Test solution for detecting content of triptolide, preparation method and detection method

Abstract

The invention discloses a test solution for detecting the content of triptolide, a preparation method and a detection method, and relates to the technical field of compound content detection. The preparation method of the test solution comprises the following steps: precisely weighing an object to be detected, adding an extraction solvent for extraction, filtering, passing the filtrate through a neutral alumina column, eluting with methanol, collecting the eluate, and recovering the eluate to be dry to obtain residues; adding a first methanol solution into the residues, dissolving, fixing the volume, and uniformly shaking; the to-be-detected substance comprises the content of the artemisia selengensis wind-dispelling capsule. The preparation method disclosed by the invention is simple to operate, the same solvent is preferably selected for extraction and elution, the redundant operation of concentrating and redissolving when different solvents are used is avoided, and the toxicity risk of the solvents is greatly reduced. The test solution for detecting the content of triptolide, provided by the invention, is suitable for HPLC (High Performance Liquid Chromatography) analysis and has a good impurity removal effect; when the test solution provided by the invention is used for detecting the content of triptolide, and specific chromatographic conditions are matched, the method has the advantage of high accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of compound content detection, and in particular to a test solution for triptolide content detection, a preparation method and a detection method. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammatory synovitis. It is considered an "immortal cancer" and requires long-term use of immunosuppressive or corticosteroid (hormone) drugs, which seriously endangers the patient's physical and mental health. Modern pharmacological studies have shown that artemisinin and its derivatives have good immunosuppressive activity, which helps to rebuild immune function, and can control immunosuppression within a certain range and reduce adverse reactions. The patent with authorization announcement number CN 103446220 B - Artemisia annua and Tripterygium wilfordii drug pair combination drug, preparation method and application shows that the extract preparation after mixing Artemisia annua and Tripterygium wilfordii in proportion is less toxic to the liver and kidneys than Tripterygium wilfordii extract, and at the same time, it is more active than the commercially available Tripterygium wilfordii drug preparation in the treatment of rheumatoid arthritis, achieving the purpose of "reducing toxicity and retaining efficacy".

[0003] Haoteng Qufeng capsule (HQC) is one of the new drug development varieties of the national major science and technology special project - research on innovative drugs of artemisinin and its derivatives. It is also a major scientific research product created by the patent inventor based on the patent (CN103446220 B). The HQC prescription is mainly composed of extracts such as Artemisia annua and Tripterygium wilfordii and auxiliary materials; it has the effects of dispelling wind and dampness, relaxing muscles and activating collaterals, and can reduce joint inflammation reactions. It is suitable for RA patients. The project team analyzed the components of HQC by UPLC-Q-TOF / MS method and showed that HQC mainly contains diterpene lactones (triptolide, triptolide, triptolide A, etc.), alkaloids (triptolide, triptolide, triptolide, etc.), flavonoids (epicatechin) and sesquiterpene lactones (artemisinin) and other compounds. Among them, triptolide is the main component of Tripterygium wilfordii for treating rheumatic pain and exerting immunosuppressive effects. It is also a potential substance that causes toxic side effects. Its therapeutic window is narrow, and the efficacy concentration and toxic concentration are close. Therefore, this component is one of the main detection indicators for most Chinese medicine preparations containing Tripterygium wilfordii.

[0004] The patent with the authorization announcement number CN 114384174 B discloses a method for determining the content of triptolide in a pharmaceutical preparation, including a sample pretreatment method for Kunxian capsules containing the triptolide preparation, which eliminates the interference of large polar components and small polar components in the preparation by extraction. However, the sample prepared by this method has a low concentration and needs to be detected with an ultra-high performance liquid phase tandem mass spectrometer (UPLC-MS / MS). Currently, the popularity of UPLC-MS / MS is far lower than that of high performance liquid phase (HPLC), and the detection cost of the former is relatively high. In addition, the multiple transfers of the organic phase (ethyl acetate) during the preparation of the test solution are likely to cause the loss of triptolide, resulting in an overall low test result (the sample recovery rate is 89.55%). Although microwave-assisted ultrasonic extraction has a higher extraction efficiency, the sample solution is also rich in a large number of interfering components. For example, the patent application publication number CN109239213A discloses a method for microwave-assisted extraction / HPLC detection of triptolide in Tripterygium wilfordii leaves. In the HPLC spectrum of the sample solution provided, other interfering peaks are large, while the peak of triptolide is extremely small. Therefore, most of the sample solutions containing Tripterygium wilfordii preparations used for HPLC analysis need to be pre-treated. Among them, neutral alumina column is currently a more common method for removing impurities from Tripterygium wilfordii medicinal materials and their preparations. However, the operation methods described in existing reports are either too complicated (for example, the patent application publication number CN 109239213A discloses a method for detecting triptolide in a preparation of Torch Flower Root, which requires 10 steps of "ultrasonic extraction-filtration-washing the filter residue twice-combining the filtrate and concentrating-methanol re-dissolving-column loading-acetone elution-eluate concentration-methanol re-dissolving-fixing the volume" when preparing the test solution), or the solvents used are highly toxic (for example, the patent application publication number CN 109239213A discloses a method for detecting triptolide in a Torch Flower Root preparation, which requires 10 steps of "ultrasonic extraction-filtration-washing the filter residue twice-combining the filtrate and concentrating-methanol re-dissolving-column loading-acetone elution-eluate concentration-methanol re-dissolving-fixing the volume" when preparing the test solution). 101339168A The method for determining the content of triptolide in Tripterygium wilfordii medicinal materials was extracted with chloroform; "Ge Dandan, Gao Jiarong, Bao Xuemei, et al. UHPLC determination of the content of triptolide and erythrin in Tripterygium wilfordii slices from different origins [J]. Guangzhou Chemical Industry, 2020, 48(16): 91-93." The extraction was carried out with acetonitrile solution and elution was carried out with dichloromethane solution; "Li Yunge, Qiao Yina, Zhang Li, et al. HPLC determination of the content of triptolide in different parts of Tripterygium wilfordii under different intercropping modes [J]. Anhui Agricultural Sciences, 2019, 47(23): 219-221. "The solution used was chloroform for extraction and ether for elution), or the solvents used in the whole process were complex and diverse (such as "Gao Li, Nie Zhongbiao, Yue Xiaohua, et al. Determination of the content of triptolide in Tripterygium wilfordii medicinal materials from different origins [J]. Shanxi Traditional Chinese Medicine, 2012, 28(08): 42-44. "First extract with 95% ethanol, concentrate on the column and then elute with ethyl acetate, and then concentrate and extract with 95% ethanol again).The patented method for the simultaneous determination of triptolide and triptolide in Tripterygium wilfordii medicinal materials or preparations with authorization announcement number CN 103336074 B established a new microemulsion capillary electrokinetic chromatography method. The test solution preparation method described in this method still requires concentration and redissolution before passing through a neutral alumina column, and as a drug detection method, the HPLC method is more applicable.

[0005] By rationally combining with Artemisia annua, the dosage of triptolide in HQC prescription is reduced, and the content of triptolide is kept in a relatively safe range while ensuring the efficacy, but this also increases the difficulty of detecting the content of triptolide in the prescription. Therefore, a method for determining the content of triptolide in HQC that is simple to operate, highly accurate and easy to popularize is urgently needed. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a test solution, a preparation method and a detection method for detecting the content of triptolide, so as to detect the content of triptolide in Haoteng Qufeng Capsules.

[0007] In order to solve the above problems, the present invention proposes the following technical solutions:

[0008] In the first aspect, the present invention provides a method for preparing a test solution for the detection of triptolide content, comprising accurately weighing the test object, adding an extraction solvent for extraction, filtering, passing the filtrate through a neutral alumina column, eluting with methanol, collecting the eluate and recovering it to dryness to obtain a residue; adding a first methanol solution to the residue to dissolve the fixed volume, and shaking well to obtain the result; the test object includes the contents of Artemisia Teng Qufeng Capsule.

[0009] A further technical solution is that the obtained test solution is filtered through a 0.2-0.5 μm filter membrane before use.

[0010] A further technical solution is that the extraction solvent is selected from at least one of methanol, anhydrous ethanol and ethyl acetate.

[0011] A further technical solution is that the ratio of the mass M of the content of the Haoteng Qufeng Capsule to the amount V of the extraction solvent is 1:25-60.

[0012] A further technical solution is that the extraction includes one or more combinations of ultrasonic extraction and heating reflux extraction.

[0013] A further technical solution is that the extraction is ultrasonic extraction.

[0014] A further technical solution is that the ultrasonic extraction time is 20-60 minutes.

[0015] A further technical solution is that the specification of the neutral alumina column is 200-300 mesh, the filling amount is 10-30g, and the column diameter is 1.0-2.0cm.

[0016] A further technical solution is that in the step of passing the filtrate through a neutral alumina column and eluting with methanol, the volume of methanol elution is 1-3 times the filling amount.

[0017] A further technical solution is that in the first methanol solution, the volume fraction of methanol is 30-90%.

[0018] In a second aspect, the present invention provides a test solution for detecting the content of triptolide, which is prepared by the preparation method described in the first aspect.

[0019] The present invention also provides application of the test solution for detecting the content of triptolide in an HPLC detection method.

[0020] In a third aspect, the present invention provides a method for detecting the content of triptolide in Haoteng Qufeng Capsule, comprising the following steps:

[0021] Prepare triptolide reference solution: add triptolide to methanol and dissolve and mix to obtain a triptolide reference solution of a preset concentration;

[0022] Preparing a test solution: the test solution is the test solution for detecting the content of triptolide described in the second aspect, or is prepared by the preparation method described in the first aspect;

[0023] Chromatographic conditions are: Chromatographic column: Welch AQ-C18 (250×4.6mm, 5μm), mobile phase: methanol-water (volume ratio 30:70); isocratic elution; detection wavelength: 200-400nm; column temperature: 28-32℃; flow rate: 0.8-1.2mL / min, injection volume 15-25μL.

[0024] Compared with the prior art, the technical effects achieved by the present invention include:

[0025] The method for preparing a test solution for the detection of the content of triptolide provided by the present invention only requires "extraction-filtration-column loading-elution-concentration-redissolution-volume setting" when preparing the test solution, eliminating the steps of washing, concentration, and re-dissolution before column loading in the existing method, significantly shortening the operation steps, and by further optimizing the types of extraction solvents, elution solvents, etc., preferably using the same solvent for extraction and elution, avoiding the tedious operations of concentration and re-dissolution when using different solvents, thereby greatly reducing the risk of solvent toxicity.

[0026] The test solution for detecting the content of triptolide provided by the present invention is suitable for HPLC analysis, has good impurity removal effect (no interference peaks around the chromatographic peak of triptolide), and the method is simple and easy to promote; the test solution provided by the present invention is used to detect the content of triptolide, and in combination with specific chromatographic conditions, it has the advantage of high accuracy.

[0027] The method for detecting the content of triptolide in the Haoteng Qufeng capsule provided by the present invention is to perform detection in combination with the above-mentioned test solution under specific chromatographic conditions. It has been verified that the sample recovery rate can reach 95.63%, with high accuracy and strong durability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The HPLC graph (220 nm) and diode array scanning 3D graph of the test solution of Example 1;

[0029] Figure 2 HPLC chart (220 nm) and diode array scanning 3D chart of the reference solution of Example 1;

[0030] Figure 3 HPLC diagram of the reference substance solution of Example 2;

[0031] Figure 4 HPLC chart of the test solution extracted with methanol in Example 2;

[0032] Figure 5 HPLC chart of the test solution extracted with ethanol in Example 2;

[0033] Figure 6 HPLC diagram of the test solution extracted with ethyl acetate in Example 2;

[0034] Figure 7 HPLC diagram of the reference substance solution of Example 3;

[0035] Figure 8 HPLC chart of the test solution reconstituted with 30% methanol solution in Example 3;

[0036] Fig. 9 HPLC chart of the test solution reconstituted with 50% methanol solution in Example 3;

[0037] Fig.10 HPLC chart of the test solution reconstituted with 70% methanol solution in Example 3;

[0038] Fig.11 HPLC chart of the test solution reconstituted with 90% methanol solution in Example 3;

[0039] Fig.12This is the HPLC chart of methanol solvent in Experiment 1;

[0040] Fig.13 This is the HPLC chart of the negative (no medicinal material) of Haoteng Qufeng Capsule in Experiment 1;

[0041] Fig.14 This is the HPLC chart of the triptolide reference solution in Experiment 1;

[0042] Fig.15 This is the HPLC chart of the test solution in Experiment 1;

[0043] Fig.16 is the regression equation of triptolide in Experiment 3;

[0044] Fig.17 The present invention provides a flow chart for preparing a test solution for detecting the content of triptolide. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments. Obviously, the embodiments described below are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0047] It should also be understood that the terms used in this description of the embodiments of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present invention. As used in the description of the embodiments of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.

[0048] Example 1

[0049] See also Fig.17 , which is a flow chart for preparing a test solution for the detection of triptolide content provided by the present invention. This embodiment provides a test solution for the detection of triptolide content, the preparation method of which is as follows Fig.17 The specific introduction is as follows:

[0050] Take 50 capsules of Haoteng Qufeng Capsules, mix the contents, take out about 3.5g, accurately weigh, put in a stoppered conical flask, accurately add 100mL of pure methanol, weigh the weight, ultrasonically treat (power 300W, frequency 40kHz) for 1h, cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 50mL of the filtrate, put it on a neutral alumina column (200-300 mesh, 20g, column diameter 1.5cm), elute with 40mL of methanol, collect the eluate, evaporate the eluate at 70℃ to obtain a residue; dissolve the residue in pure methanol and transfer it to a 10mL volumetric flask, add pure methanol to the scale, shake well, and filter through a 0.45μm filter membrane before use.

[0051] This embodiment also provides a method for detecting the content of triptolide in Haoteng Qufeng Capsule, comprising the following steps:

[0052] Preparation of triptolide reference solution: Take about 1.0 mg of triptolide reference, accurately weigh, place in a 100 mL volumetric flask, add methanol to dissolve and dilute to the scale, shake well to obtain 10 μg / mL triptolide reference solution.

[0053] The chromatographic conditions of this embodiment are:

[0054] Chromatographic column:Welch AQ-C18 (250×4.6mm, 5μm), mobile phase: methanol-water, volume ratio 30:70, isocratic elution; detection wavelength: 220nm; column temperature: 30℃; flow rate: 1mL / min, injection volume 20μL.

[0055] The test solution and reference solution of this example were used to detect the content of triptolide in Haoteng Qufeng Capsule. The results are shown in Figure 1 and Figure 2 .

[0056] Through experiments, it was found that when scanning in the wavelength range of 200-400nm, triptolide had a strong absorption at 220nm, so 220nm was finally selected as the detection wavelength.

[0057] When examining the types and ratios of mobile phases, methanol-water (volume ratio 30:70) was ultimately chosen as the mobile phase with isocratic elution.

[0058] Example 2 Investigating the effect of different extraction solvents on the test results

[0059] This embodiment provides test solutions for the detection of triptolide content obtained by using different extraction solvents, and the specific preparation method is as follows:

[0060] Take 150 capsules of Haoteng Qufeng Capsules, weigh 3 portions of the contents, each portion is about 3.5g, accurately weigh, put in a 150mL volumetric flask, accurately add 100mL of methanol, anhydrous ethanol, and ethyl acetate respectively, weigh the weight, ultrasonically treat (power 250W, frequency 100kHz) for 1h, take out, cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 50mL of the filtrate, put it on an alumina column (200-300 mesh, 20g, column diameter 1.5cm), elute with 40mL of methanol, collect the eluate, and evaporate to dryness (70℃); add an appropriate amount of pure methanol to dissolve the residue, transfer to a 10mL volumetric flask, add methanol to the scale, shake well, and filter through 0.45μm.

[0061] The test solutions for the detection of triptolide content obtained by the above three extraction solvents were respectively injected into the high performance liquid chromatograph, and the reference solution was consistent with the reference solution provided in Example 1. The chromatographic conditions of Example 1 were used for detection, and the triptolide content of different extraction solvents was calculated. The results are shown in Table 1. Figure 3-6 .

[0062] Table 1 Detection results of triptolide content in different extraction solvents

[0063]

[0064] Note: “-” means not detected.

[0065] It can be seen from the results that methanol is the best choice as the extraction solvent.

[0066] Example 3 Investigating the effect of the test solution obtained by re-dissolving the first methanol solution with different methanol contents on the test results

[0067] This embodiment provides a test solution for the detection of triptolide content obtained by re-dissolving the first methanol solution with different methanol contents, and the specific preparation method is as follows:

[0068] Take 300 capsules of Haoteng Qufeng Capsules, weigh 8 portions of the contents, each portion is about 3.5g, accurately weigh, place in a 150mL volumetric flask, accurately add 100mL of methanol, weigh the weight, ultrasonically treat (power 250W, frequency 100kHz) for 1h, take out, cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 50mL of the filtrate, put on an alumina column (200-300 mesh, 20g, column diameter 1.5cm), elute with 40mL of methanol, collect the eluate, and evaporate to dryness (70℃); add appropriate amounts of methanol solution of different volume fractions (30%, 50%, 70%, 90%) to the residue to dissolve it, transfer it to a 10mL volumetric flask, add methanol of corresponding concentration to the scale, shake well, filter, and filter at 0.45μm.

[0069] The test solutions for the detection of triptolide content obtained by re-dissolving the above-mentioned methanol solutions with different volume fractions were respectively injected into the high performance liquid chromatograph. The reference solution was consistent with the reference solution provided in Example 1. The chromatographic conditions of Example 1 were used for detection, and the triptolide content of each test solution was calculated (average). The results are shown in Table 2. Figure 7-11 .

[0070] Table 2 Test results of the test solution reconstituted with methanol solution of different volume fractions

[0071]

[0072] According to the results, the selection of 50% methanol solution is slightly better than other proportions of solvents, and no white needle-shaped crystals will be left on the bottle cap after injection. This ratio is close to the mobile phase ratio, which can avoid the appearance of starch needle crystals when the sample solution meets the mobile phase after injection, and avoid the abnormal increase of column pressure. At the same time, 50% methanol solution has good solubility for triptolide.

[0073] Example 4 Investigating the effects of test solutions obtained by different extraction methods on test results

[0074] This embodiment provides a test solution for the detection of triptolide content obtained by ultrasonic extraction, heating reflux extraction, and ultrasonic extraction and heating reflux extraction. The specific preparation method is as follows:

[0075] Take 200 capsules of Haoteng Qufeng capsules, weigh 6 portions of the contents, each about 3.5g, accurately weigh, put in a 150mL volumetric flask, accurately add 100mL of methanol, weigh the weight, and extract by ultrasonic treatment (power 300W, frequency 40kHz) for 1h, heating (70℃) reflux for 1h, ultrasonic treatment (power 300W, frequency 40kHz) for 30min + heating (70℃) reflux for 30min, respectively. After the extraction, take Take out, let cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 50 mL of the filtrate, put it on an alumina column (200-300 mesh, 20 g, column diameter 1.5 cm), elute with 40 mL of methanol, collect the eluate, and evaporate to dryness (70°C); add an appropriate amount of 50% by volume methanol solution to the residue to dissolve it, transfer it to a 10 mL volumetric flask, add 50% methanol to the scale, shake well, filter, and filter through 0.45 μm.

[0076] The test solutions for the detection of triptolide content obtained by the above-mentioned different extraction methods were respectively injected into the high performance liquid chromatograph, and the reference solution was consistent with the reference solution provided in Example 1. The chromatographic conditions of Example 1 were used for detection, and the triptolide content of each test solution was calculated (average). The results are shown in Table 3.

[0077] Table 3 Test results of test solution obtained by different extraction methods

[0078]

[0079]

[0080] According to the results in Table 3, the content of triptolide obtained by the heating reflux method is the highest, but the differences among the three methods are not large. Considering that ultrasonic extraction is more convenient, ultrasonic extraction is selected as the extraction method of the test solution in the following examples.

[0081] Example 5 Investigating the effect of different ultrasonic extraction times on the test results

[0082] This embodiment provides a test solution for the detection of triptolide content obtained by ultrasonic extraction for 20 min, 40 min, and 60 min, respectively. The specific preparation method is as follows:

[0083] Take 200 capsules of Haoteng Qufeng Capsules, weigh 6 portions of the contents, each portion is about 3.5g, accurately weigh, put in a 150mL volumetric flask, accurately add 100mL of methanol, weigh the weight, ultrasonically treat (power 300W, frequency 40kHz) for 20, 40, and 60min respectively, take out, cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 50mL of the filtrate, put on an alumina column (200-300 mesh, 20g, column diameter 1.5cm), elute with 40mL of methanol, collect the eluate, and evaporate to dryness (70℃); add an appropriate amount of 50% methanol to the residue to dissolve it, transfer it to a 10mL volumetric flask, add 50% methanol to the scale, shake well, filter, and filter through 0.45μm.

[0084] The test solutions for the detection of triptolide content obtained at the above different extraction times were respectively injected into the high performance liquid chromatograph, the reference solution was consistent with the reference solution provided in Example 1, and the chromatographic conditions of Example 1 were used for detection, and the triptolide content of each test solution was calculated (average), and the results are shown in Table 4.

[0085] Table 4 Test results of test solution obtained at different extraction times

[0086]

[0087] According to the results, ultrasonic extraction for 60 min is more effective for the extraction of triptolide.

[0088] Example 6 Investigating the effect of the amount of extraction solvent on the test results of the obtained test solution

[0089] This embodiment provides a test solution for the detection of triptolide content obtained by extraction with different amounts of methanol, and the specific preparation method is as follows:

[0090] Take 200 capsules of Haoteng Qufeng Capsules, weigh 6 portions of the contents, each portion is about 3.5g, accurately weigh, put into a 150mL volumetric flask, accurately add 100, 150, and 200mL of methanol respectively, weigh the weight, ultrasonically treat (power 300W, frequency 40kHz) for 1h, take out, cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 50, 75, and 100mL of the filtrate respectively, put on an alumina column (200-300 mesh, 20g, column diameter 1.5cm), elute with 40mL of methanol, collect the eluate, and evaporate to dryness (70℃); add an appropriate amount of 50% methanol solution to the residue to dissolve it, transfer it to a 10mL volumetric flask, add 50% methanol solution to the scale, shake well, filter, and filter through 0.45μm.

[0091] The above-mentioned test solutions for the detection of triptolide content were respectively injected into the high performance liquid chromatograph, and the reference solution was consistent with the reference solution provided in Example 1. The chromatographic conditions of Example 1 were used for detection, and the triptolide content of each test solution was calculated (average). The results are shown in Table 5.

[0092] Table 5 Test results of different methanol extraction dosages

[0093]

[0094] According to the results, there is little difference between the groups. In order to facilitate measurement and save solvent, the amount of extraction solvent used in the following examples is set to 100 mL.

[0095] Example 7 Investigating the influence of the filling amount of the alumina column on the test results during the preparation of the test solution

[0096] This example provides a test solution for the detection of triptolide content prepared by using alumina columns with different filling amounts. The specific preparation method is as follows:

[0097] Take 200 capsules of Haoteng Qufeng Capsules, weigh 6 portions of the contents, each portion is about 3.5g, accurately weigh, put in a 150mL volumetric flask, accurately add 100mL of methanol, weigh the weight, ultrasonically treat (power 300W, frequency 40kHz) for 1h, take out, cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 50mL of the filtrate, respectively, put it on alumina columns (200-300 mesh, column diameter 1.5cm) with different filling amounts (10, 20, 30g), elute with 40mL of methanol, collect the eluate, and evaporate to dryness (70℃); add appropriate amount of 50% methanol to dissolve the residue, transfer to a 10mL volumetric flask, add 50% methanol to the scale, shake well, filter, and filter at 0.45μm.

[0098] The test solutions obtained above for the detection of triptolide content were respectively injected into the high performance liquid chromatograph, and the reference solution was consistent with the reference solution provided in Example 1. The chromatographic conditions of Example 1 were used for detection, and the triptolide content of different extraction solvents was calculated. The results are shown in Table 6.

[0099] Table 6 Test results of different alumina dosages

[0100]

[0101] Filling the column with 10-30g alumina has a good effect on removing the interference peaks around triptolide, and there is no significant difference in the measured triptolide content between the experimental groups. When filling the column with 10g alumina, the filling part is shorter, when filling the column with 30g, the filling part is longer, and when filling the column with 20g, it is about one-fourth of the column length, which is more appropriate, so the following examples select 20g alumina as the preferred filling amount.

[0102] Example 8 Investigating the effect of the amount of eluent used in the process of preparing the test solution on the test results

[0103] This example provides a test solution for the detection of triptolide content prepared by using different amounts of eluent, and the specific preparation method is as follows:

[0104] Take 200 capsules of Haoteng Qufeng Capsules, weigh 6 portions of the contents, each portion is about 3.5g, accurately weigh, put in a 150mL volumetric flask, accurately add 100mL of methanol, weigh the weight, ultrasonically treat (power 300W, frequency 40kHz) for 1h, take out, cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 50mL of the filtrate, put it on an alumina column (200-300 mesh, 20g, column diameter 1.5cm), elute with 60, 40, and 20mL of methanol respectively, collect the eluate, and evaporate to dryness (70℃); add an appropriate amount of 50% methanol to dissolve the residue, transfer to a 10mL volumetric flask, add 50% methanol to the scale, shake well, filter, and filter through 0.45μm.

[0105] The test solutions for the detection of triptolide content obtained above were respectively injected into the high performance liquid chromatograph, and the reference solution was consistent with the reference solution provided in Example 1. The detection was carried out according to the chromatographic conditions of Example 1, and the triptolide content with different eluent dosages was calculated. The results are shown in Table 7.

[0106] Table 7 Test results of different eluent dosages

[0107]

[0108] According to the calculation results, there is no significant difference in the content of triptolide between the experimental groups. 40 mL of methanol, which is twice the volume of the column (20 g of alumina), was selected for elution.

[0109] According to the above experimental group, it can be known that the best preparation method of the test solution for the detection of triptolide content provided by the present invention is as follows:

[0110] Prepare the test solution: take 50 capsules of Haoteng Qufeng Capsules, mix the contents, take about 3.5g, accurately weigh, put in a stoppered conical flask, accurately add 100mL of pure methanol, weigh the weight, ultrasonically treat (power 300W, frequency 40kHz) for 1h, cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 50mL of the filtrate, put it on a neutral alumina column (200-300 mesh, 20g, column diameter 1.5cm), elute with 40mL of methanol, collect the eluate, and evaporate to dryness (70℃); dissolve the residue in 50% methanol solution by volume and transfer it to a 10mL volumetric flask, add 50% methanol solution by volume to the scale, shake well, and filter through a 0.45μm filter membrane before use.

[0111] Methodology Experiment

[0112] The method for detecting the content of triptolide in the Haoteng Qufeng Capsule of the present invention was methodologically investigated using the test solution obtained by the above-mentioned optimal preparation method.

[0113] The preparation of the triptolide reference solution is the same as the reference solution provided in Example 1.

[0114] The chromatographic conditions in the methodological experiment were the same as those in Example 1.

[0115] The negative sample of Artemisia teng qufeng capsule (without torch flower extract) was provided by Chongqing Kerui Nanhai Pharmaceutical Co., Ltd. The torch flower extract in the prescription was replaced by soluble starch of corresponding mass, and the other production processes were consistent with the normal production of Artemisia teng qufeng capsule. The preparation method of the negative sample solution of Artemisia teng qufeng capsule was the same as the preparation method of the "test solution of Artemisia teng qufeng capsule".

[0116] Experiment 1: Investigation of specificity and system adaptability

[0117] The methanol solvent, the negative sample solution of Artemisia tengqufeng capsule (without the extract of the root of huoba flower), the reference solution of triptolide and the test solution of Artemisia tengqufeng capsule were tested according to the chromatographic conditions of Example 1. Figure 12-15 .

[0118] The results show that the solvent has no interference on the sample peak, the retention time of triptolide is 46.5min, the separation degree of triptolide and its adjacent chromatographic peaks in the test solution is greater than 1.5, and the theoretical plate number is above 9700 based on the triptolide chromatographic peak. This shows that the method has good specificity and strong system adaptability.

[0119] Experiment 2: Precision test

[0120] The triptolide reference solution provided in Example 1 was injected 6 times continuously according to the chromatographic conditions of Example 1, the peak area of ​​triptolide was recorded, and the RSD was calculated. The results are shown in Table 8. This shows that the method has good precision.

[0121] Table 8 Precision test results

[0122]

[0123] Experiment 3 Linearity and detection range

[0124] Accurately weigh 4.9505 mg of triptolide reference substance, place it in a 100 mL volumetric flask, add methanol to dissolve and dilute to the mark, shake well to obtain 49.505 μg / mL triptolide reference substance solution as a stock solution (Solution No. 1). Accurately pipette 8, 6, 4, 2, 1, and 0.5 mL of Solution No. 1 into 10 mL volumetric flasks, add methanol to the mark, shake well to obtain Triptolide Reference Substance Solutions No. 2, 3, 4, 5, 6, and 7 with concentrations of 39.604, 29.703, 19.802, 9.901, 4.950, and 2.475 μg / mL, respectively.

[0125] The above-mentioned reference solution No. 1 to No. 7 was tested according to the chromatographic conditions of Example 1, and the peak area was calculated. Linear regression was performed with the peak area (Y) of triptolide as the ordinate and the concentration (X, μg / mL) as the abscissa. The results are shown in Fig.16 The results showed that triptolide had a good linear relationship in the range of 2.475-49.505 μg / mL, and the standard curve was Y=38791X-9268.4, R 2 =1.

[0126] Experiment 4: Limit of Detection and Limit of Quantitation

[0127] The No. 7 concentration reference solution in Experiment 3 was taken, continuously diluted and sampled for detection. The detection limit was investigated when the signal-to-noise ratio was 3, and the quantification limit was investigated when the signal-to-noise ratio was 10. The results are shown in Table 9.

[0128] Table 9 Results of detection limit and quantification limit

[0129]

[0130]

[0131] Experiment 5: Repeatability test

[0132] Six portions of the same batch of Haoteng Qufeng Capsules were taken, and the test solution was prepared according to the optimal preparation method. The test was carried out according to the chromatographic conditions of Example 1, the peak area of ​​triptolide was recorded, and the RSD was calculated. The results are shown in Table 10. As can be seen from the results, the detection method of the present invention complies with the provisions of Part IV (General Rule 9101) of the 2020 edition of the Chinese Pharmacopoeia that the repeatability RSD is <6 when the content is at the level of 10 μg / g, indicating that the method has good repeatability.

[0133] Table 10 Repeatability test results

[0134]

[0135] Experiment 6: Stability test

[0136] The test solution of Haoteng Qufeng Capsule was prepared according to the best preparation method, and the triptolide reference solution was prepared according to the method of Example 1. The samples were injected at 0, 4, 8, 12, 16, 20, 24h, 3d, and 5d according to the chromatographic conditions of Example 1, and the peak area of ​​triptolide was recorded and the RSD was calculated. The results are shown in Tables 11-12. It shows that the triptolide reference and samples are stable within 5 days.

[0137] Table 11 24h stability test results

[0138]

[0139] Table 12 Stability 5d test results

[0140]

[0141] Experiment 7: Sample recovery test

[0142] Take 6 portions of Haoteng Qufeng Capsules, each portion is about 0.85g, accurately weighed, and triptolide reference solution is added in a ratio of 1:1 to the triptolide content in the sample, and methanol is accurately added to make the total volume of the liquid 100mL, weighed, and ultrasonically treated (power 300W, frequency 40kHz) for 1h, taken out, cooled, and weighed again, and the lost weight was supplemented with methanol. Shake well, filter, accurately measure 50mL of the filtrate, put it on an alumina column (200-300 mesh, 20g filling column, inner diameter 1.5cm), and elute with 40mL of methanol. The eluate was collected and evaporated to dryness (70℃), and an appropriate amount of 50% methanol was added to the residue to dissolve it, transferred to a 10mL volumetric flask, 50% methanol was added to the scale, shaken well, and filtered through 0.45μm.

[0143] The filtrate was injected into a high performance liquid chromatograph, and the chromatographic conditions of Example 1 were used for detection, and the content of triptolide was calculated, and the recovery rate of the sample was calculated. The results are shown in Table 13. The recovery rate of triptolide was 93.74% to 98.4%, which meets the range requirement (80% to 115%) of 10 μg / g level for the content of the tested component in Part IV (General Rule 9101) of the 2020 edition of the Chinese Pharmacopoeia.

[0144] Table 13 Sample recovery results

[0145]

[0146] Experiment 8 Durability Test

[0147] Take Haoteng Qufeng Capsule to prepare the test solution and the triptolide reference solution. Change the mobile phase ratio, flow rate, column temperature and chromatographic column model in the chromatographic conditions in Example 1 according to the parameters in Table 14 to obtain 8 chromatographic conditions. Inject and detect according to chromatographic conditions 0 to 8, record the peak area of ​​triptolide, calculate RSD and deviation, and the results are shown in Table 15.

[0148] Table 15 Durability test results

[0149]

[0150]

[0151] The above results indicate that the chromatographic conditions in the method for detecting the content of triptolide in the Haoteng Qufeng Capsules provided by the present invention have good durability.

[0152] In summary, after methodological investigation, the method for detecting the content of triptolide in Haoteng Qufeng Capsules provided by the present invention is feasible.

[0153] The embodiment of the present invention also provides the application of the method for detecting the content of triptolide in the Artemisia Teng Qufeng Capsule in the production process of the Artemisia Teng Qufeng Capsule. For example, the method for detecting the content of triptolide in the Artemisia Teng Qufeng Capsule provided by the present invention is used to detect the content of triptolide in the Artemisia Teng Qufeng Capsule and formulate a standard.

[0154] For example, to explore the detection results and limit setting of triptolide in different batches of Haoteng Qufeng Capsules

[0155] This example provides 4 batches of Artemisia Teng Qufeng Capsules produced in pilot test by Chongqing Kerui Nanhai Pharmaceutical Co., Ltd. and 3 batches of Artemisia Teng Qufeng Capsules produced in pilot test by the pilot workshop of Chongqing Institute of Traditional Chinese Medicine, totaling 7 batches of Artemisia Teng Qufeng Capsules samples, and the above detection method is used to detect the content of triptolide in the 7 batches of samples, and the results are shown in Table 16. The results show that the content of triptolide in the 7 batches of samples is 0.0073-0.0136 mg / capsule, and the average content is 0.0073 mg / capsule.

[0156] Table 16 Content determination results of 7 batches of pilot-scale Haoteng Qufeng capsules

[0157]

[0158] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0159] The above is a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for preparing a test solution for detecting the content of triptolide, characterized in that: Accurately weigh the object to be tested, add an extraction solvent to extract, filter, pass the filtrate through a neutral alumina column, elute with methanol, collect the eluate and recover it to dryness to obtain a residue; add the first methanol solution to the residue to dissolve it to a fixed volume, and shake well to obtain; the object to be tested includes the contents of Haoteng Qufeng Capsule.

2. The preparation method according to claim 1, characterized in that The extraction solvent is selected from at least one of methanol, anhydrous ethanol and ethyl acetate.

3. The preparation method according to claim 2, characterized in that: The ratio of the mass M of the analyte to the volume V of the extraction solvent is 1:25-60.

4. The preparation method according to claim 1, characterized in that: The extraction comprises one or more combinations of ultrasonic extraction and heating reflux extraction.

5. The preparation method according to claim 1, characterized in that: The specification of the neutral alumina column is 200-300 mesh, the filling amount is 10-30g, and the column diameter is 1.0-2.0cm.

6. The preparation method according to claim 5, characterized in that: In the step of passing the filtrate through a neutral alumina column and eluting with methanol, the volume of methanol elution is 1-3 times the filling amount.

7. The preparation method according to claim 1, characterized in that: In the first methanol solution, the volume fraction of methanol is 30-90%.

8. A test solution for detecting the content of triptolide, characterized in that: Prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the test solution for detecting the content of triptolide as claimed in claim 8 in an HPLC detection method.

10. A method for detecting the content of triptolide in Haoteng Qufeng Capsule, comprising the following steps: Prepare triptolide reference solution: add triptolide to methanol and dissolve and mix to obtain a triptolide reference solution of a preset concentration; Preparation of a test solution: the test solution is the test solution for the detection of triptolide content according to claim 8, or is prepared by the preparation method according to any one of claims 1 to 7; Chromatographic conditions are: Chromatographic column: Welch AQ-C18 (250×4.6mm, 5μm), mobile phase: methanol-water, volume ratio 30:70; isocratic elution; detection wavelength: 200-400nm; column temperature: 28-32℃; flow rate: 0.8-1.2mL / min, injection volume 15-25μL.

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