CN109632990B

CN109632990B - Method for simultaneously determining content of clematis tangutica B and (R, S) -gomphrena in isatis root preparation

Info

Publication number: CN109632990B
Application number: CN201811557835.1A
Authority: CN
Inventors: 董福越; 黄远
Original assignee: Hutchison Whampoa Guangzhou Baiyunshan Chinese Medicine Co Ltd
Current assignee: Hutchison Whampoa Guangzhou Baiyunshan Chinese Medicine Co Ltd
Priority date: 2018-12-19
Filing date: 2018-12-19
Publication date: 2022-07-05
Anticipated expiration: 2038-12-19
Also published as: CN109632990A

Abstract

The invention discloses a method for simultaneously determining clematis rectus in an isatis root preparationThe method for treating B and (R, S) -Centraniliprole content adopts a high performance liquid chromatography, and the chromatographic conditions are as follows: the stationary phase takes octadecylsilane chemically bonded silica as a filler; an ultraviolet detector with the wavelength of 230-235 nm; the column temperature is 25-35 ℃; the mobile phase takes methanol as a mobile phase A and water as a mobile phase B, and a gradient elution procedure is adopted, wherein the gradient conditions are as follows: 0-3min, 97% B; 3-25min, 97-90% B; 25-27min, 90-76% B; 27-50min, 76% B; flow rate of 0.7-1.0 ml/min^‑1The method can simultaneously determine the contents of the clematis tangutica B and the epigoitrin in the isatis root preparation, has the advantages of less interference, high sensitivity, simplicity, feasibility, accuracy, rapidness, good repeatability and good stability, and can be used for quality control indexes of the isatis root preparation.

Description

Method for simultaneously determining content of clematis tangutica B and (R, S) -gomphrena in isatis root preparation

The technical field is as follows:

the invention relates to the field of traditional Chinese medicines, and in particular relates to a method for simultaneously determining the contents of clematis tangutica B and (R, S) -gomphrena in an isatis root preparation.

Background art:

because of its unique curative effect, Chinese medicine is more and more popular among people in the aspects of preventing and curing diseases, preserving health, conditioning, building body and the like. With the increasing requirements of governments, enterprises and scientific research units on the standardization of Chinese medicinal standards, the research on the quality standards of Chinese medicinal materials and prescription preparations becomes a focus of attention, the quality standards of Chinese medicinal materials and preparations thereof are mainly qualitatively identified in the past, and the quality standards are developed to the quantitative control standard for mainly measuring single effective components in the Chinese medicinal materials and then to the overall quality evaluation method for simultaneously measuring multiple components and multiple target points. The application of various novel analytical instruments also enables the quality standard of the traditional Chinese medicine to develop towards the direction of controllability, stabilization and comprehension, and plays a significant role in realizing the modernization and internationalization of the traditional Chinese medicine. Wherein, the simultaneous determination of the contents of multiple components of Chinese medicinal materials by high performance liquid chromatography has become a trend.

The radix isatidis granules are a common Chinese patent medicine for clearing heat and removing toxicity, have the effects of cooling blood and relieving sore throat, and are used for treating sore throat and dry mouth and throat caused by excessive heat in lung and stomach; the acute tonsillitis patient with the above syndrome has obvious prevention effect on influenza.

The alkaloid compound epigoitrin is accepted as a main antiviral active ingredient in isatis root medicinal materials and exists in the form of enantiomer in medicinal materials and preparations, namely (R, S) -epigoitrin, the content of the alkaloid compound epigoitrin accounts for about 0.05 percent of the crude drug amount [ see the literature of Wangrui, Yanhaiying, Yangqi, Huangshan Jun, Wangzhou. quality standard research of isatis root [ J ]. Chinese herbal medicine, 2010,41(03):478 480 ]. The lignin component is also the important material basis for the radix isatidis to exert antiviral activity, wherein the highest content is the cichorine B which accounts for 0.04 percent of the crude drug amount (see the research on the chemical components and the activities of the antiviral effective part of the radix isatidis, Huiwei Wei, Dongwei, Yangxing Yanjing, Lixiang, Liwei, 1895-1897, 2008).

The (R, S) -gomphrena and clematis tangutica B belong to different classes of compounds, are representative compounds of alkaloid and lignin components in the isatis root, but the solubility (polarity) and ultraviolet absorption characteristics of the compounds are greatly different, and no method for simultaneously measuring the contents of the (R, S) -gomphrena and the clematis tangutica B in an isatis root preparation is reported at present.

The invention content is as follows:

the invention aims to provide a method for simultaneously determining the contents of the clematis tangutica B and the (R, S) -gomphrena in an isatis root preparation, which has the advantages of less interference, high sensitivity, simplicity, feasibility, accuracy, rapidness, good repeatability and good stability and can be used as a quality control index of the isatis root preparation.

The invention is realized by the following technical scheme:

a method for simultaneously determining the contents of clematis tangutica B and (R, S) -goitrin in an isatis root preparation adopts a high performance liquid chromatography, and the chromatographic conditions are as follows: the stationary phase takes octadecylsilane chemically bonded silica as a filler; an ultraviolet detector with the wavelength of 230-235 nm; the column temperature is 25-35 ℃; the mobile phase takes methanol as a mobile phase A and water as a mobile phase B, and a gradient elution procedure is adopted, wherein the gradient conditions are as follows: 0-3min, 97% B; 3-25min, 97-90% B; 25-27min, 90-76% B; 27-50min, 76% B; flow rate of 0.7-1.0 ml/min^-1。

Wherein the column temperature is preferably 30 deg.C, the ultraviolet detector wavelength is preferably 230nm, and the flow rate is preferably 0.8ml min^-1。

Wherein the preparation of the test solution comprises the following steps:

grinding radix Isatidis preparation into fine powder, adding methanol, ultrasonic extracting, adding methanol of the same concentration to make up for lost weight, filtering, and filtering the filtrate with 0.2-0.5 μm microporous membrane to obtain test solution; the mass volume ratio of the isatis root preparation to the methanol is preferably 0.1-1g/ml, the mass concentration of the methanol is 5 wt% -50 wt%, and in addition, the mass concentration is preferably 5 wt% in consideration of economy and environmental protection. The radix Isatidis preparation comprises radix Isatidis granule, tablet, and capsule.

Wherein the preparation of the reference solution comprises the following steps: taking the clematis-Linn B reference substance, and adding methanol to obtain clematis-Linn B reference substance solution with clematis-Linn B concentration of 20-50 μ g/ml; adding methanol into (R, S) -gomphrena reference substance to obtain (R, S) -gomphrena reference substance solution with (R, S) -gomphrena concentration of 10-30 μ g/ml. Wherein the concentration of the clematisnin B is preferably 25-35 mug/ml; the concentration of (R, S) -gomphrena is preferably 20-30. mu.g/ml.

The invention has the following beneficial effects:

1) selecting 230-235nm as a measurement wavelength to ensure that the (R, S) -Notoginsen and clematisne B have stronger absorption at the wavelength, and the base line of the chromatogram is stable and has less interference; and the mobile phase methanol-water gradient elution is selected to ensure that the two compound peaks have good separation effect.

2) The method can simultaneously determine the contents of the clematis tangutica B and the epigoitrin in the isatis root preparation, has the advantages of less interference, high sensitivity, simplicity, feasibility, accuracy, rapidness, good repeatability and good stability, and can be used for quality control indexes of the isatis root preparation.

Description of the drawings:

FIG. 1 is a UV wavelength scanning spectrum of (R, S) -Centraria lobata;

FIG. 2 is a graph of the ultraviolet wavelength scanning spectrum of Canton B;

FIG. 3 is a chromatogram of the test and control samples of example 1;

FIG. 4 is a chromatogram of a test article and a control article of comparative example 1;

FIG. 5 is a chromatogram of the test article and the control article of comparative example 2.

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: method for simultaneously determining content of components B and (R, S) -gomphrena globosa in isatis root granules

1) Preparing a test solution:

grinding radix Isatidis granule, adding 1g into 10mL of 5 wt% methanol, ultrasonic extracting for 5min, supplementing lost weight with methanol of the same concentration, filtering, and filtering 25mL of filtrate with 0.45 μm or 0.22 μm microporous membrane to obtain test solution;

selection of a solvent: in the prior art, the content determination method of (R, S) -goitrin adopts water extraction (see patent 201810354557.3) or 5 wt% methanol (see the content determination method of (R, S) -goitrin in the publication of 'revised standard about isatis root granules' issued by the national pharmacopoeia committee of 3 months in 2018) for sample treatment, and the component belongs to a compound with larger polarity; in the prior art (see the literature of Chengyong, Li Xiang, Chen Jian Wei, Wangying, Yingying, HPLC method for measuring the contents of the rectilineanine B and the isolarch rosins in the isatis root granules [ J ]. modern Chinese medicine research and practice 2009.6(22):53-56) in the method for measuring the contents of the rectilineanine B, 50 wt% of methanol is adopted for sample treatment, and the component belongs to a medium-polarity compound; according to the principle of 'similar and compatible', the extraction conditions of the compounds with larger polarity difference are different. In a screening test of sample pretreatment conditions, the inventor finds that, in the isatis root preparation, in an extracting solution treated by water or 5% -50% methanol, the clematisnin B and (R, S) goitrin can be detected, and the result has no obvious difference. The two compounds with larger polarity difference can be extracted by the same solvent, and experiments prove that the water extract is not easy to pass through a microporous filter membrane, and 5 wt% of methanol is selected as the best compound in consideration of economy and environmental protection.

2) Preparation of control solutions:

taking the reference substance of clematis tangutica B, and adding 50 wt% of methanol to prepare a solution of the clematis tangutica B with the concentration of 31.8 mu g/ml.

Adding 5 wt% methanol into R, S-gomphrena reference substance to obtain solution with concentration of R, S-gomphrena 23.9 μ g/ml.

3) Selection of detection wavelength: the ultraviolet wavelength scanning of (R, S) -Noteparin and the solution of the control product of the orthorhombic clematis B at 190-400 nm by a diode array detector respectively shows that the (R, S) -Noteparin has stronger absorption at 192nm and 240nm and has the maximum absorption at 240nm (see figure 1), the orthorhombic clematis B has stronger absorption at 198.0nm, 224.0nm and 276.0nm and has larger absorption at 224.0nm and 276.0nm (see figure 2), and the ultraviolet absorption graphs of the (R, S) -Noteparin and the orthorhombic clematis B have almost no ultraviolet absorption at 224nm, 276nm and the vicinity thereof and the ultraviolet absorption at 240nm and the vicinity thereof. Therefore, it is easy to conclude that: the clematis stramineus B and (R, S) goitrin cannot simultaneously obtain larger ultraviolet absorption at the same wavelength. The inventor accidentally finds that the direct clematisn B and (R, S) -goitrin have larger ultraviolet absorption in the range of 230-235nm in a scanning wavelength test, and can realize simultaneous measurement at the same wavelength.

In order to ensure that the two compounds have larger absorption under the uniform wavelength by combining the maximum absorption characteristics of the two compounds, 230-235nm is selected as the measurement wavelength in the experiment.

4) Respectively sucking 10 μ L of the reference solution and the sample solution, injecting into high performance liquid chromatograph, and measuring to obtain chromatogram as shown in FIG. 3, wherein the retention time of standard peak in the sample to the chromatogram peak of the reference is consistent. The chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; an ultraviolet detector with the wavelength of 230 nm; the column temperature was 30 ℃, methanol was used as mobile phase a, water was used as mobile phase B, the total volume fraction of methanol and water was 100%, the flow rate was 0.8ml min-1, and gradient elution was performed as shown in table 1:

TABLE 1

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0～3	3	97
3-25	3～10	97～90
			25～27	10～24	90～76
27～50	24	76

5) Under the chromatographic conditions, the theoretical plate number is more than 10000 according to the calculation of (R, S) -Confuchun, the theoretical plate number is more than 5000 according to the calculation of a chromatographic peak of the orthodox B, and the content is calculated according to an external standard one-point method.

Comparative example 1:

published ' official notice about ' modification of the ' isatis root granules ' standard ' by adopting the national pharmacopoeia committee of 3 months in 2018, and the content of the isatis root granules is determined as follows:

preparation of a test solution: taking a proper amount of radix isatidis particles, grinding, taking about 1g, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of 5 wt% methanol into the conical flask, weighing, carrying out ultrasonic treatment for 5min, cooling, weighing again, supplementing the weight loss by 5 wt% methanol, shaking up, filtering, and taking filtrate.

Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; gradient elution is carried out according to the table 2 by taking methanol as a mobile phase A and water as a mobile phase B; the detection wavelength is 254 nm; the flow rate is 0.8 ml/min; the column temperature was 30 ℃. Respectively sucking 10 μ L of reference solution and sample solution, and measuring with high performance liquid chromatograph with theoretical plate number not less than 10000 calculated according to uridine peak.

TABLE 2

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0～3	3	97
3-20	3～10	97～90
			20～40	10～70	90～30
40～50	70	30
			50～60	3	97

The chromatograms of the test sample and the control sample are shown in FIG. 4. 254nm is the detection wavelength, the target peak of (R, S) -Noteparin can be seen in the chromatogram of the test sample, but the target peak of the clematisnin B can not be seen, the absorption value of the reference substance of the clematisnin B is very small, and the clematisnin B can not be effectively separated under the gradient elution condition of the method.

Comparative example 2:

reference documents Chengyongning, Lixiang, Chenjianwei and Wangying HPLC method for determining content of D-iron linezolin B and isolarch roside in radix Isatidis granule [ J ] modern Chinese medicine research and practice 2009.6(22):53-56 published method

Preparation of a test solution: taking a proper amount of isatis root particles, grinding, precisely weighing 1.0000g, placing in a conical flask with a plug, precisely adding 25mL of 50% methanol, carrying out ultrasonic extraction for 20min (45Hz and 600W), centrifuging (5000r/min) for 10min, taking supernatant, concentrating in a water bath at 85 ℃, fixing the volume to 2mL, and filtering with a 0.45-micron microporous filter membrane to obtain the isatis root extract.

Chromatographic conditions are as follows: the chromatographic column is an Aiglenggt TC-C18 column (4.6mm multiplied by 250mm, 5 μm); column temperature: 30 ℃; mobile phase: water (a) -methanol (B), gradient elution: 0-35 min (22% B), 35-36 min (22% B-30% B), 36-60 min (30% B); volume flow rate: 0.8 mL/min; detection wavelength: 280nm and a reference wavelength of 360 nm; sample introduction amount: 5 μ L. The chromatograms of the test sample and the control sample are shown in FIG. 5. 280nm is selected as a detection wavelength, a chromatogram of a sample can see a target peak of the clematis orthodoxa B, but a target peak of the (R, S) -Nothopoguin is not found, and a control picture of the (R, S) -Nothopoguin shows that the (R, S) -Nothopoguin has no absorption at the wavelength.

As can be seen from the comparison of example 1 with comparative examples 1 and 2, neither of the above two methods disclosed in comparative examples 1 and 2 can simultaneously determine the contents of (R, S) -gomphrena and clematisnin B in the granules of isatis root. In the invention, 230-235nm is selected as a detection wavelength, under the condition of gradient elution by the method, target peaks of (R, S) -Nothophun and clemainine B can be clearly seen in chromatograms of a reference substance and a test substance, and compared with the existing methods disclosed in comparative examples 1 and 2, the (R, S) -Nothophun and the clemainine B in the test substance have good separation degree, good peak shape and less interference peaks around the test substance. The method can simultaneously determine the content of (R, S) -gomphrena and clematis tangutica B in the isatis root granules.

Example 3: and (3) verification of methodology:

(1) stability test

Taking 1 part of isatis root particle sample solution according to example 1, injecting samples for 0, 2, 4, 8, 12 and 24 hours respectively, measuring chromatographic peaks, and recording peak areas. The results show that the peak area of (R, S) -Noteparin is 1.67%, and the peak area RSD of the clematisn B is 1.50%, which indicates that the stability of the clematisn B and the (R, S) -Noteparin in the isatis root particle test sample solution is good within 24h (the results are shown in Table 3).

TABLE 3

(2) Repeatability test

Referring to example 1, 6 portions of the test solution of the isatis root granules prepared in parallel for the same batch were taken and injected sequentially, and measured according to the high performance liquid chromatography measurement chromatographic conditions, and the RSDs of the peak areas of (R, S) -gomphrena and troostite B were recorded as 0.94% and 1.54%, respectively, indicating that the method is good in repeatability (the results are shown in table 4).

TABLE 4

(3) Sample application recovery test

A test solution was prepared by adding an appropriate amount of 6 parts of isatis root granules having a known content of ((R, S) -Noteparin 0.0209 mg/g; Nepetanine B0.0256mg/g) to respective (R, S) -Noteparin and Nepetanine B controls. The chromatographic conditions were measured by high performance liquid chromatography as in example 1, the chromatographic peak was measured, the peak area was recorded, and the amount was calculated by the external standard one-point method. The calculated average recovery rate of (R, S) -Noteparin is 100.96%, RSD is 1.58%, the average recovery rate of clematis tangutica B is 99.39%, and RSD is 1.20%, and the result shows that the accuracy is good (see the result in Table 5).

Note: the concentration of the solution of the clematis straminea B reference substance used in the content determination test is 0.0422 mg/ml.

TABLE 5

Claims

1. A method for simultaneously determining the content of clematis tangutica B and (R, S) -goitrin in an isatis root preparation is characterized in that a high performance liquid chromatography method is adopted, and the chromatographic conditions are as follows: the stationary phase takes octadecylsilane chemically bonded silica as a filler; an ultraviolet detector with the wavelength of 230-235 nm; the column temperature is 25-35 ℃; the mobile phase takes methanol as a mobile phase A and water as a mobile phase B, and a gradient elution procedure is adopted, wherein the gradient conditions are as follows: 0-3min, 97% B; 3-25min, 97-90% B; 25-27min, 90-76% B; 27-50min, 76% B; flow rate of 0.7-1.0 ml/min^-1。

2. The method for simultaneously determining the contents of clematisne B and (R, S) -gomphrena in an isatis root preparation as claimed in claim 1, wherein the ultraviolet detector has a wavelength of 230nm and a flow rate of 0.8 ml-min^-1。

3. The method for simultaneously determining the content of clematisnin B and (R, S) -gomphrenan in an isatis root preparation as claimed in claim 1 or 2, wherein the column temperature is 30 ℃.

4. The method for simultaneously determining the content of clematisnin B and (R, S) -gomphrenan in an isatis root preparation as claimed in claim 1 or 2, wherein the preparation of the test solution comprises the steps of: grinding radix Isatidis preparation into fine powder, adding methanol, ultrasonic extracting, adding methanol of the same concentration to make up for lost weight, filtering, and filtering the filtrate with 0.2-0.5 μm microporous membrane to obtain test solution.

5. The method for simultaneously determining the content of the clematisnin B and the (R, S) -goitrin in the isatis root preparation as claimed in claim 4, wherein the mass volume ratio of the isatis root preparation to the methanol is 0.1-1g/ml, and the mass concentration of the methanol is 5 wt% -50 wt%.

6. The method for simultaneously determining the content of clematisnin B and (R, S) -goitrin in an isatis root preparation according to claim 4, wherein the mass concentration of methanol is 5 wt%.

7. The method for simultaneously determining the content of the cichorine B and the (R, S) -lejaponin in the isatis root preparation as claimed in claim 4, wherein the isatis root preparation comprises isatis root granules, tablets and capsules.

8. The method for simultaneously determining the content of cichorine B and (R, S) -lejaponin in an isatis root preparation as claimed in claim 1 or 2, wherein the preparation of the control solution comprises the following steps: taking the rectilineastine B reference substance, and adding methanol to obtain rectilineastine B reference substance solution with concentration of 20-50 μ g/ml; adding methanol into (R, S) -gomphrena control to obtain (R, S) -gomphrena control solution with concentration of 10-30 μ g/ml.

9. The method for simultaneously determining the content of clematisnin B and (R, S) -goitrin in an isatis root preparation according to claim 8, wherein the concentration of clematisnin B is 25-35 μ g/ml.

10. The method for simultaneously determining the contents of cichorine B and (R, S) -goitrin in an isatis root preparation according to claim 8, wherein the concentration of (R, S) -goitrin is 20-30 μ g/ml.