Wine antifebrile dichroa quality control and detection method

Abstract

The invention belongs to the technical field of liquid phase detection, and particularly relates to a wine antifebrile dichroa quality control and detection method. According to the detection method, high performance liquid chromatography is adopted to detect the content of dichroine in a solution in a test sample, and specific chromatographic conditions are as follows: a mobile phase A is acetonitrile, a mobile phase B is an acetic acid aqueous solution with the volume fraction of 0.15-0.35%, and the pH value of the mobile phase B is adjusted to 5.5-6.5 by using triethylamine; a chromatographic column takes octadecylsilane chemically bonded silica as a stationary phase, and the detection wavelength is 223 to 227 nm. The detection method disclosed by the invention has a good linear relationship when the concentration of the febrifugine is 0.67 mu g / mL to 66.64 mu g / mL; the sensitivity is high, the precision is high, and the detection limit is lt; the concentration is 0.36 mu g / mL, and the quantification limit is 1t; 0.90 [mu] g / mL; the repetitive stability is high; the sample recovery rate is high, and the average recovery rate of the dichroine hydrochloride reference substance is more than 90%.

Description

Technical Field

[0001] This invention belongs to the field of liquid chromatography detection technology. More specifically, it relates to a quality control detection method for Changshan liquor. Background Technology

[0002] Changshan (Dichroa febrifuga Lour.) is the dried root of the plant, belonging to the Saxifragaceae family. It is harvested in autumn, the fibrous roots are removed, it is washed, and then dried. Changshan has the effects of inducing vomiting of phlegm and treating malaria, and is commonly used for phlegm retention, chest tightness, and malaria. Studies have found that the antimalarial active ingredients of Changshan are mainly quinazolone alkaloids, including three types: haloxyfopine A, B, and C. Haloxyfopine salts, when used in combination with artemisinin and its derivatives, can achieve synergistic effects and reduced toxicity in antimalarial treatment. Haloxyfopine derivatives, such as haloxyfopine ketone, are not only highly effective against coccidia, but also show great potential in the treatment of autoimmune diseases such as arthritis, scleroderma, multiple sclerosis, and nephritis. Therefore, monitoring the haloxyfopine content in Changshan samples is of great significance for the quality control of Changshan medicinal use.

[0003] Traditional Chinese medicine processing refers to the methods and techniques used in traditional Chinese medicine to process medicinal materials into prepared slices of medicinal herbs according to the requirements of traditional Chinese medicine, under the guidance of traditional Chinese medicine theory. In ancient times, it was also called "paozhi," "xiushi," or "xiuzhi." After processing, the efficacy of the medicine can be improved, the toxic side effects of the medicine can be reduced, and the medicine can be stored more easily. It is an essential procedure in the clinical use of traditional Chinese medicine. The processing methods for Changshan (Changshan) throughout history include: stir-frying until yellow (stir-frying Changshan slices over low heat until the surface is slightly yellow), charring (stir-frying Changshan slices until the surface is charred black and the inside is charred yellow), honey-roasting (mixing Changshan slices with honey and stir-frying over low heat until they are no longer sticky), vinegar-boiling (boiling Changshan slices with vinegar until the vinegar is absorbed), wheat bran-frying (stir-frying Changshan slices with wheat bran until the surface is slightly yellow), wine-roasting (mixing Changshan slices with rice wine, letting it stand until the wine is absorbed, and then stir-frying it over low heat until the surface is slightly dry), wine-stewing (placing Changshan slices with rice wine in a stewing pot, the amount of wine should be enough to cover the medicinal material, and stewing it in a double boiler until the wine is absorbed and the Changshan slices become soft), and salt-roasting (mixing Changshan slices with salt water and stir-frying it over low heat until dry). Among these, wine-roasted Changshan, also known as wine-processed Changshan, is the most widely used processing method for Changshan. However, during the processing of Changshan (a type of medicinal herb), the active substances are easily lost, which greatly weakens the efficacy of the processed medicinal material. Therefore, it is of great significance to provide a quality monitoring and testing method for Changshan and its related processed products. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the prior art and provide a method for detecting the content of datura alkaloid in datura and its processed products.

[0005] The purpose of this invention is to provide the application of the detection method in the quality monitoring of Changshan and its processed products.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] This invention provides a method for detecting the content of datura alkaloid in *Callicarpa japonica* and its processed products. The method employs high-performance liquid chromatography (HPLC) to detect the content of datura alkaloid in the test sample solution. Specific chromatographic conditions include:

[0008] Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of acetic acid with a volume fraction of 0.15–0.35%, with the pH of mobile phase B adjusted to 5.5–6.5 using triethylamine.

[0009] The elution gradient is shown in the table below:

[0010] Time (min) Mobile phase A (%) Mobile phase B (%) 0.01 8～12 88～92 15.00 8～12 88～92 15.10 100 0 20.00 100 0 20.10 8～12 88～92 30.00 8～12 88～92

[0011] The chromatographic column is a column with octadecylsilane-bonded silica gel as the stationary phase, and the detection wavelength is 223-227 nm; the test sample is Changshan or Changshan processed products.

[0012] This invention employs specific mobile phases, elution gradients, chromatographic columns, and detection wavelengths to accurately detect the content of datura alkaloids in test samples, providing a valuable means for monitoring the efficacy and quality of datura and its processed products.

[0013] In this invention, the processed Changshan products mainly refer to Changshan medicinal materials after processing, including but not limited to: Changshan processed with wine, Changshan stir-fried yellow, Changshan stir-fried charcoal, Changshan processed with honey, Changshan cooked with vinegar, Changshan stir-fried with wheat bran, Changshan processed with wine, Changshan stewed with wine, Changshan processed with salt, etc.

[0014] Preferably, the test solution is prepared by the following method: the test sample, concentrated ammonia, and chloroform are mixed in a ratio of 1g:1mL:90-110mL, sonicated, and the weight lost after sonication is replenished with chloroform. The solution is filtered, the filtrate is dried, dissolved in 45-55% methanol, filtered, and the solution is obtained.

[0015] In this invention, the concentrated ammonia water refers to an ammonia solution with a mass concentration of 25% to 28%.

[0016] Preferably, the test sample, concentrated ammonia, and chloroform are mixed in a ratio of 1g:1mL:100mL.

[0017] Preferably, the amount of methanol added is 8 to 12 mL.

[0018] Preferably, the ultrasound duration is 0.5 to 1.5 hours.

[0019] Preferably, the chromatographic conditions further include a flow rate of 0.8–1.2 mL / min.

[0020] Preferably, the chromatographic conditions further include an injection volume of 10–15 μL.

[0021] Preferably, the chromatographic conditions further include a column temperature of 30±1℃.

[0022] Preferably, the chromatographic conditions further include: the detector is an ultraviolet detector.

[0023] Furthermore, the present invention also protects the application of the detection method in the quality monitoring of Changshan and its processed products.

[0024] The present invention has the following beneficial effects:

[0025] This invention provides a method for detecting the content of datura alkaloid in *Changshan* and its processed products. High-performance liquid chromatography (HPLC) is used to detect the content of datura alkaloid in the solution of the test sample. Specific chromatographic conditions include: mobile phase A is acetonitrile; mobile phase B is a 0.15–0.35% (v / v) aqueous acetic acid solution, with the pH of mobile phase B adjusted to 5.5–6.5 using triethylamine; the chromatographic column is an octadecylsilane-bonded silica gel column as the stationary phase; and the detection wavelength is 223–227 nm. The detection method of this invention exhibits good linearity at datura alkaloid concentrations of 0.67 μg / mL to 66.64 μg / mL; it has high sensitivity and precision, with a detection limit <0.36 μg / mL and a quantitation limit <0.90 μg / mL; it has high repeatability and high recovery rate, with an average recovery rate of over 90% for datura alkaloid hydrochloride reference standard. Attached Figure Description

[0026] Figure 1 This is the chromatogram of the dichroic alkali hydrochloride reference solution.

[0027] Figure 2 Chromatograms of the reference solution and the test solution. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0029] Example 1

[0030] Preparation of reference solution: Weigh about 1 mg of Changshan alkaloid hydrochloride into a 10 mL volumetric flask, dissolve and dilute with 50% methanol and make up to volume, shake well, and then filter through a 0.45 μm microporous membrane to obtain the reference solution.

[0031] Preparation of the test solution: Weigh 0.5 g of Changshan (Changshan) powder or Changshan slices, place it in a stoppered conical flask, first add 0.5 mL of concentrated ammonia to moisten the sample powder, then accurately add 50 mL of chloroform, stopper tightly and weigh, sonicate for 1 h, cool and weigh, replenish the lost weight with chloroform, filter, collect the filtrate in an evaporating dish, place it on a water bath to evaporate to dryness, cool, dissolve in 50% methanol in a 10 mL volumetric flask and dilute to the mark, shake well, filter through a 0.45 μm microporous membrane to obtain the test solution.

[0032] The chromatographic conditions for the detection are shown in Table 1:

[0033] Table 1 Chromatographic Detection Conditions

[0034]

[0035] Testing steps:

[0036] Inject 10 μL of the test solution or reference solution and perform high-performance liquid chromatography (HPLC) detection under the above chromatographic conditions to determine the content of datura alkaloid in the samples of Changshan or Changshan medicinal slices. The results are shown in Table 2. Figure 1 and Figure 2 .

[0037] Table 2. Results of determination of alkaloid content in Changshan and wine-processed Changshan.

[0038]

[0039] Table 2 shows that the content of styraciline in Changshan slices was approximately 0.02% or 0.22 mg / g (0.22 mg of styraciline per g of Changshan slices), with an RSD of 1.64% (<3%). The content of styraciline in wine-processed Changshan was approximately 0.02% or 0.15 mg / g (0.15 mg of styraciline per g of wine-processed Changshan), with an RSD of 0.80% (<3%). The results indicate that the determination of styraciline content in this product complies with regulations.

[0040] Example 2

[0041] Preparation of reference solution: Weigh about 1 mg of Changshan alkaloid hydrochloride into a 10 mL volumetric flask, dissolve and dilute with 50% methanol and make up to volume, shake well, and then filter through a 0.45 μm microporous membrane to obtain the reference solution.

[0042] Chromatographic conditions for detection: consistent with those in Example 1.

[0043] Examining linear relationships:

[0044] Prepare standard curve solutions of reference standard solutions with concentrations of 0.67 μg / mL, 1.33 μg / mL, 6.66 μg / mL, 13.33 μg / mL, 33.32 μg / mL, and 66.64 μg / mL by precisely diluting the reference standard solution. Shake well and filter through a 0.45 μm microporous membrane to obtain the solution. Determine the concentrations according to the chromatographic conditions described above.

[0045] The experimental results are shown in Table 3. Based on the peak areas of different concentrations obtained from liquid chromatography in Table 3, a standard curve was plotted with concentration (X) as the abscissa and peak area (Y) as the ordinate. The linear regression equation for Changshan alkaloid is: Y = 46704X - 5161.1, R0 2 =1 (>0.999%), indicating that the concentration of datura alkaloid has a good linear relationship in the range of 0.67 μg / mL to 66.64 μg / mL.

[0046] Table 3 Peak areas of different concentrations of Changshan alkaloid hydrochloride reference solutions

[0047]

[0048] Example 3

[0049] Preparation of reference solution: Weigh about 1 mg of Changshan alkaloid hydrochloride into a 10 mL volumetric flask, dissolve and dilute with 50% methanol and make up to volume, shake well, and then filter through a 0.45 μm microporous membrane to obtain the reference solution.

[0050] Chromatographic conditions for detection: consistent with those in Example 1.

[0051] Precision test:

[0052] Accurately pipette the Changshan alkali hydrochloride reference solution and perform six consecutive determinations under the above chromatographic conditions. Record the peak area, and the results are shown in Table 4. The RSD value of the peak area is 0.06% (<2%), indicating that the instrument has good precision and meets the detection requirements for sample content determination.

[0053] Table 4 Precision Experiment Results

[0054]

[0055] Investigation of Limit of Detection and Limit of Quantification:

[0056] The reference solution was continuously diluted and analyzed under the chromatographic conditions described above until the signal-to-noise ratio (S / N) ≈ 3. The concentration at this point is the limit of detection (LOD), and the concentration at which the S / N ≈ 10 is the limit of quantification (LOQ). The experimental results are shown in Table 5. When the concentration is 0.36 μg / mL, the S / N is 4.73, therefore the LOD of datura oleifera according to the detection method of this invention is <0.36 μg / mL; when the concentration is 0.90 μg / mL, the S / N is 10.71, therefore the LOQ of datura oleifera is <0.90 μg / mL.

[0057] Table 5 Results of the investigation on the detection limit and quantitation limit of Changshan alkali hydrochloride.

[0058]

[0059] Example 4

[0060] Preparation of the test solution: Weigh 0.5 g of Changshan test sample powder and place it in a stoppered conical flask. First, add 0.5 mL of concentrated ammonia to moisten the sample powder, then accurately add 50 mL of chloroform. Seal tightly and weigh. Sonicate for 1 h, cool and weigh. Make up the weight loss with chloroform, filter, collect the filtrate and place it in an evaporating dish. Place it on a water bath and evaporate to dryness. Cool, dissolve in 50% methanol in a 10 mL volumetric flask and dilute to the mark. Shake well and filter through a 0.45 μm microporous membrane to obtain the test solution.

[0061] Chromatographic conditions for detection: consistent with those in Example 1.

[0062] Repeatability test:

[0063] Accurately weigh the Changshan powder and prepare 6 test solutions according to the above-mentioned test solution preparation method. Analyze the solutions under the above-mentioned chromatographic conditions and calculate the content. The experimental results are shown in Table 6.

[0064] Table 6. Repeatability test results of the Changshan liquor sample solution.

[0065]

[0066] The experimental results show that the RSD value of the content of haloxylon ammodendronine in the test solution is 3.65% (<5%), indicating that the liquid phase detection method of the present invention has good repeatability.

[0067] Stability testing:

[0068] The above-mentioned test solution was injected and analyzed at 0, 2, 4, 8, 12, 20 and 24 h after preparation, respectively, according to the above chromatographic conditions. The results are shown in Table 7.

[0069] Table 7 Results of the stability test of the test sample solution

[0070]

[0071]

[0072] Table 7 shows that the RSD values ​​of the peak area of ​​Changshanine in the Changshan test sample solution from 0 to 24 hours were 1.53%, all less than 2%, indicating that the Changshan test sample solution had good stability within 24 hours.

[0073] Example 5

[0074] Preparation of the test solution: Weigh 0.5 g of Changshan (Changshan) powder or Changshan slices, place it in a stoppered conical flask, first add 0.5 mL of concentrated ammonia to moisten the sample powder, then accurately add 50 mL of chloroform, stopper tightly and weigh, sonicate for 1 h, cool and weigh, replenish the lost weight with chloroform, filter, collect the filtrate in an evaporating dish, place it on a water bath to evaporate to dryness, cool, dissolve in 50% methanol in a 10 mL volumetric flask and dilute to the mark, shake well, filter through a 0.45 μm microporous membrane to obtain the test solution.

[0075] Chromatographic conditions for detection: consistent with those in Example 1.

[0076] Recovery test:

[0077] Nine portions of the test sample powder were accurately weighed. Two portions of test sample solutions with different amounts of datura hydrochloride were prepared according to the above test sample solution preparation method. The solutions were analyzed under the above chromatographic conditions. The recovery rate was calculated using the formula recovery rate % = (CA) / B (where A is the amount of the analyte in the test sample, B is the amount of the added reference standard, and C is the measured value). The results are shown in Table 8.

[0078] Table 8 Sample recovery rate of the test sample spiking experiment

[0079]

[0080]

[0081] The experimental results showed that the average recovery rate of the dichroa febrifuga hydrochloride reference standard was 96.75% (90%-110%), and the RSD value was 3.14% (<5%), indicating that the method had good recovery.

[0082] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for detecting the content of datura alkaloid in datura and its processed products, characterized in that, The content of datura alkaloid in the test solution was determined by high performance liquid chromatography (HPLC). Specific chromatographic conditions included: Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of acetic acid with a volume fraction of 0.15–0.35%, with the pH of mobile phase B adjusted to 5.5–6.5 using triethylamine. The elution gradient is shown in the table below: The chromatographic column is a column with octadecylsilane-bonded silica gel as the stationary phase, and the detection wavelength is 223-227 nm; the test sample is Changshan or Changshan processed products.

2. The detection method according to claim 1, characterized in that, The test solution is prepared as follows: the test sample, concentrated ammonia, and chloroform are mixed in a ratio of 1g:1mL:90-110mL, sonicated, and the weight lost after sonication is replenished with chloroform. The solution is filtered, the filtrate is dried, dissolved in 45-55% methanol, filtered, and the solution is obtained.

3. The detection method according to claim 2, characterized in that, The alcoholic beverage, concentrated ammonia, and chloroform were mixed in a ratio of 1g:1mL:100mL.

4. The detection method according to claim 2, characterized in that, The amount of methanol added is 8-12 mL.

5. The detection method according to claim 2, characterized in that, The duration of the ultrasound is 0.5 to 1.5 hours.

6. The detection method according to claim 1, characterized in that, The chromatographic conditions also include a flow rate of 0.8–1.2 mL / min.

7. The detection method according to claim 1, characterized in that, The chromatographic conditions also include an injection volume of 10–15 μL.

8. The detection method according to claim 1, characterized in that, The chromatographic conditions also include a column temperature of 30±1℃.

9. The detection method according to claim 1, characterized in that, The chromatographic conditions also include: the detector is an ultraviolet detector.

10. The application of the detection method according to any one of claims 1 to 9 in the quality control of Changshan and its processed products.

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