A method for detecting guizhifuling capsules

Abstract

The application provides a method for detecting cassia twig and poria cocos capsules, which comprises the following steps: taking cassia twig and poria cocos capsules for testing; and the chromatographic conditions of the testing comprise: a C 18 a chromatographic column with a size of 4.6*250mm and a 5um C18 column; a mobile phase: acetonitrile (B)-0.2% formic acid water (A); a gradient elution mode: 0-11min (3-3% B), 11-15min (3-10% B), 15-28min (10-15% B), 28-29min (15-16% B), 29-39min (16-20% B), 39-45min (20-23% B), 45-47min (23-23% B), 47-51min (23-35% B), 51-71min (35-43% B), 71-76min (43-95% B), 76-80min (95% B). The application has high specificity, good accuracy, precision and reproducibility, and can rapidly determine the content of various main chemical components in cassia twig and poria cocos capsules.

Description

Technical Field

[0001] This invention relates to the field of quality control of traditional Chinese medicine preparations, and in particular to a method for determining the content of the main components in Guizhi Fuling capsules. Background Technology

[0002] Guizhi Fuling Capsules (National Drug Approval Number Z10950005) are a compound preparation composed of five traditional Chinese medicines: cinnamon twig, poria cocos, peony bark, white peony root, and peach kernel. Clinically, they are mainly used to treat gynecological diseases such as chronic pelvic inflammatory disease, uterine fibroids, endometriosis, ovarian cysts, and dysmenorrhea. Modern pharmacological studies have found that Guizhi Fuling Capsules have anti-tumor, anti-inflammatory, analgesic, smooth muscle regulating, endocrine regulating, and immune-enhancing effects.

[0003] Currently, although there are studies on the content determination of Guizhi Fuling capsules, the number of components determined is small and the methods are scattered. They are not systematic and comprehensive enough to determine the content of the main components of Guizhi Fuling capsules, and cannot meet the ever-increasing quality control requirements. Summary of the Invention

[0004] In view of this, the present invention aims to establish a high-performance liquid chromatography-diode array detector (HPLC-DAD) method to determine the content of the main components in Guizhi Fuling capsules, laying the foundation for improving the quality control of Guizhi Fuling capsules.

[0005] In view of this, the present invention proposes a method for detecting Guizhi Fuling capsules, comprising the following steps:

[0006] The chromatographic conditions for testing the Guizhi Fuling capsule sample include:

[0007] 4.6×250mm, 5μm C 18 Chromatographic column; mobile phase: acetonitrile (B) - 0.2% formic acid water (A); gradient elution mode: 0–11 min (3–3% B), 11–15 min (3–10% B), 15–28 min (10–15% B), 28–29 min (15–16% B), 29–39 min (16–20% B), 39–45 min (20–23% B), 45–47 min (23–23% B), 47–51 min (23–35% B), 51–71 min (35–43% B), 71–76 min (43–95% B), 76–80 min (95% B).

[0008] Specifically, the detection is performed using a high-performance liquid chromatography-diode array detector.

[0009] Specifically, the detection conditions also include: detection wavelengths: 230nm (paeoniflorin, benzoic acid, benzoylpaeoniflorin), 254nm (4-hydroxybenzoic acid, oxidized paeoniflorin), 275nm (gallic acid, ethyl gallate, 1,2,3,4,6-O-pentagalloglucoside, cinnamic acid, paeonol), and 290nm (cinnamaldehyde).

[0010] Furthermore, the chromatographic conditions also include: a flow rate of 0.95–1.00 mL / min and a column temperature of 25–30 °C.

[0011] Specifically, the preferred flow rate is 1.0 mL / min, the column temperature is 30 °C, and the injection volume is 10 μL.

[0012] Furthermore, the preparation of the Guizhi Fuling capsule test sample is as follows: the contents of the Guizhi Fuling capsule are sieved, extracted and centrifuged to obtain the test sample solution.

[0013] Specifically, the preparation of the Guizhi Fuling capsule test sample is as follows: Take the contents of the Guizhi Fuling capsule, sieve, weigh 0.5g, add 25mL of 30-80% methanol, weigh, reflux extract for 30min, cool to room temperature, add weight, shake well, and extract at 14000r·min. -1 Centrifuge for 5 minutes to obtain the supernatant.

[0014] Specifically, the 30-80% methanol is selected from 30-70% methanol, preferably 50% methanol.

[0015] Furthermore, the aforementioned method for detecting Guizhi Fuling capsules also includes the preparation and determination of a reference solution.

[0016] Specifically, the reference standard in the reference solution may be selected from one or more of paeoniflorin, paeonol, benzoylpaeoniflorin, oxypaeoniflorin, gallic acid, 1,2,3,4,6-O-pentagalloglucoside, 4-hydroxybenzoic acid, ethyl gallate, and benzoic acid.

[0017] Furthermore, the chromatographic conditions for determining the reference solution include: 4.6 × 250 mm, 5 μm C5 18Chromatographic column; mobile phase: acetonitrile (B) - 0.2% formic acid water (A); gradient elution mode: 0–11 min (3–3% B), 11–15 min (3–10% B), 15–28 min (10–15% B), 28–29 min (15–16% B), 29–39 min (16–20% B), 39–45 min (20–23% B), 45–47 min (23–23% B), 47–51 min (23–35% B), 51–71 min (35–43% B), 71–76 min (43–95% B), 76–80 min (95% B). Detection wavelengths: 230 nm (paeoniflorin, benzoic acid, benzoylpaeoniflorin), 254 nm (4-hydroxybenzoic acid, oxidized paeoniflorin), 275 nm (gallic acid, ethyl gallate, 1,2,3,4,6-O-pentagalloglucoside, cinnamic acid, paeonol), 290 nm (cinnamaldehyde). Flow rate: 1.0 mL / min, column temperature: 30℃, injection volume: 10 μL.

[0018] Optionally, the reference solution includes a mixed reference solution of gallic acid, paeoniflorin, 1,2,3,4,6-O-pentagalloglucoside, paeonol, and cinnamaldehyde, as well as a mixed reference solution of oxidized paeoniflorin, benzoic acid, benzoyl paeoniflorin, ethyl gallate, cinnamic acid, and 4-hydroxybenzoic acid.

[0019] Specifically, the preparation of the mixed reference solution of gallic acid, paeoniflorin, 1,2,3,4,6-O-pentagalloglucoside, paeonol, and cinnamaldehyde is as follows: Accurately weigh 10.01 mg of gallic acid, 27.12 mg of paeoniflorin, 8.01 mg of 1,2,3,4,6-O-pentagalloglucoside, and 13.15 mg of paeonol, and place them together in a 25 mL volumetric flask. Accurately weigh 9.20 mg of cinnamaldehyde into a 10 mL volumetric flask, dissolve it in methanol, dilute to the mark, and shake well. Accurately transfer 2 mL of this solution into the aforementioned 25 mL volumetric flask, and dilute to the mark with 50% methanol to obtain the mixed reference stock solution of gallic acid, paeoniflorin, 1,2,3,4,6-O-pentagalloglucoside, paeonol, and cinnamaldehyde. Accurately measure 5, 4, 3, 2.5, 2, and 1 mL of the mixed reference stock solution into 5 mL volumetric flasks, dilute to volume with 50% methanol, and shake well to obtain the final product.

[0020] The preparation of the mixed reference solution of paeoniflorin oxide, benzoic acid, benzoyl paeoniflorin, ethyl gallate, cinnamic acid, and 4-hydroxybenzoic acid is as follows: Accurately weigh 11.01 mg of paeoniflorin oxide, 7.52 mg of benzoic acid, and 14.48 mg of benzoyl paeoniflorin, and place them in a 25 mL volumetric flask. Accurately weigh 15.55 mg of ethyl gallate, 13.73 mg of cinnamic acid, and 10.08 mg of 4-hydroxybenzoic acid, and place them in a 10 mL volumetric flask. Dissolve and dilute to volume with 50% methanol, and shake well. Accurately transfer 2 mL of this solution to the aforementioned 25 mL volumetric flask, and dilute to the mark with 50% methanol. Shake well to obtain the mixed reference stock solution of paeoniflorin oxide, benzoic acid, benzoyl paeoniflorin, ethyl gallate, cinnamic acid, and 4-hydroxybenzoic acid. Accurately transfer 5, 4, 3, 2.5, 2, and 1 mL of this mixed reference standard stock solution into 25 mL volumetric flasks, dilute to the mark with 50% methanol, and shake well to obtain the final product.

[0021] Take the above mixed reference solution and the test solution, inject them into a liquid chromatograph, and integrate the corresponding peak areas in the chromatogram. Calculate the content of paeoniflorin, paeonol, benzoylpaeoniflorin, oxypaeoniflorin, gallic acid, 1,2,3,4,6-O-pentagalloglucoside, 4-hydroxybenzoic acid, ethyl gallate, or benzoic acid in the test solution using the external standard method.

[0022] Compared with other determination methods, the high-performance liquid chromatography-diode array detector (HPLC-DID) technique established in this invention can determine 11 major chemical components of Guizhi Fuling capsules as shown in the table below. After methodological, intermediate precision and robustness tests, the established methodology has been proven to be reliable, laying the foundation for improving the quality control of Guizhi Fuling capsules.

[0023] The method described in this invention exhibits good linearity within the determined mass concentration range for the determination of 11 major chemical components in Guizhi Fuling capsules: paeoniflorin, paeonol, benzoylpaeoniflorin, oxypaeoniflorin, gallic acid, 1,2,3,4,6-O-pentagalloglucoside, 4-hydroxybenzoic acid, ethyl gallate, and benzoic acid. The correlation coefficients are all greater than 0.99, the average recovery rate (n=6) ranges from 96.95% to 100.84%, and the RSD ranges from 0.56% to 1.55%. Therefore, this invention demonstrates strong specificity, accuracy, precision, and reproducibility. This method can rapidly determine the content of 11 major chemical components in Guizhi Fuling capsules and is worthy of promotion, holding significant importance for further improving the quality control of Guizhi Fuling capsules. Attached Figure Description

[0024] Figure 1 The chromatogram is for gradient elution condition 1;

[0025] Figure 2The chromatogram for gradient elution condition 2 is shown.

[0026] Figure 3 The chromatogram for gradient elution condition 3 is shown.

[0027] Figure 4 Chromatograms of the sample and reference standard under gradient elution condition 4; where,

[0028] 1: Sample 230nm; 2: Sample 254nm; 3: Sample 275nm; 4: Sample 290nm; 5: Reference standard;

[0029] A: Gallic acid; B: 4-hydroxybenzoic acid; C: Oxypenoside; D: Paeoniflorin; E: Ethyl gallate; F: 1,2,3,4,6-O-pentagalloglucoside; G: Benzoic acid; H: Cinnamic acid; I: Benzoylpaeoniflorin; J: Cinnamaldehyde; K: Paeonol.

[0030] Figure 5 This is the chromatogram of the cinnamon twig-free negative preparation of the present invention.

[0031] Figure 6 This is a chromatogram of the negative chromatogram of the preparation of the present invention without white peony root and peony bark.

[0032] Figure 7 These are chromatograms obtained by the three instruments used in this invention.

[0033] Instrument 1 is an Anglient 1260 binary pump, Instrument 2 is a Thermo U3000, and Instrument 3 is an Anglient 1260 quaternary pump. Detailed Implementation

[0034] Unless otherwise specified, all conditions in this invention shall be carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the raw materials, reagents or instruments used shall be conventional products that can be obtained commercially.

[0035] It is particularly important to note that similar substitutions and modifications made to this invention are obvious to those skilled in the art, and they are all considered to be included in this invention. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0036] The following will provide a detailed description of the experimental content.

[0037] System suitability and methodological experiments for the detection of Guizhi Fuling capsules

[0038] 1. Experimental Materials

[0039] 1.1 Experimental Apparatus

[0040] Agilent 1260 binary pump HPLC system (Agilent Technologies, Inc.); Agilent 1260 quaternary pump HPLC system (Agilent Technologies, Inc.); Thermo Ultimate 3000 autosampler HPLC system with quaternary pump (Thermo Fisher Scientific); 0.0001 ppm balance (Mettler Toledo 204E); 0.0001 ppm balance (Mettler Toledo XS205DU); Centrifuge 5424R high-speed centrifuge (Eppendorf); Pacific TⅡ7 ultrapure water system (Thermo Fisher Scientific); water bath (R502B, Shanghai Shengsheng Technology Co., Ltd.); Mettler AL204 electronic analytical balance (Mettler-Toledo Shanghai Co., Ltd.); Thermo Acclaim TM 120C 18 Chromatographic column (4.6×250mm, 5μm).

[0041] 1.2 Reagents and Test Chemicals

[0042] Paeoniflorin (batch number 110736-201842, content calculated as 97.4%), benzoic acid (batch number 100419-201703, content calculated as 99.9%), gallic acid (batch number 110831-201605, content calculated as 90.8%), cinnamaldehyde (batch number 110710-201821, content calculated as 99.6%), paeonol (batch number 110708-201407, content calculated as 99.6%). The following were purchased from the National Institutes for Food and Drug Control (NIFDC) at 9.9% purity: 4-hydroxybenzoic acid (batch number 120M53353V, content 99.9%) and ethyl gallate (batch number MKBH3037V, content 96%): SIGMA-ALDRICH; 1,2,3,4,6-O-pentagalloyl glucose (batch number P3432E7C1, content 98%): Shanghai Acmec Biochemical; paeoniflorin oxide (batch number 180502, content 98%) and cinnamic acid (batch number 180607, content 98.8%): Nanjing Senbeijia Biotechnology Co., Ltd.; benzoyl paeoniflorin (batch number 18020501, content 98%): Chengdu Pufeide Biotechnology Co., Ltd.; methanol and acetonitrile were of chromatographic grade, formic acid was of analytical grade, and ultrapure water was used. Guizhi Fuling Capsules (batch numbers 171001, 181001, 180901, produced by Jiangsu Kangyuan Pharmaceutical Co., Ltd.).

[0043] 2. Methods and Results

[0044] 2.1 Preparation of the test solution

[0045] 2.1.1 Investigation of the preparation method of the test sample

[0046] (1) Investigation of extraction solvent

[0047] Preparation of the test solution: Accurately weigh approximately 0.25 g of the contents of this product (passed through a No. 4 sieve). Extract in parallel with 30%, 50%, 70%, and 25 mL extraction solvents, respectively. Sonicate for 30 min, cool, weigh, and replenish the lost weight with methanol. Shake well, centrifuge at 14000 r / min for 5 min, and collect the supernatant to obtain the test solution. Inject two parallel injections under the chromatographic conditions described in section 2.3.2, and take the average peak area. The extraction efficiency is evaluated by the ratio of peak area (S) to sample weight (g) (S / g). The results are shown in Table 1.

[0048] Table 1 Results of the extraction solvent investigation

[0049]

[0050]

[0051] Results: Table 1 shows that the RSD value of F(S / g) is 2.42% > 2%. The S / g value is the highest when the extraction solvent is 50% methanol. Furthermore, impurities are present near the gallic acid peak when 90% methanol and pure methanol are used as extraction solvents. Therefore, 50% methanol was selected as the extraction solvent.

[0052] (2) Examination of extraction methods

[0053] Preparation of the test solution: Accurately weigh approximately 0.25 g of the contents of this product (passed through a No. 4 sieve) and place it in a stoppered conical flask. Accurately add 25 mL of 50% methanol and weigh again. Extract by ultrasonication and reflux for 30 min each. Cool and weigh again. Make up the weight loss with 50% methanol, shake well, and centrifuge at 14000 r / min for 5 min. Collect the supernatant to obtain the test solution. Prepare two parallel solutions and inject them twice under the chromatographic conditions in section 2.3.2. Take the average peak area. The extraction efficiency is evaluated by the ratio of peak area (S) to sample weight (g) (S / g). The results are shown in Table 2.

[0054] Table 2 Results of the Extraction Method Examination

[0055]

[0056] Results: Table 2 shows that the RSD values ​​of B(S / g) and F(S / g) are 2.73% and 5.45% respectively, which are greater than 2%, and reflux extraction is more efficient than ultrasonic extraction. Therefore, reflux extraction was chosen.

[0057] (3) Examination of reflux time

[0058] Preparation of the mixed reference solution: Accurately weigh appropriate amounts of gallic acid reference standard (A), 4-hydroxybenzoic acid reference standard (B), paeoniflorin oxide reference standard (C), paeoniflorin reference standard (D), ethyl gallate reference standard (E), 1,2,3,4,6-O-pentagalloglucoside reference standard (F), benzoic acid reference standard (G), cinnamic acid reference standard (H), benzoylpaeoniflorin reference standard (I), cinnamaldehyde reference standard (J), and paeonol reference standard (K). Prepare a mixed reference solution containing A (76.1 μg / mL), B (4.3 μg / mL), C (29.6 μg / mL), D (255.2 μg / mL), E (11.3 μg / mL), F (61.6 μg / mL), G (24.5 μg / mL), H (10.4 μg / mL), I (36.6 μg / mL), J (56.7 μg / mL), and K (94.0 μg / mL) by adding 50% methanol.

[0059] Preparation of the test solution: Accurately weigh approximately 0.25 g of the contents of this product (passed through a No. 4 sieve), add 25 mL of 50% methanol, and reflux for extraction for 10, 20, 30, 40, and 50 min respectively. Cool, weigh, and replenish the lost weight with methanol. Shake well and centrifuge at 14000 r / min for 5 min. Collect the supernatant to obtain the test solution. Prepare two parallel aliquots and inject them twice under the chromatographic conditions described in section 2.3.2. Take the average peak area. Calculate the content using the single-point external standard method. The results are shown in Table 3.

[0060] Table 3 Results of the reflux time investigation

[0061]

[0062] Results: Table 3 shows that the RSD values ​​of the extraction efficiencies of each component under reflux extraction for 10, 20, 30, 40 and 50 min are all <2%, which means that the length of reflux time has little effect on the extraction efficiency of each component under reflux extraction. However, the extraction efficiency of components C and F increases with the extension of reflux time. Considering all factors, reflux extraction for 30 min is selected.

[0063] (4) Investigation on the multiple extraction solvent

[0064] Preparation of the test solution: Accurately weigh approximately 0.5 g, 0.33 g, 0.25 g, and 0.20 g of the contents of this product (passed through a No. 4 sieve), place them in stoppered conical flasks, accurately add 25 mL of 50% methanol, reflux for 30 min, cool, weigh, replenish the lost weight with methanol, shake well, centrifuge at 14000 r / min for 5 min, and collect the supernatant to obtain the test solution. Prepare two parallel solutions, inject two parallel injections under the chromatographic conditions in section 2.3.2, and take the average peak area. Calculate the content using the external standard one-point method. The results are shown in Table 4.

[0065] Table 4 Results of Solvent Dosage Comparison

[0066]

[0067]

[0068] Results: As can be seen from Table 4, the content of each component to be tested is not significantly affected by the solvent volume ratio. Therefore, 50 times the volume of 50% methanol solution was selected for extraction.

[0069] 2.1.2 Final preparation method of the test solution

[0070] Weigh approximately 0.5g of the contents of Guizhi Fuling capsules (passed through a No. 4 sieve), accurately add 25mL of 50% methanol, reflux for 30min, cool, weigh again, replenish the lost weight with methanol, shake well, centrifuge at 14000r / min for 5min, and take the supernatant to obtain the test solution.

[0071] 2.2 Preparation of reference solution

[0072] 1) Preparation of a mixed reference solution of gallic acid, paeoniflorin, 1,2,3,4,6-O-pentagalloglucoside, paeonol, and cinnamaldehyde.

[0073] Accurately weigh 10.01 mg of gallic acid, 27.12 mg of paeoniflorin, 8.01 mg of 1,2,3,4,6-O-pentagalloglucoside, and 13.15 mg of paeonol, and place them together in a 25 mL volumetric flask. Accurately weigh 9.20 mg of cinnamaldehyde into a 10 mL volumetric flask, dissolve it in methanol, dilute to the mark, and shake well. Accurately transfer 2 mL of this solution to the aforementioned 25 mL volumetric flask, and dilute to the mark with 50% methanol to obtain a mixed reference stock solution of gallic acid, paeoniflorin, 1,2,3,4,6-O-pentagalloglucoside, paeonol, and cinnamaldehyde. Accurately measure 5, 4, 3, 2.5, 2, and 1 mL of the mixed reference stock solution into separate 5 mL volumetric flasks, dilute to the mark with 50% methanol, and shake well to obtain the series of reference solutions 1.

[0074] 2) Preparation of a mixed reference solution of paeoniflorin oxide, benzoic acid, benzoylpaeoniflorin, ethyl gallate, cinnamic acid, and 4-hydroxybenzoic acid.

[0075] Accurately weigh 11.01 mg of paeoniflorin oxide, 7.52 mg of benzoic acid, and 14.48 mg of benzoyl paeoniflorin, and place them in a 25 mL volumetric flask. Accurately weigh 15.55 mg of ethyl gallate, 13.73 mg of cinnamic acid, and 10.08 mg of 4-hydroxybenzoic acid, and place them in a 10 mL volumetric flask. Dissolve and dilute to volume with 50% methanol, and shake well. Accurately transfer 2 mL of this solution to the aforementioned 25 mL volumetric flask, and dilute to the mark with 50% methanol. Shake well to obtain a mixed reference stock solution of paeoniflorin oxide, benzoic acid, benzoyl paeoniflorin, ethyl gallate, cinnamic acid, and 4-hydroxybenzoic acid. Accurately transfer 5, 4, 3, 2.5, 2, and 1 mL of this mixed reference stock solution to 25 mL volumetric flasks, and dilute to the mark with 50% methanol. Shake well to obtain a series of reference solutions 2.

[0076] 2.3 Chromatographic conditions

[0077] 2.3.1 Optimization of chromatographic conditions

[0078] Chromatographic conditions

[0079] Column: Thermo Acclaim TM 120C18 column (4.6mm × 250mm, 5μm)

[0080] Column temperature: 30℃

[0081] Flow rate: 1 mL / min

[0082] Injection volume: 10 μL

[0083] Detection wavelengths: 230nm (paeoniflorin, benzoic acid, benzoylpaeoniflorin), 254nm (4-hydroxybenzoic acid, oxidized paeoniflorin), 275nm (gallic acid, ethyl gallate, 1,2,3,4,6-O-pentagalloglucoside, cinnamic acid, paeonol), 290nm (cinnamaldehyde).

[0084] (1) Gradient elution conditions 1

[0085]

[0086] like Figure 1 As shown, peak B (4-hydroxybenzoic acid) and peak C (oxidosperidin) in the chromatogram of the test solution could not be separated. The report shows that the resolution of peak C was only 0.98, and the resolution of peak E (ethyl gallate) was 1.44 < 1.5. Therefore, further optimization of chromatographic conditions is required.

[0087] (2) Gradient elution conditions 2

[0088]

[0089] like Figure 2 As shown, in the chromatogram of the test solution, peak 6 (1,2,3,4,6-O-pentagalloglucoside) and peak 7 (benzoic acid) co-eluted, while peak 8 (cinnamic acid) and peak 9 (benzoylpaeoniflorin) were not separated. The chromatographic conditions need further optimization.

[0090] (3) Gradient elution conditions 3

[0091]

[0092] like Figure 3 As shown, in the chromatogram of the test solution, peak 6 (1,2,3,4,6-O-pentagalloglucopyranoside) and peak 7 (benzoic acid) still co-eluted, so the chromatographic conditions need to be further optimized.

[0093] (4) Gradient elution conditions 4

[0094]

[0095] like Figure 4 As shown, all chromatographic peaks in the chromatogram of the test solution are well separated.

[0096] 2.3.2 Final chromatographic conditions

[0097] Thermo Acclaim TM 120C 18 Chromatographic column (4.6×250mm, 5μm); mobile phase: acetonitrile (B)-0.2% formic acid water (A), gradient elution: 0–11 min (3–3% B), 11–15 min (3–10% B), 15–28 min (10–15% B), 28–29 min (15–16% B), 29–39 min (16–20% B), 39–45 min (20–23% B), 45–47 min (23–23% B), 47–51 min (23–35% B), 51–71 min (35–43% B), 71–76 min (43–95% B), 76–80 min (95% B). Flow rate: 1 mL / min. -1 Column temperature: 30℃; Injection volume: 10μL; Detection wavelengths: 230nm (paeoniflorin, benzoic acid, benzoylpaeoniflorin), 254nm (4-hydroxybenzoic acid, oxidized paeoniflorin), 275nm (gallic acid, ethyl gallate, 1,2,3,4,6-O-pentagalloglucoside, cinnamic acid, paeonol), 290nm (cinnamaldehyde). See the chromatograms of the samples and reference standards below. Figure 4 .

[0098] 2.4 Methodological Examination

[0099] 2.4.1 Specificity Test

[0100] The qualitative analysis results show that 4-hydroxybenzoic acid and paeonol are not specific, while gallic acid, paeoniflorin oxidase, paeoniflorin, ethyl gallate, 1,2,3,4,6-O-pentagalloylglucose, benzoic acid, and benzoylpaeoniflorin are derived from white peony root and peony bark, and cinnamic acid and cinnamaldehyde are derived from cinnamon twig.

[0101] Negative control preparation A (without cinnamon twig) and negative control preparation B (without white peony root and moutan bark) were prepared according to the method described in section "2.1". 10 μL each of negative control solutions A and B, and the mixed control solution prepared in section "2.2", were accurately injected into the high-performance liquid chromatograph. The results showed that no impurity peaks appeared at the retention time of the analyte in the chromatogram of the negative control samples, indicating good specificity. See [link to relevant documentation]. Figure 5 and Figure 6 .

[0102] The results showed that the cinnamon twig-free negative control preparation showed no interference from J (cinnamaldehyde 290 nm) and H (cinnamic acid 275 nm) at the corresponding retention times in the chromatograms. The white peony root and peony bark negative control preparations showed no interference from gallic acid (A), paeoniflorin oxide (C), paeoniflorin (D), ethyl gallate (E), 1,2,3,4,6-O-pentagalloglucoside (G), benzoic acid (H), or benzoylpeoniflorin (I) at the corresponding retention times in the chromatograms.

[0103] 2.4.2 System Suitability Test

[0104] System suitability was examined at the beginning of the method validation. The mixed reference solution was precisely pipetted and injected into the high-performance liquid chromatograph (HPLC). The injection was repeated six times, with 10 μL injected each time. Peak areas were measured, and RSDs were calculated. The RSD values ​​were all less than 0.6%. The test solution was precisely pipetted 10 μL and injected twice consecutively. The resolution R between the analyte and adjacent chromatographic peaks was greater than 1.5. The results are shown in Table 5.

[0105] Table 5. Results of System Suitability Tests (n=6)

[0106]

[0107] 2.4.3 Examination of Linear Relationships

[0108] The preparation of the series of reference solutions is the same as in section "2.2" above. Two parallel injections are performed under the chromatographic conditions described in section "2.3.2," and the average peak area is taken. The injection concentration (μg·mL⁻¹) is used as the reference standard. -1Using the x-axis (X) and the y-axis (Y), regression calculations were performed, and each component showed a good linear relationship within a certain concentration range. The results are shown in Table 6.

[0109] Table 6 Results of Linearity Examination

[0110]

[0111] 2.4.4 Repeatability Test

[0112] Six portions of the contents of Guizhi Fuling Capsules (batch number 171001) were accurately weighed. Test solutions were prepared according to the method described in section "2.1". The content was determined under the chromatographic conditions described in section "2.3.2", with two parallel injections. The average peak area was used to calculate the content using the regression equation. See Table 7. The RSD values ​​of all components were less than 0.5%, indicating good repeatability of the method.

[0113] Table 7 Results of repeatability tests (n=6)

[0114]

[0115] 2.4.5 Precision Test

[0116] Take one sample solution and inject it six times consecutively under the chromatographic conditions in section “2.3.2”. Calculate the RSD value of the peak area of ​​each component. The results show that the RSD value of the peak area of ​​each component is less than 1%, indicating that the instrument has good precision. See Table 8.

[0117] Table 8. Precision test results (n=6)

[0118]

[0119]

[0120] 2.4.6 Stability Test

[0121] One sample of the test solution was injected at 0, 6, 12, 18, 24, 30, and 42 hours. The RSD values ​​of the peak areas of each component were all <0.5%, indicating that the test solution was stable within 42 hours. See Table 9.

[0122] Table 9. Stability test results (n=7)

[0123]

[0124] 2.4.7 Spiking Recovery Test

[0125] Accurately weigh 13.50 mg of benzoylpaeoniflorin, 7.10 mg of benzoic acid, and 10.29 mg of paeoniflorin oxide, and place them together in a 20 mL volumetric flask. Dissolve and dilute to volume with 50% methanol, and shake well. Accurately transfer 3 (low), 4 (medium), and 5 (high) mL of this solution into separate 25 mL volumetric flasks. Accurately weigh 10.51 mg of ethyl gallate, 10.57 mg of cinnamic acid, and 7.49 mg of 4-hydroxybenzoic acid, and place them together in a 25 mL volumetric flask. Dissolve and dilute to volume with 50% methanol, and shake well. Accurately transfer 10 mL of this solution into a separate 25 mL volumetric flask, and dilute to the mark with 50% methanol. Shake well. Take 2 (low), 3 (medium), and 4 (high) mL of this solution and place them into the aforementioned 25 mL low, medium, and high volumetric flasks, respectively. Dilute to volume with 50% methanol to obtain mixed reference solution 3.

[0126] Accurately weigh 13.04 mg of cinnamaldehyde reference standard and place it in a 25 mL volumetric flask. Dissolve and dilute to volume with methanol, then shake well. Accurately weigh 9.07 mg of gallic acid, 22.85 mg of paeoniflorin, 7.12 mg of 1,2,3,4,6-O-pentagalloglucoside, and 10.92 mg of paeonol. Place them together in a 20 mL volumetric flask, add 4 mL of the prepared cinnamaldehyde solution, dissolve and dilute to volume with 50% methanol, and shake well to obtain a high-concentration mixed reference standard solution. Accurately weigh 9.06 mg of gallic acid, 23.40 mg of paeoniflorin, 7.17 mg of 1,2,3,4,6-O-pentagalloglucoside, and 11 mg of paeonol. 26 mg of cinnamaldehyde was placed in a 25 mL volumetric flask, and 3.5 mL of prepared cinnamaldehyde solution was added. The solution was dissolved and diluted to volume with 50% methanol, and the mixture was shaken well to obtain a mixed reference solution of intermediate concentration. 7.03 mg of gallic acid, 12.07 mg of paeoniflorin, 5.60 mg of 1,2,3,4,6-O-pentagalloglucoside, and 8.10 mg of paeonol were accurately weighed and placed in a 25 mL volumetric flask. 2 mL of prepared cinnamaldehyde solution was added, and the solution was dissolved and diluted to volume with 50% methanol, and the mixture was shaken well to obtain a mixed reference solution of low concentration. Finally, four mixed reference solutions of high, medium, and low concentrations were obtained for later use.

[0127] Take approximately 0.25g of the contents of Guizhi Fuling Capsules (batch number 171001) with known content, accurately weigh it, and add 5mL of the above-prepared mixed reference solution 3 of high, medium and low concentrations respectively. Prepare sample solutions according to the method under "2.1", and prepare 3 copies of each concentration in parallel.

[0128] Accurately weigh approximately 0.25 g of the contents of Guizhi Fuling Capsules (batch number 171001), and add 5 mL of the prepared mixed reference solutions of high, medium, and low concentrations as described above. Prepare sample solutions according to the method in section "2.1", with three replicates prepared for each concentration.

[0129] Inject the sample according to the chromatographic conditions described in section "2.3.2", using two parallel injections. The average peak area was then used in the regression equation to calculate the content. The average recovery rates of each component ranged from 96.95% to 100.84%, with RSD values ​​all less than 2%, meeting the recovery limits specified in the 2015 edition of the Chinese Pharmacopoeia. This indicates that the established method has good accuracy; see Table 10 for details.

[0130] Table 10 Results of the recovery test

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] 2.5 Durability Test

[0139] 2.5.1 Intermediate Precision

[0140] Table 11 Intermediate Precision Experiment Personnel and Instruments

[0141]

[0142] Using Thermo Acclaim™ 120C 18 (4.6×250mm, 5μm) chromatographic column. The effect of random variation on precision was investigated: different analysts used different instruments to determine the samples on different days. The content was calculated using the external standard two-point method, and the sample peak area was between the peak areas of the two reference standards. The RSD values ​​of each component were calculated. The results showed that the RSD of each component was less than 4%, and the resolution R was greater than 1.5, indicating that random variation had a small impact on precision. The results are shown in the table below. Figure 7 See Table 12.

[0143] Table 12 Intermediate Precision Results

[0144]

[0145] 2.5.2 Column robustness

[0146] Three identical chromatographic columns were used, namely Thermo Acclaim. TM 120C18 (4.6×250mm, 5μm) The robustness of the chromatographic column was investigated. The RSD values ​​of the contents were calculated, and the results showed that the RSD of each component was less than 5%, indicating that different chromatographic columns of the same type had little impact on the determination results of the contents of each component, as shown in Table 13.

[0147] Table 13 Results of the column robustness test

[0148]

[0149] 2.5.3 Column Temperature and Flow Rate

[0150] Table 14 Column Temperature and Flow Rate Durability Test

[0151]

[0152] Chromatographic conditions can be: fixed column type Thermo Acclaim TM 120C 18 (4.6 × 250 mm, 5 μm). The column temperature was fixed at 30 °C. The flow rate could be between 0.95 and 1.00 mL / min. -1 Fluctuations. The instrument can switch between Angilent 1260 binary pump, Thermo U3000 and Angilent 1260 quaternary pump.

[0153] 2.6 Sample Content Determination

[0154] Take this product (batch numbers 171001, 181001, 180901), prepare two sample solutions in parallel according to the method in section "2.1", inject the two solutions in parallel according to the chromatographic conditions in section "2.3.2", take the average peak area and substitute it into the regression equation to calculate the content. The results are shown in Table 15.

[0155] Table 15 Results of content determination in three batches of samples (mg·g) -1 )

[0156]

[0157] 3 Discussion

[0158] Selection of components for analysis: The experiment selected several components with proven activity, primarily 11 components from white peony root, moutan bark, and cinnamon twig. Since the triterpenoid acid content in Poria cocos is relatively low, UPLC-QQQ-MS / MS was chosen for detection.

[0159] Optimization of chromatographic conditions: Due to the varying maximum absorption wavelengths of the analytes, a DAD detector was used to separate the components into four groups based on ±5 nm of their maximum absorption wavelengths. By altering the mobile phase gradient, two pairs of difficult-to-separate chromatographic peaks—paeoniflorin and 4-hydroxybenzoic acid, and 1,2,3,4,6-O-pentagalloglucoside and benzoic acid—were separated.

[0160] Optimization of sample extraction conditions: The RSD values ​​of extraction efficiencies for each component under different extraction conditions show that the extraction method has a significant impact on the extraction efficiency of 1,2,3,4,6-O-pentagalloglucopyranose (RSD > 5%). Since 1,2,3,4,6-O-pentagalloglucopyranose is a glycoside, reflux extraction may be superior to ultrasonic extraction. Furthermore, it was found that this component could not pass through a 0.22 μm microporous membrane, suggesting possible adsorption. When different concentrations of methanol (30–100%) were used as extraction solvents, 90% methanol and pure methanol were found to have more impurity peaks; therefore, 50% methanol was the preferred extraction solvent. Other extraction conditions had a relatively small impact on the extraction efficiency of the other 10 components (RSD < 3%). After comprehensive consideration, reflux extraction with 50% methanol for 30 min was ultimately selected, followed by centrifugation and collection of the supernatant.

[0161] Methodological evaluation: After evaluation of methodology, intermediate precision, and robustness, the established methodology was proven to be reliable.

[0162] Determination methods for the content of 11 major chemical components in Guizhi Fuling capsules

[0163] A method for determining 11 main components in Guizhi Fuling capsules using high performance liquid chromatography-diode array detector (HPLC-DID) includes the following steps:

[0164] 1. Preparation of the test solution

[0165] Weigh approximately 0.5g of the contents of Guizhi Fuling capsules (passed through a No. 4 sieve), accurately weigh, add 25mL of 50% methanol, weigh again, reflux for 30min, cool to room temperature, add methanol to make up the weight, shake well, and extract at 14000r·min. -1 Centrifuge for 5 minutes, and the supernatant is the sample solution.

[0166] 2. Preparation of reference solution

[0167] 1) Preparation of a mixed reference solution of gallic acid, paeoniflorin, 1,2,3,4,6-O-pentagalloglucoside, paeonol, and cinnamaldehyde.

[0168] Accurately weigh 10.01 mg of gallic acid, 27.12 mg of paeoniflorin, 8.01 mg of 1,2,3,4,6-O-pentagalloglucoside, and 13.15 mg of paeonol, and place them together in a 25 mL volumetric flask. Accurately weigh 9.20 mg of cinnamaldehyde into a 10 mL volumetric flask, dissolve it in methanol, dilute to the mark, and shake well. Accurately transfer 2 mL of this solution to the aforementioned 25 mL volumetric flask, and dilute to the mark with 50% methanol to obtain a mixed reference stock solution of gallic acid, paeoniflorin, 1,2,3,4,6-O-pentagalloglucoside, paeonol, and cinnamaldehyde. Accurately measure 5, 4, 3, 2.5, 2, and 1 mL of the mixed reference stock solution into separate 5 mL volumetric flasks, dilute to the mark with 50% methanol, and shake well to obtain the series of reference solutions 1.

[0169] 2) Preparation of a mixed reference solution of paeoniflorin oxide, benzoic acid, benzoylpaeoniflorin, ethyl gallate, cinnamic acid, and 4-hydroxybenzoic acid.

[0170] Accurately weigh 11.01 mg of paeoniflorin oxide, 7.52 mg of benzoic acid, and 14.48 mg of benzoyl paeoniflorin, and place them in a 25 mL volumetric flask. Accurately weigh 15.55 mg of ethyl gallate, 13.73 mg of cinnamic acid, and 10.08 mg of 4-hydroxybenzoic acid, and place them in a 10 mL volumetric flask. Dissolve and dilute to volume with 50% methanol, and shake well. Accurately transfer 2 mL of this solution to the aforementioned 25 mL volumetric flask, and dilute to the mark with 50% methanol. Shake well to obtain a mixed reference stock solution of paeoniflorin oxide, benzoic acid, benzoyl paeoniflorin, ethyl gallate, cinnamic acid, and 4-hydroxybenzoic acid. Accurately transfer 5, 4, 3, 2.5, 2, and 1 mL of this mixed reference stock solution to 25 mL volumetric flasks, and dilute to the mark with 50% methanol. Shake well to obtain a series of reference solutions 2.

[0171] 3 Chromatographic conditions

[0172] ThermoAcclaim TM 120C 18 Chromatographic column (4.6×250mm, 5μm); mobile phase: acetonitrile (B)-0.2% formic acid water (A), gradient elution: 0–11 min (3–3% B), 11–15 min (3–10% B), 15–28 min (10–15% B), 28–29 min (15–16% B), 29–39 min (16–20% B), 39–45 min (20–23% B), 45–47 min (23–23% B), 47–51 min (23–35% B), 51–71 min (35–43% B), 71–76 min (43–95% B), 76–80 min (95% B). Flow rate: 1 mL / min. -1Column temperature: 30℃; Injection volume: 10μL; Detection wavelengths: 230nm (paeoniflorin, benzoic acid, benzoylpaeoniflorin), 254nm (4-hydroxybenzoic acid, oxidized paeoniflorin), 275nm (gallic acid, ethyl gallate, 1,2,3,4,6-O-pentagalloglucoside, cinnamic acid, paeonol), 290nm (cinnamaldehyde). See the chromatograms of the samples and reference standards below. Figure 4 .

[0173] In the above examples, all compounds exhibited good linearity within the measured concentration range, and the average recovery rates met the requirements. The method provided by this invention demonstrates good repeatability, the instrument has good precision, and the components in the sample solution remained stable within 24 hours. These research results provide valuable insights for the qualitative and quantitative study of trace components in complex components or complex matrices of traditional Chinese medicine, and also provide data support for safe and rational drug use in clinical practice.

[0174] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications 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. A method for detecting Guizhi Fuling capsules, characterized in that, Includes the following steps: The chromatographic conditions for testing the Guizhi Fuling capsule sample include: 4.6×250mm, 5μm C 18 Chromatographic column; Mobile phase: Acetonitrile B - 0.2% formic acid water A; Gradient elution mode: 0–11 min, 3–3% B; 11–15 min, 3–10% B; 15–28 min, 10–15% B; 28–29 min, 15–16% B; 29–39 min, 16–20% B; 39–45 min, 20–23% B; 45–47 min, 23–23% B; 47–51 min, 23–35% B; 51–71 min, 35–43% B; 71–76 min, 43–95% B; 76–80 min, 95% B; The preparation of the Guizhi Fuling capsule test sample was as follows: The contents of the Guizhi Fuling capsule were sieved, weighed (0.5 g), and 25 mL of 30-80% methanol was added. The mixture was weighed, extracted by reflux for 30 min, cooled to room temperature, the weight was added, and the mixture was shaken well. The extraction was then performed at 14000 r·min. -1 Centrifuge for 5 minutes and collect the supernatant. The method also includes the preparation and determination of reference solutions; the reference solutions include a mixed reference solution of gallic acid, paeoniflorin, 1,2,3,4,6-O-pentagalloglucoside, paeonol, and cinnamaldehyde, as well as a mixed reference solution of oxidized paeoniflorin, benzoic acid, benzoyl paeoniflorin, ethyl gallate, cinnamic acid, and 4-hydroxybenzoic acid. The detection conditions also include: the detection wavelength for paeoniflorin, benzoic acid, and benzoylpaeoniflorin is 230 nm; the detection wavelength for 4-hydroxybenzoic acid and oxidized paeoniflorin is 254 nm; the detection wavelength for gallic acid, ethyl gallate, 1,2,3,4,6-O-pentagalloglucoside, cinnamic acid, and paeonol is 275 nm; and the detection wavelength for cinnamaldehyde is 290 nm.

2. The method according to claim 1, characterized in that, The chromatographic conditions also include: a flow rate of 0.95–1.0 mL / min and a column temperature of 25–30 °C.

3. The method according to claim 1, characterized in that, The flow rate was 1.0 mL / min, and the column temperature was 30 °C.

4. The method according to claim 3, characterized in that, The 30-80% methanol is selected from 50% methanol.

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