Detection method and use of the content and related substances of a brucine patch

Abstract

The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting the content and related substances of brucine patch. The detection of the brucine content and the determination of related substances in the brucine hydrogel patch are important contents of drug quality research. Completely extracting the component to be measured is a prerequisite for accurately determining the content. The present invention includes (1) a method for extracting brucine from the hydrogel patch; (2) a method for analyzing and detecting the content and related substances of brucine, and the method is high performance liquid chromatography. The present invention provides technical support for the quality research of brucine hydrogel patch, and at the same time provides a reference for the research on the content and related substances of other alkaline alkaloid hydrogel patches.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for detecting the content and related substances of brucine patch. Background Art

[0002] Strychnos nux-vomica is bitter and warm in nature; it is highly toxic. It has the effects of dredging collaterals to relieve pain and dissipating stasis to reduce swelling. It is used for traumatic injuries, fracture swelling and pain, rheumatoid arthritis, numbness and paralysis, etc. The main active ingredients of Strychnos nux-vomica are total alkaloids, including brucine, strychnine, vomicine, novacine, loganin, icajine, etc.; among them, brucine is the active ingredient for anti-inflammatory analgesia and treating arthritis of Strychnos nux-vomica.

[0003] The raw material drug brucine in this article is extracted and separated from processed Strychnos nux-vomica, with a purity of over 98%, and almost free of highly toxic substance strychnine. Brucine hydrogel patch has a large drug loading capacity, accurate dosage, sustained release of the drug, and can exert its efficacy persistently. In the treatment of osteoarthritis, compared with the traditional Chinese medicine Strychnos nux-vomica, the raw material drug brucine has significantly reduced toxicity and greatly improved efficacy. Brucine hydrogel patch, because it is a topical drug, has less absorption into the blood and a high local drug concentration in the joint, which not only improves the local efficacy but also reduces the systemic toxic and side effects. Therefore, brucine hydrogel patch has important clinical value in the treatment of osteoarthritis.

[0004] At present, there is no report on the content detection and related substance research of brucine hydrogel patch. The detection of brucine content and the determination of related substances in brucine hydrogel patch are important contents of drug quality research. Completely extracting the component to be measured is the premise for accurate content determination.

[0005] Extracting brucine and related substances from brucine hydrogel patch needs to overcome technical difficulties and find suitable extraction conditions; at the same time, the HPLC mobile phase system for detecting brucine content in the Chinese Pharmacopoeia cannot completely separate brucine and related substances. It is necessary to develop a method for detecting the content and related substances of brucine patch to ensure product quality. Summary of the Invention

[0006] The purpose of the present invention is to provide an analytical method for detecting the content and related substances in brucine hydrogel patch.

[0007] Another purpose of the present invention is to provide the application of the analytical method for detecting the content and related substances in brucine patch in controlling the product quality of brucine patch.

[0008] The present invention provides a method for analyzing and detecting the content and related substances of brucine gel patch, including (1) a method for extracting brucine from the gel patch; (2) a method for analyzing and detecting the content and related substances of brucine, and the analytical detection method used is high performance liquid chromatography.

[0009] First, prepare samples for the determination of the content of brucine patch and the inspection of related substances. Extracting brucine and related impurities from the gel patch is a necessary condition for sample detection.

[0010] The method for extracting brucine from the gel patch in step (1) is as follows:

[0011] The extraction study includes extraction solvent, solvent dosage, solvent pH value, extraction method, extraction time, extraction temperature, etc.

[0012] The extraction solvents investigated include aqueous solutions, mixed solutions of inorganic salt solutions with different concentrations and different types and methanol.

[0013] The pH value range of the solution is 2.0 - 5.0, and the pH regulators are organic acids and inorganic acids;

[0014] The organic acids are selected from linear or branched saturated fatty acids, unsaturated fatty acids, and aromatic organic acids.

[0015] The organic acids are selected from formic acid, acetic acid, citric acid, tartaric acid, and malic acid; the inorganic acids are selected from hydrochloric acid, phosphoric acid, and sulfuric acid.

[0016] The extraction methods investigated include ultrasonic and stirring;

[0017] The extraction time is 5h - 20h with water bath stirring;

[0018] The extraction temperature range is 30°C - 70°C.

[0019] Take an appropriate amount of brucine gel patch, remove the protective layer, cut it into small pieces, and sequentially place it in a 200 - 250 ml volumetric flask containing an appropriate amount of solvent to prevent adhesion. Place a rotor, stir at 30 - 70°C for 5 - 20h, then ultrasonic for 0.5 - 3h, let it cool, take out the rotor, dilute to the scale with solvent, shake well, and filter through a 0.45μm filter membrane. Prepare a solution containing about 0.2mg (related substances) / 0.1mg (content) of brucine per 1ml.

[0020] The extraction solvent is a mixed solution of inorganic salt solution and methanol;

[0021] The extraction solvent is preferably a mixed solution of 0.9 - 10% inorganic salt solution and methanol;

[0022] The extraction solvent is more preferably a mixed solution of 4 - 10% inorganic salt solution and methanol.

[0023] The inorganic salts in the extraction solvent are selected from: chlorides, iodides, phosphates, sulfates, nitrates, nitrites, borates, thiosulfates, sulfites, bisulfites, etc. For example, 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, etc.

[0024] The inorganic salts are more preferably sodium chloride, phosphate, sulfate; most preferably sodium chloride.

[0025] The inorganic salt solution and the methanol mixture are mixed in any ratio, and the preferred ratio is 80:20.

[0026] The research results show that when the extraction solvent is an aqueous solution of inorganic salt and methanol at a ratio of 4:1, the solvent pH value is 2 - 5, the extraction method includes stirring for 5 - 16 hours and ultrasonic treatment for 0.5 - 1 hour, and the extraction temperature is 40 - 60 °C, the extraction effect is optimal.

[0027] The method for analyzing and detecting the content of brucine and related substances in step (2),

[0028] Acetic acid, triethylamine, phosphoric acid, sodium heptanesulfonate, and sodium dihydrogen phosphate were studied as mobile phase regulators; the pH value range of the mobile phase is 3.80 - 4.10.

[0029] The method for analyzing and detecting the content of brucine and related substances, and the method is high performance liquid chromatography. The liquid chromatography conditions used are as follows:

[0030] Chromatographic column: octadecylsilane-bonded silica gel column, preferably Agilent ZORBAX SB-C18, 250 mm × 4.6 mm, 5 μm; Kromasil C18, 250 mm × 4.6 mm, 5 μm; Ultimate LP-C18, 250 mm × 4.6 mm, 5 μm; Waters XBridge C18, 250 mm × 4.6 mm, 5 μm, etc.;

[0031] Mobile phase for related substances: acetonitrile - equal volume mixture of 0.01 mol / L sodium heptanesulfonate and 0.02 mol / L potassium dihydrogen phosphate solution = 15:85 - 25:75; the preferred ratio is 18:82. The pH of the mobile phase is adjusted to 3.90 - 4.10 with 5% phosphoric acid;

[0032] Mobile phase for content: acetonitrile - equal volume mixture of 0.01 mol / L sodium heptanesulfonate and 0.02 mol / L potassium dihydrogen phosphate solution = 15:85 - 25:75; the preferred ratio is 21:79. The pH of the mobile phase is adjusted to 3.80 - 4.10 with 5% phosphoric acid;

[0033] The detection wavelength is 255 - 280 nm;

[0034] The flow rate is 0.5 - 2.0 ml / min;

[0035] The column temperature is 25 - 40 °C;

[0036] The injection volume is 5 - 60 μl.

[0037] The brucine and related substances in the brucine plaster of the present invention are:

[0038]

[0039] Description of the Drawings

[0040] Figure 1 The chromatogram obtained under the chromatographic conditions for the determination of the content of processed semen strychni in Volume I of Chinese Pharmacopoeia (2020 Edition);

[0041] Figure 2 The chromatogram obtained under the chromatographic conditions in the reference literature;

[0042] Figure 3 The chromatogram obtained under the chromatographic conditions established in this study - pH 3.90;

[0043] Figure 4 The chromatogram obtained under the chromatographic conditions established in this study - pH 4.00;

[0044] Figure 5 The chromatogram obtained under the chromatographic conditions established in this study - pH 4.10. Detailed Description of the Invention

[0045] The preparation methods of the samples for the content determination and related substance inspection of the brucine plasters in Examples 1 - 19 are as follows:

[0046] In Example 1, 1 g of the paste was placed in a 25 - ml volumetric flask, 20 ml of an aqueous solution (pH adjusted to 2.0 with acetic acid) was added, sonicated for 3 hours, stirred overnight (15 h) in a 60 - degree water bath (stirring speed was medium), sonicated for 1 h, and made up to the mark with methanol. The extraction recovery rate of brucine was 99.7%.

[0047] The above method can completely extract the brucine in the hydrogel plaster, but sonication for 3 hours is too intense for the extraction method and is not suitable for the operation of routine test samples. Therefore, the extraction method needs to be optimized.

[0048] Example 2: Take 1 g of the paste, place it in a 25-ml volumetric flask, add 20 ml of 0.9% sodium chloride solution (pH adjusted to 2.0 with formic acid), ultrasonicate for 1 hour, add 5 ml of methanol, stir in a water bath at 60 °C overnight (15 h), and make up the volume with methanol. The extraction recovery rate of brucine is measured to be 99.6%.

[0049] In this experiment, the ultrasonication time was reduced, and the results showed no significant difference, indicating that replacing water with 0.9% sodium chloride solution is beneficial to the extraction of brucine from the hydrogel patch.

[0050] Examples 3 - 5: Take 1 g of the paste and place it in a 25-ml volumetric flask, add 20 ml of 0.9% sodium chloride solution (pH adjusted to different values with phosphoric acid), ultrasonicate for 1 h, add 5 ml of methanol, stir in a water bath at 60 °C overnight (15 h), and make up the volume with methanol. The details are as follows:

[0051]

[0052] Examples 6 - 14: Take a 7×10 cm hydrogel patch (equivalent to 7 g of the paste), weigh it, cut it into small pieces of 3 cm×1.5 cm, place them in a 200-ml volumetric flask, add inorganic salt solutions with different concentrations (pH adjusted to 3.0 with formic acid)-methanol (80:20) to make up the volume to 200 ml, stir in a water bath, and ultrasonicate for 0.5 h. The details are as follows:

[0053]

[0054]

[0055] Note: Since the prescription process is relatively mature, in this experiment, the gel paste was not used for the samples, but the formed hydrogel patch was used. After the patch was cut into pieces and then extracted, and at the same time, the extraction scale was enlarged. These changes did not have an obvious impact on the extraction.

[0056] Adjust the pH value of the extraction solution to 3.0 and increase the inorganic salt concentration to investigate the extraction effect. From the results of Examples 6 - 8, Example 9, and Examples 12 - 13, it can be seen that within a certain range, increasing the inorganic salt concentration can indeed improve the extraction recovery rate of brucine, and an inorganic salt concentration of 4% is selected.

[0057] On the premise that other conditions remain unchanged, continue to increase the pH value of the extraction solution (pH value < 7) (Example 14), and the recovery rate of brucine hardly changes. Fix the composition of the extraction solution, that is, 4% sodium chloride solution (pH adjusted to 3.0 with formic acid)-methanol (80:20), and investigate the influence of different extraction times on the extraction recovery rate. From the results of Examples 8 and 9, it can be seen that the extraction recovery rates RAD% of stirring in a water bath overnight (15 h) and for 5 h are 0.7, with no obvious change. To save time costs, the extraction time is determined to be 5 h.

[0058] Fix the composition of the extraction solution and the extraction time, i.e., the extraction solution is 4% sodium chloride solution (adjust the pH value to 3.0 with formic acid)-methanol (80:20), and the extraction time is 5 h. Investigate the effect of different extraction temperatures on the extraction recovery rate. From the results of Examples 9 and 10, it can be seen that there is no obvious difference between extraction at 60 °C and 40 °C.

[0059] Fix the pH value of the extraction solution (pH 3.0), the extraction time (5 h) and the extraction temperature (60 °C), and investigate the extraction effect of the extraction solvent without methanol. From the results of Example 11, it can be seen that the recovery rate of the solvent without methanol is lower than that with methanol, and the optimal sample preparation method for the content is the method used in Examples 8 and 9.

[0060] To ensure that the extraction method is applicable to the preparation of related substance samples, the extraction of impurities was studied.

[0061] Example 15 Take about 25 mg of the raw material test sample, weigh it accurately, place it in a 25 mL volumetric flask, dissolve it with methanol and dilute it to the mark, and shake well; accurately measure 5 mL, place it in a 10 mL volumetric flask, and dilute it to the mark with water.

[0062] Examples 16-17 Take the gel patch 7*10 cm (equivalent to 7 g of paste), weigh it, cut it into small pieces of 3 cm * 1.5 cm, place it in a 200 mL volumetric flask, add 4% sodium chloride solution (adjust the pH value to 5.0 with hydrochloric acid)-methanol (80:20) to make up to 200 mL, stir in a water bath, and ultrasonicate for 0.5 h. The results are as follows:

[0063] 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

[0064] Note: In Example 16, stir in a water bath for 5 h, and in Example 17, stir in a water bath overnight (15 h).

[0065] From the above results, it can be seen that: compared with the raw drug (Example 15), among the gel patches (Examples 16-17), except for the unknown impurities with RRT 0.18 and RRT 0.81 and the known impurity 8, the rest of the impurities all come from the raw drug, and the normalization results of each impurity have no obvious difference from those of the raw drug, and there is also no difference in the results after prolonging the stirring time, indicating that the sample extraction is complete.

[0066] Examples 18-19 The preparation method and detection results of the samples for the release degree of brucine patch are as follows:

[0067] Take this product, cut it into circular samples with a diameter of 30 mm, remove the protective layer, make the adhesive surface of this product face upward, place it in the following release media, take the release solution at different times, filter it, and take the subsequent filtrate for determination.

[0068]

[0069]

[0070] The above experiments show that the release rate of brucine gel plaster in 2% sodium chloride solution is much greater than that in pure water medium, proving that the addition of sodium chloride also promotes the release of brucine from the gel plaster without adjusting the pH value by adding acid.

[0071] The screening methods for the content determination and related substances inspection of the brucine plaster in Examples 20 - 21 are as follows:

[0072] In Example 20, the content analysis method of semen strychni in Part I of the Chinese Pharmacopoeia (2020 Edition) was selected. An octadecylsilane chemically bonded silica column (250 mm × 4.6 mm, 5 μm) was used; the mobile phase was a mixed solution of acetonitrile - 0.01 mol / L sodium heptanesulfonate and 0.02 mol / L sodium dihydrogen phosphate in equal volume (adjusted to pH 2.85 with 5% phosphoric acid) (21:79) for elution, the flow rate was 1 ml / min; the detection wavelength was 260 nm, the column temperature was 30 °C, and the injection volume was 10 μl. The chromatogram is shown in the appendix Figure 1 as follows.

[0073] The resolution of each peak is not good. This method is not applicable to the detection of related substances, but can be used for content determination.

[0074] In Example 21, the following chromatographic conditions were selected: an octadecylsilane chemically bonded silica column (4.6 × 150 mm, 5 μm) was used; mobile phase A of the chromatographic column was acetonitrile, and mobile phase B was an aqueous solution of 0.2% triethylamine with 0.2% acetic acid; the flow rate was 1 ml / min; the detection wavelength was 260 nm, the column temperature was 30 °C, and the injection volume was 10 μl. Gradient elution was performed.

[0075] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 5 95 20 21 79 25 60 40 35.01 10 90 35 10 90

[0076] The chromatogram is shown in the appendix Figure 2 as follows.

[0077] Among them, the resolution of the impurity peaks at RT 17.058 min and RT 17.375 min is insufficient.

[0078] In Example 22, an octadecylsilane chemically bonded silica column (250 mm × 4.6 mm, 5 μm) was used; the mobile phase was a mixed solution of acetonitrile - 0.01 mol / L sodium heptanesulfonate and 0.02 mol / L sodium dihydrogen phosphate in equal volume (adjusted to pH 3.90 with 5% phosphoric acid) (18:82) for elution, the flow rate was 1 ml / min; the detection wavelength was 260 nm, the column temperature was 30 °C, and the injection volume was 40 μl. The chromatogram is shown in the appendix Figure 3 as follows.

[0079] Example 23 An octadecylsilyl silica gel column (250 mm × 4.6 mm, 5 μm) was selected; the mobile phase was an equal-volume mixed solution of acetonitrile - 0.01 mol / L sodium heptanesulfonate and 0.02 mol / L sodium dihydrogen phosphate (adjusted to pH 4.0 with 5% phosphoric acid) (18:82) for elution, the flow rate was 1 ml / min; the detection wavelength was 260 nm, the column temperature was 30 °C, and the injection volume was 40 μl. The chromatogram is as attached Figure 4 as shown.

[0080] Example 24 An octadecylsilyl silica gel column (250 mm × 4.6 mm, 5 μm) was selected; the mobile phase was an equal-volume mixed solution of acetonitrile - 0.01 mol / L sodium heptanesulfonate and 0.02 mol / L sodium dihydrogen phosphate (adjusted to pH 4.10 with 5% phosphoric acid) (18:82) for elution, the flow rate was 1 ml / min; the detection wavelength was 260 nm, the column temperature was 30 °C, and the injection volume was 40 μl. The chromatogram is as attached Figure 5 as shown. The resolution of each peak was good, and the method was applicable.

[0081] In summary, for the method of extracting brucine from the hydrogel patch, Examples 8, 9, 10, 12, 13, and 14 were relatively good. The release rate study of the brucine patch showed that the addition of sodium chloride promoted the release of brucine from the hydrogel patch. For the methods of analyzing and detecting the content and related substances of brucine, Examples 22, 23, and 24 were relatively good, and the resolution between brucine and related substances and among related substances was good.

[0082] The methods for analyzing and detecting the content and related substances of the brucine hydrogel patch are important components of its quality research. Among them, the detection of content and related substances is the basis for formulating its quality standard, and the quality standard is the criterion for examining whether its preparation products are qualified. Therefore, the methods for analyzing and detecting the content and related substances of the brucine hydrogel patch are crucial factors related to the quality of the entire preparation product. The above research provides technical support for the quality research of the brucine hydrogel patch and also provides a reference for the research on the content and related substances of other alkaline alkaloid hydrogel patches.

Claims

1. An analytical and detection method for the content of brucine and related substances in a brucine plaster, characterized in that, Including: (1) extracting brucine and related substances from the cataplasm; (2) a method for analyzing and detecting the content of brucine and related substances, and the method is high performance liquid chromatography. The related substances in the brucine cataplasm are as follows: The extraction of brucine and related substances from the cataplasm in step (1) is as follows: after stirring with the extraction solvent at 30 - 70 °C for 5 - 20 h, ultrasonic treatment for 0.5 - 3 h, and sampling; the extraction solvent is a mixed solution of an inorganic salt solution and methanol; the inorganic salt solution is adjusted with an acid, and the pH value is 2.0 - 5.

0. The method is high performance liquid chromatography. The chromatographic conditions are as follows: Chromatographic column: octadecylsilyl bonded silica gel column. Mobile phase for related substances: acetonitrile - a mixed solution of 0.01 mol / L sodium heptanesulfonate and 0.02 mol / L potassium dihydrogen phosphate in equal volume = 15:85 - 25:75; the mobile phase is adjusted to pH 3.90 - 4.10 with 5% phosphoric acid. Mobile phase for brucine content: acetonitrile - a mixed solution of 0.01 mol / L sodium heptanesulfonate and 0.02 mol / L potassium dihydrogen phosphate in equal amount and equal volume = 15:85 - 25:75; the mobile phase is adjusted to pH 3.80 - 4.10 with 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 °C. The injection volume is 5 - 60 μl.

2. The method for analyzing and detecting the content of brucine and related substances in a brucine cataplasm according to claim 1, characterized in that: the extraction solvent in step (1) is a mixed solution of 0.9 - 10% inorganic salt solution and methanol; the inorganic salts are selected from: chlorides, iodides, phosphates, sulfates, nitrates, nitrites, borates, thiosulfates, sulfites or bisulfites.

3. The method for analyzing and detecting the content of brucine and related substances in a brucine cataplasm according to claim 1 or 2, characterized in that: the extraction solvent in step (1) is a mixed solution of 4 - 10% inorganic salt solution and methanol, and the inorganic salts are sodium chloride, phosphate or sulfate.

4. The method for analyzing and detecting the content of brucine and related substances in a brucine cataplasm according to claim 1 or 2, characterized in that: the acid used to adjust the pH value of the extraction solvent in step (1) is selected from straight-chain or branched-chain saturated fatty acids, unsaturated fatty acids, aromatic organic acids or inorganic acids.

5. The method for analyzing and detecting the content of brucine and related substances in a brucine cataplasm according to claim 4, characterized in that: the acid used to adjust the pH value of the extraction solvent in step (1), the organic acids are selected from formic acid, acetic acid, citric acid, tartaric acid or citric acid; the inorganic acids are selected from hydrochloric acid, phosphoric acid or sulfuric acid.

6. The analytical detection method for the content of brucine and related substances in a brucine plaster as described in claim 1, characterized in that, The chromatographic conditions are as follows: Chromatographic column: selected from Agilent ZORBAX SB-C18, 250 mm × 4.6 mm, 5 μm; Kromasil C18, 250 mm × 4.6 mm, 5 μm; Ultimate LP-C18, 250 mm × 4.6 mm, 5 μm; or Waters XBridge C18, 250 mm × 4.6 mm, 5 μm; Mobile phase for related substances: acetonitrile - equal volume mixture of 0.01 mol / L sodium heptanesulfonate and 0.02 mol / L potassium dihydrogen phosphate = 18:82; the pH of the mobile phase is adjusted to 3.90 - 4.10 with 5% phosphoric acid; Mobile phase for brucine content: acetonitrile - equal volume and equal amount mixture of 0.01 mol / L sodium heptanesulfonate and 0.02 mol / L potassium dihydrogen phosphate = 21:79; the pH of the mobile phase is adjusted to 3.80 - 4.10 with 5% phosphoric acid; Detection wavelength is 255 - 280 nm; Flow rate is 0.5 - 2.0 ml / min; Column temperature is 25 - 40 °C Injection volume is 5 - 60 μl.

7. Use of the analytical detection method for the content of brucine and related substances in a brucine plaster as described in any one of claims 1 - 2 in controlling the product quality of the brucine plaster.

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