector or a diode array detector is adopted to detect the wavelength of 200-300nm; the flow rate is 0.8ml/min-1.2ml/min; the number of theoretical plates is not less than 3000 calculated by the digitoxin D;
s2 preparation of reference substance solution
Taking reference substance ginsenoside Rg 1 Ginsenoside Rb and ginsenoside Rb 1 Notoginsenoside R 1 Precisely weighing digitoxin D, and adding methanol to obtain ginsenoside Rg containing 0.3 mg/lml 1 Ginsenoside Rb 1 Notoginsenoside R 1 And 30 mu g of a solution of digitoxin D;
s3, preparation of test solution
Taking the content of the Heptamine capsule, grinding, weighing 0.2g, precisely weighing, placing in a conical flask with a plug, precisely adding 20ml of methanol, weighing, ultrasonically extracting for 50min or refluxing for 3 h, weighing to supplement loss, cooling, adding 10g of diatomite, shaking, centrifuging at 4500r/min for 40min, filtering the supernatant, and taking the subsequent filtrate;
s4-determination
Respectively sucking 10-20 μ l of each of the reference solution and the test solution, injecting into high performance liquid chromatograph, and measuring to obtain measurement result.
2. The method for simultaneously detecting the contents of a plurality of active ingredients in a Xueping capsule according to claim 1, wherein: the Xueping capsule is prepared from the following seven medicinal materials: dried rehmannia root, cooked rhubarb, garden burnet root, pseudo-ginseng, cuttlebone, india madder root and cattail pollen charcoal.
3. The method for simultaneously detecting the contents of a plurality of active ingredients in a Xueping capsule according to claim 1, wherein: the flow rate is 1.0ml/min; the column temperature is 30 ℃; the detection wavelength was 203nm.
4. The method for simultaneously detecting the contents of a plurality of active ingredients in a Xueping capsule according to claim 1, wherein: in the step S3, a purifying agent is added before centrifugation in the preparation of the test solution, wherein the purifying agent is diatomite; after centrifugation, filtration is filter head filtration, and the aperture of the filter head is 0.22 mu m.
5. The method for simultaneously detecting the content of a plurality of active ingredients in a Xueping capsule according to claim 1, wherein: preparing a test solution: weighing 0.2g, precisely weighing, placing in a conical flask with a plug, precisely weighing 20ml of methanol, weighing, ultrasonically extracting for 50min, weighing to supplement loss, cooling, adding 10g of diatomite, shaking, centrifuging at 4500r/min for 40min, filtering supernatant, and collecting subsequent filtrate.
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