A HPLC detection method for simultaneously determining the contents of multiple components in Cannabis capsule

Abstract

The application belongs to the technical field of content detection, and discloses a kind of HPLC detection methods for simultaneously determining the content of multiple components in semen Cannabis capsules.The detection method includes mixing reference solution, chrysophanol and methanol to obtain mixed reference solution.The reference solution includes physcion solution, magnolol solution, houpo chrysophanol solution, emodin solution, rhein solution, nobiletin solution, aloe emodin solution, hesperidin solution and paeoniflorin solution;The contents of semen Cannabis capsules are mixed with methanol, and the test solution is obtained after heating and refluxing;The mixed reference solution and test solution are respectively sucked and injected into high performance liquid chromatograph.The detection method can simultaneously determine the content of multiple index components, can comprehensively reflect the quality of the preparation, and the test method has excellent precision, repeatability, stability and high sample recovery rate.

Description

Technical Field

[0001] The present 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 Maren Capsules. Background Art

[0002] Maren Capsules are included in the转正标准 of the newly approved traditional Chinese medicine standards of the Ministry of Health. It is a traditional Chinese medicine compound preparation processed from 7 herbs, namely Cannabis Seed, Prepared Rhubarb, Bitter Apricot Seed, Fructus Aurantii Immaturus (fried), Magnolia Officinalis (processed with ginger), and Paeonia Lactiflora (fried) (part of the prepared rhubarb is the raw medicinal powder). It has the effect of moistening the intestines and promoting defecation, and is mainly used clinically for intestinal dryness and constipation, etc., with remarkable curative effects. However, its current quality standard only includes the description of appearance, thin-layer chromatography identification (Fructus Aurantii Immaturus (fried) and Paeonia Lactiflora (fried)), and the inspection items of capsules, lacking the content determination item; relevant literature only focuses on the content determination of the active ingredients in prepared rhubarb, without covering other herbs, and the quality control indicators are not comprehensive.

[0003] Traditional Chinese medicine compound preparations are formulated according to the principles of "emperor, minister, assistant, and guide", and their efficacy is often the result of the combined action of multiple active ingredients. The determination of quality markers should be mainly based on the emperor herb, taking into account the minister, assistant, and guide herbs. In Maren Capsules, Cannabis Seed for moistening the intestines and promoting defecation is the emperor herb; rhubarb for promoting defecation and clearing heat, bitter apricot seed for descending qi and moistening the intestines, and paeonia lactiflora for nourishing yin and regulating the interior are the minister herbs; fructus aurantii immaturus and magnolia officinalis for descending qi and breaking stagnation, promoting defecation by purging; the whole formula jointly plays the role of moistening the intestines, clearing heat, and promoting qi and defecation.

[0004] Therefore, it is of great significance to develop an HPLC detection method for simultaneously determining the contents of multiple components in Maren Capsules, so as to comprehensively control and evaluate the quality of Maren 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 Maren Capsules, and solve the problem that the prior art cannot simultaneously determine the contents of multiple components in Maren Capsules.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

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

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

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

[0010] 2) Mix the content of Maren Capsules with methanol, and obtain a test solution after heating under reflux;

[0011] 3) Pipette the mixed reference solution and the test solution separately into the 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 as mobile phase A, phosphoric acid solution as mobile phase B;

[0014] Gradient elution procedure:

[0015] At 0 min, mobile phase A was 14% and mobile phase B was 86%.

[0016] After 10 minutes, mobile phase A was 14% and mobile phase B was 86%.

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

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

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

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

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

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

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

[0024] 95 min, mobile phase A was 80%, mobile phase B was 20%.

[0025] Preferably, in step 1), the concentrations of the emodin methyl ether solution and the magnolol solution are 0.21–0.22 mg / mL, 0.30–0.31 mg / mL, 0.42–0.43 mg / mL, 0.20–0.21 mg / mL, 0.30–0.31 mg / mL, 0.39–0.40 mg / mL, 0.23–0.24 mg / mL, 0.40–0.41 mg / mL, and 0.51–0.52 mg / mL, respectively.

[0026] The solvent for the reference solution is methanol.

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

[0028] Preferably, in step 1), the concentrations of emodin methyl ether, magnolol, and honokiol in the mixed reference solution are 43.4–43.9 μg / mL, 9.1–9.2 μg / mL, 8.3–8.5 μg / mL, 19.7–20.7 μg / mL, 14.3–15.2 μg / mL, 19.7–19.9 μg / mL, 23.5–24.0 μg / mL, 58.3–61.5 μg / mL, and 148.5–153.8 μg / mL, respectively.

[0029] Preferably, in step 2), the ratio of the contents of the hemp seed capsule to methanol is 1g:45-55mL;

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

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

[0032] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0033] The detection method described in this invention can simultaneously determine the content of multiple indicator components in hemp seed capsules, which can comprehensively reflect the quality of the preparation and make up for the deficiencies of existing quality control indicators. Moreover, the test method described in this invention has excellent detection performance, such as high precision, repeatability, stability and high recovery rate. Finally, this method can be applied to the research and development, production quality control and quality supervision and inspection of hemp seed capsules, providing a powerful technical means for drug quality assurance and has significant practical value. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0035] Figure 1The chromatogram is of the mixed reference solution obtained in Example 1, wherein 1 is paeoniflorin, 2 is hesperidin, 3 is aloe-emodin, 4 is noriheptacortin, 5 is rhein, 6 is emodin, 7 is honokiol, 8 is honokiol, 9 is rhein, and 10 is emodin methyl ether.

[0036] Figure 2 The chromatogram is of the test solution obtained in Example 1, wherein 1 is paeoniflorin, 2 is hesperidin, 3 is aloe-emodin, 4 is norihesperidin, 5 is rhein, 6 is emodin, 7 is honokiol, 8 is honokiol, 9 is rhein, and 10 is emodin methyl ether.

[0037] Figure 3 The chromatogram is of the cathode sample solution lacking white peony (fried) obtained in Example 1, wherein 2 is hesperidin, 3 is aloe-emodin, 4 is norihesperidin, 5 is rhein, 6 is emodin, 7 is honokiol, 8 is honokiol, 9 is rhein, and 10 is emodin methyl ether.

[0038] Figure 4 The chromatogram is of the cathode sample solution lacking Citrus aurantium (fried) obtained in Example 1, wherein 1 is paeoniflorin, 3 is aloe-emodin, 5 is rhein, 6 is emodin, 7 is magnolol, 8 is magnolol, 9 is emodin, and 10 is emodin methyl ether.

[0039] Figure 5 The chromatogram is of the cathode sample solution lacking cooked rhubarb obtained in Example 1, wherein 1 is paeoniflorin, 2 is hesperidin, 4 is norihesperidin, 7 is honokiol, and 8 is honokiol.

[0040] Figure 6 The chromatogram is of the cathode sample solution lacking Magnolia officinalis (processed with ginger) obtained in Example 1, wherein 1 is paeoniflorin, 2 is hesperidin, 3 is aloe-emodin, 4 is noriheptacortin, 5 is rhein, 6 is emodin, 9 is chrysophanol, and 10 is emodin methyl ether. Detailed Implementation

[0041] This invention provides an HPLC detection method for simultaneously determining the content of multiple components in hemp seed capsules, comprising the following steps:

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

[0043] The reference solutions include emodin methyl ether solution, magnolol solution, honokiol solution, emodin solution, rhein solution, nobiletin solution, aloe-emodin solution, hesperidin solution, and paeoniflorin solution;

[0044] 2) Mix the contents of the hemp seed capsules with methanol, heat under reflux to obtain the test solution;

[0045] 3) Pipette the mixed reference solution and the test solution separately into the 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 procedure:

[0049] At 0 min, mobile phase A was 14% and mobile phase B was 86%.

[0050] After 10 minutes, mobile phase A was 14% and mobile phase B was 86%.

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

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

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

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

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

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

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

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

[0059] In this 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, and 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, and even more preferably 0.85–0.9 mL / min; the chromatographic column is preferably… The NT-C18 chromatographic column has dimensions of 250 mm × 4.6 mm and a diameter of 5 μm.

[0060] In this invention, regarding the selection of wavelength, a diode array detector was used to scan 10 components: emodin methyl ether, rhein, honokiol, honokiol, rhein, rhein, nobiletin, aloe-emodin, hesperidin, and paeoniflorin, obtaining spectra (200–400 nm). The results showed that the maximum absorption wavelengths of emodin methyl ether, rhein, honokiol, honokiol, rhein, 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 a wavelength of 230 nm, therefore 230 nm was selected as the detection wavelength.

[0061] In this invention, regarding the selection of the mobile phase, mobile phase systems of 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 chromatographic peaks of the 10 components were best in the acetonitrile-0.1% phosphoric acid mobile phase system. The acetonitrile-0.05-0.15% phosphoric acid solution mobile phase system was further investigated, and the peak shapes of the chromatographic peaks of the 10 components did not change significantly.

[0062] In this invention, the selection of the chromatographic column was examined. The effects of NT-C18 (250mm×4.6mm, 5μm) column, column II [Thermo Acclaim-C18 (250mm×4.6mm, 5μm)], and column III [phenomenex Luna-C18 (250mm×4.6mm, 5μm)] on the separation of 10 analytes were investigated. At a flow rate of 1.0 mL / min, norepinephrine and rhein were poorly separated in other columns, with the best separation effect observed in column I (resolution 1.4). When the flow rate was reduced to 0.9 mL / min, the resolution reached 1.6. The other eight analytes were well separated in all three columns (flow rates of 1.0 or 0.9 mL / min). The effects of the chromatographic conditions described in this invention on the determination of the 10 analytes were also investigated in different high-performance liquid chromatographs (WatersArc, Agilent 1260). All 10 analytes achieved good separation, demonstrating good robustness.

[0063] In this invention, in step 1), the concentration of the emodin methyl ether solution is preferably 0.21–0.22 mg / mL, more preferably 0.212–0.218 mg / mL, and even 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 even more preferably 0.304–0.306 mg / mL; and the concentration of the magnolol solution is… The preferred concentration is 0.42–0.43 mg / mL, more preferably 0.422–0.428 mg / mL, and even more preferably 0.425–0.426 mg / mL. The preferred concentration of the rhein solution is 0.20–0.21 mg / mL, more preferably 0.202–0.208 mg / mL, and even more preferably 0.204–0.205 mg / mL. The preferred concentration of the rhein solution is 0.30–0.31 mg / mL. The concentration of the noriheptacortin solution is preferably 0.303–0.307 mg / mL, more preferably 0.304–0.306 mg / mL; the concentration of the noriheptacortin solution is preferably 0.39–0.40 mg / mL, further preferably 0.391–0.397 mg / mL, more preferably 0.394–0.396 mg / mL; and the concentration of the aloe-emodin solution is preferably 0.23–0.24 mg / mL, more preferably 0.234–0.239 mg / mL. L, more preferably 0.235-0.238 mg / mL, the concentration of hesperidin solution is preferably 0.40-0.41 mg / mL, further preferably 0.402-0.407 mg / mL, more preferably 0.403-0.405 mg / mL, the concentration of paeoniflorin solution is preferably 0.51-0.52 mg / mL, further preferably 0.512-0.518 mg / mL, more preferably 0.514-0.516 mg / mL;

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

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

[0066] In this invention, in step 1), the concentration of emodin methyl ether in the mixed reference 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 magnolol 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; and the concentration of rhein is preferably 14.3–15.2 μg / mL, more preferably… The concentration of the herbicide is preferably 14.5–15.0 μg / mL, more preferably 14.6–14.8 μg / mL; the concentration of the herbicide is preferably 19.7–19.9 μg / mL, even more preferably 19.75–19.85 μg / mL, even more preferably 19.78–19.82 μg / mL; and the concentration of the herbicide is preferably 23.5–24.0 μg / mL, even more preferably 23.6–23.9 μg / mL. More preferably, the concentration of hesperidin is 23.7–23.8 μg / mL; more preferably, the concentration of hesperidin is 58.3–61.5 μg / mL; even more preferably, it is 58.6–60.4 μg / mL; more preferably, it is 58.7–59.4 μg / mL; and more preferably, the concentration of paeoniflorin is 148.5–153.8 μg / mL; even more preferably, it is 148.7–152.8 μg / mL; and even more preferably, it is 148.6–151.4 μg / mL.

[0067] In this invention, in step 2), the ratio of the contents of the hemp seed capsule to methanol is preferably 1g:45-55mL, more preferably 1g:47-53mL, and even more preferably 1g:48-50mL.

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

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

[0070] In this invention, in step 2), the contents of the hemp seed capsule are ground before being mixed with methanol. The particle size of the ground contents 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 grinding speed is preferably 800-1000 rpm, more preferably 850-950 rpm, and even more preferably 900 rpm. This grinding speed can ensure the grinding effect and avoid over-grinding that leads to component loss.

[0071] In this invention, regarding the selection of the test sample solution preparation process, the peak shape and extraction rate of 10 components were used as indicators. Methanol, ethanol, 70% methanol, 70% ethanol, 50% methanol, or 50% ethanol were used as extraction solvents. Two extraction methods were investigated: ultrasonic extraction (experimentation times 30 min, 40 min, 50 min, 60 min) and reflux extraction (experimentation times 30 min, 45 min, 60 min, 90 min) were performed at 500 W power and 45 kHz. It was found that the peak shape of the test sample extracted with different ethanol concentrations was slightly inferior to that extracted with different methanol concentrations. Under heating and reflux extraction, the extraction rate of the 10 components was the highest with methanol as the extraction solvent. After 60 min of reflux extraction, the 10 components were basically completely extracted.

[0072] In this invention, the effective components of rhubarb (aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether), the effective component of white peony root (paeoniflorin), and the effective components of immature bitter orange (hesperidin and hesperidin) and magnolia bark (honokiol and magnolol) were selected as indicator components. To conduct a more comprehensive evaluation of the quality of hemp seed capsules, the indicator components of hemp seed, trigonelline and amygdalin, were also investigated. It was found that trigonelline had poor retention in chromatographic columns packed with octadecylsilane-bonded silica gel, but good retention in chromatographic columns packed with amino-bonded silica gel. Amygdalin had good separation in a methanol-water mobile phase system. Both of these components cannot share the same chromatographic column or mobile phase system with other components. Therefore, rhein methyl ether, rhein, magnolol, honokiol, rhein, rhein, novohesperidin, aloe-rhein, hesperidin, and paeoniflorin were ultimately identified as the index components for content determination.

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

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

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

[0076] The electric thermostatic water bath was purchased from Beijing Yongguangming Instrument Co., Ltd., model number DZKW-D-2.

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

[0078] The tangeretin was purchased from Shanghai Shifeng Biotechnology Co., Ltd., batch number S625092451, with a purity of 99.27%.

[0079] The aloe-emodin was purchased from Jiangxi Baicaoyuan Biotechnology Co., Ltd., batch number 000248-202402, with a purity of 98.37%.

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

[0081] Phosphoric acid, formic acid, and acetic acid were purchased from Chengdu Xiya Chemical Co., Ltd., and were of chromatographic grade.

[0082] Methanol and acetonitrile were purchased from Tedia, USA, and were of chromatographic purity.

[0083] The water is Wahaha purified water;

[0084] All six types of hemp seed capsules were purchased from Hunan Lebang Pharmaceutical Co., Ltd., with production batch numbers 240801, 240302, 240602, 240202, 231101 and 230901, and a dosage of 0.35g / capsule.

[0085] Example 1

[0086] Preparation of reference solution: Accurately weigh 4.38 mg of rhein methyl ether, 6.11 mg of magnolol, 8.41 mg of magnolol, 4.12 mg of rhein, 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 into 20 mL volumetric flasks, dissolve in methanol and dilute to the mark to obtain the reference solution;

[0087] Preparation of mixed reference solution: Accurately weigh 4.62 mg of rhein and place it in a 20 mL volumetric flask. Then, accurately pipette 4 mL of rhein methyl ether solution, 0.6 mL of magnolol solution, 0.4 mL of magnolol solution, 2 mL of rhein solution, 1 mL of rhein solution, 1 mL of nobiletin solution, 2 mL of aloe-rhein solution, 3 mL of hesperidin solution, and 6 mL of paeoniflorin solution into the same volumetric flask to obtain the mixed reference solution.

[0088] Preparation of a series of mixed reference solutions: Accurately pipette 5 mL, 4 mL, 3 mL, 2 mL, 1 mL and 0.5 mL of the mixed reference solution into 5 mL volumetric flasks and dilute to volume with methanol.

[0089] Linearity assessment: Accurately pipette 10 μL of the solution from each of the volumetric flasks and perform chromatographic determination. The chromatographic conditions are as follows: detection wavelength 230 nm, mobile phase flow rate 0.9 mL / min, column temperature 30 ℃, mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and gradient elution program as shown in Table 1.

[0090] Table 1 Gradient elution program

[0091] 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 x-axis, denoted as "X", and the peak area as the y-axis, denoted as "Y", a linear regression was performed 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] 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 Sichuan tangerine peel extract 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 rhein Y = 42.96X + 62.42 0.9998 22.96～229.6 rhein methyl ether Y = 39.54X - 6.945 0.9998 4.345～43.45

[0095] Preparation of test solution: Take 10 capsules each of batches 240801, 240302, 240602, 240202, 231101 and 230901 of hemp seed capsules, grind the contents at 1000 rpm until the particle size is 0.15 mm, accurately weigh 1 g of the ground contents and place them in a stoppered conical flask with 50 mL of methanol, heat and reflux at 90 °C for 1 h, then cool to room temperature, weigh, make up the lost weight with methanol, filter, and the sample solution is obtained;

[0096] Content determination: Take 10 μL of each of the above sample solutions and perform chromatographic determination according to the above chromatographic conditions. Substitute the obtained peak areas into the regression equation shown in Table 2, and the results are shown in Table 3.

[0097] Table 3. Detection results of the content of each component in hemp seed capsules

[0098]

[0099]

[0100] Example 2

[0101] Follow the preparation methods for the reference solution and mixed reference solution in Example 1;

[0102] Preparation of a series of mixed reference solutions: Accurately pipette 5 mL, 4 mL, 3 mL, 2 mL, 1 mL and 0.5 mL of the mixed reference solution into 5 mL volumetric flasks and dilute to volume with methanol.

[0103] Linearity assessment: Accurately pipette 10 μL of the solution from each of the volumetric flasks and perform chromatographic determination. The chromatographic conditions are as follows: detection wavelength 220 nm, mobile phase flow rate 0.8 mL / min, column temperature 35 ℃, mobile phase: acetonitrile as mobile phase A, 0.15% phosphoric acid solution as mobile phase B, and gradient elution program as shown in Table 1.

[0104] Using the concentration of each component as the x-axis, denoted as "X", and the peak area as the y-axis, denoted as "Y", a linear regression is performed to obtain the regression equation.

[0105] Preparation of test solution: Take 10 capsules each from batches 240801, 240302, 240602, 240202, 231101 and 230901 of hemp seed capsules, grind the contents at 1000 rpm until the particle size is 0.1 mm, accurately weigh 1 g of the ground contents and place them in a stoppered conical flask with 50 mL of methanol, heat and reflux at 95 °C for 1 h, then cool to room temperature, weigh, make up the lost weight with methanol, filter, and the sample solution is obtained;

[0106] Content determination: Accurately pipette 10 μL of the above sample solution and perform chromatographic determination according to the above chromatographic conditions. Substitute the obtained peak area into the regression equation obtained from the linear relationship study to calculate the content of each component.

[0107] Example 3

[0108] Follow the preparation methods for the reference solution and mixed reference solution in Example 1;

[0109] Preparation of a series of mixed reference solutions: Accurately pipette 5 mL, 4 mL, 3 mL, 2 mL, 1 mL and 0.5 mL of the mixed reference solution into 5 mL volumetric flasks and dilute to volume with methanol.

[0110] Linearity assessment: Accurately pipette 10 μL of the solution from each of the volumetric flasks and perform chromatographic determination. The chromatographic conditions are as follows: detection wavelength 230 nm, mobile phase flow rate 0.9 mL / min, column temperature 25 ℃, mobile phase: acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B, and gradient elution program as shown in Table 1.

[0111] Using the concentration of each component as the x-axis, denoted as "X", and the peak area as the y-axis, denoted as "Y", a linear regression is performed to obtain the regression equation.

[0112] Preparation of test solution: Take 10 capsules each of batches 240801, 240302, 240602, 240202, 231101 and 230901 of hemp seed capsules, grind the contents at 800 rpm until the particle size is 0.12 mm, accurately weigh 1 g of the ground contents and place them in a stoppered conical flask with 50 mL of methanol, heat at 90 °C under reflux for 55 min, then cool to room temperature, weigh, make up the lost weight with methanol, filter, and the sample solution is obtained;

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

[0114] Exclusivity test:

[0115] Follow the preparation methods for the reference solution and mixed reference solution in Example 1;

[0116] Take 10 hemp seed capsules, grind the contents at 1000 rpm until the particle size is 0.15 mm, take 1 g of the ground contents and 50 mL of methanol and put them in a stoppered conical flask, heat at 90 °C and reflux for 1 h, then cool to room temperature, weigh, make up the lost weight with methanol, filter, and the sample solution is obtained.

[0117] According to the preparation method of hemp seed capsules in Chinese patent CN 101642499A, hemp seed capsule samples lacking white peony (fried), hemp seed capsule samples lacking immature bitter orange (fried), hemp seed capsule samples lacking magnolia bark (ginger-processed) and hemp seed capsule samples lacking prepared rhubarb were prepared respectively. Then, the corresponding cathode sample solutions were prepared according to the test sample solution preparation method.

[0118] 10 μL of the mixed reference solution, 10 μL of the test solution, and 10 μL of each cathode sample solution were injected into the high performance liquid chromatograph and detected under the chromatographic conditions of Example 1.

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

[0120] Precision testing:

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

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

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

[0124] Table 4. Relative standard deviations of each component

[0125] rhein methyl ether 0.10 rhein 0.11 magnolol 0.57 honokiol 0.41 emodin 0.23 rhein 0.24 Sichuan tangerine peel extract 0.16 Aloe-emodin 0.18 hesperidin 0.27 paeoniflorin 1.33

[0126] As can be seen from Table 4, the test method described in this invention has good precision.

[0127] Repeatability testing:

[0128] Take 60 capsules of hemp seed capsules from batch 240602 and divide them into six groups. Perform the following steps on each group of hemp seed capsules: take the contents and grind them at 1000 rpm until the particle size is 0.15 mm. Take 1 g of the ground contents and put them in a stoppered conical flask with 50 mL of methanol. Heat and reflux at 90 °C for 1 h. Then cool to room temperature, make up the lost weight with methanol, filter, and the sample solution is obtained.

[0129] 10 μL of each of the six groups was injected into a high-performance liquid chromatograph (HPLC) and analyzed under the chromatographic conditions of Example 1. The results were substituted into the regression equation shown in Table 2 to calculate the content of each component. The average value of the six groups was taken. The results were as follows: the average content of emodin methyl ether was 1.346 mg / g (RSD 1.59%), the average content of rhein was 9.251 mg / g (RSD 1.64%), the average content of magnolol was 0.1547 mg / g (RSD 1.46%), and the average content of magnolol was 0.07535 mg / g (RSD 1.44%). The average content of emodin was... The average content of rhein was 0.7478 mg / g (RSD 1.39%), the average content of chrysophanol was 0.2540 mg / g (RSD 2.13%), the average content of noriheptacortin was 0.2229 mg / g (RSD 2.28%), the average content of aloe-emodin was 0.7538 mg / g (RSD 1.92%), the average content of hesperidin was 1.885 mg / g (RSD 1.13%), and the average content of paeoniflorin was 6.024 mg / g (RSD 1.03%). Therefore, the test method described in this invention has good repeatability.

[0130] Stability test:

[0131] The test solution from the first group in the repeatability test was taken, and 10 μL was injected into the high-performance liquid chromatograph at 0 h, 3 h, 9 h, 15 h, 24 h, and 30 h, respectively, and the peak areas were measured according to the chromatographic conditions of Example 1. The peak areas were recorded. The results were as follows: the RSD of the peak areas of rhein methyl ether was 1.18%, rhein was 1.18%, magnolol was 0.59%, magnolol was 1.11%, rhein was 0.23%, chrysophanic acid was 1.65%, nobiletin was 1.89%, aloe-rhein was 1.23%, hesperidin was 1.63%, and paeoniflorin was 1.81%. This shows that the peak areas of the test solution can still be stably measured after standing at room temperature for 30 h.

[0132] Recovery rate test:

[0133] Take six portions of hemp seed capsules from batch 240602, each weighing 0.5g, and place each of the six samples into a 50mL volumetric flask.

[0134] To each volumetric flask, add 6 mL of a methanol solution containing 0.5169 mg / mL paeoniflorin, 3 mL of a methanol solution containing 0.3602 mg / mL hesperidin, 1.5 mL of a methanol solution containing 0.2510 mg / mL aloe-emodin, 0.3 mL of a methanol solution containing 0.3689 mg / mL norepene terpenoid, 0.4 mL of a methanol solution containing 0.2964 mg / mL rhein, 1.5 mL of a methanol solution containing 0.2506 mg / mL emodin, 0.1 mL of a methanol solution containing 0.4197 mg / mL magnolol, 0.2 mL of a methanol solution containing 0.3789 mg / mL magnolol, 15 mL of a methanol solution containing 0.3008 mg / mL rhein, and 3 mL of a methanol solution containing 0.2282 mg / mL emodin methyl ether, respectively, and dilute to volume with methanol.

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

[0136] Table 5 Results of recovery rate tests for each component.

[0137]

[0138]

[0139]

[0140] As shown in Table 5, the detection method of the present invention has a high recovery rate and good accuracy.

[0141] In summary, the HPLC detection method for simultaneously determining the content of multiple components in hemp seed capsules established in this invention exhibits excellent performance in terms of specificity, precision, repeatability, stability, and recovery rate. It can be used for the quality control and evaluation of hemp seed capsules, providing reliable technical support for the research, development, production, and quality supervision of hemp seed capsules.

[0142] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An HPLC method for simultaneously determining the content of multiple components in hemp seed capsules, characterized in that, Includes the following steps: 1) Mix the reference solution, rhein, and methanol to obtain a mixed reference solution; The reference solutions include emodin methyl ether solution, magnolol solution, honokiol solution, emodin solution, rhein solution, nobiletin solution, aloe-emodin solution, hesperidin solution, and paeoniflorin solution; 2) Mix the contents of the hemp seed capsules with methanol, heat under reflux to obtain the test solution; 3) Pipette the mixed reference solution and the test solution separately into the high-performance liquid chromatograph for detection; The chromatographic conditions are 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; chromatographic column: Pntulips® NT-C18 column, specifications: 250mm×4.6mm, 5μm; Gradient elution procedure: At 0 min, mobile phase A was 14% and mobile phase B was 86%. After 10 minutes, mobile phase A was 14% and mobile phase B was 86%. 20 min, mobile phase A is 20%, mobile phase B is 80%; 25 min, mobile phase A is 20%, mobile phase B is 80%; 35 min, mobile phase A is 30%, mobile phase B is 70%; 55 min, mobile phase A is 45%, mobile phase B is 55%; 65 min, mobile phase A is 55%, mobile phase B is 45%; 75 min, mobile phase A is 70%, mobile phase B is 30%; 90 min, mobile phase A is 80%, mobile phase B is 20%; 95 min, mobile phase A is 80%, mobile phase B is 20%.

2. The HPLC detection method for simultaneously determining the content of multiple components in hemp seed capsules according to claim 1, characterized in that, In step 1), the concentrations of the emodin methyl ether solution and the magnolol solution are 0.21-0.22 mg / mL, 0.30-0.31 mg / mL, 0.42-0.43 mg / mL, 0.20-0.21 mg / mL, 0.30-0.31 mg / mL, 0.39-0.40 mg / mL, 0.23-0.24 mg / mL, 0.40-0.41 mg / mL, and 0.51-0.52 mg / mL, respectively. The solvent for the reference solution is methanol.

3. The HPLC detection method for simultaneously determining the content of multiple components in hemp seed capsules according to claim 2, characterized in that, In step 1), the concentration of rhein in the mixed reference solution is 0.23~0.24 mg / mL.

4. The HPLC detection method for simultaneously determining the content of multiple components in hemp seed capsules according to claim 3, characterized in that, In step 1), the concentrations of emodin methyl ether, magnolol, and honokiol in the mixed reference solution are 43.4–43.9 μg / mL, 9.1–9.2 μg / mL, 8.3–8.5 μg / mL, 19.7–20.7 μg / mL, 14.3–15.2 μg / mL, 19.7–19.9 μg / mL, 23.5–24.0 μg / mL, 58.3–61.5 μg / mL, and 148.5–153.8 μg / mL, respectively.

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

6. The HPLC detection method for simultaneously determining the content of multiple components in hemp seed capsules according to claim 5, characterized in that, In the chromatographic conditions, the concentration of the phosphoric acid solution is 0.05~0.15%.

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