HPLC (High Performance Liquid Chromatography) detection method for simultaneously determining contents of various components in fructus cannabis capsule

Abstract

The invention belongs to the technical field of content detection, and discloses an HPLC (High Performance Liquid Chromatography) detection method for simultaneously determining the contents of various components in a fructus cannabis capsule. The detection method comprises the following steps: mixing a reference substance solution, chrysophanol and methanol to obtain a mixed reference substance solution; the reference substance solution comprises an emodin monomethyl ether solution, a magnolol solution, a honokiol solution, an emodin solution, a rhein solution, a nobiletin solution, an aloe-emodin solution, a hesperidin solution and a paeoniflorin solution; mixing the fructus cannabis capsule content with methanol, and heating and refluxing to obtain a test solution; respectively sucking the mixed reference substance solution and the test solution, and injecting into a high performance liquid chromatograph. According to the detection method, the content of multiple index components can be measured at the same time, the quality of the preparation can be comprehensively reflected, and the detection method has excellent precision, repeatability and stability and high sample adding recovery rate.

Description

Technical Field

[0001] The invention relates to the technical field of content detection, and in particular to an HPLC detection method for simultaneously determining the contents of multiple components in hemp seed capsules. Background Art

[0002] Hemp Seed Capsule is included in the second volume of the Ministry's Standards for the Regularization of New Drugs. It is a Chinese medicine compound preparation made from the extraction of seven herbs: hemp seed, cooked rhubarb, bitter almond, immature bitter orange (fried), magnolia bark (processed with ginger), and white peony root (fried). (The cooked rhubarb part is the original raw powder of the herbal medicine). It has the effect of moistening the intestines and relieving constipation. It is mainly used clinically for constipation and other symptoms, and has a significant effect. However, its current quality standards only include properties, thin layer identification (immature bitter orange (fried) and white peony root (fried)) and capsule inspection items, and lack content determination items; the relevant literature also only conducts content determination on the active ingredients in cooked rhubarb, without involving other medicinal materials, and the quality control indicators are not comprehensive.

[0003] Traditional Chinese medicine compound preparations are based on the formula of "monarch, minister, assistant and messenger", and their efficacy is often the result of the combined action of multiple active ingredients. The determination of quality markers should focus on monarch drugs, while taking into account minister, assistant and messenger drugs. In Ma Ren Capsule, hemp seed moistens the intestines and relieves constipation as the monarch drug; rhubarb relieves constipation and relieves heat, bitter almonds lower qi and moisten the intestines, and white peony nourishes yin and harmonizes the interior, which are all minister drugs; Zhishi and Magnolia bark lower qi and break up stagnation, lower qi and relieve constipation; the whole formula has the effect of moistening the intestines and relieving heat, promoting qi and relieving constipation.

[0004] Therefore, it is of great significance to develop an HPLC detection method that can simultaneously determine the content of multiple components in Ma Ren Capsules, so as to comprehensively control and evaluate the quality of Ma Ren Capsules. Summary of the invention

[0005] The present invention aims to provide an HPLC detection method for simultaneously determining the contents of multiple components in hemp seed capsules, thereby solving the problem that the prior art cannot simultaneously determine the contents of multiple components in hemp seed capsules.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The present invention provides an HPLC detection method for simultaneously determining the contents of multiple components in hemp seed capsules, comprising the following steps:

[0008] 1) mixing the reference substance solution, chrysophanol and methanol to obtain a mixed reference substance solution;

[0009] The reference substance solutions include physaccharin methyl ether solution, magnolol solution, honokiol solution, rhein solution, rhein solution, nobiletin solution, aloe-rhein solution, hesperidin solution and paeoniflorin solution;

[0010] 2) Mixing the contents of the Hemp Seed Capsule with methanol, heating and refluxing to obtain a test solution;

[0011] 3) Absorb the mixed reference substance solution and the test solution respectively, and inject them into a high performance liquid chromatograph for detection;

[0012] The chromatographic conditions are as follows:

[0013] Detection wavelength: 200 - 250 nm; Column temperature: 25 - 35 °C; Mobile phase flow rate: 0.8 - 1 mL / min; Mobile phase: Acetonitrile is mobile phase A, and phosphoric acid solution is mobile phase B;

[0014] Gradient elution program:

[0015] At 0 min, mobile phase A is 14%, and mobile phase B is 86%;

[0016] At 10 min, mobile phase A is 14%, and mobile phase B is 86%;

[0017] At 20 min, mobile phase A is 20%, and mobile phase B is 80%;

[0018] At 25 min, mobile phase A is 20%, and mobile phase B is 80%;

[0019] At 35 min, mobile phase A is 30%, and mobile phase B is 70%;

[0020] At 55 min, mobile phase A is 45%, and mobile phase B is 55%;

[0021] At 65 min, mobile phase A is 55%, and mobile phase B is 45%;

[0022] At 75 min, mobile phase A is 70%, and mobile phase B is 30%;

[0023] At 90 min, mobile phase A is 80%, and mobile phase B is 20%;

[0024] At 95 min, mobile phase A is 80%, and mobile phase B is 20%.

[0025] Preferably, in the step 1), the concentration of physcion solution is 0.21 - 0.22 mg / mL, the concentration of magnolol solution is 0.30 - 0.31 mg / mL, the concentration of honokiol solution is 0.42 - 0.43 mg / mL, the concentration of emodin solution is 0.20 - 0.21 mg / mL, the concentration of rhein solution is 0.30 - 0.31 mg / mL, the concentration of nobiletin solution is 0.39 - 0.40 mg / mL, the concentration of aloe-emodin solution is 0.23 - 0.24 mg / mL, the concentration of hesperidin solution is 0.40 - 0.41 mg / mL, and the concentration of paeoniflorin solution is 0.51 - 0.52 mg / mL;

[0026] The solvent of the reference substance solution is methanol.

[0027] Preferably, in the step 1), the concentration of chrysophanol in the mixed reference substance solution is 0.23 - 0.24 mg / mL.

[0028] Preferably, in the step 1), the concentration of physcion in the mixed reference substance solution is 43.4 - 43.9 μg / mL, the concentration of magnolol is 9.1 - 9.2 μg / mL, the concentration of honokiol is 8.3 - 8.5 μg / mL, the concentration of emodin is 19.7 - 20.7 μg / mL, the concentration of rhein is 14.3 - 15.2 μg / mL, the concentration of nobiletin is 19.7 - 19.9 μg / mL, the concentration of aloe-emodin is 23.5 - 24.0 μg / mL, the concentration of hesperidin is 58.3 - 61.5 μg / mL, and the concentration of paeoniflorin is 148.5 - 153.8 μg / mL.

[0029] Preferably, in the step 2), the dosage ratio of the content of Maren Capsule to methanol is 1 g: 45 - 55 mL;

[0030] The temperature of the heating reflux is 80 - 100 °C, and the time of the heating reflux is 50 - 70 min.

[0031] Preferably, in the chromatographic conditions, the concentration of the phosphoric acid solution is 0.05 - 0.15%.

[0032] From the above technical solutions, it can be seen that compared with the prior art, the present invention has the following beneficial effects:

[0033] The detection method of the present invention can simultaneously determine the contents of multiple index components in Maren Capsule, can more comprehensively reflect the quality of the preparation, and makes up for the deficiencies of the existing quality control indicators; moreover, the test method of the present invention has excellent precision, repeatability, stability, and high sample addition recovery rate and other good detection performances; finally, this method can be applied to the research and development, production quality control, quality supervision and inspection and other links of Maren Capsule, providing a powerful technical means for drug quality assurance, and having remarkable practicability. Description of the Drawings

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0035] Figure 1Chromatogram of the mixed reference substance solution obtained in Example 1, where 1 is paeoniflorin, 2 is hesperidin, 3 is aloe-emodin, 4 is nobiletin, 5 is rhein, 6 is emodin, 7 is honokiol, 8 is magnolol, 9 is chrysophanol, and 10 is physcion;

[0036] Figure 2 Chromatogram of the test sample solution obtained in Example 1, where 1 is paeoniflorin, 2 is hesperidin, 3 is aloe-emodin, 4 is nobiletin, 5 is rhein, 6 is emodin, 7 is honokiol, 8 is magnolol, 9 is chrysophanol, and 10 is physcion;

[0037] Figure 3 Chromatogram of the cathode sample solution obtained in Example 1 without stir-fried white peony root, where 2 is hesperidin, 3 is aloe-emodin, 4 is nobiletin, 5 is rhein, 6 is emodin, 7 is honokiol, 8 is magnolol, 9 is chrysophanol, and 10 is physcion;

[0038] Figure 4 Chromatogram of the cathode sample solution obtained in Example 1 without stir-fried immature bitter orange, where 1 is paeoniflorin, 3 is aloe-emodin, 5 is rhein, 6 is emodin, 7 is honokiol, 8 is magnolol, 9 is chrysophanol, and 10 is physcion;

[0039] Figure 5 Chromatogram of the cathode sample solution obtained in Example 1 without prepared rhubarb, where 1 is paeoniflorin, 2 is hesperidin, 4 is nobiletin, 7 is honokiol, and 8 is magnolol;

[0040] Figure 6 Chromatogram of the cathode sample solution obtained in Example 1 without ginger-processed magnolia bark, where 1 is paeoniflorin, 2 is hesperidin, 3 is aloe-emodin, 4 is nobiletin, 5 is rhein, 6 is emodin, 9 is chrysophanol, and 10 is physcion. Detailed implementation mode

[0041] The present invention provides an HPLC detection method for simultaneously determining the contents of multiple components in Maren Capsules, comprising the following steps:

[0042] 1) Mix the reference substance solution, chrysophanol, and methanol to obtain a mixed reference substance solution;

[0043] The reference substance solution includes physcion solution, magnolol solution, honokiol solution, emodin solution, rhein solution, nobiletin solution, aloe-emodin solution, hesperidin solution, and paeoniflorin solution;

[0044] 2) Mix the content of Maren Capsules with methanol, and obtain a test sample solution after heating under reflux;

[0045] 3) Pipette the mixed reference solution and the test solution respectively, and inject them into a high performance liquid chromatograph for detection;

[0046] The chromatographic conditions are as follows:

[0047] Detection wavelength: 200 - 250 nm; Column temperature: 25 - 35 °C; Mobile phase flow rate: 0.8 - 1 mL / min; Mobile phase: Acetonitrile as mobile phase A, phosphoric acid solution as mobile phase B;

[0048] Gradient elution program:

[0049] At 0 min, mobile phase A is 14%, mobile phase B is 86%;

[0050] At 10 min, mobile phase A is 14%, mobile phase B is 86%;

[0051] At 20 min, mobile phase A is 20%, mobile phase B is 80%;

[0052] At 25 min, mobile phase A is 20%, mobile phase B is 80%;

[0053] At 35 min, mobile phase A is 30%, mobile phase B is 70%;

[0054] At 55 min, mobile phase A is 45%, mobile phase B is 55%;

[0055] At 65 min, mobile phase A is 55%, mobile phase B is 45%;

[0056] At 75 min, mobile phase A is 70%, mobile phase B is 30%;

[0057] At 90 min, mobile phase A is 80%, mobile phase B is 20%;

[0058] At 95 min, mobile phase A is 80%, mobile phase B is 20%.

[0059] In the present invention, the detection wavelength is preferably 210 - 240 nm, more preferably 220 - 230 nm; the column temperature is preferably 27 - 32 °C, more preferably 28 - 31 °C, even more preferably 29 - 30 °C; the mobile phase flow rate is preferably 0.82 - 0.96 mL / min, more preferably 0.84 - 0.94 mL / min, even more preferably 0.85 - 0.9 mL / min; the chromatographic column is preferably NT-C18 chromatographic column, with a specification of 250 mm × 4.6 mm, 5 μm.

[0060] In the present invention, regarding the selection of wavelength, a diode array detector was used to scan 10 components including physcion, chrysophanol, magnolol, honokiol, emodin, rhein, nobiletin, aloe-emodin, hesperidin, and paeoniflorin, and a spectrogram (200 - 400 nm) was obtained. As a result, the maximum absorption wavelengths of physcion, chrysophanol, magnolol, honokiol, emodin, rhein, nobiletin, aloe-emodin, hesperidin, and paeoniflorin were 224 nm, 224 nm, 290 nm, 292 nm, 222 nm, 230 nm, 334 nm, 226 nm, 284 nm, and 230 nm respectively. By comparison, it can be seen that the above 10 components all have good absorption at 230 nm wavelength. Therefore, 230 nm was selected as the detection wavelength.

[0061] In the present invention, regarding the selection of the mobile phase, mobile phase systems such as methanol - water, methanol - 0.1% phosphoric acid, methanol - 0.1% formic acid, acetonitrile - water, acetonitrile - 0.1% phosphoric acid, and acetonitrile - 0.1% formic acid were investigated. It was found that the peak shapes of the chromatograms of 10 components were the best in the acetonitrile - 0.1% phosphoric acid mobile phase system. Then, the mobile phase system of acetonitrile - 0.05 - 0.15% phosphoric acid solution was investigated, and the results showed that the peak shapes of the chromatograms of 10 components did not change significantly.

[0062] In the present invention, regarding the selection of the chromatographic column, the effects of chromatographic column NT - C18 (250 mm × 4.6 mm, 5 μm), chromatographic column II [Thermo Acclaim - C18 (250 mm × 4.6 mm, 5 μm)], and chromatographic column III [phenomenex Luna - C18 (250 mm × 4.6 mm, 5 μm)] on the separation of 10 components to be measured were investigated. At a flow rate of 1.0 mL / min, it was found that the separation of nobiletin and rhein was poor in other chromatographic columns, and the separation effect was the best in chromatographic column I (separation degree was 1.4). When the flow rate was reduced to 0.9 mL / min, the separation degree of the two reached 1.6, while the other 8 components could be well separated in the three chromatographic columns (flow rate 1.0 or 0.9 mL / min); according to the chromatographic conditions described in the present invention, the effects of the determination of 10 components were detected in different high - performance liquid chromatographs (Waters Arc, Agilent 1260), and the 10 components were all well separated, with good durability.

[0063] In the present invention, in the step 1), the concentration of the physcion solution is preferably 0.21 - 0.22 mg / mL, more preferably 0.212 - 0.218 mg / mL, and still more preferably 0.215 - 0.216 mg / mL; the concentration of the magnolol solution is preferably 0.30 - 0.31 mg / mL, more preferably 0.302 - 0.308 mg / mL, and still more preferably 0.304 - 0.306 mg / mL; the concentration of the honokiol solution is preferably 0.42 - 0.43 mg / mL, more preferably 0.422 - 0.428 mg / mL, and still more preferably 0.425 - 0.426 mg / mL; the concentration of the emodin solution is preferably 0.20 - 0.21 mg / mL, more preferably 0.202 - 0.208 mg / mL, and still more preferably 0.204 - 0.205 mg / mL; the concentration of the rhein solution is preferably 0.30 - 0.31 mg / mL, more preferably 0.303 - 0.307 mg / mL, and still more preferably 0.304 - 0.306 mg / mL; the concentration of the nobiletin solution is preferably 0.39 - 0.40 mg / mL, more preferably 0.391 - 0.397 mg / mL, and still more preferably 0.394 - 0.396 mg / mL; the concentration of the aloe-emodin solution is preferably 0.23 - 0.24 mg / mL, more preferably 0.234 - 0.239 mg / mL, and still more preferably 0.235 - 0.238 mg / mL; the concentration of the hesperidin solution is preferably 0.40 - 0.41 mg / mL, more preferably 0.402 - 0.407 mg / mL, and still more preferably 0.403 - 0.405 mg / mL; the concentration of the paeoniflorin solution is preferably 0.51 - 0.52 mg / mL, more preferably 0.512 - 0.518 mg / mL, and still more preferably 0.514 - 0.516 mg / mL;

[0064] The solvent of the reference substance solution is preferably methanol.

[0065] In the present invention, in the step 1), the concentration of chrysophanol in the mixed reference substance solution is preferably 0.23 - 0.24 mg / mL, more preferably 0.231 - 0.238 mg / mL, and still more preferably 0.234 - 0.236 mg / mL.

[0066] In the present invention, in the step 1), the concentration of physcion in the mixed reference substance solution is preferably 43.4 - 43.9 μg / mL, more preferably 43.45 - 43.8 μg / mL, and even more preferably 43.5 - 43.7 μg / mL; the concentration of magnolol is preferably 9.1 - 9.2 μg / mL, more preferably 9.12 - 9.18 μg / mL, and even more preferably 9.14 - 9.16 μg / mL; the concentration of honokiol is preferably 8.3 - 8.5 μg / mL, more preferably 8.35 - 8.45 μg / mL, and even more preferably 8.36 - 8.42 μg / mL; the concentration of emodin is preferably 19.7 - 20.7 μg / mL, more preferably 19.8 - 20.4 μg / mL, and even more preferably 20.1 - 20.3 μg / mL; the concentration of rhein is preferably 14.3 - 15.2 μg / mL, more preferably 14.5 - 15.0 μg / mL, and even more preferably 14.6 - 14.8 μg / mL; the concentration of nobiletin is preferably 19.7 - 19.9 μg / mL, more preferably 19.75 - 19.85 μg / mL, and even more preferably 19.78 - 19.82 μg / mL; the concentration of aloe-emodin is preferably 23.5 - 24.0 μg / mL, more preferably 23.6 - 23.9 μg / mL, and even more preferably 23.7 - 23.8 μg / mL; the concentration of hesperidin is preferably 58.3 - 61.5 μg / mL, more preferably 58.6 - 60.4 μg / mL, and even more preferably 58.7 - 59.4 μg / mL; the concentration of paeoniflorin is preferably 148.5 - 153.8 μg / mL, more preferably 148.7 - 152.8 μg / mL, and even more preferably 148.6 - 151.4 μg / mL.

[0067] In the present invention, in the step 2), the dosage ratio of the content of Maren Capsule to methanol is preferably 1 g:45 - 55 mL, more preferably 1 g:47 - 53 mL, and even more preferably 1 g:48 - 50 mL;

[0068] The temperature of heating under reflux is preferably 80 - 100 °C, more preferably 85 - 95 °C, and even more preferably 90 - 92 °C, and the time of heating under reflux is preferably 50 - 70 min, more preferably 55 - 65 min, and even more preferably 60 - 62 min.

[0069] In the present invention, in the chromatographic conditions, the concentration of the phosphoric acid solution is preferably 0.05 - 0.15%, more preferably 0.08 - 0.14%, and even more preferably 0.1 - 0.12%.

[0070] In the present invention, in step 2), the content of Maren Capsules is ground before being mixed with methanol. The particle size of the ground content is preferably 0.1 - 0.15 mm, more preferably 0.11 - 0.14 mm, and even more preferably 0.12 - 0.13 mm. This particle size can improve the extraction efficiency. The rotation speed of grinding is preferably 800 - 1000 rpm, more preferably 850 - 950 rpm, and even more preferably 900 rpm. This grinding rotation speed can ensure the grinding effect and avoid component loss caused by over-grinding.

[0071] In the present invention, regarding the selection of the preparation process of the test solution, when preparing the test solution, taking the peak shapes and extraction rates of 10 component chromatographic peaks as indicators, methanol, ethanol, 70% methanol, 70% ethanol, 50% methanol or 50% ethanol were respectively used as extraction solvents, and ultrasonic extraction (the investigation time was 30 min, 40 min, 50 min, 60 min) and reflux extraction (the investigation time was 30 min, 45 min, 60 min, 90 min) were carried out at a power of 500 W and a frequency of 45 KHz. It was found that the peak shapes of the test solutions extracted with different ethanol concentrations were slightly inferior to those extracted with different methanol concentrations. Under heating reflux extraction, the extraction rates of 10 components were the highest when methanol was used as the extraction solvent. When refluxing for 60 min, 10 components were basically completely extracted.

[0072] In the present invention, the effective components of the ministerial drug rhubarb (aloe-emodin, rhein, emodin, chrysophanol, physcion), the effective component of white peony root (paeoniflorin), the effective components of the adjuvant drug immature bitter orange (hesperidin, nobiletin), and the effective components of magnolia bark (honokiol, magnolol) were selected as index components. In order to more comprehensively evaluate the quality of Maren Capsules, the index components trigonelline of Cannabis sativa L. and amygdalin of bitter almond in Maren Capsules were also investigated. It was found that trigonelline had poor retention in the chromatographic column filled with octadecylsilane-bonded silica gel and had better retention in the chromatographic column filled with amino-bonded silica gel. Amygdalin had good resolution in the mobile phase methanol-water system. The two could not share the same chromatographic column or mobile phase system with other components. Therefore, physcion, chrysophanol, magnolol, honokiol, emodin, rhein, nobiletin, aloe-emodin, hesperidin, and paeoniflorin were finally determined as the index components for content determination.

[0073] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0074] In the present invention, the high-performance liquid chromatograph was purchased from Agilent Company, model Agilent 1260, equipped with a diode array detector;

[0075] The chromatographic column is NT-C18 chromatographic column, with a specification of 250 mm × 4.6 mm, 5 μm;

[0076] The electrothermal constant temperature water bath was purchased from Beijing Yongguang Instrument Co., Ltd., model DZKW-D-2;

[0077] The ultrasonic cleaner was purchased from Kunshan Ultrasonic Instrument Co., Ltd., model KQ-500DE;

[0078] Nobiletin was purchased from Shanghai Shifeng Biotechnology Co., Ltd., batch number S625092451, purity 99.27%;

[0079] Aloe-emodin was purchased from Jiangxi Baicaoyuan Biotechnology Co., Ltd., batch number 000248-202402, purity 98.37%;

[0080] Physcion, paeoniflorin, hesperidin, rhein, honokiol, magnolol, emodin, and chrysophanol were all purchased from the National Institutes for Food and Drug Control. The production batch number of physcion is 110758-201817, purity 99.2%, the production batch number of paeoniflorin is 110736-202018, purity 96.7%, the production batch number of hesperidin is 110721-202019, purity 95.3%, the production batch number of rhein is 110757-202308, purity 95.0%, the production batch number of honokiol is 110730-201915, purity 99.8%, the production batch number of magnolol is 110729-20236, purity 99.2%, the production batch number of emodin is 110756-201913, purity 96.0%, and the production batch number of chrysophanol is 110796-201922, purity 99.4%;

[0081] Phosphoric acid, formic acid, and acetic acid were purchased from Chengdu Xiya Chemical Industry Co., Ltd., chromatographically pure;

[0082] Methanol and acetonitrile were purchased from Tedia Company in the United States, chromatographically pure;

[0083] The water was Wahaha purified water;

[0084] Six kinds of Maren capsules were all purchased from Hunan Lebang Pharmaceutical Co., Ltd., production batch numbers were 240801, 240302, 240602, 240202, 231101, and 230901 respectively, 0.35 g / capsule.

[0085] Example 1

[0086] Preparation of reference substance solution: Accurately weigh 4.38 mg of physcion, 6.11 mg of magnolol, 8.41 mg of honokiol, 4.12 mg of emodin, 6.04 mg of rhein, 7.94 mg of nobiletin, 4.79 mg of aloe-emodin, 8.16 mg of hesperidin, and 10.25 mg of paeoniflorin respectively, place them in a 20 mL volumetric flask, dissolve with methanol and make up to the mark to obtain the reference substance solution;

[0087] Preparation of mixed reference substance solution: Accurately weigh 4.62 mg of chrysophanol, place it in a 20 mL volumetric flask, then accurately pipette 4 mL of physcion solution, 0.6 mL of magnolol solution, 0.4 mL of honokiol solution, 2 mL of emodin solution, 1 mL of rhein solution, 1 mL of nobiletin solution, 2 mL of aloe-emodin solution, 3 mL of hesperidin solution, and 6 mL of paeoniflorin solution into this volumetric flask to obtain the mixed reference substance solution;

[0088] Preparation of series of mixed reference substance solutions: Accurately pipette 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, and 0.5 mL of the mixed reference substance solution respectively, place them in 5 mL volumetric flasks, and make up to the mark with methanol;

[0089] Investigation of linear relationship: Accurately pipette 10 μL of the solutions in the above volumetric flasks respectively for chromatographic determination. The chromatographic conditions are as follows: detection wavelength 230 nm, mobile phase flow rate 0.9 mL / min, column temperature 30 °C, mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and the gradient elution program is shown in Table 1.

[0090] Table 1 Gradient elution program

[0091] Time / min Mobile phase A / % Mobile phase B / % 0 14 86 10 14 86 20 20 80 25 20 80 35 30 70 55 45 55 65 55 45 75 70 30 90 80 20 95 80 20

[0092] Using the concentration of each component as the abscissa, denoted as "X", and the peak area as the ordinate, denoted as "Y", perform linear regression to obtain the regression equation, as shown in Table 2.

[0093] Table 2 Linear relationship between the concentration of each component and the peak area

[0094] Component Regression equation r <![CDATA[Linear range / μg·mL -1 > Paeoniflorin Y = 13.56X + 11.92 0.9994 14.87～148.7 Hesperidin Y = 21.76X + 18.18 0.9993 5.832～58.32 Aloe-emodin Y = 52.82X + 6.667 0.9998 2.356～23.56 Nobiletin Y = 31.25X + 3.453 0.9998 1.971～19.71 Rhein Y = 55.80X + 3.642 0.9998 1.435～14.35 Emodin Y = 41.69X + 4.938 0.9998 1.978～19.78 Honokiol Y = 36.49X + 1.460 0.9998 0.8393～8.393 Magnolol Y = 39.40X + 3.316 0.9997 0.9082～9.082 Chrysophanol Y = 42.96X + 62.42 0.9998 22.96～229.6 Physcion Y = 39.54X - 6.945 0.9998 4.345～43.45

[0095] Preparation of test solution: Take 10 capsules of Maren Capsules of batches 240801, 240302, 240602, 240202, 231101, and 230901 respectively, take the contents and grind them to a particle size of 0.15 mm at a speed of 1000 rpm. Accurately weigh 1 g of the ground contents and place it in a stoppered conical flask with 50 mL of methanol, heat under reflux at 90 °C for 1 h, then cool to room temperature, weigh, make up the reduced weight with methanol, and filter to obtain the sample solution;

[0096] Content determination: Pipette 10 μL of each of the above sample solutions, perform chromatographic determination under the above chromatographic conditions, substitute the obtained peak areas into the regression equation shown in Table 2, and calculate the results as shown in Table 3.

[0097] Table 3 Detection results of the contents of various components in Maren Capsules

[0098]

[0099]

[0100] Example 2

[0101] Operate according to the preparation methods of the reference substance solution and the mixed reference substance solution in Example 1;

[0102] Preparation of a series of mixed reference substance solutions: Precisely pipette 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, and 0.5 mL of the mixed reference substance solution respectively, place them in 5-mL volumetric flasks, and make up the volume with methanol;

[0103] Investigation of linear relationship: Precisely pipette 10 μL of the solutions in the above volumetric flasks respectively for chromatographic determination. The chromatographic conditions are as follows: detection wavelength 220 nm, mobile phase flow rate 0.8 mL / min, column temperature 35 °C, mobile phase: acetonitrile as mobile phase A, 0.15% phosphoric acid solution as mobile phase B, and the gradient elution program is shown in Table 1.

[0104] Using the concentration of each component as the abscissa, denoted as "X", and the peak area as the ordinate, denoted as "Y", perform linear regression to obtain the regression equation.

[0105] Preparation of the test solution: Take 10 capsules of Maren Capsules of batches 240801, 240302, 240602, 240202, 231101, and 230901 respectively, take the contents and grind them to a particle size of 0.1 mm at a rotation speed of 1000 rpm. Precisely weigh 1 g of the ground contents and place it in a stoppered conical flask with 50 mL of methanol, heat under reflux at 95 °C for 1 h, then cool to room temperature, weigh, make up the reduced weight with methanol, and filter to obtain the sample solution;

[0106] Content determination: Precisely pipette 10 μL of each of the above sample solutions, perform chromatographic determination under the above chromatographic conditions, substitute the obtained peak areas into the regression equation obtained from the investigation of linear relationship, and calculate the content results of each component.

[0107] Example 3

[0108] Operate according to the preparation methods of the reference substance solution and the mixed reference substance solution in Example 1;

[0109] Preparation of a series of mixed reference substance solutions: Accurately pipette 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, and 0.5 mL of the mixed reference substance solution respectively, and place them in 5-mL volumetric flasks. Dilute to the mark with methanol.

[0110] Investigation of linear relationship: Accurately pipette 10 μL of the solutions in the above volumetric flasks respectively for chromatographic determination. The chromatographic conditions are as follows: detection wavelength 230 nm, mobile phase flow rate 0.9 mL / min, column temperature 25 °C. Mobile phase: acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B. The gradient elution program is shown in Table 1.

[0111] Using the concentration of each component as the abscissa, denoted as "X", and the peak area as the ordinate, denoted as "Y", perform linear regression to obtain the regression equation.

[0112] Preparation of test solution: Take 10 capsules of Maren Capsules of batches 240801, 240302, 240602, 240202, 231101, and 230901 respectively. Take the contents and grind them at a speed of 800 rpm until the particle size is 0.12 mm. Accurately weigh 1 g of the ground contents and place it in a stoppered conical flask with 50 mL of methanol. Heat under reflux at 90 °C for 55 min, then cool to room temperature, weigh, and make up the reduced weight with methanol. Filter to obtain the sample solution.

[0113] Content determination: Accurately pipette 10 μL of the above sample solution respectively, perform chromatographic determination according to the above chromatographic conditions, substitute the obtained peak area into the regression equation obtained from the investigation of linear relationship, and calculate the content results of each component.

[0114] Specificity test:

[0115] Operate according to the preparation methods of the reference substance solution and the mixed reference substance solution in Example 1.

[0116] Randomly take 10 Maren Capsules, take the contents and grind them at a speed of 1000 rpm until the particle size is 0.15 mm. Take 1 g of the ground contents and place it in a stoppered conical flask with 50 mL of methanol. Heat under reflux at 90 °C for 1 h, then cool to room temperature, weigh, and make up the reduced weight with methanol. Filter to obtain the sample solution.

[0117] According to the preparation method of Maren Capsules in Chinese Patent CN 101642499A, prepare Maren Capsule samples lacking Paeonia lactiflora (fried), Fructus Aurantii Immaturus (fried), Magnolia officinalis (processed with ginger), and Rheum palmatum (prepared) respectively, and then prepare the corresponding cathode sample solutions according to the preparation method of the test solution.

[0118] Respectively pipette 10 μL of the mixed reference substance solution, 10 μL of the test substance solution, and 10 μL of each cathode sample solution, and inject them into a high-performance liquid chromatograph respectively for detection under the chromatographic conditions of Example 1.

[0119] The detection results are as Figures 1 - 6 shown. It can be seen from Figures 1 - 6 that the retention times of the chromatographic peaks of each component in the chromatogram of the test substance solution are the same as those of the reference substance, and the resolution between adjacent components is ≥1.5. There are no corresponding chromatographic peaks at the same retention time in each negative sample, indicating that the negative has no interference and the method has good specificity.

[0120] Precision test:

[0121] Repeat the preparation steps of the reference substance solution and the mixed reference substance solution in Example 1;

[0122] Add 1 mL of the mixed reference substance solution to a 5 mL volumetric flask and make up the volume with methanol; add 2 mL of the mixed reference substance solution to a 10 mL volumetric flask and make up the volume with methanol;

[0123] Respectively pipette 10 μL of the solution in the 5 mL volumetric flask and 10 μL of the solution in the 10 mL volumetric flask, and perform chromatographic detection under the chromatographic conditions described in Example 1. The relative standard deviations (RSD) of each component are shown in Table 4.

[0124] Table 4 Relative standard deviations of each component

[0125] Component RSD / (%) Physcion 0.10 Chrysophanol 0.11 Magnolol 0.57 Honokiol 0.41 Emodin 0.23 Rhein 0.24 Nobiletin 0.16 Aloe-emodin 0.18 Hesperidin 0.27 Paeoniflorin 1.33

[0126] It can be seen from Table 4 that the precision of the test method described in the present invention is good.

[0127] Repeatability test:

[0128] Take 60 capsules of Maren Capsules of batch 240602, divide them into six groups, and perform the following steps on each group of Maren Capsules: Take the contents and grind them to a particle size of 0.15 mm at a speed of 1000 rpm. Take 1 g of the ground contents and place it in a stoppered conical flask with 50 mL of methanol. Heat under reflux at 90 °C for 1 h, then cool to room temperature, make up the reduced weight with methanol, and filter to obtain the sample solution;

[0129] 10 μL was taken from each of the six groups and injected into a high performance liquid chromatograph for detection under the chromatographic conditions of Example 1. The obtained results were substituted into the regression equation shown in Table 2 to calculate the contents of each component. The average values of the results obtained from the six groups were as follows: the average content of physcion was 1.346 mg / g (RSD was 1.59%), the average content of chrysophanol was 9.251 mg / g (RSD was 1.64%), the average content of magnolol was 0.1547 mg / g (RSD was 1.46%), the average content of honokiol was 0.07535 mg / g (RSD was 1.44%), the average content of emodin was 0.7478 mg / g (RSD was 1.39%), the average content of rhein was 0.2540 mg / g (RSD was 2.13%), the average content of nobiletin was 0.2229 mg / g (RSD was 2.28%), the average content of aloe-emodin was 0.7538 mg / g (RSD was 1.92%), the average content of hesperidin was 1.885 mg / g (RSD was 1.13%), and the average content of paeoniflorin was 6.024 mg / g (RSD was 1.03%). It can be seen that the test method described in the present invention has good repeatability.

[0130] Stability test:

[0131] The test solution of the first group in the repeatability test was taken, and 10 μL was taken and injected into a high performance liquid chromatograph for injection and determination under the chromatographic conditions of Example 1 at 0 h, 3 h, 9 h, 15 h, 24 h, and 30 h of standing, respectively. The peak areas were recorded. The obtained results were as follows: the RSD of the peak area of physcion was 1.18%, the RSD of the peak area of chrysophanol was 1.18%, the RSD of the peak area of magnolol was 0.59%, the RSD of the peak area of honokiol was 1.11%, the RSD of the peak area of emodin was 0.23%, the RSD of the peak area of rhein was 1.65%, the RSD of the peak area of nobiletin was 1.89%, the RSD of the peak area of aloe-emodin was 1.23%, the RSD of the peak area of hesperidin was 1.63%, and the RSD of the peak area of paeoniflorin was 1.81%. It can be seen that the peak area can still be stably measured when the test solution is placed at room temperature for 30 h.

[0132] Spiked recovery test:

[0133] Six portions of Maren capsules of batch 240602, each 0.5 g, were taken and placed in 50 mL volumetric flasks respectively;

[0134] Add 6 mL of a methanol solution containing 0.5169 mg / mL of paeoniflorin, 3 mL of a methanol solution containing 0.3602 mg / mL of hesperidin, 1.5 mL of a methanol solution containing 0.2510 mg / mL of aloe-emodin, 0.3 mL of a methanol solution containing 0.3689 mg / mL of nobiletin, 0.4 mL of a methanol solution containing 0.2964 mg / mL of rhein, 1.5 mL of a methanol solution containing 0.2506 mg / mL of emodin, 0.1 mL of a methanol solution containing 0.4197 mg / mL of honokiol, 0.2 mL of a methanol solution containing 0.3789 mg / mL of magnolol, 15 mL of a methanol solution containing 0.3008 mg / mL of chrysophanol, and 3 mL of a methanol solution containing 0.2282 mg / mL of physcion to each volumetric flask at the level of 100% of the content of each component, and make up the volume with methanol;

[0135] Take 10 μL of the solution in each volumetric flask respectively, detect according to the chromatographic conditions described in Example 1, and calculate the recovery rates of each component. The calculation results are shown in Table 5.

[0136] Table 5 Test results of the recovery rates of each component added

[0137]

[0138]

[0139]

[0140] As can be seen from Table 5, the detection method described in the present invention has a high recovery rate of the sample added and good accuracy.

[0141] In summary, the HPLC detection method established by the present invention for simultaneously determining the contents of multiple components in Maren Capsules is excellent in terms of specificity, precision, repeatability, stability, and recovery rate of the sample added, and can be used for the quality control and evaluation of Maren Capsules, providing reliable technical support for the research and development, production, and quality supervision of Maren Capsules.

[0142] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A HPLC detection method for simultaneously determining the contents of multiple components in Ma Ren Capsules, characterized in that: The steps include: 1) mixing the reference substance solution, chrysophanol and methanol to obtain a mixed reference substance solution; The reference substance solutions include physaccharin methyl ether solution, magnolol solution, honokiol solution, rhein solution, rhein solution, nobiletin solution, aloe-rhein solution, hesperidin solution and paeoniflorin solution; 2) Mixing the contents of the Hemp Seed Capsule with methanol, heating and refluxing to obtain a test solution; 3) Pipette the mixed reference solution and test solution separately and inject them into high performance liquid chromatography for detection; The chromatographic conditions were as follows: Detection wavelength: 200-250nm; column temperature: 25-35℃; mobile phase flow rate: 0.8-1mL / min; mobile phase: acetonitrile as mobile phase A, phosphoric acid solution as mobile phase B; Gradient elution program: 0 min, mobile phase A was 14%, mobile phase B was 86%; 10 min, mobile phase A was 14%, mobile phase B was 86%; 20 min, mobile phase A: 20%, mobile phase B: 80%; 25 min, mobile phase A: 20%, mobile phase B: 80%; 35 min, mobile phase A: 30%, mobile phase B: 70%; 55 min, mobile phase A: 45%, mobile phase B: 55%; 65 min, mobile phase A: 55%, mobile phase B: 45%; 75 min, mobile phase A: 70%, mobile phase B: 30%; 90 min, mobile phase A: 80%, mobile phase B: 20%; 95min, mobile phase A was 80%, mobile phase B was 20%.

2. The HPLC detection method for simultaneously determining the contents of multiple components in Ma Ren Capsules according to claim 1 is characterized in that: In the step 1), the concentration of the physophanol methyl ether solution is 0.21-0.22 mg / mL, the concentration of the magnolol solution is 0.30-0.31 mg / mL, the concentration of the honokiol solution is 0.42-0.43 mg / mL, the concentration of the rhein solution is 0.20-0.21 mg / mL, the concentration of the rhein solution is 0.30-0.31 mg / mL, the concentration of the nobiletin solution is 0.39-0.40 mg / mL, the concentration of the aloe-emodin solution is 0.23-0.24 mg / mL, the concentration of the hesperidin solution is 0.40-0.41 mg / mL, and the concentration of the paeoniflorin solution is 0.51-0.52 mg / mL; The solvent of the reference solution is methanol.

3. The HPLC detection method for simultaneously determining the contents of multiple components in Ma Ren Capsules according to claim 2 is characterized in that: In the step 1), the concentration of chrysophanol in the mixed reference solution is 0.23-0.24 mg / mL.

4. The HPLC detection method for simultaneously determining the contents of multiple components in Ma Ren Capsules according to claim 3 is characterized in that: In the step 1), the concentration of physaccharin methyl ether in the mixed reference solution is 43.4-43.9 μg / mL, the concentration of magnolol is 9.1-9.2 μg / mL, the concentration of honokiol is 8.3-8.5 μg / mL, the concentration of rhein is 19.7-20.7 μg / mL, the concentration of rhein is 14.3-15.2 μg / mL, the concentration of nobiletin is 19.7-19.9 μg / mL, the concentration of aloe-emodin is 23.5-24.0 μg / mL, the concentration of hesperidin is 58.3-61.5 μg / mL, and the concentration of paeoniflorin is 148.5-153.8 μg / mL.

5. The HPLC detection method for simultaneously determining the contents of multiple components in Ma Ren Capsules according to claim 1 is characterized in that: In the step 2), the ratio of the contents of the hemp seed capsule to methanol is 1 g: 45-55 mL; The heating reflux temperature is 80-100° C., and the heating reflux time is 50-70 min.

6. The HPLC detection method for simultaneously determining the contents of multiple components in Ma Ren Capsules according to claim 5 is characterized in that: In the chromatographic conditions, the concentration of the phosphoric acid solution is 0.05-0.15%.

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