阅读说明：本技术 一测多评法测定丹红化瘀制剂成分含量的方法 (Method for determining contents of components of Danhong Huayu preparation by one-test-multiple-evaluation method ) 是由 唐清 匡艳辉 林娟 王德勤 李楚源 于 2019-11-14 设计创作，主要内容包括：本发明公开了一种一测多评法测定丹红化瘀制剂成分含量的方法,以新橙皮苷为内标,建立新橙皮苷与参素钠、原儿茶醛、丹酚酸D、柚皮苷、丹酚酸B之间的相对校正因子,通过只测定新橙皮苷的含量,用相对校正因子计算出另外五种成分的含量,建立全面的可控制丹红化瘀制剂的质量标准。(The invention discloses a method for determining the component content of a danhong Huayu preparation by a one-test-multiple-evaluation method, which takes neohesperidin as an internal standard, establishes relative correction factors between the neohesperidin and sodium ginsengenin, protocatechualdehyde, salvianolic acid D, naringin and salvianolic acid B, calculates the content of other five components by only determining the content of the neohesperidin and using the relative correction factors, and establishes a comprehensive quality standard for controlling the danhong Huayu preparation.)

1. A method for determining the contents of the components of a danhonghuayu preparation by a multi-evaluation method is characterized in that relative correction factors among neohesperidin, sodium ginseng extract, protocatechualdehyde, salvianolic acid D, naringin and salvianolic acid B are established by taking the neohesperidin as an internal standard, the contents of other five components are calculated by using the relative correction factors by only determining the content of the neohesperidin, and the contents are determined by adopting a liquid chromatography, wherein the test conditions of the chromatography and the system applicability are as follows: a C18 chromatography column; acetonitrile is taken as a mobile phase A, 0.2-0.8 wt% formic acid aqueous solution is taken as a mobile phase B, a gradient elution procedure is adopted, and the gradient conditions are as follows: 0-25 min, 5-12% of A; 25-30 min, 12-15% A; 30-70 min, 15-25% A; the flow rate is 0.8-1.2mL/min, the detection wavelength is 280-290 nm, and the column temperature is 28-32 ℃.

2. The method for determining the contents of the components of the Danhong Huayu preparation according to claim 1, wherein the detection wavelength is 286nm, 0.5 wt% formic acid aqueous solution is used as the mobile phase B, the flow rate is 1mL/min, and the column temperature is 30 ℃.

3. The method for determining the contents of the components of the Danhong Huayu preparation according to claim 1 or 2, wherein the relative correction factor F is calculated as follows:

in the formula, A_SPeak area, W, of neohesperidin control_SIs the concentration or quality of neohesperidin as control, A_KFor a certain object to be measuredPeak area of the component; w_KIs the concentration or mass of a certain component to be measured.

4. The method for determining the contents of the components of a preparation for removing blood stasis containing red sage root according to claim 1 or 2, wherein the relative correction factors of danshensu sodium, protocatechualdehyde, salvianolic acid D, naringin and salvianolic acid B are in the ranges of 0.1-15, 0.1-15 and 0.1-15, respectively.

5. The method for determining the contents of the components of a preparation for removing blood stasis containing danhong Huayu according to claim 1 or 2, wherein the relative correction factors of danshensu sodium, protocatechuic aldehyde, salvianolic acid D, naringin and salvianolic acid B are 3.173 + -0.30, 0.461 + -0.30, 1.728 + -0.30, 1.252 + -0.30 and 1.498 + -0.30, respectively.

6. The method for determining the contents of the components of a preparation for removing blood stasis containing danshensu according to claim 1 or 2, wherein the relative correction factors of danshensu sodium, protocatechuic aldehyde, salvianolic acid D, naringin and salvianolic acid B are 3.173 ± 0.15, 0.461 ± 0.15, 1.728 ± 0.15, 1.252 ± 0.15 and 1.498 ± 0.15, respectively.

7. The method for determining the contents of the components of the Danhong Huayu preparation according to claim 1 or 2, wherein the preparation of the test solution comprises the steps of: taking a proper amount of the Danhonghuayu preparation, putting the Danhonghuayu preparation into a volumetric flask or a conical flask, adding 50 wt% -90 wt% of methanol aqueous solution until each 1ml of the solution contains 0.1ml of the Danhonghuayu preparation, shaking up, and filtering through a microporous filter membrane to obtain a test solution.

8. The method for determining the contents of the components of the Danhong Huayu preparation according to claim 7, wherein the concentration of the methanol aqueous solution is 80 wt%.

9. The method for determining the content of the components of the Danhong Huayu preparation according to the one-test-multiple-evaluation method of claim 1 or 2, wherein the Danhong Huayu preparation comprises any one of Danhong Huayu oral liquid, Danhong Huayu soup, Danhong Huayu tablet, Danhong Huayu granule, Danhong Huayu capsule, Danhong Huayu dripping pill and Danhong Huayu paste.

10. The method for determining the content of the components of the Danhong Huayu preparation according to claim 1 or 2, wherein the preparation of the control solution comprises the following steps: respectively and accurately weighing appropriate amount of 6 reference substances, and preparing into mixed standard solution of sodium danshensu, protocatechualdehyde, salvianolic acid D, naringin, neohesperidin and salvianolic acid B with certain concentration with 80 wt% methanol water solution.

The technical field is as follows:

the invention relates to a medicine detection method, in particular to a method for determining the content of components of a Danhong Huayu preparation by a multi-evaluation method.

Background art:

the QAMS (quantitative analysis of multi-components by single-marker) is a quality control method which can determine only a certain representative component (effective, cheap and easily available) and calculate the content of other effective components to be detected so that the calculated value and the actual value of the effective components meet the requirement of quantitative methodology. The method for one-test-multiple-evaluation can better solve the bottleneck problem that a reference substance is lacked in the quality control of the traditional Chinese medicine, can better finish the quantitative evaluation of unstable components, and has the advantages of low detection cost and high analysis efficiency.

The Danhong Huayu preparation mainly comprises seven raw material medicines of salvia miltiorrhiza, angelica, szechuan lovage rhizome, peach seed, safflower, radix bupleuri and bitter orange, and comprises but is not limited to orally taken liquid, Danhong Huayu decoction or Danhong Huayu tablet, Danhong Huayu granule, Danhong Huayu capsule, Danhong Huayu dropping pill and other pharmaceutically acceptable dosage forms.

The Danhong Huayu oral liquid is a unique variety (Chinese medicine standard Z10960051) of Guangzhou Baiyunshan and Megaoku traditional Chinese medicine limited company, the quality standard of the Danhong Huayu oral liquid is recorded in the first part of the 'Chinese pharmacopoeia' of 2015 edition at present, and the quality standard comprises thin-layer identification of medicinal materials such as salvia miltiorrhiza, radix bupleuri, angelica, ligusticum wallichii and the like and determination of protocatechuic aldehyde content by an HPLC method. At present, no research report of a Danhong Huayu preparation one-test multiple evaluation method is found.

The Danhong Huayu preparation is prepared from a plurality of medicinal materials, the active ingredients are complex, and whether a one-test-and-multiple-evaluation method is suitable or not and how to analyze the active ingredients in the Danhong Huayu preparation becomes a problem. The inventor researches the above and selects several components (neohesperidin, sodium ginseng element, protocatechualdehyde, salvianolic acid D, naringin and salvianolic acid B) with clear pharmacological activity and rich content for determination. In developing a one-test-multiple-evaluation method, the selection of a target compound will seriously affect the reliability and practicability of the assay method, and no scientific method for screening the target compound exists at present. In a multi-scale study, the choice of which component is used as the internal standard is also a critical issue, since the analyte content is calculated from the relative calibration factor between the internal standard and the other compounds, and the stability of the relative calibration factor affects the accuracy of the final content measurement. The inventor researches the above and finds a stable compound as an internal standard substance, and finds that the compound is particularly suitable for a multi-evaluation method of a danhong stasis-removing preparation, thereby solving the related technical problems.

The invention content is as follows:

aiming at the defects of the existing single-component and multi-index content determination method, the invention provides a method for determining the content of the components of the danhonghuayu preparation by a one-measurement multi-evaluation method, which has high practicability, simple operation, cost saving and stability and controllability.

The invention is realized by the following technical scheme:

a method for determining the contents of components of a danhong Huayu preparation by a multi-evaluation method comprises the steps of establishing relative correction factors among neohesperidin, sodium danshensu, protocatechualdehyde, salvianolic acid D, naringin and salvianolic acid B by taking neohesperidin as an internal standard, calculating the contents of other five components by using the relative correction factors by only determining the content of the neohesperidin, and determining by adopting a liquid chromatography, wherein the test conditions of the chromatography and the system applicability are as follows: a C18 chromatography column; the mobile phase uses acetonitrile as mobile phase A, 0.2-0.8 wt% and preferably 0.5 wt% formic acid water solution as mobile phase B, and adopts a gradient elution procedure, and the gradient conditions are as follows: 0-25 min, 5-12% of A; 25-30 min, 12-15% A; 30-70 min, 15-25% A; the flow rate is 0.8-1.2mL/min, preferably 1 mL/min; the detection wavelength is 280 nm-290 nm, preferably 286nm, the column temperature is 28-32 ℃, and preferably 30 ℃.

The content of sodium danshensu, protocatechualdehyde, salvianolic acid D, salvianolic acid B and naringin is calculated by the following formula:

the content (mg/g) is (C)_S/W)×(A_U/A_S)×V×D×F

C_SConcentration of neohesperidin control (mg/mL);

w sample size (g);

A_Upeak areas of other components in the sample;

A_Speak area of neohesperidin control;

v volume of extraction solvent (mL);

d, dilution times;

f relative correction factor;

wherein the calculation formula of the relative correction factor F is as follows:

in the formula, A_SPeak area, W, of neohesperidin control_SIs the concentration or quality of neohesperidin as control, A_KThe peak area of a certain component to be measured; w_KIs the concentration or mass of a certain component to be measured.

Preferably, the relative correction factors of sodium danshensu, protocatechualdehyde, salvianolic acid D, naringin and salvianolic acid B are respectively 0.1-15, 0.1-15 and 0.1-15.

Preferably, the relative correction factors of the danshensu sodium, the protocatechualdehyde, the salvianolic acid D, the naringin and the salvianolic acid B are 3.173 +/-0.30, 0.461 +/-0.30, 1.728 +/-0.30, 1.252 +/-0.30 and 1.498 +/-0.30 respectively.

Particularly preferably, the relative correction factors of the salvianic acid A sodium, the protocatechualdehyde, the salvianolic acid D, the naringin and the salvianolic acid B are 3.173 +/-0.15, 0.461 +/-0.15, 1.728 +/-0.15, 1.252 +/-0.15 and 1.498 +/-0.15 respectively.

Preparation of a test solution: placing the Danhonghuayu preparation in a volumetric flask or a conical flask, adding 50-90 wt% methanol aqueous solution, preferably 80 wt% methanol aqueous solution until each 1ml of the solution contains 0.1ml of the Danhonghuayu preparation, shaking up, and filtering with a microporous membrane to obtain a test solution. The microporous filter membrane is a 0.45 mu m microporous filter membrane.

The Danhong Huayu preparation mainly comprises seven raw materials of salvia miltiorrhiza, angelica, szechuan lovage rhizome, peach seed, safflower, radix bupleuri and bitter orange, and comprises but is not limited to a Danhong Huayu oral liquid, a Danhong Huayu decoction, a Danhong Huayu tablet, a Danhong Huayu granule, a Danhong Huayu capsule, a Danhong Huayu dropping pill, a Danhong Huayu paste and other pharmaceutically acceptable preparations.

Preparation of control solutions: respectively and accurately weighing appropriate amount of 6 reference substances, and preparing into mixed standard solution of sodium danshensu, protocatechualdehyde, salvianolic acid D, naringin, neohesperidin and salvianolic acid B with certain concentration with 80 wt% methanol water solution.

The invention has the following beneficial effects: according to the invention, neohesperidin is taken as an internal standard, relative correction factors among neohesperidin, sodium ginseng element, protocatechualdehyde, salvianolic acid D, naringin and salvianolic acid B are established, the contents of the other five components are calculated by using the relative correction factors through only measuring the content of neohesperidin, the method is simple, convenient, rapid, comprehensive, accurate and high in sensitivity, the detection cost and time are saved, the working efficiency is improved, the comprehensive quality standard for controlling the salvia miltiorrhiza and safflower stasis removing preparation is established, and the detection result is more stable and controllable.

Description of the drawings:

FIG. 1 is an HPLC chart of the combination of the control and Danhong Huayu oral liquid;

wherein, a, an HPLC chart of a mixed reference substance, b, an HPLC chart of the danhong Huayu oral liquid, 1, sodium danshensu, 2, protocatechualdehyde, 3, salvianolic acid D, 4, naringin, 5, neohesperidin, 6 and salvianolic acid D.

The specific implementation mode is as follows:

the following is a further description of the invention and is not intended to be limiting.

Example 1:

the Danhong Huayu preparation of the embodiment is an oral liquid of Danhong Huayu

The instrument comprises the following steps: 2695 HPLC (high Performance liquid chromatography) by Waters corporation, USA; daian U3000; a chromatographic column: waters Xbridge (250 mm. times.4.6 mm, 5 μm); one hundred thousand balances (sartorius, germany) model CP 225D; model BT 214D ten thousandth balance (sartorius, Germany)

Materials: 30 batches of Danhong Huayu oral liquid samples are collected in experiments and are all provided by Guangzhou Baiyun mountain and Megaku Chinese medicine limited company, sodium danshensu (batch No. 110855-201614, purity: 98.1%), protocatechualdehyde (batch No. 110810-201608, purity: 99.3%), hesperidin (batch No. 110721-201115, purity: 95.3%, batch No. 110721, purity: 96.2%), neohesperidin (batch No. 111857-201703, purity: 99.2%), salvianolic acid B (batch No. 111562-201716, purity: 94.1%), naringin (batch No. Y-006-171216, purity: > 98%) which is purchased from China pharmaceutical product verification institute, and naringin (batch No. Y-006-171216, purity: 98%) is purchased from Douglas biological technology, Inc. Acetonitrile as chromatographically pure (Merck, Germany); formic acid was chromatographically pure (komi european corporation); methanol was analytically pure (Guangzhou chemical laboratories, Inc.); the water is distilled water (Guangzhou Drechsler food and beverage Co., Ltd.).

The method and the result are as follows:

chromatographic conditions are as follows: waters Xbridge (250 mm. times.4.6 mm, 5 μm); the mobile phase is acetonitrile (A) -0.5% formic acid aqueous solution (B), gradient elution is carried out (0-25 min, 5% → 12% A; 25-30 min, 12% → 15% A; 30-70 min, 15% → 25% A; flow rate is 1.0 mL/min; detection wavelength 286nm, column temperature 30 ℃, sample injection amount 10 uL. under the chromatographic conditions, the separation degree of each component to be detected is better, as shown in figure 1. in the invention, the peak emergence time of salvianolic acid D and naringin is close, and the components can be better separated on a Waters Xbridge chromatographic column (the separation degree is more than 1.5).

Preparation of control solutions: respectively and accurately weighing appropriate amount of 6 reference substances, and preparing into mixed standard substance solutions with concentrations of sodium danshensu, protocatechualdehyde, salvianolic acid D, naringin, neohesperidin and salvianolic acid B of about 400, 25, 70 and 25 μ g/mL respectively by using 80 wt% methanol water solution.

Preparation of a test solution: precisely measuring 1ml of Danhonghuayu oral liquid, placing in a 10ml volumetric flask, adding 80 wt% methanol water solution to scale, shaking, filtering with 0.45 μm micropore, and collecting the subsequent filtrate.

Selection of internal standard:

the neohesperidin is a component which has higher content and good separation degree and is positioned in the middle part of a chromatogram in a peak position, and the neohesperidin is selected as an internal standard substance to reduce the errors of relative retention time and relative peak area of other chromatographic peaks.

Salvianolic acid B was selected as an internal standard, one-test-multiple-evaluation content analysis was performed, the relative retention time is shown in Table 1, and the calculated 12-lot content is shown in Table 2. As can be seen from the table, when salvianolic acid B was used as an internal standard, the peak area was small and the content was low, so that errors were likely to occur in the calculation of the content of other substances, and the relative average deviation (%) between the external standard method of protocatechuic aldehyde and the one-test-multiple evaluation method was large. Therefore, it is not suitable to select salvianolic acid B as the internal standard.

Therefore, in the invention, the neohesperidin of the flavonoid is finally selected as the internal standard substance by comparing with the phenolic acid component. The neohesperidin is found through experimental research to be a component which is higher in content, good in separation degree and has a peak position in the middle part of a chromatogram, and the neohesperidin serving as an internal standard substance can reduce errors of relative retention time and relative peak area of other chromatographic peaks. The salvianolic acid B is selected as an internal standard to perform one-test-multiple-evaluation content analysis, and because the peak area is small and the content is low, errors are easy to generate when the content of other substances is calculated, and the relative average deviation is large. Therefore, neohesperidin with relatively stable structure and controllable error of research result is finally selected as the internal standard substance.

TABLE 1 relative Retention time results for different instruments

TABLE 2 determination results (mg/ml) of 6 components in the Danhong Huayu oral liquid by different methods

Methodology investigation:

and (3) linear relation investigation: the mixed control solutions were precisely aspirated by 0.5, 1, 2, 5, 10, 15, and 20. mu.L, respectively, and injected into a liquid chromatograph, and the results of regression analysis were determined under the above chromatographic conditions using the peak area A as the ordinate and the sample amount C as the abscissa, and are shown in Table 3.

TABLE 3 regression equation and Linear Range for six Components

And (3) precision test: precisely sucking 10 μ L of the mixed reference substance, continuously sampling for 6 times according to the above chromatographic conditions, recording peak areas of sodium danshensu, protocatechualdehyde, salvianolic acid D, naringin, neohesperidin and salvianolic acid B, and calculating RSD. As a result, the peak areas RSD (n ═ 6) of the respective components were 1.23%, 1.49%, 0.68%, 1.62%, 2.21%, and 0.42%, respectively, indicating that the precision of the apparatus was good.

And (3) stability test: the same test solution of the danhonghuayu oral liquid is respectively injected and analyzed for 0, 2, 4, 6, 8, 12, 24 and 48 hours, the RSD of the peak areas of the danshensu sodium, the protocatechualdehyde, the salvianolic acid D, the naringin, the neohesperidin and the salvianolic acid B are respectively calculated to be 1.24 percent, 1.57 percent, 1.29 percent, 0.59 percent, 2.41 percent and 0.61 percent, and the result shows that the test solution is stable in 48 hours.

And (3) repeatability test: 6 parts of test solution is prepared from the same batch of the oral liquid for reducing the red blood stasis, the average contents of sodium danshensu, protocatechualdehyde, salvianolic acid D, naringin, neohesperidin and salvianolic acid B in the 6 parts of test solution are determined to be 4.2894, 0.4200, 0.8409, 1.7650, 1.0760 and 0.3774mg/ml according to the sample injection analysis of the chromatographic conditions, and the RSD is respectively 1.33%, 1.16%, 1.35%, 1.34%, 1.22% and 1.23%, and the result shows that the method has good repeatability.

Sample recovery rate test: precisely measuring 0.5ml and 6 parts of the same batch of Danhonghuayu oral liquid respectively, adding 1ml of 80 wt% methanol solution containing danshensu sodium, protocatechuic aldehyde, salvianolic acid D, naringin, neohesperidin and salvianolic acid B with the concentrations of 2.145, 0.21, 0.3921, 0.8645, 0.5415 and 0.1905mg/ml, preparing a test sample according to a test sample solution preparation method, carrying out sample injection analysis according to the chromatographic conditions, measuring the content of each component, and calculating the sample addition recovery rate. The average sample recovery rate of each component is as follows: 97.12%, 101.34%, 102.27%, 100.73%, 101.37% and 92.54% of RSD are respectively 3.12%, 2.88%, 2.37%, 2.24%, 2.86% and 3.65%, and the result shows that the method has good accuracy.

Calculating the relative correction factor of the component to be detected:

precisely sucking and mixing 0.5, 1, 2, 5, 10, 15 and 20 μ l of the reference substance solution, performing sample injection analysis according to the chromatographic conditions, and recording the peak area of each chromatographic peak. Using neohesperidin as internal standard, and calculating relative correction factor (f) ═ A according to formula_i×C_r)/(A_r×C_i)[A_iPeak area of the internal standard; a. the_rIs the peak area of the component to be measured; c_iConcentration as an internal standard; c_rThe concentration of the component to be measured), the relative correction factors of danshensu sodium, protocatechualdehyde, salvianolic acid D, naringin and salvianolic acid B at different concentrations are calculated, and the results are shown in Table 4.

TABLE 4 relative correction factor determination results of six components in DANHONGHUAYU oral liquid

One-test-multiple-evaluation durability evaluation:

influence of different instruments relative correction factors: the results of the relative correction factors of sodium danshensu, protocatechualdehyde, salvianolic acid D, naringin and salvianolic acid B on Waters e2695 and U3000 instruments are respectively examined, the results are shown in Table 5, the results show that the RSD of each component relative to the correction factor is less than 4%, and that different instruments have no significant influence on the relative correction factor of each component.

TABLE 5 Effect of different instruments on the relative correction factor of each component

Positioning chromatographic peaks of components to be detected:

precisely sucking the mixed reference solution, and respectively measuring and calculating the relative retention values of the components to be measured, namely the sodium danshensu, the protocatechuic aldehyde, the salvianolic acid D, the naringin and the salvianolic acid B in two instruments, namely a Waters e2695 instrument and a U3000 instrument. The results show that different brands of chromatographs have less influence on the relative retention time (RSD < 5%), see table 6, and therefore, the chromatographic peaks of the components can be located according to the relative retention method.

TABLE 6 relative retention time results for different instruments

Comparison of the results of the multi-evaluation method and the external standard method:

sucking 30 batches of the test solution 10 μ L of the oral liquid for red blood stasis removal, analyzing the sample injection, and calculating the contents of sodium danshensu, protocatechualdehyde, salvianolic acid D, naringin and salvianolic acid B by a one-test-multiple evaluation method and an external standard method, wherein the results are shown in Table 7. The results show that the contents measured by the two methods have no obvious difference (RAD is less than 4 percent), which indicates that the one-test-multiple-evaluation method can be used for measuring the contents of multiple components of the Danhong Huayu oral liquid.

TABLE 7 determination results (mg/ml) of 6 components in the Danhong Huayu oral liquid by different methods

Example 2:

reference example 1 is repeated except that the Danhong Huayu oral liquid is replaced with Danhong Huayu decoction.

The active ingredients of the Danhong Huayu decoction consist of seven raw materials of danshen root, Chinese angelica, Szechuan lovage rhizome, peach kernel, safflower, Chinese thorowax root and bitter orange under the item of the Danhong Hua Huayu oral liquid of 677 pages of the first part of Chinese pharmacopoeia of 2015 edition:

the preparation method comprises the following steps: decocting seven raw materials such as red sage root and the like in water twice, adding 8-10 times of water for the first time, decocting for half an hour, adding 5-8 times of water for the second time, decocting for 20 minutes, combining filtrates of the two times, and filtering to obtain the Danhong Huayu decoction. Or concentrating into soft extract to obtain DANHONGHUAYU paste.

Example 3:

reference example 1 is repeated except that the oral liquid is replaced by a tablet.

The active ingredients of the Danhong Huayu tablet comprise seven raw materials of danshen root, Chinese angelica, Szechuan lovage rhizome, peach seed, safflower, Chinese thorowax root and bitter orange under the item of Danhong Hua Huayu oral liquid on the first 677 pages of Chinese pharmacopoeia 2015 edition:

the preparation method comprises the following steps: decocting Saviae Miltiorrhizae radix in water twice, each for half an hour, filtering, mixing filtrates, concentrating at low temperature to obtain soft extract or fluid extract with relative density, drying, adding appropriate amount of adjuvant, mixing, granulating, and drying to obtain the final product. Or making into tablet or coating with film to obtain DANHONGHUAYU tablet.

Example 4:

reference example 1 is carried out with the difference that the Danhong Huayu oral liquid is replaced by Danhong Huayu dripping pills.

The active ingredients of the Danhong Huayu tablet comprise seven raw materials of danshen root, Chinese angelica, Szechuan lovage rhizome, peach seed, safflower, Chinese thorowax root and bitter orange under the item of Danhong Hua Huayu oral liquid on the first 677 pages of Chinese pharmacopoeia 2015 edition:

the preparation method comprises the following steps: decocting Saviae Miltiorrhizae radix in water twice, half an hour each time, filtering, mixing filtrates, concentrating at low temperature to obtain soft extract or concentrating to obtain fluid extract with relative density, drying, adding appropriate amount of adjuvant, mixing, making into pill, and drying to obtain DANHONGHUAYU dripping pill.