Rapid quantitative detection method of tetrandrine and fangchunolin in radix stephaniae tetrandrae

Abstract

This invention provides a rapid quantitative detection method for tetrandrine and fangchinorline in Stephania tetrandra, relating to the field of high-performance liquid chromatography (HPLC) analysis technology. The method employs a single reference standard, either tetrandrine or fangchinorline, for HPLC-UV high-performance liquid chromatography analysis of Stephania tetrandra samples at a detection wavelength of 218–234 nm. Since tetrandrine and fangchinorline have similar UV absorption wavelengths at 218–234 nm, this invention enables rapid quantitative detection of tetrandrine and fangchinorline, achieving the effect of simultaneously determining the content of tetrandrine and fangchinorline using a single reference standard without the need for correction factors. Compared with existing methods for detecting tetrandrine and fangchinorline in Stephania tetrandra, this method has the advantages of being rapid, simple, and environmentally friendly.

Description

Technical Field

[0001] This invention relates to the field of high performance liquid chromatography analysis technology, and in particular to a rapid quantitative detection method for fangchinine and fangchinorline in Stephania tetrandra. Background Technology

[0002] Fangji is the dried root of *Stephania tetrandra* S. Moore, a plant in the Menispermaceae family. Fangji has the effects of dispelling wind and relieving pain, promoting diuresis and reducing swelling. It is used for rheumatic pain, edema, beriberi, dysuria, and eczema / sores.

[0003] Modern pharmaceutical research shows that Stephania tetrandra contains various chemical components, including alkaloids, flavonoids, sterols, and organic acids. Alkaloids are the main active components of Stephania tetrandra, exhibiting good therapeutic effects in various aspects, including anti-inflammatory, analgesic, antipyretic, diuretic, antimicrobial, tumor cell growth inhibition, organ protection, antibacterial, and neuroprotective effects. The Chinese Pharmacopoeia published in mainland China uses tetrandrine and fangchinorline as quality control indicators, employing 2% hydrochloric acid with methanol under reflux for 30 minutes, followed by approximately 30 minutes of liquid chromatography analysis. The Hong Kong Standards for Chinese Medicinal Herbs published in the Hong Kong Special Administrative Region of the People's Republic of China uses 50% ethanol with two ultrasonic extractions for 60 minutes, followed by approximately 20 minutes of liquid chromatography analysis. The total extraction time for both methods is over 60 minutes, and both require two reference standards for quantification.

[0004] Therefore, it is both necessary and urgent to develop a rapid, environmentally friendly, and low-cost green analytical method for fangchi root powder and fangchinorline, so as to provide a technical basis for the quality evaluation technology of fangchi.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a rapid and quantitative method for detecting fangchiine and fangchinorline in Stephania tetrandra powder. Compared with existing methods for detecting fangchiine and fangchinorline in Stephania tetrandra powder, this method has the advantages of being rapid, simple, and environmentally friendly.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0008] According to one aspect of the present invention, a rapid quantitative detection method for tetrandrine and tebufenozide in Stephania tetrandra root powder is provided, the detection method comprising the following steps:

[0009] (A) The sample of Stephania tetrandra to be tested is pretreated to obtain the solution to be tested;

[0010] (B) The test solution was analyzed by HPLC-UV high performance liquid chromatography to obtain the quantitative detection results of tetrandrine and tebufenozide;

[0011] The detection and analysis were performed using a single reference standard with isoabsorption wavelength quantification, and the detection wavelength was 218–234 nm.

[0012] The single reference standard is tetrandrine or tebufenozide.

[0013] This invention provides a rapid quantitative detection method for tetrandrine and fangchinorline in Stephania tetrandra. This method innovatively employs a single reference standard (tetrandrine or fangchinorline) and utilizes HPLC-UV high-performance liquid chromatography at a detection wavelength of 218–234 nm to analyze Stephania tetrandra samples. Since tetrandrine and fangchinorline have similar UV absorption wavelengths at 218–234 nm, this method can rapidly obtain quantitative detection results for both tetrandrine and fangchinorline. It achieves the effect of simultaneously determining the content of tetrandrine and fangchinorline using a single reference standard without the need for correction factors. Compared with existing methods for detecting tetrandrine and fangchinorline in Stephania tetrandra, this method has the advantages of being rapid, simple, and environmentally friendly.

[0014] Preferably, the HPLC-UV high-performance liquid chromatography detection wavelength in step (B) is 224 nm.

[0015] It should be noted that the analytical principle of HPLC-UV high-performance liquid chromatography is based on the separation of compounds by a chromatographic column, and the determination of content by a UV detector based on the response of the analyte at a specific wavelength. To ensure the sensitivity of the analytical method, existing methods typically use the maximum absorption wavelength of tetrandrine and fangchinorline as the detection wavelength. Because tetrandrine and fangchinorline have different UV responses at their maximum absorption wavelengths, a correction factor is required to achieve the quantification of multiple compounds by a single reference standard.

[0016] If the wavelengths with the same ultraviolet absorption of tetrandrine and tebufenozide are used as the detection wavelengths, it may be possible to simultaneously determine the content of tetrandrine and tebufenozide using a single reference standard without using a correction factor.

[0017] Through long-term research, the applicant discovered that tetrandrine and fangchinorhinone have the same ultraviolet absorption wavelength at a detection wavelength of 224 nm. Therefore, this application creatively employs a single reference standard (tetrandrine or fangchinorhinone) using HPLC-UV high-performance liquid chromatography to detect and analyze Stephania tetrandra samples. This method can rapidly obtain quantitative detection results for tetrandrine and fangchinorhinone, achieving the effect of simultaneously determining the content of tetrandrine and fangchinorhinone using a single reference standard without the need for correction factors. Compared with existing methods for detecting tetrandrine and fangchinorhinone in Stephania tetrandra, this method has the advantages of being rapid, simple, and environmentally friendly.

[0018] In a preferred embodiment of the present invention, step (A) preprocessing includes:

[0019] The sample of Stephania tetrandra to be tested was placed in a methanol solution containing 2% hydrochloric acid, and then extracted by ultrasonication to obtain the test solution.

[0020] In the above preferred embodiment, the ratio of the sample to be tested (Stephania tetrandra) to the methanol solution containing 2% hydrochloric acid is 0.5:10-50 g / ml, preferably 0.5:10 g / ml;

[0021] In the preferred embodiment described above, the parameter conditions for ultrasonic extraction satisfy at least one of the following:

[0022] Extraction power 380W, extraction frequency 37kHz, extraction time 10-30min.

[0023] In a preferred embodiment of the present invention, the chromatographic column of the HPLC-UV high-performance liquid chromatography in step (B) is a core-shell type column.

[0024] Preferably, the chromatographic column is a CORTECS T3 (4.6×150mm, 2.7μm) core-shell chromatographic column;

[0025] As a preferred embodiment, core-shell chromatographic columns offer advantages such as high column efficiency, low column pressure, and fast analysis speed, making them a suitable liquid chromatography technique for rapid analysis and detection in the industrialization of traditional Chinese medicine. Unlike conventional silica gel columns, which are entirely porous packing materials, core-shell chromatographic packing materials consist of a non-porous silica gel core and an outer porous core-shell layer, forming a surface-porous microsphere. This porous core-shell layer results in a shorter solvent diffusion path and a smaller theoretical plate height, thus achieving higher column efficiency. Simultaneously, core-shell packing materials exhibit better permeability and lower back pressure, allowing for higher analytical flow rates. It can achieve rapid analysis similar to ultra-high performance liquid chromatography (UHPLC) on conventional liquid chromatography, improving industrial-scale detection efficiency and reducing detection costs without increasing investment in industrial equipment.

[0026] In a preferred embodiment of the present invention, the column temperature of the chromatographic column is 25-35°C, preferably 30°C.

[0027] In a preferred embodiment of the present invention, the elution method of the HPLC-UV high performance liquid chromatography is isocratic elution.

[0028] In the preferred embodiment described above, the isocratic elution uses ethanol and 0.3% phosphoric acid solution as the mobile phase, the isocratic elution time is 10 min, and the flow rate is 0.8-1.2 mL / min, preferably 1 mL / min.

[0029] In the preferred embodiment described above, the volume ratio of ethanol to 0.3% phosphoric acid solution is 15:85 to 21:79, preferably 18:82.

[0030] As a preferred embodiment, this application uses ethanol and 0.3% phosphoric acid solution as the mobile phase, which has the advantages of being green and environmentally friendly. Typically, the mobile phase used in reversed-phase liquid chromatography is more polar than its hydrophobic stationary phase. Therefore, the mobile phase is usually a binary solvent composed of polar organic solvents such as methanol and acetonitrile, and water. This generates a large amount of waste that needs to be treated, posing a potential threat to the ecological environment and the safety of operators. Ethanol, as an ideal green alternative solvent in chromatography, has lower toxicity and volatility than acetonitrile and methanol, a lower vapor pressure, and higher stability in mobile phase mixtures. Furthermore, the cost of treating ethanol waste is relatively low.

[0031] In a preferred embodiment of the present invention, the detection and analysis method includes:

[0032] The contents of tetrandrine and fangchinorline in the test solution were determined by HPLC-UV high performance liquid chromatography at a wavelength of 218–234 nm; subsequently, the total amounts of tetrandrine and fangchinorline were calculated.

[0033] Preferably, the detection and analysis method includes: using HPLC-UV high-performance liquid chromatography at a wavelength of 224 nm to detect the content of tetrandrine and fangchinorline in the test solution; subsequently, the total amount of tetrandrine and fangchinorline is obtained.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] This invention provides a rapid quantitative detection method for tetrandrine and fangchinorline in Stephania tetrandra. This method innovatively employs a single reference standard (tetrandrine or fangchinorline) and utilizes HPLC-UV high-performance liquid chromatography at a detection wavelength of 218–234 nm to analyze Stephania tetrandra samples. Since tetrandrine and fangchinorline have similar UV absorption at 218–234 nm, this method can rapidly obtain quantitative detection results for both tetrandrine and fangchinorline. It achieves the effect of simultaneously determining the content of tetrandrine and fangchinorline using a single reference standard without the need for correction factors. Compared with existing methods for detecting tetrandrine and fangchinorline in Stephania tetrandra, this method has the advantages of being rapid, simple, and environmentally friendly. Attached Figure Description

[0036] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0037] Figure 1 The image shows the UV full scan of the tetrandrine and tetrandrine reference solutions of equal mass concentration provided in Example 4 of this invention.

[0038] Figure 2 The above are UV full scan images of the equimolar concentration tetrandrine and tetrandrine reference solutions provided in Example 4 of this invention.

[0039] Figure 3 The HPLC chromatograms of the mixed reference standard working solution and the test solution provided in Example 5 of this invention are shown below.

[0040] Figure 4 The linear regression analysis diagram of tetrandrine provided in Example 6 of this invention;

[0041] Figure 5 The linear regression analysis diagram of tetrandrine provided in Example 6 of the present invention. Detailed Implementation

[0042] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] The technical solution of the present invention will be further described below with reference to the embodiments.

[0044] Note: The instruments, reagents, and materials used in the following embodiments of this application include:

[0045] Vanquish Core high-performance liquid chromatograph (Thermo Fisher Scientific), XP-6 part per million balance (Mettler-Toledo Instruments, Inc., USA), XPE205 part per million balance (Mettler-Toledo Instruments, Inc., USA), XM-500UVF ultrasonic cleaner (Xiaomei Ultrasonic Instruments Co., Ltd.).

[0046] Methanol (analytical grade, Guangdong Guanghua Technology Co., Ltd.), hydrochloric acid (superior grade, Guangdong Guanghua Technology Co., Ltd.), phosphoric acid (chromatographic grade, Shanghai Aladdin Reagent Co., Ltd.), ethanol (chromatographic grade, Maclean's), acetonitrile (chromatographic grade, Shanghai Xingke High Purity Solvent Co., Ltd.), tetrandrine (Chengdu Pusi Biotechnology Co., Ltd., batch number PS020463, purity 99.92%), and tetrandrine (Chengdu Pusi Biotechnology Co., Ltd., batch number PS020129, purity 98.58%).

[0047] Example 1

[0048] A rapid quantitative detection method for fangchiine and fangchinorin in Stephania tetrandra root powder, the detection method comprising the following steps:

[0049] 1. Preparation of reference solution:

[0050] Accurately weigh the tetrandrine reference standard, dissolve it in methanol, and dilute to 500 mL. Mix well to obtain a reference solution with a concentration of 29.481 μmol / L.

[0051] 2. Preparation of the test solution:

[0052] Accurately weigh approximately 0.5 g of Stephania tetrandra powder (passed through a No. 6 sieve), place it in a 100 mL Erlenmeyer flask, add 10 mL of 2% hydrochloric acid methanol, extract by sonication (380 W power, 37 kHz frequency) for 10 min, shake well, aspirate the supernatant, filter through a 0.22 μm organic filter membrane, and prepare for analysis.

[0053] 3. The content of tetrandrine in Stephania tetrandra was determined by detecting the test solution at a wavelength of 224 nm using HPLC-UV high-performance liquid chromatography; the content of fangchinorline in Stephania tetrandra was determined by detecting the test solution at a wavelength of 224 nm using HPLC-UV high-performance liquid chromatography.

[0054] Furthermore, the total amount of tetrandrine and tebufenozide was obtained;

[0055] The chromatographic conditions for the HPLC-UV high-performance liquid chromatography are as follows:

[0056] The chromatographic column was a CORTECS T3 (4.6×150mm, 2.7μm), the column temperature was 30℃, the mobile phase was ethanol:0.3% phosphoric acid (18:82), isocratic elution was performed for 10 min, the flow rate was 1 mL / min, the injection volume was 1 μL, and the detection wavelength was 224 nm.

[0057] Example 2

[0058] A rapid quantitative detection method for fangchiine and fangchinorin in Stephania tetrandra root powder, the detection method comprising the following steps:

[0059] 1. Preparation of reference solution:

[0060] Accurately weigh the tetrandrine reference standard, dissolve it in methanol, and dilute to 500 mL. Mix well to obtain a reference solution with a concentration of 29.662 μmol / L.

[0061] 2. Preparation of the test solution:

[0062] Accurately weigh approximately 0.5 g of Stephania tetrandra powder (passed through a No. 6 sieve), place it in a 100 mL Erlenmeyer flask, add 10 mL of 2% hydrochloric acid methanol, extract by sonication (380 W power, 37 kHz frequency) for 10 min, shake well, aspirate the supernatant, filter through a 0.22 μm organic filter membrane, and prepare for analysis.

[0063] 3. The content of tetrandrine in Stephania tetrandra was determined by detecting the test solution at a wavelength of 224 nm using HPLC-UV high-performance liquid chromatography; the content of fangchinorline in Stephania tetrandra was determined by detecting the test solution at a wavelength of 224 nm using HPLC-UV high-performance liquid chromatography.

[0064] Furthermore, the total amount of tetrandrine and tebufenozide was obtained;

[0065] The chromatographic conditions for the HPLC-UV high-performance liquid chromatography are as follows:

[0066] The chromatographic column was a CORTECS T3 (4.6×150mm, 2.7μm), the column temperature was 30℃, the mobile phase was ethanol:0.3% phosphoric acid (18:82), isocratic elution was performed for 10 min, the flow rate was 1 mL / min, the injection volume was 1 μL, and the detection wavelength was 224 nm.

[0067] Example 3

[0068] This embodiment analyzes the pretreatment process parameters of this application based on the detection method of Embodiment 2:

[0069] (1) Replace “2% hydrochloric acid methanol” in the test solution preparation step of Example 2 with “2% hydrochloric acid ethanol”, and the rest is the same as in Example 2.

[0070] By comparing the extraction efficiency of 2% hydrochloric acid methanol (Example 2) and the above-mentioned 2% hydrochloric acid ethanol, the results showed that the relative deviation of the content measured by the two methods was 8.0%, which was significant. Therefore, this application selected 2% hydrochloric acid methanol as the extraction solvent. The detection results are shown in Table 1:

[0071] Table 1. Content measured under different extraction solvents:

[0072]

[0073] (2) The extraction method of "ultrasonication" in the preparation step of the test sample solution in Example 2 was replaced by the "heating reflux" method. Implementation methods with different ultrasonic times and different extraction solvent volumes were also provided for comparison with Example 2. (See Table 2 below for specific parameters and conditions.)

[0074] By comparing the extraction rates of ultrasound and reflux heating, no significant difference was found. Considering the simplicity of the method, ultrasound was chosen as the extraction method.

[0075] By comparing the effects of different extraction solvent volumes (10 mL, 25 mL, 50 mL) on the extraction rates of tetrandrine and tetrandrine, it was found that the solid-liquid ratio had little effect on the extraction rate of the target compounds. Based on the principles of saving solvent and environmental protection, and taking into account the chromatographic peak height of the analytes, an extraction solvent volume of 10 mL was adopted.

[0076] The study of ultrasonic extraction time (10 min, 20 min, 30 min) revealed that ultrasonic extraction for 10 min was sufficient to fully extract the analyte.

[0077] Table 2. Content measured under different extraction conditions:

[0078]

[0079] In summary, the extraction method selected in this invention is as follows: take about 0.5g of Stephania tetrandra powder (passed through a No. 6 sieve), place it in a stoppered conical flask, add 10mL of 2% hydrochloric acid methanol, and sonicate for 10min.

[0080] Example 4

[0081] This embodiment determines the isoabsorption detection wavelength of the chromatography of this application based on the detection method of Embodiment 2:

[0082] (1) Take the stock solution of the reference standard and dilute it with the mobile phase to prepare reference standard solutions of 19.83 μg / mL tebufenozide and 19.77 μg / mL fenbufenozide. Scan the solutions at a wavelength of 200-400 nm using a UV spectrophotometer. It was found that there was no intersection of the same absorbance values ​​in the obtained UV full scan. Therefore, the experiment was changed to use reference standard solutions of tebufenozide and fenbufenozide with the same molar concentration.

[0083] Figure 1 The image shows a UV full scan of the equivalent mass concentration of tetrandrine and tetrandrine reference solutions provided in this embodiment.

[0084] (2) Take the reference stock solution and dilute it with the mobile phase to obtain reference solutions with equimolar concentrations of 29.481 μmol / L for tetrandrine and 29.662 μmol / L for tebufenozide. Scan the solution in the same way and find that the intersection of the isoabsorbance values ​​of the two reference solutions is 224 nm.

[0085] Figure 2 The image shows the UV full scan of the equimolar concentration tetrandrine and tetrandrine reference solutions provided in this embodiment.

[0086] (3) The high performance liquid chromatography was confirmed using the chromatographic conditions of Example 2. The results showed that at a wavelength of 224 nm, the peak areas of equimolar concentrations of tetrandrine and tetrandrine reference solutions were equal, both with high response, good peak shape, and stable baseline. Therefore, 224 nm was selected as the isoabsorption detection wavelength (see Table 3).

[0087] Table 3 shows a comparison of the peak areas of tetrandrine and tebufenozide at equimolar concentrations under different wavelengths.

[0088] Table 3:

[0089]

[0090] Example 5: Localization of chromatographic peaks

[0091] (i) The test solution was prepared using the method provided in (1) of this embodiment, and then analyzed using the chromatographic conditions in (3) of this embodiment. Six test samples were measured in parallel. Using tetrandrine as the reference peak (S), the average relative retention time of tetrandrine was calculated to be 0.750, and the RSD was 0.1%. This indicates that tetrandrine can be used as the reference peak, and the relative retention time can be used to locate the chromatographic peak of tetrandrine.

[0092] (1) Preparation of reference solution

[0093] Accurately weigh tebufenozide reference standard and tetrandrine reference standard, dissolve them in methanol, and dilute to volume in a 500 mL volumetric flask. Mix well to obtain a mixed solution of reference standards with concentrations of 29.481 μmol / L and 29.662 μmol / L, respectively.

[0094] (2) Preparation of test solution

[0095] Accurately weigh approximately 0.5 g of Stephania tetrandra powder (passed through a No. 6 sieve), place it in a 100 mL Erlenmeyer flask, add 10 mL of 2% hydrochloric acid methanol, extract by sonication (380 W power, 37 kHz frequency) for 10 min, shake well, aspirate the supernatant, filter through a 0.22 μm organic filter membrane, and prepare for analysis.

[0096] (3) Chromatographic conditions

[0097] The chromatographic column was a CORTECS T3 (4.6×150mm, 2.7μm), the column temperature was 30℃, the mobile phase was ethanol:0.3% phosphoric acid (18:82), isocratic elution was performed for 10 min, the flow rate was 1 mL / min, the injection volume was 1 μL, and the detection wavelength was 224 nm.

[0098] (ii) Prepare the test solution using the methods (1) and (2) above. Take the mixed reference working solution and the test solution, and inject them for analysis according to the chromatographic conditions (3) above. The chromatogram is shown in [reference]. Figure 3 .

[0099] Figure 3 The HPLC chromatograms of the mixed reference standard working solution and the test solution provided in this embodiment are shown. Figure 3 In the diagram, A is the chromatogram of the mixed reference standard working solution, and B is the chromatogram of the test solution. Figure 3 1 is tetrandrine, and 2 is tetrandrine.

[0100] Example 6 Methodological Investigation

[0101] (I) Linearity, Limit of Detection, and Limit of Quantitation:

[0102] Prepare reference stock solutions of tetrandrine and tetrandrine according to the method in Example 5 (1) above, and dilute them stepwise with methanol to obtain reference solutions with concentrations of 393.396, 196.698, 78.679, 39.340, 19.670, 7.868, 3.934, 1.574 μmol / L and 299.367, 149.684, 59.873, 29.937, 14.968, 5.987, 2.994, 1.197 μmol / L.

[0103] The analysis was performed under the chromatographic conditions described in Example 5 (3). Linear regression was performed with the peak area Y of each analyte as the ordinate and the molar concentration X (μmol / L) as the abscissa. The limit of detection (LOD) and limit of quantitation (LOQ) of the analytical method were determined with the concentrations having a signal-to-noise ratio ≥3 and ≥10, respectively.

[0104] Figure 4 The linear regression analysis diagram of tetrandrine provided in this embodiment;

[0105] Figure 5The linear regression analysis diagram of tetrandrine provided in this embodiment;

[0106] Tetrandrine and tetrandrine showed good linearity within their respective linear ranges, with a correlation coefficient r = 1.0000. The LOD of tetrandrine was 1.078 μmol / L and the LOQ was 1.197 μmol / L, while the LOD of tetrandrine was 1.338 μmol / L and the LOQ was 1.416 μmol / L (Table 4).

[0107] Table 4. Linear range, linear equation, correlation coefficient, limit of detection and limit of quantitation for tetrandrine and tetrandrine.

[0108] Table 4:

[0109]

[0110] (II) Precision and stability:

[0111] The test solution was prepared according to the method in Example 5(1) above, and the precision of the method was evaluated by the relative standard deviation of the results of six parallel determinations on the same day. The results showed that the total precision of tetrandrine and tebufenozide was 1.9%, indicating that the method had good repeatability.

[0112] Table 5 Repeatability Experiments

[0113]

[0114] The test solution was placed at room temperature and injected for analysis at 0, 6, and 12 hours after preparation. The RSD values ​​of the peak areas of tetrandrine and tebufenozide were 1.6% and 1.7%, respectively, indicating that the test solution was stable within 12 hours.

[0115] Table 6 Solution stability experiments:

[0116]

[0117] (III) Recovery Rate:

[0118] Select a sample with known content of Stephania tetrandra for a spiking recovery test. Accurately weigh 0.25 g of the sample, add tetrandrine and tetrandrine reference standards to the sample, prepare the spiked solution of the test sample according to the method in Example 5 (1) above, set 3 concentrations, and perform 3 parallel determinations for each concentration, and calculate the recovery rate of tetrandrine and tetrandrine.

[0119] The results showed that the recovery rates of tetrandrine were 99.2%–101.8% and that of tebufenozide were 100.2%–101.9%, with average recoveries of 100.4% and 101.0%, respectively, and RSDs of 0.9% and 0.6%, respectively, indicating that the method had good accuracy (Tables 7–8).

[0120] Table 7 Accuracy Test Results (Stephania tetrandra):

[0121]

[0122] Table 8. Accuracy Test Results (Stephanorhinone):

[0123]

[0124] (iv) Durability:

[0125] The study investigated the impact of minor changes in chromatographic conditions, including mobile phase ratio, column temperature, flow rate, and column type, on the assay results. The results showed that minor changes in analytical conditions resulted in a change of no more than 3.0% in the content of the test sample solution; detailed results are shown in Table 9.

[0126] Table 9 Durability Test:

[0127]

[0128]

[0129] Example 7: Analysis of Multiple Batches of Samples

[0130] The test solution was prepared according to the method in (1) of Example 5 above. The content of 14 batches of Stephania tetrandra samples was determined according to the chromatographic conditions in (3) of Example 5 above. Two parallel determinations were performed on each batch, and the content was calculated by external standard method and isoabsorption wavelength method respectively.

[0131] The external standard method uses tetrandrine and tetrandrine as reference standards, and calculates the contents of tetrandrine and tetrandrine in the samples respectively.

[0132] The isoabsorption wavelength method was used with tetrandrine as a reference to calculate the contents of tetrandrine and tetrandrine in the samples. The results are shown in Table 10.

[0133] Table 10 Comparison of multiple batches of samples:

[0134]

[0135] By calculating the relative error (RE) between the two methods:

[0136] RE = (WEAW - WESM) / WESM * 100% (where WEAW is the content measured by the isoabsorption wavelength method, and WESM is the content measured by the external standard method);

[0137] The results of the two methods for determining the content of tetrandrine in 14 batches of Stephania tetrandra samples showed a relative error of less than 2.0%, indicating that there was no significant difference between the two methods. The isoabsorption wavelength method can be used to determine the content of the two analytes in Stephania tetrandra.

[0138] In summary, this application, through the study of the ultraviolet absorption characteristics of fangchinorin and tetrandrine, screened out their molar ultraviolet isoabsorption wavelengths, and based on this, established an analytical method for determining the content of fangchinorin and tetrandrine in Stephania tetrandra using HPLC-UV isoabsorption wavelength method. This truly achieves the simultaneous determination of fangchinorin and tetrandrine using only one reference standard. Compared with the methods reported in the Chinese Pharmacopoeia and other literature, this method has the advantages of being rapid, simple, and environmentally friendly. The establishment of this method provides a basis for improving the quality evaluation technology of Stephania tetrandra and its related products, and also provides a reference for the development of rapid and green HPLC quantitative analysis methods for other medicinal materials.

[0139] The differences and advantages of the technical solution in this application compared with existing testing methods are shown in Table 11.

[0140] Table 11:

[0141]

[0142] As shown in the table above, previous literature reports mostly used the external standard method to determine the content of Stephania tetrandra (Table 11). Most of these methods suffer from long analysis times, high consumption of organic solvents, and high consumption of reference standards (requiring two reference standards for quantification). This method, through optimization of sample preparation and chromatographic analysis conditions, completes the determination of fangchinine and fangchinorline content in Stephania tetrandra within 10 minutes under conventional laboratory conditions (ordinary liquid chromatography system and ultrasonic device). The entire analytical process consumes only 10 mL of methanol, 1.8 mL of ethanol, and one reference standard. Compared with previously reported methods, this method significantly improves sample detection efficiency and reduces detection costs and environmental pollution.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A rapid quantitative detection method for fangchinine and fangchinorline in Stephania tetrandra, characterized in that, The detection method includes the following steps: (A) The sample of Stephania tetrandra to be tested is pretreated to obtain the solution to be tested; (B) The test solution was analyzed by HPLC-UV high performance liquid chromatography to obtain the quantitative detection results of tetrandrine and tebufenozide; The detection and analysis were performed using a single reference standard for quantification at the same absorption wavelength, and the HPLC-UV high-performance liquid chromatography detection wavelength was 224 nm. The single reference standard is tetrandrine or tebufenozide.

2. The rapid quantitative detection method for fangchiine and tetrandrine in Stephania tetrandra according to claim 1, characterized in that, Step (A) preprocessing includes: The sample of Stephania tetrandra to be tested was placed in a methanol solution containing 2% hydrochloric acid, and then extracted by ultrasonication to obtain the test solution.

3. The rapid quantitative detection method for fangchiine and tetrandrine in Stephania tetrandra according to claim 2, characterized in that, The ratio of the sample to be tested (Stephania tetrandra) to a methanol solution containing 2% hydrochloric acid is 0.5:10~50 g / ml.

4. The rapid quantitative detection method for fangchiine and tetrandrine in Stephania tetrandra according to claim 3, characterized in that, The ratio of the sample to be tested (Stephania tetrandra) to the methanol solution containing 2% hydrochloric acid was 0.5:10 g / ml.

5. The rapid quantitative detection method for fangchiine and tetrandrine in Stephania tetrandra according to claim 2, characterized in that, The parameters for ultrasonic extraction must satisfy at least one of the following: Extraction power 380 W, extraction frequency 37 kHz, extraction time 10~30 min.

6. The rapid quantitative detection method for fangchiine and tetrandrine in Stephania tetrandra according to claim 1, characterized in that, In step (B), the HPLC-UV high-performance liquid chromatography column is a core-shell column.

7. The rapid quantitative detection method for fangchiine and tetrandrine in Stephania tetrandra according to claim 6, characterized in that, In step (B), the HPLC-UV high-performance liquid chromatography column is a CORTECS T3 core-shell column.

8. The rapid quantitative detection method for fangchiine and tetrandrine in Stephania tetrandra according to claim 6, characterized in that, The column temperature of the chromatographic column is 25~35℃.

9. The rapid quantitative detection method for fangchiine and tetrandrine in Stephania tetrandra according to claim 8, characterized in that, The column temperature of the chromatographic column is 30℃.

10. The rapid quantitative detection method for fangchiine and tetrandrine in Stephania tetrandra according to claim 1, characterized in that, The elution method for the HPLC-UV high performance liquid chromatography is isocratic elution; The isocratic elution was performed using ethanol and 0.3% phosphoric acid solution as the mobile phase, with an isocratic elution time of 10 min and a flow rate of 0.8~1.2 mL / min.

11. The rapid quantitative detection method for fangchiine and tetrandrine in Stephania tetrandra according to claim 10, characterized in that, The isocratic elution was performed using ethanol and 0.3% phosphoric acid solution as the mobile phase, with an isocratic elution time of 10 min and a flow rate of 1 mL / min.

12. The rapid quantitative detection method for fangchiine and tetrandrine in Stephania tetrandra according to claim 10, characterized in that, The volume ratio of ethanol to 0.3% phosphoric acid solution is 15:85 to 21:

79.

13. The rapid quantitative detection method for fangchiine and tetrandrine in Stephania tetrandra according to claim 12, characterized in that, The volume ratio of ethanol to 0.3% phosphoric acid solution is 18:

82.

14. The rapid quantitative detection method for fangchiine and tetrandrine in Stephania tetrandra according to claim 1, characterized in that, The detection and analysis methods include: The contents of tetrandrine and fangchinorline in the test solution were determined by HPLC-UV high performance liquid chromatography at a wavelength of 224 nm; subsequently, the total amounts of tetrandrine and fangchinorline were obtained.

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