CN114624352A

CN114624352A - Method for detecting content of strychnine emplastrum and related substances and application

Info

Publication number: CN114624352A
Application number: CN202110730629.1A
Authority: CN
Inventors: 胡杰; 薛春美; 吕玉芹
Original assignee: Beijing Increasepharm Corp ltd; Hainan Selection Pharmaceutical Co ltd
Current assignee: Sailing Pharmaceutical Technology Group Co ltd
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2022-06-14

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting the content of strychnine emplastrum and related substances. The detection of the content of strychnine in the strychnine gel emplastrum and the determination of related substances are important contents for the research of the medicine quality, and the premise of accurately determining the content is to completely extract the components to be detected. The invention comprises (1) a method for extracting strychnine from gel plaster; (2) a method for analyzing and detecting the strychnine content and related substances is high performance liquid chromatography. The invention provides technical support for the quality research of the strychnine gel emplastrum, and provides reference for the content of other alkaline alkaloid gel emplastrums and the research of related substances.

Description

Content of strychnine emplastrum, related substance detection method and application

Technical Field

The invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for detecting the content of strychnine emplastrum and related substances.

Background

Nux vomica is bitter in property and warm; it is toxic. Has effects of dredging collaterals, relieving pain, resolving hard mass and relieving swelling. Can be used for treating traumatic injury, fracture, rheumatism, numbness, and paralysis. The main effective component of semen Strychni is total alkaloids including strychnine, loganin, and ecamine; wherein, the strychnine is the effective component of nux vomica for resisting inflammation, relieving pain and treating arthritis.

The brucine as the raw material medicine is obtained by extracting and separating from the processed nux vomica, the purity reaches more than 98 percent, and the strychnine hardly contains any toxic substance. The strychnine gel emplastrum has large drug-loading rate, accurate dosage, slow-release drug release and long-lasting drug effect. In the aspect of treating osteoarthritis, the toxicity of the bulk drug strychnine is greatly reduced and the drug effect is greatly improved compared with the traditional Chinese medicine nux vomica. The strychnine gel plaster has less blood invasion and high local medicine concentration in joints because of being an external medicine, thereby improving the local medicine effect and reducing the toxic and side effects of the whole body. Therefore, the strychnine gel emplastrum has important clinical value in the aspect of treating osteoarthritis.

At present, no report on content detection of strychnine gel plaster and related substance research exists. The detection of the content of strychnine in the strychnine gel emplastrum and the determination of related substances are important contents of medicine quality research, and the premise of accurately determining the content is to completely extract the components to be detected.

Extracting strychnine and related substances from the strychnine gel plaster needs to overcome the technical problem and find proper extraction conditions; meanwhile, the brucine and related substances cannot be completely separated by an HPLC mobile phase system for detecting the content of the brucine in Chinese pharmacopoeia. The content of strychnine emplastrum and a detection method of related substances are required to be developed to ensure the product quality.

Disclosure of Invention

The invention aims to provide a method for analyzing and detecting the content and related substances in strychnine gel emplastrum.

The invention also aims to provide the application of the analytical detection method of the content and related substances in the strychnine emplastrum in the aspect of controlling the quality of the strychnine emplastrum product.

The invention provides a method for analyzing and detecting the content of strychnine gel emplastrum and related substances, which comprises the following steps of (1) extracting strychnine from the gel emplastrum; (2) a method for analyzing and detecting strychnine content and related substances is high performance liquid chromatography.

First, the content measurement of strychnine emplastrum and the preparation of samples for the examination of related substances were performed. Extraction of strychnine and related impurities from gel plaster is a necessary condition for sample detection.

The method for extracting the strychnine from the gel plaster in the step (1) comprises the following steps:

the extraction research comprises an extraction solvent, the dosage of the solvent, the pH value of the solvent, an extraction mode, extraction time, extraction temperature and the like.

The extraction solvent considers the mixed solution of aqueous solution, inorganic salt solutions with different concentrations and different types and methanol.

The pH value range of the solution is 2.0-5.0, and the pH regulator is organic acid and inorganic acid;

the organic acid is selected from straight chain or branched chain saturated fatty acid, unsaturated fatty acid, and aromatic organic acid.

The organic acid is selected from formic acid, acetic acid, citric acid, tartaric acid and citric acid; the inorganic acid is selected from hydrochloric acid, phosphoric acid and sulfuric acid.

The extraction mode inspects the ultrasound and the stirring;

the extraction time is 5-20h by water bath stirring;

the extraction temperature range is 30-70 ℃.

Taking a proper amount of strychnine gel emplastrum, removing the protective layer, cutting into small pieces, sequentially putting into a 200-ml measuring flask filled with a proper amount of solvent for preventing adhesion, putting into a rotor, stirring at 30-70 ℃ for 5-20h, performing ultrasonic treatment for 0.5-3h, cooling, taking out the rotor, adding the solvent to dilute to a scale, shaking uniformly, and filtering with a 0.45-micrometer filter membrane. Making into solution containing brucine 0.2mg (related substance)/0.1 mg (content) per 1 ml.

The extraction solvent is a mixed solution of an inorganic salt solution and methanol;

the extraction solvent is preferably a mixed solution of 0.9-10% inorganic salt solution and methanol;

the extraction solvent is more preferably a mixture of 4-10% inorganic salt solution and methanol.

The inorganic salt in the extraction solvent is selected from: chlorides, iodides, phosphates, sulfates, nitrates, nitrites, borates, thiosulfates, sulfites, bisulfites, and the like. Such as sodium chloride, sodium iodide, sodium phosphate, calcium phosphate, sodium sulfate, magnesium sulfate, calcium sulfate, sodium nitrate, sodium nitrite, sodium borate, sodium thiosulfate, sodium sulfite, magnesium sulfite, sodium bisulfite, and the like.

The inorganic salt is more preferably sodium chloride, phosphate, sulfate; most preferred is sodium chloride.

The inorganic salt solution and the methanol mixed solution are mixed in any proportion, and the preferred proportion is 80: 20.

Research results show that the extraction solvent is inorganic salt aqueous solution and methanol at a ratio of 4:1, the pH value of the solvent is 2-5, the extraction mode comprises stirring for 5-16 hours, ultrasonic treatment for 0.5-1 hour, and the extraction effect is optimal when the extraction temperature is 40-60 ℃.

The method for analyzing and detecting the strychnine content and related substances in the step (2),

the mobile phase regulator researches acetic acid, triethylamine, phosphoric acid, sodium heptanesulfonate and sodium dihydrogen phosphate; the pH value of the mobile phase is 3.80-4.10.

A method for analyzing and detecting the strychnine content and related substances is high performance liquid chromatography. The liquid chromatography conditions used were as follows:

a chromatographic column: octadecylsilane bonded silica gel column, preferably Agilent ZORBAX SB-C18, 250mm × 4.6mm, 5 μm; kromasil C18, 250 mm. times.4.6 mm, 5 μm; ultimate LP-C18, 250mm × 4.6mm, 5 μm; waters Xbridge C18, 250mm × 4.6mm, 5 μm, etc.;

related substances mobile phase: equal-volume mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L potassium dihydrogen phosphate is 15:85-25: 75; preferably 18: 82. Adjusting the pH of the mobile phase to 3.90-4.10 by using 5% phosphoric acid;

content of mobile phase: equal volume of mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L potassium dihydrogen phosphate is 15:85-25: 75; preferably in a ratio of 21: 79. Adjusting the pH of the mobile phase to 3.80-4.10 by using 5% phosphoric acid;

the detection wavelength is 255-280 nm;

the flow rate is 0.5-2.0 ml/min;

the column temperature is 25-40 ℃;

the injection volume is 5-60 μ l.

The strychnine and related substances in the strychnine emplastrum provided by the invention are as follows:

drawings

FIG. 1 is a chromatogram obtained by using chromatographic conditions for measuring the content of semen Strychni preparata in the first part of Chinese pharmacopoeia 2020 edition;

FIG. 2 is a spectrum obtained using chromatographic conditions in the reference;

FIG. 3 is a graph obtained using chromatographic conditions established in this study-pH 3.90;

FIG. 4 is a graph, pH 4.00, obtained using chromatographic conditions established in this study;

FIG. 5 is a graph, pH 4.10, obtained using chromatographic conditions established in this study.

Detailed Description

Examples 1-19 determination of the content of strychnine in the plaster and preparation of samples for examination of the relevant substances are as follows:

example 1g of paste was taken and put into a 25ml measuring flask, 20ml of water solution (acetic acid to adjust pH to 2.0) was added and ultrasonic treatment was carried out for 3 hours, the mixture was stirred overnight (15 hours) in a 60 ℃ water bath (stirring speed is medium), ultrasonic treatment was carried out for 1 hour, and volume was fixed with methanol. The extraction recovery rate of strychnine is 99.7%.

The method can completely extract the strychnine in the gel plaster, but the ultrasonic treatment is carried out for 3 hours, and the extraction mode is too strong, so that the method is not suitable for daily sample inspection operation. There is therefore a need to optimize the extraction process.

Example 21 g of paste is taken and placed in a 25ml measuring flask, 20ml of 0.9% sodium chloride solution (formic acid to adjust the pH value to 2.0) is added, ultrasonic treatment is carried out for 1 hour, 5ml of methanol is added, water bath stirring is carried out at 60 ℃ overnight (15h), and the volume is determined by methanol. The extraction recovery rate of strychnine is measured to be 99.6%.

The test reduces the ultrasonic time, and the result has no obvious difference, which shows that the 0.9 percent sodium chloride solution is used for replacing water, thereby being beneficial to extracting the strychnine in the gel plaster.

Example 3-5 taking 1g of the paste, placing the paste in a 25ml measuring flask, adding 20ml of 0.9% sodium chloride solution (phosphoric acid is adjusted to different pH values), carrying out ultrasonic treatment for 1h, adding 5ml of methanol, stirring in a water bath at 60 ℃ overnight (15h), and adding methanol to fix the volume. The method comprises the following specific steps:

examples 6-14 gel patches 7 x 10cm (corresponding to 7g of paste) were weighed, cut into 3cm x 1.5cm pieces, placed in 200ml measuring flasks, charged to 200ml with different concentrations of inorganic salt solution (formic acid to adjust the pH to 3.0) -methanol (80:20), stirred in a water bath and sonicated for 0.5 h. The method comprises the following specific steps:

note: because the prescription process is mature, the sample in the experiment does not use the gel paste any more, but the formed gel plaster is adopted, the plaster is cut into pieces and then extracted, the extraction scale is enlarged, and the changes do not have obvious influence on the extraction.

Adjusting the pH value of the extraction solution to 3.0, increasing the concentration of inorganic salt, and inspecting the extraction effect. From the results of examples 6 to 8, 9, and 12 to 13, it was found that the increase in the concentration of the inorganic salt within a certain range indeed improves the extraction yield of strychnine, and the concentration of the inorganic salt was selected to be 4%.

Under the premise of keeping other conditions unchanged, the pH value of the extracting solution is continuously increased (the pH value is less than 7) (example 14), and the recovery rate of the strychnine is almost unchanged. The extract composition, i.e. 4% sodium chloride solution (formic acid adjusted to pH3.0) -methanol (80:20) was fixed and the effect of different extraction times on the extraction recovery was examined. From the results of examples 8 and 9, it can be seen that the extraction recovery rate RAD% was 0.7 with no significant change between the overnight stirring in water bath (15h) and the 5h extraction, and the extraction time was determined to be 5h for saving the time cost.

The composition and extraction time of the extract solution were fixed, i.e. the extract solution was 4% sodium chloride solution (pH adjusted to 3.0 with formic acid) -methanol (80:20), the extraction time was 5h, and the effect of different extraction temperatures on the extraction recovery rate was examined. As is clear from the results of examples 9 and 10, the extraction at 60 ℃ is not significantly different from that at 40 ℃.

The pH value (pH3.0), extraction time (5h) and extraction temperature (60 ℃) of the extract were fixed, and the extraction effect of the extraction solvent without methanol was examined. From the results of example 11, it can be seen that the solvent recovery without methanol is lower than that with methanol, and the optimal content sample preparation method is that used in examples 8 and 9.

In order to ensure that the extraction method is simultaneously suitable for preparation of related substance samples, the impurity extraction condition is researched.

Example 15 about 25mg of the raw material sample is taken, precisely weighed, placed in a 25mL measuring flask, dissolved in methanol and diluted to the scale, and shaken up; precisely measuring 5ml, placing in a 10ml measuring flask, and adding water to dilute to scale.

Examples 16-17 gel patches 7 x 10cm (corresponding to 7g of paste) were weighed, cut into 3cm x 1.5cm pieces, placed in a 200ml measuring flask, added with 4% sodium chloride solution (hydrochloric acid to adjust the pH to 5.0) -methanol (80:20) to 200ml, stirred in a water bath and sonicated for 0.5 h. The results are as follows:

impurity name/RRT	Example 15	Example 16	Example 17
				0.18	/	0.032	0.031
Impurity 8	/	0.650	0.725
				0.46	0.097	0.089	0.088
0.60	0.032	0.030	0.030
				0.69	0.043	0.044	0.044
Impurity 4	0.670	0.555	0.572
				0.81	/	0.023	0.022
Impurity 1	0.073	0.086	0.073
				Impurity 7	0.018	0.028	0.020
Strychnine	98.238	97.670	97.609
				Impurity 3	0.050	0.047	0.047
1.54	0.022	0.024	0.021
				Impurity 2	0.756	0.721	0.718
Number of impurities	9	12	12

Note: example 16 was stirred in a water bath for 5h and example 17 was stirred in a water bath overnight (15 h).

From the above results, it can be seen that: compared with the bulk drugs (example 15), in the gel plaster (examples 16-17), except unknown impurities and known impurities 8 of RRT0.18 and RRT0.81, all the other impurities are derived from the bulk drugs, the normalization result of each impurity has no obvious difference from the bulk drugs, and the result of prolonging the stirring time has no difference, which indicates that the sample is completely extracted.

Examples 18-19 methods of preparing samples of the release rates of strychnine emplastrum and the results of the tests are as follows:

taking the product, cutting into circular sample with diameter of 30mm, removing the protective layer to make the adhesive surface of the product face upward, placing in the release medium, respectively taking the release solution at different times, filtering, and measuring the subsequent filtrate.

The experiments show that the release degree of the strychnine gel plaster in a 2% sodium chloride solution is far greater than that of a pure water medium, and the addition of the sodium chloride also promotes the release of the strychnine from the gel plaster under the condition that the pH value is not adjusted by adding acid.

Examples 20-21 assay of strychnine-containing plasters and examination of related substances the following were screened:

example 20 the content of strychnos nux-vomica prepared in the first department of the Chinese pharmacopoeia 2020 edition was analyzed by octadecylsilane bonded silica gel column (250 mm. times.4.6 mm, 5 μm); eluting with a mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L sodium dihydrogen phosphate (pH adjusted to 2.85 with 5% phosphoric acid) (21:79) at flow rate of 1 ml/min; the detection wavelength is 260nm, the column temperature is 30 ℃, and the injection volume is 10 ul. The chromatogram is shown in figure 1. :

the separation degree of each peak is poor, and the method is not suitable for detecting related substances, but can be used for measuring the content.

Example 21 the following chromatographic conditions were selected: bonding a silica gel column (4.6X 150mm, 5um) with octadecylsilane; the chromatographic column mobile phase A is acetonitrile, and the mobile phase B is 0.2% acetic acid and 0.2% triethylamine water solution; the flow rate is 1 ml/min; the detection wavelength is 260nm, the column temperature is 30 ℃, and the injection volume is 10 ul. Gradient elution.

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	5	95
20	21	79
			25	60	40
35.01	10	90
			35	10	90

The chromatogram is shown in FIG. 2.

Wherein, the impurity peak separation degrees of RT17.058min and RT17.375min are not enough.

Example 22 the selection of the use of an octadecylsilane bonded silica gel column (250 mm. times.4.6 mm, 5 μm); eluting with a mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L sodium dihydrogen phosphate (pH adjusted to 3.90 with 5% phosphoric acid) (18:82) at flow rate of 1 ml/min; the detection wavelength is 260nm, the column temperature is 30 ℃, and the injection volume is 40 ul. The chromatogram is shown in FIG. 3.

Example 23 elected to use an octadecylsilane bonded silica gel column (250 mm. times.4.6 mm, 5 μm); eluting with a mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L sodium dihydrogen phosphate (pH adjusted to 4.0 with 5% phosphoric acid) (18:82) at flow rate of 1 ml/min; the detection wavelength is 260nm, the column temperature is 30 ℃, and the injection volume is 40 ul. The chromatogram is shown in FIG. 4.

Example 24 selection of the use of an octadecylsilane bonded silica gel column (250 mm. times.4.6 mm, 5 μm); eluting with a mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L sodium dihydrogen phosphate (pH adjusted to 4.10 with 5% phosphoric acid) (18:82) at flow rate of 1 ml/min; the detection wavelength is 260nm, the column temperature is 30 ℃, and the injection volume is 40 ul. The chromatogram is shown in FIG. 5. The separation degree of each peak is good, and the method is applicable.

In summary, the method for extracting strychnine from the gel patch, preferably according to examples 8, 9, 10, 12, 13 and 14, shows that the addition of sodium chloride promotes the release of strychnine from the gel patch. The method for analyzing and detecting the content of the strychnine and related substances is better in examples 22, 23 and 24, wherein the separation degree between the strychnine and the related substances is good.

The analytical detection method of the content and related substances of the strychnine gel emplastrum is an important component of the quality research of the strychnine gel emplastrum, wherein the detection of the content and the related substances is the basis for setting the quality standard of the strychnine gel emplastrum, and the quality standard is a guideline for inspecting whether a preparation product is qualified or not. Therefore, the analytical detection method of the content of the strychnine gel emplastrum and related substances is a crucial factor in the quality of the whole preparation product. The research provides technical support for the quality research of the strychnine gel emplastrum, and simultaneously provides reference for the content research of other alkaline alkaloid gel emplastrums and the research of related substances.

Claims

1. A method for analyzing and detecting the content and related substances in strychnine emplastrum is characterized in that,

comprises (1) extracting strychnine and related substances from gel plaster; (2) a method for analyzing and detecting the content of strychnine and related substances is high performance liquid chromatography.

2. The method for analyzing and detecting the content and related substances in the strychnine emplastrum as claimed in claim 1, is characterized by comprising the following steps: the extraction of strychnine from the gel plaster in the step (1) is as follows: stirring for 5-20h at 30-70 deg.C with extraction solvent, ultrasonic treating for 0.5-3h, and sampling; the extraction solvent is a mixed solution of an inorganic salt solution and methanol; the inorganic salt solution is adjusted by acid, and the pH value is 2.0-5.0.

3. The method for analyzing and detecting the content and related substances in the strychnine emplastrum as claimed in claim 1 or 2, which is characterized by comprising the following steps: the extraction solvent in the step (1) is a mixed solution of 0.9-10% of inorganic salt solution and methanol; the inorganic salt is selected from: chloride, iodide, phosphate, sulfate, nitrate, nitrite, borate, thiosulfate, sulfite, bisulfite.

4. The method for analyzing and detecting the content and related substances in the strychnine emplastrum as claimed in claim 3, is characterized by comprising the following steps: the extraction solvent in the step (1) is a mixed solution of 4-10% of inorganic salt solution and methanol, and the inorganic salt is preferably sodium chloride, phosphate and sulfate; the ratio of inorganic salt solution to methanol is preferably 80: 20.

5. The method for analyzing and detecting the content and related substances in the strychnine emplastrum as claimed in claim 1 or 2, which is characterized by comprising the following steps: the acid used for adjusting the pH value of the extraction solvent in the step (1) is selected from straight chain or branched chain saturated fatty acid, unsaturated fatty acid, aromatic organic acid and inorganic acid.

6. The method for analyzing and detecting the content and related substances in the strychnine emplastrum as claimed in claim 5, is characterized by comprising the following steps: the acid used for adjusting the pH value of the extraction solvent in the step (1), wherein the organic acid is selected from formic acid, acetic acid, citric acid, tartaric acid and citric acid; the inorganic acid is selected from hydrochloric acid, phosphoric acid and sulfuric acid.

7. The method for analyzing and detecting related substances in the strychnine emplastrum as claimed in claim 1, wherein the chromatographic conditions are as follows:

a chromatographic column: octadecylsilane chemically bonded silica gel column;

related substances mobile phase: equal-volume mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L potassium dihydrogen phosphate is 15:85-25: 75; adjusting the pH of the mobile phase to 3.90-4.10 by using 5% phosphoric acid;

content of mobile phase: equal volume of mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L potassium dihydrogen phosphate is 15:85-25: 75; adjusting the pH of the mobile phase to 3.80-4.10 by using 5% phosphoric acid;

the detection wavelength is 255-280 nm;

the flow rate is 0.5-2.0 ml/min;

the column temperature is 25-40 ℃;

the injection volume is 5-60 μ l.

8. The method for analyzing and detecting related substances in the strychnine emplastrum as claimed in claim 7, wherein the chromatographic conditions are as follows:

and (3) chromatographic column: agilent ZORBAX SB-C18, 250mm × 4.6mm, 5 μm; kromasil C18, 250mm × 4.6mm, 5 μm; ultimate LP-C18, 250mm × 4.6mm, 5 μm; waters Xbridge C18, 250 mm. times.4.6 mm, 5 μm;

related substances mobile phase: equal-volume mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L potassium dihydrogen phosphate is 18: 82; adjusting the pH of the mobile phase to 3.90-4.10 by using 5% phosphoric acid;

content of mobile phase: equal volume of mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L potassium dihydrogen phosphate is 21: 79; adjusting the pH of the mobile phase to 3.80-4.10 by using 5% phosphoric acid;

the detection wavelength is 255-280 nm;

the flow rate is 0.5-2.0 ml/min;

the column temperature is 25-40 DEG C

The injection volume is 5-60 μ l.

9. The method for analyzing and detecting related substances in the strychnine emplastrum as claimed in claim 1 or 2, wherein the related substances in the strychnine emplastrum are:

10. use of the analytical detection method for the content and related substances in strychnine emplastrum as claimed in any one of claims 1-2 for controlling the quality of strychnine emplastrum products.