Method for detecting multiple index components in compound lactation-producing granules

Abstract

The invention provides a method for detecting multiple index components in compound lactation-producing granules, and relates to the technical field of analysis and detection. According to the method, a high performance liquid chromatography analysis method is adopted for detection, and high performance liquid chromatography conditions comprise that a C18 chromatographic column is adopted, acetonitrile is used as a mobile phase A, a 0.1 v / v% phosphoric acid solution is used as a mobile phase B, gradient elution is carried out, and the detection wavelengths are 209-211 nm, 249-251 nm and 315-317 nm. The method is good in specificity, linear relation, stability, precision and accuracy, various index components can be qualitatively and quantitatively detected at the same time in one-time analysis, reference is provided for quality control of the compound yuanling granules, in addition, the method is high in detection efficiency, reagent consumables, manpower and time cost used for detection are reduced, and the method is suitable for large-scale popularization and application. Good popularization and application values are realized.

Description

Technical Field

[0001] The embodiments of the present invention relate to the technical field of analytical detection, and particularly to a method for detecting multiple index components in compound Chanruling granules. Background Art

[0002] Traditional Chinese medicine believes that there are deficiency and excess syndromes in the case of insufficient lactation in women. Deficiency syndromes are mostly due to qi and blood deficiency after childbirth, resulting in insufficient source of milk production; excess syndromes are caused by stagnation of liver qi and stagnation of milk. "Treatise on Women's Diseases" states that "for the failure of milk secretion after childbirth, it is advisable to strengthen the spleen and stomach to nourish the source of transformation". Therefore, clinically, the therapeutic principles of supplementing qi and nourishing blood, assisting with promoting lactation or soothing the liver and relieving depression, regulating qi and promoting lactation are mostly adopted. Compound Chanruling granules are composed of 9 herbs including Codonopsis pilosula, Astragalus membranaceus, Angelica sinensis, Ligusticum chuanxiong, stir-fried Vaccaria segetalis, Bupleurum chinense, Tetrapanax papyriferus, Rhaponticum uniflorum, and stir-fried Sus scrofa linnaeus, and have the effects of tonifying qi and blood, assisting in biochemistry, and promoting lactation. This formula is modified from "Tongru Dan" in "Fu Qingzhu's Treatise on Women's Diseases" and "Xiaru Yongquan Powder" in "Formulas of the Imperial Medical Office of the Qing Dynasty". In the formula, Codonopsis pilosula is sweet and flat, benefiting qi, strengthening the spleen and nourishing the lungs, and promoting the production of body fluid and blood; Astragalus membranaceus is sweet and warm, tonifying qi, strengthening yang and consolidating the exterior, arresting sweating and consolidating the collapse, and expelling pus by supporting healthy qi; the combination of the two herbs is the monarch drug for tonifying qi and blood and reinforcing the middle and consolidating the collapse. Angelica sinensis is sweet, nourishing, moistening, pungent and warm, good at nourishing blood and promoting blood circulation; Ligusticum chuanxiong is pungent and warm, good at promoting blood circulation and qi movement, to regulate qi mechanism; the two herbs work together to assist the monarch drug in warming and dispersing qi movement, nourishing blood and promoting blood circulation as the minister drug. Stir-fried Vaccaria segetalis is bitter, cathartic, flat, promoting blood circulation and dredging the channels and promoting lactation; Tetrapanax papyriferus is sweet, light, slightly cold and promoting diuresis, good at clearing heat and promoting qi and promoting lactation; Rhaponticum uniflorum is bitter and cold, clearing and discharging, both clearing heat and detoxifying and dissipating carbuncles, and promoting lactation; stir-fried Sus scrofa linnaeus is salty, softening, slightly cold, with the power of detoxifying and dissipating carbuncles and anti-inflammatory and promoting lactation; the combination of them is used as the assistant drug for dissipating stasis and carbuncles and promoting lactation. Bupleurum chinense soothes the liver and promotes the rise of yang, and promotes qi movement as the envoy drug. The combination of all herbs has the effects of tonifying qi and blood, opening orifices and relieving depression, promoting the production of body fluid and promoting lactation.

[0003] Compound Chanruling granules are brownish-yellow granules, and their preparation method is as follows: Weigh 1500 g of Codonopsis pilosula, 1500 g of Astragalus membranaceus, 1200 g of Angelica sinensis, 800 g of Ligusticum chuanxiong, 1200 g of stir-fried Vaccaria segetalis, 800 g of Bupleurum chinense, 1500 g of Tetrapanax papyriferus, 1000 g of Rhaponticum uniflorum, and 500 g of stir-fried Sus scrofa linnaeus, a total of 9 herbs, add 13 times the amount of water (v / m), heat under reflux for 90 min, repeat 3 times, combine the filtrates, concentrate the filtrates, dry, pulverize to obtain an extract powder, add 0.8 times the amount of auxiliary materials (starch: dextrin = 0.8:1.0 (m / m)), mix evenly, use 72.0% ethanol as the wetting agent, granulate by wet method, dry at 60 °C, size the granules, and pack to obtain compound Chanruling granules. Currently, an effective quality control standard has not been established. Therefore, studying a method for detecting multiple index components in compound Chanruling granules is of great significance for the quality control of compound Chanruling granules. Summary of the Invention

[0004] To this end, the embodiments of the present invention provide a method for detecting multiple index components in Compound Chanruling Granules. Compound Chanruling Granules are developed from a total of 9 Chinese herbal medicines, namely Codonopsis pilosula, Astragalus membranaceus, Angelica sinensis, Ligusticum chuanxiong, Stir-fried Vaccaria segetalis, Bupleurum chinense, Tetrapanax papyriferus, Rhaponticum uniflorum, and Stir-fried Pig's Hoof Nail. Codonopside and Calycosin are the main quality control index components of the monarch drugs Codonopsis pilosula and Astragalus membranaceus respectively. Ferulic acid is an important component of the ministerial drug Angelica sinensis, and the ministerial drug Ligusticum chuanxiong also contains ferulic acid. Saikosaponin α is the main component of the envoy drug Bupleurum chinense, and β-Ecdysterone is the index component of the adjuvant drug Rhaponticum uniflorum. Therefore, the present invention selects these five compounds, namely Codonopside, Calycosin, Ferulic acid, Saikosaponin α, and β-Ecdysterone, as the index components of Compound Chanruling Granules.

[0005] To achieve the above object, the embodiments of the present invention provide the following technical solutions:

[0006] According to the first aspect of the embodiments of the present invention, the present invention provides a method for detecting multiple index components in Compound Chanruling Granules, which is detected by high performance liquid chromatography analysis method. The high performance liquid chromatography conditions include:

[0007] Using a C 18 chromatographic column, the detection wavelengths are 209 - 211 nm, 249 - 251 nm, and 315 - 317 nm. Acetonitrile is used as mobile phase A, and 0.1 v / v% phosphoric acid solution is used as mobile phase B for gradient elution. The gradient elution program is as follows:

[0008] 0 - 10 min, mobile phase A is 8% - 18%, and mobile phase B is 92% - 82%;

[0009] 10 - 20 min, mobile phase A is 18% - 22%, and mobile phase B is 82% - 78%;

[0010] 20 - 30 min, mobile phase A is 22% - 40%, and mobile phase B is 78% - 60%;

[0011] 30 - 40 min, mobile phase A is 40% - 56%, and mobile phase B is 60% - 44%;

[0012] 40 - 45 min, mobile phase A is 56%, and mobile phase B is 44%;

[0013] 45 - 50 min, mobile phase A is 56% - 50%, and mobile phase B is 44% - 50%;

[0014] 50 - 52 min, mobile phase A is 50% - 8%, and mobile phase B is 50% - 92%;

[0015] 52 - 58 min, mobile phase A is 8%, and mobile phase B is 92%;

[0016] The multiple index components include lobetyolin, calycosin, ferulic acid, saikosaponin a and β -ecdysterone.

[0017] Further, the high performance liquid chromatography conditions also include:

[0018] Column temperature: 20 - 30 °C; Flow rate: 0.8 - 1.2 mL / min; Injection volume: 8 - 12 μL.

[0019] Further, the chromatographic column is packed with octadecylsilyl silica gel, with a specification of 4.6×250 mm, 5 μm; and / or,

[0020] Detection wavelengths: 210 nm, 250 nm and 316 nm; and / or,

[0021] Column temperature: 25 °C; Flow rate: 1.0 mL / min; Injection volume: 10 μL.

[0022] Further, Fufang Chanruling Granules are mixed with a solvent, subjected to ultrasonic treatment, shaken well, filtered to obtain a test solution; wherein, the solvent is one or a combination of two of methanol and ethanol, and the mass - volume ratio of Fufang Chanruling Granules to the solvent is 1 g: 18 - 22 mL.

[0023] Further, the conditions of the ultrasonic treatment are: 300 - 600 W, 20 - 40 min;

[0024] The filtration is carried out using a 0.22 μm filter membrane;

[0025] After the ultrasonic treatment, it also includes placing the ultrasonic system to room temperature, and the room temperature is: 20 - 30 °C.

[0026] Further, the detection method includes the following steps:

[0027] 1) Sample preparation

[0028] Preparation of the test solution: Fufang Chanruling Granules and methanol are mixed at a mass - volume ratio of 1 g: 20 ml, ultrasonically treated at 500 W for 30 min, taken out and placed at room temperature, shaken well, and filtered through a 0.22 μm filter membrane to obtain the test solution;

[0029] Preparation of the mixed reference solution: Take reference substances of lobetyolin, calycosin, ferulic acid, saikosaponin a, β -ecdysterone, accurately weigh, dissolve with methanol and make up the volume to obtain a series of mixed reference solutions with different concentrations;

[0030] 2) Detect the above - mentioned test solution and mixed reference solution respectively according to the high performance liquid chromatography conditions, compare the retention times of the two for qualitative analysis, and use the external standard method for quantitative analysis.

[0031] Furthermore, in the series of mixed reference solutions of different concentrations, the concentration range of codonopsis pilosula is 5.635-180.320 μg / mL, the concentration range of calycosin is 5.033-161.056 μg / mL, the concentration range of ferulic acid is 2.516-80.512 μg / mL, the concentration range of saikosaponin α is 2.528-80.896 μg / mL, and the concentration range of β-ecdysterone is 6.893-220.576 μg / mL.

[0032] Furthermore, the raw materials of the compound Chanruling granules include Codonopsis pilosula, Astragalus membranaceus, Angelica sinensis, Ligusticum chuanxiong, stir-fried Vaccaria segetalis, Bupleurum chinense, Smilax glabra, Rhizoma Cyperi, and roasted pig's hoof.

[0033] According to a second aspect of an embodiment of the present invention, the present invention provides use of any of the above methods in quality control of Compound Chanruling Granules.

[0034] The embodiments of the present invention have the following advantages:

[0035] The present invention adopts specific high performance liquid chromatography conditions to simultaneously perform qualitative and quantitative detection of five index components in the compound Chanruling granules, namely, codonopsis pilosula, calycosin, ferulic acid, saikosaponin α, and β-ecdysterone in one analysis. The method has good specificity, linear relationship, stability, precision and accuracy, and provides a reference for the quality control of the compound Chanruling granules. In addition, the present invention has high detection efficiency, reduces the reagent consumables, manpower and time costs used in the detection, and has good promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0037] Figure 1 HPLC chromatogram of the mixed reference substance provided by the present invention;

[0038] Figure 2 HPLC chromatogram of the negative test sample provided by the present invention;

[0039] Figure 3 HPLC chromatogram of the test sample provided by the present invention;

[0040] Figure 4 The standard curve of dangshen glycoside provided by the present invention;

[0041] Figure 5The standard curve graph of calycosin provided by the present invention;

[0042] Figure 6 The standard curve graph of ferulic acid provided by the present invention;

[0043] Figure 7 The standard curve graph of saikosaponin α provided by the present invention;

[0044] Figure 8 The standard curve graph of β-ecdysterone provided by the present invention;

[0045] Figure 9 The HPLC chromatogram of the mixed reference substances in Comparative Example 1 provided by the present invention.

[0046] Figure 10 The HPLC chromatogram of the mixed reference substances in Comparative Example 2 provided by the present invention. Specific Embodiments

[0047] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0048] Instruments and Experimental Reagents

[0049] 1. Instruments: Agilent 1260 (DAD) type high performance liquid chromatograph (Agilent Technologies, USA), LQ-C3002 type electronic balance (Shenzhen Feiya Weighing Apparatus Co., Ltd.), XSE1050DU type electronic balance (Mettler-Toledo Group Co., Ltd.), FA2004 type electronic analytical balance (Shanghai Shangping Instrument Co., Ltd.), YM-060S type Yumeng ultrasonic cleaner (Shenzhen Fangao Microelectronics Co., Ltd.), HP-01 type vacuum pump (Bonsi Instrument Technology (Shanghai) Co., Ltd.).

[0050] 2. Information of drugs and reference substances is shown in Table 1.

[0051] Table 1 Information Table of Reference Substances

[0052]

[0053] 3. Reagents: Acetonitrile is chromatographically pure (Anhui Tiandi High Purity Solvents Co., Ltd.), phosphoric acid is chromatographically pure (Tianjin Kemiou Technology Co., Ltd.), methanol is analytically pure, and water is pure water.

[0054] Example 1

[0055] This example provides a method for detecting multiple target components in Compound Chanruling Granules:

[0056] 1. Preparation of test solution

[0057] Take an appropriate amount of Compound Chanruling Granules, grind them finely, accurately weigh 2.5 g, place them in a conical flask, accurately add 50 ml of methanol, weigh, ultrasonically treat (power 500 W, frequency 40 kHz) for 30 min, take out and let it cool to room temperature, make up the lost weight with methanol, shake well, filter through a 0.22 μm filter membrane, and take the subsequent filtrate to obtain test sample No. 1.

[0058] 2. Preparation of mixed reference substance solution

[0059] Take reference substances of lobetyolin, calycosin, ferulic acid, saikosaponin a, and β-ecdysterone, accurately weigh them, dissolve them with methanol and make up the volume to prepare a series of mixed reference substance solutions with different concentrations. In a series of mixed reference substance solutions with different concentrations, the concentration range of lobetyolin is 5.635 - 180.320 μg / mL, the concentration range of calycosin is 5.033 - 161.056 μg / mL, the concentration range of ferulic acid is 2.516 - 80.512 μg / mL, the concentration range of saikosaponin a is 2.528 - 80.896 μg / mL, and the concentration range of β-ecdysterone is 6.893 - 220.576 μg / mL.

[0060] 3. High performance liquid chromatography determination

[0061] Use high performance liquid chromatography to detect test sample solution No. 1 and mixed reference substance solution respectively, and compare their retention times for qualitative analysis. Detect a series of mixed reference substance solutions with different concentrations to obtain the absorption peak areas of lobetyolin, calycosin, ferulic acid, saikosaponin a, and β-ecdysterone in the mixed reference substance solution. Take the concentration of each target component in the mixed reference substance solution as the abscissa (X-axis), and the peak area of each target component as the ordinate (Y-axis) to plot a standard curve, and obtain the standard curve regression equations of lobetyolin, calycosin, ferulic acid, saikosaponin a, and β-ecdysterone. Then detect test sample No. 1 to obtain the absorption peak areas of lobetyolin, calycosin, ferulic acid, saikosaponin a, and β-ecdysterone, and substitute them into the corresponding standard curve regression equations to calculate the concentrations of lobetyolin, calycosin, ferulic acid, saikosaponin a, and β-ecdysterone.

[0062] The detection conditions for high performance liquid chromatography are as follows: The chromatographic column is Agilent XDB-C 18Column (4.6×250 mm, 5 μm); column temperature 25 °C; detection wavelengths 210 nm, 250 nm and 316 nm; flow rate 1.0 mL / min. Mobile phase A is acetonitrile and mobile phase B is 0.1% (v / v) phosphoric acid solution. Gradient elution is performed. The gradient elution program is as follows:

[0063] 0 - 10 min, mobile phase A is 8% - 18% and mobile phase B is 92% - 82%;

[0064] 10 - 20 min, mobile phase A is 18% - 22% and mobile phase B is 82% - 78%;

[0065] 20 - 30 min, mobile phase A is 22% - 40% and mobile phase B is 78% - 60%;

[0066] 30 - 40 min, mobile phase A is 40% - 56% and mobile phase B is 60% - 44%;

[0067] 40 - 45 min, mobile phase A is 56% and mobile phase B is 44%;

[0068] 45 - 50 min, mobile phase A is 56% - 50% and mobile phase B is 44% - 50%;

[0069] 50 - 52 min, mobile phase A is 50% - 8% and mobile phase B is 50% - 92%;

[0070] 52 - 58 min, mobile phase A is 8% and mobile phase B is 92%.

[0071] Example 2

[0072] This example provides a method for detecting multiple index components in Compound Chanruling Granules:

[0073] 1. Preparation of test solution

[0074] Take an appropriate amount of Compound Chanruling Granules, grind them finely, accurately weigh 2.5 g, place them in a conical flask, accurately add 48 ml of ethanol, weigh, perform ultrasonic treatment (power 500 W, frequency 40 kHz) for 20 min, take it out and let it cool to room temperature, make up the lost weight with ethanol, shake well, filter through a 0.22 μm filter membrane, and take the subsequent filtrate to obtain test solution No. 2.

[0075] 2. Preparation of mixed reference substance solution

[0076] Take reference substances of lobetyolin, calycosin, ferulic acid, saikosaponin α, and β - ecdysterone, accurately weigh them, dissolve them with ethanol and make up the volume, and prepare a series of mixed reference substance solutions with different concentrations according to the steps in Example 1.

[0077] 3. Determination by High Performance Liquid Chromatography

[0078] The test samples No. 2 and the mixed reference solution were detected according to the method in Step 3 of Example 1. Qualitative analysis was carried out by comparing the retention times, and the contents of lobetyolin, calycosin, ferulic acid, saikosaponin α, and β-ecdysterone were calculated based on the peak areas.

[0079] The detection conditions for high performance liquid chromatography were as follows: the chromatographic column was Agilent XDB-C 18 column (4.6×250 mm, 5 μm); the column temperature was 20 °C; the detection wavelengths were 209 nm, 249 nm, and 315 nm; the flow rate was 1.0 mL / min. Mobile phase A was acetonitrile, and mobile phase B was 0.1% (v / v) phosphoric acid solution. Gradient elution was carried out according to the procedure in Step 3 of Example 1.

[0080] Example 3

[0081] This example provides a method for detecting multiple index components in Compound Chanruling Granules:

[0082] 1. Preparation of test sample solution

[0083] An appropriate amount of Compound Chanruling Granules was taken, ground finely, and 2.5 g was accurately weighed and placed in a conical flask. 55 ml of methanol was accurately added, and the weight was weighed. It was ultrasonically treated (power 500 W, frequency 40 kHz) for 40 min, taken out and allowed to cool to room temperature. Ethanol was added to make up for the lost weight, shaken well, filtered through a 0.22 μm filter membrane, and the subsequent filtrate was taken to obtain test sample No. 3.

[0084] 2. Preparation of mixed reference solution

[0085] Reference substances of lobetyolin, calycosin, ferulic acid, saikosaponin α, and β-ecdysterone were accurately weighed, dissolved in ethanol and made up to volume, and a series of mixed reference solutions with different concentrations were prepared according to the method in Step 2 of Example 1.

[0086] 3. Detection

[0087] The test sample No. 3 and the mixed reference solution were detected according to the method in Step 3 of Example 1. Qualitative analysis was carried out by comparing the retention times, and the contents of lobetyolin, calycosin, ferulic acid, saikosaponin α, and β-ecdysterone were calculated based on the peak areas.

[0088] The detection conditions for high performance liquid chromatography were as follows: the chromatographic column was Agilent XDB-C 18Column (4.6×250 mm, 5 μm); column temperature 30°C; detection wavelengths 211 nm, 251 nm, and 317 nm; flow rate 1.0 mL / min. Mobile phase A is acetonitrile, and mobile phase B is 0.1% (v / v) phosphoric acid solution. Gradient elution is carried out according to the procedure in step 3 of Example 1.

[0089] Example 4

[0090] Specificity investigation

[0091] Take the compound Chanruling granules with batch number 20231006, and prepare the test solution according to the method in step 1 of Example 1; take the compound Chanruling granules lacking Codonopsis pilosula, Angelica sinensis, Ligusticum chuanxiong, Astragalus membranaceus, Bupleurum chinense, and Rhaponticum uniflorum, and prepare the negative test solution according to the method in step 1 of Example 1; respectively take the reference substances of lobetyolin, calycosin, ferulic acid, saikosaponin α, and β-ecdysterone, accurately weigh them, dissolve them in methanol and make up the volume, then take 1 mL of each and mix and make up the volume to prepare a mixed reference substance solution. The concentration of lobetyolin in the mixed reference substance solution is 0.1 mg / mL, the concentration of calycosin is 0.02 mg / mL, the concentration of ferulic acid is 0.04 mg / mL, the concentration of saikosaponin α is 0.04 mg / mL, and the concentration of β-ecdysterone is 0.05 mg / mL.

[0092] Perform HPLC detection on the above mixed reference substance solution, negative test solution, and test solution according to the chromatographic conditions in step 3 of Example 1. The detection results are qualitatively analyzed according to the retention time. The results are shown in Figure 1 、 Figure 2 and Figure 3 , the chromatographic peaks of each component in the mixed reference substance can be clearly separated from each other, the peak shape is symmetrical. Among them, calycosin, lobetyolin, and saikosaponin α have a greater response at 210 nm, β-ecdysterone has a greater response at 250 nm, and ferulic acid has a greater response at 316 nm; there is no obvious absorption peak at the retention time position of the target substance to be detected in the negative control; the chromatogram of the test sample has good resolution, has obvious peaks at the corresponding retention time, the peaks to be detected are symmetrical and obvious, and there is no obvious tailing phenomenon. The results show that the negative sample has no interference on the test sample and the specificity is good.

[0093] Example 5

[0094] 1. Investigation of linear relationship

[0095] Accurately weigh appropriate amounts of the reference substances of lobetyolin, calycosin, ferulic acid, saikosaponin α, and β-ecdysterone. According to the method in Step 2 of Example 1, add methanol to prepare a series of mixed reference substance solutions with different concentrations. According to the chromatographic conditions in Step 3 of Example 1, accurately pipette 10 μL of the mixed reference substance solution and inject it into the ultra-high performance liquid chromatograph. Using the concentrations of each target component in the mixed reference substance solution as the abscissa and the peak areas of each target component as the ordinate, plot the standard curves of lobetyolin, calycosin, ferulic acid, saikosaponin α, and β-ecdysterone, and obtain the corresponding standard regression equations, correlation coefficients, and linear ranges. The results are shown in Table 2, and the specific curve diagram is shown in Figures 4 - 8 .

[0096] Table 2 Standard curve data of five target components

[0097]

[0098]

[0099] 2. Investigation of stability

[0100] Take the compound chanruling granules with batch number 20231006, prepare the test solution according to the method in Step 1 of Example 1, and according to the chromatographic conditions in Step 3 of Example 1, inject samples for analysis at 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h respectively to obtain the chromatographic peak areas of lobetyolin, calycosin, ferulic acid, saikosaponin α, and β-ecdysterone. The results are shown in Table 3. The results show that the RSDs of the chromatographic peak areas of lobetyolin, calycosin, ferulic acid, saikosaponin α, and β-ecdysterone within 24 h are all less than 2%, indicating that the test solution has good stability within 24 h.

[0101] Table 3 Chromatographic peak area data of each target component of the test solution from 0 to 24 h

[0102]

[0103]

[0104] 3. Investigation of precision

[0105] 3.1 Investigation of instrument precision

[0106] Weigh appropriate amounts of lobetyolin, calycosin, ferulic acid, saikosaponin α, and β-ecdysterone reference substances accurately, and prepare a mixed reference substance solution with a concentration of 0.045 mg / mL for lobetyolin, 0.05 mg / mL for calycosin, 0.025 mg / mL for ferulic acid, 0.025 mg / mL for saikosaponin α, and 0.07 mg / mL for β-ecdysterone according to the method in Step 2 of Example 1. According to the chromatographic conditions in Step 3 of Example 1, inject the sample continuously for 6 times, and the results are shown in Table 4. The results show that the RSD of the peak areas of the 5 index components for 6 consecutive injections is less than 2%, indicating good instrument precision.

[0107] Table 4 Data of Five Index Components in the Mixed Reference Substance for 6 Consecutive Injections

[0108]

[0109]

[0110] 3.2 Investigation of Repeatability

[0111] Take the compound Chanruling granules sample with batch number 20231013, and prepare 6 portions of test solution in parallel according to the method in Step 1 of Example 1. According to the chromatographic conditions in Step 3 of Example 1, determine each index component, and the results are shown in Table 5. The results show that the RSD of the contents of the 5 index components is less than 1%, all meeting the requirements of the Chinese Pharmacopoeia (2020 Edition), indicating good method repeatability.

[0112] Table 5 Determination Results of Repeatability Experiment (mg / g)

[0113]

[0114] 3.3 Investigation of Intermediate Precision

[0115] Different experimenters take the compound Chanruling granules sample of the same batch number (20231006), prepare 3 portions of test solution each according to the method in Step 1 of Example 1, and inject and analyze them on the same HPLC according to the chromatographic conditions in Step 3 of Example 1. The results are shown in Table 6. The results show that the RSD of the contents of the 5 index components in the compound Chanruling granules is less than 2%, indicating good intermediate precision.

[0116] Table 6 Results of Intermediate Precision (mg / g)

[0117]

[0118] 4. Sample Recovery

[0119] Accurately weigh 9 portions of the compound Chanruling granules samples with known concentrations (batch number 20231006), and add the mixed reference substance solutions of five target components at three levels of 50%, 100%, and 150% of the content, with 3 replicates for each level. Prepare the test solution according to the method in Step 1 of Example 1, and inject and analyze respectively according to the chromatographic conditions in Step 3 of Example 1. The results are shown in Table 7. It can be seen from Table 7 that the spike recoveries of the 5 target components all meet the recovery limits specified in the Chinese Pharmacopoeia (Volume IV) of 2020 edition, indicating that the accuracy of this determination method is good.

[0120] Table 7 Results of spike recoveries

[0121]

[0122]

[0123] 5. Investigation of different batches of chromatographic columns

[0124] Take the compound Chanruling granules samples of batch number 20231006, prepare the test solution by the method in Step 1 of Example 1, and compare the effects of 3 different batches of chromatographic columns of the same model Agilent XDB-C 18 column (4.6×250mm, 5μm) on the content changes of 5 target components in the test solution. The results are shown in Table 8. It can be seen from the results that the RSDs of the contents of the 5 target components measured by 3 different batches of chromatographic columns of the same model Agilent XDB-C 18 column (4.6×250mm, 5μm) are all less than 2%, indicating that this model of chromatographic column is suitable for the content determination of 5 target components in the samples.

[0125] Table 8 Investigation results of different batches of chromatographic columns

[0126]

[0127] Comparative Example 1

[0128] The difference between this comparative example and Example 1 is only that in the chromatographic conditions of Step (3), acetonitrile is used as mobile phase A and 0.1% (v / v) phosphoric acid solution is used as mobile phase B for isocratic elution.

[0129] Prepare the mixed reference substance solution according to the method of Example 4 and conduct HPLC detection. The detection chromatogram is shown in Figure 9 . The results show that although the components to be detected can be basically separated, the retention times of the active components are very close, the resolution is poor, and the peak width of a single chromatographic peak is large. This method is not suitable for detecting multiple target components in the more complex compound Chanruling granules.

[0130] Comparative Example 2

[0131] The difference between this comparative example and Example 1 is only that in step (3), the chromatographic conditions use acetonitrile as mobile phase A and 0.1% (v / v) phosphoric acid solution as mobile phase B for gradient elution.

[0132] The program of gradient elution is as follows:

[0133] 0 - 15 min, mobile phase A is 5% - 15%, mobile phase B is 95% - 85%;

[0134] 15 - 25 min, mobile phase A is 15% - 25%, mobile phase B is 85% - 75%;

[0135] 25 - 35 min, mobile phase A is 25% - 35%, mobile phase B is 75% - 65%;

[0136] 35 - 40 min, mobile phase A is 35% - 45%, mobile phase B is 65% - 55%;

[0137] 40 - 45 min, mobile phase A is 45% - 60%, mobile phase B is 55% - 40%;

[0138] 45 - 50 min, mobile phase A is 60% - 80%, mobile phase B is 40% - 20%;

[0139] 50 - 55 min, mobile phase A is 80% - 95%, mobile phase B is 20% - 5%;

[0140] 55 - 58 min, mobile phase A is 95% - 5%, mobile phase B is 5% - 95%.

[0141] Prepare the mixed reference substance solution according to the method of Example 4 and conduct HPLC detection. The detection chromatogram is shown in Figure 10 . The results show that: the retention times of the first 4 peaks to be measured are relatively close, and the peaks to be detected are close to other substance peaks, with poor resolution. This method is not suitable for detecting multiple target components in the more complex compound Chanruling granules.

[0142] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A method for detecting multiple index components in compound Chanruling granules, characterized in that, Detection is carried out by using the high performance liquid chromatography analysis method. The high performance liquid chromatography conditions include: Using column C 18 chromatographic column, detection wavelengths of 209 - 211 nm, 249 - 251 nm and 315 - 317 nm, acetonitrile as mobile phase A, 0.1 v / v% phosphoric acid solution as mobile phase B, gradient elution was performed, and the gradient elution program was as follows: From 0 to 10 min, mobile phase A is 8% - 18%, and mobile phase B is 92% - 82%; From 10 to 20 min, mobile phase A is 18% - 22%, and mobile phase B is 82% - 78%; From 20 to 30 min, mobile phase A is 22% - 40%, and mobile phase B is 78% - 60%; From 30 to 40 min, mobile phase A is 40% - 56%, and mobile phase B is 60% - 44%; From 40 to 45 min, mobile phase A is 56%, and mobile phase B is 44%; From 45 to 50 min, mobile phase A is 56% - 50%, and mobile phase B is 44% - 50%; From 50 to 52 min, mobile phase A is 50% - 8%, and mobile phase B is 50% - 92%; From 52 to 58 min, mobile phase A is 8%, and mobile phase B is 92%; The multiple index components include lobetyolin, calycosin, ferulic acid, saikosaponin α and β - ecdysterone.

2. The detection method for multiple index components in the compound prolactin granules according to claim 1, characterized in that The high performance liquid chromatography conditions further include: Column temperature is 20 - 30°C; flow rate is 0.8 - 1.2 mL / min; injection volume is 8 - 12 μL.

3. The detection method for multiple index components in the compound prolactin - promoting granule according to claim 1, characterized in that The chromatographic column is packed with octadecylsilyl bonded silica gel, with a specification of 4.6×250 mm, 5 μm; and / or, Detection wavelengths are 210 nm, 250 nm and 316 nm; and / or, Column temperature is 25°C; flow rate is 1.0 mL / min; injection volume is 10 μL.

4. The detection method for various index components in the compound prolactin granules according to claim 1, characterized in that, The compound prolactin - promoting granule is mixed with a solvent, ultrasonically treated, shaken well, and filtered to obtain a test solution; wherein, the solvent is one or a combination of two of methanol or ethanol, and the mass - volume ratio of the compound prolactin - promoting granule to the solvent is 1 g:18 - 22 mL.

5. The detection method for multiple index components in the compound prolactin - promoting granule according to claim 4, characterized in that The conditions for the ultrasonic treatment are: 300 - 600 W, 20 - 40 min; The filtration is carried out using a 0.22 μm filter membrane; After the ultrasonic treatment, it further includes cooling the ultrasonic system to room temperature, and the room temperature is: 20 - 30°C.

6. The detection method for multiple index components in the compound Chanruling granules according to any one of claims 1-5, characterized in that, The detection method includes the following steps: 1) Sample preparation Preparation of the test solution: The compound prolactin - promoting granule is mixed with methanol at a mass - volume ratio of 1 g:20 ml, ultrasonically treated at 500 W for 30 min, taken out and cooled to room temperature, shaken well, and filtered through a 0.22 μm filter membrane to obtain the test solution; Preparation of the mixed reference solution: Take reference substances of lobetyolin, calycosin, ferulic acid, saikosaponin α, β - ecdysterone, accurately weigh them, dissolve them with methanol and make up the volume to prepare a series of mixed reference solutions with different concentrations; 2) Detect the above - mentioned test solution and mixed reference solution respectively according to the high performance liquid chromatography conditions, compare the retention times of the two for qualitative analysis, and use the external standard method for quantitative analysis.

7. The detection method for multiple index components in the compound prolactin granules according to claim 6, characterized in that, In the series of mixed reference solutions of different concentrations, the concentration range of codonopsis pilosula is 5.635-180.320 μg / mL, the concentration range of calycosin is 5.033-161.056 μg / mL, the concentration range of ferulic acid is 2.516-80.512 μg / mL, the concentration range of saikosaponin α is 2.528-80.896 μg / mL, and the concentration range of β-ecdysterone is 6.893-220.576 μg / mL.

8. The detection method for multiple index components in the compound Chanyuanling granules according to claim 6, characterized in that, The raw materials of the compound Chanruling granules include Codonopsis pilosula, Astragalus membranaceus, Angelica sinensis, Chuanxiong, stir-fried Vaccaria segetalis, Bupleurum chinense, Thunbergia scoparia, Rhizoma Cyperi, and roasted pig's hoof.

9. Use of the method according to any one of claims 1 to 8 in the quality control of Fufang Chanruling Granules.

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