A method for simultaneously detecting aloin, aloe-emodin and indirubin in new compound aloe capsules

Abstract

The invention discloses a method for simultaneously detecting aloin, aloe-emodin and indirubin in a new compound aloe capsule, and belongs to the field of medical technology. The invention analyzes the structure of each component in the new compound aloe capsule and its physical and chemical properties, determines that the new compound aloe capsule is quality controlled by aloin, aloe-emodin and indirubin, screens out universal chromatographic conditions through a large number of experiments, and detects according to the following conditions: chromatographic column: C18ODS, 4.6×250mm; detection wavelength: 300nm; mobile phase A: 0.08% phosphoric acid aqueous solution; mobile phase B: acetonitrile; column oven: 30°C; flow rate: 0.8mL / min; gradient elution. The detection method provided by the invention can realize the effective separation of the above three components, and the method has high detection sensitivity, good stability, good reproducibility, high detection accuracy, saves manpower and time costs, can improve efficiency, and can better control the quality of the new compound aloe capsule.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a method for simultaneously detecting aloin, aloe-emodin and indirubin in a new compound aloe capsule. Background Art

[0002] New Compound Aloe Vera Capsule is a national protected Chinese medicine. It is a compound preparation made from three medicinal materials: aloe vera, indigo and amber. It has the effects of regulating the liver and kidney, clearing away heat and moistening the intestines, and calming the mind. It is used to treat habitual constipation, dry stools, or abdominal distension and abdominal pain caused by constipation for several days. Aloe vera and indigo are the main ingredients of the new compound aloe vera capsule. The main active ingredients in aloe vera are aloin and aloe emodin, and indirubin is the active ingredient in indigo.

[0003] The quality control of the existing new compound aloe capsule is relatively simple. Due to the large difference in polarity of the main ingredients in the new compound aloe capsule, the National Food and Drug Administration issued the drug standard (revised) in 2015. Thin layer chromatography was used to identify indigo, indirubin and aloe medicinal materials, and high performance liquid chromatography was used to detect the content of aloin, the main ingredient of the new compound aloe capsule. In order to simplify the detection method, some literatures proposed to control the quality of the new compound aloe capsule by measuring the content of a certain ingredient (such as indirubin or aloin) in the new compound aloe capsule. However, as a compound Chinese medicine preparation, the new compound aloe capsule is too thin to use only one ingredient for quality control and is easy to add indicator ingredients artificially, which cannot fully reflect the quality of the new compound aloe capsule. The existing methods are either cumbersome, time-consuming and labor-intensive, and different methods need to be used to detect different ingredients, or only detect a certain ingredient of the new compound aloe capsule, with poor accuracy, unable to guarantee the quality of the capsule, and there is a blind spot in quality control. Therefore, the content determination method of the new compound aloe vera capsule has room for further optimization and improvement. For Chinese patent medicines, especially compound medicines, quality control through multiple indicators is also an inevitable trend to improve the quality control standards of Chinese medicines. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a method for simultaneously detecting aloin, aloe-emodin and indirubin in a new compound aloe capsule, so as to solve the problems that the existing detection methods are cumbersome, time-consuming and labor-intensive, and the quantification of a single component cannot fully reflect the quality of the new compound aloe capsule.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention discloses a method for simultaneously detecting aloin, aloe-emodin and indirubin in a new compound aloe capsule. The detection method comprises the following steps:

[0007] S1. Preparation of reference solution

[0008] Accurately weigh aloin, aloe-emodin and indirubin reference substances that have been dried to constant weight to prepare a mixed standard stock solution, and store it at 4°C;

[0009] S2. Preparation of test solution

[0010] Remove the capsule shell of the new compound aloe vera capsule, dissolve, sonicate and extract to prepare the new compound aloe vera capsule test solution;

[0011] S3. Establishment of linear regression equation

[0012] Take the mixed standard stock solution of S1 to prepare a series of mixed reference solutions, filter through a 0.45 μm microporous membrane, and inject into the high performance liquid chromatograph in sequence, perform gradient elution, and use the high performance liquid chromatograph workstation to perform linear regression analysis on each chemical component and calculate the linear regression equation;

[0013] S4. Content determination

[0014] Take the new compound aloe vera capsule test solution of S2, inject it into the high performance liquid chromatograph, perform gradient elution, analyze the results using the high performance liquid chromatograph workstation, substitute the peak area into the linear regression equation obtained in S3, and calculate the contents of aloin, aloe-emodin and indirubin in the test solution;

[0015] Among them, in S3 and S4, the mobile phase of the high performance liquid chromatograph is 0.08% phosphoric acid aqueous solution-acetonitrile.

[0016] Preferably, 0.08% phosphoric acid aqueous solution is phase A, acetonitrile is phase B, and the gradient elution conditions are as follows:

[0017]

[0018] Preferably, the specific preparation method of S1 reference solution is: accurately weigh aloin, aloe-emodin and indirubin dried to constant weight, prepare mixed standard stock solutions with concentrations of 100 μg / mL, 118 μg / mL and 100 μg / mL respectively using methanol solution, store at 4°C, and filter through a 0.45 μm microporous membrane before injection.

[0019] Preferably, in S2, DMF is used for dissolution.

[0020] Preferably, in S2, ultrasonic extraction is followed by further water washing and re-extraction.

[0021] Preferably, in S3 and S4, the detection wavelength of the high performance liquid chromatograph is 300 nm.

[0022] Preferably, in S3 and S4, the column temperature of the high performance liquid chromatograph is 30°C.

[0023] Preferably, in S3 and S4, the gradient elution flow rate of the high performance liquid chromatograph is 0.8 mL / min.

[0024] Preferably, in S3 and S4, the chromatographic column of the high performance liquid chromatograph is Hypersil C18 ODS, 4.6×250 mm

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The invention provides a method for simultaneously detecting aloin, aloe-emodin and indirubin in a new compound aloe capsule. According to the components in the new compound aloe capsule and their physical and chemical properties, it is determined that aloin, aloe-emodin and indirubin are used to control the quality of the new compound aloe capsule, so as to solve the problem that the polarity difference of the main components of the new compound aloe capsule is large and multiple components cannot be separated at one time. The elution effects of four different elution systems, namely water-methanol, water-acetonitrile, 0.08% phosphoric acid aqueous solution-acetonitrile and 0.1% phosphoric acid aqueous solution-acetonitrile, under different gradients are compared. The result shows that when 0.08% phosphoric acid aqueous solution-acetonitrile is used as the mobile phase, the separation effect of aloin, aloe-emodin and indirubin from impurity peaks in the new compound aloe capsule is better. The invention provides a method for simultaneously qualitatively and quantitatively detecting aloin, aloe-emodin and indirubin in a new compound aloe capsule, which can simultaneously realize effective separation and content detection of the three components of aloin, aloe-emodin and indirubin, overcomes the quality control blind spot problem of detecting a single component of the new compound aloe capsule in the prior art, is easy to detect artificially added components, and overcomes the problem of needing to use different methods to detect different components in the prior art. 0.08% phosphoric acid aqueous solution-acetonitrile is used as a mobile phase, the mobile phase system is optimized, and the problem that the polarity difference of the main components is large and the elution of the three components cannot be realized simultaneously is solved. After methodological investigation, the method is simple to operate, has strong specificity, high detection sensitivity, good stability, good reproducibility, high detection accuracy, saves manpower, material and time costs, can improve detection efficiency, and can objectively, comprehensively and accurately evaluate the quality of the new compound aloe vera capsule extract, and better control the quality of the new compound aloe vera capsule, which is of great significance to the quality control, quality improvement and clinical efficacy of the new compound aloe vera capsule in the future.

[0027] Furthermore, after determining the optimal mobile phase composition, the present invention screened the best gradient elution program through a large number of experiments. The experiment found that with 0.08% phosphoric acid aqueous solution as phase A and acetonitrile as phase B, when using 0-10 min, 80%A; 10-15 min, 80%→75%A; 15-20 min, 75%→70%A; 20-25 min, 70%→62%A; 25-30 min, 62%→55%A; 30-35 min, 55%→45%A; 35-40 min, 45%→35%A; 40-50 min, 35%A, good separation of each chromatographic peak can be achieved.

[0028] Furthermore, the present invention conducts experimental comparisons on different extraction solvents (methanol, ethanol, chloroform, DMF), and the results show that it is difficult to simultaneously extract aloin, aloe-emodin and indirubin, three components with greatly different polarities, using common solvents such as methanol and water. DMF has good dissolving and extraction capabilities and can extract all three components at the same time, but the dissolving ability of DMF is too strong, and the obtained components are too many and difficult to separate all of them. Therefore, further water washing and extraction are required after extraction with DMF.

[0029] Furthermore, the present invention screened the flow rate (1 mL / min, 0.8 mL / min, 0.5 mL / min). Since the new compound aloe capsule contains many ingredients, the separation effect of aloin, aloe-emodin and indirubin from the impurity peaks is poor when the flow rate is high. If the flow rate is too low, the time required is long. Finally, a flow rate of 0.8 mL / min was selected for gradient elution. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the HPLC chromatogram of the mixed reference solution of the present invention; wherein, peak 1 is aloin, peak 2 is aloe-emodin, and peak 3 is indirubin;

[0031] Figure 2 is a standard curve diagram of aloin of the present invention;

[0032] Figure 3 is the aloe-emodin standard curve diagram of the present invention;

[0033] Figure 4 is the indirubin standard curve diagram of the present invention;

[0034] Figure 5 The HPLC chromatogram of the novel compound aloe capsule of the present invention is shown in the figure; wherein, peak 1 is aloin, peak 2 is aloe-emodin, and peak 3 is indirubin. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments 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 should fall within the scope of protection of the present invention.

[0036] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0037] The following is combined with Figure 1-5 The present invention is described in further detail:

[0038] The instruments used in the present invention are as follows:

[0039] Shimadzu-LabSolutions (Shimadzu LC-20, Japan); BSA224S 1 / 10,000 electronic balance (Sartorius (Shanghai) Trading Co., Ltd.); GL2202-1SCN 1 / 100 electronic balance (Sartorius (Shanghai) Trading Co., Ltd.); KQ-300DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0040] The reagents and materials used in the present invention are as follows:

[0041] The purity of aloin (YJ-110787), aloe-emodin (A575400), and indirubin (I521350) were all HPLC ≥ 98%; methanol (chromatographic grade); acetonitrile (chromatographic grade); phosphoric acid; New Compound Aloe Vera Capsules (Hebei Wanbang Fulin Pharmaceutical Co., Ltd., batch number: LH201917, LH201901, LH201918).

[0042] The present invention provides a method for simultaneously detecting aloin, aloe-emodin and indirubin in a new compound aloe capsule, comprising the following steps:

[0043] 1. Preparation of reference solution

[0044] Accurately weigh aloin, aloe-emodin and indirubin reference substances that have been dried to constant weight to prepare a mixed standard stock solution, and store it at 4°C;

[0045] 2. Preparation of test solution

[0046] Remove the capsule shell of the new compound aloe vera capsule, dissolve, sonicate and extract to prepare the new compound aloe vera capsule test solution;

[0047] 3. Establishment of linear regression equation

[0048] Take the mixed standard stock solution of step 1, prepare mixed reference solution of series concentration, filter through 0.45 μm microporous membrane, inject into high performance liquid chromatograph in sequence, gradient elute, use high performance liquid chromatograph workstation, take series reference concentration as abscissa X, corresponding peak area as ordinate Y, perform linear regression analysis on each chemical component and calculate linear regression equation;

[0049] 4. Content Determination

[0050] Take the new compound aloe vera capsule test solution prepared in step 2, inject it into the high performance liquid chromatograph, perform gradient elution, analyze the results using the high performance liquid chromatograph workstation, substitute the peak area into the linear regression equation of step 3, and calculate the contents of aloin, aloe-emodin and indirubin in the test solution.

[0051] The specific detection steps of a method for simultaneously detecting aloin, aloe-emodin and indirubin in a new compound aloe capsule provided by the present invention are as follows:

[0052] 1. Preparation of reference solution

[0053] Accurately weigh 0.0050 g of aloin, 0.0059 g of aloe-emodin, and 0.0050 g of indirubin that have been dried to constant weight, place them in a 50 mL volumetric flask, add methanol to the mark to prepare mixed standard stock solutions with concentrations of 100 μg / mL, 118 μg / mL, and 100 μg / mL, respectively. Store at 4 °C and filter through a 0.45 μm microporous filter before injection.

[0054] 2. Preparation of test solution

[0055] Accurately weigh 1.0000 g of the new compound aloe capsule without the capsule shell, accurately measure 25 mL of N, N-dimethylformamide (DMF) to dissolve, ultrasonic extraction for 1 h, naturally cool to room temperature, filter, take 10 mL of the filtrate, add five times the amount of water to dilute, extract with 20 mL of ethyl acetate, repeat the extraction twice, combine the upper liquid, and prepare the new compound aloe capsule test solution. Before injection, pass it through a 0.45 μm microporous filter membrane.

[0056] 3. Establishment of linear regression equation

[0057] Take the mixed standard stock solution prepared in step 1, add methanol to gradually dilute it into a series of mixed standard solutions, the concentrations of aloin obtained are as follows: 10, 20, 40, 80 and 100 μg / mL; the concentrations of aloe-emodin are as follows: 11.8, 23.6, 47.2, 94.4 and 118 μg / mL; the concentrations of indirubin are as follows: 10, 20, 40, 80 and 100 μg / mL. The prepared solution was filtered through a 0.45 μm microporous filter membrane and injected into Shimadzu-LabSolutions (Shimadzu LC-20, Japan) in turn, and gradient eluted. The liquid chromatography conditions are as follows:

[0058] Column: Hypersil C18 ODS, 4.6 × 250 mm

[0059] Detection wavelength: 300 nm

[0060] Column temperature: 30°C

[0061] Flow rate: 0.8 mL / min

[0062] Injection volume: 10 μL

[0063] Mobile phase: 0.08% phosphoric acid in water (phase A) and acetonitrile (phase B)

[0064] Gradient elution conditions are shown in Table 1:

[0065] Table 1 Gradient elution conditions

[0066]

[0067] The obtained HPLC chromatogram is as follows Figure 1 The peak areas of the three series of reference substances are shown in Table 2:

[0068] Table 2 Peak areas of three series of reference substances

[0069]

[0070] With the concentration of the series of reference substances as the abscissa X and the corresponding peak area as the ordinate Y, the linear regression equations of the three reference substances are shown in Table 3:

[0071] Table 3 Linear regression equation of reference substance

[0072]

[0073] The obtained standard curves of aloin, aloe-emodin and indirubin are shown in Figure 2-Figure 4 .

[0074] 4. Content Determination

[0075] The instruments used, the preparation methods of the reference solution, the preparation methods of the test solution, and the liquid chromatography conditions were the same as above. Five batches of the new compound aloe vera capsule test solution prepared in step 2 were injected into Shimadzu-LabSolutions (Shimadzu LC-20, Japan) for analysis. The HPLC chromatogram obtained is shown in the figure below. Figure 5 , Substitute the peak area into the linear regression equation obtained in step 3 to calculate the contents of aloin, aloe-emodin and indirubin in the test solution. The results are shown in Table 4:

[0076] Table 4 Contents of aloin, aloe-emodin and indirubin

[0077]

[0078] A method for simultaneously detecting aloin, aloe-emodin and indirubin in a new compound aloe capsule provided by the present invention was investigated by methodology:

[0079] (1) Precision

[0080] One portion of the reference solution prepared according to the above method was analyzed according to the above detection method, and parallel injection was performed 6 times with an injection volume of 10 μL. The peak area and retention time of the sample HPLC spectrum were analyzed and the RSD value was calculated. The results are shown in Table 5. The calculated RSD was less than 3%, indicating that the parallel injection precision of the equipment was good.

[0081] Table 5 Peak area and retention time of precision study

[0082]

[0083] (2) Repeatability

[0084] Six test solutions were prepared according to the above method and analyzed according to the above detection method. The injection volume was 10 μL. The peak area and retention time of the sample HPLC spectrum were analyzed and the RSD value was calculated. The results are shown in Table 6. The calculated RSD was less than 3%. The results showed that the sample chromatographic peak had good reproducibility and the method had good repeatability.

[0085] Table 6 Peak area and retention time of repeatability study

[0086]

[0087] (3) Stability

[0088] One sample solution was prepared according to the above method, and analyzed at 0, 2, 4, 8, 12 and 24 h according to the above detection method. The injection volume was 10 μL. The peak area and retention time of the sample HPLC spectrum were analyzed and the RSD value was calculated. The results are shown in Table 7. The calculated RSD was less than 3%, indicating that the chromatographic peak of the new compound aloe capsule test solution at room temperature within 24 h had almost no change and good stability.

[0089] Table 7 Peak areas and retention times of stability studies

[0090]

[0091] (4) Sample recovery rate

[0092] Accurately add the reference substance in an amount equal to that of the component to be tested, and prepare 6 test solutions according to the above method. Analyze according to the above detection method, the injection volume is 10 μL, and the peak area and retention time of the sample HPLC spectrum are analyzed and the RSD value is calculated. The results are shown in Table 8. The calculated RSD is less than 3%, among which the sample recovery rate of aloin is between 98.333% and 103.380%, and the RSD is 1.898%; the sample recovery rate of aloe-emodin is between 97.070% and 101.623%, and the RSD is 1.620%; the sample recovery rate of indirubin is between 99.702% and 104.450%, and the RSD is 1.667%, indicating that the method has good accuracy.

[0093] Table 8 Recovery rates of three components

[0094]

[0095] A system adaptability test was conducted on a method for simultaneously detecting aloin, aloe-emodin and indirubin in a new compound aloe capsule provided by the present invention:

[0096] The test solution was prepared according to the above method, and the sample was analyzed according to the above detection method. The durability of three brands of chromatographic columns, Extend C18, 4.6×250 mm, Hypersil C18 ODS, 4.6×250 mm, and Kromasil 100-5-C18, 4.6×250 mm, was investigated. The content was calculated by analyzing the peak area and retention time of the sample HPLC spectrum, and the RSD was calculated. The results are shown in Table 9. The calculated RSDs are all less than 5%. The RSDs of aloin, aloe-emodin, and indirubin are 2.597%, 1.742%, and 2.351%, respectively, indicating that the three components are well separated in different chromatographic columns, the determination results are basically consistent, and the durability is good.

[0097] Table 9 Durability test results of different brands of chromatographic columns

[0098]

[0099] Example 1

[0100] The optimization comparison of the test solution preparation method was carried out as follows.

[0101] 1. Preparation of reference solution

[0102] The preparation steps of the reference solution are the same as the above-mentioned specific detection steps.

[0103] 2. Preparation of test solution

[0104] Accurately weigh 1.0000 g of the new compound aloe vera capsule without the capsule shell, accurately measure 25 mL of DMF, methanol, ethanol, and chloroform respectively, and treat for 1 h according to the method shown in Table 10. After the treatment, naturally cool to room temperature, make up the loss with the solvent used, filter, and pass through a 0.45 μm microporous filter membrane before injection.

[0105] Table 10 Optimization of test solution preparation method

[0106]

[0107] 3. Content determination

[0108] Take the test solution prepared with different solvents in Example 1 and inject it into Shimadzu-LabSolutions (Shimadzu LC-20, Japan) for analysis. The liquid chromatography conditions are the same as the above specific detection steps. The final extraction method is selected through a comprehensive investigation of the target component content and the overall chromatogram. The results are shown in Table 11:

[0109] Table 11 Contents of aloin, aloe-emodin and indirubin in new compound aloe capsules dissolved in different solvents

[0110]

[0111] The results show that the ingredients in the new compound aloe capsule are relatively complex, and some ingredients are difficult to separate due to their similar polarity. When methanol, ethanol and chloroform are used as solvents, the non-target component peaks are high and the separation is poor, causing the overall baseline of the chromatogram to drift, and aloe-emodin has no obvious peak. Direct injection using DMF ultrasonic extraction will also have the same problem, but DMF can increase the extraction amount of aloe-emodin, so we chose to use DMF as the solvent for extraction. Considering the safety of the experiment and the ease of operation, we chose to use ultrasonic extraction.

[0112] The preparation method of the test solution is as follows: accurately weigh 1.0000 g of the new compound aloe capsule without the capsule shell, accurately measure 25 mL of DMF for ultrasonic treatment for 1 h, naturally cool to room temperature after the treatment, take 10 mL of the filtrate after re-weighting, add 5 times the amount of water to dilute, and then use ethyl acetate for extraction, 20 mL each time, and extract twice. Combine the upper extracts, filter, and pass through a 0.45 μm microporous filter membrane before injection.

[0113] Example 2

[0114] The comparison of mobile phases in different liquid chromatography conditions was carried out as follows.

[0115] 1. Preparation of reference solution

[0116] The preparation steps of the reference solution are the same as the above-mentioned specific detection steps.

[0117] 2. Preparation of test solution

[0118] The preparation steps of the test solution are the same as the above specific detection steps.

[0119] 3. Content determination

[0120] Take 5 batches of test solution and inject them into Shimadzu-LabSolutions (Shimadzu LC-20, Japan) for analysis. The HPLC conditions are as follows. Compare the elution effects of different mobile phase systems. The four mobile phase systems are as follows:

[0121] Column: Hypersil C18 ODS, 4.6 × 250 mm

[0122] Detection wavelength: 300 nm

[0123] Column temperature: 30 °C

[0124] Flow rate: 0.8 mL / min

[0125] Injection volume: 10 μL

[0126] Mobile phase: (1) 0.08% phosphoric acid aqueous solution (phase A) and acetonitrile (phase B)

[0127] (2) 0.1% phosphoric acid aqueous solution (phase A) and acetonitrile (phase B)

[0128] (3) Water (Phase A) and Acetonitrile (Phase B)

[0129] (4) Water (Phase A) and Methanol (Phase B)

[0130] The gradient elution conditions were the same as those in the above specific detection steps.

[0131] Substitute the peak area into the linear regression equation obtained in the above specific detection step 3, and calculate the average value of the content of the new compound aloe capsule under the four mobile phase conditions. The results are shown in Table 12:

[0132] Table 12 Contents of aloin, aloe-emodin and indirubin when injected with different mobile phases

[0133]

[0134] The results showed that when the mobile phase (1) was used, i.e., 0.08% phosphoric acid aqueous solution (phase A) and acetonitrile (phase B), the contents of aloin, aloe-emodin, and indirubin in the new compound aloe capsule were the highest and the overall peak elution was good. Therefore, 0.08% phosphoric acid aqueous solution (phase A) and acetonitrile (phase B) were selected as the mobile phase.

[0135] Example 3

[0136] The comparison of flow rates under different liquid chromatography conditions was carried out as follows.

[0137] 1. Preparation of reference solution

[0138] The preparation steps of the reference solution are the same as those of the above-mentioned specific detection method.

[0139] 2. Preparation of test solution

[0140] The preparation steps of the test solution are the same as those of the above-mentioned specific detection method.

[0141] 3. Content determination

[0142] Take 5 batches of the new compound aloe vera capsule test solution prepared in step 2 and inject it into Shimadzu-LabSolutions (Shimadzu LC-20, Japan) for analysis. The liquid chromatography conditions are as follows, and the samples are injected at three different flow rates:

[0143] Column: Hypersil C18 ODS, 4.6 × 250 mm

[0144] Detection wavelength: 300 nm

[0145] Column temperature: 30 °C

[0146] Flow rate: 1.0 mL / min; 0.8 mL / min; 0.5 mL / min

[0147] Injection volume: 10 μL

[0148] Mobile phase: 0.08% phosphoric acid in water (phase A) and acetonitrile (phase B)

[0149] The gradient elution conditions are the same as the above specific detection method:

[0150] Substitute the peak area into the linear regression equation obtained in step 3 of the above specific detection method, calculate the average value of the content of the new compound aloe capsule at three flow rates, and the results are shown in Table 13:

[0151] Table 13 Contents of aloin, aloe-emodin and indirubin at different flow rates

[0152]

[0153] The results showed that when the flow rate was 0.8 mL / min, the chromatographic peaks of aloin, aloe-emodin and indirubin in the new compound aloe capsules had better peak shapes, and the separation from impurities was better than that at 1 mL / min. The measured contents of the three components were higher and the required time was shorter than that at 0.5 mL / min. Therefore, 0.8 mL / min was selected as the elution flow rate.

[0154] The above experimental results show that the method provided by the present invention for simultaneously detecting aloin, aloe-emodin and indirubin in the new compound aloe capsule has high sensitivity, good stability, high precision and good repeatability, can objectively, comprehensively and accurately evaluate the quality of the new compound aloe capsule, and realize the control of multiple index components in the new compound aloe capsule. It can be used to improve the quality standard of the new compound aloe capsule, which is of great significance for ensuring clinical efficacy.

[0155] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for simultaneously detecting aloin, aloe-emodin and indirubin in a new compound aloe capsule, characterized in that: The detection method includes the following steps: S1. Preparation of reference solution Accurately weigh aloin, aloe-emodin and indirubin reference substances that have been dried to constant weight to prepare a mixed standard stock solution, and store it at 4°C; S2. Preparation of test solution Remove the capsule shell of the new compound aloe vera capsule, dissolve, ultrasonicate, wash with water and extract to prepare the new compound aloe vera capsule test solution; wherein DMF is used for dissolution; S3. Establishment of linear regression equation Take the mixed standard stock solution of S1 to prepare a series of mixed reference solutions, filter through a 0.45 μm microporous membrane, and inject into the high performance liquid chromatograph in sequence, perform gradient elution, and use the high performance liquid chromatograph workstation to perform linear regression analysis on each chemical component and calculate the linear regression equation; S4. Content determination Take the new compound aloe vera capsule test solution of S2, inject it into the high performance liquid chromatograph, perform gradient elution, analyze the results using the high performance liquid chromatograph workstation, substitute the peak area into the linear regression equation obtained in S3, and calculate the contents of aloin, aloe-emodin and indirubin in the test solution; Among them, in S3 and S4, the mobile phase of the high performance liquid chromatograph is 0.08% phosphoric acid aqueous solution-acetonitrile; 0.08% phosphoric acid aqueous solution is phase A, acetonitrile is phase B, and the gradient elution conditions are as follows: ； In S3 and S4, the gradient elution flow rate of the HPLC was 0.8 mL / min, the detection wavelength was 300 nm, and the column temperature was 30 °C.

2. The method for simultaneously detecting aloin, aloe-emodin and indirubin in a new compound aloe capsule according to claim 1, characterized in that: The specific preparation method of S1 reference solution is as follows: accurately weigh aloin, aloe-emodin and indirubin dried to constant weight, and use methanol solution to prepare mixed standard stock solutions with concentrations of 100 μg / mL, 118 μg / mL and 100 μg / mL, respectively. Store at 4°C and filter through a 0.45 μm microporous filter before injection.

3. The method for simultaneously detecting aloin, aloe-emodin and indirubin in a new compound aloe capsule according to claim 1, characterized in that: In S3 and S4, the HPLC column was Hypersil C18 ODS, 4.6 × 250 mm.

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